PA00XCSB.Pdf

Leidos Proprietary

1. EXECUTIVE SUMMARY

A summary of efforts for the planned, ongoing, and completed projects for the Malaria Vaccine Development Program (MVDP) contract for this reporting period are herein detailed. A compiled Gantt chart including activities associated with each of the projects has been created and included as an attachment to this report. Ongoing projects that will continue through FY2019 include two vaccine development projects, the CSP vaccine development project (CSP Vaccine) and liver stage vaccine development project (Liver Stage Vaccine), as well as the clinical study with RH5 (RH5.1 Clinical Study), the latter to assess long-term immunogenicity in RH5.1/AS01 vaccinees. Of note is that while both the CSP and the liver stage vaccine development projects were initiated as epitope-based projects, these have since been realigned to target whole proteins; therefore, the project names have also been realigned to remove “epitope-based.” Expansion of work on the RCR complex into a vaccine development project (RCR Complex) occurred in early FY2019 and this project will continue until the end date of the contract. Lastly, a new project, the RH5.1 human monoclonal antibody identification and development project (RH5.1 Human mAb), was initiated in early FY2019 and will continue until the end date of the contract. Two projects were completed in FY2019, the blood stage epitope-based vaccine development project and the PD1 blockade inhibitor project (PD1 Block Inh). Leidos continues to seek collaborators for information exchange under NDA, reagent exchange under MTA, and collaboration under CRADA, to expand our body of knowledge and access to reagents with minimal cost to the program. This type of collaboration benefits the malaria research community and extends the utility of the contract. The information contained herein is intended to provide technical detail regarding activities conducted within the specified reporting period; however, at the request of USAID, Leidos retains data in quarterly reports for activities completed within the relevant fiscal year. Of note is that GO (Gene Ontology) standards have been adopted for protein naming conventions.

2. CONTRACTS MANAGEMENT AND ADMINISTRATION

2.1. CURRENT BUDGET SUMMARY The budget summary for FY19 is provided in Table 2.1-1. Table 2.1-2 provides FY19 to-date costs against FY19 Annual Work Plan Estimates. Detailed subcontractor spending is provided in the associated Q4 financial report.

MVDP Quarterly Report 2 Leidos Proprietary

MVDP Quarterly Report 3 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product CONTRACTS AND ADMINISTRATION Signed: 5/28/15 Modifications: MOD 1 2/22/16 MOD 2 3/4/16 MOD 3 8/16/16 MOD 4 2/13/17 MVDP Operations Manager key personnel Prime Contract MOD 5 5/1/17 approval notice received 9/13/19. MOD 6 7/18/17 MOD 7 9/28/17 MOD 8 1/09/18 MOD 9 12/20/18 MOD 10 3/1/19 Contractor – Employee Non- Disclosure/Conflict Signed Non-Disclosure/COI Agreements i. Delivered of Interest (COI) are located on SharePoint. Agreements Delivered: 09/3/19 ii. Annual Work Plan Approved: Q1 FY19 Delivered: 01/14/19 vi. Quarterly Reports Q2 FY19 Delivered: 04/15/19 Upcoming Reports: 01/14/2020 Q3 FY19 Delivered: 07/15/19 Q1 FY19 Delivered: 01/14/19 Quarterly Financial vii. Q2 FY19 Delivered: 04/15/19 Upcoming Reports: 01/14/2020 Reports Q3 FY19 Delivered: 07/15/19 Delivered: 10/14/19 viii. Annual Report/Q4 Report Approved: Delivered: The Small Business Small Business H.10.a Subcontracting Plan was Subcontracts Subcontracting Plan included in Leidos’ proposal. Due: Annually in October; Individual Subcontract H.10.b.1 submitted via eSRS by Leidos’ Upcoming Reports: 10/30/18 Reports Small Business Liaison office. Due: Annually in October; Summary Subcontract Upcoming Report: 10/30/19 H.10.b.2 submitted via eSRS by Leidos’ Report Small Business Liaison office. Final Overall Contract ix. N/A Upcoming Report: 5/27/20 Report Held: See Table 2.4-1. Upcoming Meetings: Regularly Biweekly USAID–Leidos C.4.4 Ad hoc meetings held: See scheduled biweekly meetings are held the Update Meetings Table 2.4-1. first and third Wednesdays of each month. Contract Administration Meetings with No actions in the reporting G.4.C Contracting Officer’s period. Representative (COR)

MVDP Quarterly Report 4 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product Due: All contracts and Upon approval of all future deliverables, USAID Development administration deliverables will Leidos will upload to the DEC. At xiv. Experience Information be uploaded to the DEC after contract completion, Leidos will upload all 30 days from approval. task-associated documents as well. ELEMENT 1: PROTOCOL DEVELOPMENT/WHITE PAPERS See section 3.2 regarding abstract/poster xii. Publications/Posters Q1 FY2019: 4 posters submissions. C.3.2.2 New Project Proposals None in the reporting period ELEMENT 2: IMPLEMENTATION OF RESEARCH AND DEVELOPMENT PROJECTS CSP Vaccine Development Project Project Plan approved by USAID on Final Individual Project iii. Approved 1/29/16. descriptions Revised Project Plan approved 04/26/2017. Q1 FY2019: procurement activities Subcontract/Task Order completed for IMV, JHU, VaxDesign, and Awards: VLP Biotech, JHU, VLP Biotech. ADARC, EpiVax, Precision Subcontracts: Q2 FY2019: procurement completed for Antibody, ImmunoVaccine, xii. Procurement of Materials, VaxDesign and ongoing for JHU. VaxDesign Supplies, and Services Q3 FY2019: procurement completed for Other Procurement JHU (passive transfer study). Vehicles: CPC Scientific, Q4 FY2019: procurement completed for Vaxine JHU (passive transfer study) Final Individual Project iv. — Report VLP Biotech Quarterly Reports VLP162 + DPX4 Stability Study Documentation Individual Project Data Provided herein, circulated via email v. (ImmunoVaccine) Sets and/or uploaded to SharePoint. VaxDesign Data Slides CSP Project Update Slides JHU Study Data See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. ix. Other/Ad Hoc Reports — RH5.1/AS01 Vaccine Clinical Study Final Individual Project Plan approved by USAID on iii. Approved Project descriptions 2/17/16. Q1 FY2019: procurement for recently approved activities is ongoing for Oxford. Subcontract/Task Order Procurement of Materials, Q2 FY2019: Oxford procurement xii. Awards: University of Oxford, Supplies, and Services completed. EpiVax Q3 FY2019: none Q4 FY2019: none Final Individual Project iv. — Report

MVDP Quarterly Report 5 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product Schedule and regulatory approvals for late Individual Project Data Provided herein, circulated via email, v. immunogenicity time point Set and/or uploaded to SharePoint. Oxford quarterly reports Oxford data updates See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. ix. Other/Ad Hoc Reports — Trial registered on September 5, 2016 xii. Registration of Trials Completed (ClinicalTrials.gov) Liver Stage Vaccine Development Project Project Plan approved by USAID on Final Individual 6/13/16. Attachment 1 - CD8 platform iii. Approved Project descriptions scouting plan approved by USAID on 6/6/19. Q2 FY2019: procurement completed for Subcontract Awards: SGI (kickoff held 3/27/2019) and GeoVax EpiVax, VaxDesign, Precision Procurement of Materials, (kickoff held 3/7/19). xii. Antibody, Multimeric Supplies, and Services Q3 FY2019: procurement completed for BioTherapeutics, ADARC, ADARC (MVA construct testing). GeoVax Q4 FY2019: none Final Individual Project iv. — Report Individual Project Data VaxDesign, ADARC and Provided herein, circulated via email v. Set GeoVax data sets and/or uploaded to SharePoint. See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. Data report for CRADA with ix. Other/Ad Hoc Reports Leidos/NMRC data exchange NMRC RCR Complex Vaccine Development Project-WEHI

Final Individual Project Project Plan approved by USAID on iii. Approved descriptions 3/21/2019. Q1 FY2019: procurement ongoing with WEHI, SGI, and Precision Antibody Q2 FY2019: Procurement completed for IMV (W2), SGI (kickoff meeting held Pending: WEHI (W3), IMV 3/27/19) and Precision Antibody; ongoing (W3) for WEHI (W2) Procurement of Materials, Q3 FY2019: Procurement completed for xii. Subcontract/Task Order Supplies, and Services Awards: WEHI (W2 in Q3), WEHI (W2); Kickoff meeting with ImmunoVaccine (W2 in Q2), Precision Antibody held in May 2019. SGI and Precision Antibody task order discontinued. Q4 FY2019: Procurement ongoing for WEHI Q4 FY2019: Procurement completed for IMV (W3).

MVDP Quarterly Report 6 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product Final Individual Project iv. — Report Preliminary ELISA dataset for Study W1 Repeat ELISA dataset for Study W1 The preliminary ELISA dataset was GIA for Study W1 circulated to USAID and the repeat ELISA Preliminary ELISA dataset for was discussed in the biweekly meeting and Individual Project Data Study W2 provided herein. v. Set Repeat ELISA dataset for Study An IPT meeting was conducted to discuss W2 Test Bleed WEHI W1/W2 ELISA and GIA results on ELISA dataset for Study W2 September 17, 2019 (US/UK)/September Terminal Bleed 18, 2019 (AUS). Repeat RH5 ELISA for Study W2 GIA-LDH for Study W1 See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-University of Oxford Final Individual Project Project Plan approved by USAID on iii. Approved descriptions 2/13/19. Q1 FY2019: procurement for recently approved activities is ongoing with Oxford Procurement of Materials, Subcontract Award: Q2 FY2019: Oxford procurement xii. Supplies, and Services University of Oxford completed Q3 FY2019: none Q4 FY2019: none Final Individual Project iv. — Report Oxford Quarterly Reports Individual Project Data v. VLP Development Data Provided herein or circulated via email. Set RCR Stability-Matrix-M Data See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. ix. Other/Ad Hoc Reports — RH5.1 Human mAb Identification and Development Final Individual Project Project Plan approved by USAID (regular iii. Approved descriptions elements only) on 1/29/19. Q2 FY2019: Oxford procurement Procurement of Materials, Subcontract Award: xii. completed Supplies, and Services University of Oxford Q3 FY2019: none Final Individual Project iv. — Report Individual Project Data v. Oxford data sets mAb cloning update provided herein Set

MVDP Quarterly Report 7 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. ix. Other/Ad Hoc Reports — Blood-Stage Epitope Vaccine Development Project Plan approved by USAID on Final Individual iii. Approved 3/24/2016. Addendum 1 approved on Project descriptions 6/23/16. Subcontracts Awards: Agilvax, VLP Biotech, Expres2ion, NYBC, Precision Antibody, Swiss TPH Procurement of Materials, xii. MTAs: WEHI (Alan Cowman), No procurement activities for FY2019 Supplies, and Services Swiss TPH (Gerd Pluschke), University of Oxford (Simon Draper), Wellcome Trust Sanger Institute (Gavin Wright) Draft delivered to USAID on 01/31/19. Final Individual Project iv. Approved USAID feedback received on 3/14/19. Report Final Approval 5/29/19. Individual Project Data v. None during reporting period. Set See section 3.2 regarding abstract/poster xii. Publications None during reporting period. submissions. ix. Other/Ad Hoc Reports — PD1 Blockade Inhibitor

Final Individual Project Project Plan approved by USAID on iii. Approved descriptions 1/19/2017.

Procurement of Materials, Subcontract Awards: Final data from second RH5/Alum study xii. Supplies, and Services ADARC, University of Oxford delivered to USAID on Feb 12 2019. Final Individual Project Final Project Report delivered to USAID iv. Approved Report on 6/10/19 Individual Project Data v. None during reporting period. Set Sent to USAID on 6/10/19 for clearance. xii. Publications Submitted to PLOS ONE. Clearance received 6/19/19. ix. Other/Ad Hoc Reports — ELEMENT 3: SCG ANNUAL MEETING SUPPORT Planning for the FY19 meeting began in Q1 x. SCG Annual Meeting Event Date: May 1 – 2, 2019 of FY19.

MVDP Quarterly Report 8 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product Venue: College Park Marriott Hotel x.a-e. Logistic Support Travel: Leidos Travel Lodging: College Park Marriott Hotel ELEMENT 4: PROCURMENT OF MATERIALS, SUPPLIES AND SERVICES The repository is used to store and distribute materials for conduct of the MVDP program. In preparation for MVDP Reagents SriSai Biopharmaceutical transition plan development, Leidos will xi. Repository Solutions submit a plan with multiple options to USAID so that USAID can determine/approve the final disposition of the repository material. *Each Roman numeral crosswalks to a deliverable, as called out by the prime contract (AID-OAA-C-15-00071).

2.3. MANAGEMENT TOOLS Leidos’ SharePoint document repository is a Fiscal Information Security Management Act−compliant, web-based tool that provides access to program data/documentation, deliverables, work products, and schedules. This type of interface is an especially important information exchange among study sites as vaccines progress through milestones. Leidos granted folder permissions to subcontractor technical leads and customer points of contact, commensurate with their roles, allowing direct updates to their respective folders (Table 2.3-1). Transparency is achieved via USAID access to program folders. Leidos has uploaded documents relevant to the current reporting period to SharePoint.

Table 2.3-1. Management Tools Tool Description Location

Cloud-based solution for exchanging and SharePoint https://vector.leidos.com/sites/ITLSO/MVDP storage of documents Provide USAID OCONUS line to call 1-855-462-5367 Conference Phone Scientific Consultant Group members 1778004 Lines and CONUS line to communicate with 2013235 Leidos

2.4. TEAM MEETINGS Leidos also achieves transparency by routine copying of designated customer staff regarding email communications and teleconferences. As required, Leidos has set a standing biweekly meeting with USAID to review our MVDP activities (see Table 2.4-1). Ad hoc discussions to ensure positive study outcomes have been implemented. Leidos uses standard business tools (e.g., email, phone, teleconference, and desktop sharing) to communicate with staff and customers. Meeting agendas and summaries/minutes are available in the “Meeting Materials” folder on the MVDP SharePoint site (https://vector.leidos.com/sites/ITLSO/MVDP/Deliverables/Meeting Materials).

MVDP Quarterly Report 9 Leidos Proprietary

Table 2.4-1. Team Meetings

Meeting Date Topic October 3, 2018 Leidos/USAID Biweekly Teleconference October 25, 2018 Leidos/USAID Biweekly Teleconference October 30, 2018 Face-to-face meeting at ASTMH - Rh5 Complex Project November 7, 2018 Leidos/USAID Biweekly Teleconference November 21, 2018 Leidos/USAID Biweekly Teleconference November 27, 2018 EpiVax / Leidos Discussion / USAID RH5 Analysis December 12, 2018 Leidos/USAID Biweekly Teleconference January 9, 2019 Leidos/USAID Biweekly Teleconference January 23, 2019 Leidos/USAID Biweekly Teleconference January 28, 2019 Oxford/Leidos/USAID Joint Meeting - RH5.1 Clinical Study GIA Data Discussion February 6, 2019 Leidos/USAID Biweekly Teleconference February 19,2019 Leidos/USAID Biweekly Teleconference March 6,2019 Leidos/USAID Biweekly Teleconference March 20,2019 Leidos/USAID Biweekly Teleconference April 3, 2019 Leidos/USAID Biweekly Teleconference April 17, 2019 Leidos/USAID Biweekly Teleconference May 1, 2019 SCG Meeting May 15, 2019 Leidos/USAID Biweekly Teleconference June 5, 2019 Leidos/USAID Biweekly Teleconference June 19, 2019 Leidos/USAID Biweekly Teleconference July 10, 2019 Leidos/USAID Biweekly Teleconference July 24, 2019 Leidos/USAID Biweekly Teleconference August 7, 2019 Leidos/USAID Biweekly Teleconference August 21, 2019 Leidos/USAID Biweekly Teleconference September 17, 2019 Oxford/WEHI/Leidos/USAID IPT Meeting September 18, 2019 Leidos/USAID Biweekly Teleconference September 19, 2019 Leidos/USAID/NMRC NDA Meeting

3. ELEMENT 1 ACTIVITIES

3.1. PUBLICATIONS In FY2019, Leidos anticipates development of manuscripts for several projects. Status of these efforts, including the collaborators/subcontractors involved as well as the targeted journal, will be provided in this section.

MVDP Quarterly Report 10

Leidos Proprietary

3.3. NEW PROJECTS No new projects were proposed in Q3 FY2019. However, an alternative mRNA-based vaccine approach was proposed in Q3 FY2019 (see Section 4.1 for additional information). The FY2020 Annual Workplan (AWP) was submitted on September 3, 2019. While no new projects were proposed in the AWP, several new efforts were proposed for ongoing projects. For the Liver Stage Vaccine Development Project, two mRNA vaccine development efforts and assessment T cell simulation of PBMCs (from vaccinees) with peptides containing class I epitopes in collaboration with NMRC were included. For the CSP Vaccine Development Project, assessment of T and B cell responses via the MIMIC platform for optimized CSP VLPs formulated in DPX4 was included. In Q4 FY2019, approval was received for the two mRNA vaccine development efforts. USAID did not approve assessment of B cell responses to CSP VLPs in the MIMIC platform. Approval decisions by USAID for the other efforts are anticipated in early Q1 FY2020.

4. ELEMENT 2 ACTIVITIES

Upon identification of viable vaccine projects to support development from proof-of-principle testing, manufacturing, and clinical trial evaluation, Leidos drafts a detailed plan and protocols, identifies subcontractors, and executes the plan. Ongoing and planned projects are described in this section, as well as adjuvants, immunostimulators, and platforms slated for use in FY2019.

4.1. ADJUVANT/DELIVERY PLATFORMS Adjuvant, immunostimulators, and delivery platforms identified through scouting efforts and slated for use in FY2019 are detailed in this section.

4.1.1 ADJUVANTS The DepoVaxTM platform is in use for the CSP and RCR complex projects. AdvaxTM and Addvax are in use for the RCR complex project. Use of Matrix-MTM is slated for the RCR complex project.

4.1.1.1 DepoVax The DepoVax platform, developed by ImmunoVaccine Inc., contains lipids, cholesterol, oil, emulsifier and an immunostimulant (e.g., cGAMP, polyI:C, and/or Pam3Cys). This lipid-in-oil platform is designed to present antigen(s) and adjuvant(s) at a long lasting depot that effectively attracts antigen-presenting cells (APCs) and from which antigen is released over an extended period of time, from weeks to months. DepoVax promotes Th2 responses and enhances Th1 immune responses without triggering regulatory T cells. DepoVax has been used in the clinic as part of a Phase I/II study for a cancer vaccine (clinicaltrials.gov identifier: NCT01095848). Of note is that there are no aqueous components in this formulation; therefore, antigen is lyophilized for use with DepoVax and components are mixed and emulsified prior to administration using materials provided as part of an administration kit. Leidos executed a purchase order with ImmunoVaccine for formulation and provision of adjuvants for preliminary efficacy studies.

4.1.1.2 Advax Advax is a microparticle-based adjuvant comprised of microcrystalline delta inulin, a plant-derived polysaccharide developed by Vaxine. Of note is that delta inulin is insoluble at body temperature. This adjuvant generates Th1 and Th2 immune responses and activates the alternative complement pathway. However, Advax does not activate nuclear factor-kappa B (NFkB) so an inflammatory response is not seen with this adjuvant. This adjuvant has been assessed in multiple clinical studies. Advax can be combined with immunostimulators. Vaxine has developed an Advax + CpG formulation, which is also of interest for this project. Note that the CpG in this formulation is proprietary to Vaxine. Leidos

MVDP Quarterly Report 13 Leidos Proprietary

executed an NDA with Vaxine, gathered information regarding this adjuvant, and is currently working to place a purchase order with Vaxine to access the adjuvant for preliminary efficacy studies.

4.1.1.3 AddaVax AddaVax (the research grade version of the human-use adjuvant MF59) is a squalene-based oil-in water nano-emulsion that induces both cellular (Th1) and humoral (Th2) responses through the recruitment and activation of antigen presenting cells. This adjuvant is commercially available.

4.1.1.4 Matrix-M Matrix-M is a saponin-based adjuvant comprised of purified saponin, synthetic cholesterol, and a phospholipid patented by Novavax. This adjuvant generates both cell-mediated and antibody-mediated immune responses, and has the potential to increase immune response duration as shown in numerous clinical trials (Shinde et al., 2018). The Draper group has an access agreement to use this adjuvant for malaria vaccine development.

4.1.2 IMMUNOSTIMULATORS CpG in combination with Advax is in use for the RCR complex project.

4.1.2.1 CpG ODN 7909 is a class B CpG that induces a strong Th1 response and has been assessed in many clinical studies. This molecule is a TLR9 agonist. CpG is commercially available through a variety of vendors.

4.1.3 PLATFORMS The WHcAg VLP platform is in use for the CSP project. The SGI and GeoVax platforms are slated for use in liver stage project. The SGI and SpyTag/SpyCatcher VLP platforms are slated for use in the RCR complex project.

4.1.3.1 WHcAg VLP The woodchuck hepatitis B core antigen (WHcAg) VLP platform, developed by VLP Biotech, is based on the core protein the of woodchuck hepatitis B virus. The core proteins self-assemble into VLPs with 240 copies of the antigen per VLP. This platform can accommodate multiple foreign sequence insertions, with long insertions possible at the N and C-termini. Inserts within the surface-exposed loop are possible, which is particularly beneficial for B cell epitopes as the VLP configuration permits cross- linking of B cells. Studies with this platform have shown it to be equal or more immunogenic than HBcAg for both B cell and T cell responses, not significantly cross-reactive with the HBcAg for B cell responses and only partially cross-reactive with HBcAg for T cell (CD4) responses, and function as a vaccine carrier platform for heterologous, B cell epitopes (Billaud et al., 2005a; Billaud et al., 2005b). In consideration of cost, WHcAg VLPs can be easily expressed at high levels in E. coli. Note that this platform has not yet been tested in the clinic.

4.1.3.2 SpyTag/SpyCatcher VLP Platform To alleviate the pitfalls of more traditional VLP development, the groups of Draper, Biswas and Howarth at the University of Oxford (Brune et al., 2016) developed the SpyTag/SpyCatcher “plug-and-display” VLP platform, which employs use of the SpyTag peptide and SpyCatcher protein (originally generated by splitting the CnaB2 domain from the Streptococcus pyogenes fibronectin-binding protein FbaB (Zakeri et al., 2012)) to decorate the VLP surface with antigen. SpyTag-linked antigen and SpyCatcher-linked VLP carrier (resulting from the genetic fusion of SpyCatcher to VLP coat protein monomers followed by expression and self-assembly) are required for the production of VLPs using this platform. Mixing of these two components results in the spontaneous formation of an irreversible bond between the SpyTag-Antigen and SpyCatcher-VLPs, yielding VLPs decorated with the antigen of interest. The SpyTag/SpyCatcher platform has been used to generate VLPs displaying a variety of malaria-related antigens (e.g. CIDR, Pfs25, CSP) as well as self-antigens and antigens related to cancer, tick-borne

MVDP Quarterly Report 14 Leidos Proprietary

encephalitis, and tuberculosis (Brune and Howarth, 2018). VLPs generated using this platform can be administered in the presence or absence of adjuvant, and studies with Pfs25-AP205 VLPs showed a higher anti-Pfs25 response than Pfs25 alone or Pfs25-SpyTag. Additionally, Pfs25-AP205 VLPs formulated in AddaVax yielded a slight increase in the anti-Pfs25 response when compared to the same VLPs without adjuvant (Brune et al., 2016). This platform has not yet been assessed in the clinic.

4.1.3.3 SGI RNA-based Platform Recent advances in mRNA vaccine delivery have elevated these platforms to the point at which feasibility of mRNA-based vaccines has been demonstrated in the clinic (Pascolo, 2008). Further advances in this technology have led to the development of platforms such as the self-amplifying RNA- based replicon from Synthetic Genomics Inc. (SGI). This technology has been shown to out-perform standard mRNA in terms of protein expression levels and duration of expression (Bahl et al., 2017; Brazzoli et al., 2016; Hekele et al., 2013). The SGI replicon technology comes encapsulated within a proprietary lipid nanoparticle that ensures the replicon is taken up by cells and provides an adjuvant effect. In addition to the antigen(s) of interest (both mono and poly-cistronic replicons are possible), SGI’s replicon encodes a unique RNA polymerase (engine) that replicates the delivered RNA construct into thousands of copies, which are then translated using the cell’s translational machinery. While protein expression with traditional mRNA typically peaks in a day and decays over 5-7 days, protein expression with the replicon is more durable, lasting approximately 1 month. On May 9, 2019, SGI informed Leidos that they were acquired and that the RNA vaccine organization would be closed, effective immediately. Therefore, the SGI platform will no longer be available to the MVDP.

4.1.3.4 GeoVax MVA Platform Modified Vaccinia virus Ankara (MVA)-based vaccines have been widely tested in the clinic and are known to generate high cellular responses (Gilbert, 2013). The main drawback of these platforms has been that immunogenicity is greater when these vectors are used to boost pre-existing T cell responses. However, GeoVax’s 4th generation MVA-VLP platform requires no immune response priming due to improved transgene stability during manufacture and elevated levels of expression compared to the parent platform. This is evidenced by a clinical study with GeoVax’s MVA-based HIV vaccine, where cellular (both CD8 and CD4) and humoral responses were seen in humans administered the MVA-VLP only (Goepfert et al., 2014). Such responses in animal models have also been seen (Brault et al., 2017). Also of note is that this platform does not require adjuvant.

4.1.3.5 Alternative RNA Approach During the May 15, 2019 biweekly meeting, Leidos proposed and alternative RNA approach (via collaboration with the Leidos Innovations Center (LInC), Promosome, TriLink, and Acuitas) to replace the SGI RNA platform approach planned for the Liver Stage Vaccine and RCR Complex Vaccine Projects. On May 16, 2019, USAID declined the use of this strategy as part of the CD8 Platform Scouting Attachment to the Liver Stage Project Plan (and instead elected to move forward only with the GeoVax platform) and requested a “one-pager” summarizing the alternative RNA approach for the RCR Complex Vaccine Project. On May 23, 2019, Leidos provided a summary of the alternative RNA approach to USAID for review noting applicability of the approach to addressing protein expression concerns with the SPECT2 mRNA in the MIMIC system (Liver Stage Vaccine Project). Use of this approach was proposed as part of the FY2020 AWP. A summary of the alternative RNA approach follows. Leidos and Promosome will work together to design and test RESCUE modifications to best increase expression of the proteins in the RCR complex. This will include: a) transiently transfecting mammalian cell lines with RESCUE-modified sequences and b) evaluating expression levels of each candidate protein by semi-quantitative Western blot analyses to identify the optimum modifications. The optimum RESCUE-modified sequences will be incorporated into plasmids for RNA production by TriLink. TriLink

MVDP Quarterly Report 15 Leidos Proprietary

will incorporate modified nucleotides (one-methylpseudouridine-5’-triphosphate in place of UTP) and cap mRNA to increase mRNA stability. Leidos will perform quality control studies with the TriLink generated mRNAs. The resulting mRNAs will be encapsulated by Acuitas, using their lipid nanoparticle technology, and/or formulated in a DPX cationic emulsion-based adjuvant compatible with mRNA-based vaccines.

4.2. CSP VACCINE DEVELOPMENT PROJECT: AMY NOE AND JAYNE CHRISTEN The CSP vaccine development project is a multi-year effort that will continue through the end of the contract in May 2020. Previously, the WHcAg VLP platform formulated in DPX4 was down-selected with completion of preliminary efficacy assessments using the VLP162 parent construct. Further, T cell epitope assessments of PfCSP were previously completed and these data, along with recently published data were used to develop a VLP construct optimization strategy that was initiated in FY2018. The work performed in Q1 FY2019 includes initiation of CSP class II T cell epitope immunogenicity assessments at VaxDesign, continuation of the construct optimization activities, and initiation of a repeat confirmatory efficacy study with the VLP162/DPX4 formulation. The work performed in Q2 FY2019 includes completion of CSP class II T cell epitope immunogenicity assessments at VaxDesign, continuation of the construct optimization activities, and completion of a repeat confirmatory efficacy study with the VLP162/DPX4 formulation. The work performed in Q3 FY2019 includes VLP purification and conduct of rabbit immunizations for the planned passive transfer study, as well as ICS down-selection and continuation of the construct optimization activities. The work performed in Q4 FY2019 includes conduct of two passive transfer studies, continuation of construct development activities, design and (contingent) approval of the first efficacy (active vaccination/challenge) study to assess optimized VLPs, and proposal of a study design/plan to assess optimized VLPs in the MIMIC platform. Further, VLP purification activities for the active vaccination/challenge study were initiated at the end of this quarter and will continue into Q1 FY2020.

4.2.1 T CELL EPITOPE ASSESSMENTS Previously, in silico T cell epitope assessments of PfCSP were performed by EpiVax to identify CSP class I and class II epitopes predicted to bind HLA molecules. This in silico analysis also included identification of additional predicted class II epitopes from a set of CSP sequence variants with the ultimate goal of achieving a VLP-based CSP vaccine with broad Pf strain coverage. A total of four predicted class II epitopes (C1-C4) were identified in the PfCSP 3D7 protein sequence and nine predicted class II epitopes were found in the sequence variants (ICS2-ICS10), the latter all overlapping CSP region 2 (R2) and termed immunogenic consensus sequences (ICS) by EpiVax. Of note is that one of the ICS (ICS8) is a sequence match for the class II epitope predicted to overlap the PfCSP 3D7 R2 (i.e., C3). In order to validate the EpiVax in silico HLA predictions, in vitro HLA binding assessments were previously performed for the four PfCSP 3D7 predicted class II epitopes and a subset of the ICS deemed of high interest (i.e., ICS2, ICS5, ICS7, and ICS8). Although the ability of a sequence to bind HLA is one of the main factors influencing immunogenicity, it is not the only factor. Therefore, in order to assess immunogenicity of the different ICS to further aid in selection of ICS for incorporation into WHcAg VLP, in Q1 FY2019, VaxDesign initiated immunogenicity assessments of the ICS using PBMCs from HLA- matched donors. Preliminary data from VaxDesign suggest that a subset of the ICS appears to have elevated responsiveness across the HLA types. The full data set was received from VaxDesign at the end of Q2 FY2019 and was circulated to USAID on March 20, 2019. Several ICS induced multifunctional responses across a broad range of HLA types; ICS5, ICS7, ICS8, and ICS9 were the most promiscuous and immunogenic of the set tested. Based on magnitude of response, ICS2 may also be of interest. An IPT meeting to review the data was held on April 17, 2019. On April 18, 2019, USAID noted their approval regarding moving forward with addition of ICS9, ICS5, ICS7, ICS2, to the VLP (in addition to

MVDP Quarterly Report 16 Leidos Proprietary the already included ICS8). These data were used to down-select the specific ICS for inclusion in the optimized WHcAg VLP construct(s).

4.2.2 CONSTRUCT OPTIMIZATION Optimization of the VLP162 construct by VLP Biotech is based on a cloning plan developed and initiated in FY2018, and includes insertion of sequences from the conserved R1 region of PfCSP (which contains CSP cleavage site, KKLKQP), alternative sequences for the NANP repeat loop region, and incorporation of additional class II epitopes from the N- and C-terminal regions of PfCSP 3D7 as well as from the R2 region of heterologous CSP strains (selected based on results of the above described T cell epitope assessments. USAID approved expansion of the loop insert effort to include development of VLPs with five novel loop inserts (further described below). Procurement associated with expansion of the loop insert effort (further described below) began at the end of Q4 FY2018 and was completed in Q1 FY2019. The optimization activities completed in FY2018 include insertion of PfCSP 3D7 amino acids 58-78 into the VLP N-terminal insertion site and extension of the PfCSP 3D7 C-terminal insert to incorporate much of the predicted class II epitope regions in the C-terminus of PfCSP 3D7 (i.e., amino acids 313- 387). The parent VLPs resulting from these efforts are VLP407 and VLP411 (Table 4.2-1). Further, several attempts at extending the C-terminal insert to include different ICS were made at the end of FY2018 with limited success; therefore, Leidos enlisted the assistance of a protein modeler to provide feedback on the sequence configurations with increased stability and a higher probability of soluble expression. ICS sequence cloning efforts were temporarily paused for input from the modeler. In Q2 FY2019, the modeler found that addition of ICS to the C-terminal insert, CS(313-387), was problematic for VLP assembly, as the hydrophobic sequences interfere with RNA binding at the protamine binding site (located at the C-terminus of the VLP backbone sequence, N-terminal of the CSP insert). As RNA binding is necessary for VLP assembly, the modeler evaluated other sites for ICS insertion during Q3 FY2019. In early Q3 FY19, USAID approved moving forward with addition of ICS9, ICS5, ICS7, ICS2, to the VLP. With input from the modeler, at the end of Q3 FY2019, VLP Biotech successfully added three of the ICS (2, 5, and 7) and was working on a VLP to add the fourth (i.e., ICS9). In Q4 FY2019, VLP Biotech successfully added all four ICS sequences (ICS 2, 5, 7, and 9 – termed the “ICS string”) to a VLP, VLP449 (Table 4.2-1). Per information from the modeler and based on the highly hydrophobic nature of the ICS string, this insert must be positioned at the C-terminus of the VLP. Based on several unsuccessful attempts to include both CS(313-387) and the ICS in the C-terminal insertion site, as well as input from the modeler, options for relocating the CS(313-387) insert are being explored. These initial efforts include VLP459 and VLP460 (Table 4.2-1). In Q1 FY2019, the strategy for construction of the loop insert clones was developed and DNA cloning initiated. A list of the planned clones is provided in Table 4.2-1; the NPDP and NVDP sequences are shown in bold to facilitate sequence comparison among the loop inserts. Of note is that VLP Biotech recommended a conservative approach to developing the loop insert clones whereby both VLP407 and VLP411 were used as the parent for different constructs. This approach was recommended by VLP Biotech due to concerns regarding the less than optimal expression levels for VLP411 as well as the possibility of lower immunogenicity directed to the loop insert when CSP B cell epitopes are placed in both the loop and N-terminal regions of the VLP. Specifications for the new VLPs, including the alternative loop inserts, are shown in Table 4.2-1. In Q2 FY2019, construction of these new VLPs (VLP430 – VLP436) was completed; all of the VLPs assembled and demonstrated soluble expression. USAID down-selected VLP430 – VLP434 for progression to the passive transfer study (as described below). An IPT meeting was held on February 14, 2019, to discuss these data as well as plan for the passive transfer study. Production of sufficient quantities of these VLPs was completed in early Q3 FY2019 and immunizations begun shortly thereafter. Of note that the B cell epitope inserts NANPNVDP(NANP)3 and NPDPNANPNVDP(NANP)3 were named B1 and B2 in Q4 FY2019.

MVDP Quarterly Report 17 Leidos Proprietary

Table 4.2-1. VLP162 Optimization – Alternative Loop Inserts

VLP ID N-term Insert Loop Insert (position 78) C-term Insert Assembly/Expression VLP162 None NANPNVDP(NANP)3 318-377 Yes, soluble VLP407 None NANPNVDP(NANP)3 313-387 Yes, soluble VLP411 58-76 NANPNVDP(NANP)3 313-387 Yes, soluble VLP430 None KQPADGNPDPNANPN 313-387 Yes, soluble VLP431 None PADGNPDPNANPNVD 313-387 Yes, soluble VLP432 None NPDPNANPNVDPNAN 313-387 Yes, soluble VLP433 None NPDPNANPNVDP(NANP)3 313-387 Yes, soluble KLKQPADGNPDPNANPNVDP VLP434 None 313-387 Yes, soluble (NANP)3 VLP435 58-76 NPDPNANPNVDPNAN 313-387 Yes, soluble VLP436 58-76 NPDPNANPNVDP(NANP)3 313-387 Yes, soluble VLP449 None NANPNVDP(NANP)3 ICS String Yes, soluble VLP459 CS(58-B2-387) NANPNVDP(NANP)3 ICS String TBD VLP460 None CS(B2+-387) ICS String TBD B2+ includes several amino acids N-terminal of the NPDP but does not include the C2 cluster (i.e., 58-76)

4.2.3 PASSIVE TRANSFER STUDIES Rabbits were selected as the animal model for collection of passive transfer sera to ensure a sufficient volume of sera would be available within the necessary timeframe. Of note is that, in addition to the VLPs selected for the passive transfer study (VLP430 – VLP434), USAID requested production of rabbit sera for VLP435 and VLP436. Two rabbits were immunized per VLP and an equal volume of sera pooled from the two rabbits for the passive transfer. All VLPs were formulated in ISA720 (50% by volume) and two immunizations were given with a 6-week interval (April 4, 2019 and May 15, 2019). The test bleed was performed at week 5 (May 9, 2019) and the terminal bleed was at week 11 (June 20, 2019). Test bleed sera ELISA titer assessments (circulated to USAID on June 5, 2019) are shown in Figure 4.2-1. The sera were assessed for titers to the WHcAg VLP backbone (panel A), recombinant CSP (panel B), an NANP peptide (panel C), and an NVDP peptide (panel D). Titers were comparable for the two rabbits in each group. Rabbits immunized with VLP430 and VLP431 demonstrated lower titers to the NVDP peptide. Titers with VLP435 and VLP436 (both contain an N-terminal insert) were comparable to those with VLP432 and VLP433, indicating that the N-terminal insert does not appear to impact overall titer.

MVDP Quarterly Report 18

Leidos Proprietary

4.2.4 CONFIRMATORY EFFICACY EVALUATION Based on the positive results of the preliminary efficacy assessments with VLP162 in DPX4, Leidos moved this formulation forward to confirmatory efficacy evaluation to conduct a more stringent evaluation of efficacy including use of a greater number of mice per cohort, assessment of individual (rather than pooled) mouse samples, and to conduct cellular response evaluations using mouse PBMCs. The report for this study was received by Leidos at the beginning of Q4 FY2018 and the data were circulated to USAID. Unfortunately, several animals that received the VLP162/DPX4 formulation died during the study. Upon investigation, ImmunoVaccine informed Leidos in Q1 FY2019 that sterility tested showed a microbial contaminant in the formulated material. While VLP production includes a sterile filtration step just prior to aliquoting, the identified microbial contaminant (Microbacterium sp.) is known to be able to pass through a 0.2 μm filter. Subsequently, Leidos requested that VLP Biotech perform a series of sterility testing to further evaluate this finding as well as to implement sterility testing for VLP lots that will be used for efficacy assessments (including passive transfer and/or challenge studies). Results of the sterility testing by VLP Biotech showed no microbial growth. Results of the sterility testing by ImmunoVaccine demonstrated no microbial growth in the unformulated VLP162 but found microbial growth in the DPX4 formulated VLP162. ImmunoVaccine is currently evaluating excipients for evidence of bacterial contamination. A repeat confirmatory efficacy study was initiated in late December 2018 (Table 4.2-6).

Table 4.2-6. VLP162 Confirmatory Efficacy Study 2 – DPX4 Formulation

# mice, Study Day Cohort # Test Sample R strain -5 0 21 35 42 60 65 67 1 Naïve - 10, C57Bl/6 none None none

2 VLP162 (20 µg, no adjuvant) IM 10, C57Bl/6 √ √ √ 3 bleed

3 VLP162 (20 µg) + DPX4 IM 10, C57Bl/6 - √ None √ leed 1 leed B 2 Bleed Bleed Pre 4 VLP162 (20 µg) + DPX4 IM 10, C57Bl/6 √ √ √ Challenge 5 VLP162 (50 µg) + ISA 720 IM 10, C57Bl/6 √ √ √

The study findings were received and circulated to USAID in March 2019. In the VLP162 + ISA 720 group positive control group, only 20% sterile protection was seen (Figure 4.2-2). In the VLP162 + DPX groups, 30% protection was seen in the 2-dose group and 10% protection was seen in the 3-dose group (cohorts 3 and 4, respectively, Table 4.2-6). No sterile protection was seen in the naïve or unadjuvanted VLP162 groups. Good titers to NANP were seen with the VLP162 + DPX4 and VLP162 + ISA720 formulations. Of note is that the 2-dose group performed incrementally better for three measures: sterile protection, ELISA titer, and average patency day (day 4.7 for the 2-dose group and day 4.4 for the 3-dose cohort). Based on this finding, a 2-dose regimen may be considered for future studies confirmatory efficacy assessments with the optimized constructs.

MVDP Quarterly Report 21 Leidos Proprietary

Figure 4.2-2. Confirmatory Efficacy Study 2 Results – ELISA Titers and Protection

Based on data from the alternative loop insert passive transfer studies, the following confirmatory efficacy study design (Table 4.2-7), the first to including optimized VLPs, was approved by USAID on September 26, 2019. This study is planned for Q1 FY2020.

Table 4.2-7. Optimized VLP Confirmatory Efficacy Study Design – DPX4 Formulations

# mice, Study Day Cohort # Test Sample R strain -5 0 21 35 42 56 65 1 Naïve - 10, C57Bl/6 none none none 2 VLP407 (20ug) in DPX IM 10, C57Bl/6 √ √ √

3 VLP433 (20ug) in DPX4 IM 10, C57Bl/6 √ √ √ bleed - leed 1 leed

4 VLP407 (10ug) + VLP449 (10ug) in DPX4 IM 10, C57Bl/6 √ √ B √ 2 Bleed Pre Challenge 5 VLP433(10ug) + VLP449 (10ug) in DPX4 IM 10, C57Bl/6 √ √ √ 6 VLP162 (50ug) in ISA 720 (Pos Ctrl) IM 10, C57Bl/6 √ √ √

MVDP Quarterly Report 22 Leidos Proprietary

4.2.5 PRELIMINARY STABILITY ASSESSMENTS OF VLP162 + DPX4 In Q2 FY2018, ImmunoVaccine began work to conduct a 6-month stability study on the VLP162 + DPX4 formulation in order to (1) confirm that the formulation is stable while it is maintained for animals studies, (2) evaluate refrigerated storage (the material is currently stored frozen), and (3) provide preliminary data regarding stability of the WHcAg VLPs in the DepoVax platform. This study was completed in Q1 FY2019 and results for the 0, 1, 3, and 6-month time points are shown in Table 4.2-8 and Table 4.2-9. It was noted after the 0-month time point testing that the VLP162 content did not conform to the specification. Additional testing performed by IMV indicated precipitation occurring during the formulation and/or freeze-drying process. This same issue was found in a retain sample from a previous lot. As this material is representative of that used in efficacy assessments, the decision was made to continue with the stability program. Formulation optimization is possible; however, this will not be performed until the fully optimized VLP has been developed and assessed for precipitation with DPX4. In addition to the VLP162 content not complying with the target specification, it was also noted that viscosity increased over the 6-month period at both temperatures. All other results were within the established specification at the 6-month time point. ImmunoVaccine concluded that further formulation development is needed to improve stability of the VLP formulation.

MVDP Quarterly Report 23

Leidos Proprietary

MVDP Quarterly Report 28 Leidos Proprietary

Table 4.4-2. A Summary of Published Analysis of Five Proteins Selected for Development and Characterized Proteins

Table 4.4-2 provides a summary of characteristics of the five proteins selected for analysis (1) (Arevalo-Herrera et al., 2016) (2) (Crompton et al., 2010) (3) (Dent et al., 2015) (4) (Le Roch et al., 2003). Based on microarray using P. falciparum (5) (Le Roch et al., 2004). Based on microarray or mass spectral; analysis using P. falciparum (6) (Lindner et al., 2013). Based on mass spectral results using P. falciparum, proteins were ranked for abundance from 1 to 1991, 1 being the most abundant. (7) PlasmoDB (http://plasmodb.org/plasmo/) (8) (Tarun et al., 2008). Based on mass spectral analysis using P. yoelii

MVDP Quarterly Report 29 Leidos Proprietary

4.4.1 LIVER STAGE PLAN MILESTONE I.3 Analysis of the immune response to PFD0430c protein began on August 27, 2018, with AmpTec starting the synthesis the mRNA. In mid-November 2018 AmpTec provided the mRNA encoding PFD0430c (SPECT2-mRNA) that VaxDesign is using to transfect cells to express the protein in situ. VaxDesign began developing the transfection of dendritic cells in late November 2018. Transfection of dendritic cells with the SPECT2-mRNA was initially problematic, resulting in extensive cell death. VaxDesign has observed this cytotoxic effect of mRNA on dendritic cells before. As potential solutions to this issue VaxDesign evaluated transfecting feeder cells (African green monkey kidney VERO cells and human endothelium EA.hy926 cells) to provide antigen to the dendritic cells, and to optimize transfection of the dendritic cells while reducing cell death. Transfection efficiency was monitored using PCR with primers for SPECT2 to detect intracellular mRNA. As an indication for protein expression by transfected cells, VaxDesign evaluated T cell stimulation. This was performed by initially priming T cells with the dendritic cells presenting the antigen from the feeder cells transfected with SPECT2-mRNA, or with dendritic cells directly transfected with the SPECT2-mRNA. The recall (i.e., restimulation) immune response of T cells was evaluated using the two class II epitopes derived from SPECT2 that were previously demonstrated to elicit immunity in the same donor when primed with the peptides. Demonstration of a recall response will indicate SPECT2 is being translated from the mRNA and the protein is processed to provide the peptides at levels sufficient to prime the T cells. Feeder cells were efficiently transfected without significant cytotoxicity, providing a potential solution to the cytotoxicity seen in the transfected dendritic cells. By modifying the culture media and transfecting rested dendritic cells, dendritic cells were also efficiently transfected while reducing cytotoxicity. That is, when monocytes were treated with cytokines to derive the dendritic cells, the cells were allowed to rest overnight before transfection with 4 µg SPECT2-mRNA/million cells verses transfecting within two hours after deriving the dendritic cells. Collectively, these modifications to the dendritic cells’ transfection doubled the viability of the dendritic cells following transfection. With the improved viability, transfected dendritic cells were used to evaluate antigen presentation in the MIMIC model. To further optimize transfection with the intent to maximize expression of SPECT2, different amounts of mRNA were tested in dendritic cells. VaxDesign evaluated using 2, 10, or 50 µg of mRNA/ million cells, and found that using 50 µg SPECT2-mRNA increased transfection nearly 100-fold relative to using 2 µg. While increasing the amount of mRNA did increase cytotoxicity, the number of viable transfected dendritic cells are sufficient for carrying out antigen presentation to the T cells, and 50 µg of mRNA was be used to transfect the dendritic cells in the MIMIC model. VaxDesign used T cell stimulation for the class I and class II epitopes (Table 4.4-3) as an approach to demonstrate effective transfection resulting in expression of the protein. Donor PBMC stimulated with peptides did demonstrate recall of immune response when restimulated with the same peptides; however, priming using SPECT2-mRNA transfected cells did not result in recall of the T cell response when the T cells were restimulated with either the peptides or using SPECT2-mRNA transfected cells. Figure 4.4-1 provides representative results for the class II peptides. To improve the possibility of measuring SPECT2-specific responses in the CD8+ MIMIC assay, SPECT2 peptide pools were used to prime the T cells, thereby stimulating the proliferation of SPECT2 responsive T cells and increasing the number of potentially responsive T cells for subsequent restimulation with the SPECT2 encoding mRNA. Overall, this approach resulted in the mRNA construct producing more reactivity in the CD8+ T cell assay compared to the CD4+ T cell assay. However, the distribution of the dual-functional effector population in the mRNA transfected conditions had a dispersal pattern that is indicative of non-specific activation rather than an epitope-specific response. The lack of a multi-functional response by the mRNA restimulated peptide-primed samples also indicated that the restimulation response is not SPECT2-specific. The lack of restimulation by SPECT2-mRNA transfected cells indicates that the level of protein expression is too low to prime the T cells.

MVDP Quarterly Report 30 Leidos Proprietary

To enable a direct demonstration of protein expression Leidos developed polyclonal sera in mice to peptides derived from SPECT2, with the first vaccinations performed by Precision Antibody the first week of March 2019 and sera provided to VaxDesign on May 9, 2019. The peptides used to develop the sera include CL2-19 and CL2-20, as well as two additional peptides that EpiVax predicted were class II epitopes in SPECT2 (i.e., ISDYTKSLSAEAKVSC, DKDIIKILTNADTVTKC). To test expression, SPECT2-mRNA was transfected at 10 µg / 2 million dendritic cells, or dendritic cells were pulsed with the peptides CL2-19 and CL2-20 at 5µg, 2.5 µg, 1 µg, 0.5 µg or 0.25 µg per mL. After eight hours incubation, cells were washed, fixed, and permeabilized to enable detection of intracellular and extracellular bound peptides by subsequent treatment with the mouse antisera (used at 1:15,000) and goat anti-mouse AF647 that provide a fluorescent signal for detection using Celigo imaging cytometry. Controls were: dendritic cells that did not receive antigen, dendritic cells pulsed with 5 µg/mL of peptides but without probing with mouse sera, and dendritic cells pulsed with 5 µg/mL of peptides and probed with naïve mouse sera. Dendritic cells treated with 5 µg to 1 µg peptide demonstrated significant detectable fluorescence, but no fluorescence was detected by dendritic cells transfected with SPECT2-mRNA. While it is possible that the mouse antisera do not bind to the SPECT2 protein, this result indicates the respective peptides are not detectable when the protein is expressed from this mRNA. With this result, the current analysis at VaxDesign is being concluded with the final report. A higher level of expression is needed to evaluate the antigen processing for SPECT2. In the FY2020 AWP, contract Leidos has proposed application of technologies developed by Promosome to redesign the SPECT2-mRNA, and purification of the mRNA to remove double-stranded mRNA as a way of potentially increasing protein expression to the levels needed for evaluation of antigen processing by VaxDesign. This work was approved by USAID at the end of in Q4 FY2019.

Figure 4.4-1. Recall IFN-γ response in the MIMIC model “Pool” contains the peptides CL2-19 and CL2-20; “mRNA” is SPECT2-mRNA.

MVDP Quarterly Report 31 Leidos Proprietary

Table 4.4-3. Peptides derived from PFD0430c Peptide Location in Peptide Peptide Sequence PFD0430c Designation* 29 – 37 RLNNRSIYK CL1-64 72 – 81 KISSFINQRK CL1-65 237 – 245 HTSVFPGLY CL1-66 257 – 265 NPLGEADSL CL1-67 285 – 293 GIANDLSTL CL1-68 357 – 365 RSKKTFLVK CL1-70 371 – 379 YTIGLPPYI CL1-71 440 – 448 YESQLGGKI CL1-72 523 – 531 NLFEWSKTV CL1-73 534 – 542 HPMPINIKL CL1-74 550 – 558 DSDDLKESY CL1-75 570 – 579 GLSPHDTMQK CL1-76 386 – 394 AYKNAVNEL CL1-77 224 – 233 DENTLENRNI CL1-78 360 – 382 Ac-KTFLVKSNCVKYTIGL PPYIPWD-amide CL2-19 445 – 461 GGKITKIINVSTSSIEQ-amide CL2-20 *Peptides designated with CL1-X target class I, and peptides designated with CL2-X target class II

Activity 2017 2018 2019 Oct No Dec Jan Feb Mar Apr Ma Jun Jul Au Sep Oct No Dec Jan Feb Mar Apr Ma Jun Jul Au Sep Peptide Synthesis Class II Peptide Experiments Class II Peptide Analysis Class I Peptide Experiments Class I Peptide Analysis Protein Selection mRNA Synthesis Transfection Development Antibody Development Protein Expression Development CD4 & CD8 Response Data Analysis Figure 4.4-2. Schedule for Analysis by MIMIC of Epitopes from New Proteins

MVDP Quarterly Report 32 Leidos Proprietary

May 29, 2019 and USAID approval received on June 6, 2019. A final PDF of the Liver Stage Vaccine Project Plan CD8 Platform Scouting Attachment was provided to USAID on June 10, 2019. Initial proof- of-concept studies previously performed with this model found that SYVPSAEQI integrated into the MagaVax platform elicited CD8 T cell responses in mice. These data are detailed in the FY2018 USAID MVDP Annual Report. The decision to proceed to a challenge study with one or both MagaVax constructs was dependent on the elevation of T cells responses in the MagaVax compared to that of the positive control (AdPyCS). Based on USAID’s initial specified Acceptance Criterion where constructs will be selected for further study only if they perform considerably better than the positive control at a historically non-protective dose or equal to or better than the positive control that would be expected to be protective, the decision was made not to proceed with challenge studies at this time. The other two platforms that Leidos initially planned to evaluate were Synthetic Genomics, Inc., (SGI) self- replicating RNA replicon and GeoVax’s modified Vaccinia virus (MVA)-VLP. However, in Q3 FY2019, SGI informed Leidos that their RNA platform would no longer be available due to acquisition of the company. USAID elected not to include an RNA platform as part of CD8 platform scouting at this time (refer to section 4.1 for additional details). This change was incorporated into the CD8 Platform Scouting Attachment approved by USAID in Q3 FY2019. Further, the experimental approach and design for the GeoVax platform was also detailed in the approved attachment. The kick-off meeting with GeoVax was held on March 7, 2019.

4.4.1.2 Optimizing the Number of Mosquito Bites for Infection Prior to starting the platform scouting analysis with the GeoVax MVA-VLP, the number of mosquito bites required to ensure malaria infection in this model needed to be determined. This study informed the targeted infectious exposure that will be used for the subsequent studies at ADARC.

On December 10, 2018, of Q1 FY2019, USAID Table 4.4-4. Bite Titration Study approved the study design (Table 4.4-4). In brief, BALB/c (n = 7) mice were exposed to various Cohort # Test Sample numbers of mosquitos infected with Py 17XNL. 1 No bites Following exposure, individual mosquitos were 2 2 bites dissected to check for infectivity. Parasitemia in the 3 4 bites mice was assessed via blood smears. Results of this 4 6 bites study were delivered to USAID on Feb 11 in Q2 FY2019 and are shown in Table 4.4-5. Of note, 5 8 bites each mouse was anesthetized and placed on its abdomen for a 3-minute exposure to allow the mosquitos to bite. All mice that were exposed to either 6 or 8 mosquitos were infected. In addition, the overall percentage of mosquitos positive for sporozoites was ~75%. Based on the data Leidos and USAID agreed to move forward with using at least 6 bites/mouse to ensure that all mice are infected.

MVDP Quarterly Report 33 Leidos Proprietary

Table 4.4-5. Bite Titration Study Results

4.4.1.3 Titration of AdPyCS Immunization for Mosquito Bite Challenge In addition to optimizing the number of mosquito bites for infection prior to evaluating the GeoVax MVA-VLP platform, ADARC will also titrate the AdPyCS immunization to determine the dose that gives 40-50% protection following mosquito bite challenge. On December 10, 2018 of Q1 FY2019, USAID approved the study design (Table 4.4-6). In brief, BALB/c (n = 7) mice were immunized IM with various doses of AdPyCS in the hind limb without adjuvant. At day 10 post-immunization mice were challenged via 6 mosquitos infected with Py 17XNL. Following bite challenge individual mosquitos were dissected to check for infectivity. Parasitemia in the mice was assessed via blood smears. Results of this study were delivered to USAID on March 24 in Q2 FY2019 and are shown in Table 4.4-7. The percentage of dissected mosquitos positive for sporozoites was ~88% overall and ADARC visibly confirmed (mosquitoes got swollen with blood) greater than 4 bites per mouse. No protection was seen with either 107, 108 or 109 AdPyCS. Only a single mouse was protected at 1010 (14%) while 3 out of 7 (43%) mice were protected with 1011 AdPyCS. Based on these study results ADARC suggest using 1011 AdPyCS as a positive control in the GeoVax proof-of-concept studies using PyCS.

MVDP Quarterly Report 34 Leidos Proprietary

Table 4.4-6. AdPyCS Dosing Study Cohort # Test Sample Route # Mice, Strain Day 0 Day 10

1 Naïve none 7 BALB/c none 2 107 AdPyCS IM 7 BALB/c √ 3 108 AdPyCS IM 7 BALB/c √ 4 109 AdPyCS IM 7 BALB/c √ 5 1010 AdPyCS IM 7 BALB/c √ 6 1011 AdPyCS IM 7 BALB/c √ 6 bites/mouse

Table 4.4-7. AdPyCS Titration Study Results

Number of infected parasitemia + or - Mouse ID Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 % Protection mosquitos 1-1 + + + ++ (>1%) ++ ++ 1-2 - + + + ++ ++ 36/42 1-3 + + ++ ++ ++ ++ (visibly None 1-4 - + + + ++ ++ comfirmed 88% 0% 1-5 + + + ++ ++ ++ that >4 bites 1-6 - - + + ++ ++ per mouse) 1-7 - + + + ++ ++ 2-1 - - + ++ ++ ++ 2-2 - + + + ++ ++ 2-3 + + ++ ++ ++ ++ AdPyCS 10^7 vp 2-4 + + ++ ++ ++ ++ 38/42 91% 0% /mouse 2-5 - - + + ++ ++ 2-6 - - + + ++ ++ 2-7 + + + ++ ++ ++ 3-1 - - + + ++ ++ 3-2 - + + + + ++ 3-3 - - + + ++ ++ AdPyCS 10^8 vp 3-4 + + + ++ ++ ++ 37/42 88% 0% /mouse 3-5 - + + + + ++ 3-6 - - + + + ++ 3-7 - - + + ++ ++ 4-1 - + + + ++ ++ 4-2 - - + + + ++ 4-3 - - - - ++ ++ AdPyCS 10^9 vp 4-4 - + + + + ++ 36/42 86% 0% /mouse 4-5 - - - - ++ ++ 4-6 - - - - + + 4-7 - - + + + ++ 5-1 - - - - + + 5-2 - - - - + ++ 5-3 ------AdPyCS 10^10 vp 5-4 - - - + ++ ++ 37/42 88% 14% /mouse 5-5 - - - + + ++ 5-6 - - - - + + 5-7 - - - - + + 6-1 - - - - + ++ 6-2 - - - - + + 6-3 ------AdPyCS 10^11 vp 6-4 - - - - + ++ 38/42 91% 43% /mouse 6-5 ------6-6 - - - - + + 6-7 ------

4.4.1.4 Construction of MVA-PyCS Vaccine In Q3 FY2019, GeoVax began work to construct a recombinant MVA-VLP vaccine expressing PyCS. GeoVax obtained a synthetic PyCS gene with restriction sites so the sequence could be inserted into a shuttle vector. The shuttle vector includes recombination sites to enable recombination into the MVA- VLP vector. The shuttle plasmid also contains a GFP reporter to allow rapid isolation of recombinant viruses. The synthetic PyCS gene was inserted into the shuttle vector by standard molecular biology

MVDP Quarterly Report 35 Leidos Proprietary

techniques. Two versions of the PyCS construct were generated, both designed to express proteins that will assemble in VLPs but differing in the length of the tail for the Marburg VP40 transmembrane sequence, as GeoVax has seen differences in expression of certain antigens using different versions of the Marburg VP40. One version has an abbreviated cytoplasmic tail, 8 amino acids (CVLP-1) and a second with a longer cytoplasmic tail, 69 amino acids (CVLP-2). Prior to generating the vaccine constructs, the transient expression of PyCS in the shuttle plasmids was examined by western blot. In brief, duck fibroblast (DF1) cells were infected with MVA-MARVP40 recombinant virus (expressing the Marburg virus VP40 matrix protein) and transfected with one of the two different shuttle plasmids (CVLP-1 or CVLP-2) designed to interact with VP40. The virus was harvested 48 hours post transfection. Culture supernatants were aspirated, clarified by centrifugation, and frozen. Cells were chemically lysed and the lysates clarified by centrifugation. Both the media and the cell lysates were analyzed by western blot for the expression of Marburg VP40 (as an internal control) and for CS protein. Results of transient expression with the shuttle plasmids (Figure. 4.4-2) were provided to USAID on May 12, 2019. The anti-VP40 blot demonstrates approximately equivalent expression of VP40 in both cell lysates and supernatants across all constructs including the MVA-VP40 only (Figure. 4.4-2 Upper Panel). Anti-PyCS antibody (clone 2F6; provided by Dr. Moryia Tsuji) demonstrates expression of CS from all constructs (Figure. 4.4-2 Lower Panel). These data suggest the CVLP-1 construct with the shorter cytoplasmic tail gives superior secretion.

Figure 4.4-3. Transient Expression of PyCS in MVA-VLP Platform by Western Blot Analysis

In addition to the western blot analysis GeoVax in Q4 FY2019 also performed immunoprecipitation (IP) to determine whether the PyCS was incorporated into the Marburg virus matrix VLPs (Figure. 4.4-4). These results were provided to USAID on July 3, 2019. In brief, DF1 cells were infected with MVA- VP40 virus (expressing the Marburg virus VP40 matrix protein) and transfected with the two different shuttle plasmids expressing the different versions of PyCS, VLP-1 and VLP-2. Forty-eight hours post transfection the media was taken and clarified by centrifugation. Supernatants were used for the IP assay. IP was performed using either anti-PyCS Ab (mAb clone 2F6; provided by Dr. Moryia Tsuji) or an anti-VP40 Ab. Following the pull down the samples were probed with anti-PyCS Ab on a western blot. PyCS was pulled down by the anti-PyCS, as well as anti-VP40 Ab, suggesting that PyCS is incorporated into VP40 VLPs (Figure. 4.4-4).

MVDP Quarterly Report 36 Leidos Proprietary

Figure 4.4-4. Analysis of VLP Formation by Immunoprecipitation

With transient expression of PyCS demonstrated (Figure 4.4-3 and Figure 4.4-4), GeoVax transfected the MVA-PyCS shuttle plasmids into chick embryo fibroblast (CEF) cells infected with the MVA-VLP vector. MVA-PyCS recombinant virus was then selected based on the expression of GFP, which is present in the shuttle vectors and initial recombinants but is removed during clone purification. Virus selection is then followed by a repetition of multiple clone purification by limited dilution to purify the recombinant virus from the parental virus and to remove the GFP sequence from the rescued recombinant virus. GeoVax purified the recombinant virus in early Q4 FY2019. A Research Seed Stock (RSS) of the MVA-VLP-PyCS vaccine was generated in CEF cells. In brief, the virus was inoculated into CEF cells at an appropriate Multiplicity of Infection (MOI) and allowed to replicate. The virus was harvested and then infected into fresh flasks. After infection, the cells were scraped into the media, pelleted by centrifugation, resuspended into the appropriate amount of fresh media, and subjected to three freeze-thaw cycles. The cell debris was removed by centrifugation and the supernatant containing the released virus was aliquoted. Using the RSS, GeoVax produced preparations of the MVA-VLP-PyCS vaccine and the control vaccine for use in animal studies. In brief, GeoVax infected cultures of CEF cells with the MVA-VLP-PyCS RSS and the control vaccine stock. The cells were harvested by centrifugation and homogenized by sonication to release the virus. The homogenates were clarified by centrifugation. The virus was then purified, concentrated, and formulated in the final buffer (1X PBS, 7.5% [w/v] sucrose, pH 7.3) by sucrose pad ultracentrifugation. The purified virus was titrated. The MVA-PyCS vaccine was titrated following GeoVax’s standard TCID50 method. The result was measured as 4.27x108 TCID50/mL. Using GeoVax’s standard plaque assay method the MVA-PyCS vaccine was measured as 7.2 x108 PFU/mL. Following titration, the virus was diluted with formulation buffer to the target concentration of 1 × 108 TCID50/mL and dispensed into sterile vials.

MVDP Quarterly Report 37 Leidos Proprietary

To confirm that the purified virus contained the PyCS sequence, PCR analysis was performed in Q4 FY2019 using primers specific for the insert and the shuttle plasmid (containing the sequence-verified PyCP construct) as a control. The amplification band from the virus was at the same size as the one from the shuttle plasmid, confirming correct insertion of the PyCP open reading frame (Figure 4.4-5). These results were provided to USAID on August 6, 2019.

Figure 4.4-5. Confirmation of PyCS insert by PCR

To establish that the purified virus expresses PyCS protein, both media and cell lysates were analyzed by western blot in Q4 2019. Results of the PyCS expression by the purified virus (Figure. 4.4-6) were provided to USAID on September 7, 2019. The anti-PyCS blot (mAb clone 2F6; provided by Dr. Moryia Tsuji) demonstrates expression of PyCS in both cell lysates and supernatants with greater expression in the latter (Figure. 4.4-6). PyCS was not detected in the control MVA or MVA-VP40 viruses.

MVDP Quarterly Report 38 Leidos Proprietary

Figure 4.4-6. Confirmation of PyCS expression by Western Blot

To further confirm expression of PyCS antigen, the purified virus was analyzed by transmission electron microscopy (EM) and immunogold staining in Q4 FY2019. Results of the EM analysis (Figure. 4.4-7) were provided to USAID on August 6 and September 7, 2019. In brief, cultured DF1 cells were infected with MVA-PyCS at an MOI of 1 for 24 hours. The cells were fixed and stained with primary anti-PyCSP 2F6 Ab (provided by Dr. Moryia Tsuji) followed by secondary immunogold anti-mouse Ab. The cells were imaged on a TEM at Emory University. In addition to MVA-PyCS, MVA-MARV (positive control for VLP formation), MVA-VP40 (negative control for PyCS staining) and MVA-CSP (comparator VLP from Burnet Institute) were analyzed. Positive Ab staining was seen in cells infected with MVA-PyCS and VLP formation was evident for MVA-PyCS with filamentous particles visible outside the cells. A summary of the EM analysis with the different MVAs are in Table 4.4-8. On August 20, 2019 GeoVax shipped the vaccines (MVA-PyCS and MVA control) to ADARC for testing. ADARC received the vaccines on August 21, 2019.

Figure 4.4-7. Electron Microscopy Analysis of MVA-PyCS Vaccine

MVDP Quarterly Report 39 Leidos Proprietary

Table 4.4-8. Summary of Electron Microscopy Analysis

4.4.1.5 MVA-PyCS Vaccine Challenge Study With completion and delivery of the MVA-VLP vaccine, a study to evaluate the protective efficacy or delay in patency was commenced at ADARC with the first immunization administered on September 10, 2019. The approved study design is shown in Table 4.4-9. Results of the challenge study are anticipated in Q1 FY2020.

Table 4.4-9. MVA-PyCS Platform Challenge Study Design

4.4.2 CSP RNA VACCINE DEVELOPMENT Development of an mRNA-based CSP vaccine construct by the Leidos LInC group was proposed as part of the FY2020 AWP. This was approved by USAID in late Q4 FY2019. Development of an MTA with WRAIR is in process.

MVDP Quarterly Report 40 Leidos Proprietary

vaccine candidate in animal models and good titers were seen in humans immunized with RH5.1/AS01, no sterile protection was found upon CHMI. WEHI has previously provided data indicating that two additional proteins in the RH5 complex perform better in the GIA as compared to RH5 when the proteins are formulated with IFA (70% for RH5 compared to 80% and 90% for CyRPA and RIPR, respectively). However, it is unclear if the same GIA activity is possible with human-use adjuvants (thus Study W1 was initiated) and/or if the moderate increases in GIA seen with CyRPA and RIPR will translate to achievement of sterile protection in humans with those proteins administered alone. Further, Dr. Simon Draper (Oxford) indicated that GIA activity was similar among the individual RH5 complex proteins in their hands (data not received). Current information suggests that blood stage proteins can act synergistically to achieve higher GIA activity; therefore, a multi-antigen RH5 complex vaccine may be more efficacious than a single protein vaccine. Thus, the RCR Complex Project Plan will test the following hypotheses: (1) Targeting single and/or multiple proteins in the RCR complex can interrupt the mechanism of merozoite invasion of erythrocytes during the blood stage infection of P. falciparum and (2) Targeting multiple proteins in the complex may be more effective than targeting a single protein in the complex. To address these hypotheses, Leidos developed two parallel paths under the overall RCR Complex Project Plan as well as a workflow diagram to illustrate the interconnectedness of the projects conducted at Oxford and WEHI. Per the USAID-approved project plan, there are a total of three approaches for development of an RCR complex vaccine including recombinant protein, VLP, and RNA approaches. In addition, specific studies have been included in the workflow to facilitate down-selection (e.g., studies to identify a test protein dose and to compare different adjuvants). Based on immunogenicity assessments (e.g., ELISA and GIA activity), constructs/formulations will be downselected for comparison in a penultimate study. These interdependencies and decision points are shown in the RCR Complex Vaccine Project Workflow Figure 4.5-1, where decision points are shown as to be determined (TBD). The workflow diagram was updated to incorporate the decision by USAID on February 19, 2019, to proceed with a dose-ranging study using antigens formulated in DPX4 at WEHI. To incorporate the decision made by USAID on June 3, 2019 to not proceed with an RNA approach for the RCR Complex Vaccine Development Project (see sections 4.1 and 4.5.2), the workflow diagram has been updated to indicate that the mRNA Approach is “Not Funded.” Based on the evolution of this project, USAID approval of the project as a whole was provided prior to approval of the project plans. The project plans for both the work at WEHI and Oxford were approved in Q2 FY2019.

MVDP Quarterly Report 41 Leidos Proprietary

Figure 4.5-1. RCR Complex Vaccine Project Workflow. Down-selection is based on analysis of sera samples via GIA (pLDH and flow cytometry methods) and Quantitative ELISA

On October 30, 2018, an IPT Meeting was held at ASTMH in New Orleans. This face-to-face meeting was attended by Simon Draper’s group (Oxford), Alan Cowman’s group (WEHI), Leidos and USAID. The following outcomes of the meeting are listed below. 1) Addition of dose-ranging study with Matrix-M to the project plan entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine- University of Oxford.” 2) Change in animal model from mice to rats. Studies will be conducted with rats (except Study W1, which had already begun) to obtain higher bleed volumes, enable estimates of variance due to individual animal differences, and avoid non-specific GIA issues. 3) Incorporation of an analytical plan. Leidos consulted with an in-house statistician for guidance in power analysis and sample size determination.

4.5.1 WEHI: CECILLE BROWNE/AMY NOE Leidos submitted a draft plan (RH5 Complex Vaccine Development Project Plan) to USAID in August 2018 that included Study W1 (evaluating different human-use adjuvants) and follow-on studies to be performed by WEHI. These follow-on studies include 1) immunogenicity of double and triple protein combinations, 2) immunogenicity of monocistronic and polycistronic RNA replicons encoding complex proteins, and 3) development of reagent antibodies that specifically recognize the RCR complex. In December 2018, Leidos submitted to USAID a revised project plan with a new title: RCR Complex Project Plan - WEHI. The revised project plan includes an optional dose titration study in rats to bridge Study W1 (conducted in mice) for follow-on studies described in the project plan. Prior to approval of the project plan, USAID approved a study design for Study 1 (later renamed to Study W1) to evaluate the immunogenicity (ELISA and GIA) of individual proteins in the RCR complex (RH5, RIPR, and CyRPA) in combination with human-use adjuvants in mice. In Q4 FY2018, Leidos completed procurement efforts with WEHI to conduct this study and managed the logistics to ship DPX-formulated proteins from IMV to WEHI. Study W1 was initiated in October 2018.

MVDP Quarterly Report 42 Leidos Proprietary

During the IPT meeting in October 2018, USAID raised the concern that the 20 µg immunization dose used in Study W1 may be too high to distinguish between adjuvant groups. Oxford noted that they previously found maxed ELISA responses for RH5.1 at concentrations higher than 16 ng with Matrix-M. However, WEHI noted that they have previously used up to 200 µg of RH5 and routinely use a 20 µg dose of this protein in rabbits. Based on this discussion, Leidos recommended the performance of terminal bleeds after two doses instead of three (two weeks post second immunization). The revised study design for Study W1 is shown in Table 4.5-1. A preliminary immunogenicity test (ELISA) was completed by WEHI in December 2018, and result of this preliminary ELISA was submitted to USAID in December 2018. Results show that DPX4 gave superior titers compared to the Advax/CpG and AddaVax, which were the other two human-use adjuvants tested in the study (Figure 4.5-2 and Table 4.5-2). In this ELISA, all DPX4 and IFA dilutions gave O.D. readings that are too high such that titers could only be estimated (Table 4.5.2). The ELISA was repeated to further dilute serum samples and allow interpolation of titers using a four-parameter logistic (4PL) curve fit. GIA and repeat ELISA were performed in February 2019, and GIA results were received in April 2019. Results of the second ELISA also show that DPX4 induced higher titers compared to Advax/CpG and AddaVax; in this second ELISA, O.D. readings generated dilution curves that allowed interpolation of OD1 titers (Figure 4.5-3 and Table 4.5-3). GIA results using the FACS method show that purified IgG from mice immunized with antigens formulated with DPX4 consistently inhibited parasite growth for all three antigens, while purified IgG from mice immunized with antigens formulated with Advax+CpG or AddaVax resulted in variable parasite growth inhibition (Figure 4.5-4). To confirm that the purified IgG used in GIA bind to the respective proteins that they were raised against, ELISA was performed. ELISA results show that the purified IgG samples (used in GIA) recognized the respective antigens that they were raised against (Figure 4.5-5). WEHI also established the LDH method of GIA and tested available W1 samples using this method. GIA-LDH results show correlation with the GIA- FACS method (Figure 4.5-6). Overall, the result of Study W1 led to the selection of DPX4 as the adjuvant to use in follow-on studies. In February 2019, USAID approved the initiation of Study W2 (Dose-ranging Study in Rats) before completion of Study W1 GIA and DPX4 will be used as adjuvant. The study design for W2 is shown in Table 4.5-4.

Table 4.5-4. Study W2 – Dose-Ranging Study in Rats # rats, Study Day Cohort Formulation R strain -2 0 28 42 56 70 1 RH5.1 protein, 20 µg + DPX4 IM 6, Wistar √ √ √ 2 RH5.1 protein, 2 µg + DPX4 IM 6, Wistar √ √ √

3 RH5.1 protein, 0.2 µg + DPX4 IM 6, Wistar √ √ √ 4 CyRPA protein, 2 µg + DPX4 IM 6, Wistar √ √ √ bleed 5 CyRPA protein, 0.2 µg + DPX4 IM 6, Wistar - √ √ √ Pre 6 RIPR protein, 2 µg + DPX4 IM 6, Wistar √ √ bleed Test √ Terminal Bleed 7 RIPR protein, 0.2 µg + DPX4 IM 6, Wistar √ √ √ 8 Adjuvant alone (Negative Control) IM 6, Wistar √ √ √

Procurement with IMV for Study W2 was completed in Q2, and formulation of individual proteins in DPX4 was completed in Q3. WEHI initiated rat immunizations in May 2019. In the first immunization, a technical error occurred resulting in the administration of higher doses of proteins in rats. Instead of 20 µg, 2 µg and 0.2 µg doses, rats received 36 µg, 3.6 µg and 0.36 µg, respectively. Leidos reviewed

MVDP Quarterly Report 43 Leidos Proprietary

with WEHI the protocol provided by IMV (included with the formulation shipment) to ensure that protocol steps are followed and that rats are immunized with protein amounts indicated in the study design. The second immunization was completed on June 13 using the planned doses of 20 µg, 2 µg and 0.2 µg. The final immunization and terminal bleeds were completed on July 11 and July 25, respectively. WEHI performed ELISA on individual rat samples (test and terminal bleeds). Results of ELISA using terminal bleed samples are shown in Figure 4.5.7. The 2 µg and 0.2 µg protein amounts showed differentiation with all three proteins. The 2 µg and 20 µg protein amounts of RH5.1 showed less differentiation. The dilution curves for individual animals are shown in Figure 4.5-8. GIA for Study W2 is anticipated to be completed in the first week of October 2019. Note that due to issues with their flow cytometer, WEHI will not able to perform the GIA-FACS method for Study W2. Instead, WEHI will test W2 samples using the LDH method. WEHI anticipates that a new flow cytometer will be ready by the time samples generated in Study W3 become available. An IPT meeting was conducted on September 17, 2019 in which Study W1 and W2 results were discussed. The team will continue discussion regarding protein doses to use in Study W3. Upon completion of GIA, a final decision on protein doses for W3 will be made. The study design for W3 is shown in Table 4.5-5. Final dose assignments for W3 will be based on GIA results from study W2 and will be determined in October 2019 to allow time for formulation manufacturing at IMV and for shipment to WEHI. WEHI will begin immunizations in December 2019.

Table 4.5-1. Study W1. Immunogenicity Study in Mice of Individual RCR Proteins in Three Human use Adjuvants and IFA

Cohort Study Day Test Sample R # mice, strain # -2 0 28 42 1 RIPR (20 µg) + IFA IM 6, BALB/c √ √ 2 RH5.1 (20 µg) + IFA IM 6, BALB/c √ √ 3 CyRPA (20 µg) + IFA IM 6, BALB/c √ √ 4 RIPR (20 µg) + AddaVax IM 6, BALB/c √ √

5 RH5.1 (20 µg) + AddaVax IM 6, BALB/c √ √ 6 CyRPA (20 µg) + AddaVax IM 6, BALB/c √ √ bleed 7 RIPR (20 µg) + Advax+CpG IM 6, BALB/c - √ √ 8 RH5.1 (20 µg) + Advax+CpG IM 6, BALB/c Pre √ √ Terminal bleed 9 CyRPA (20 µg) + Advax+CpG IM 6, BALB/c √ √ 10 RIPR (20 µg) + DPX4 IM 6, BALB/c √ √ 11 RH5.1 (20 µg) + DPX4 IM 6, BALB/c √ √ 12 CyRPA (20 µg) + DPX4 IM 6, BALB/c √ √

MVDP Quarterly Report 44 Leidos Proprietary

Figure 4.5-2. W1 First ELISA - Preliminary immunogenicity Results of Individual RCR Proteins in Four Adjuvants. Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

Table 4.5-2. Preliminary Estimates of Immunogenicity Titers (at O.D. = 1.0)

RH5.1 RIPR CyRPA DPX4 >51,200 >51,200 30,000 IFA 25,000 >51,200 >51,200 Advax+CpG 3,200 8,800 400 AddaVax 600 3,200 2,400

MVDP Quarterly Report 45 Leidos Proprietary

Figure 4.5-3. W1 Second ELISA - Immunogenicity Results of Individual RCR Proteins in Four Adjuvants (repeat ELISA). Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

Table 4.5-3. Immunogenicity Titers Derived from 4PL Curve Fit (at O.D. = 1.0); Second ELISA

RH5 RIPR CyRPA DPX4 288,479 1,302,740 29,107 IFA 185,188 537,314 47,561 Advax+CpG 25,087 14,541 < 1,000 AddaVax 3,365 15,180 < 1,000

MVDP Quarterly Report 46 Leidos Proprietary

Figure 4.5-4. W1 GIA Results (FACS Method) – IgG was purified from pooled mice sera from terminal bleeds (2 weeks post second immunization). Purified IgG samples were serially diluted and tested by GIA using the flow cytometry method.

MVDP Quarterly Report 47 Leidos Proprietary

Figure 4.5-6. W1 GIA LDH Method compared to GIA FACS Method – Purified IgG samples (that were used in GIA) were serially diluted and tested by GIA using the LDH method. GIA LDH Results are compared to results using GIA FACS methods. Note that due to insufficient samples, the RH5 + AddaVax sample and CyRPA + Advax/CpG sample were not tested in the LDH method.

MVDP Quarterly Report 48 Leidos Proprietary

Figure 4.5-7. Study W2 ELISA – A) OD1 titers were determined using 4PL Curve fit based on average of absorbances per dilution per cohort at terminal bleed. Each cohort (each bar) represents six rats. B) OD1 titers were determined using 4PL Curve fit based on dilution curves of individual rats. Each circle represents one rat. Note that for some rats, OD1 titers could not be determined - 3 rats in RH5.1 0.2 µg test and terminal bleed sets, 5 rats in CyRPA 0.2 µg test bleed set and 1 rat in CyRPA 2 µg test bleed set.

MVDP Quarterly Report 49 Leidos Proprietary

MVDP Quarterly Report 50 Leidos Proprietary

Figure 4.5-8. W2 ELISA Dilution Curves - Immunogenicity Results of Individual RCR Proteins formulated at different proteins amounts in DPX4 in rats. Individual sera from test and terminal bleeds were serially diluted and tested by ELISA. A) RH5, B) Ripr, and C) CyRPA.

Table 4.5-5. Study W3 Protein Combination Study in Rats

# rats, Study Day Cohort Formulation R strain -2 0 28 42 56 70 1 RH5.1 + DPX4 IM 6, Wistar √ √ √ 2 CyRPA + DPX4 IM 6, Wistar √ √ √ 3 Ripr + DPX4 IM 6, Wistar √ √ √

4 RH5.1 + Ripr + DPX4 IM 6, Wistar √ √ √

5 RH5.1 + CyRPA + DPX4 IM 6, Wistar √ √ √ Bleed 6 Ripr + CyRPA + DPX4 IM 6, Wistar - √ √ √ Pre RH5.1 + CyRPA + Ripr + DPX4 bleed Test 7 IM 6, Wistar √ √ √ Terminal Bleed Dose #1 By Molar Ratio RH5.1 + CyRPA + Ripr + DPX4 8 IM 6, Wistar √ √ √ Dose #2 By Mass 9 DPX4 alone (Negative Control) IM 6, Wistar √ √ √

4.5.1.1 Timeline for RCR Complex Project WEHI An updated timeline for RCR Project Plan WEHI is shown in Table 4.5-6.

MVDP Quarterly Report 51 Leidos Proprietary

Table 4.5-6. Timeline for RCR Project WEHI

4.5.2 SYNTHETIC GENOMICS INC. (SGI) USAID approved the study design for Study W4 (Replicon Study in Rats). SGI would prepare monocistronic and polycistronic RNA replicons encoding the RCR proteins. Procurement with SGI was completed, and a kickoff meeting was held on March 27, 2019. However, on May 9, 2019, SGI informed Leidos that their RNA platform would no longer be available due to acquisition of the company. On May 15, 2019, Leidos proposed an alternate RNA strategy to replace the SGI platform. However, on June 3, 2019, USAID notified Leidos that the alternate RNA approach would not be funded for the RCR project due to time limitations.

4.5.3 PRECISION ANTIBODY The procurement process was completed with Precision Antibody in Q2. Precision Antibody will develop an antibody reagent that specifically recognizes the RCR complex. The antibody reagent was originally planned for the detection of complex formation in cultures transfected with RNA replicons in Study W4, a study that has been discontinued. Although Study W4 (RNA study) will no longer be conducted, USAID has agreed to continue with the RCR antibody development. In the first phase of this project, Precision Antibody will evaluate RCR stability in the adjuvant used in monoclonal antibody development. The goal of the first phase is to ensure that the immunogen is in a complex formation when administered to mice. RCR stability evaluation, which will begin in Q3 (April 2019), will be performed using immunoprecipitation followed by Western Blotting and/or Size exclusion chromatography. If the RCR complex is stable in adjuvant, Precision Antibody will use the RCR as immunogen to generate monoclonal antibodies using Precision’s established workflow. If the RCR complex is not stable in the first adjuvant, another adjuvant will be tested. If the RCR complex is not stable in the second adjuvant, a decision will be made whether or not to continue with monoclonal antibody development.

MVDP Quarterly Report 52 Leidos Proprietary

Precision Antibody performed two rounds of immunoprecipitation with anti-RH5 antibody (hR5.016 from Oxford), which can only recognize RH5 when in complex with CyRPA. In both rounds, the three proteins were found to be present in the immunoprecipitate suggesting that the complex may be intact in Ribi adjuvant. However, also in both rounds, the positive control (no Ribi) showed no immunoprecipitated protein; thus a definitive conclusion cannot be made. Several approaches were recommended by Precision to investigate the positive control. However, due to time and funding limitations, rather than investigate the positive control, Precision was directed to move ahead with immunization of mice. The test bleed of the 4-5 animals will inform whether antibodies to RCR complex (to be coated in an ELISA plate) have been induced. If no reactivity is observed, boost immunization will be performed. If reactivity is still not observed after the boost, a decision will be made whether to move forward with hybridoma development.

4.5.4 UNIVERSITY OF OXFORD (OXFORD): JAYNE CHRISTEN/AMY NOE On December 17, 2018 of Q1 FY2019, Leidos submitted a draft project plan entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford” to USAID. Presented in the plan were the study designs for four in-life studies (Studies O1-O4) as well as the description of two reagent/assay development activities (i.e., quantitative ELISA and VLPs decorated with RCR recombinant proteins). USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Leidos circulated the updated project plan to USAID on February 5, 2019; USAID approved the project plan on February 13, 2019. A kick-off meeting was held on March 4, 2019. An overview of the development plan for this project is provided in Table 4.5-7.

Table 4.5-7. Overview of the RCR Complex Vaccine Development Project Plan-Oxford

Phase Milestone Activities • Humoral response assessments Study O1 1. Dose-Ranging Study • GIA assessment • Generation of quantitative ELISA control sera 2. Assess immunogenicity of individual • Humoral response assessments Study O2 proteins and double/triple protein • GIA assessment mixtures 3. Establish quantitative method to • Calibration-free concentration analysis method Assay assess antigen-specific IgG levels for • Affinity purification method Development the RCR proteins • Technology transfer to WEHI • Generation and expression of RH5-SpyTag, CyRPA-SpyTag, and RIPR-SpyTag 4. Generation and characterization of VLP • Conjugation of single antigen-SpyTag or RCR- single antigen VLPs and RCR complex Development SpyTag complexes to HBsAg-SpyCatcher VLP VLPs carrier • Protein purification and characterization 5. Assess immunogenicity of individual • Humoral response assessments Study O3 VLPs, VLP combinations, and RCR • GIA assessment complex VLPs 6. Head-to-Head immunogenicity • Humoral response assessments Study O4 analyses for all relevant • GIA assessment immunogens/formulations As noted in the appropriate sections below, USAID has approved both the VLP development and the Study O1 design (Table 4.5-8). The draft design of Study O2 (Table 4.5-9, recombinant proteins) has also been approved; however, determination of protein concentration is pending results of Study O1. The final study design will be provided to USAID with the data from Study O1 for final approval. The

MVDP Quarterly Report 53 Leidos Proprietary

Study O3 (VLPs) design is pending approval. Additionally, the Study O4 (all relevant immunogens/formulations) design is pending adjuvant, antigen, and construct approval. Leidos initiated procurement activities in November 2018 of Q1 FY2019 and completed activities in February 2019 of Q2 FY2019.

4.5.4.1 Dose-Ranging Study (Study O1) On November 30, 2018 of Q1 FY2019, USAID approved the design of Study O1. In Study O1, the immunogenicity of RH5, CyRPA and RIPR proteins as well as the ability of antibodies induced to elicit parasite growth inhibition will be evaluated individually in rats (Table 4.5-8). Study O1 was initiated during Q4 FY2019 and is expected to be completed in Q1 FY2020.

Table 4.5-8. Study O1 – Dose-Ranging Study

Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 (2 µg) + Matrix-M IM 6, Wistar √ √ √

2 RH5.1 (200 ng) + Matrix-M IM 6, Wistar √ √ √

3 RH5.1 (20 ng) + Matrix-M IM 6, Wistar √ √ √

4 CyRPA (2 µg) + Matrix-M IM 6, Wistar bleed √ √ √ - Pre 5 RIPR (2 µg) + Matrix-M IM 6, Wistar √ √ bleed Test √ Terminal Bleed 6 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √ 7 Negative Control IM 3, Wistar ** **Terminal Bleed

4.5.4.2 Immunogenicity Study of Individual Proteins and Double/Triple Protein Mixtures (Study O2) USAID approved the draft design of Study O2 on October 4, 2018 of Q1 FY2019 (Table 4.5-9); however, determination of protein concentration is pending results of Study O1. The final study design will be provided to USAID with the data from Study O1 for final approval. Activities will be expanded on during the course of project reporting. Note that discussions to include additional cohorts to the design of Study O2 were initiated during Q4 FY2019. To allow for a head-to-head comparison of DPX4 and Matrix-M, it has been proposed to include five additional cohorts (RH5 + DPX4, RIPR + DPX4, CyRPA + DPX4, Reconstituted RCR complex + DPX4, DPX4 only). Based on recent data from Oxford, the addition of one cohort for the RIPR-EGF (5-8) fragment has also been proposed. An updated study design was circulated to USAID on September 27, 2019.

MVDP Quarterly Report 54 Leidos Proprietary

Table 4.5-9. Study O2 – Individual Proteins and Double/Triple Protein Mixtures

Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 2 CyRPA + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 3 RIPR + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 4 RH5.1 + RIPR + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ RH5.1 + CyRPA + Matrix-M 5 IM 6, Wistar √ √ √ (Dose TBD)

RIPR + CyRPA + Matrix-M (Dose 6 IM 6, Wistar √ √ √ TBD) bleed RH5.1 + CyRPA + RIPR + Matrix-M - Pre 7 (Dose TBD) IM 6, Wistar √ √ bleed Test √

Equivalent Molar Ratio Terminal Bleed RH5.1 + CyRPA + RIPR + Matrix-M 8 (Dose TBD) IM 6, Wistar √ √ √ Equivalent Protein Concentration Reconstituted RCR + Matrix-M 9 IM 6, Wistar √ √ √ (Dose TBD) 10 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √

During Q4 FY2019, the stability of the purified, reconstituted RCR complex was evaluated in Matrix-M. As shown in Figure 4.5-9, 2 or 10 µg of RCR complex were formulated in TBS or Matrix-M and incubated overnight at 4°C. Immunoprecipitation was performed using the human R5.016 monoclonal antibody, which specifically recognizes RH5 when in complex, and samples were analyzed by SDS-PAGE. SDS-PAGE results revealed protein bands of expected molecular weight for each protein of the RCR complex, which indicates that the RCR complex is stable in TBS and Matrix-M. Additionally, these results show that the RCR complex can be precipitated by hR5.016.

Figure 4.5-9. Stability of RCR Complex in Matrix-M

MVDP Quarterly Report 55 Leidos Proprietary

4.5.4.3 Development of Quantitative ELISA In conversations and approval of the FY2019 annual work plan, USAID indicated support of pursuing the development of quantitative ELISAs at Oxford with subsequent assay transfer to WEHI. Activities will be expanded on during the course of project reporting.

4.5.4.4 VLP Development USAID approved the development of SpyCatcher-HBsAg VLPs decorated with single antigens or the RCR complex on October 4, 2018 of Q1 FY2019. VLP development began in Q3 FY2019 with the production of SpyCatcher-HBsAg VLPs and initiation of cloning efforts for RH5-SpyTag, CyRPA-SpyTag, and RIPR-SpyTag. SpyCatcher-HBsAg VLPs were characterized by dynamic light scattering (DLS), transmission election microscopy (TEM), SDS-PAGE, and Western Blot (Figure 4.5-10). Analysis by DLS (Figure 4.5-10A) and TEM (Figure 4.5-10B) yielded expected results for VLP diameter (~26 nm) and VLP morphology. SDS-PAGE analysis revealed a single protein band of expected molecular weight (MW) at 37 kDa while Western Blot analysis using an anti-HBsAg antibody identified multiple bands in the VLP preparation that are consistent with protein monomers and dimers (Figure 4.5-10C).

Figure 4.5-10. Characterization of SpyCatcher-HBsAg VLPs

MVDP Quarterly Report 56 Leidos Proprietary

suggest that the RCR (CyRPA-SpyTag) complex can be conjugated onto the SpyCatcher-HBsAg VLPs (Figure 4.5-11B). In this experiment, the RCR (CyRPA-SpyTag) complex was incubated with SpyCatcher-HBsAg VLPs overnight at 4°C in Tris Buffered Saline (20mM Tris-HCl, 150mM NaCl, pH 7.4). Four different molar ratios of RCR:HBsAg were evaluated (0.5:1, 1:1, 1.5:1, and 2:1). As shown in Figure 4.5.11B, all molar ratios resulted in the formation of the RCR-HBsAg VLPs (yellow rectangle). However, using a molar ratio of 1:1 or higher resulted in the absence of unconjugated HBsAg VLPs (no red rectangle). Optimization of the conjugation reaction is ongoing at the time of this writing.

Figure 4.5-11. Characterization of RCR (CyRPA-SpyTag) (A) and Preliminary Conjugation of RCR-SpyTag Complex to SpyCatcher-HBsAg VLPs (B)

All cloning efforts for RH5-SpyTag, RIPR-SpyTag, and CyRPA-SpyTag (both N- and C-term SpyTag) were completed during Q4 FY2019. Stable S2 cell lines have been generated for RIPR-SpyTag and large scale purification of both RH5-SpyTag and RIPR-SpyTag is ongoing at the time of this writing. Both N- and C-term CyRPA-SpyTag were successfully conjugated to SpyCatcher-HBsAg VLPs. However, N- term CyRPA-SpyTag:HBsAg VLPs cannot form a complex with RH5 and RIPR at the surface of the VLP. Moreover, C-term CyRPA-SpyTag:HBsAg VLPs precipitate upon incubation with RH5 and RIPR. RCR complex formation at the surface of the SpyCatcher-HBsAg VLPs is being explored at the time of this writing.

4.5.4.5 Immunogenicity Study of Individual VLPs, VLP Combinations, and RCR Complex VLPs (Study O3) A study design and description were presented in the draft project plan, entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford” which was originally submitted to USAID on December 17, 2018 of FY2019. USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Study O3 is pending approval by USAID at the time of this writing.

4.5.4.6 Head-to-Head Immunogenicity Analyses of All Relevant Immunogens/Formulations (Study O4) A study design and description were presented in the draft project plan, entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford” which was originally submitted to USAID on December 17, 2018 of FY2019. USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Study O4 is pending approval by USAID at the time of this writing.

MVDP Quarterly Report 57 Leidos Proprietary

4.5.4.7 Updated Schedule: RCR Complex Project Oxford The initial project schedule provided by Oxford (included in the Project Plan) had a Study O1 start date in February 2019 and completion date in May 2020. Approval of the Project Plan was received from USAID on February 13, 2019. Of note is that due to the number of rats involved in the planned (USAID-approved) studies for this project, it is necessary for Oxford to subcontract the in life portions. Oxford finalized their subcontract in March 2019. Further, study approval by Novavax is needed prior to provision of Matrix-M. Oxford received study approval from Novavax in May 2019 and a signed MTA in June 2019. Based on this updated timeframe, Leidos requested a revised schedule from Oxford. The project schedule shown in Table 4.5-10 was provided by Oxford in Q4 FY2019. Note that Study O1 started in July 2019, and Oxford communicated that Noble Life Sciences can run Study O2 and Study O3 in parallel.

Table 4.5-10. Project Timeline Received from Oxford

4.6. RH5.1 HUMAN MAB IDENTIFICATION AND DEVELOPMENT PROJECT: VIN KOTRAIAH/CECILLE BROWNE The RH5.1 human mAb identification and development project is new for FY2019. Procurement efforts were started in Q1 FY2019 and are anticipated to be finalized in Q2 FY2019. Leidos (working with Oxford) provided the synopsis for this project to USAID in August 2018 and included this project in the FY2019 Annual Work Plan (submitted to USAID on September 1, 2018). In response to their review of the FY2019 annual work plan, USAID indicated their interest in funding portions of this proposal. On December 5, 2018, Leidos submitted to USAID a draft Project Plan on RH5.1 Human mAb Isolation and Development to USAID. USAID reviewed the plan and provided their comments on December 14, 2018. Leidos updated the plan based on feedback from USAID and submitted a final plan on December 19, 2018. USAID approved elements in the plan relating to mAb development, screening and characterization to be performed in the Draper lab on January 29, 2019. A decision on the R5.016 immunogen work was deferred. A kick-off meeting was held on March 4, 2019 where Oxford presented a slide deck titled “High-throughput cloning of RH5-specific mAbs,” that contained the VAC063 data as well as their initial list of suggested VAC063 samples to use in this project. This presentation was circulated to USAID and an IPT meeting was held on March 18, 2019 to discuss the VAC063 data and to finalize sample selections. Additional sample selection updates were received from Oxford and circulated to USAID on March 22, 2019. Initiation of the technical work is pending USAID approval of sample selection, anticipated in early Q3 FY2019. As anticipated, a priority list of 20 samples was approved by USAID on April 1, 2019 (Table 4.6-2) and progress to date is described below. An overview of the development plan for this project is provided in Table 4.6-1 followed by a brief summary of the proposed project.

MVDP Quarterly Report 58 Leidos Proprietary

Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project

Phases Milestones Activities • Single B cell isolation (memory B cells (mBC) or plasmablasts) B Cell Isolation and • Reverse transcription and paired heavy and light chain Cloning nested PCR • DNA purification and cloning into heavy and light chain B Cell Isolation and vectors mAb Development • Transfection of heavy and light chain vectors into HEK293 mAb Expression and cells Purification • Harvest of supernatant and screening by ELISA • Affinity purification of antibodies from supernatant • Sequencing of heavy and light chain variable regions mAb Sequencing • Determination of sequence maturation and germline alleles ELISA Titer and • Determination of ELISA titers Protein Mapping • Protein region mapping using a panel of RH5 variants GIA • Growth Inhibition Assay (using pLDH method) mAb Screening Epitope Similarity • Determination of similarity of B cell epitopes recognized by Screen antibodies using competition assay and SPR/BLI mAb Down-Selection • Monoclonal antibody down-selection mAb Affinity • Determination of antibody affinity by SPR mAb mAb Inhibition of • Assessment of inhibitory activity of antibodies on RCR, Characterization Complex Formation RH5-P113 and RH5-Basigin complexes mAb Down-Selection • Selection of monoclonal antibodies for structural studies Computational • Identification of sequence variants of the immunogen that Assessments are stable and have the right conformation R5.016 Immunogen Production of Select • Epitope grafting and production of selected immunogens Design (optional) Immunogen Designs Immunogen Down- • SPR screening for binding to R5.016 mAb Selection Production of • Production and QC of down-selected immunogen R5.016 Immunogen Immunogen Production Coupling of (optional) • Conjugation of down-selected immunogens to VLPs Immunogen to VLP Formulation of • Selection and procurement of adjuvant Immunogen:VLP • Formulation of immunogen R5.016 Immunogen • Rat immunizations with RH5.1 comparator Rat Immunization Testing (optional) • Collection of sera Immunogenicity and • Humoral response and GIA activity with R5.016 mAb GIA assessments comparator • Recombinant antibody expression and purification Antibody Production • Fab fragment generation New mAb • Screening for Fab:RH5 co-crystals Structure Crystal Screening • Cryoprotection of crystals Determination • X-ray diffraction studies (optional) Crystal Structure • Model building and refinement Determination • Epitope delineation

MVDP Quarterly Report 59 Leidos Proprietary

Oxford proposed identification of human RH5 mAbs and conduct of a B cell analysis using VAC063 clinical samples. This project will involve surveying the B cell and Ab repertories from vaccinated subjects utilizing both B cell cloning and serum immunomics techniques in order to understand the epitopes recognized by human anti-RH5 sera, how these epitopes might contribute to functional GIA, and gather information on differences in repertoire based on dose/immunization regimen/CHMI. As part of this effort, Oxford also proposed evaluating novel immune mechanisms of protection based on the sterile protection and reduced parasite growth rate seen with unvaccinated individuals after CHMI. These activities have been incorporated into the project proposal and will be expanded on during the course of project reporting. The initial project schedule provided by Oxford (included in the Project Plan) had a start date of December 2018 and a completion date in May 2020. Approval of the Project Plan (regular elements only) was received from USAID on January 29, 2019. Per USAID request, Leidos directed Oxford to perform a sample selection inclusive of the RH5.1 clinical trial GIA data. Oxford conducted the same in March 2019, once the RH5.1 clinical study GIA data were available for analysis. Approval of sample selection by USAID is anticipated in early Q3 FY2019. Based on this updated timeframe, Leidos requested a revised schedule from Oxford, shown in Figure 4.6-1. Note that the updated project schedule begins in April 2019 and a completion date in September 2020, extending beyond the MVDP POP end date. Leidos recommends holding an IPT meeting in Q1 FY2020 to determine the project stopping point.

Figure 4.6-1. Project Timeline Received from Oxford

The priority list of 20 samples approved by USAID on April 1, 2019 is shown in Table 4.6-2. Note that the first 20 samples were approved with the Oxford group aiming to deliver roughly 10 RH5-specific mAbs from each of these 20 samples and characterize them. In the “Group” column of Table 4.6-2, the samples are color coded by the VAC063 Group number to which they belong. Six samples drawn from Group 7, 10 drawn from Group 5 and 4 from Group 3 constitute the total of 20 approved samples. In the “Volunteer” column, the volunteer IDs are color coded by the selection criterion. The color yellow indicates that the volunteer was selected on the basis of In Vivo Growth Inhibition (IVGI) only. The green colored cells indicate that the volunteers were selected on the basis of IVGI and ELISA, Avidity or GIA. The blue colored cells indicate that the volunteers were selected on the basis of ELISA, Avidity or GIA. Note that Oxford’s slide summary with the ELISA, Avidity, GIA and IVGI data for all the VAC063 volunteers was provided to USAID in Q2 FY2019. On June 17, 2019, Oxford notified Leidos that volunteer 01-809 (01-032) from Group 7 with a priority number of 7 (Table 4.6-2) had withdrawn consent and that Oxford could no longer use this volunteer’s samples. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement. The consent withdrawal and proposed replacement sample were communicated to USAID in the bi-weekly meeting on June 19, 2019.

MVDP Quarterly Report 60 Leidos Proprietary

Table 4.6-2. Priority list of samples with their VAC063 Group number and Volunteer IDs are shown. Availability of PBMC samples from the indicated day of collection are also shown. The time-points at which the ELISA, Avidity and in vitro GIA data are available are also shown. Lastly, the in vivo growth inhibition (IVGI) is shown for the samples from Groups 5 and 7.

C-1 - day before challenge; C+28 - 28 days after challenge; DOD – day of diagnosis; IVGI was calculated as % reduction in parasite multiplication rate (PMR) in individual vaccinees versus the mean PMR of the control group. Volunteer 01-809 (01- 032) from Group 7 with a priority number of 7 withdrew consent in June 2019. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement.

Table 4.6-3 depicts the progress as of June 24, 2019. A naming convention was developed to track the mAbs isolated in this project. Each sample in the priority list has been assigned a letter of the alphabet (shown in column “Patient ID (cloning)”) and antibodies isolated from a particular sample will have a designation that begins with the letter assigned to that sample. For example, a mAb identified as BD5 indicates that it has been isolated from a Patient ID B sample and D5 indicates the well ID for which the particular B cell was sorted into during single B cell sorting by flow cytometry using the RH5-specific tetramer probes. In some instances, samples were subjected to multiple rounds of tetramer-based sorting. In these cases, the first repeat sorting is indicated using an apostrophe after Patient ID letter

MVDP Quarterly Report 61 Leidos Proprietary

and second repeat is indicated by an apostrophe followed by the number 2 after the Patient ID letter (see Patient ID H for example). Table 4.6-3 shows the number of RH5-specific mAbs that have been isolated and cloned to date (“RH5-specific mAbs cloned”). A total of 18 mAbs have been isolated from three patient samples; all of these mAbs were isolated prior to the start of the USAID-funded project during test runs of the overall process. Of the three patient samples, only the patient ID “B” sample is present in the priority approved list of samples for the mAb identification and development project; the other two patient samples are not on the priority approved list. Sixteen of the 18 mAbs have been purified and characterized in ELISA and GIA assays; 2 mAbs could not be produced in sufficient quantities for characterization studies. Of note is that sorting of B cells from six additional samples from the priority list was completed in Q3 FY2019 as shown in Table 4.6-3; mAb cloning for these is ongoing.

Table 4.6-3. Summary of progress in Q3 FY2019. The blank cells under the RH5-specific mAbs cloned column indicate that the cloning is pending.

The current set of 16 mAbs was characterized in ELISA using four different RH5-based plate antigens (Figure 4.6-2). All four antigens have a C-tag for purification and proper folding of RH5.1 and RH5.2 following purification has been verified using dot blots and mAbs previously characterized as recognizing conformational epitopes. Briefly, conformational mAbs were used to develop dot blots of denatured (boiled) protein, normal protein and “no protein” for each plate antigen. Only the natively expressed protein was recognized by the mAbs indicating proper folding of the recombinant plate antigen. Proper folding of the Bundle antigen has been confirmed by the Higgins Lab at Oxford and further details on the confirmation are awaited.

Figure 2.6-2. RH5 plate antigens used in ELISA. Apart from RH5.1 (native and denatured) and the N- terminus alone antigen, an additional antigen called RH5.2 in which the N-terminus and intrinsic loop are deleted was used as a plate antigen. Of note is that Oxford also uses the notation “SV3” or “delta NL” when referring to

MVDP Quarterly Report 62 Leidos Proprietary the RH5.2 antigen; however, Leidos will use RH5.2 for consistency. A plate antigen called Bundle which displays the epitope of the R5.016 mAb isolated from the VAC057 trial (described in Alanine et al., Cell 2019) on a non- RH5 protein scaffold was also used as plate antigen. For the ELISA assessments, cut-off thresholds were used for determining binding versus non-binding by the mAbs, particularly regarding how “weak” binders are identified above background. Cut-off OD values were designated depending on the background typically experienced. Two measurements of background were used – control mAbs on PBS-coated wells and casein (the blocking buffer/diluent used for all samples) on antigen-coated wells. The background values for the former never exceeded 0.09 OD and the latter OD value on RH5.1coated wells never exceeded a value of 0.1. Therefore, a value below an OD of 0.1 was regarded 'at or below background' and therefore negative for binding. The ELISA data for the 16 mAbs along with control mAbs are shown in Figure 4.6-3. Also shown on each graph are OD values for control mAbs, R5.016, h4BA7 and LB12. R5.016 is known to bind a conformational epitope in the bundle region, h4BA7 is a linear epitope in the intrinsic loop, and LB12 is known to bind a linear epitope in the N-terminus region. All of these control mAbs worked as expected except LB12, which was slightly positive for SV3 (i.e., RH5 delta NL) and strongly positive for Bundle. Oxford is currently evaluating the aliquot of LB12 used in these experiments. All mAbs with the exception of BC8 appear to bind conformational epitopes on RH5.

Figure 4.6-3. ELISA data for purified mAbs from patient IDs A, B and C. Also shown on each graph are OD data for control mAbs (R5.016, h4BA7 and LB12).

GIA assays have been carried out by the LDH method and the sixteen mAbs were initially screened at 2 mg/ml final concentration. The 7 (of the 9) mAbs that showed high levels of growth inhibition at 2 mg/ml were next tested for dose-response. Both of these data sets are shown in Figure 4.6-4. The

MVDP Quarterly Report 63 Leidos Proprietary

mAb BD5 from the fractional dose Group 3 volunteer (Priority number 19) showed nearly 80% inhibition even at the lowest concentration tested (<0.1 mg/mL). Based on the ELISA data (Figure 4.6- 3) the mAb BD5 epitope seems to overlap that of R5.016.

Figure 4.6-4. GIA data for the sixteen purified mAbs from patient IDs A, B, and C at 2 mg/ml (left panel) and for select mAbs in dose titration (right panel)

In addition to the work proposed by Oxford, an immune repertoire survey of RH5.1 vaccinees was also proposed (summarized in the section below). The final decision on this work is pending analysis of the clinical study GIA data. The BD5 mAb was further tested in the GIA using an 8-point 4-fold dilution series. The data are shown in Figure 4.6-5. The IC50 was calculated as approximately 2 µg/mL making it the most potent human RH5 antibody discovered by the Oxford Figure 4.6-5. Dose titration GIA data for BD5 mAb group. In September, Oxford informed Leidos of a change in the naming convention for the mAbs isolated in this project. Instead of using letters to refer to the volunteer, they will now use the volunteer priority numbers as shown in the first column of Table 4.6-2. The name of the mAb will now consist of the priority number followed by the sort location of the B cell on a 96-well plate (Row and Column number). Oxford also reported that PCRs were working again and that the PCRs from cells sorted from 3 volunteers have been completed. Ligation and transformation efficiencies were low but they were planning to switch to a restriction enzyme from NEB to increase the efficiency. The Oxford update slide deck was sent to USAID 9/16/2019.

MVDP Quarterly Report 64 Leidos Proprietary

be made until after receipt and review of the GIA data from the RH5.1/AS01 clinical study. A brief summary of the proposed project is provided below. This project aims to use novel sequencing technologies (Boutz et al., 2014; DeKosky et al., 2016; Lavinder et al., 2015; Lavinder et al., 2014; Saphire et al., 2018; Williams et al., 2017) to identify the B cell and IgG immune repertoires for select RH5.1 vaccinees from the VAC063 clinical study and would involve collaboration between the Draper lab at Oxford and Drs. Georgiou and Ippolito at the University of Texas Austin. The humoral response to vaccination involves the generation of short-lived plasmablasts that peak approximately 8 days after immunization and the generation of broadly diverse memory B cells and long-lived plasma cells that secrete high-affinity mAbs in the germinal centers of the lymph nodes. The three iterative germinal center processes of somatic hypermutation of germline variable region DNA sequences, clonal expansion and selection of high affinity B cell receptor variants are involved in the evolution of the humoral response to vaccination. The data generated through this project will help us understand the evolution of the humoral response in vaccinees in a cohort over time as well as before and after primary and/or secondary CHMI. In addition, the evolution of the humoral response can be compared between dose cohorts and between different immunization regimens. Through this approach, we may for example, identify clusters of antibody or BCR sequences that encode highly functional mAbs but are present in very low frequency in the vaccinees. Such information together with structural information on how these mAbs recognize the cryptic epitopes on RH5.1 could mean that the RH5.1 immunogen has the potential to be engineered in the future, to elicit greater expansion of these highly functional mAbs and thereby better protective efficacy. In discussions with USAID, they indicated that the decision to fund this project would not be made until after review of the VAC063 GIA data. There are no Q1 FY2019 updates to report for this section. Preliminary sample selections for this project also were discussed in the IPT meeting on March 18, 2019. In further discussions, USAID tasked Leidos with following up with Drs. Georgiou and Ippolito regarding the sample selection and the use of pre-immune samples in their work. Additional sample selection updates were received from Oxford and circulated to USAID on March 22, 2019. A priority list of 20 samples was approved by USAID on April 1, 2019. On May 1, 2019, Leidos met with UTA and Oxford to discuss the clinical study data and the Oxford sample selection. During this call, Leidos noted the importance of scoping the UTA project so that it could be completed within the prime contract POP. Based on this and the factors included below, UTA indicated that it would be best to proceed with assessment of the five fractional dose group volunteer samples in the approved priority list of 20 samples. Oxford concurred with this selection. The factors considered are as follows:

• A sufficient number of samples should be tested to obtain a complete picture of the anti-RH5 antibody responses for the specific question(s) asked.

• Sample selection should facilitate publication of the data.

• Based on timeframe, with receipt of the sample in June, the number of samples screened should be seven or less.

• As the highest levels of immunogenicity (ELISA and GIA) are seen in samples from the fractional dose group, UTA noted that they anticipate this group being the richest source of antibodies.

• Each sample selected should have 2 vials of cells available for shipment to UTA (Oxford later confirmed that there are sufficient cells for the five selected fractional dose group volunteers).

MVDP Quarterly Report 65

Leidos Proprietary

Figure 4.7-2. Summary of the observed additive, inhibitory or synergistic mAb combinations. The RCR complex and the different mAbs against the three complex proteins are depicted in panel A. RH5 protein is depicted in blue and anti-RH5 mAbs are indicated by the R5 in their names. CyRPA is depicted in orange and anti- CyRPA mAbs are indicated by Cy in their names. RIPR is depicted in green and the mAb with RP in its name is the anti-RIPR 3H7 mAb. The GIA activity of the pair-wise combinations of RP.012 (3H7) with CyRPA mAbs and with RH5 mAbs is depicted in Panel B as additive (Add), antagonistic (minus 2x, for example) or synergistic (3x, for example). Due to the ongoing work with 3H7 and interesting data originating from Oxford, Leidos was approved by USAID to determine the sequence of the variable regions of the 3H7 mAb and to create expression plasmids from which a recombinant form of 3H7 could be produced. The project was launched in Q4 FY2019. The determination of the amino acid sequences of peptide fragments of light chain and heavy chain by LC/MS was completed in August 2019. The corresponding nucleotide sequences were then incorporated along with constant regions of each chain into separate expression cassettes via gene synthesis. Both of these cassettes have been cloned into a pcDNA3.1 expression vector. Small-scale expression analysis of the recombinant mAb from these vectors is currently underway.

4.8. PD1 BLOCKADE INHIBITOR RESEARCH ACTIVITIES: TIM PHARES In malaria-infected individuals, PD1 is expressed by T cells suggesting PD1-mediated T cell dysfunction may contribute to the lack of long-lived protective immunity against the parasite. Further, in a rodent malaria model PD1 severely dampened T cell proliferation and function mediating chronic infection. These data argue that blocking PD1 inhibition may establish a long lasting amnestic response following vaccination. Blockade of the PD1:PDL1 pathway has been a successful and impressive immunotherapy for cancer. Moreover, recent studies are exploring the application to “adjuvant-ize” PD1 blockade inhibitors in prophylactic vaccination. Thus the PD1 Blockade Adjuvant Project Plan was written and approved by USAID in January 2017. The goal of the plan is to determine whether targeting PD1 inhibition enhances immunity against malaria. An overview of the development plan for this project is provided in Table 4.8-1. In Q1 FY2018, USAID requested this project be closed out at the end of FY2018 following the completion of the assessment of PD1 expression on CD8 T cells and Treg generation subsequent to AdPyCS immunization and completion of two separate RH5.1/Alum studies. The assessment of PD1 expression and Treg generation was completed in Q2 FY2018. The in-life portion of the initial RH5.1/Alum study was completed in Q1 FY2018 with T cell ELISPOT data provided in Q1 FY2018, anti- RH5.1Ab titers provided in Q3 FY2018 and GIA results provided in Q4 FY2018. The in-life portion of the second RH5.1/Alum study was completed in Q4 FY2018 with T cell ELISPOT data provided in Q4 FY2018. Anti-RH5.1Ab titers and GIA results for study two were completed in Q2 FY2019. All technical work on this project was completed in Q2 FY2019. A draft of the PD1 Blockade Inhibitor Project Final Report was provided to USAID on April 11, 2019 and USAID provided feedback on May 7, 2019. Leidos circulated an updated version of the project report to USAID on May 18, 2019, which USAID approved on May 29, 2019. Leidos circulated the final version (PDF) of the final project report

MVDP Quarterly Report 68 Leidos Proprietary on June 10, 2019. A draft of the PD1 manuscript was provided to USAID on May 28, 2019 and USAID provided feedback on June 5, 2019. Leidos circulated the updated manuscript on June 10, 2019, which was submitted for USAID clearance (clearance received June 19, 2019). The manuscript has been submitted to PLOS ONE. The PD1 blockers used in these studies include, but are not limited to, commercially available PD1 or PDL1 mAb and several PD1 peptide blockers identified by Leidos Life Sciences. These PD1 peptide blockers (~20 amino acids) bind to both human and mouse PD1 and have shown efficacy in other experimental models. The three PD1 peptide blockers currently being tested in these studies are: I. PD1 peptide combo that consists of four separate PD1 peptide blockers; II. Single PD1 peptide blocker, LD01, that is microbiome-derived; and III. Single PD1 peptide that is a derivative of LD01, named LD02.

Table 4.8-1. Overview of the PD1 Blockade Adjuvant Project Plan

Phase Milestone Task

PD1 Blockade 1. Assess the effect of PD1 blockers on Immunogenicity and efficacy testing with Studies vaccine efficacy liver and blood stage antigen

2. Assess PD1 expression and Treg Adjuvant Determine whether PD1 blockers modify generation following immunization with or Formulation Studies PD1 expression and/or Tregs without PD1 blockers

4.8.1 MILESTONE 1: ASSESS THE EFFECT OF PD1 PEPTIDE BLOCKERS ON VACCINE EFFICACY A subcontract for testing the effects of PD1 blockade on vaccine efficacy using a recombinant replication-defective adenovirus expressing the P. yoelii circumsporozoite (CS) protein (AdPyCS) with ADARC was executed in January 2017 and activities have been initiated and are described below. Subcontracting activity with University of Oxford was initiated in August 2017 and has been completed. Studies at University of Oxford assessed the efficacy of PD1 peptide blockers on both cellular and humoral immune responses to the blood stage antigen RH5 (RH5.1 formulated in alum).

4.8.1.1 Assess the Effect of PD1 Peptide Blockers on Immunogenicity of AdPyCS To test whether immunogenicity is enhanced following PD1 blockade, BALB/c mice (n = 5) were immunized IM with 1010 virus particles of AdPyCS in the hind limb without adjuvant. At days 1, 3, 5 and 7 post-immunization mice were treated IP with 200 µg of peptide(s) or monoclonal antibodies (mAbs) shown in Table 4.8-2. In addition to the test peptides, ovalbumin (OVA) peptide was also included as a control peptide. At day 12 post-immunization immunogenicity was assessed by measuring the number of splenic CS-specific, IFN-γ secreting CD8 T cells using the ELISPOT assay after stimulation with the H- 2kd restricted CD8 epitope SYVPSAEQI. Results of the immunogenicity studies are detailed in the FY2018 USAID MVDP Annual Report.

MVDP Quarterly Report 69 Leidos Proprietary

Table 4.8-2. Cohorts for AdPyCS + PD1 Blockade Immunogenicity Study

Cohort # Test Sample 1 AdPyCS alone 2 AdPyCS + PD1 Peptide Combo 3 AdPyCS + Ova Peptide 4 AdPyCS + LD01 Peptide 5 AdPyCS + LD02 Peptide 6 AdPyCS + anti-PD1 mAb 7 AdPyCS + anti-PDL1 mAb

4.8.1.2 Optimizing Dose of AdPyCS Immunization Prior to evaluating the effects of PD1 blockade on protection, studies to determine a suboptimal AdPyCS dose against Py were carried out. BALB/c (n = 4-5) mice were immunized IM with various doses (Table 4.8-3) of AdPyCS in the hind limb without adjuvant. At day 12 post-immunization mice were challenged with ≥200 Py 17XNL sporozoites intravenously (IV). Parasitemia was assessed via blood smears beginning at day 3. Based on data from three dosage studies, Leidos and USAID agreed to use the suboptimal dose of 109 AdPyCS for immunization in studies assessing the effects of PD1modulation on protection (4.8.1.3) as the 109 dose is at the threshold of protection. Results of the individual dosage studies are detailed in the FY2018 USAID MVDP Annual Report.

4.8.1.3 Assess the Effect of PD1 Peptide Table 4.8-3. AdPyCS Dosing Study Blockers on Protection with AdPyCS With completion of 4.8.1.1 and 4.8.1.2 Cohort # Test Sample experiments, a study to evaluate whether 1 Naïve protective efficacy is enhanced following PD1 7 blockade was commenced and completed in June 2 10 AdPyCS 2017. BALB/c mice (n = 9-10) were immunized IM 3 108 AdPyCS with 109 virus particles of AdPyCS in the hind limb 4 109 AdPyCS without adjuvant. At days 1, 3, 5 and 7 post- 10 immunization mice were treated IP with 200µg of 5 10 AdPyCS peptide(s) or mAbs shown in Table 4.8-2. In 6 1011 AdPyCS addition to the testing peptides, OVA peptide was also included as a control peptide. At day 12 post- immunization mice were challenged with ~50-75 Py 17XNL sporozoites IV. Parasitemia was assessed via blood smears beginning at day three. Results of the two challenge studies are detailed in the FY2018 USAID MVDP Annual Report.

4.8.1.4 Assess PD1 Expression on CD8 T Cells and Treg Generation Subsequent to AdPyCS Immunization Increases in immunogenicity and protection of AdPyCS immunization following PD1 inhibition suggest PD1 directly or indirectly, through the development of Treg, negatively impacts the immune response. To assess whether PD1 expression and/or Treg generation is elevated subsequent to AdPyCS immunization two studies were conducted in Q1 FY2018 and Q2 FY2018 by ADARC. Detailed data can be found in the FY2018 USAID MVDP Annual Report.

MVDP Quarterly Report 70 Leidos Proprietary

4.8.1.5 Assess the Effect of PD1 Peptide Blockers on Immunogenicity of RH5 As stated above, subcontracting activity with University of Oxford was completed and a study was initiated in August 2017. To test whether RH5.1 protein immunogenicity is enhanced following PD1 blockade BALB/c mice (n = 4) were immunized with 60 ng RH5.1 protein in 250 μg Alhydrogel. All immunizations are administered IM into both hind limbs. At days 1, 3, 6 and 8 post-immunization mice were treated IP with 200 µg of peptide(s) (LD01, LD02 or combo) or anti-PD1 mAb (Table 4.8-4). As shown in Table 4.8-4 peptide(s) or anti-PD1 mAb treatment occurs either after each immunization, only after the first immunization or after the second and third immunization to identify the optimal dosing schedule. At days 27, 55 and 63 post-immunization mice were bled to assess Ab levels via ELISA and GIA respectively. At day 63 post-immunization T cell responses were assessed by measuring the number of splenic RH5.1-specific, IFN-γ secreting T cells (spot-forming units =SFU) using the ELISPOT assay after stimulation with pooled (n = 7) RH5 peptides (10 µg/mL (Table 4.8-5)) or RH5.1 protein (5 µg/ml). The study was completed in Q1 FY2018 and T cell and humoral immunity assessments were completed by Q4 FY2018. Results of this study are detailed in the FY2018 USAID MVDP Annual Report.

Table 4.8-4. Groups for RH5.1/Alum + PD1 Blockade Immunogenicity Study

No Groups Test Sample 0 1 3 6 8 27 28 29 31 34 36 55 56 57 59 62 63 mice RH5.1 protein +  1 4      alum  RH5.1 protein +  2 alum + PD1 peptide 4           Combo RH5.1 protein +  3 alum + PD1 peptide 4                  Combo RH5.1 protein +  4 4          alum + LD01 peptide  RH5.1 protein +  5 4                 alum + LD01 peptide  RH5.1 protein +  6 4          alum + LD02 peptide  RH5.1 protein +  7 4                 alum + LD02 peptide  RH5.1 protein +  8 alum + anti-PD1 4           mAb RH5.1 protein +  9 alum + anti-PD1 4                  mAb RH5.1 protein +  10 alum + PD1 peptide 4              Combo RH5.1 protein +  11 4             alum + LD01 peptide   IM injection of RH5.1/Alum  Bleed for GIA/ELISA assay  IP injection of PD1 blocking peptides and mAb  Assess T responses via ELISPOT

MVDP Quarterly Report 71 Leidos Proprietary

Table 4.8-5. PfRH5 Specific Peptides Used to Stimulate T Cells

Epitope Sequence of Peptide Position in PfRH5

A7 TYDKVKSKCNDIKNDLIATI T216-I235

C6 NIANSIDILQEKEGHLDFVI N156-I175

C9 NLNKKMGSYIYIDTIKFIHK N396-K415

D9 YIDTIKFIHKEMKHIFNRIE Y406-E425

F6 KHLSYNSIYHKSSTYGKCIA K186-A205

F10 NDVPIKMEYFQTYKKNKPLT N506-T525

G10 DVPIKMEYFQTYKKNKPLTQ D507-Q526

To confirm that RH5.1 protein immunogenicity is enhanced following PD1 blockade, BALB/c mice (n = 6) were again immunized with 60 ng RH5.1 protein in 250 μg Alhydrogel. All immunizations are administered IM into both hind limbs. At days 1, 3, 6 and 8 post-immunization mice were treated IP with 200 µg of peptide(s) (LD01, LD02, combo peptides or OVA (negative peptide)) or anti-PD1 mAb or isotype control mAb (Table 4.8-6). As shown in Table 4.8-6 peptide(s) or mAb treatment occurs either after each immunization or after the second and third immunization. The in-life portion of the second RH5.1/Alum study was completed in Q4 FY2018 with T cell ELISPOT data provided in Q4 FY2018. These results are detailed in the FY2018 USAID MVDP Annual Report. Results of the serum anti-RH5.1 total IgG titers and GIA were completed in Q2 FY2019 and delivered to USAID on February 12, 2019. Assessment of serum anti-RH5.1 total IgG titers via ELISA at day 63 post-immunization (final bleed) showed that the majority of the cohorts, including the RH5 + Alum alone cohort (Group 1), measured 102 or below (Figure 4.8-1). Based on the low titers, the first two bleeds were not tested. Low RH5.1-specific IgG titers in this study resulted in less than 20% inhibition for all groups (1-13) tested at the highest IgG concentration (data not shown), resembling the negative mouse serum (Figure 4.8-2). The discrepancies between Study 1 and Study 2 were discussed with Oxford and they confirmed that the same batches of Alum and RH5.1 protein were used for both studies. Further, as shown in Figure 4.8-2, the GIA results with RH5.1 protein + polyI:C (Group 14 and 15), which ran in parallel to the second study, indicates the RH5.1 protein in the second study was immunogenic. While the cohorts of mice were different, Oxford’s animal unit did not report any health concerns during routine screening/monitoring between studies; however, in the second study three mice were either culled or found dead during the study. All technical work on this project was completed in Q2 FY2019. A project report was be provided to and approved by USAID in Q3 FY2019.

MVDP Quarterly Report 72

Leidos Proprietary

Figure 4.8-2. GIA Activity of Mouse Serum. Pooled serum from Group 14 and Group 15 at day 63 post-immunization was tested in GIA with cultured P. falciparum parasites using the LDH method. Dashed line indicates 50% inhibition.

5. ELEMENT 3 ACTIVITIES: AMY NOE/JESSICA SMITH

5.1. 2018 SCG MEETING FEEDBACK AND LEIDOS RESPONSES During Q1 FY2019 USAID distributed the 2018 SCG Meeting Report to Leidos. Based on the feedback provided, Leidos addressed comments from the 2018 SCG Meeting Report in the FY19 Q1 Quarterly Report to clarify gaps in understanding related to the Leidos MVDP program overview and partnerships, the RH5.1 clinical study immunogenicity assessments using EpiVax peptides, and the liver stage vaccine development project sections. Per request from USAID received during review of the FY19 Q2 Quarterly Report, this information has been deleted herein and from subsequent FY19 Quarterly Reports. Please refer to the FY19 Q1 Quarterly Report for these details.

5.2.1 SCG MEETING READ AHEAD MATERIALS At the end of Q2 FY2019, Leidos provide 2019 SCG read-ahead materials to USAID for the following projects:

• CSP Vaccine Development Project

• Liver Stage Vaccine Development Project

• Blood Stage Epitope Vaccine Development Project

• PD1 Modulation Project On April 2, 2019 input from Oxford with respect to the read ahead materials for the three remaining projects (bulleted below) was received and provided to USAID also on April 2, 2019.

• RH5.1 Clinical Study

• RH5.1 Human mAb Identification and Development Project

• RCR Complex Vaccine Development Project

MVDP Quarterly Report 74 Leidos Proprietary

5.2.2 SCG MEETING PRESENTATIONS Per USAID request, during Q3 FY2019, three Leidos MVDP project presentations were prepared for the 2019 SCG meeting as follows:

• RH5.1 Clinical Study

• RH5.1 Human mAb Identification and Development Project

• RCR Complex Vaccine Development Project Per USAID request, Leidos arranged an IPT meeting with the University of Oxford (held on April 23, 2019) to walkthrough the RH5.1 Clinical Study and RH5.1 Human mAb Identification and Development Project presentations.

5.3. SCG MEETING EXECUTION The SCG meeting was held May 1-2, 2019. 50 invitees attended the Day 1 open session with 25 attending the SCG Meeting dinner. Planning for the 2019 meeting began in mid-December 2018 with the RFP process to the NOVA/MD area. The College Park Marriott Hotel in Hyattsville, MD has been selected for the May 2019 meeting. Leidos prepared a registration site using CVENT and invitations were sent out mid-March. At the end of Q2, three invitees declined and 27 accepted. During Q4 FY2019 USAID provided preliminary guidance regarding the 2020 SCG meeting. Formal planning will begin in Q1 FY2020 once dates are finalized with the SCG members.

6. ELEMENT 4 ACTIVITIES

6.1. MVDP REAGENTS REPOSITORY: JESSICA SMITH SriSai Biopharmaceutical Solutions (SBS) maintained, received, and distributed the reagents/materials needed for the ongoing and future studies during Q3 FY2019. SBS furnishes all the necessary services, management, qualified personnel, materials, equipment, facilities, and travel required for a biologics repository and inventory management services related to cGMP and non-GMP vaccines and associated products. SBS offers Controlled Room Temperature Storage (15 to 30°C), Controlled Room Temperature with humidity control, Refrigerated Storage (2 to 8°C), Freezer Storage (-20 ±10°C), Ultra-low Freezer Storage (-80 ±10°C), Low Freezer Storage (-30±10°C) and Vapor Phase Liquid Nitrogen (≤ -140°C), as well as the option to set units at a customer-defined temperature. The current inventory consists of standard storage temperatures ranging from -80°C to 4°C. Monthly inventories and reports were received from SBS in Oct – Sep 2019.

7. LEIDOS POINTS OF CONTACT

In Q2 FY2019, USAID approved a key personnel change for program manager from Gabriel Gutierrez to Amy Noe. As Amy Noe previously served as Operations Manager, Leidos is actively looking for a replacement that meets the contract requirements for this position. In Q4 FY2019, USAID approved the key personnel change for Operations Manager, Shannon K. Robinson.

MVDP Quarterly Report 75 Leidos Proprietary

7.1. PROGRAM MANAGER Amy Noe, Ph.D., MBA Leidos Life Sciences 5202 Presidents Court Suite 110 Frederick, MD 21703-8398 Phone: 858-826-6105 Mobile: 858-201-9176

7.2. OPERATIONS MANAGER Shannon Robinson, MBA, PMP Leidos LInC 10260 Campus Point Drive, MS C-4 San Diego, CA 92121 Phone: (858) 826-6034

7.3. CONTRACTS MANAGER Casey Koontz Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0435 Fax: 301-846-0794

8. APPENDIX 1: LITERATURE CITED

Arevalo-Herrera, M., Lopez-Perez, M., Dotsey, E., Jain, A., Rubiano, K., Felgner, P.L., Davies, D.H., Herrera, S., 2016. Antibody Profiling in Naive and Semi-immune Individuals Experimentally Challenged with Plasmodium vivax Sporozoites. PLoS Negl Trop Dis 10, e0004563. Bahl, K., Senn, J.J., Yuzhakov, O., Bulychev, A., Brito, L.A., Hassett, K.J., Laska, M.E., Smith, M., Almarsson, O., Thompson, J., Ribeiro, A.M., Watson, M., Zaks, T., Ciaramella, G., 2017. Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Mol Ther 25, 1316-1327. Billaud, J.N., Peterson, D., Barr, M., Chen, A., Sallberg, M., Garduno, F., Goldstein, P., McDowell, W., Hughes, J., Jones, J., Milich, D., 2005a. Combinatorial approach to hepadnavirus-like particle vaccine design. J Virol 79, 13656-13666. Billaud, J.N., Peterson, D., Schodel, F., Chen, A., Sallberg, M., Garduno, F., Goldstein, P., McDowell, W., Hughes, J., Jones, J., Milich, D., 2005b. Comparative antigenicity and immunogenicity of hepadnavirus core proteins. J Virol 79, 13641-13655. Boutz, D.R., Horton, A.P., Wine, Y., Lavinder, J.J., Georgiou, G., Marcotte, E.M., 2014. Proteomic identification of monoclonal antibodies from serum. Anal Chem 86, 4758-4766. Brault, A.C., Domi, A., McDonald, E.M., Talmi-Frank, D., McCurley, N., Basu, R., Robinson, H.L., Hellerstein, M., Duggal, N.K., Bowen, R.A., Guirakhoo, F., 2017. A Zika Vaccine Targeting NS1 Protein Protects Immunocompetent Adult Mice in a Lethal Challenge Model. Sci Rep 7, 14769. Brazzoli, M., Magini, D., Bonci, A., Buccato, S., Giovani, C., Kratzer, R., Zurli, V., Mangiavacchi, S., Casini, D., Brito, L.M., De Gregorio, E., Mason, P.W., Ulmer, J.B., Geall, A.J., Bertholet, S., 2016. Induction of Broad-Based Immunity and Protective Efficacy by Self-amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin. J Virol 90, 332-344. Brune, K.D., Howarth, M., 2018. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front Immunol 9, 1432.

MVDP Quarterly Report 76 Leidos Proprietary

Brune, K.D., Leneghan, D.B., Brian, I.J., Ishizuka, A.S., Bachmann, M.F., Draper, S.J., Biswas, S., Howarth, M., 2016. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234. Crompton, P.D., Kayala, M.A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G.E., Molina, D.M., Burk, C.R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D.L., Liang, X., Doumbo, O.K., Miller, L.H., Doolan, D.L., Baldi, P., Felgner, P.L., Pierce, S.K., 2010. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A 107, 6958-6963. DeKosky, B.J., Lungu, O.I., Park, D., Johnson, E.L., Charab, W., Chrysostomou, C., Kuroda, D., Ellington, A.D., Ippolito, G.C., Gray, J.J., Georgiou, G., 2016. Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires. Proc Natl Acad Sci U S A 113, E2636-2645. Dent, A.E., Nakajima, R., Liang, L., Baum, E., Moormann, A.M., Sumba, P.O., Vulule, J., Babineau, D., Randall, A., Davies, D.H., Felgner, P.L., Kazura, J.W., 2015. Plasmodium falciparum Protein Microarray Antibody Profiles Correlate With Protection From Symptomatic Malaria in Kenya. J Infect Dis 212, 1429-1438. Gilbert, S.C., 2013. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 31, 4241-4246. Goepfert, P.A., Elizaga, M.L., Seaton, K., Tomaras, G.D., Montefiori, D.C., Sato, A., Hural, J., DeRosa, S.C., Kalams, S.A., McElrath, M.J., Keefer, M.C., Baden, L.R., Lama, J.R., Sanchez, J., Mulligan, M.J., Buchbinder, S.P., Hammer, S.M., Koblin, B.A., Pensiero, M., Butler, C., Moss, B., Robinson, H.L., Group, H.S., National Institutes of, A., Infectious Diseases, H.I.V.V.T.N., 2014. Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis 210, 99-110. Hekele, A., Bertholet, S., Archer, J., Gibson, D.G., Palladino, G., Brito, L.A., Otten, G.R., Brazzoli, M., Buccato, S., Bonci, A., Casini, D., Maione, D., Qi, Z.Q., Gill, J.E., Caiazza, N.C., Urano, J., Hubby, B., Gao, G.F., Shu, Y., De Gregorio, E., Mandl, C.W., Mason, P.W., Settembre, E.C., Ulmer, J.B., Craig Venter, J., Dormitzer, P.R., Rappuoli, R., Geall, A.J., 2013. Rapidly produced SAM((R)) vaccine against H7N9 influenza is immunogenic in mice. Emerg Microbes Infect 2, e52. Lavinder, J.J., Horton, A.P., Georgiou, G., Ippolito, G.C., 2015. Next-generation sequencing and protein mass spectrometry for the comprehensive analysis of human cellular and serum antibody repertoires. Curr Opin Chem Biol 24, 112-120. Lavinder, J.J., Wine, Y., Giesecke, C., Ippolito, G.C., Horton, A.P., Lungu, O.I., Hoi, K.H., DeKosky, B.J., Murrin, E.M., Wirth, M.M., Ellington, A.D., Dorner, T., Marcotte, E.M., Boutz, D.R., Georgiou, G., 2014. Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc Natl Acad Sci U S A 111, 2259-2264. Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., Yan, S.F., Williamson, K.C., Holder, A.A., Carucci, D.J., Yates, J.R., 3rd, Winzeler, E.A., 2004. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res 14, 2308-2318. Le Roch, K.G., Zhou, Y., Blair, P.L., Grainger, M., Moch, J.K., Haynes, J.D., De La Vega, P., Holder, A.A., Batalov, S., Carucci, D.J., Winzeler, E.A., 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-1508. Lindner, S.E., Swearingen, K.E., Harupa, A., Vaughan, A.M., Sinnis, P., Moritz, R.L., Kappe, S.H., 2013. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol Cell Proteomics 12, 1127-1143. Pascolo, S., 2008. Vaccination with messenger RNA (mRNA). Handb Exp Pharmacol, 221-235.

MVDP Quarterly Report 77 Leidos Proprietary

Saphire, E.O., Schendel, S.L., Fusco, M.L., Gangavarapu, K., Gunn, B.M., Wec, A.Z., Halfmann, P.J., Brannan, J.M., Herbert, A.S., Qiu, X., Wagh, K., He, S., Giorgi, E.E., Theiler, J., Pommert, K.B.J., Krause, T.B., Turner, H.L., Murin, C.D., Pallesen, J., Davidson, E., Ahmed, R., Aman, M.J., Bukreyev, A., Burton, D.R., Crowe, J.E., Jr., Davis, C.W., Georgiou, G., Krammer, F., Kyratsous, C.A., Lai, J.R., Nykiforuk, C., Pauly, M.H., Rijal, P., Takada, A., Townsend, A.R., Volchkov, V., Walker, L.M., Wang, C.I., Zeitlin, L., Doranz, B.J., Ward, A.B., Korber, B., Kobinger, G.P., Andersen, K.G., Kawaoka, Y., Alter, G., Chandran, K., Dye, J.M., Viral Hemorrhagic Fever Immunotherapeutic, C., 2018. Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection. Cell 174, 938-952 e913. Shinde, V., Fries, L., Wu, Y., Agrawal, S., Cho, I., Thomas, D.N., Spindler, M., Lindner, E., Hahn, T., Plested, J., Flyer, D., Massare, M.J., Zhou, B., Fix, A., Smith, G., Glenn, G.M., 2018. Improved Titers against Influenza Drift Variants with a Nanoparticle Vaccine. N Engl J Med 378, 2346-2348. Tarun, A.S., Peng, X., Dumpit, R.F., Ogata, Y., Silva-Rivera, H., Camargo, N., Daly, T.M., Bergman, L.W., Kappe, S.H., 2008. A combined transcriptome and proteome survey of malaria parasite liver stages. Proc Natl Acad Sci U S A 105, 305-310. Williams, L.D., Ofek, G., Schatzle, S., McDaniel, J.R., Lu, X., Nicely, N.I., Wu, L., Lougheed, C.S., Bradley, T., Louder, M.K., McKee, K., Bailer, R.T., O'Dell, S., Georgiev, I.S., Seaman, M.S., Parks, R.J., Marshall, D.J., Anasti, K., Yang, G., Nie, X., Tumba, N.L., Wiehe, K., Wagh, K., Korber, B., Kepler, T.B., Munir Alam, S., Morris, L., Kamanga, G., Cohen, M.S., Bonsignori, M., Xia, S.M., Montefiori, D.C., Kelsoe, G., Gao, F., Mascola, J.R., Moody, M.A., Saunders, K.O., Liao, H.X., Tomaras, G.D., Georgiou, G., Haynes, B.F., 2017. Potent and broad HIV-neutralizing antibodies in memory B cells and plasma. Sci Immunol 2. Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., Howarth, M., 2012. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697.

MVDP Quarterly Report 78

Leidos Proprietary

1. EXECUTIVE SUMMARY

A summary of efforts for the completed, ongoing, and planned projects for the Malaria Vaccine Development Program (MVDP) contract for this reporting period are detailed within. A compiled Gantt chart including activities associated with each of the projects has been created and included as an attachment to this report. Ongoing projects that will continue through FY2019 include two vaccine development projects, the CSP vaccine development project (CSP Vaccine) and liver stage vaccine development project (Liver Stage Vaccine), as well as the clinical study with Rh5 (RH5.1 Clinical Study), the latter to assess long-term immunogenicity in RH5.1 vaccinees. Of note is that while both the CSP and the liver stage vaccine development projects were initiated as epitope-based projects, these have since been realigned to target whole proteins/protein domains; therefore, the project names have also been realigned to remove “epitope-based”. Expansion of work on the RCR complex into a vaccine development project (RCR Complex) occurred FY2019 and this project will continue through the fiscal year. Lastly, a new project, the RH5.1 human monoclonal antibody identification and development project (RH5.1 Human mAb), was initiated in FY2019 and will continue through the fiscal year. Two projects will be completed in FY2019, the blood stage epitope-based vaccine development project and the PD-1 blockade inhibitor project (PD-1 Block Inh). Leidos continues to seek collaborators for information exchange under NDA, reagent exchange under MTA, and collaboration under CRADA, to expand our body of knowledge and access to reagents with minimal cost to the program. This type of collaboration benefits the malaria research community and extends the utility of the contract. The information contained herein is intended to provide technical detail regarding activities conducted within the specified reporting period; however, at the request of USAID, Leidos will retain data in quarterly reports for activities completed within the relevant fiscal year.

2. CONTRACTS MANAGEMENT AND ADMINISTRATION

2.1. Current Budget Summary The budget summary for the quarter is provided in Table 2.1-1. Table 2.1-1. Contract Budget Costs are based on B.4 Contract No. AID-OAA-C-15-000071. Table 2.1-2 provides FY19 to-date costs against FY19 Annual Work Plan Estimates. Detailed subcontractor spending is provided in the

MVDP Quarterly Report 2 Leidos Proprietary

MVDP Quarterly Report 3 Leidos Proprietary

2.2. Deliverables Summary In Table 2.2-1 below, we provide the deliverables for which Leidos is responsible under the prime contract with respect to the tasks assigned by USAID. All deliverables are assigned according to the four (4) “Elements” that are outlined in the prime contract.

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes

CONTRACTS AND ADMINISTRATION Signed: 5/28/15 Modifications: MOD 1 2/22/16 MOD 2 3/4/16 MOD 3 8/16/16 Prime Contract MOD 4 2/13/17 MOD 5 5/1/17 MOD 6 7/18/17 MOD 7 9/28/17 MOD 8 1/09/18 MOD 9 12/20/18 Contractor – Employee Non- Signed Non-Disclosure/COI i. Disclosure /Conflict of Interest Delivered Agreements are located on (COI) Agreements SharePoint. Delivered: 08/31/2018 ii. Annual Work Plan Upcoming Plan: 09/01/2019 Approved: 11/08/2018 Upcoming Reports: vi. Quarterly Reports Q1 FY19 Delivered: 01/14/19 04/14/2019 Quarterly Financial Upcoming Reports: vii. Q1 FY19 Delivered: 01/14/19 Reports 04/14/2019 Delivered: 10/15/18 viii. Annual Report/Q4 Report Upcoming Reports: 10/15/19 Approved: 12/03/2018 Delivered: The Small Business Small Business H.10.a Subcontracting Plan was included Subcontracts Subcontracting Plan in Leidos’ proposal. Due: Annually in October; Individual Subcontract Upcoming Reports: 10/30/18 H.10.b.1 submitted via eSRS by Leidos’ Reports Small Business Liaison office. Due: Annually in October; Summary Subcontract H.10.b.2 submitted via eSRS by Leidos’ Upcoming Report: 10/30/18 Report Small Business Liaison office. ix. Final Overall Contract Report N/A Upcoming Report: 5/27/20 Upcoming Meetings: Held: See Table 2.4-1. Regularly scheduled biweekly Biweekly USAID–Leidos C.4.4 Ad hoc meetings held: See meetings are held the first and Update Meetings Table 2.4-1. third Wednesdays of each month. Contract Administration Meetings with No actions in the reporting G.4.C Contracting Officer’s period. Representative (COR)

MVDP Quarterly Report 4 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes Upon approval of all future Due: All contracts and deliverables, Leidos will upload USAID Development administration deliverables will to the DEC. At contract xiv. Experience Information be uploaded to the DEC after completion, Leidos will upload 30 days from approval. all task-associated documents as well. ELEMENT 1: PROTOCOL DEVELOPMENT/WHITE PAPERS/ See section 3.2 regarding xii. Publications/Posters Q1 FY2019: 4 posters abstract/poster submissions. C.3.2.2 New Project Proposals None in the reporting period ELEMENT 2: IMPLEMENTATION OF RESEARCH AND DEVELOPMENT PROJECTS CSP Vaccine Development Project Project Plan approved by Final Individual Project USAID on 1/29/16. iii. Approved descriptions Revised Project Plan approved 04/26/2017. Subcontract/Task Order Awards: VLP Biotech, JHU, Q1 FY2019: procurement for Subcontracts: ADARC, EpiVax, Precision recently approved activities was xii Procurement of Materials, Antibody, ImmunoVaccine completed for IMV, JHU, Supplies, and Services Other Procurement Vehicles: VaxDesign, and VLP Biotech. CPC Scientific, Vaxine iv. Final Individual Project Report — VLP Biotech Quarterly Reports Provided herein, circulated via v. Individual Project Data Sets VLP162 + DPX4 Stability Study email and/or uploaded to Documentation (ImmunoVaccine) SharePoint. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RH5.1/AS01 Vaccine Clinical Study Final Individual Project Plan approved by iii. Approved Project descriptions USAID on 2/17/16. Q1 FY2019: procurement for Procurement of Materials, Subcontract Award: University xii recently approved activities is Supplies, and Services of Oxford ongoing for Oxford. iv. Final Individual Project Report — Schedule and regulatory approvals Provided herein, circulated via v. Individual Project Data Set for late immunogenicity time email, and/or uploaded to point SharePoint. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — Trial registered on September xii. Registration of Trials Completed 5, 2016 (ClinicalTrials.gov)

MVDP Quarterly Report 5 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes

Liver Stage Vaccine Development Project Project Plan approved by USAID on 6/13/16. Final Individual Attachment 1 - CD8 platform iii. Approved Project descriptions scouting is in preparation with the draft to be circulated to USAID in early Q2 FY2019. Pending: ADARC, SGI, GeoVax Q1 FY2019: procurement for Procurement of Materials, Subcontract Awards: EpiVax, recently approved activities is xii. Supplies, and Services VaxDesign, Multimeric ongoing with ADARC, SGI, and BioTherapeutics. GeoVax iv. Final Individual Project Report — v. Individual Project Data Set None during reporting period. Provided herein. xii. Publications None during reporting period. ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-WEHI WEHI Study 1 approved in Q1 FY2018. RCR Project Plan – Final Individual Project WEHI submitted to USAID in iii. Under revision descriptions December 11, 2018. USAID provided edits and comments on December 18, 2018.

Pending: WEHI, SGI, Precision Q1 FY2019: procurement for Procurement of Materials, Antibody recently approved activities is xii. Supplies, and Services Subcontract/Task Order ongoing with WEHI, SGI, and Awards: WEHI, ImmunoVaccine Precision Antibody iv. Final Individual Project Report — Preliminary ELISA dataset for Circulated to USAID and v. Individual Project Data Set Study W1 provided herein. xii. Publications None during reporting period. ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-University of Oxford Project Plan submitted to Final Individual Project USAID on December 17, 2018. iii. Under revision descriptions USAID provided comments and edits on December 21, 2018. Q1 FY2019: procurement for Procurement of Materials, Pending: University of Oxford xii. recently approved activities is Supplies, and Services Subcontract Award: ongoing with Oxford iv. Final Individual Project Report — v. Individual Project Data Set None during reporting period. xii. Publications None during reporting period. ix. Other/Ad Hoc Reports —

MVDP Quarterly Report 6 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes

Blood-Stage Epitope Vaccine Development Project Plan approved by Final Individual USAID on 3/24/2016. iii. Approved Project descriptions Addendum 1 approved on 6/23/16. Subcontracts Awards: Agilvax, VLP Biotech, Expres2ion, NYBC, Precision Antibody, Swiss TPH Procurement of Materials, No procurement activities for xii. MTAs: WEHI (Alan Cowman), Supplies, and Services Q1 FY2019 Swiss TPH (Gerd Pluschke), University of Oxford (Simon Draper), Wellcome Trust Sanger Institute (Gavin Wright) Draft to be circulated in early iv. Final Individual Project Report Under development Q2 FY2019 v. Individual Project Data Set None during reporting period. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — PD-1 Blockade Inhibitor Final Individual Project Project Plan approved by USAID iii. Approved descriptions on 1/19/2017.

Procurement of Materials, Subcontract Awards: ADARC, No procurement activities for xii. Supplies, and Services University of Oxford Q1 FY2019. iv. Final Individual Project Report — v. Individual Project Data Set None during reporting period. xii. Publications None during reporting period. ix. Other/Ad Hoc Reports — ELEMENT 3: SCG ANNUAL MEETING SUPPORT Planning for the FY19 meeting x. SCG Annual Meeting Event Date: May 1 – 2, 2019 began in Q1 of FY19. Venue: TBD x.a-e. Logistic Support Travel: Leidos Travel Lodging: TBD

MVDP Quarterly Report 7 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes

ELEMENT 4: PROCURMENT OF MATERIALS, SUPPLIES AND SERVICES The repository is used to store and distribute materials xi. MVDP Reagents Repository SriSai Biopharmaceutical Solutions for conduct of the MVDP program.

*Each Roman numeral crosswalks to a deliverable, as called out by the prime contract (AID-OAA-C-15-00071).

2.3. Management Tools Leidos’ SharePoint document repository is a Fiscal Information Security Management Actcompliant, web-based tool that provides access to program data/documentation, deliverables, work products, and schedules. This type of interface is an especially important information exchange among study sites as vaccines progress through milestones. Leidos granted folder permissions to subcontractor technical leads and customer points of contact, commensurate with their roles, allowing direct updates to their respective folders (Table 2.3-1). Transparency is achieved via USAID access to program folders. Leidos has uploaded documents relevant to the current reporting period to SharePoint.

Table 2.3-1. Management Tools

Tool Description Location

Cloud-based solution for exchanging and SharePoint https://vector.leidos.com/sites/ITLSO/MVDP storage of documents

Provide USAID OCONUS line to call 1-855-462-5367 Conference Phone Scientific Consultant Group members and 1778004 Lines CONUS line to communicate with Leidos 2013235

2.4. Team Meetings Leidos also achieves transparency by routine copying of designated customer staff regarding email communications and teleconferences. As required, Leidos has set a standing biweekly meeting with USAID to review our MVDP activities (see Table 2.4-1). Ad hoc discussions to ensure positive study outcomes have been implemented. Leidos uses standard business tools (e.g., email, phone, teleconference, and desktop sharing) to communicate with staff and customers. Meeting agendas and summaries/minutes are available in the “Meeting Materials” folder on the MVDP SharePoint site (https://vector.leidos.com/sites/ITLSO/MVDP/Deliverables/Meeting Materials).

Table 2.4-1. Team Meetings

MVDP Quarterly Report 8 Leidos Proprietary

Table 2.4-1. Team Meetings

Meeting Date Topic November 21, 2018 Leidos/USAID Biweekly Teleconference November 27, 2018 EpiVax / Leidos Discussion / USAID RH5 Analysis December 12, 2018 Leidos/USAID Biweekly Teleconference

3. Element 1 Activities

3.1. Publications In FY2019, Leidos anticipates development of manuscripts for several projects. Status of these efforts, including the collaborators/subcontractors involved as well as the targeted journal, will be provided in this section.

3.2. Abstract Submissions/Poster Presentations Leidos and its subcontractors have made several presentations at scientific meetings/conferences in FY2019. The list of abstracts/presentations for FY2019 can be found in Table 3.2-1. These presentations/posters are used to inform the community of the current activities as well as to identify new collaborators and/or projects.

Table 3.2-1. Meeting/Conference Abstract Submissions/Presentations

Meeting/Conference Date(s) Title

In silico T cell epitope prediction identifies high value ASTMH Annual Meeting Oct 28-Nov 1, 2018 RH5 epitopes that correlate with response in 2018 (Q1 FY2019) vaccinees

Efficacy of the Novel Plasmodium falciparum Blood- ASTMH Annual Meeting Oct 28-Nov 1, 2018 Stage Vaccine RH5.1/AS01B in a Phase I/IIa Clinical 2018 (Q1 FY2019) Trial

American Association of Modeling HLA binding and “self” conservation using in Pharmaceutical Scientists Nov 4-7, 2018 (Q1 FY2019) silico tools predicts immunogenic T cell epitopes in (AAPS) PharmSci 360 2018 vaccinated individuals

A green Trition X-114 extraction protocol for the Eastern Analytical Nov 12-14, 2018 separation of virus-like particles from lipid-based Symposium (EAS) 2018 (Q1 FY2019) preparations

3.3. New Projects No new projects were proposed in Q1 FY2019.

4. Element 2 Activities

MVDP Quarterly Report 9 Leidos Proprietary

4.1. Adjuvant/Delivery Platforms Adjuvant, immunostimulators, and delivery platforms identified through scouting efforts and slated for use in FY2019 are detailed in this section.

4.1.1 Adjuvants The DepoVaxTM platform is in use for the CSP and RCR complex projects. AdvaxTM and Addvax are in use for the RCR complex project. Use of Matrix-MTM is slated for the RCR complex project.

4.1.2 Immunostimulators CpG in combination with Advax is in use for the RCR complex project.

MVDP Quarterly Report 10 Leidos Proprietary

4.1.3 Platforms The WhcAg VLP platform is in use for the CSP project. The SGI and GeoVax platforms are slated for use in liver stage project. The SGI and SpyTag/SpyCatcher VLP platforms are slated for use in the RCR complex project.

4.1.3.1 WhcAg VLP The woodchuck hepatitis B core antigen (WHcAg) VLP platform, developed by VLP Biotech, is based on the core protein the of woodchuck hepatitis B virus. The core proteins self-assemble into VLPs with 240 copies of the antigen per VLP. This platform can accommodate multiple foreign sequence insertions, with long insertions possible at the N and C-termini. Inserts within the surface-exposed loop are possible, which is particularly beneficial for B cell epitopes as the VLP configuration permits cross- linking of B cells. Studies with this platform have shown it to be equal or more immunogenic than HBcAg for both B cell and T cell responses, not significantly cross-reactive with the HBcAg for B cell responses and only partially cross-reactive with HBcAg for T cell (CD4) responses, and function as a vaccine carrier platform for heterologous, B cell epitopes (Billaud et al., 2005a; Billaud et al., 2005b). In consideration of cost, WHcAg VLPs can be easily expressed at high levels in E. coli. Note that this platform has not yet been tested in the clinic.

MVDP Quarterly Report 11 Leidos Proprietary

4.2. CSP Vaccine Development Project: Amy Noe and Jayne Christen The CSP vaccine development project is a multi-year effort that will continue through FY2019. Previously, the WHcAg VLP platform formulated in DPX4 was downselected with completion of preliminary efficacy assessments using the VLP162 parent construct. Further, T cell epitope assessments of PfCSP were previously completed and these data, along with recently published data were used to develop a VLP construct optimization strategy that was initiated in FY2018. The work performed in Q1 FY2019 includes initiation of CSP class II T cell epitope immunogenicity assessments at VaxDesign, continuation of the construct optimization activities, and initiation of a repeat confirmatory efficacy study with the VLP162/DPX4 formulation.

4.2.1 T Cell Epitope Assessments Previously, in silico T cell epitope assessments of PfCSP were performed by EpiVax to identify CSP class I and class II epitopes predicted to bind HLA molecules. This in silico analysis also included identification of additional predicted class II epitopes from a set of CSP sequence variants with the ultimate goal of achieving a VLP-based CSP vaccine with broad Pf strain coverage. A total of four predicted class II epitopes (C1-C4) were identified in the PfCSP 3D7 protein sequence and nine predicted class II epitopes were found in the sequence variants (ICS2-ICS10), the latter all overlapping CSP region 2 (R2) and termed immunogenic consensus sequences (ICS) by EpiVax. Of note is that one of the ICS (ICS8) is a sequence match for the class II epitope predicted to overlap the PfCSP 3D7 R2 (i.e., C3). In order to validate the EpiVax in silico HLA predictions, in vitro HLA binding assessments were previously performed for the four PfCSP 3D7 predicted class II epitopes and a subset of the ICS deemed of high interest (i.e., ICS2, ICS5, ICS7, and ICS8). Although the ability of a sequence to bind HLA is one of the main factors influencing immunogenicity, it is not the only factor. Therefore, in order to assess immunogenicity of the different ICS to further aid in selection of ICS for incorporation into WHcAg VLP, in Q1 FY2019, VaxDesign initiated immunogenicity assessments of the ICS using PBMCs from HLA- matched donors. Preliminary data from VaxDesign suggests that a subset of the ICS appears to have elevated responsiveness across the HLA types. The full data set is anticipated from VaxDesign in Q2 FY2019. These data will be used to downselect the specific ICS for inclusion in the optimized WHcAg VLP construct(s).

4.2.2 Construct Optimization Optimization of the VLP162 construct by VLP Biotech is based on a cloning plan developed and initiated in FY2018, and includes insertion of sequences from the conserved R1 region of PfCSP (which contains CSP cleavage site, KKLKQP), alternative sequences for the NANP repeat loop region, and incorporation of additional class II epitopes from the N- and C-terminal regions of PfCSP 3D7 as well as from the R2 region of heterologous CSP strains (selected based on results of the above described T cell epitope assessments. USAID approved expansion of the loop insert effort to include development of VLPs with five novel loop inserts (further described below). Procurement associated with expansion of the loop

MVDP Quarterly Report 12 Leidos Proprietary insert effort (further described below) began at the end of Q4 FY2018 and was completed in Q1 FY2019. The optimization activities completed in FY2018 include insertion of PfCSP 3D7 amino acids 58-78 into the VLP N-terminal insertion site and extension of the PfCSP 3D7 C-terminal insert to incorporate much of the predicted class II epitope regions in the C-terminus of PfCSP 3D7 (i.e., amino acids 313- 387). The parent VLPs resulting from these efforts are VLP407 and VLP411 (Table 4.2-1). Further, several attempts at extending the C-terminal insert to include different ICS were made at the end of FY2018 with limited success; therefore, Leidos enlisted the assistance of a protein modeler to provide feedback on the sequence configurations with increased stability and a higher probability of soluble expression. ICS sequence cloning efforts have been paused until input from the modeler is received (anticipated in Q2 FY2019). In addition, the data from VaxDesign (as described above) will be included in the overall analysis of ICS to include in the construct(s). In Q1 FY2019, the strategy for construction of the loop insert clones was developed and DNA cloning initiated. A list of the planned clones is provided in Table 4.2-1; the NPDP and NVDP sequences are shown in bold to facilitate sequence comparison among the loop inserts. Of note is that VLP Biotech recommended a conservative approach to developing the loop insert clones whereby both VLP407 and VLP411 were used as the parent for different constructs. This approach was recommended by VLP Biotech due to concerns regarding the less than optimal expression levels for VLP411 as well as the possibility of lower immunogenicity directed to the loop insert when CSP B cell epitopes are placed in both the loop and N-terminal regions of the VLP. Additional VLPs may be cloned based on assembly/expression of the VLPs shown in Table 4.2-1. USAID will be involved in the decision regarding which VLPs will progress to the passive transfer study. Expression levels for this initial set of clones are anticipated in Q2 FY2019 and will be used to determine next steps.

Table 4.2-1. VLP162 Optimization – Loop Inserts VLP ID N-term Insert Loop Insert (position 78) C-term Insert Assembly/Expression VLP407 None NANPNVDP(NANP)3 318-387 Yes, soluble VLP411 58-76 NANPNVDP(NANP)3 313-387 Yes, soluble VLP430 None KQPADGNPDPNANPN 313-387 TBD VLP431 None PADGNPDPNANPNVD 313-387 TBD VLP432 None NPDPNANPNVDPNAN 313-387 TBD VLP433 None NPDPNANPNVDPNANPNANPNANP 313-387 TBD KLKQPADGNPDPNANPNVDPNANP VLP434 None 313-387 TBD NANPNANP VLP435 58-76 NPDPNANPNVDPNAN 313-387 TBD VLP436 58-76 NPDPNANPNVDPNANPNANPNANP 313-387 TBD

4.2.3 Confirmatory Efficacy Evaluation Based on the positive results of the preliminary efficacy assessments with VLP162 in DPX4, Leidos moved this formulation forward to confirmatory efficacy evaluation to conduct a more stringent evaluation of efficacy including use of a greater number of mice per cohort, assessment of individual (rather than pooled) mouse samples, and to conduct cellular response evaluations using mouse PBMCs. The report for this study was received by Leidos at the beginning of Q4 FY2018 and the data were circulated to USAID. Unfortunately, several animals that received the VLP162/DPX4 formulation died during the study. Upon investigation, ImmunoVaccine informed Leidos in Q1 FY2019 that sterility tested showed a microbial contaminant in the formulated material. Subsequently, Leidos requested that

MVDP Quarterly Report 13 Leidos Proprietary

VLP Biotech perform a series of sterility testing to further evaluate this finding as well as to implement sterility testing for VLP lots that will be used for efficacy assessments (including passive transfer and/or challenge studies). Results of the sterility testing are anticipated in Q2 FY2019. Further, a repeat confirmatory efficacy study was initiated in late December 2018 (Table 4.2-2). The study report is anticipated in Q3 FY2019.

Table 4.2-2. VLP162 Confirmatory Efficacy Study 2 – DPX4 Formulation # mice, Study Day Cohort # Test Sample R strain -52 0 21 35 42 56 63* 67

1 Naïve - 10, C57Bl/6 none None none

2 VLP162 (20 µg, no adjuvant) IM 10, C57Bl/6 √ √ √ 3 VLP162 (20 µg) + DPX4 IM 10, C57Bl/6 √ none √

PBMC 3

Challenge 4 VLP162 (20 µg) + DPX4 IM 10, C57Bl/6 bleed/PBMC 1 √ √ √

Test bleed 1 Test

4 VLP162 (50 µg) + ISA 720 IM 10, C57Bl/6 Pre √ √ √

Test bleed 2/PBMCTest

4.2.4 Preliminary Stability Assessments of VLP162 + DPX4 In Q2 FY2018, ImmunoVaccine began work to conduct a 6-month stability study on the VLP162 + DPX4 formulation in order to (1) confirm that the formulation is stable while it is maintained for animals studies, (2) evaluate refrigerated storage (the material is currently stored frozen), and (3) provide preliminary data regarding stability of the WHcAg VLPs in the DepoVax platform. This study was completed in Q1 FY2019 and results for the 0, 1, 3, and 6-month time points are shown in Table 4.2-3 and Table 4.2-4. It was noted after the 0-month time point testing that the VLP162 content did not conform to the specification. Additional testing performed by IMV indicated precipitation occurring during the formulation and/or freeze-drying process. This same issue was found in a retain sample from a previous lot. As this material is representative of that used in efficacy assessments, the decision was made to continue with the stability program. Formulation optimization is possible; however, this will not be performed until the fully optimized VLP has been developed and assessed for precipitation with DPX4. In addition to the VLP162 content not complying with the target specification, it was also noted that viscosity increased over the 6-month period at both temperatures. All other results were within the established specification at the 6-month time point. ImmunoVaccine concluded that further formulation development is needed to improve stability of the VLP formulation.

MVDP Quarterly Report 14

Leidos Proprietary

Table 4.4-2 a summary of published analysis of five proteins selected for development and characterized proteins

(1) Arevalo Table 4.4-2 provides a summary of characteristics of the five proteins selected for analysis -Herrera et al., 2016 (2) Crompton et al., 2010 (3) Dent et al., 2015 (4) Le Roch et al., 2003. Based on microarray using P. falciparum (5) Le Roch et al., 2004. Based on microarray or mass spectral; analysis using P. falciparum. (6) Lindner et al., 2013. Based on mass spectral results using P. falciparum, proteins were ranked for abundance from 1 to 1991, 1 being the most abundant. (7) PlasmoDB (http://plasmodb.org/plasmo/) (8) Tarun et al., 2008. Based on mass spectral analysis using P. yoelii

MVDP Quarterly Report 18 Leidos Proprietary

4.4.1 Liver Stage Plan Milestone I.3 Analysis of the immune response to PFD0430c protein began on August 27, 2018, with AmpTec starting the synthesis the mRNA. In mid-November 2018 AmpTec provided the mRNA encoding PFD0430c that VaxDesign is using to transfect cells to express the protein in situ. VaxDesign began developing the transfection of dendritic cells in late November 2018. Transfection of dendritic cells with the PFD0430c mRNA has been problematic, resulting in extensive cell death. VaxDesign has observed this cytotoxic effect of mRNA on dendritic cells before. To circumvent this issue, VaxDesign will attempt to transfect feeder cells to express the protein in the MIMIC system. Once cells are successfully transfected, analysis of the immune response will start with the class II epitopes and progress to the class I epitopes (Table 4.4-3). The analysis is planned for completion by the end of February 2019 (Figure 4.4-1).

Table 4.4-3. Peptides derived from PFD0430c

Peptide Location Peptide Peptide Sequence in PFD0430c Designation* 29 - 37 RLNNRSIYK CL1-64 72 - 81 KISSFINQRK CL1-65 237 - 245 HTSVFPGLY CL1-66 257 - 265 NPLGEADSL CL1-67 285 - 293 GIANDLSTL CL1-68 357 - 365 RSKKTFLVK CL1-70 371 - 379 YTIGLPPYI CL1-71 440 - 448 YESQLGGKI CL1-72 523 - 531 NLFEWSKTV CL1-73 534 - 542 HPMPINIKL CL1-74 550 - 558 DSDDLKESY CL1-75 570 - 579 GLSPHDTMQK CL1-76 386 - 394 AYKNAVNEL CL1-77 224 - 233 DENTLENRNI CL1-78 360 - 382 Ac-KTFLVKSNCVKYTIGL PPYIPWD-amide CL2-19 445 - 461 GGKITKIINVSTSSIEQ-amide CL2-20 *Peptides designated with CL1-X target class I, and peptides designated with CL2-X target class II

Activity 2017 2018 2019 Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Peptide Synthesis Class II Peptide Experiments Class II Peptide Analysis Class I Peptide Experiments Class I Peptide Analysis Protein Selection mRNA Synthesis mRNA /Epitope Analysis

MVDP Quarterly Report 19 Leidos Proprietary

Figure 4.4-1. Schedule for Analysis by MIMIC of Epitopes from New Proteins

4.4.1.2 Optimizing the number of mosquito bites for infection. Prior to starting the platform scouting analysis with the SGI RNA Replicon and GeoVax MVA-VLP, the number of mosquito bites required to ensure malaria infection in this model will be determined. This study will inform the targeted infectious exposure that will be used for the subsequent studies at ADARC.

Table 4.4-4. Bite Titration Study On December 10, 2018 of Q1 FY2019, USAID approved the study design (Table 4.4-4). In Cohort # Test Sample brief, BALB/c (n = 7) mice will be exposed to various numbers of mosquitos infected with Py 1 No bites 17XNL. Following exposure, individual mosquitos will be dissected to check for 2 2 bites infectivity. Parasitemia in the mice will be 3 4 bites assessed via blood smears. Results of this study are anticipated in Q2 FY2019. 4 6 bites

5 8 bites

4.4.1.3 Titration of AdPyCS immunization for mosquito bite challenge. In addition to optimizing the number of mosquito bites for infection prior to evaluating the SGI RNA Replicon and GeoVax MVA-VLP platforms, ADARC will also titrate the AdPyCS immunization to determine the dose that gives 40-50% protection following mosquito bite challenge. On December 10, 2018 of Q1 FY2019, USAID approved the study design (Table 4.4-5). In brief, BALB/c (n = 7) mice will be immunized IM with various doses of AdPyCS in the hind limb without adjuvant. At day 10 post- immunization mice will be challenged via mosquitos infected with Py 17XNL. Following bite challenge individual mosquitos will be dissected to check for infectivity. Parasitemia in the mice will be assessed via blood smears. Results of this study are anticipated in Q2 FY2019.

MVDP Quarterly Report 20 Leidos Proprietary

Table 4.4-5. AdPyCS Dosing Study

Cohort # Test Sample Route # Mice, Strain Day 0 Day 10

1 Naïve none 7 BALB/c none 7

2 10 AdPyCS IM 7 BALB/c √

3 10 AdPyCS IM 7 BALB/c √ 4

- 4 109 AdPyCS IM 7 BALB/c √ 4

4. 10 5 10 AdPyCS IM 7 BALB/c √ Challenge:

Mosquito Bite 11

“x” number“x” of bites 6 10 AdPyCS IM 7 BALB/c √ determined in Table

4.5. RCR Complex Vaccine Development Project: Cecille Browne (WEHI), Jayne Christen (Oxford), and Amy Noe This project is based on evaluation of proteins in the RCR complex as vaccine candidates by comparison of the individual and combined proteins. Although high GIA activity was observed with the RH5.1 vaccine candidate in animal models and good titers were seen in humans immunized with RH5.1/AS01, no sterile protection was found upon CHMI. WEHI has previously provided data indicating that two additional proteins in the Rh5 complex perform better in the GIA as compared to Rh5 when the proteins are formulated with IFA (70% for Rh5 compared to 80% and 90% for CyRPA and Ripr, respectively). However, it is unclear if the same GIA activity is possible with human-use adjuvants (thus Study W1 was initiated) and/or if the moderate increases in GIA seen with CyRPA and Ripr will translate to achievement of sterile protection in humans with those proteins administered alone. Further, Dr. Simon Draper (Oxford) indicated that GIA activity was similar among the individual Rh5 complex proteins in their hands (data not received). It is known that blood stage proteins can act synergistically to achieve higher GIA activity; therefore, a multi-antigen Rh5 complex vaccine may be more efficacious than a single protein vaccine. Thus, the RCR Complex Project Plan will test the following hypotheses: (1) Targeting single and/or multiple proteins in the RCR complex can interrupt the mechanism of merozoite invasion of erythrocytes during the blood stage infection of P. falciparum and (2) Targeting multiple proteins in the complex may be more effective than targeting a single protein in the complex. To address these hypotheses, Leidos developed two parallel paths under the overall RCR Complex Project Plan as well as a workflow diagram to illustrate the interconnectedness of the projects conducted at Oxford and WEHI. Overall, there are a total of three approaches for development of an RCR complex vaccine including recombinant protein, VLP, and RNA approaches. In addition, specific studies have been included in the workflow to facilitate down-selection (e.g., studies to identify a test protein dose and to compare different adjuvants). Based on immunogenicity assessments (e.g., ELISA and GIA activity), constructs/formulations will be down-selected for comparison in a penultimate study. These interdependencies and decision points are shown in the RCR Complex Vaccine Project Workflow Figure 4.5-1, where decision points are shown as to be determined (TBD). Based on the evolution of this project, USAID approval of the project as a whole was provided prior to approval of the project plans. The project plans for both the work at WEHI and Oxford are currently under revision. Also of note is that USAID has approved some but not all of the study designs, as described below.

MVDP Quarterly Report 21 Leidos Proprietary

Figure 4.5-1. RCR Complex Vaccine Project Workflow. Down-selection is based on analysis of sera samples via GIA (pLDH and flow cytometry methods) and Quantitative ELISA On October 30, 2018, an IPT Meeting was held at ASTMH in New Orleans. This face-to-face meeting was attended by Simon Draper’s group (Oxford), Alan Cowman’s group (WEHI), Leidos and USAID. The following outcomes of the meeting are listed below. 1) Addition of dose-ranging study with Matrix-M to the project plan entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine- University of Oxford.” 2) Change in animal model from mice to rats. Studies will be conducted with rats (except Study W1, which had already begun) to obtain higher bleed volumes, allow individual animal assessments, and avoid non-specific GIA issues. 3) Incorporation of an analytical plan. Leidos consulted with an in-house statistician for guidance in power analysis and sample size determination.

4.5.1 WEHI: Cecille Browne/Amy Noe Leidos submitted a draft plan (Rh5 Complex Vaccine Development Project Plan) to USAID in August 2018 that included Study W1 (evaluating different human-use adjuvants) and follow-on studies to be performed by WEHI. These follow-on studies include 1) immunogenicity of double and triple protein combinations, 2) immunogenicity of monocistronic and polycistronic RNA replicons encoding complex proteins, and 3) development of reagent antibodies that specifically recognize the RCR complex. In December 2018, Leidos submitted to USAID a revised project plan with a new title: RCR Complex Project Plan - WEHI. The revised project plan includes an optional dose titration study in rats to bridge Study W1 (conducted in mice) to follow-on studies described in the project plan. Prior to approval of the project plan, USAID approved a study design for Study 1 (later renamed to Study W1) to evaluate the immunogenicity (ELISA and GIA) of individual proteins in the RCR complex (Rh5, Ripr, and CyRPA) in combination with human-use adjuvants in mice. In Q4 FY2018, Leidos completed procurement efforts with WEHI to conduct this study and managed the logistics to ship DPX-formulated proteins from IMV to WEHI. Study W1 was initiated in October 2018.

MVDP Quarterly Report 22 Leidos Proprietary

Table 4.5-1. Study W1. Immunogenicity study in mice of individual RCR Proteins in three human use adjuvants and IFA

Study Day Cohort # Test Sample R # mice, strain -2 0 28 42 1 Ripr (20 µg) + IFA IM 6, BALB/c √ √ 2 RH5.1 (20 µg) + IFA IM 6, BALB/c √ √ 3 CyRPA (20 µg) + IFA IM 6, BALB/c √ √ 4 Ripr (20 µg) + AddaVax IM 6, BALB/c √ √

5 RH5.1 (20 µg) + AddaVax IM 6, BALB/c √ √ 6 CyRPA (20 µg) + AddaVax IM 6, BALB/c √ √

bleed 7 Ripr (20 µg) + Advax+CpG IM 6, BALB/c - √ √ 8 RH5.1 (20 µg) + Advax+CpG IM 6, BALB/c Pre √ √

Terminal bleedTerminal 9 CyRPA (20 µg) + Advax+CpG IM 6, BALB/c √ √ 10 Ripr (20 µg) + DPX4 IM 6, BALB/c √ √ 11 RH5.1 (20 µg) + DPX4 IM 6, BALB/c √ √ 12 CyRPA (20 µg) + DPX4 IM 6, BALB/c √ √

MVDP Quarterly Report 23 Leidos Proprietary

Figure 4.5-2. Preliminary immunogenicity results of individual RCR proteins in four adjuvants. Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

Table 4.5-2. Preliminary estimates of immunogenicity titers (at O.D. = 1.0)

RH5.1 Ripr CyRPA DPX4 >51,200 >51,200 30,000 IFA 25,000 >51,200 >51,200 Advax+CpG 3,200 8,800 400 AddaVax 600 3,200 2,400

4.5.2 University of Oxford (Oxford): Jayne Christen/Amy Noe On December 17, 2018 of Q1 FY2019, Leidos submitted a draft project plan, entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford,” to USAID. Presented in the plan were the study designs for four in-life studies (Studies O1-O4) as well as the description of two reagent/assay development activities (i.e. quantitative ELISA and VLPs decorated with RCR recombinant proteins). USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Leidos is preparing the next iteration of the project plan and expects to submit to USAID for review in early Q2 FY2019. An overview of the development plan for this project is provided in Table 4.5-3.

MVDP Quarterly Report 24 Leidos Proprietary

Table 4.5-3. Overview of the RCR Complex Vaccine Development Project Plan-Oxford

Phase Milestone Activities  Humoral response assessments Study O1 1. Dose-Ranging Study  GIA assessment  Generation of quantitative ELISA control sera 2. Assess immunogenicity of individual  Humoral response assessments Study O2 proteins and double/triple protein mixtures  GIA assessment

3. Establish quantitative method to assess  Calibration-free concentration analysis method Assay antigen-specific IgG levels for the RCR  Affinity purification method Development proteins  Technology transfer to WEHI  Generation and expression of RH5-SpyTag, CyRPA- SpyTag, and Ripr-SpyTag 4. Generation and characterization of single VLP Development  Conjugation of single antigen-SpyTag or RCR-SpyTag antigen VLPs and RCR complex VLPs complexes to HBsAg-SpyCatcher VLP carrier  Protein purification and characterization 5. Assess immunogenicity of individual VLPs,  Humoral response assessments Study O3 VLP combinations, and RCR complex VLPs  GIA assessment

6. Head-to-Head immunogenicity analyses for  Humoral response assessments Study O4 all relevant immunogens/formulations  GIA assessment

As noted in the appropriate sections below, USAID has approved both the VLP development and the Study O1 design (Table 4.5-4). The draft design of Study O2 (Table 4.5-5, recombinant proteins) has also been approved; however, determination of protein concentration is pending results of Study O1. The final study design will be provided to USAID with the data from Study O1 for final approval. The Study O3 (VLPs) design is pending approval. Additionally, the Study O4 (all relevant immunogens/formulations) design is pending adjuvant, antigen, and construct approval. Leidos initiated procurement activities in November 2018 of Q1 FY2019 and activities are ongoing.

4.5.2.1 Dose-Ranging Study (Study O1) On November 30, 2018 of Q1 FY2019, USAID approved the design of Study O1. In Study O1, the immunogenicity of Rh5, CyRPA and Ripr proteins as well as the ability of antibodies induced to elicit parasite growth inhibition will be evaluated individually in rats (Table 4.5-4). Leidos expects initiation of Study O1 during Q2 FY2019.

Table 4.5-4. Study O1 – Dose-Ranging Study

Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 (2 µg) + Matrix-M IM 6, Wistar √ √ √

2 RH5.1 (200 ng) + Matrix-M IM 6, Wistar √ √ √

3 RH5.1 (20 ng) + Matrix-M IM 6, Wistar √ √ √

4 CyRPA (2 µg) + Matrix-M IM 6, Wistar bleed √ √ √

5 Ripr (2 µg) + Matrix-M IM 6, Wistar Pre √ √ √

Test bleed

6 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √ BleedTerminal 7 Negative Control IM 3, Wistar ** ** Terminal Bleed

MVDP Quarterly Report 25 Leidos Proprietary

4.5.2.2 Immunogenicity Study of Individual Proteins and Double/Triple Protein Mixtures (Study O2) USAID approved the draft design of Study O2 on October 4, 2018 of Q1 FY2019 (Table 4.5-5); however, determination of protein concentration is pending results of Study O1. The final study design will be provided to USAID with the data from Study O1 for final approval. Activities will be expanded on during the course of project reporting.

Table 4.5-5. Study O2 – Individual Proteins and Double/Triple Protein Mixtures

Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 2 CyRPA + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 3 Ripr + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 4 RH5.1 + Ripr + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ RH5.1 + CyRPA + Matrix-M 5 IM 6, Wistar √ √ √ (Dose TBD)

6 Ripr + CyRPA + Matrix-M (Dose TBD) IM 6, Wistar √ √ √

RH5.1 + CyRPA + Ripr + Matrix-M bleed

- 7 (Dose TBD) IM 6, Wistar √ √ √

Pre Equivalent Molar Ratio Test bleed

Terminal BleedTerminal RH5.1 + CyRPA + Ripr + Matrix-M 8 (Dose TBD) IM 6, Wistar √ √ √ Equivalent Protein Concentration Reconstituted RCR + Matrix-M 9 IM 6, Wistar √ √ √ (Dose TBD) 10 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √

4.5.2.3 Development of Quantitative ELISA In conversations and approval of the FY2019 annual work plan, USAID indicated support of pursuing the development of quantitative ELISAs at Oxford with subsequent assay transfer to WEHI. Activities will be expanded on during the course of project reporting.

4.5.2.4 VLP Development USAID approved the development of HBsAg-SpyCatcher VLPs decorated with single antigens or the RCR complex on October 4, 2018 of Q1 FY2019. Activities will be expanded on during the course of project reporting.

4.5.2.5 Immunogenicity Study of Individual VLPs, VLP Combinations, and RCR Complex VLPs (Study O3) A study design and description were presented in the draft project plan, entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford” which was originally submitted to USAID on December 17, 2018 of FY2019. USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Study O3 is pending approval by USAID at the time of this writing.

4.5.2.6 Head-to-Head Immunogenicity Analyses of all Relevant Immunogens/Formulations (Study O4) A study design and description were presented in the draft project plan, entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford” which was originally

MVDP Quarterly Report 26 Leidos Proprietary

submitted to USAID on December 17, 2018 of FY2019. USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Study O4 is pending approval by USAID at the time of this writing.

4.6. RH5.1 Human mAb Identification and Development Project: Vin Kotraiah/Cecille Browne The RH5.1 human mAb identification and development project is new for FY2019. Procurement efforts were started in Q1 FY2019 and are anticipated to be finalized in Q2 FY2019. Leidos (working with Oxford) provided the synopsis for this project to USAID in August 2018 and included this project in the FY2019 Annual Work Plan (submitted to USAID on September 1, 2018). In response to their review of the FY2019 annual work plan, USAID indicated their interest in funding portions of this proposal. On December 5, 2018, Leidos submitted to USAID a draft Project Plan on RH5.1 Human mAb Isolation and Development to USAID. USAID reviewed the plan and provided their comments on December 14, 2018. Leidos updated the plan based on feedback from USAID and submitted a final plan on December 19, 2018. An overview of the development plan for this project is provided in Table 4.6-1 followed by a brief summary of the proposed project.

Table 4.6-1. Overview of the development plan for the RH5.1 human mAb isolation and development project

Phases Milestones Activities  Single B cell isolation (memory B cells (mBC) or plasmablasts) B Cell Isolation and  Reverse transcription and paired heavy and light chain nested PCR Cloning  DNA purification and cloning into heavy and light chain vectors B Cell Isolation and  Transfection of heavy and light chain vectors into HEK293 cells mAb Expression and  Harvest of supernatant and screening by ELISA mAb Development Purification  Affinity purification of antibodies from supernatant  Sequencing of heavy and light chain variable regions mAb Sequencing  Determination of sequence maturation and germline alleles ELISA Titer and  Determination of ELISA titers Protein Mapping  Protein region mapping using a panel of Rh5 variants GIA  Growth Inhibition Assay (using pLDH method) mAb Screening Epitope Similarity  Determination of similarity of B cell epitopes recognized by antibodies Screen using competition assay and SPR/BLI mAb Downselection  Monoclonal antibody down-selection mAb Affinity  Determination of antibody affinity by SPR mAb mAb Inhibition of  Assessment of inhibitory activity of antibodies on RCR, Rh5-P113 andr Characterization Complex Formation Rh5-Basigin complexes mAb Downselection  Selection of monoclonal antibodies for structural studies Computational  Identification of sequence variants of the immunogen that are stable assessments and have the right conformation R5.016 Immunogen Production of select  Epitope grafting and production of selected immunogens Design (optional) immunogen designs Immunogen  SPR screening for binding to R5.016 mAb downselection Production of  Production and QC of downselected immunogen R5.016 Immunogen immunogen Production Coupling of (optional)  Conjugation of downselected immunogens to VLPs immunogen to VLP

MVDP Quarterly Report 27 Leidos Proprietary

Table 4.6-1. Overview of the development plan for the RH5.1 human mAb isolation and development project

Phases Milestones Activities Formulation of  Selection and procurement of adjuvant immunogen:VLP  Formulation of immunogen  Rat immunizations with RH5.1 comparator R5.016 Immunogen Rat immunization Testing (optional)  Collection of sera Immunogenicity and  Humoral response and GIA activity with R5.016 mAb comparator GIA assessments

 Recombinant antibody expression and purification Antibody production  Fab fragment generation New mAb  Screening for Fab:Rh5 co-crystals Structure Crystal screening Determination  Cryoprotection of crystals (optional)  X-ray diffraction studies Crystal structure  Model building and refinement determination  Epitope delineation

Oxford proposed identification of human Rh5 mAbs and conduct of a B cell analysis using VAC063 clinical samples. This project will involve surveying the B cell and Ab repertories from vaccinated subjects utilizing both B cell cloning and serum immunomics techniques in order to understand the epitopes recognized by human anti-Rh5 sera, how these epitopes might contribute to functional GIA, and gather information on differences in repertoire based on dose/immunization regimen/CHMI. As part of this effort, Oxford also proposed evaluating novel immune mechanisms of protection based on the sterile protection and reduced parasite growth rate seen with unvaccinated individuals after CHMI. These activities have been incorporated into the project proposal and will be expanded on during the course of project reporting. In addition to the work proposed by Oxford, an immune repertoire survey of RH5.1 vaccinees was also proposed (summarized in the section below). The final decision on this work is pending analysis of the clinical study GIA data.

4.6.1 Immune Repertoire Survey of Select VAC063 Vaccinees In Q4 FY2018, USAID requested “project synopses” for several of the proposed projects including the VAC063 Immune Repertoire Survey Project. Leidos (working with the University of Texas Austin and Oxford) provided the synopsis for this project to USAID in August 2018 and included this project in the FY2019 Annual Work Plan (submitted to USAID on September 1, 2018). In their response to the proposed annual work plan, USAID indicated that a decision regarding funding for this project will not be made until after receipt and review of the GIA data from the RH5.1/AS01 clinical study. A brief summary of the proposed project is provided below. This project aims to use novel sequencing technologies to identify the B cell and IgG immune repertoires for select RH5.1 vaccinees from the VAC063 clinical study and would involve collaboration between the Draper lab at Oxford and Drs. Georgiou and Ippolito at the University of Texas Austin. The humoral response to vaccination involves the generation of short-lived plasmablasts that peak approximately 8 days after immunization and the generation of broadly diverse memory B cells and long- lived plasma cells that secrete high-affinity mAbs in the germinal centers of the lymph nodes. The three iterative germinal center processes of somatic hypermutation of germline variable region DNA sequences, clonal expansion and selection of high affinity B cell receptor variants are involved in the

MVDP Quarterly Report 28

Leidos Proprietary

4.7.1 HLA binding analysis for select PreVax epitopes In the first phase of project, protein sequences of five selected blood stage antigens derived from Plasmodium falciparum had been screened in the PreVax analysis for potential immunogenicity using the EpiMatrix system. As part of this analysis, a total of 8,076 9-mer and 10-mer peptides were screened for Class I T cell epitope content. EpiVax ranked the peptides based on EpiMatrix Score and homology with human sequences, as well as common flags for peptide synthesis and solubility. EpiVax and Leidos then selected the top candidates among each of the six most common Class I HLA alleles (A*0101, A*0201, A*0301, A*2402, B*0702, B*4403), for in vitro testing in HLA binding assays. Also as part of the EpiMatrix analysis, putative Class II T cell epitopes were identified, and several high- scoring T cell epitope clusters were selected for further testing in HLA binding assays. The objective was to confirm the predicted HLA binding propensity of the Class I and II peptides in in vitro HLA binding assays using soluble HLA proteins based on the ability of the peptides to compete with a high binding control peptide.

4.7.2 Class I In the first phase of project described in Section 5.1, protein sequences of five selected blood stage antigens derived from P. falciparum were screened for potential immunogenicity using the EpiMatrix system. As part of this analysis, a total of 8,076 9-mer and 10-mer peptides were screened for Class I T cell epitope content. EpiVax ranked the peptides based on EpiMatrix Score and homology with human sequences, as well as common flags for peptide synthesis and solubility. EpiVax and Leidos then selected the top candidates among each of the six most common Class I HLA alleles (A*0101, A*0201, A*0301, A*2402, B*0702, B*4403), yielding a total of 206 peptides for in vitro testing.

4.7.2.1 Methods Synthesis of Peptides The selected peptides were produced by 9-fluoronylmethoxycarbonyl (Fmoc) synthesis at 21st Century Biochemicals. The peptides were designed with a C-terminal hydroxyl group cap and delivered >85% pure as ascertained by HPLC, mass spectrometry, and UV scan (ensuring purity, mass, and spectrum, respectively). HLA Binding Assay The cell-free class I HLA binding assay in use at Pure Protein, LLC allows for in vitro quantification of peptide-HLA binding affinity in a competition format. In this assay, a fluorescently labeled, high binding, control peptide is loaded onto 384-well ½-area plates along with unlabeled experimental peptides. Unlike the protocol for soluble HLA DR alleles, which are supplied empty, to remove endogenous peptides and render the HLA A and B molecules receptive to binding, HLA is heated at 53°C for 15 minutes and immediately added to the assay. The mixture is then allowed to stand for three days. Once the mixture has reached steady equilibrium (at 72 hours), displacement of the high binding control peptide is measured through Fluorescence Polarization. Binding of experimental peptides is expressed as the percent inhibition of the labeled control peptide (experimental fluorescence / control fluorescence, multiplied by 100). The percent inhibition values for each experimental peptide (across a range of molar concentrations) are used to calculate the concentration which inhibits 50% of the labeled control peptide’s specific binding. This value is referred to as the peptide’s IC50. Peptides were assayed against a panel of six common Class I HLA alleles. They are: A*0101, A*0201, A*0301, A*2402, and B*0702 and B*4402. Test peptides are solvated in DMSO at 10 mM and further diluted in an aqueous buffer. For each experimental peptide, samples are prepared at a high

MVDP Quarterly Report 30

Leidos Proprietary

Table 4.7-8 Leidos R9 Class II T Cell Epitope Clusters (Synthesized)

Cluster Human JMX # Peptide Name Peptide Sequence Score HMLGY Score 1 AMA1_1-17_001A* MRKLYCVLLLSAFEFTY-amide 19.42 1.18 2 AMA1_12-29_002A** AFEFTYMINFGRGQNYWE-amide 16.08 0.00 3 AMA1_284-299_004A** Ac-FQNYTYLSKNVVDNWE-amide 13.39 0.22 4 AMA1_364-377_006A* Ac-KEGFKNKNASMIKS-amide 17.75 0.00 5 AMA1_364-386_006 Ac-KEGFKNKNASMIKSAFLPTGAFK-amide 24.86 0.73 6 AMA1_372-386_006B* ASMIKSAFLPTGAFK-amide 11.92 1.83 7 AMA1_417-436_007A* TCLINNSSYIATTALSHPIE-amide 28.63 1.05 8 AMA1_558-578_009 Ac-LATILMVYLYKRKGNAEKYDK-amide 27.98 0.29 9 CYRPA_240-257_005A* HFYVGDNINNVKNVNFIE-amide 12.34 0.78 10 CYRPA_336-355_008B* Ac-DENRTLFIYSSSQGIYNIHT-amide 23.60 0.29 11 EBA175_4-20_001A* NISIYFFASFFVLYFAK-amide 20.61 0.31 12 EBA175_11-28_001B* Ac-ASFFVLYFAKARNEYDIK-amide 21.69 0.43 13 EBA175_64-80_002C* Ac-ENKLDILNNSKFNKRWK-amide 15.18 0.00 14 EBA175_119-140_004A* Ac-KQNKYVPINAVRVSRILSFLDS-amide 21.85 1.38 15 EBA175_206-219_006A* Ac-DHFIEASKKESQLL-amide 16.25 0.33 16 EBA175_293-309_008A* Ac-EKLWEAMLSEHKNNINN-amide 10.55 0.22 17 EBA175_516-538_010 Ac-ILAIAIYESRILKRKYKNKDDKE-amide 12.17 1.00 18 EBA175_556-575_011A* Ac-GTDYWNDLSNRKLVGKINTN-amide 14.63 0.45 19 EBA175_687-707_012D* Ac-YQEYQKGNNYKMYSEFKSIK-amide 15.12 0.67a 20 EBA175_747-763_014 Ac-DVPISIIRNNEQTSQEA-amide 13.47 0.25 21 EBA175_1108-1124_015A* Ac-YEDIVLKSHMNRESDD-amide 12.66 0.00 22 EBA175_1262-1278_016A* Ac-KHGFHTMNNLHGDGVSE-amide 18.98 0.89 23 EBA175_1297-1313_017A* SGNVLNMRSNNNNFNNI-amide 34.14 0.44 24 EBA175_1304-1321_017B* Ac-RSNNNNFNNIPSRYNLYD-amide 9.65 0.14 25 RIPR_61-84_002 Ac-DYAYLNKYVYTILNRDSTEKIKTF-amide 17.88 0.50 26 RIPR_414-432_006A* Ac-VSCYRVSFNLNKLKYVTES-amide 30.57 0.28 27 RIPR_470-486_007C* YDYLCVFNNQNITSDKN-amide 25.02 0.00 28 RIPR_485-508_008A** Ac-KNSHLHSNIPSLYNSSILPDINKS-amide 19.26 2.00 a 29 RIPR_507-522_009B* Ac-KSKFHLISRNSRTNQY-amide 25.17 0.76 30 RIPR_561-577_011B* Ac-KKVEIFRSRFSSKLQCQ-amide 26.84 1.43 31 RIPR_662-676_014 Ac-DNPYISKYGNKLCER-amide 12.88 0.00 32 RIPR_704-718_015A** Ac-EEGYKNVKGKCVPDNK-amide 10.37 0.00 33 RIPR_802-817_017 Ac-KEHYYRSSRGECILND-amide 11.01 0.50 34 RIPR_1045-1065_019 Ac-NEIFHTIIYLKKKIGNSVIYD-amide 26.67 0.65 35 Epivax 44/RH5_79-97 Ac-HSTYIKSYLNTNVNDGLKY-amide 18.60 0.25 36 Epivax 45/RH5_93-110* Ac-DGLKYLFIPSHNSFIKKY-amide 31.73 0.13 37 Epivax 46/RH5_179-196* Ac-YTFLDYYKHLSYNSIYHK-amide 24.63 0.69 38 Epivax 47/RH5_187-205* Ac-HLSYNSIYHKSSTYGKCIA-amide 29.68 0.35 39 Epivax 48/RH5_305-326* Ac-DEYNTKKKKLIKCIKNHENDFN-amide 14.34 1.55 40 Epivax 49/RH5_360-381* Ac-YDEYIHKLILSVKSKNLNKDLS-amide 49.71 1.93 41 Epivax 50/RH5_390-407* SELLLTNLNKKMGSYIYI-amide 24.28 2.06 42 Epivax 51/RH5_453-473 Ac-DELLKRILDMSNEYSLFITSD-amide 11.03 0.80 43 Epivax 52/RH5_488-504* Ac-KHLNNIFHHLIYVLQMK-amide 28.01 0.25 44 Epivax 53/RH5_512-525* Ac-EYFQTYKKNKPLTQ-amide 28.98 2.73 a 45 Epivax 54/RH5_145-162** LQYHFKELSNYNIANSID-amide 17.27 0.08 46 Epivax 55/RH5_382-397 DMTNILQQSELLLTNL-amide 16.91 5.50 a a JanusMatrix Human Homology Score changed since initial analysis as a result of updates to UniProt reviewed human proteome.

By design, we prioritized epitope clusters that covered multiple HLA alleles (as indicated by high EpiMatrix Cluster Scores), had limited potential for cross-reactivity with human sequences (as indicated

MVDP Quarterly Report 38 Leidos Proprietary by low Human JanusMatrix Homology Scores), and had limited polymorphism in field isolates. In Table 4.7-2 above, peptides that were trimmed from their original, computer generated sequences are marked with a single asterisk(*), and peptides with added charge are marked with a double asterisk(**).

4.7.3.1 Methods Peptide synthesis The selected peptides were produced by 9-fluoronylmethoxycarbonyl (Fmoc) synthesis at 21st Century Biochemicals. The peptides were designed with N-terminal acetyl group (“Ac-“ in Table 4.7-8, above) and C-terminal amino group (“-amide” in Table 4.7-8, above) caps and delivered >85% pure as ascertained by HPLC, mass spectrometry and UV scan (ensuring purity, mass, and spectrum, respectively). Based on advice received from 21st Century Biochemicals, eleven peptides were synthesized without an N-terminal acetyl group to facilitate synthesis and purification. In order to establish a net charge, Lysine flanking residues (shown in red in Table 4.7-8) were also added to several peptides. In all cases, the amino acid content of each peptide was determined to enable reconstitution at highly accurate molarity. Table 4.7-9, below, lists selected peptides that were not able to be synthesized or purified to completion.

Table 4.7-9 Leidos Class II T Cell Epitope Clusters (Failed to Synthesize or Purify)

Human JMX HMLGY Peptide Name Peptide Sequence Cluster Score Score AMA1_153-169_003A** KKGIIIENSNTTFLTPV 25.61 0.14 AMA1_541-560_008A* TYDKMKIIIASSAAVAVLAT 51.66 1.39 CYRPA_31-53_002 RHVFIRTELSFIKNNVPCIRDMF 25.53 0.83 CYRPA_222-242_004 GVQYFFLRPYISKNDLSFHFY 20.7 0.62 CYRPA_261-284_006 EKDLEFVCSNRDFLKDNKVLQDVS 18.4 2.23 CYRPA_301-319_007A** AECYIFFNNENSILIKPEK 53.91 0.23 RIPR_1-24_001A* MFRIFFTLLIIILIKKTSAIDLIE 99.43 1.44 RIPR_146-164_003A* THIIIYYISQPLLLEPHV 37.52 1.11 RIPR_182-201_004A* GMYISLRSVHVHTHNAILQQ 29.59 0.39 RIPR_231-253_005 LTHYLFFINIQYQCISPLNLQEN 25.68 0.19 RIPR_526-547_010 NISMLEIQNEISSHNSNQFSTD 13.66 0.09 RIPR_627-644_013 NDQYLFVSYSCSNLCNKC 17.33 0.36 RIPR_742-756_016 SERFVLENGVCICAN 10.5 0.8 RIPR_1007-1032_018A*,** IEFSYIYNQIIWKINNSKESYVFYYD 32.08 0.3

HLA Binding Assay The binding assay in use at EpiVax yields an indirect measure of peptide-MHC affinity (Steere et al 2006). In this assay, a fluorescently labeled, high binding, control peptide, and soluble HLA molecules are loaded onto a 96-well plate along with unlabeled experimental peptides. Unlike binding assays for HLA A and B alleles, where a heating step is required to remove a stabilizing endogenous peptide and render the binding groove ready to accept test peptides, soluble HLA DR alleles are supplied empty and do not require a heating step. The mixture is then allowed to stand overnight. Once the mixture has reached steady equilibrium (at 24 hours), the HLA-peptide complexes are captured on an ELISA plate coated with anti-human DR antibody and labeled control peptides are detected with a Europium-linked probe. Time-resolved fluorescence measuring bound labeled control peptide is assessed by a SpectraMax M5

MVDP Quarterly Report 39

Leidos Proprietary range do tend to bind fairly frequently when they occur within the context of an epitope cluster. In this study, peptides scoring between 1.28 and 1.64 bound roughly three quarters of the time. By contrast peptides predicted to bind at the standard EpiMatrix threshold (scores >1.64) bound roughly 79% of the time. Peptide AMA1_12-29_002A, which bound to all eight alleles including DRB1*0701 and DRB1*1301 with high to very high affinity is a good example (Figure 4.7-4). Although this peptide has no significant EpiMatrix hits for either DRB1*0701 or DRB1*1301, there are elevated Z-scores in frames 15 and 17, both of which contain EpiBars. We believe that strong, well-centered EpiBars reflect a potent HLA-DR binding potential that, in our experience, has often extended beyond only those alleles with significant Z- scores. EpiMatrix Cluster Detail Report File: LEIDOS-R9-SYNTHESIZED-CLUSTERS Sequence: 02_AMA1_12-29_002A Frame Frame Hydro- DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*0901 DRB1*1101 DRB1*1301 DRB1*1501 AA Sequence Hits Start Stop phobicity Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score Z-Score 12 AFEFTYMIN 20 0.53 -0.75 -2.42 -0 89 -1.32 -0.64 -0.66 -0 87 -2.09 0.51 0 13 FEFTYMINF 21 0.64 -0.06 -0.05 0.36 1 03 0.52 0.92 -0.16 0.73 0.01 0 14 EFTYMINFG 22 0.29 -1.46 -1.1 -0 62 -0.52 0.29 0.06 -0 38 -0.53 -0.33 0 15 FTYMINFGR 23 0.18 2.43 1.64 1.21 1 26 1.62 1.93 2.14 1.41 2.16 5 16 TYMINFGRG 24 -0.18 -0.23 -0 5 -0 25 -0.35 -0.16 -0.93 0.27 -1.23 -0.25 0 17 YMINFGRGQ 25 -0.49 2.17 1.03 2.15 1 58 1.7 1.61 2.56 0 56 0.76 4 18 MINFGRGQN 26 -0.73 0.19 -0.03 -0.47 0.19 1.29 0.39 0.78 0.11 1.55 0 19 INFGRGQNY 27 -1.09 1.23 1.92 0.81 0 31 -0.21 0.32 0.04 0 24 1.27 1 20 NFGRGQNYW 28 -1.69 -1.01 -1.63 -2 09 0.15 -1.16 -1.02 -1 24 -1.31 -1.57 0 21 FGRGQNYWE 29 -1.69 0.43 1.18 1.02 0.3 1.38 0.32 0.51 0 57 0.85 0

Summarized Results DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*0901 DRB1*1101 DRB1*1301 DRB1*1501 Total Maximum Single Z score 2.43 1.92 2.15 1 58 1.7 1.93 2.56 1.41 2.16 -- Sum of Significant Z scores 4.6 3.56 2.15 0 1.7 1.93 4.7 0 2.16 20 8 Count of Significant Z Scores 2 2 1 0 1 1 2 0 1 10 Total Assessments Performed 90 Hydrophobicity -0.58 EpiMatrix Score 11.51 EpiMatrix Score (w/o flanks) 16.08 Figure 4.7-4. EpiMatrix Cluster report for AMA1_12-29_002A. N- and C-terminal flanking residues are important to the stability of peptide binding in the open-ended binding groove of HLA-DR. Thus, we sometimes observe lower affinity interactions for EpiBars that are poorly centered within a longer peptide sequence. For example, peptide EpiVax 53/RH5_512-525 occurs at the C-terminus of the RH5 antigen and no natural C-terminal flanking residues exist for the EpiBar in frame 517 (Figure 4.7-5). Although some dose-dependent inhibition of control peptide binding was observed, this peptide bound to only three alleles with low to medium affinity.

MVDP Quarterly Report 44 Leidos Proprietary

Figure 4.7-5. EpiMatrix Cluster report for EpiVax 53/RH5_512-525. Considering each of the experimental peptides independently, we can see that all 46 of the experimental peptides (100%) were found to bind to at least two HLA DR alleles. Thirty-seven of the 46 test peptides (80%) bound to at least four different alleles, and 22 peptides (48%) bound to all eight alleles. Overall, 202 peptide-allele combinations bound with moderate to very high affinity.

4.7.4 Discussion EpiVax had carried out a PreVAX analysis on five blood stage epitope vaccine antigens and has subsequently determined the binding affinity for select predicted epitopes towards recombinant HLA proteins in vitro as reported in this QR. The goal was to identify predicted class I and II epitopes with verified high affinity binding towards the HLA alleles for each of the five antigens. These high affinity binding epitopes are likely to be presented on antigen-presenting cells by the respective HLA alleles through HLA-facing residues on the epitopes which will then enable interaction with cognate TCRs via the TCR facing residues on the residues. At least one class I epitope with a good z-score and minimal or no homology to human sequences that showed moderate to very high binding affinity towards the tested HLA alleles was identified for each of the 5 blood stage antigens. These results indicate that it may be possible to attain broad population coverage with any of the five antigens for at least one class I epitope. Similarly, it may be possible to attain broad population coverage for class II alleles; 2-5 peptides were validated as binders to all 8 class II HLA alleles for each of the 5 blood stage antigens. These findings provide a starting resource for designing epitope-based blood stage malaria vaccines. Of course, the ability of these epitopes to elicit a T cell response in malaria-exposed human PBMCs remains to be verified. Along these lines, a set of epitopes from RH5 have been examined for their ability to elicit a recall response from VAC063 vaccinees PBMCs. In these experiments, the PBMCs from RH5.1 vaccinated individuals were stimulated with peptides corresponding to the predicted epitopes. These data will be presented in a separate report related to the RH5.1 VAC063 clinical trial project. To our knowledge, this is the first comprehensive analysis of epitope content in these blood stage antigens. The IEDB resource lists a limited set of T cell epitopes that have been reported in literature for AMA1 and EBA175. There are no epitopes in IEDB for the other three antigens. Warnae et al (2016) used in silico tools to predict T cell epitopes in RH5 and EBA175 RII domain but did not validate them in either MHC binding assay or T cell response assays.

MVDP Quarterly Report 45 Leidos Proprietary

An epitope-based blood stage malaria vaccine would need to consist of the predicted Class I and II epitopes presented here and that have been subsequently validated in T cell assays. In addition, they would need to be combined with B cell epitopes in order to elicit a robust amnestic humoral response through the CD4+ helper T cell response as well as a CD8+ response. The CD8 response could potentially be relevant for blood-stage antigens that are known to be expressed in the liver stage as well, such as AMA-1 (Silvie et al 2004). Also, parasite-specific CD8+ responses have been shown to be important for humoral immune response in a mouse model of chronic malaria [Horne-Debets et al 2013 and 2016]. The epitope analysis represented here could also inform the development of next generation of antigen- based vaccines. The Janus Matrix analysis of the five antigens scored the homology of predicted Class I and Class II epitopes to human protein sequences. Sequences with high human homology are likely to be recognized as self via interaction with Tregs to down-regulate the overall immune response to the vaccine antigen. These sequences could be engineered out from the whole antigen context to reduce homology to human sequences and thus boost the overall immune response to the vaccine antigen. In terms of overall protein-wide EpiMatrix scores and number of predicted epitopes found, CyRPA, RH5 and Ripr had good overall scores and indicating their epitope-rich nature. These data reiterate the importance of these proteins as vaccine candidates.

4.8. PD-1 Blockade Inhibitor Research Activities: Tim Phares In malaria-infected individuals, PD-1 is expressed by T cells suggesting PD-1-mediated T cell dysfunction may contribute to the lack of long-lived protective immunity against the parasite. Further, in a rodent malaria model PD-1 severely dampened T cell proliferation and function mediating chronic infection. These data argue that blocking PD-1 inhibition may establish a long lasting amnestic response following vaccination. Blockade of the PD-1:PD-L1 pathway has been a successful and impressive immunotherapy for cancer. Moreover, recent studies are exploring the application to “adjuvant-ize” PD-1 blockade inhibitors in prophylactic vaccination. Thus the PD-1 Blockade Adjuvant Project Plan was written and approved by USAID in January 2017. The goal of the plan is to determine whether targeting PD-1 inhibition enhances immunity against malaria. An overview of the development plan for this project is provided in Table 4.8-1. In Q1 FY2018, USAID requested this project be closed out at the end of FY2018 following the completion of the assessment of PD-1 expression on CD8 T cells and Treg generation subsequent to AdPyCS immunization and completion of two separate RH5.1/Alum studies. The assessment of PD-1 expression and Treg generation was completed in Q2 FY2018. The in-life portion of the initial RH5.1/Alum study was completed in Q1 FY2018 with T cell ELISPOT data provided in Q1 FY2018, anti- RH5.1Ab titers provided in Q3 FY2018 and GIA results provided in Q4 FY2018. The in-life portion of the second RH5.1/Alum study was completed in Q4 FY2018 with T cell ELISPOT data provided in Q4 FY2018. Anti-RH5.1Ab titers and GIA results for study two are expected in Q1 FY2019. The PD-1 blockers used in these studies include, but are not limited to, commercially available PD-1 or PD-L1 mAb and several PD-1 peptide blockers identified by Leidos Life Sciences. These PD-1 peptide blockers (~20 amino acids) bind to both human and mouse PD-1 and have shown efficacy in other experimental models. The three PD-1 peptide blockers currently being tested in these studies are: I. PD-1 peptide combo that consists of four separate PD-1 peptide blockers; II. Single PD-1 peptide blocker, LD01, that is microbiome-derived; and III. Single PD-1 peptide that is a derivative of LD01, named LD02.

MVDP Quarterly Report 46 Leidos Proprietary

Table 4.8-1. Overview of the PD-1 Blockade Adjuvant Project Plan

Phase Milestone Task PD-1 Blockade 1. Assess the effect of PD-1 blockers on vaccine Immunogenicity and efficacy testing with Studies efficacy liver and blood stage antigen Adjuvant 2. Assess PD-1 expression and T generation reg Determine whether PD-1 blockers modify Formulation following immunization with or without PD-1 PD1 expression and/or T Studies blockers regs

4.8.1 Milestone 1: Assess the effect of PD-1 peptide blockers on vaccine efficacy A subcontract for testing the effects of PD-1 blockade on vaccine efficacy using a recombinant replication-defective adenovirus expressing the P. yoelii circumsporozoite (CS) protein (AdPyCS) with ADARC was executed in January 2017 and activities have been initiated and are described below. Subcontracting activity with University of Oxford was initiated in August 2017 and has been completed. Studies at University of Oxford assessed the efficacy of PD-1 peptide blockers on both cellular and humoral immune responses to the blood stage antigen Rh5 (RH5.1 formulated in alum).

4.8.1.1 Assess the effect of PD-1 peptide blockers on immunogenicity of AdPyCS To test whether immunogenicity is enhanced following PD-1 blockade, BALB/c mice (n = 5) were immunized IM with 1010 virus particles of AdPyCS in the hind limb without adjuvant. At days 1, 3, 5 and 7 post-immunization mice were treated IP with 200 µg of peptide(s) or monoclonal antibodies (mAbs) shown in Table 4.8-2. In addition to the test peptides, ovalbumin (OVA) peptide was also included as a control peptide. At day 12 post-immunization immunogenicity was assessed by measuring the number of splenic CS-specific, IFN-γ secreting CD8 T cells using the ELISPOT assay after stimulation with the H- 2kd restricted CD8 epitope SYVPSAEQI. Results of the immunogenicity studies are detailed in the FY2018 USAID MVDP Annual Report.

Table 4.8-2. Cohorts for AdPyCS + PD-1 Blockade Immunogenicity Study Cohort # Test Sample 1 AdPyCS alone 2 AdPyCS + PD-1 Peptide Combo 3 AdPyCS + Ova Peptide 4 AdPyCS + LD01 Peptide 5 AdPyCS + LD02 Peptide 6 AdPyCS + anti-PD-1 mAb 7 AdPyCS + anti-PD-L1 mAb

4.8.1.2 Optimizing dose of AdPyCS immunization Prior to evaluating the effects of PD-1 blockade on protection, studies to determine a suboptimal AdPyCS dose against Py were carried out. BALB/c (n = 4-5) mice were immunized IM with various doses (Table 4.8-3) of AdPyCS in the hind limb without adjuvant. At day 12 post-immunization mice were challenged with ≥200 Py 17XNL sporozoites intravenously (IV). Parasitemia was assessed via blood smears beginning at day 3.

Based on data from three dosage studies, Leidos and USAID agreed to use the suboptimal dose of 109 AdPyCS for immunization in studies assessing the effects of PD-1modulation on protection (4.8.1.3) as the 109 dose is at the threshold of protection. Results of the individual dosage studies are detailed in the FY2018 USAID MVDP Annual Report.

MVDP Quarterly Report 47 Leidos Proprietary

Table 4.8-3. AdPyCS Dosing Study

Cohort # Test Sample 1 Naïve 2 107 AdPyCS 3 108 AdPyCS 4 109 AdPyCS 5 1010 AdPyCS 6 1011 AdPyCS

4.8.1.3 Assess the effect of PD-1 peptide blockers on protection with AdPyCS With completion of 4.8.1.1 and 4.8.1.2 experiments, a study to evaluate whether protective efficacy is enhanced following PD-1 blockade was commenced and completed in June 2017. BALB/c mice (n = 9- 10) were immunized IM with 109 virus particles of AdPyCS in the hind limb without adjuvant. At days 1, 3, 5 and 7 post-immunization mice were treated IP with 200µg of peptide(s) or mAbs shown in Table 4.8-2. In addition to the testing peptides, OVA peptide was also included as a control peptide. At day 12 post-immunization mice were challenged with ~50-75 Py 17XNL sporozoites IV. Parasitemia was assessed via blood smears beginning at day three. Results of the two challenge studies are detailed in the FY2018 USAID MVDP Annual Report.

4.8.1.4 Assess PD-1 expression on CD8 T cells and Treg generation subsequent to AdPyCS Immunization Increases in immunogenicity and protection of AdPyCS immunization following PD-1 inhibition suggest PD-1 directly or indirectly, through the development of Treg, negatively impacts the immune response. To assess whether PD-1 expression and/or Treg generation is elevated subsequent to AdPyCS immunization two studies were conducted in Q1 FY2018 and Q2 FY2018 by ADARC. Detailed data can be found in the FY2018 USAID MVDP Annual Report.

4.8.1.5 Assess the effect of PD-1 peptide blockers on immunogenicity of Rh5 As stated above, subcontracting activity with University of Oxford was completed and a study was initiated in August 2017. To test whether RH5.1 protein immunogenicity is enhanced following PD-1 blockade BALB/c mice (n = 4) were immunized with 60 ng RH5.1 protein in 250 μg Alhydrogel. All immunizations are administered IM into both hind limbs. At days 1, 3, 6 and 8 post-immunization mice were treated IP with 200 µg of peptide(s) (LD01, LD02 or combo) or anti-PD-1 mAb (Table 4.8-4). As shown in Table 4.8-4 peptide(s) or anti-PD-1 mAb treatment occurs either after each immunization, only after the first immunization or after the second and third immunization to identify the optimal dosing schedule. At days 27, 55 and 63 post-immunization mice were bled to assess Ab levels via ELISA and GIA respectively. At day 63 post-immunization T cell responses were assessed by measuring the number of splenic RH5.1-specific, IFN-γ secreting T cells (spot-forming units =SFU) using the ELISPOT assay after stimulation with pooled (n = 7) Rh5 peptides (10 µg/mL (Table 4.8-5)) or RH5.1 protein (5 µg/ml). The study was completed in Q1 FY2018 and T cell and humoral immunity assessments were completed by Q4 FY2018. Results of this study are detailed in the FY2018 USAID MVDP Annual Report.

MVDP Quarterly Report 48 Leidos Proprietary

Table 4.8-4. Groups for RH5.1/Alum + PD-1 Blockade Immunogenicity Study No Groups Test Sample 0 1 3 6 8 27 28 29 31 34 36 55 56 57 59 62 63 mice RH5.1 protein +  1 4      alum  RH5.1 protein +  2 alum + PD-1 4           peptide Combo RH5.1 protein +  3 alum + PD-1 4                  peptide Combo RH5.1 protein +  4 alum + LD01 4           peptide RH5.1 protein +  5 alum + LD01 4                  peptide RH5.1 protein +  6 alum + LD02 4           peptide RH5.1 protein +  7 alum + LD02 4                  peptide RH5.1 protein +  8 alum + anti-PD-1 4           mAb RH5.1 protein +  9 alum + anti-PD-1 4                  mAb RH5.1 protein +  10 alum + PD-1 4              peptide Combo RH5.1 protein +  11 alum + LD01 4              peptide

 IM injection of RH5.1/Alum  Bleed for GIA/ELISA assay  IP injection of PD-1 blocking peptides and mAb  Assess T responses via ELISPOT

Table 4.8-5. PfRH5 Specific Peptides Used to Stimulate T Cells Epitope Sequence of Peptide Position in PfRH5 A7 TYDKVKSKCNDIKNDLIATI T216-I235 C6 NIANSIDILQEKEGHLDFVI N156-I175 C9 NLNKKMGSYIYIDTIKFIHK N396-K415 D9 YIDTIKFIHKEMKHIFNRIE Y406-E425 F6 KHLSYNSIYHKSSTYGKCIA K186-A205 F10 NDVPIKMEYFQTYKKNKPLT N506-T525 G10 DVPIKMEYFQTYKKNKPLTQ D507-Q526

MVDP Quarterly Report 49 Leidos Proprietary

To confirm that RH5.1 protein immunogenicity is enhanced following PD-1 blockade BALB/c mice (n = 6) were again immunized with 60 ng RH5.1 protein in 250 μg Alhydrogel. All immunizations are administered IM into both hind limbs. At days 1, 3, 6 and 8 post-immunization mice were treated IP with 200 µg of peptide(s) (LD01, LD02, combo peptides or OVA (negative peptide)) or anti-PD-1 mAb or isotype control mAb (Table 4.8-6). As shown in Table 4.8-6 peptide(s) or mAb treatment occurs either after each immunization or after the second and third immunization. The in-life portion of the second RH5.1/Alum study was completed in Q4 FY2018 with T cell ELISPOT data provided in Q4 FY2018. These results are detailed in the FY2018 USAID MVDP Annual Report. Results of the serum anti-RH5.1 total IgG titers and GIA are expected in early Q2 FY2019. Table 4.8-6. Groups for RH5.1/Alum + PD-1 Blockade Immunogenicity Confirmatory Study

MVDP Quarterly Report 50 Leidos Proprietary

5. Element 3 Activities: Amy Noe/Jessica Smith

5.1. 2018 SCG Meeting Feedback and Leidos Responses During Q1 FY2019 USAID distributed the 2018 SCG Meeting Report to Leidos. Based on the feedback provided, Leidos has addressed three comments from the report below.

5.1.1 Program Overview and Partnerships The report highlighted three areas of concern from the SCG members regarding Program Overview and Partnerships. The specific statement from the report is as follows: The SCG noted the continued high level of engagement of Leidos as a contracting partner for the program. In order to realize the full potential of the collaboration, the SCG emphasized that:

 The milestones and go/no-go criteria for the research projects should be presented.  In the interests of transparency, the commercial and personal relationships between Leidos and their other partners should continue to be reviewed regularly.  MVDP staff should give careful consideration to the level of technical detail envisioned for all presentations and involve appropriate presenters (in person or through teleconferencing) to ensure that the SCG has the information required to fully evaluate each project. With regard to the first and third bullets, Leidos notes that USAID reviewed and approved all presentations prior to the annual meeting. Not only were the slides reviewed by USAID, but also, Leidos walked through the presentations with the USAID technical advisors. Leidos updated project presentations based on the USAID comments and edits received, and resubmitted updated presentations for approval prior to the meeting. It is also important to note that during the process of developing the invitee list, Leidos asked USAID if any Leidos subcontract personnel should be present at the SCG meeting. The only Leidos subcontractors selected by USAID for attendance at the meeting were from the University of Oxford. With regard to the second bullet, it is important to relay to both USAID and the SCG that this comment denotes gaps in the understanding by the SCG of the requirements for government contractors set forth in the FAR, including the heavy regulation of procurement processes as well as the mandatory DCAA (Defense Contract Audit Agency) audits that occur for all subcontracts established by Leidos. At the request of USAID, Leidos previously walked through its business processes as a government contractor as well as the MVDP-specific project development process during both the 2016 and 2017 SCG meetings. This walk-through included discussion of the MVDP contract-mandated project approval process, which includes review and approval of all project plans by USAID. It is important to note that not only do the project plans outline the study plans/designs but also detail potential vendors and their capabilities. Further, as part of the project development process and in order to give USAID the best opportunity to understand and evaluate the different technologies available, Leidos commonly arranges meetings between potential vendors and the USAID technical advisors to discuss details of the specific vendor technology. Lastly, given the makeup of the SCG and relatively broad distribution of the SCG Meeting Report, Leidos is highly concerned that such misunderstandings and the resulting discussion/comments may negatively impact current and future contract awards for Leidos (both inside and outside the malaria field) as the SCG and invited guests include key decision makers across multiple government agencies (i.e., NIAID, WRAIR, NMRC, and USAID) as well as important collaborators/partners across academia, industry, and non-profit organizations.

MVDP Quarterly Report 51 Leidos Proprietary

5.1.2 RH5.1 Clinical Study Immunogenicity Assessments Using Peptides Generated from EpiVax Predictions There was also a comment in the report concerning the previously conducted assessments to evaluate immunogenicity of EpiVax predicted class II epitopes using PBMCs collected from RH5.1 vaccinees. The specific comment is as follows: “With regards to the T cell analyses of Rh5 vaccines and in particular the predictive value of the EpiVax suite of algorithms, the SCG recommends that the algorithm’s ability to predict not only the hotspots but also the cold spots is validated. While this may have been validated in other systems, it is just good science to include these analyses in this data package.” It is important to clarify to both USAID and the SCG that the “predictive value of the EpiVax suite of algorithms” is solely based on peptide binding to HLA molecules. The EpiVax tools are not designed to predict immunogenicity nor does EpiVax make such claims. While peptide binding to HLA is a major contributing factor to immunogenicity, it is by no means the only factor. Therefore, “validation” of the EpiVax in silico data for the predicted Rh5 class II epitopes is based on in vitro determination of peptide binding to HLA (which was previously conducted and reported to USAID) and not assessment of immunogenicity. It is also important to note that these analyses, both the cellular response assessments conducted by Oxford and the subsequent iTEM/J-iTEM analysis by EpiVax, were not scoped under the MVDP. Rather, they were performed gratis by both collaborators to evaluate if peptides derived from the set of EpiVax predicted epitopes could elicit immune responses in vaccinees. As such, this study was not designed to evaluate immunogenicity of Rh5 sequences where EpiVax did not predict epitope content (i.e., “cold spots”). While both Leidos and EpiVax agree that such data are important, particularly with regard to establishing a positivity cut-off for this data set, Oxford indicated that there were insufficient cells to establish this value. Note that these are the same cell sets slated for use in the RH5.1 human mAb identification and development project. To help compensate for the lack of establishment of a positivity cut-off by Oxford, EpiVax evaluated two positivity thresholds for the iTEM/J-iTEM analysis.

5.1.3 Liver Stage Vaccine Development Project Leidos also noted several misunderstandings in the SCG Meeting Report summary of the Liver Stage Vaccine Development Project. The first of these misunderstandings is in regards to the reasons for down selection from five to three proteins. The specific statement is as follows: “The two additional gene sequences that were on the list last year, Pf14_0301 and PFE1025C, had been found to lack the ability to bind to all the human HLA alleles tested and were deprioritized due to that shortcoming.” Leidos notes that context is essential when understanding this downselection. Given the available resources to assess the five proteins, only a subset of predicted epitopes for each protein could be evaluated in vitro for HLA binding. For HLA Pf14_0301 and PFE1025C, it was found that none of the predicted epitopes tested for one of the HLA alleles bound. However, as only a relatively small subset of predicted epitopes were assessed across all of the different HLA alleles, the statement that these proteins, “lack the ability to bind to all the human HLA alleles tested” is incorrect, as it implies that the whole repertoire of predicted epitopes was assessed. Further, the wording of this statement suggests that the two deprioritized proteins were deficient in epitopes that bound to all six HLA alleles rather than to only a single HLA allele. It is important to note that Leidos reviewed the downselection process during the 2017 SCG Meeting and indicated that for each class I HLA allele, ~25 epitopes were selected across all five proteins, providing a total of 120 predicted epitopes selected for in vitro HLA binding assessments. Further, this same presentation included data that showed over 1400 epitopes were predicted across the five proteins. Thus, less than 9% of the total number of epitopes that were predicted were ultimately tested for HLA binding. While time did not permit presenting these data again

MVDP Quarterly Report 52 Leidos Proprietary during the 2018 SCG Meeting, it was clearly stated in the 2018 SCG presentation that only a fraction of the total number of predicted epitopes were tested for HLA binding. Thus, Pf14_0301 and PFE1025C were only eliminated from progression in this project because epitopes to test immune response in the MIMIC system had not been identified. These data are insufficient to indicate that these proteins are deficient as antigens. The second set of misunderstandings is in regards to the specific statement below: “There was extensive discussion around the general concept that immunogenicity does not necessarily imply protection, and that these targets need to be validated further as appropriate targets for vaccine development. It is an enormous and risky leap to start with gene sequences that appear to be transcribed (from earlier work identifying them as liver stage mRNA transcripts) and assume that they are appropriately expressed and available as protein antigens accessible to the host immune system.” While Leidos agrees with the statements that, “immunogenicity does not necessarily imply protection”, “targets need to be validated”, and it is a risk to “start with gene sequences…and assume that they are appropriately expressed”, the context of these statements is incorrect when applied to the liver stage project. Leidos notes that the last statement is the most concerning as the 2018 presentation to the SCG clearly documented that, based upon antibody development during infection with Plasmodium, the three selected proteins had previously been demonstrated to be expressed in humans. Further, Leidos stated during that same presentation that Tarun et al. had demonstrated four of the proteins in the liver phase of infection in mice infected with P. yoelii using mass spectral analysis (SPECT2 is the exception). Tarun’s work is the only publication of a mass screening approach to identify translated (i.e., expressed) proteins in the liver phase of malaria. Thus, Leidos is making USAID aware of the gap between what was presented by Leidos on the liver stage project at the 2018 meeting and the resulting report by the SCG. With regard to the SCG’s comments on immunogenicity and target validation, Leidos notes that the role of an epitope or antigen in protective immunity can only be determined in context of vaccine studies designed to test that particular epitope or antigen. Indeed, Leidos had proposed demonstrating that an immune response could be elicited by human cells since animal models are not good indicators of a broad human T cell response, and originally had in its plans to test protective immunity in humans for selected epitopes. Leidos limited the evaluation of protective immunity to be in humans only when the approach was for an epitope-based vaccine. As the approach was expanded to the whole protein as an antigen, Leidos continued to support first demonstrating the T cell response using human T cells, but proposed testing protective immunity to the specific proteins in mouse models. The second slide in the 2018 SCG presentation by Leidos indicated this proposed testing following the testing of the response by human cells in the MIMIC model. Leidos notes it concern that the SCG report raises this flag regarding target validation but does not mention the planned work toward this goal that Leidos conveyed during the meeting. The third set of misunderstandings is in regards to the specific statements below: “The SCG had numerous suggestions for criteria that might be considered in order to validate the selected genes as potential vaccine candidates. These include: - First, attempts to make knockouts in rodent malaria parasites, preferably inducible knockouts, should be performed. An ideal vaccine candidate would be essential for life in at least one stage of parasite development. The concern was raised that any antigen that could be knocked out with subsequent survival of the parasite would be a very risky candidate indeed, as the parasite has already proven its vast potential to utilize alternate pathways and for adaptation away from increased immune pressure.

MVDP Quarterly Report 53 Leidos Proprietary

- Confirmation of the presence of protein expression in the parasite is absolutely essential. - Since the genes are reportedly highly conserved across malaria species, preliminary work in the mouse model to confirm that these antigens are immunogenic as well as protective is highly desirable.

- Ideally, these candidates should be expressed in a malaria-appropriate expression system as a precursor to vaccine development, with careful confirmation of folding, tertiary structure, absence of inappropriate glycosylation and scalability. Creation of the protein will be required for further development in order to create the appropriate antigen and other reagents for assessing immunogenicity, even if another platform choice is subsequently made for vaccine delivery. However, this kind of expensive effort should perhaps await the efforts suggested above to ensure that the genes in question actually translate into meaningful vaccine candidates.” With regard to the first bullet, Leidos does not agree that a gene must be essential to be a good vaccine candidate, with the F1 antigen in Yersinia pestis being just one example of a non-essential antigen that has proven to be one of the best vaccine candidates. Further, Zhang et al. (doi: 10.1126/science.aap7847) recently reported results identifying essential genes in P. falciparum using saturation mutagenesis. The report identified the vast majority of essential genes are housekeeping genes (such as those used for RNA metabolism); whereas genes associated with host/parasite interactions tend to be redundant and individually are not essential. Therefore, Leidos notes its concerns regarding the knock out strategy proposed by the SGC. With that understanding, Leidos has provided the results from Zhang et al in the recent project report summarizing the finding for the five selected proteins. With regard to the second bullet, Leidos assumes “expression” means translated. Please see the above discussion of this gap in the SCG’s understanding of the basis for how proteins were selected for the liver stage project. With regard to the third bullet, Leidos agrees with this recommendation and has proposed this work to USAID as part of the Liver Stage Project Plan Attachment 1, which, at the time of this writing is currently under review by USAID. With regard to the forth bullet, Leidos agrees with this statement and has proposed this work to USAID as part of the Liver Stage Project Plan Attachment 1, which, at the time of this writing is currently under review by USAID. Further, Leidos has noted to USAID that the lack of reagents for these novel protein is of high concern. As such, Leidos supports demonstrating protective efficacy before undertaking this type of development.

5.2. 2019 SCG Meeting Planning The USAID Scientific Consultants Group (SCG) provides advice on specific issues and areas related to the USAID MVDP direction and strategy. The SCG is charged with ensuring the application of the highest standards of technical and scientific excellence, assisting in the development of overall program strategy and goals, providing strategic advice and recommendations, and identifying new opportunities for innovative and impactful research. The SCG meeting will be held May 1-2, 2019. Planning for the 2019 meeting began in mid-December 2018 with the RFP process to the NOVA/MD area.

6. Element 4 Activities

6.1. MVDP Reagents Repository: Jessica Smith SriSai Biopharmaceutical Solutions (SBS) maintained, received, and distributed the reagents/materials needed for the ongoing and future studies during Q1 FY19. SBS furnishes all the necessary services, management, qualified personnel, materials, equipment, facilities, and travel required for a biologics

MVDP Quarterly Report 54 Leidos Proprietary repository and inventory management services related to cGMP and non-GMP vaccines and associated products. SBS offers Controlled Room Temperature Storage (15 to 30°C), Controlled Room Temperature with humidity control, Refrigerated Storage (2 to 8°C), Freezer Storage (-20 ±10°C), Ultra-low Freezer Storage (-80 ±10°C), Low Freezer Storage (-30±10°C) and Vapor Phase Liquid Nitrogen (≤ -140°C), as well as the option to set units at a customer-defined temperature. The current inventory consists of standard storage temperatures ranging from -80°C to 4°C.

7. Leidos Points of Contact

7.1. Program Manager Gabriel M. Gutierrez, Ph.D. Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0426 Mobile: 240-529-2811 Fax: 301-846-0794 [email protected]

7.2. Operations Manager Amy Noe, Ph.D., MBA Leidos Life Sciences 4161 Campus Point Court, M/S E3 San Diego, CA 92121 Phone: 858-826-6105 Mobile: 858-201-9176

7.3. Contracts Manager Casey Koontz Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0435 Fax: 301-846-0794

8. Appendix 1: Literature Cited

MVDP Quarterly Report 55 Leidos Proprietary

Brault, A.C., Domi, A., McDonald, E.M., Talmi-Frank, D., McCurley, N., Basu, R., Robinson, H.L., Hellerstein, M., Duggal, N.K., Bowen, R.A., Guirakhoo, F., 2017. A Zika Vaccine Targeting NS1 Protein Protects Immunocompetent Adult Mice in a Lethal Challenge Model. Sci Rep 7, 14769. Brazzoli, M., Magini, D., Bonci, A., Buccato, S., Giovani, C., Kratzer, R., Zurli, V., Mangiavacchi, S., Casini, D., Brito, L.M., De Gregorio, E., Mason, P.W., Ulmer, J.B., Geall, A.J., Bertholet, S., 2016. Induction of Broad-Based Immunity and Protective Efficacy by Self-amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin. J Virol 90, 332-344. Brune, K.D., Howarth, M., 2018. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front Immunol 9, 1432. Brune, K.D., Leneghan, D.B., Brian, I.J., Ishizuka, A.S., Bachmann, M.F., Draper, S.J., Biswas, S., Howarth, M., 2016. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234. Crompton, P.D., Kayala, M.A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G.E., Molina, D.M., Burk, C.R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D.L., Liang, X., Doumbo, O.K., Miller, L.H., Doolan, D.L., Baldi, P., Felgner, P.L., Pierce, S.K., 2010. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A 107, 6958-6963. Dent, A.E., Nakajima, R., Liang, L., Baum, E., Moormann, A.M., Sumba, P.O., Vulule, J., Babineau, D., Randall, A., Davies, D.H., Felgner, P.L., Kazura, J.W., 2015. Plasmodium falciparum Protein Microarray Antibody Profiles Correlate With Protection From Symptomatic Malaria in Kenya. J Infect Dis 212, 1429-1438. Gilbert, S.C., 2013. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 31, 4241-4246. Goepfert, P.A., Elizaga, M.L., Seaton, K., Tomaras, G.D., Montefiori, D.C., Sato, A., Hural, J., DeRosa, S.C., Kalams, S.A., McElrath, M.J., Keefer, M.C., Baden, L.R., Lama, J.R., Sanchez, J., Mulligan, M.J., Buchbinder, S.P., Hammer, S.M., Koblin, B.A., Pensiero, M., Butler, C., Moss, B., Robinson, H.L., Group, H.S., National Institutes of, A., Infectious Diseases, H.I.V.V.T.N., 2014. Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis 210, 99-110. Hekele, A., Bertholet, S., Archer, J., Gibson, D.G., Palladino, G., Brito, L.A., Otten, G.R., Brazzoli, M., Buccato, S., Bonci, A., Casini, D., Maione, D., Qi, Z.Q., Gill, J.E., Caiazza, N.C., Urano, J., Hubby, B., Gao, G.F., Shu, Y., De Gregorio, E., Mandl, C.W., Mason, P.W., Settembre, E.C., Ulmer, J.B., Craig Venter, J., Dormitzer, P.R., Rappuoli, R., Geall, A.J., 2013. Rapidly produced SAM((R)) vaccine against H7N9 influenza is immunogenic in mice. Emerg Microbes Infect 2, e52. Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., Yan, S.F., Williamson, K.C., Holder, A.A., Carucci, D.J., Yates, J.R., 3rd, Winzeler, E.A., 2004. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res 14, 2308-2318. Le Roch, K.G., Zhou, Y., Blair, P.L., Grainger, M., Moch, J.K., Haynes, J.D., De La Vega, P., Holder, A.A., Batalov, S., Carucci, D.J., Winzeler, E.A., 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-1508. Lindner, S.E., Swearingen, K.E., Harupa, A., Vaughan, A.M., Sinnis, P., Moritz, R.L., Kappe, S.H., 2013. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol Cell Proteomics 12, 1127- 1143. Morse, D., Webster, W., Kalanon, M., Langsley, G., McFadden, G.I., 2016. Plasmodium falciparum Rab1A Localizes to Rhoptries in Schizonts. PLoS One 11, e0158174. Pascolo, S., 2008. Vaccination with messenger RNA (mRNA). Handb Exp Pharmacol, 221-235. Shinde, V., Fries, L., Wu, Y., Agrawal, S., Cho, I., Thomas, D.N., Spindler, M., Lindner, E., Hahn, T., Plested, J., Flyer, D., Massare, M.J., Zhou, B., Fix, A., Smith, G., Glenn, G.M., 2018. Improved Titers against Influenza Drift Variants with a Nanoparticle Vaccine. N Engl J Med 378, 2346-2348. Tarun, A.S., Peng, X., Dumpit, R.F., Ogata, Y., Silva-Rivera, H., Camargo, N., Daly, T.M., Bergman, L.W., Kappe, S.H., 2008. A combined transcriptome and proteome survey of malaria parasite liver stages. Proc Natl Acad Sci U S A 105, 305-310. Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., Howarth, M., 2012. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697.

MVDP Quarterly Report 56

Leidos Proprietary

1. EXECUTIVE SUMMARY

A summary of efforts for the planned, ongoing, and completed projects for the Malaria Vaccine Development Program (MVDP) contract for this reporting period are detailed within. A compiled Gantt chart including activities associated with each of the projects has been created and included as an attachment to this report. Ongoing projects that will continue through FY2019 include two vaccine development projects, the CSP vaccine development project (CSP Vaccine) and liver stage vaccine development project (Liver Stage Vaccine), as well as the clinical study with RH5 (RH5.1 Clinical Study), the latter to assess long-term immunogenicity in RH5.1 vaccinees. Of note is that while both the CSP and the liver stage vaccine development projects were initiated as epitope-based projects, these have since been realigned to target whole proteins; therefore, the project names have also been realigned to remove “epitope-based”. Expansion of work on the RCR complex into a vaccine development project (RCR Complex) occurred in early FY2019 and this project will continue through the fiscal year. Lastly, a new project, the RH5.1 human monoclonal antibody identification and development project (RH5.1 Human mAb), was initiated in early FY2019 and will continue through the fiscal year. Two projects will be completed in FY2019, the blood stage epitope-based vaccine development project and the PD1 blockade inhibitor project (PD1 Block Inh). Leidos continues to seek collaborators for information exchange under NDA, reagent exchange under MTA, and collaboration under CRADA, to expand our body of knowledge and access to reagents with minimal cost to the program. This type of collaboration benefits the malaria research community and extends the utility of the contract. The information contained herein is intended to provide technical detail regarding activities conducted within the specified reporting period; however, at the request of USAID, Leidos will retain data in quarterly reports for activities completed within the relevant fiscal year. Of note is that GO (Gene Ontology) standards have been adopted for protein naming conventions.

2. CONTRACTS MANAGEMENT AND ADMINISTRATION

2.1. CURRENT BUDGET SUMMARY The budget summary for the quarter is provided in Table 2.1-1. Table 2.1-2 provides FY19 to-date costs against FY19 Annual Work Plan Estimates. Detailed subcontractor spending is provided in the

MVDP Quarterly Report 2 Leidos Proprietary

MVDP Quarterly Report 3 Leidos Proprietary

2.2. DELIVERABLES SUMMARY In Table 2.2-1 below, we provide the deliverables for which Leidos is responsible under the prime contract with respect to the tasks assigned by USAID. All deliverables are assigned according to the four (4) “Elements” that are outlined in the prime contract.

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes

CONTRACTS AND ADMINISTRATION Signed: 5/28/15 Modifications: MOD 1 2/22/16 MOD 2 3/4/16 MOD 3 8/16/16 MOD 4 2/13/17 Prime Contract MOD 5 5/1/17 MOD 6 7/18/17 MOD 7 9/28/17 MOD 8 1/09/18 MOD 9 12/20/18 MOD 10 3/1/19 Contractor – Employee Non- Signed Non-Disclosure/COI i. Disclosure /Conflict of Interest Delivered Agreements are located on (COI) Agreements SharePoint. Delivered: 08/31/2018 ii. Annual Work Plan Upcoming Plan: 09/01/2019 Approved: 11/08/2018 Q1 FY19 Delivered: 01/14/19 Upcoming Reports: vi. Quarterly Reports Q2 FY19 Delivered: 04/15/19 07/14/2019 Quarterly Financial Q1 FY19 Delivered: 01/14/19 Upcoming Reports: vii. Reports Q2 FY19 Delivered: 04/15/19 07/14/2019 Delivered: 10/15/18 viii. Annual Report/Q4 Report Upcoming Reports: 10/15/19 Approved: 12/03/2018 Delivered: The Small Business Small Business H.10.a Subcontracting Plan was included in Subcontracts Subcontracting Plan Leidos’ proposal. Due: Annually in October; Individual Subcontract Upcoming Reports: 10/30/18 H.10.b.1 submitted via eSRS by Leidos’ Small Reports Business Liaison office. Due: Annually in October; Summary Subcontract H.10.b.2 submitted via eSRS by Leidos’ Small Upcoming Report: 10/30/18 Report Business Liaison office. ix. Final Overall Contract Report N/A Upcoming Report: 5/27/20 Upcoming Meetings: Regularly Held: See Table 2.4-1. Biweekly USAID–Leidos Update scheduled biweekly meetings are C.4.4 Ad hoc meetings held: See Meetings held the first and third Table 2.4-1. Wednesdays of each month. Contract Administration G.4.C Meetings with Contracting No actions in the reporting period. Officer’s Representative (COR)

MVDP Quarterly Report 4 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes Upon approval of all future Due: All contracts and deliverables, Leidos will upload to USAID Development Experience administration deliverables will be the DEC. At contract xiv. Information uploaded to the DEC after 30 days completion, Leidos will upload all from approval. task-associated documents as well. ELEMENT 1: PROTOCOL DEVELOPMENT/WHITE PAPERS See section 3.2 regarding xii. Publications/Posters Q1 FY2019: 4 posters abstract/poster submissions. C.3.2.2 New Project Proposals None in the reporting period ELEMENT 2: IMPLEMENTATION OF RESEARCH AND DEVELOPMENT PROJECTS CSP Vaccine Development Project Project Plan approved by USAID Final Individual Project on 1/29/16. iii. Approved descriptions Revised Project Plan approved 04/26/2017. Subcontract/Task Order Q1 FY2019: procurement Awards: VLP Biotech, JHU, activities completed for IMV, Subcontracts: ADARC, EpiVax, Precision JHU, VaxDesign, and VLP xii. Procurement of Materials, Antibody, ImmunoVaccine, Biotech. Supplies, and Services VaxDesign Q2 FY2019: procurement Other Procurement Vehicles: completed for VaxDesign and CPC Scientific, Vaxine ongoing for JHU. iv. Final Individual Project Report — VLP Biotech Quarterly Reports VLP162 + DPX4 Stability Study Provided herein, circulated via v. Individual Project Data Sets Documentation (ImmunoVaccine) email and/or uploaded to VaxDesign Data Slides SharePoint. CSP Project Update Slides See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RH5.1/AS01 Vaccine Clinical Study Final Individual Project Plan approved by USAID iii. Approved Project descriptions on 2/17/16. Q1 FY2019: procurement for Subcontract/Task Order recently approved activities is Procurement of Materials, xii. Awards: University of Oxford, ongoing for Oxford. Supplies, and Services EpiVax Q2 FY2019: Oxford procurement completed. iv. Final Individual Project Report — Schedule and regulatory approvals Provided herein, circulated via for late immunogenicity time point v. Individual Project Data Set email, and/or uploaded to Oxford quarterly reports SharePoint. Oxford data updates

MVDP Quarterly Report 5 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — Trial registered on September 5, xii. Registration of Trials Completed 2016 (ClinicalTrials.gov) Liver Stage Vaccine Development Project Project Plan approved by USAID on 6/13/16. Attachment 1 - CD8 Final Individual iii. Approved platform scouting is in Project descriptions preparation and expected to be completed in Q3 FY2019. Q2 FY2019: Mosquito Bite titration study data provided to Subcontract Awards: EpiVax, USAID on Feb 11 2019. AdPyCS Procurement of Materials, VaxDesign, Multimeric xii. titration study data provided to Supplies, and Services BioTherapeutics, ADARC, SGI, USAID on March 24 2019. GeoVax Kickoff meetings with GeoVax and SGI in March Q2 FY2019. iv. Final Individual Project Report — v. Individual Project Data Set None during reporting period. Provided herein. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-WEHI Final Individual Project Project Plan approved by USAID iii. Approved descriptions on 3/21/2019.

Q1 FY2019: procurement for recently approved activities is ongoing with WEHI, SGI, and Precision Antibody Q2 FY2019: (Procurement for Study W2 - IMV was completed Pending: WEHI and formulation is scheduled to Procurement of Materials, Subcontract/Task Order initiate in April 2019. xii. Supplies, and Services Awards: WEHI, ImmunoVaccine, Procurement for Study W2- SGI and Precision Antibody WEHI is ongoing. Procurement for Study W4 – SGI is completed; kickoff meeting was held on March 27, 2019. Procurement for Precision Antibody was completed and RCR complex evaluation will initiate in April 2019) iv. Final Individual Project Report —

MVDP Quarterly Report 6 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes The preliminary ELISA dataset was circulated to USAID and the Preliminary ELISA dataset for Study repeat ELISA was discussed in the v. Individual Project Data Set W1 biweekly meeting and provided Repeat ELISA dataset for Study W1 herein.

See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-University of Oxford Final Individual Project Project Plan approved by USAID iii. Approved descriptions on 2/13/19. Q1 FY2019: procurement for recently approved activities is Procurement of Materials, Subcontract Award: University xii. ongoing with Oxford Supplies, and Services of Oxford Q2 FY2019: Oxford procurement completed iv. Final Individual Project Report — v. Individual Project Data Set None during reporting period. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RH5.1 Human mAb Identification and Development Project Plan approved by USAID Final Individual Project iii. Approved (regular elements only) on descriptions 1/29/19. Procurement of Materials, Subcontract Award: University Q2 FY2019: Oxford xii. Supplies, and Services of Oxford procurement completed iv. Final Individual Project Report — v. Individual Project Data Set None during reporting period. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — Blood-Stage Epitope Vaccine Development Project Plan approved by USAID Final Individual iii. Approved on 3/24/2016. Addendum 1 Project descriptions approved on 6/23/16. Subcontracts Awards: Agilvax, VLP Biotech, Expres2ion, NYBC, Precision Antibody, Swiss TPH Procurement of Materials, MTAs: WEHI (Alan Cowman), No procurement activities for xii. Supplies, and Services Swiss TPH (Gerd Pluschke), Q1 FY2019 University of Oxford (Simon Draper), Wellcome Trust Sanger Institute (Gavin Wright)

MVDP Quarterly Report 7 Leidos Proprietary

Table 2.2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes Draft delivered to USAID on iv. Final Individual Project Report Under development 01/31/2019. USAID feedback received on 3/14/2019. v. Individual Project Data Set None during reporting period. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — PD1 Blockade Inhibitor Final Individual Project Project Plan approved by USAID iii. Approved descriptions on 1/19/2017.

Final data from second Procurement of Materials, Subcontract Awards: ADARC, xii. RH5/Alum study delivered to Supplies, and Services University of Oxford USAID on Feb 12 2019. Draft anticipated in early Q3 iv. Final Individual Project Report Under development FY2019 v. Individual Project Data Set None during reporting period. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — ELEMENT 3: SCG ANNUAL MEETING SUPPORT Planning for the FY19 meeting x. SCG Annual Meeting Event Date: May 1 – 2, 2019 began in Q1 of FY19. Venue: College Park Marriott Hotel x.a-e. Logistic Support Travel: Leidos Travel Lodging: College Park Marriott Hotel ELEMENT 4: PROCURMENT OF MATERIALS, SUPPLIES AND SERVICES The repository is used to store xi. MVDP Reagents Repository SriSai Biopharmaceutical Solutions and distribute materials for conduct of the MVDP program. *Each Roman numeral crosswalks to a deliverable, as called out by the prime contract (AID-OAA-C-15-00071).

MVDP Quarterly Report 8 Leidos Proprietary

Table 2.3-1. Management Tools Tool Description Location Cloud-based solution for exchanging and SharePoint https://vector.leidos.com/sites/ITLSO/MVDP storage of documents Provide USAID OCONUS line to call 1-855-462-5367 Conference Phone Scientific Consultant Group members and 1778004 Lines CONUS line to communicate with Leidos 2013235

Table 2.4-1. Team Meetings

Meeting Date Topic January 9, 2019 Leidos/USAID Biweekly Teleconference January 23, 2019 Leidos/USAID Biweekly Teleconference January 28, 2019 Oxford/Leidos/USAID Joint Meeting - RH5.1 Clinical Study GIA Data Discussion February 6, 2019 Leidos/USAID Biweekly Teleconference February 19, 2019 Leidos/USAID Biweekly Teleconference March 6, 2019 Leidos/USAID Biweekly Teleconference March 20, 2019 Leidos/USAID Biweekly Teleconference

3. ELEMENT 1 ACTIVITIES

3.2. ABSTRACT SUBMISSIONS/POSTER PRESENTATIONS Leidos and its subcontractors have made several presentations at scientific meetings/conferences in FY2019. The list of abstracts/presentations for FY2019 can be found in Table 3.2-1. These presentations/posters are used to inform the community of the current activities as well as to identify new collaborators and/or projects.

MVDP Quarterly Report 9

Leidos Proprietary

3.3. NEW PROJECTS No new projects were proposed in Q2 FY2019.

4. ELEMENT 2 ACTIVITIES

4.1. ADJUVANT/DELIVERY PLATFORMS Adjuvant, immunostimulators, and delivery platforms identified through scouting efforts and slated for use in FY2019 are detailed in this section.

MVDP Quarterly Report 11 Leidos Proprietary

4.1.2 IMMUNOSTIMULATORS CpG in combination with Advax is in use for the RCR complex project.

MVDP Quarterly Report 12 Leidos Proprietary

4.2. CSP VACCINE DEVELOPMENT PROJECT: AMY NOE AND JAYNE CHRISTEN The CSP vaccine development project is a multi-year effort that will continue through FY2019. Previously, the WHcAg VLP platform formulated in DPX4 was downselected with completion of preliminary efficacy assessments using the VLP162 parent construct. Further, T cell epitope assessments of PfCSP were previously completed and these data, along with recently published data were used to develop a VLP construct optimization strategy that was initiated in FY2018. The work performed in Q1 FY2019 includes initiation of CSP class II T cell epitope immunogenicity assessments at VaxDesign, continuation of the construct optimization activities, and initiation of a repeat confirmatory efficacy study with the VLP162/DPX4 formulation. The work performed in Q2 FY2019 includes completion of CSP class II T cell epitope immunogenicity assessments at VaxDesign, continuation of the construct optimization activities, and completion of a repeat confirmatory efficacy study with the VLP162/DPX4 formulation.

MVDP Quarterly Report 13 Leidos Proprietary interest (i.e., ICS2, ICS5, ICS7, and ICS8). Although the ability of a sequence to bind HLA is one of the main factors influencing immunogenicity, it is not the only factor. Therefore, in order to assess immunogenicity of the different ICS to further aid in selection of ICS for incorporation into WHcAg VLP, in Q1 FY2019, VaxDesign initiated immunogenicity assessments of the ICS using PBMCs from HLA- matched donors. Preliminary data from VaxDesign suggest that a subset of the ICS appears to have elevated responsiveness across the HLA types. The full data set was received from VaxDesign at the end of Q2 FY2019 and was circulated to USAID on March 20, 2019. An IPT meeting to review the data will be scheduled for Q3 FY2019. These data will be used to downselect the specific ICS for inclusion in the optimized WHcAg VLP construct(s). Several ICS induced multifunctional responses across a broad range of HLA types; ICS5, ICS7, ICS8, and ICS9 were the most promiscuous and immunogenic of the set tested. Based on magnitude of response, ICS2 may also be of interest. A comprehensive analysis incorporating the EpiVax data will be performed in Q3 FY2019.

4.2.2 CONSTRUCT OPTIMIZATION Optimization of the VLP162 construct by VLP Biotech is based on a cloning plan developed and initiated in FY2018, and includes insertion of sequences from the conserved R1 region of PfCSP (which contains CSP cleavage site, KKLKQP), alternative sequences for the NANP repeat loop region, and incorporation of additional class II epitopes from the N- and C-terminal regions of PfCSP 3D7 as well as from the R2 region of heterologous CSP strains (selected based on results of the above described T cell epitope assessments. USAID approved expansion of the loop insert effort to include development of VLPs with five novel loop inserts (further described below). Procurement associated with expansion of the loop insert effort (further described below) began at the end of Q4 FY2018 and was completed in Q1 FY2019. The optimization activities completed in FY2018 include insertion of PfCSP 3D7 amino acids 58-78 into the VLP N-terminal insertion site and extension of the PfCSP 3D7 C-terminal insert to incorporate much of the predicted class II epitope regions in the C-terminus of PfCSP 3D7 (i.e., amino acids 313- 387). The parent VLPs resulting from these efforts are VLP407 and VLP411 (Table 4.2-1). Further, several attempts at extending the C-terminal insert to include different ICS were made at the end of FY2018 with limited success; therefore, Leidos enlisted the assistance of a protein modeler to provide feedback on the sequence configurations with increased stability and a higher probability of soluble expression. ICS sequence cloning efforts have been paused until input from the modeler is received. In Q2 FY2019, the modeler found that addition of ICS to the C-terminal insert is problematic for VLP assembly, as the hydrophobic sequences interfere with RNA binding at the protamine binding site (located at the C-terminus of the VLP backbone sequence, N-terminal of the CSP insert). As RNA binding is necessary for VLP assembly, the modeler is currently evaluating other sites for ICS insertion. In addition, the data from VaxDesign (as described above) will be included in the overall analysis of ICS to include in the construct(s). In Q1 FY2019, the strategy for construction of the loop insert clones was developed and DNA cloning initiated. A list of the planned clones is provided in Table 4.2-1; the NPDP and NVDP sequences are shown in bold to facilitate sequence comparison among the loop inserts. Of note is that VLP Biotech recommended a conservative approach to developing the loop insert clones whereby both VLP407 and VLP411 were used as the parent for different constructs. This approach was recommended by VLP Biotech due to concerns regarding the less than optimal expression levels for VLP411 as well as the possibility of lower immunogenicity directed to the loop insert when CSP B cell epitopes are placed in both the loop and N-terminal regions of the VLP. Specifications for the new VLPs, including the alternative loop inserts, are shown in Table 4.2-1. In Q2 FY2019, construction of these new VLPs (VLP430 – VLP436) was completed; all of the VLPs assembled and demonstrated soluble expression. USAID downselected VLP430 – VLP434 for progression to the passive transfer study (as described below). An IPT meeting was held on February 14, 2019, to discuss these data as well as plan for the

MVDP Quarterly Report 14 Leidos Proprietary passive transfer study. Production of sufficient quantities of these VLP (for rabbit immunizations) will be completed in early Q3 FY2019.

Table 4.2-1. VLP162 Optimization – Alternative Loop Inserts

VLP ID N-term Insert Loop Insert (position 78) C-term Insert Assembly/Expression VLP162 None NANPNVDP(NANP)3 318-377 Yes, soluble VLP407 None NANPNVDP(NANP)3 313-387 Yes, soluble VLP411 58-76 NANPNVDP(NANP)3 313-387 Yes, soluble VLP430 None KQPADGNPDPNANPN 313-387 Yes, soluble VLP431 None PADGNPDPNANPNVD 313-387 Yes, soluble VLP432 None NPDPNANPNVDPNAN 313-387 Yes, soluble NPDPNANPNVDPNANPNA VLP433 None 313-387 Yes, soluble NPNANP KLKQPADGNPDPNANPNV VLP434 None 313-387 Yes, soluble DPNANPNANPNANP VLP435 58-76 NPDPNANPNVDPNAN 313-387 Yes, soluble NPDPNANPNVDPNANPNA VLP436 58-76 313-387 Yes, soluble NPNANP

4.2.3 PASSIVE TRANSFER STUDIES In early March 2019, USAID approved the passive transfer design shown in Table 4.2-2 to determine if antisera raised by VLPs containing alternative loop inserts will provide protection upon passive transfer challenge. Sera generated from immunization with VLP162 will be used as a positive control (pos ctrl) and sera generated from immunization with VLP407 will serve as the baseline control (base ctrl). Pb/PfCSP (full-length, labeled) parasites will be used for challenge (via bite) and the readouts will be via survival liver load assessment (fluorescence) followed by sterile protection. For this study, rabbits will be used for sera generation to provide sufficient quantities for the study as well as additional sera in the event an additional passive transfer study is desired. Per USAID request, rabbit immune sera will be generated to VLP435 and VLP436; however, these sera will not be assessed in the approved passive transfer study. Sera generation will be performed at the beginning of Q3 FY2019 and the passive transfer study is slated to begin in May 2019.

MVDP Quarterly Report 15

Leidos Proprietary was seen in the 2-dose group and 10% protection was seen in the 3-dose group (cohorts 3 and 4, respectively, Table 4.2-3). No sterile protection was seen in the naïve or unadjuvanted VLP162 groups. Good titers to NANP were seen with the VLP162 + DPX4 and VLP162 + ISA720 formulations. Of note is that the 2-dose group performed incrementally better for three measures: sterile protection, ELISA titer, and average patency day (day 4.7 for the 2-dose group and day 4.4 for the 3-dose cohort). Based on this finding, a 2-dose regimen will be considered for the confirmatory efficacy assessments with the optimized constructs. A root cause investigation of the low sterile protection results will be initiated in Q3 FY2019.

Figure 4.2-1. Confirmatory Efficacy Study 2 Results – ELISA Titers and Protection

4.2.5 PRELIMINARY STABILITY ASSESSMENTS OF VLP162 + DPX4 In Q2 FY2018, ImmunoVaccine began work to conduct a 6-month stability study on the VLP162 + DPX4 formulation in order to (1) confirm that the formulation is stable while it is maintained for animals studies, (2) evaluate refrigerated storage (the material is currently stored frozen), and (3) provide preliminary data regarding stability of the WHcAg VLPs in the DepoVax platform. This study was completed in Q1 FY2019 and results for the 0, 1, 3, and 6-month time points are shown in Table 4.2-4 and Table 4.2-5. It was noted after the 0-month time point testing that the VLP162 content did not conform to the specification. Additional testing performed by IMV indicated precipitation occurring during the formulation and/or freeze-drying process. This same issue was found in a retain sample from a previous lot. As this material is representative of that used in efficacy assessments, the decision was made to continue with the stability program. Formulation optimization is possible; however, this will not be performed until the fully optimized VLP has been developed and assessed for precipitation with DPX4. In addition to the VLP162 content not complying with the target specification, it was also noted that viscosity increased over the 6-month period at both temperatures. All other results were within the established specification at the 6-month time point. ImmunoVaccine concluded that further formulation development is needed to improve stability of the VLP formulation.

MVDP Quarterly Report 17

Leidos Proprietary

4.3.3 PHASE I CLINICAL STUDY GIA DATA ANALYSIS Although Oxford received the GIA data from the GIA Reference Center in Q1 FY2019, they noted that inconsistencies in the data need to be assessed. The GIA data were received from Oxford in late January 2019 and an IPT meeting was held to discuss these data on January 28, 2019. Compared to the previous clinical study with the viral vectored RH5 (study not funded by USAID, data not shown), relatively high GIA levels were achieved with the RH5.1/AS01B formulation (Figure 4.3-1). These data influenced the decision to move forward with the project to develop and isolate human RH5 monoclonal antibodies (as described in the subject project section below).

Figure 4.3-1. RH5.1 Clinical Study Median GIA Results at 10 mg/mL (A) and 2.5 mg/mL (B) of Purified IgG

4.4. LIVER STAGE VACCINE DEVELOPMENT PROJECT: KEN TUCKER/TIM PHARES Table 4.4-1 provides an outline summarizing the liver stage project plan. As previously reported, the project progressed from an initial in silico analysis of 100 proteins to inform selection of five proteins for further analysis. Table 4.4-2 provides a summary of characteristics of the five proteins selected for analysis, and includes additional proteins tested in vaccines for comparison (i.e., CelTOS, CSP, and TRAP). Further in silico analysis of class I and class II epitopes in each protein and evaluation of the epitopes binding to the cognate HLA alleles resulted in the selection of just three proteins for evaluation of immune response to the epitopes using an in vitro model of the human immune system (i.e., PF08_0081, PF14_0593, and PFD0430c). The first stage of the in vitro biological testing (milestone 1.3 of the plan evaluating immunity to peptides in Table 4.4-1) was completed. Funding for the second stage of the in vitro biological testing (evaluating immunity to the protein) was limited to test one protein (PFD0430c). Currently the project is continuing with the in vitro biological testing of PFD0430c (SPECT2). Prior work with the epitope-based vaccine EP1300 did not provide interpretable data, so did not inform potential design of an epitope-based vaccine. Concurrent with the study using EP1300, USAID directed that the liver stage vaccine should not target an epitope-based vaccine but should target a whole protein as the antigen. Further work to perform delivery platform scouting studies based on whole protein or proteins domains is being negotiated with USAID.

MVDP Quarterly Report 20

Leidos Proprietary

Table 4.4-2. A Summary of Published Analysis of Five Proteins Selected for Development and Characterized Proteins

Table 4.4-2 provides a summary of characteristics of the five proteins selected for analysis (1) (Arevalo-Herrera et al., 2016) (2) (Crompton et al., 2010) (3) (Dent et al., 2015) (4) (Le Roch et al., 2003). Based on microarray using P. falciparum (5) (Le Roch et al., 2004). Based on microarray or mass spectral; analysis using P. falciparum. (6) (Lindner et al., 2013). Based on mass spectral results using P. falciparum, proteins were ranked for abundance from 1 to 1991, 1 being the most abundant. (7) PlasmoDB (http://plasmodb.org/plasmo/) (8) (Tarun et al., 2008). Based on mass spectral analysis using P. yoelii

MVDP Quarterly Report 22 Leidos Proprietary

4.4.1 LIVER STAGE PLAN MILESTONE I.3 Analysis of the immune response to PFD0430c protein began on August 27, 2018, with AmpTec starting the synthesis the mRNA. In mid-November 2018 AmpTec provided the mRNA encoding PFD0430c (SPECT2-mRNA) that VaxDesign is using to transfect cells to express the protein in situ. VaxDesign began developing the transfection of dendritic cells in late November 2018. Transfection of dendritic cells with the SPECT2-mRNA was initially problematic, resulting in extensive cell death. VaxDesign has observed this cytotoxic effect of mRNA on dendritic cells before. As potential solutions to this issue VaxDesign evaluated transfecting feeder cells (African green monkey kidney VERO cells and human endothelium EA.hy926 cells) to provide antigen to the dendritic cells, and to optimize transfection of the dendritic cells while reducing cell death. Transfection efficiency was monitored using PCR with primers for SPECT2 to detect intracellular mRNA. As an indication for protein expression by transfected cells, VaxDesign evaluated T cell stimulation. This was performed by initially priming T cells with the dendritic cells presenting the antigen from the feeder cells transfected with SPECT2-mRNA, or with dendritic cells directly transfected with the SPECT2-mRNA. The recall (i.e., restimulation) immune response of T cells was evaluated using the two class II epitopes derived from SPECT2 that were previously demonstrated to elicit immunity in the same donor when primed with the peptides. Demonstration of a recall response indicates SPECT2 is being translated from the mRNA and the protein is processed to provide the peptides at levels sufficient to prime the T cells. Feeder cells were efficiently transfected without significant cytotoxicity, providing a potential solution to the cytotoxicity seen in the transfected dendritic cells. By modifying the culture media and transfecting rested dendritic cells, dendritic cells were also efficiently transfected while reducing cytotoxicity. That is, when monocytes were treated with cytokines to derive the dendritic cells, the cells were allowed to rest overnight before transfection with 4 µg SPECT2-mRNA/million cells verses transfecting within two hours after deriving the dendritic cells. Collectively, these modifications to the dendritic cells’ transfection doubled the viability of the dendritic cells following transfection. With the improved viability, transfected dendritic cells were used to evaluate antigen presentation in the MIMIC model. To further optimize transfection with the intent to maximize expression of SPECT2, different amounts of mRNA were tested in dendritic cells. VaxDesign evaluated using 2, 10, or 50 µg of mRNA/ million cells, and found that using 50 µg SPECT2-mRNA increased transfection nearly 100-fold relative to using 2 µg. While increasing the amount of mRNA did increase cytotoxicity, the number of viable transfected dendritic cells are sufficient for carrying out antigen presentation to the T cells, and 50 µg of mRNA will be used to transfect the dendritic cells in the MIMIC model. VaxDesign is using T cell stimulation for the class II epitopes (Table 4.4-3, peptides CL2-19 and CL2-20) as an approach to demonstrate effective transfection resulting in expression of the protein. Antigen expression was not detected using either the transfected feeder cells or the transfected dendritic cells. Restimulation using SPECT2-mRNA transfected cells also did not result in T cell stimulation. However, the approach to demonstrate protein expression is indirect. It is not possible to conclude if the lack of T cell restimulation is due to a lack of protein expression or a lack of processing of the protein resulting in the presentation of the epitopes to the T cells. To enable a direct demonstration of protein expression Leidos began developing antibodies to peptides derived from SPECT2, with the first vaccinations starting the first week of March 2019. This antibody will be used to directly test for SPECT2 expression in the transfected cells. The peptides used to develop the antibody include CL2-19 and CL2-20, as well as two additional peptides that EpiVax predicted were class II epitopes in SPECT2 (i.e., ISDYTKSLSAEAKVSC, DKDIIKILTNADTVTKC). Antibody is planned to be available to VaxDesign by May1, 2019, but is dependent upon detecting high titers of peptide specific antibody as determined using enzyme linked immunosorbent assays with the peptides as the antigen.

MVDP Quarterly Report 23 Leidos Proprietary

Once protein expression is confirmed, analysis of the immune response will start with the class II epitopes and progress to the class I epitopes (Table 4.4-3). The analysis is planned for completion by the end of July 2019 (Figure 4.4-1).

Table 4.4-3. Peptides derived from PFD0430c

Peptide Location in Peptide Peptide Sequence PFD0430c Designation* 29 - 37 RLNNRSIYK CL1-64 72 - 81 KISSFINQRK CL1-65 237 - 245 HTSVFPGLY CL1-66 257 - 265 NPLGEADSL CL1-67 285 - 293 GIANDLSTL CL1-68 357 - 365 RSKKTFLVK CL1-70 371 - 379 YTIGLPPYI CL1-71 440 - 448 YESQLGGKI CL1-72 523 - 531 NLFEWSKTV CL1-73 534 - 542 HPMPINIKL CL1-74 550 - 558 DSDDLKESY CL1-75 570 - 579 GLSPHDTMQK CL1-76 386 - 394 AYKNAVNEL CL1-77 224 - 233 DENTLENRNI CL1-78 360 - 382 Ac-KTFLVKSNCVKYTIGL PPYIPWD-amide CL2-19 445 - 461 GGKITKIINVSTSSIEQ-amide CL2-20 *Peptides designated with CL1-X target class I, and peptides designated with CL2-X target class II

Activity 2017 2018 2019 Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Peptide Synthesis Class II Peptide Experiments Class II Peptide Analysis Class I Peptide Experiments Class I Peptide Analysis Protein Selection mRNA Synthesis Transfection Development Antibody Development Protein Expression Development mRNA /Epitope Analysis Figure 4.4-1. Schedule for Analysis by MIMIC of Epitopes from New Proteins

MVDP Quarterly Report 24 Leidos Proprietary

4.4.1.1 Platform Scouting Studies - CD8 T Cell Responses In FY2018 USAID requested an addendum for the Liver Stage Vaccine Project to perform CD8 T cell response platform scouting studies using a well-studied animal model of malaria, the H-2kd restricted class I epitope (SYVPSAEQI) of P. yoelii (Py) CSP that is known to be protective upon Py challenge. The initial addendum to the project plan was delivered January 26, 2018, as an attachment to the CSP project. Based on comments received from USAID on March 21, 2018, Leidos revised the addendum to only include assessment of CD8 platform scouting studies. An updated version of the addendum was submitted to USAID in Q2 FY2019. Concerns with an appropriate positive control for assessment of Pf proteins has delayed completion of the addendum and an updated draft will be submitted to USAID in April 2019. Initial proof-of-concept studies previously performed with this model found that SYVPSAEQI integrated into the MagaVax platform elicited CD8 T cell responses in mice. These data are detailed in the FY2018 USAID MVDP Annual Report. The decision to proceed to a challenge study with one or both MagaVax constructs was dependent on the elevation of T cells responses in the MagaVax compared to that of the positive control (AdPyCS). Based on USAID’s initial specified Acceptance Criterion where constructs will be selected for further study only if they perform considerably better than the positive control at a historically non-protective dose or equal to or better than the positive control that would be expected to be protective, the decision was made not to proceed with challenge studies at this time. The other two platforms that Leidos will evaluate are Synthetic Genomics Inc (SGI) self-replicating RNA replicon and GeoVax’s modified Vaccinia virus (MVA)-VLP. The experimental approach and design for these two platforms will be detailed in the updated addendum in Q3 FY2019. Kick-off meetings with GeoVax and SGI were held on March 7, 2019, and March 27, 2019, respectively.

4.4.1.2 Optimizing the Number of Mosquito Bites for Infection. Prior to starting the platform scouting analysis with the SGI RNA Replicon and GeoVax MVA-VLP, the number of mosquito bites required to ensure malaria infection in this model needed to be determined. This study informed the targeted infectious exposure that will be used for the subsequent studies at ADARC.

On December 10, 2018 of Q1 FY2019, USAID Table 4.4-4. Bite Titration Study approved the study design (Table 4.4-4). In brief, BALB/c (n = 7) mice were exposed to various Cohort # Test Sample numbers of mosquitos infected with Py 17XNL. 1 No bites Following exposure, individual mosquitos were 2 2 bites dissected to check for infectivity. Parasitemia in 3 4 bites the mice was assessed via blood smears. Results of this study were delivered to USAID on Feb 11 in 4 6 bites Q2 FY2019 and are shown in Table 4.4-5. Of 5 8 bites note, each mouse was anesthetized and placed on its abdomen for a 3-minute exposure to allow the mosquitos to bite. All mice that were exposed to either 6 or 8 mosquitos were infected. In addition, the overall percentage of mosquitos positive for sporozoites was ~75%. Based on the data Leidos and USAID agreed to move forward with using at least 6 bites/mouse to ensure that all mice are infected.

MVDP Quarterly Report 25 Leidos Proprietary

Table 4.4-5. Bite Titration Study Results

4.4.1.3 Titration of AdPyCS Immunization for Mosquito Bite Challenge. In addition to optimizing the number of mosquito bites for infection prior to evaluating the SGI RNA Replicon and GeoVax MVA-VLP platforms, ADARC will also titrate the AdPyCS immunization to determine the dose that gives 40-50% protection following mosquito bite challenge. On December 10, 2018 of Q1 FY2019, USAID approved the study design (Table 4.4-6). In brief, BALB/c (n = 7) mice were immunized IM with various doses of AdPyCS in the hind limb without adjuvant. At day 10 post- immunization mice were challenged via 6 mosquitos infected with Py 17XNL. Following bite challenge individual mosquitos were dissected to check for infectivity. Parasitemia in the mice was assessed via blood smears. Results of this study were delivered to USAID on March 24 in Q2 FY2019 and are shown in Table 4.4-7. The percentage of dissected mosquitos positive for sporozoites was ~88% overall and ADARC visibly confirmed (mosquitoes got swollen with blood) greater than 4 bites per mouse. No protection was seen with either 107, 108 or 109 AdPyCS. Only a single mouse was protected at 1010 (14%) while 3 out of 7 (43%) mice were protected with 1011 AdPyCS. Based on these study results ADARC suggest using 1011 AdPyCS as a positive control in the GeoVax and SGI proof-of- concept studies using PyCS.

MVDP Quarterly Report 26 Leidos Proprietary

Table 4.4-6. AdPyCS Dosing Study

Cohort # Test Sample Route # Mice, Strain Day 0 Day 10 1 Naïve none 7 BALB/c none 2 107 AdPyCS IM 7 BALB/c √ 3 108 AdPyCS IM 7 BALB/c √ 4 109 AdPyCS IM 7 BALB/c √ 5 1010 AdPyCS IM 7 BALB/c √ 6 bites/mouse 6 6 1011 AdPyCS IM 7 BALB/c √

Table 4.4-7. AdPyCS Titration Study Results

MVDP Quarterly Report 27 Leidos Proprietary

4.5. RCR COMPLEX VACCINE DEVELOPMENT PROJECT: CECILLE BROWNE (WEHI), JAYNE CHRISTEN (OXFORD), AND AMY NOE This project is based on evaluation of proteins in the RCR complex as vaccine candidates by comparison of the individual and combined proteins. Although high GIA activity was observed with the RH5.1 vaccine candidate in animal models and good titers were seen in humans immunized with RH5.1/AS01, no sterile protection was found upon CHMI. WEHI has previously provided data indicating that two additional proteins in the RH5 complex perform better in the GIA as compared to RH5 when the proteins are formulated with IFA (70% for RH5 compared to 80% and 90% for CyRPA and RIPR, respectively). However, it is unclear if the same GIA activity is possible with human-use adjuvants (thus Study W1 was initiated) and/or if the moderate increases in GIA seen with CyRPA and RIPR will translate to achievement of sterile protection in humans with those proteins administered alone. Further, Dr. Simon Draper (Oxford) indicated that GIA activity was similar among the individual RH5 complex proteins in their hands (data not received). Current information suggests that blood stage proteins can act synergistically to achieve higher GIA activity; therefore, a multi-antigen RH5 complex vaccine may be more efficacious than a single protein vaccine. Thus, the RCR Complex Project Plan will test the following hypotheses: (1) Targeting single and/or multiple proteins in the RCR complex can interrupt the mechanism of merozoite invasion of erythrocytes during the blood stage infection of P. falciparum and (2) Targeting multiple proteins in the complex may be more effective than targeting a single protein in the complex. To address these hypotheses, Leidos developed two parallel paths under the overall RCR Complex Project Plan as well as a workflow diagram to illustrate the interconnectedness of the projects conducted at Oxford and WEHI. Per the USAID-approved project plan, there are a total of three approaches for development of an RCR complex vaccine including recombinant protein, VLP, and RNA approaches. In addition, specific studies have been included in the workflow to facilitate down-selection (e.g., studies to identify a test protein dose and to compare different adjuvants). Based on immunogenicity assessments (e.g., ELISA and GIA activity), constructs/formulations will be downselected for comparison in a penultimate study. These interdependencies and decision points are shown in the RCR Complex Vaccine Project Workflow Figure 4.5-1, where decision points are shown as to be determined (TBD). The workflow diagram was updated to incorporate the decision by USAID on February 19, 2019, to proceed with a dose-ranging study using antigens formulated in DPX4 at WEHI. Based on the evolution of this project, USAID approval of the project as a whole was provided prior to approval of the project plans. The project plans for both the work at WEHI and Oxford were approved in Q2 FY2019.

MVDP Quarterly Report 28 Leidos Proprietary

Figure 4.5-1. RCR Complex Vaccine Project Workflow. Down-selection is based on analysis of sera samples via GIA (pLDH and flow cytometry methods) and Quantitative ELISA

On October 30, 2018, an IPT Meeting was held at ASTMH in New Orleans. This face-to-face meeting was attended by Simon Draper’s group (Oxford), Alan Cowman’s group (WEHI), Leidos and USAID. The following outcomes of the meeting are listed below. 1) Addition of dose-ranging study with Matrix-M to the project plan entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine- University of Oxford.” 2) Change in animal model from mice to rats. Studies will be conducted with rats (except Study W1, which had already begun) to obtain higher bleed volumes, enable estimates of variance due to individual animal differences , and avoid non-specific GIA issues. 3) Incorporation of an analytical plan. Leidos consulted with an in-house statistician for guidance in power analysis and sample size determination.

4.5.1 WEHI: CECILLE BROWNE/AMY NOE Leidos submitted a draft plan (RH5 Complex Vaccine Development Project Plan) to USAID in August 2018 that included Study W1 (evaluating different human-use adjuvants) and follow-on studies to be performed by WEHI. These follow-on studies include 1) immunogenicity of double and triple protein combinations, 2) immunogenicity of monocistronic and polycistronic RNA replicons encoding complex proteins, and 3) development of reagent antibodies that specifically recognize the RCR complex. In December 2018, Leidos submitted to USAID a revised project plan with a new title: RCR Complex Project Plan - WEHI. The revised project plan includes an optional dose titration study in rats to bridge Study W1 (conducted in mice) to follow-on studies described in the project plan. Prior to approval of the project plan, USAID approved a study design for Study 1 (later renamed to Study W1) to evaluate the immunogenicity (ELISA and GIA) of individual proteins in the RCR complex (RH5, RIPR, and CyRPA) in combination with human-use adjuvants in mice. In Q4 FY2018, Leidos completed procurement efforts with WEHI to conduct this study and managed the logistics to ship DPX-formulated proteins from IMV to WEHI. Study W1 was initiated in October 2018.

MVDP Quarterly Report 29 Leidos Proprietary

Table 4.5-1. Study W1. Immunogenicity Study in Mice of Individual RCR Proteins in Three Human use Adjuvants and IFA

Cohort # mice, Study Day Test Sample R # strain -2 0 28 42 1 RIPR (20 µg) + IFA IM 6, BALB/c √ √ 2 RH5.1 (20 µg) + IFA IM 6, BALB/c √ √ 3 CyRPA (20 µg) + IFA IM 6, BALB/c √ √ 4 RIPR (20 µg) + AddaVax IM 6, BALB/c √ √

5 RH5.1 (20 µg) + AddaVax IM 6, BALB/c √ √ 6 CyRPA (20 µg) + AddaVax IM 6, BALB/c √ √

7 RIPR (20 µg) + Advax+CpG IM 6, BALB/c - bleed √ √ 8 RH5.1 (20 µg) + Advax+CpG IM 6, BALB/c Pre √ √ Terminalbleed 9 CyRPA (20 µg) + Advax+CpG IM 6, BALB/c √ √ 10 RIPR (20 µg) + DPX4 IM 6, BALB/c √ √ 11 RH5.1 (20 µg) + DPX4 IM 6, BALB/c √ √ 12 CyRPA (20 µg) + DPX4 IM 6, BALB/c √ √

MVDP Quarterly Report 30 Leidos Proprietary

Figure 4.5-2. First ELISA - Preliminary immunogenicity results of individual RCR proteins in four adjuvants. Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

Table 4.5-2. Preliminary Estimates of Immunogenicity Titers (at O.D. = 1.0)

RH5.1 RIPR CyRPA DPX4 >51,200 >51,200 30,000 IFA 25,000 >51,200 >51,200 Advax+CpG 3,200 8,800 400 AddaVax 600 3,200 2,400

MVDP Quarterly Report 31 Leidos Proprietary

Figure 4.5-3. Second ELISA - Immunogenicity results of individual RCR proteins in four adjuvants (repeat ELISA). Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

Table 4.5-3. Immunogenicity Titers Derived from 4PL Curve Fit (at O.D. = 1.0); Second ELISA RH5 RIPR CyRPA DPX4 288,479 1,302,740 29,107 IFA 185,188 537,314 47,561 Advax+CpG 25,087 14,541 < 1,000 AddaVax 3,365 15,180 < 1,000

MVDP Quarterly Report 32 Leidos Proprietary

Table 4.5-4. Study W2 – Dose-Ranging Study in Rats

# rats, Study Day Cohort Formulation R strain -2 0 28 42 56 70 1 RH5.1 protein, 20 µg + DPX4 IM 6, Wistar √ √ √

2 RH5.1 protein, 2 µg + DPX4 IM 6, Wistar √ √ √

3 RH5.1 protein, 0.2 µg + DPX4 IM 6, Wistar √ √ √ 4 CyRPA protein, 2 µg + DPX4 IM 6, Wistar √ √ √ 5 CyRPA protein, 0.2 µg + DPX4 IM 6, Wistar - bleed √ √ √ Pre 6 RIPR protein, 2 µg + DPX4 IM 6, Wistar √ √ Testbleed √ TerminalBleed RIPR protein, 0.2 µg + DPX4 IM 6, Wistar √ √ √ 7 Adjuvant alone (Negative Control) IM 6, Wistar √ √ √

Table 4.5-5. Study W4 – Immunogenicity Study using RNA Replicons in Rats Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RIPR monocistronic mRNA (10 µg) IM 6, Wistar √ √ √ 2 RH5 monocistronic mRNA (10 µg) IM 6, Wistar √ √ √ 3 CyRPA monocistronic mRNA (10 µg) IM 6, Wistar √ √ √

RH5, CyRPA, RIPR monocistronic

4 mRNAs, admix (total of 10 µg; 3.3 µg IM 6, Wistar √ √ √ per monocistronic replicon)

RH5, CyRPA, RIPR monocistronic - bleed

5 mRNAs, admix (total of 30 µg; 10 µg IM 6, Wistar Pre √ √ √ Testbleed

per monocistronic replicon) TerminalBleed RH5, CyRPA, and RIPR polycistronic 6 IM 6, Wistar √ √ √ mRNA (10 µg) Lipid formulation control 7 IM 6, Wistar √ √ √ (Negative control)

4.5.1.1 Updated Schedule: RCR Complex Project WEHI An updated timeline for RCR Project Plan WEHI is shown in Table 4.5-6. Note that this project is expected to extend beyond the MVDP POP, and decisions will need to be made regarding mechanism for continuing the project after May 2020.

MVDP Quarterly Report 33 Leidos Proprietary

Table 4.5-6. Updated Timeline for RCR Project Plan WEHI

4.5.3 PRECISION ANTIBODY The procurement process was completed with Precision Antibody in Q2. Precision Antibody will develop an antibody reagent that specifically recognize the RCR complex. The antibody reagent would be used to detect complex formation in cultures transfected with RNA replicons. In the first phase of this project, Precision Antibody will evaluate RCR stability in the adjuvant used in monoclonal antibody development. The goal of the first phase is to ensure that the immunogen is in a complex formation when administered to mice. RCR stability evaluation, which will begin in Q3 (April 2019), will be performed using immunoprecipitation followed by Western Blotting and/or Size exclusion chromatography. If the RCR complex is stable in adjuvant, Precision Antibody will use the RCR as immunogen to generate monoclonal antibodies using Precision’s established workflow. If the RCR complex is not stable in the first adjuvant, another adjuvant will be tested. If the RCR complex is not stable in the second adjuvant, a decision will be made whether or not to continue with monoclonal antibody development.

4.5.4 UNIVERSITY OF OXFORD (OXFORD): JAYNE CHRISTEN/AMY NOE On December 17, 2018 of Q1 FY2019, Leidos submitted a draft project plan, entitled “Development of a Plasmodium falciparum RCR Protein Complex Vaccine-University of Oxford,” to USAID. Presented in

MVDP Quarterly Report 34 Leidos Proprietary the plan were the study designs for four in-life studies (Studies O1-O4) as well as the description of two reagent/assay development activities (i.e., quantitative ELISA and VLPs decorated with RCR recombinant proteins). USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Leidos circulated the updated project plan to USAID on February 5, 2019; USAID approved the project plan on February 13, 2019. A kick-off meeting was held on March 4, 2019. An overview of the development plan for this project is provided in Table 4.5-7.

Table 4.5-7. Overview of the RCR Complex Vaccine Development Project Plan-Oxford

As noted in the appropriate sections below, USAID has approved both the VLP development and the Study O1 design (Table 4.5-8). The draft design of Study O2 (Table 4.5-9, recombinant proteins) has also been approved; however, determination of protein concentration is pending results of Study O1. The final study design will be provided to USAID with the data from Study O1 for final approval. The Study O3 (VLPs) design is pending approval. Additionally, the Study O4 (all relevant immunogens/formulations) design is pending adjuvant, antigen, and construct approval. Leidos initiated procurement activities in November 2018 of Q1 FY2019 and completed activities in February 2019 of Q2 FY2019.

4.5.4.1 Dose-Ranging Study (Study O1) On November 30, 2018 of Q1 FY2019, USAID approved the design of Study O1. In Study O1, the immunogenicity of RH5, CyRPA and RIPR proteins as well as the ability of antibodies induced to elicit parasite growth inhibition will be evaluated individually in rats (Table 4.5-8). Leidos expects initiation of Study O1 during Q3 FY2019.

MVDP Quarterly Report 35 Leidos Proprietary

Table 4.5-8. Study O1 – Dose-Ranging Study Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 (2 µg) + Matrix-M IM 6, Wistar √ √ √

2 RH5.1 (200 ng) + Matrix-M IM 6, Wistar √ √ √

3 RH5.1 (20 ng) + Matrix-M IM 6, Wistar √ √ √ 4 CyRPA (2 µg) + Matrix-M IM 6, Wistar √ √ √ - bleed

5 RIPR (2 µg) + Matrix-M IM 6, Wistar Pre √ √ √ Testbleed

6 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √ TerminalBleed 7 Negative Control IM 3, Wistar ** **Terminal Bleed

Table 4.5-9. Study O2 – Individual Proteins and Double/Triple Protein Mixtures Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 2 CyRPA + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 3 RIPR + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ 4 RH5.1 + RIPR + Matrix-M (Dose TBD) IM 6, Wistar √ √ √ RH5.1 + CyRPA + Matrix-M 5 IM 6, Wistar √ √ √

(Dose TBD)

RIPR + CyRPA + Matrix-M (Dose 6 IM 6, Wistar √ √ √ TBD) bleed

RH5.1 + CyRPA + RIPR + Matrix-M - bleed

7 (Dose TBD) IM 6, Wistar Pre √ √ √ Test

Equivalent Molar Ratio TerminalBleed RH5.1 + CyRPA + RIPR + Matrix-M 8 (Dose TBD) IM 6, Wistar √ √ √ Equivalent Protein Concentration Reconstituted RCR + Matrix-M 9 IM 6, Wistar √ √ √ (Dose TBD) 10 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √

MVDP Quarterly Report 36 Leidos Proprietary

4.5.4.4 VLP Development USAID approved the development of HBsAg-SpyCatcher VLPs decorated with single antigens or the RCR complex on October 4, 2018 of Q1 FY2019. Activities will be expanded on during the course of project reporting.

4.5.4.7 Updated Schedule: RCR Complex Project Oxford The initial project schedule provided by Oxford (included in the Project Plan) had a Study O1 start date in February 2019 and completion date in May 2020. Approval of the Project Plan was received from USAID on February 13, 2019. Of note is that due to the number of rats involved in the planned (USAID-approved) studies for this project, it is necessary for Oxford to subcontract the in life portions. Oxford finalized their subcontract in March 2019. Further, study approval by Novavax is needed prior to provision of Matrix-M. Based on this updated timeframe, Leidos requested a revised schedule from Oxford, shown in Figure 4.6-1. Note that the updated project schedule has a Study O1 start date in April 2019 and a completion date in August 2020, extending beyond the MVDP POP end date. Leidos recommends holding an IPT meeting in Q1 FY2020 to determine the project stopping point.

Table 4.5-10. Project Timeline Received from Oxford

MVDP Quarterly Report 37 Leidos Proprietary

2018. Leidos updated the plan based on feedback from USAID and submitted a final plan on December 19, 2018. USAID approved elements in the plan relating to mAb development, screening and characterization to be performed in the Draper lab on January 29, 2019. A decision on the R5.016 immunogen work was deferred. A kick-off meeting was held on March 4, 2019 where Oxford presented a slide deck titled “High-throughput cloning of RH5-specific mAbs” that contained the VAC063 data as well as their initial list of suggested VAC063 samples to use in this project. This presentation was circulated to USAID and an IPT meeting was held on March 18, 2019 to discuss the VAC063 data and to finalize sample selections. Additional sample selection updates were received from Oxford and circulated to USAID on March 22, 2019. Initiation of the technical work is pending USAID approval of sample selection, anticipated in early Q3 FY2019. An overview of the development plan for this project is provided in Table 4.6-1 followed by a brief summary of the proposed project.

Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project Phases Milestones Activities • Single B cell isolation (memory B cells (mBC) or plasmablasts) B Cell Isolation and • Reverse transcription and paired heavy and light chain nested PCR Cloning • DNA purification and cloning into heavy and light chain vectors B Cell Isolation and • Transfection of heavy and light chain vectors into HEK293 cells mAb Expression and mAb Development • Harvest of supernatant and screening by ELISA Purification • Affinity purification of antibodies from supernatant • Sequencing of heavy and light chain variable regions mAb Sequencing • Determination of sequence maturation and germline alleles ELISA Titer and • Determination of ELISA titers Protein Mapping • Protein region mapping using a panel of RH5 variants GIA • Growth Inhibition Assay (using pLDH method) mAb Screening Epitope Similarity • Determination of similarity of B cell epitopes recognized by Screen antibodies using competition assay and SPR/BLI mAb Downselection • Monoclonal antibody down-selection mAb Affinity • Determination of antibody affinity by SPR mAb mAb Inhibition of • Assessment of inhibitory activity of antibodies on RCR, RH5-P113 Characterization Complex Formation and RH5-Basigin complexes mAb Downselection • Selection of monoclonal antibodies for structural studies Computational • Identification of sequence variants of the immunogen that are assessments stable and have the right conformation R5.016 Immunogen Production of select • Epitope grafting and production of selected immunogens Design (optional) immunogen designs Immunogen • SPR screening for binding to R5.016 mAb downselection Production of • Production and QC of downselected immunogen R5.016 Immunogen immunogen Production Coupling of (optional) • Conjugation of downselected immunogens to VLPs immunogen to VLP Formulation of • Selection and procurement of adjuvant R5.016 Immunogen immunogen:VLP • Formulation of immunogen Testing (optional) • Rat immunizations with RH5.1 comparator Rat immunization • Collection of sera

MVDP Quarterly Report 38 Leidos Proprietary

Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project Phases Milestones Activities Immunogenicity and • Humoral response and GIA activity with R5.016 mAb comparator GIA assessments • Recombinant antibody expression and purification Antibody production • Fab fragment generation New mAb • Screening for Fab:RH5 co-crystals Structure Crystal screening • Cryoprotection of crystals Determination • X-ray diffraction studies (optional) Crystal structure • Model building and refinement determination • Epitope delineation

Figure 4.6-1. Project Timeline Received from Oxford In addition to the work proposed by Oxford, an immune repertoire survey of RH5.1 vaccinees was also proposed (summarized in the section below). The final decision on this work is pending analysis of the clinical study GIA data.

MVDP Quarterly Report 39 Leidos Proprietary proposed annual work plan, USAID indicated that a decision regarding funding for this project will not be made until after receipt and review of the GIA data from the RH5.1/AS01 clinical study. A brief summary of the proposed project is provided below. This project aims to use novel sequencing technologies (Boutz et al., 2014; DeKosky et al., 2016; Lavinder et al., 2015; Lavinder et al., 2014; Saphire et al., 2018; Williams et al., 2017) to identify the B cell and IgG immune repertoires for select RH5.1 vaccinees from the VAC063 clinical study and would involve collaboration between the Draper lab at Oxford and Drs. Georgiou and Ippolito at the University of Texas Austin. The humoral response to vaccination involves the generation of short-lived plasmablasts that peak approximately 8 days after immunization and the generation of broadly diverse memory B cells and long-lived plasma cells that secrete high-affinity mAbs in the germinal centers of the lymph nodes. The three iterative germinal center processes of somatic hypermutation of germline variable region DNA sequences, clonal expansion and selection of high affinity B cell receptor variants are involved in the evolution of the humoral response to vaccination. The data generated through this project will help us understand the evolution of the humoral response in vaccinees in a cohort over time as well as before and after primary and/or secondary CHMI. In addition, the evolution of the humoral response can be compared between dose cohorts and between different immunization regimens. Through this approach, we may for example, identify clusters of antibody or BCR sequences that encode highly functional mAbs but are present in very low frequency in the vaccinees. Such information together with structural information on how these mAbs recognize the cryptic epitopes on RH5.1 could mean that the RH5.1 immunogen has the potential to be engineered in the future, to elicit greater expansion of these highly functional mAbs and thereby better protective efficacy. In discussions with USAID, they indicated that the decision to fund this project would not be made until after review of the VAC063 GIA data. There are no Q1 FY2019 updates to report for this section. Preliminary sample selections for this project also were discussed in the IPT meeting on March 18, 2019. In further discussions, USAID tasked Leidos with following up with Drs. Georgiou and Ippolito regarding the sample selection and the use of pre-immune samples in their work.

4.7. BLOOD STAGE EPITOPE-BASED VACCINE DEVELOPMENT PROJECT: VIN KOTRAIAH The blood stage epitope-based vaccine development project focused on identifying highly inhibitory B cell epitopes from proteins in the RCR complex (RH5, CyRPA, and RIPR) as well as in EBA175 and AMA1. Antibodies that inhibit parasite invasion of erythrocytes in a growth inhibition assay (GIA) have been identified for a number of blood-stage vaccine candidates of high interest. Leidos developed a plan to utilize these inhibitory antibodies to identify the matching mimotopes from which an epitope-based vaccine could be derived. The mimotope vaccine concept works by providing the epitopes to stimulate development of the associated inhibitory antibodies. An overview of the development plan for this project is provided in Table 4.7-1. In Q2 FY2018, USAID decided to pause work on this project following completion of preliminary assessments of VLPs in mice for immunogenicity and GIA (Milestone 1.4) and VLP Prototyping (2.1). The GIA testing was completed in June 2018. The remaining activities have consisted of testing the re- purified anti-RIPR mAb (3H7) in the GIA assay (completed in Q4 FY2018) and completing the HLA binding assays on the EpiVax predicted T cell epitopes in the blood stage antigens. The final reports on the HLA binding assays were received in Q1 FY2019. All technical work on this project was completed in Q1 FY2019. A draft project report was provided to USAID on Jan 31, 2019, and feedback was provided by USAID on March 14, 2019. Leidos provided an updated version of the project report to USAID on March 29, 2019.

MVDP Quarterly Report 40

Leidos Proprietary

Table 4.8-1. Overview of the PD1 Blockade Adjuvant Project Plan

Phase Milestone Task PD1 Blockade 1. Assess the effect of PD1 blockers on Immunogenicity and efficacy testing with Studies vaccine efficacy liver and blood stage antigen Adjuvant 2. Assess PD1 expression and T generation reg Determine whether PD1 blockers modify Formulation following immunization with or without PD1 PD1 expression and/or T Studies blockers regs

Table 4.8-2. Cohorts for AdPyCS + PD1 Blockade Immunogenicity Study Cohort # Test Sample 1 AdPyCS alone 2 AdPyCS + PD1 Peptide Combo 3 AdPyCS + Ova Peptide 4 AdPyCS + LD01 Peptide 5 AdPyCS + LD02 Peptide 6 AdPyCS + anti-PD1 mAb 7 AdPyCS + anti-PDL1 mAb

MVDP Quarterly Report 42 Leidos Proprietary

4.8.1.3 Assess the Effect of PD1 Peptide Table 4.8-3. AdPyCS Dosing Study Blockers on Protection with AdPyCS With completion of 4.8.1.1 and 4.8.1.2 Cohort # Test Sample experiments, a study to evaluate whether protective efficacy is enhanced following PD1 1 Naïve blockade was commenced and completed in June 2 107 AdPyCS 2017. BALB/c mice (n = 9-10) were immunized 9 3 108 AdPyCS IM with 10 virus particles of AdPyCS in the hind 9 limb without adjuvant. At days 1, 3, 5 and 7 post- 4 10 AdPyCS immunization mice were treated IP with 200µg of 5 1010 AdPyCS peptide(s) or mAbs shown in Table 4.8-2. In 6 1011 AdPyCS addition to the testing peptides, OVA peptide was also included as a control peptide. At day 12 post-immunization mice were challenged with ~50-75 Py 17XNL sporozoites IV. Parasitemia was assessed via blood smears beginning at day three. Results of the two challenge studies are detailed in the FY2018 USAID MVDP Annual Report.

MVDP Quarterly Report 43 Leidos Proprietary

Table 4.8-4. Groups for RH5.1/Alum + PD1 Blockade Immunogenicity Study

No Groups Test Sample 0 1 3 6 8 27 28 29 31 34 36 55 56 57 59 62 63 mice RH5.1 protein +  1 4      alum  RH5.1 protein +  2 alum + PD1 4           peptide Combo RH5.1 protein +  3 alum + PD1 4                  peptide Combo RH5.1 protein +  4 alum + LD01 4           peptide RH5.1 protein +  5 alum + LD01 4                  peptide RH5.1 protein +  6 alum + LD02 4           peptide RH5.1 protein +  7 alum + LD02 4                  peptide RH5.1 protein +  8 alum + anti-PD1 4           mAb RH5.1 protein +  9 alum + anti-PD1 4                  mAb RH5.1 protein +  10 alum + PD1 4              peptide Combo RH5.1 protein +  11 alum + LD01 4              peptide

 IM injection of RH5.1/Alum  Bleed for GIA/ELISA assay  IP injection of PD1 blocking peptides and mAb  Assess T responses via ELISPOT

Table 4.8-5. PfRH5 Specific Peptides Used to Stimulate T Cells

Epitope Sequence of Peptide Position in PfRH5 A7 TYDKVKSKCNDIKNDLIATI T216-I235 C6 NIANSIDILQEKEGHLDFVI N156-I175 C9 NLNKKMGSYIYIDTIKFIHK N396-K415 D9 YIDTIKFIHKEMKHIFNRIE Y406-E425 F6 KHLSYNSIYHKSSTYGKCIA K186-A205 F10 NDVPIKMEYFQTYKKNKPLT N506-T525 G10 DVPIKMEYFQTYKKNKPLTQ D507-Q526

MVDP Quarterly Report 44

Leidos Proprietary

Table 4.8-6. Groups for RH5.1/Alum + PD1 Blockade Immunogenicity Confirmatory Study

Figure 4.8-2. GIA Activity of Mouse Serum. Pooled serum from Group 14 and Group 15 at day 63 post- immunization was tested in GIA with cultured P. falciparum parasites using the LDH method. Dashed line indicates 50% inhibition.

MVDP Quarterly Report 46 Leidos Proprietary

5. Element 3 Activities: Amy Noe/Jessica Smith

5.2. 2019 SCG MEETING PLANNING The USAID Scientific Consultants Group (SCG) provides advice on specific issues and areas related to the USAID MVDP direction and strategy. The SCG is charged with ensuring the application of the highest standards of technical and scientific excellence, assisting in the development of overall program strategy and goals, providing strategic advice and recommendations, and identifying new opportunities for innovative and impactful research. The SCG meeting will be held May 1-2, 2019. Planning for the 2019 meeting began in mid-December 2018 with the RFP process to the NOVA/MD area. The College Park Marriott Hotel in Hyattsville, MD has been selected for the May 2019 meeting. Leidos prepared a registration site using CVENT and invitations were sent out mid-March. At the end of Q2, three invitees declined and 27 accepted. A registration report was provided to USAID on April1.

5.2.1 SCG MEETING READ AHEAD MATERIALS At the end of Q2 FY2019, Leidos provide 2019 SCG read-ahead materials to USAID for the following projects:

• CSP Vaccine Development Project

• Liver Stage Vaccine Development Project

• Blood Stage Epitope Vaccine Development Project

• PD1 Modulation Project Input from Oxford on read ahead materials for the three remaining projects is anticipated in early Q3 FY2019. These will then be provided to USAID.

6. ELEMENT 4 ACTIVITIES

6.1. MVDP REAGENTS REPOSITORY: JESSICA SMITH SriSai Biopharmaceutical Solutions (SBS) maintained, received, and distributed the reagents/materials needed for the ongoing and future studies during Q2 FY2019. SBS furnishes all the necessary services, management, qualified personnel, materials, equipment, facilities, and travel required for a biologics repository and inventory management services related to cGMP and non-GMP vaccines and associated products. SBS offers Controlled Room Temperature Storage (15 to 30°C), Controlled Room Temperature with humidity control, Refrigerated Storage (2 to 8°C), Freezer Storage (-20 ±10°C), Ultra-low Freezer Storage (-80 ±10°C), Low Freezer Storage (-30±10°C) and Vapor Phase Liquid Nitrogen (≤ -140°C), as well as the option to set units at a customer-defined temperature. The current inventory consists of standard storage temperatures ranging from -80°C to 4°C. Monthly inventories and reports were received from SBS in January, February, and March.

MVDP Quarterly Report 47 Leidos Proprietary

7. LEIDOS POINTS OF CONTACT

7.1. PROGRAM MANAGER Amy Noe, Ph.D., MBA Leidos Life Sciences 5202 Presidents Court Suite 110 Frederick, MD 21703-8398 Phone: 858-826-6105 Mobile: 858-201-9176

7.2. ACTING OPERATIONS MANAGER Jessica L. Smith Leidos Life Sciences 5202 Presidents Court Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0475 7.3. CONTRACTS MANAGER Casey Koontz Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0435 Fax: 301-846-0794

8. APPENDIX 1: LITERATURE CITED

MVDP Quarterly Report 48 Leidos Proprietary

Brazzoli, M., Magini, D., Bonci, A., Buccato, S., Giovani, C., Kratzer, R., Zurli, V., Mangiavacchi, S., Casini, D., Brito, L.M., De Gregorio, E., Mason, P.W., Ulmer, J.B., Geall, A.J., Bertholet, S., 2016. Induction of Broad-Based Immunity and Protective Efficacy by Self-amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin. J Virol 90, 332-344. Brune, K.D., Howarth, M., 2018. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front Immunol 9, 1432. Brune, K.D., Leneghan, D.B., Brian, I.J., Ishizuka, A.S., Bachmann, M.F., Draper, S.J., Biswas, S., Howarth, M., 2016. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234. Crompton, P.D., Kayala, M.A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G.E., Molina, D.M., Burk, C.R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D.L., Liang, X., Doumbo, O.K., Miller, L.H., Doolan, D.L., Baldi, P., Felgner, P.L., Pierce, S.K., 2010. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A 107, 6958-6963. DeKosky, B.J., Lungu, O.I., Park, D., Johnson, E.L., Charab, W., Chrysostomou, C., Kuroda, D., Ellington, A.D., Ippolito, G.C., Gray, J.J., Georgiou, G., 2016. Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires. Proc Natl Acad Sci U S A 113, E2636-2645. Dent, A.E., Nakajima, R., Liang, L., Baum, E., Moormann, A.M., Sumba, P.O., Vulule, J., Babineau, D., Randall, A., Davies, D.H., Felgner, P.L., Kazura, J.W., 2015. Plasmodium falciparum Protein Microarray Antibody Profiles Correlate With Protection From Symptomatic Malaria in Kenya. J Infect Dis 212, 1429-1438. Gilbert, S.C., 2013. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 31, 4241-4246. Goepfert, P.A., Elizaga, M.L., Seaton, K., Tomaras, G.D., Montefiori, D.C., Sato, A., Hural, J., DeRosa, S.C., Kalams, S.A., McElrath, M.J., Keefer, M.C., Baden, L.R., Lama, J.R., Sanchez, J., Mulligan, M.J., Buchbinder, S.P., Hammer, S.M., Koblin, B.A., Pensiero, M., Butler, C., Moss, B., Robinson, H.L., Group, H.S., National Institutes of, A., Infectious Diseases, H.I.V.V.T.N., 2014. Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis 210, 99-110. Hekele, A., Bertholet, S., Archer, J., Gibson, D.G., Palladino, G., Brito, L.A., Otten, G.R., Brazzoli, M., Buccato, S., Bonci, A., Casini, D., Maione, D., Qi, Z.Q., Gill, J.E., Caiazza, N.C., Urano, J., Hubby, B., Gao, G.F., Shu, Y., De Gregorio, E., Mandl, C.W., Mason, P.W., Settembre, E.C., Ulmer, J.B., Craig Venter, J., Dormitzer, P.R., Rappuoli, R., Geall, A.J., 2013. Rapidly produced SAM((R)) vaccine against H7N9 influenza is immunogenic in mice. Emerg Microbes Infect 2, e52. Lavinder, J.J., Horton, A.P., Georgiou, G., Ippolito, G.C., 2015. Next-generation sequencing and protein mass spectrometry for the comprehensive analysis of human cellular and serum antibody repertoires. Curr Opin Chem Biol 24, 112-120. Lavinder, J.J., Wine, Y., Giesecke, C., Ippolito, G.C., Horton, A.P., Lungu, O.I., Hoi, K.H., DeKosky, B.J., Murrin, E.M., Wirth, M.M., Ellington, A.D., Dorner, T., Marcotte, E.M., Boutz, D.R., Georgiou, G., 2014. Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc Natl Acad Sci U S A 111, 2259-2264. Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., Yan, S.F., Williamson, K.C., Holder, A.A., Carucci, D.J., Yates, J.R., 3rd, Winzeler, E.A., 2004. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res 14, 2308-2318. Le Roch, K.G., Zhou, Y., Blair, P.L., Grainger, M., Moch, J.K., Haynes, J.D., De La Vega, P., Holder, A.A., Batalov, S., Carucci, D.J., Winzeler, E.A., 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-1508. Lindner, S.E., Swearingen, K.E., Harupa, A., Vaughan, A.M., Sinnis, P., Moritz, R.L., Kappe, S.H., 2013. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol Cell Proteomics 12, 1127-1143. Pascolo, S., 2008. Vaccination with messenger RNA (mRNA). Handb Exp Pharmacol, 221-235.

MVDP Quarterly Report 49 Leidos Proprietary

MVDP Quarterly Report 50

Leidos Proprietary

1. EXECUTIVE SUMMARY

2. CONTRACTS MANAGEMENT AND ADMINISTRATION

MVDP Quarterly Report 2 Leidos Proprietary

MVDP Quarterly Report 3 Leidos Proprietary

Table 2.1-2. Annual Cost Summary Estimated Costs* Actual Costs FY2019 Adjusted # Project Name FY2019 Annual Q3 FY2019 FY2019 Costs Annual WP WP Costs Costs To Date** Costs 1 CSP Vaccine $556,100 $661,038 $192,586 $425,439 RH5.1 Clinical 2 $745,385 $845,384 $167,910 $265,835 Study 3 RCR Complex $1,131,500 $684,791 $274,615 $274,615 4 Liver Stage Vaccine $1,111,530 $1,295,200 $40,555 $302,684 Blood Stage 5 $0 $0 $12,725 $122,113 Epitope 6 Repository $90,000 $90,000 $17,128 $38,490 7 PD-1 Block Inh $33,000 $33,000 $0 $48,521 8 RH5.1 Human mAb $803,000 $883,991 $0 $0 TOTAL $4,470,515 $4,493,404 $705,518 $1,477,697

*Adjusted costs will be updated each quarter to show actual budgets. Initial costs are a rough order of magnitude and provide an estimate for each project. True cost are actualized on the procurement process begins. **Includes trailing FY18 expenses invoiced in FY19 Q1

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product CONTRACTS AND ADMINISTRATION Signed: 5/28/15 Modifications: MOD 1 2/22/16 MOD 2 3/4/16 MOD 3 8/16/16 Awaiting final decision on Operations MOD 4 2/13/17 Manager (Key Personnel) Prime Contract MOD 5 5/1/17 appointment. Documentation MOD 6 7/18/17 submitted May 15. MOD 7 9/28/17 MOD 8 1/09/18 MOD 9 12/20/18 MOD 10 3/1/19 MOD 11 (Pending)

MVDP Quarterly Report 4 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product Contractor – Employee Signed Non-Disclosure/COI i. Non- Disclosure/Conflict of Delivered Agreements are located on Interest (COI) Agreements SharePoint. Delivered: 08/31/2018 ii. Annual Work Plan Upcoming Plan: 09/01/2019 Approved: 11/08/2018 Q1 FY19 Delivered: 01/14/19 vi. Quarterly Reports Q2 FY19 Delivered: 04/15/19 Upcoming Reports: 01/14/2020 Q3 FY19 Delivered: 07/15/19 Q1 FY19 Delivered: 01/14/19 Quarterly Financial vii. Q2 FY19 Delivered: 04/15/19 Upcoming Reports: 01/14/2020 Reports Q3 FY19 Delivered: 07/15/19 Delivered: 10/15/18 viii. Annual Report/Q4 Report Upcoming Reports: 10/15/19 Approved: 12/03/2018 Delivered: The Small Business Small Business H.10.a Subcontracting Plan was included Subcontracts Subcontracting Plan in Leidos’ proposal. Due: Annually in October; Individual Subcontract H.10.b.1 submitted via eSRS by Leidos’ Upcoming Reports: 10/30/18 Reports Small Business Liaison office. Due: Annually in October; Summary Subcontract Upcoming Report: 10/30/19 H.10.b.2 submitted via eSRS by Leidos’ Report Small Business Liaison office. Final Overall Contract ix. N/A Upcoming Report: 5/27/20 Report Upcoming Meetings: Regularly Held: See Table 2.4-1. Biweekly USAID–Leidos scheduled biweekly meetings are held C.4.4 Ad hoc meetings held: See Update Meetings the first and third Wednesdays of each Table 2.4-1. month. Contract Administration Meetings with Contracting No actions in the reporting G.4.C Officer’s Representative period. (COR) Upon approval of all future Due: All contracts and deliverables, Leidos will upload to the USAID Development administration deliverables will be xiv. DEC. At contract completion, Leidos Experience Information uploaded to the DEC after 30 will upload all task-associated days from approval. documents as well. ELEMENT 1: PROTOCOL DEVELOPMENT/WHITE PAPERS See section 3.2 regarding xii. Publications/Posters Q1 FY2019: 4 posters abstract/poster submissions. C.3.2.2 New Project Proposals None in the reporting period

MVDP Quarterly Report 5 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product ELEMENT 2: IMPLEMENTATION OF RESEARCH AND DEVELOPMENT PROJECTS CSP Vaccine Development Project Project Plan approved by USAID on Final Individual Project 1/29/16. iii. Approved descriptions Revised Project Plan approved 04/26/2017. Subcontract/Task Order Q1 FY2019: procurement activities Awards: VLP Biotech, JHU, completed for IMV, JHU, VaxDesign, Subcontracts: ADARC, EpiVax, Precision and VLP Biotech. xii. Procurement of Materials, Antibody, ImmunoVaccine, Q2 FY2019: procurement completed Supplies, and Services VaxDesign for VaxDesign and ongoing for JHU. Other Procurement Vehicles: Q3 FY2019: procurement completed CPC Scientific, Vaxine for JHU (passive transfer study). Final Individual Project iv. — Report VLP Biotech Quarterly Reports VLP162 + DPX4 Stability Study Provided herein, circulated via email v. Individual Project Data Sets Documentation (ImmunoVaccine) and/or uploaded to SharePoint. VaxDesign Data Slides CSP Project Update Slides See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RH5.1/AS01 Vaccine Clinical Study Final Individual Project Plan approved by USAID on iii. Approved Project descriptions 2/17/16. Q1 FY2019: procurement for recently approved activities is ongoing for Subcontract/Task Order Procurement of Materials, Oxford. xii. Awards: University of Oxford, Supplies, and Services Q2 FY2019: Oxford procurement EpiVax completed. Q3 FY2019: none Final Individual Project iv. — Report Schedule and regulatory approvals for late immunogenicity time Provided herein, circulated via email, v. Individual Project Data Set point and/or uploaded to SharePoint. Oxford quarterly reports Oxford data updates See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — Trial registered on September 5, 2016 xii. Registration of Trials Completed (ClinicalTrials.gov)

MVDP Quarterly Report 6 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product Liver Stage Vaccine Development Project Project Plan approved by USAID on Final Individual 6/13/16. Attachment 1 - CD8 iii. Approved Project descriptions platform scouting plan approved by USAID on 6/6/19. Q2 FY2019: procurement completed Subcontract Awards: EpiVax, for SGI (kickoff held 3/27/2019) and Procurement of Materials, VaxDesign, Multimeric xii. GeoVax (kickoff held 3/7/19). Supplies, and Services BioTherapeutics, ADARC, SGI, Q3 FY2019: procurement completed GeoVax for ADARC (MVA construct testing). Final Individual Project iv. — Report Mosquito Bite titration study data provided to USAID on Feb 11 2019. AdPyCS titration study data provided v. Individual Project Data Set ADARC and GeoVax data sets to USAID on March 24 2019. GeoVax MVA-MARVP construct update provided herein. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-WEHI

Final Individual Project Project Plan approved by USAID on iii. Approved descriptions 3/21/2019.

Q1 FY2019: procurement ongoing with WEHI, SGI, and Precision Antibody Pending: WEHI Q2 FY2019: Procurement completed Procurement of Materials, Subcontract/Task Order for IMV (W2), SGI (kickoff meeting xii. Supplies, and Services Awards: WEHI, ImmunoVaccine, held 3/27/19) and Precision Antibody; SGI and Precision Antibody ongoing for WEHI (W2) Q3 FY2019: Procurement completed for WEHI (W2); Kickoff meeting with Precision Antibody held in May 2019. Final Individual Project iv. — Report The preliminary ELISA dataset was Preliminary ELISA dataset for circulated to USAID and the repeat Study W1 ELISA was discussed in the biweekly v. Individual Project Data Set Repeat ELISA dataset for Study meeting and provided herein. IPT W1 meeting will be scheduled to discuss GIA for Study W1 GIA results. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions.

MVDP Quarterly Report 7 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product ix. Other/Ad Hoc Reports — RCR Complex Vaccine Development Project-University of Oxford Final Individual Project Project Plan approved by USAID on iii. Approved descriptions 2/13/19. Q1 FY2019: procurement for recently approved activities is ongoing Procurement of Materials, Subcontract Award: University with Oxford xii. Supplies, and Services of Oxford Q2 FY2019: Oxford procurement completed Q3 FY2019: none Final Individual Project iv. — Report v. Individual Project Data Set None during reporting period. See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — RH5.1 Human mAb Identification and Development Final Individual Project Project Plan approved by USAID iii. Approved descriptions (regular elements only) on 1/29/19. Q2 FY2019: Oxford procurement Procurement of Materials, Subcontract Award: University xii. completed Supplies, and Services of Oxford Q3 FY2019: none Final Individual Project iv. — Report v. Individual Project Data Set Oxford data sets mAb cloning update provided herein See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — Blood-Stage Epitope Vaccine Development Project Plan approved by USAID on Final Individual iii. Approved 3/24/2016. Addendum 1 approved on Project descriptions 6/23/16. Subcontracts Awards: Agilvax, VLP Biotech, Expres2ion, NYBC, Precision Antibody, Swiss TPH Procurement of Materials, MTAs: WEHI (Alan Cowman), No procurement activities for Q1-Q3 xii. Supplies, and Services Swiss TPH (Gerd Pluschke), FY2019 University of Oxford (Simon Draper), Wellcome Trust Sanger Institute (Gavin Wright) Draft delivered to USAID on Final Individual Project iv. Approved 01/31/19. USAID feedback received Report on 3/14/19. Final Approval 5/29/19. v. Individual Project Data Set None during reporting period.

MVDP Quarterly Report 8 Leidos Proprietary

Table 2.2-1. Deliverable Summary

Deliverable/Work ID* Actions/Status Notes Product See section 3.2 regarding xii. Publications None during reporting period. abstract/poster submissions. ix. Other/Ad Hoc Reports — PD1 Blockade Inhibitor

Final Individual Project Project Plan approved by USAID on iii. Approved descriptions 1/19/2017.

Final data from second RH5/Alum Procurement of Materials, Subcontract Awards: ADARC, xii. study delivered to USAID on Feb 12 Supplies, and Services University of Oxford 2019. Final Individual Project Final Project Report delivered to iv. Approved Report USAID on 6/10/19 v. Individual Project Data Set None during reporting period. Sent to USAID on 6/10/19 for xii. Publications Submitted to PLOS ONE. clearance. Clearance received 6/19/19. ix. Other/Ad Hoc Reports — ELEMENT 3: SCG ANNUAL MEETING SUPPORT Planning for the FY19 meeting began x. SCG Annual Meeting Event Date: May 1 – 2, 2019 in Q1 of FY19. Venue: College Park Marriott Hotel x.a-e. Logistic Support Travel: Leidos Travel Lodging: College Park Marriott Hotel ELEMENT 4: PROCURMENT OF MATERIALS, SUPPLIES AND SERVICES The repository is used to store and distribute materials for conduct of the MVDP program. In preparation for transition plan MVDP Reagents xi. SriSai Biopharmaceutical Solutions development, Leidos will submit a plan Repository with multiple options to USAID so that USAID can determine/approve the final disposition of the repository material. *Each Roman numeral crosswalks to a deliverable, as called out by the prime contract (AID-OAA-C-15-00071).

MVDP Quarterly Report 9 Leidos Proprietary respective folders (Table 2.3-1). Transparency is achieved via USAID access to program folders. Leidos has uploaded documents relevant to the current reporting period to SharePoint.

Table 2.3-1. Management Tools

Tool Description Location

Cloud-based solution for exchanging and SharePoint https://vector.leidos.com/sites/ITLSO/MVDP storage of documents

Provide USAID OCONUS line to call 1-855-462-5367 Conference Phone Scientific Consultant Group members and 1778004 Lines CONUS line to communicate with Leidos 2013235

Table 2.4-1. Team Meetings

Meeting Date Topic April 3, 2019 Leidos/USAID Biweekly Teleconference April 17, 2019 Leidos/USAID Biweekly Teleconference May 1, 2019 SCG Meeting May 15, 2019 Leidos/USAID Biweekly Teleconference June 5, 2019 Leidos/USAID Biweekly Teleconference June 19, 2019 Leidos/USAID Biweekly Teleconference

3. ELEMENT 1 ACTIVITIES

MVDP Quarterly Report 10

Leidos Proprietary

3.3. NEW PROJECTS No new projects were proposed in Q3 FY2019. However, an alternative mRNA-based vaccine approach was proposed in Q3 FY2019 (see Section 4.1 for additional information).

4. ELEMENT 2 ACTIVITIES

4.1. ADJUVANT/DELIVERY PLATFORMS Adjuvant, immunostimulators, and delivery platforms identified through scouting efforts and slated for use in FY2019 are detailed in this section.

MVDP Quarterly Report 13 Leidos Proprietary

4.1.2 IMMUNOSTIMULATORS CpG in combination with Advax is in use for the RCR complex project.

MVDP Quarterly Report 14 Leidos Proprietary

4.1.3.5 Alternative RNA Approach During the May 15, 2019 biweekly meeting, Leidos proposed and alternative RNA approach (via collaboration with the Leidos Innovations Center (LInC), Promosome, TriLink, and Acuitas) to replace the SGI RNA platform approach planned for the Liver Stage Vaccine and RCR Complex Vaccine Projects. On May 16, 2019, USAID both declined the use of this strategy as part of the CD8 Platform Scouting Attachment to the Liver Stage Project Plan (and instead elected to move forward only with the GeoVax platform) and requested a “one-pager” summarizing the alternative RNA approach for the RCR Complex Vaccine Project. On May 23, 2019, Leidos provided a summary of the alternative RNA approach to USAID for review noting applicability of the approach to addressing protein expression concerns with the SPECT2 mRNA in the MIMIC system (Liver Stage Vaccine Project). A summary of the alternative RNA approach follows. Leidos and Promosome will work together to design and test RESCUE modifications to best increase expression of the proteins in the RCR complex. This will include: a) transiently transfecting mammalian cell lines with RESCUE-modified sequences and b) evaluating expression levels of each candidate protein by semi-quantitative Western blot analyses to identify the optimum modifications. The optimum RECUE-modified sequences will be incorporated into plasmids for RNA production by TriLink. TriLink will incorporate modified nucleotides (one-methylpseudouridine-5’-triphosphate in place of UTP) and cap mRNA to increase mRNA stability. Leidos will perform quality control studies with the TriLink generated mRNAs. The resulting mRNAs will be encapsulated by Acuitas, using their lipid nanoparticle technology, and/or formulated in a DPX cationic emulsion-based adjuvant compatible with mRNA-based vaccines.

MVDP Quarterly Report 15 Leidos Proprietary

MVDP Quarterly Report 16 Leidos Proprietary

The optimization activities completed in FY2018 include insertion of PfCSP 3D7 amino acids 58-78 into the VLP N-terminal insertion site and extension of the PfCSP 3D7 C-terminal insert to incorporate much of the predicted class II epitope regions in the C-terminus of PfCSP 3D7 (i.e., amino acids 313- 387). The parent VLPs resulting from these efforts are VLP407 and VLP411 (Table 4.2-1). Further, several attempts at extending the C-terminal insert to include different ICS were made at the end of FY2018 with limited success; therefore, Leidos enlisted the assistance of a protein modeler to provide feedback on the sequence configurations with increased stability and a higher probability of soluble expression. ICS sequence cloning efforts have been paused until input from the modeler is received. In Q2 FY2019, the modeler found that addition of ICS to the C-terminal insert is problematic for VLP assembly, as the hydrophobic sequences interfere with RNA binding at the protamine binding site (located at the C-terminus of the VLP backbone sequence, N-terminal of the CSP insert). As RNA binding is necessary for VLP assembly, the modeler is currently evaluating other sites for ICS insertion. In early Q3 FY19, USAID approved moving forward with addition of ICS9, ICS5, ICS7, ICS2, to the VLP. With input from the modeler, at the end of Q3 FY2019, VLP Biotech successfully added three of the ICS (2, 5, and 7) and was working on a VLP to add the fourth (i.e., ICS9). In Q1 FY2019, the strategy for construction of the loop insert clones was developed and DNA cloning initiated. A list of the planned clones is provided in Table 4.2-1; the NPDP and NVDP sequences are shown in bold to facilitate sequence comparison among the loop inserts. Of note is that VLP Biotech recommended a conservative approach to developing the loop insert clones whereby both VLP407 and VLP411 were used as the parent for different constructs. This approach was recommended by VLP Biotech due to concerns regarding the less than optimal expression levels for VLP411 as well as the possibility of lower immunogenicity directed to the loop insert when CSP B cell epitopes are placed in both the loop and N-terminal regions of the VLP. Specifications for the new VLPs, including the alternative loop inserts, are shown in Table 4.2-1. In Q2 FY2019, construction of these new VLPs (VLP430 – VLP436) was completed; all of the VLPs assembled and demonstrated soluble expression. USAID downselected VLP430 – VLP434 for progression to the passive transfer study (as described below). An IPT meeting was held on February 14, 2019, to discuss these data as well as plan for the passive transfer study. Production of sufficient quantities of these VLPs was completed in early Q3 FY2019 and immunizations begun shortly thereafter.

Table 4.2-1. VLP162 Optimization – Alternative Loop Inserts

VLP ID N-term Insert Loop Insert (position 78) C-term Insert Assembly/Expression VLP162 None NANPNVDP(NANP)3 318-377 Yes, soluble VLP407 None NANPNVDP(NANP)3 313-387 Yes, soluble VLP411 58-76 NANPNVDP(NANP)3 313-387 Yes, soluble VLP430 None KQPADGNPDPNANPN 313-387 Yes, soluble VLP431 None PADGNPDPNANPNVD 313-387 Yes, soluble VLP432 None NPDPNANPNVDPNAN 313-387 Yes, soluble NPDPNANPNVDPNANPNA VLP433 None 313-387 Yes, soluble NPNANP KLKQPADGNPDPNANPNVD VLP434 None 313-387 Yes, soluble PNANPNANPNANP VLP435 58-76 NPDPNANPNVDPNAN 313-387 Yes, soluble NPDPNANPNVDPNANPNA VLP436 58-76 313-387 Yes, soluble NPNANP

MVDP Quarterly Report 17 Leidos Proprietary

Rabbits were selected as the animal model for collection of passive transfer sera to ensure a sufficient volume of sera would be available within the necessary timeframe. Of note is that, in addition to the VLPs selected for the passive transfer study (VLP430 – VLP434), USAID requested production of rabbit sera for VLP435 and VLP436. Two rabbits were immunized per VLP and an equal volume of sera pooled from the two rabbits for the passive transfer. All VLPs were formulated in ISA720 (50% by volume) and two immunizations were given with a 6-week interval (April 4, 2019 and May 15, 2019). The test bleed was performed at week 5 (May 9, 2019) and the terminal bleed was at week 11 (June 20, 2019). Test bleed sera ELISA titer assessments (circulated to USAID on June 5, 2019) are shown in Figure 4.2-1. The sera were assessed for titers to the WHcAg VLP backbone (panel A), recombinant CSP (panel B), an NANP peptide (panel C), and an NVDP peptide (panel D). Titers were comparable for the two rabbits in each group. Rabbits immunized with VLP430 and VLP431 demonstrated lower titers to the NVDP peptide. Titers with VLP435 and VLP436 (both contain an N-terminal insert) were comparable to those with VLP432 and VLP433, indicating that the N-terminal insert does not appear to impact overall titer.

Figure 4.2-1. Passive Transfer Sera ELISA Titers – Test Bleed

MVDP Quarterly Report 18

Leidos Proprietary

The study findings were received and circulated to USAID in March 2019. In the VLP162 + ISA 720 group positive control group, only 20% sterile protection was seen (Figure 4.2-1). In the VLP162 + DPX groups, 30% protection was seen in the 2-dose group and 10% protection was seen in the 3-dose group (cohorts 3 and 4, respectively, Table 4.2-3). No sterile protection was seen in the naïve or unadjuvanted VLP162 groups. Good titers to NANP were seen with the VLP162 + DPX4 and VLP162 + ISA720 formulations. Of note is that the 2-dose group performed incrementally better for three measures: sterile protection, ELISA titer, and average patency day (day 4.7 for the 2-dose group and day 4.4 for the 3-dose cohort). Based on this finding, a 2-dose regimen will be considered for the confirmatory efficacy assessments with the optimized constructs. Analysis of the flow cytometry data will be available in early Q4 FY2019.

Figure 4.2-2. Confirmatory Efficacy Study 2 Results – ELISA Titers and Protection

4.2.5 PRELIMINARY STABILITY ASSESSMENTS OF VLP162 + DPX4 In Q2 FY2018, ImmunoVaccine began work to conduct a 6-month stability study on the VLP162 + DPX4 formulation in order to (1) confirm that the formulation is stable while it is maintained for animals studies, (2) evaluate refrigerated storage (the material is currently stored frozen), and (3) provide preliminary data regarding stability of the WHcAg VLPs in the DepoVax platform. This study was completed in Q1 FY2019 and results for the 0, 1, 3, and 6-month time points are shown in Table 4.2-4 and Table 4.2-5. It was noted after the 0-month time point testing that the VLP162 content did not conform to the specification. Additional testing performed by IMV indicated precipitation occurring during the formulation and/or freeze-drying process. This same issue was found in a retain sample from a previous lot. As this material is representative of that used in efficacy assessments, the decision was made to continue with the stability program. Formulation optimization is possible; however, this will not be performed until the fully optimized VLP has been developed and assessed for precipitation with DPX4. In addition to the VLP162 content not complying with the target specification, it was also noted that viscosity increased over the 6-month period at both temperatures. All other results were within the established specification at the 6-month time point. ImmunoVaccine concluded that further formulation development is needed to improve stability of the VLP formulation.

MVDP Quarterly Report 20

Leidos Proprietary

Table 4.4-2. A Summary of Published Analysis of Five Proteins Selected for Development and Characterized Proteins

Table 4.4-2 provides a summary of characteristics of the five proteins selected for analysis (1) (Arevalo-Herrera et al., 2016) (2) (Crompton et al., 2010) (3) (Dent et al., 2015) (4) (Le Roch et al., 2003). Based on microarray using P. falciparum (5) (Le Roch et al., 2004). Based on microarray or mass spectral; analysis using P. falciparum. (6) (Lindner et al., 2013). Based on mass spectral results using P. falciparum, proteins were ranked for abundance from 1 to 1991, 1 being the most abundant. (7) PlasmoDB (http://plasmodb.org/plasmo/) (8) (Tarun et al., 2008). Based on mass spectral analysis using P. yoelii

MVDP Quarterly Report 25 Leidos Proprietary

4.4.1 LIVER STAGE PLAN MILESTONE I.3 Analysis of the immune response to PFD0430c protein began on August 27, 2018, with AmpTec starting the synthesis the mRNA. In mid-November 2018 AmpTec provided the mRNA encoding PFD0430c (SPECT2-mRNA) that VaxDesign is using to transfect cells to express the protein in situ. VaxDesign began developing the transfection of dendritic cells in late November 2018. Transfection of dendritic cells with the SPECT2-mRNA was initially problematic, resulting in extensive cell death. VaxDesign has observed this cytotoxic effect of mRNA on dendritic cells before. As potential solutions to this issue VaxDesign evaluated transfecting feeder cells (African green monkey kidney VERO cells and human endothelium EA.hy926 cells) to provide antigen to the dendritic cells, and to optimize transfection of the dendritic cells while reducing cell death. Transfection efficiency was monitored using PCR with primers for SPECT2 to detect intracellular mRNA. As an indication for protein expression by transfected cells, VaxDesign evaluated T cell stimulation. This was performed by initially priming T cells with the dendritic cells presenting the antigen from the feeder cells transfected with SPECT2-mRNA, or with dendritic cells directly transfected with the SPECT2-mRNA. The recall (i.e., restimulation) immune response of T cells was evaluated using the two class II epitopes derived from SPECT2 that were previously demonstrated to elicit immunity in the same donor when primed with the peptides. Demonstration of a recall response indicates SPECT2 is being translated from the mRNA and the protein is processed to provide the peptides at levels sufficient to prime the T cells. Feeder cells were efficiently transfected without significant cytotoxicity, providing a potential solution to the cytotoxicity seen in the transfected dendritic cells. By modifying the culture media and transfecting rested dendritic cells, dendritic cells were also efficiently transfected while reducing cytotoxicity. That is, when monocytes were treated with cytokines to derive the dendritic cells, the cells were allowed to rest overnight before transfection with 4 µg SPECT2-mRNA/million cells verses transfecting within two hours after deriving the dendritic cells. Collectively, these modifications to the dendritic cells’ transfection doubled the viability of the dendritic cells following transfection. With the improved viability, transfected dendritic cells were used to evaluate antigen presentation in the MIMIC model. To further optimize transfection with the intent to maximize expression of SPECT2, different amounts of mRNA were tested in dendritic cells. VaxDesign evaluated using 2, 10, or 50 µg of mRNA/ million cells, and found that using 50 µg SPECT2-mRNA increased transfection nearly 100-fold relative to using 2 µg. While increasing the amount of mRNA did increase cytotoxicity, the number of viable transfected dendritic cells are sufficient for carrying out antigen presentation to the T cells, and 50 µg of mRNA will be used to transfect the dendritic cells in the MIMIC model. VaxDesign is using T cell stimulation for the class II epitopes (Table 4.4-3, peptides CL2-19 and CL2-20) as an approach to demonstrate effective transfection resulting in expression of the protein. While donor PBMC stimulated with peptides did demonstrate recall of immune response when restimulated with the same peptides, priming using SPECT2-mRNA transfected cells did not result in recall of the T cell response when the T cells were restimulated with either the peptides or using SPECT2-mRNA transfected cells (Figure 4.4-1). The lack of restimulation by SPECT2-mRNA transfected cells indicates that the level of protein expression is too low to prime the T cells. However, the approach to demonstrate protein expression is indirect. It is not possible to conclude if the lack of T cell restimulation is due to a lack of protein expression or a lack of processing of the protein resulting in the presentation of the epitopes to the T cells. To enable a direct demonstration of protein expression Leidos developed polyclonal sera in mice to peptides derived from SPECT2, with the first vaccinations performed by Precision Antibody the first week of March 2019 and sera provided to VaxDesign on May 9, 2019. This polyclonal sera is being used in flow cytometry to directly test for SPECT2 expression in the transfected cells. The peptides used to develop the sera include CL2-19 and CL2-20, as well as two additional peptides that EpiVax predicted were class II epitopes in SPECT2 (i.e., ISDYTKSLSAEAKVSC, DKDIIKILTNADTVTKC).

MVDP Quarterly Report 26 Leidos Proprietary

Figure 4.4-1. Recall IFN-γ response in the MIMIC model. “Pool” contains the peptides CL2-19 and CL2-20; “mRNA” is SPECT2-mRNA. The analysis is planned for completion by the end of July 2019 (Figure 4.4-2). Even if protein is detected, it appears that the level of expression is not high enough to prime T cells; therefore, a higher level of expression is needed to evaluate the antigen processing for SPECT2. In Q3 FY2019, Leidos proposed application of technologies developed by Promosome to redesign the SPECT2-mRNA as a way of potentially increasing protein expression to the levels needed for evaluation of antigen processing by VaxDesign.

MVDP Quarterly Report 27 Leidos Proprietary

4.4.1.1 Platform Scouting Studies - CD8 T Cell Responses In FY2018 USAID requested an addendum for the Liver Stage Vaccine Project to perform CD8 T cell response platform scouting studies using a well-studied animal model of malaria, the H-2kd restricted class I epitope (SYVPSAEQI) of P. yoelii (Py) CSP that is known to be protective upon Py challenge. The initial addendum to the project plan was delivered January 26, 2018, as an attachment to the CSP project. Based on comments received from USAID on March 21, 2018, Leidos revised the attachment to only include assessment of CD8 platform scouting studies. An updated version of the attachment was submitted to USAID in Q2 FY2019. Concerns with an appropriate positive control for assessment of Pf proteins had delayed completion of the attachment. An updated draft was submitted to USAID on May 6, 2019 and USAID provided feedback on May 22, 2019. An updated attachment was submitted on May 29, 2019 and USAID approval received on June 6, 2019. A final PDF of the Liver Stage Vaccine Project Plan CD8 Platform Scouting Attachment was provided to USAID on June 10, 2019. Initial proof- of-concept studies previously performed with this model found that SYVPSAEQI integrated into the MagaVax platform elicited CD8 T cell responses in mice. These data are detailed in the FY2018 USAID MVDP Annual Report. The decision to proceed to a challenge study with one or both MagaVax constructs was dependent on the elevation of T cells responses in the MagaVax compared to that of the positive control (AdPyCS). Based on USAID’s initial specified Acceptance Criterion where constructs will be selected for further study only if they perform considerably better than the positive control at a historically non-protective dose or equal to or better than the positive control that would be expected to be protective, the decision was made not to proceed with challenge studies at this time. The other two platforms that Leidos initially planned to evaluate were Synthetic Genomics Inc (SGI) self-replicating RNA replicon and GeoVax’s modified Vaccinia virus (MVA)-VLP. However, in Q3 FY2019, SGI informed Leidos that their RNA platform would no longer be available due to acquisition of the company. USAID elected not to include an RNA platform as part of CD8 platform scouting at this time (refer to section 4.1 for additional details). This change was incorporated into the CD8 Platform Scouting Attachment approved by USAID in Q3 FY2019. Further, the experimental approach and design for the GeoVax platform was also detailed in the approved attachment. The kick-off meeting with GeoVax was held on March 7, 2019.

4.4.1.2 Optimizing the Number of Mosquito Bites for Infection. Prior to starting the platform scouting analysis with the GeoVax MVA-VLP, the number of mosquito bites required to ensure malaria infection in this model needed to be determined. This study informed the targeted infectious exposure that will be used for the subsequent studies at ADARC.

MVDP Quarterly Report 28 Leidos Proprietary

On December 10, 2018 of Q1 FY2019, USAID Table 4.4-4. Bite Titration Study approved the study design (Table 4.4-4). In brief, Cohort # Test Sample BALB/c (n = 7) mice were exposed to various numbers of mosquitos infected with Py 17XNL. 1 No bites Following exposure, individual mosquitos were 2 2 bites dissected to check for infectivity. Parasitemia in the 3 4 bites mice was assessed via blood smears. Results of this 4 6 bites study were delivered to USAID on Feb 11 in Q2 5 8 bites FY2019 and are shown in Table 4.4-5. Of note, each mouse was anesthetized and placed on its abdomen for a 3-minute exposure to allow the mosquitos to bite. All mice that were exposed to either 6 or 8 mosquitos were infected. In addition, the overall percentage of mosquitos positive for sporozoites was ~75%. Based on the data Leidos and USAID agreed to move forward with using at least 6 bites/mouse to ensure that all mice are infected.

Table 4.4-5. Bite Titration Study Results

Number of infected Mouse ID Day 3 Day 4 Day 5 Day 6 Day 7 mosquitos 1-1 - - + + + 2/2 1-2 - - - - - 1/2 1-3 - - - + + 1/2 2 bites 11/14 1-4 - + + + + 2/2 /mouse (78.6%) 1-5 - - + + + 2/2 1-6 - - - - - 1/2 1-7 + + + + + 2/2 2-1 - - - - - 2/4 2-2 + + + + + 4/4 2-3 + + + + + 3/4 4 bites 21/28 2-4 + + + + + 3/4 /mouse (75%) 2-5 - - + + + 2/4 2-6 + + + + + 4/4 2-7 - + + + + 3/4 3-1 + + + + + 5/6 3-2 - + + + + 4/6 3-3 + + + + + 4/6 6 bites 31/42 3-4 + + + + + 5/6 /mouse (73.8%) 3-5 + + + + + 6/6 3-6 - + + + + 4/6 3-7 - + + + + 3/6 4-1 + + + + + 6/8 4-2 + + + + + 7/8 4-3 - + + + + 5/8 8 bites 41/56 4-4 + + + + + 6/8 /mouse (73.2%) 4-5 - + + + + 4/8 4-6 + + + + + 6/8 4-7 + + + + + 7/8 4.4.1.3 Titration of AdPyCS Immunization for Mosquito Bite Challenge. In addition to optimizing the number of mosquito bites for infection prior to evaluating the GeoVax MVA-VLP platform, ADARC will also titrate the AdPyCS immunization to determine the dose that gives 40-50% protection following mosquito bite challenge. On December 10, 2018 of Q1 FY2019, USAID approved the study design (Table 4.4-6). In brief, BALB/c (n = 7) mice were immunized IM with various doses of AdPyCS in the hind limb without adjuvant. At day 10 post-immunization mice were challenged via 6 mosquitos infected with Py 17XNL. Following bite challenge individual mosquitos were

MVDP Quarterly Report 29 Leidos Proprietary dissected to check for infectivity. Parasitemia in the mice was assessed via blood smears. Results of this study were delivered to USAID on March 24 in Q2 FY2019 and are shown in Table 4.4-7. The percentage of dissected mosquitos positive for sporozoites was ~88% overall and ADARC visibly confirmed (mosquitoes got swollen with blood) greater than 4 bites per mouse. No protection was seen with either 107, 108 or 109 AdPyCS. Only a single mouse was protected at 1010 (14%) while 3 out of 7 (43%) mice were protected with 1011 AdPyCS. Based on these study results ADARC suggest using 1011 AdPyCS as a positive control in the GeoVax proof-of-concept studies using PyCS.

Table 4.4-6. AdPyCS Dosing Study Cohort # Test Sample Route # Mice, Strain Day 0 Day 10

1 Naïve none 7 BALB/c none 2 107 AdPyCS IM 7 BALB/c √ 3 108 AdPyCS IM 7 BALB/c √ 4 109 AdPyCS IM 7 BALB/c √ 5 1010 AdPyCS IM 7 BALB/c √ 6 1011 AdPyCS IM 7 BALB/c √ bites/mouse 6

Table 4.4-7. AdPyCS Titration Study Results

MVDP Quarterly Report 30 Leidos Proprietary

4.4.1.4 Construction of MVA-PyCS vaccine In Q3 FY2019, GeoVax began work to construct a recombinant MVA-VLP vaccine expressing PyCS. GeoVax obtained a synthetic PyCS gene with restriction sites so the sequence could be inserted into a shuttle vector. The shuttle vector includes recombination sites to enable recombination into the MVA- VLP vector. The shuttle plasmid also contains a GFP reporter to allow rapid isolation of recombinant viruses. The synthetic PyCS gene was inserted into the shuttle vector by standard molecular biology techniques. Two versions of the PyCS construct were generated, both designed to express proteins that will assemble in VLPs but differing in the length of the tail for the Marburg VP40 transmembrane sequence, as GeoVax has seen differences in expression of certain antigens using different versions of the Marburg VP40. One version has an abbreviated cytoplasmic tail, 8 amino acids (CVLP-1) and a second with a longer cytoplasmic tail, 69 amino acids (CVLP-2). Prior to generating the vaccine constructs, the transient expression of PyCS in the shuttle plasmids was examined by western blot. In brief, duck fibroblast (DF1) cells were infected with MVA-MARVP40 recombinant virus (expressing the Marburg virus VP40 matrix protein) and transfected with one of the two different shuttle plasmids (CVLP-1 or CVLP-2) designed to interact with VP40. The virus was harvested 48 hours post transfection. Culture supernatants were aspirated, clarified by centrifugation, and frozen. Cells were chemically lysed and the lysates clarified by centrifugation. Both the media and the cell lysates were analyzed by western blot for the expression of Marburg VP40 (as an internal control) and for CS protein. Results of transient expression with the shuttle plasmids (Figure. 4.4-2) were provided to USAID on May 12, 2019. The anti-VP40 blot demonstrates approximately equivalent expression of VP40 in both cell lysates and supernatants across all constructs including the MVA-VP40 only (Figure. 4.4-2 Upper Panel). Anti-PyCS antibody (clone 2F6; provided by Dr. Moryia Tsuji) demonstrates expression of CS from all constructs (Figure. 4.4-2 Lower Panel). These data suggest the CVLP-1 construct with the shorter cytoplasmic tail gives superior secretion.

Figure 4.4-3. Transient Expression of PyCS in MVA-VLP Platform by Western Blot Analysis With transient expression of PyCS demonstrated (Figure. 4.4-3), GeoVax has transfected the MVA- PyCS shuttle plasmids into chick embryo fibroblast (CEF) cells infected with the MVA-VLP vector. MVA-PyCS recombinant virus was then selected based on the expression of GFP, which is present in the shuttle vectors and initial recombinants but is removed during clone purification. Virus selection is then followed by a repetition of multiple clone purification by limited dilution to purify the recombinant from the parental virus and to remove the GFP sequence from the rescued recombinant virus. GeoVax is expecting to have purification completed by early Q4 FY2019.

MVDP Quarterly Report 31 Leidos Proprietary

4.5. RCR COMPLEX VACCINE DEVELOPMENT PROJECT: CECILLE BROWNE (WEHI), JAYNE CHRISTEN (OXFORD), AND AMY NOE This project is based on evaluation of proteins in the RCR complex as vaccine candidates by comparison of the individual and combined proteins. Although high GIA activity was observed with the RH5.1 vaccine candidate in animal models and good titers were seen in humans immunized with RH5.1/AS01, no sterile protection was found upon CHMI. WEHI has previously provided data indicating that two additional proteins in the RH5 complex perform better in the GIA as compared to RH5 when the proteins are formulated with IFA (70% for RH5 compared to 80% and 90% for CyRPA and RIPR, respectively). However, it is unclear if the same GIA activity is possible with human-use adjuvants (thus Study W1 was initiated) and/or if the moderate increases in GIA seen with CyRPA and RIPR will translate to achievement of sterile protection in humans with those proteins administered alone. Further, Dr. Simon Draper (Oxford) indicated that GIA activity was similar among the individual RH5 complex proteins in their hands (data not received). Current information suggests that blood stage proteins can act synergistically to achieve higher GIA activity; therefore, a multi-antigen RH5 complex vaccine may be more efficacious than a single protein vaccine. Thus, the RCR Complex Project Plan will test the following hypotheses: (1) Targeting single and/or multiple proteins in the RCR complex can interrupt the mechanism of merozoite invasion of erythrocytes during the blood stage infection of P. falciparum and (2) Targeting multiple proteins in the complex may be more effective than targeting a single protein in the complex. To address these hypotheses, Leidos developed two parallel paths under the overall RCR Complex Project Plan as well as a workflow diagram to illustrate the interconnectedness of the projects conducted at Oxford and WEHI. Per the USAID-approved project plan, there are a total of three approaches for development of an RCR complex vaccine including recombinant protein, VLP, and RNA approaches. In addition, specific studies have been included in the workflow to facilitate down-selection (e.g., studies to identify a test protein dose and to compare different adjuvants). Based on immunogenicity assessments (e.g., ELISA and GIA activity), constructs/formulations will be downselected for comparison in a penultimate study. These interdependencies and decision points are shown in the RCR Complex Vaccine Project Workflow Figure 4.5-1, where decision points are shown as to be determined (TBD). The workflow diagram was updated to incorporate the decision by USAID on February 19, 2019, to proceed with a dose-ranging study using antigens formulated in DPX4 at WEHI. To incorporate the decision made by USAID on June 3, 2019 to not proceed with an RNA approach for the RCR Complex Vaccine Development Project (see sections 4.1 and 4.5.2), the workflow diagram has been updated to indicate that the mRNA Approach is “Not Funded.” Based on the evolution of this project, USAID approval of the project as a whole was provided prior to approval of the project plans. The project plans for both the work at WEHI and Oxford were approved in Q2 FY2019.

MVDP Quarterly Report 32 Leidos Proprietary

Figure 4.5-1. RCR Complex Vaccine Project Workflow. Down-selection is based on analysis of sera samples via GIA (pLDH and flow cytometry methods) and Quantitative ELISA

MVDP Quarterly Report 33 Leidos Proprietary

During the IPT meeting in October 2018, USAID raised the concern that the 20 µg immunization dose used in Study W1 may be too high to distinguish between adjuvant groups. Oxford noted that they previously found maxed ELISA responses for RH5.1 at concentrations higher than 16 ng with Matrix-M. However, WEHI noted that they have previously used up to 200 µg of RH5 and routinely use a 20 µg dose of this protein in rabbits. Based on this discussion, Leidos recommended the performance of terminal bleeds after two doses instead of three (two weeks post second immunization). The revised study design for Study W1 is shown in Table 4.5-1. A preliminary immunogenicity test (ELISA) was completed by WEHI in December 2018, and result of this preliminary ELISA was submitted to USAID in December 2018. Results show that DPX4 gave superior titers compared to the Advax/CpG and AddaVax, which were the other two human-use adjuvants tested in the study (Figure 4.5-2 and Table 4.5-2). However, since all DPX4 and IFA dilutions gave O.D. readings greater than 1.0, titers could only be estimated (Table 4.5.2). The ELISA will be repeated to further dilute these serum samples and allow interpolation of titers using a four-parameter logistic (4PL) curve fit. GIA and repeat ELISA were performed in February 2019, and GIA results were received in April 2019. Results of the second ELISA also show that DPX4 induced higher titers compared to Advax/CpG and AddaVax. In the second ELISA, dilution curves reached O.D. readings lower than 1.0 and this allowed the interpolation of titers using a 4PL curve fit (Figure 4.5-3 and Table 4.5-3). GIA using purified serum IgG from Study W1was completed in April 2019. GIA results show that IgG purified from sera from mice immunized with each of the three antigens inhibited parasite growth as measured by flow cytometry (Figure 4.5-4). Purified IgG from mice immunized with DPX formulations with all three antigens inhibited parasite growth, while purified IgG from Advax + CpG or AddaVax formulations inhibited growth with 1 or 2 of the individual proteins. Binding of the purified IgG (used in GIA) to antigens that they were raised against was demonstrated by ELISA (Figure 4.5-5). In February 2019, USAID approved the initiation of Study W2 (Dose Ranging Study) in rats before completion of Study W1 GIA; DPX will be used as adjuvant. The study design for W2 is shown in Table 4.5-4. Thus, procurement with IMV for Study W2 was completed in Q2, and formulation of individual proteins in DPX4 was completed in Q3. WEHI initiated rat immunizations in May 2019. In the first immunization, a technical error occurred resulting in the administration of higher doses of proteins in rats. Instead of 20 µg, 2 µg and 0.2 µg doses, rats received 36 µg, 3.6 µg and 0.36 µg, respectively. Leidos reviewed with WEHI the protocol provided by IMV (included with the formulation shipment) to ensure that protocol steps are followed and that rats are immunized with protein amounts indicated in the study design. The second immunization was completed on June 13 using the planned doses of 20 µg, 2 µg and 0.2 µg. The final immunization and terminal bleeds are scheduled on July 11 and July 25, respectively. On March 21, 2019, USAID approved RCR Project Plan WEHI and the study design for Study W4 (Table 4.5-5). Replicon generation is estimated to be completed in Q3/Q4. Procurement for Study W4 at WEHI will be initiated in Q3. In Q3, SGI had communicated that they could no longer produce replicons for the RCR project. Leidos submitted an alternate plan for W4 to USAID; however, this alternate plan was not approved. Thus, W4 of the RCR project will not be conducted.

MVDP Quarterly Report 34 Leidos Proprietary

Table 4.5-1. Study W1. Immunogenicity Study in Mice of Individual RCR Proteins in Three Human use Adjuvants and IFA

5 RH5.1 (20 µg) + AddaVax IM 6, BALB/c √ √ 6 CyRPA (20 µg) + AddaVax IM 6, BALB/c √ √

Figure 4.5-2. First ELISA - Preliminary immunogenicity Results of Individual RCR Proteins in Four Adjuvants. Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

MVDP Quarterly Report 35 Leidos Proprietary

Table 4.5-2. Preliminary Estimates of Immunogenicity Titers (at O.D. = 1.0)

RH5.1 RIPR CyRPA DPX4 >51,200 >51,200 30,000 IFA 25,000 >51,200 >51,200 Advax+CpG 3,200 8,800 400 AddaVax 600 3,200 2,400

Figure 4.5-3. Second ELISA - Immunogenicity Results of Individual RCR Proteins in Four Adjuvants (repeat ELISA). Pooled mice sera from terminal bleeds (2 weeks post second immunization) were serially diluted and tested by ELISA. Dilution curves: IFA (blue), DPX4 (red), Advax+CpG (green) and AddaVax (black). Gray lines depict all pre-bleed samples.

Table 4.5-3. Immunogenicity Titers Derived from 4PL Curve Fit (at O.D. = 1.0); Second ELISA

RH5 RIPR CyRPA DPX4 288,479 1,302,740 29,107 IFA 185,188 537,314 47,561 Advax+CpG 25,087 14,541 < 1,000 AddaVax 3,365 15,180 < 1,000

MVDP Quarterly Report 36 Leidos Proprietary

Figure 4.5-4. W1 GIA Results – IgG was purified from pooled mice sera from terminal bleeds (2 weeks post second immunization). Purified IgG samples were serially diluted and tested by GIA using the flow cytometry method.

Figure 4.5-5. W1 ELISA of purified IgG – Purified IgG samples (that were used in GIA) were serially diluted from a starting concentration of 10 mg/ml. Dilutions were tested on plates coated with the indicated antigen (ELISA). Figure legends represent the cohort of mice that were immunized with the indicated antigen combined with adjuvant.

MVDP Quarterly Report 37 Leidos Proprietary

Table 4.5-4. Study W2 – Dose-Ranging Study in Rats

# rats, Study Day Cohort Formulation R strain -2 0 28 42 56 70 1 RH5.1 protein, 20 µg + DPX4 IM 6, Wistar √ √ √ 2 RH5.1 protein, 2 µg + DPX4 IM 6, Wistar √ √ √ 3 µ IM 6, Wistar √ √ √ RH5.1 protein, 0.2 g + DPX4

4 CyRPA protein, 2 µg + DPX4 IM 6, Wistar √ √ √ 5 CyRPA protein, 0.2 µg + DPX4 IM 6, Wistar - bleed √ √ √ Pre Testbleed

6 RIPR protein, 2 µg + DPX4 IM 6, Wistar √ √ √ TerminalBleed RIPR protein, 0.2 µg + DPX4 IM 6, Wistar √ √ √ 7 Adjuvant alone (Negative Control) IM 6, Wistar √ √ √

Table 4.5-5. Study W4 – Immunogenicity Study using RNA Replicons in Rats

Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RIPR monocistronic mRNA (10 µg) IM 6, Wistar √ √ √ 2 RH5 monocistronic mRNA (10 µg) IM 6, Wistar √ √ √ 3 CyRPA monocistronic mRNA (10 µg) IM 6, Wistar √ √ √

RH5, CyRPA, RIPR monocistronic

4 mRNAs, admix (total of 10 µg; 3.3 µg IM 6, Wistar √ √ √ per monocistronic replicon)

RH5, CyRPA, RIPR monocistronic - bleed Pre 5 mRNAs, admix (total of 30 µg; 10 µg IM 6, Wistar √ √ Testbleed √

per monocistronic replicon) TerminalBleed RH5, CyRPA, and RIPR polycistronic 6 IM 6, Wistar √ √ √ mRNA (10 µg) Lipid formulation control 7 IM 6, Wistar √ √ √ (Negative control)

MVDP Quarterly Report 38 Leidos Proprietary

Table 4.5-6. Updated Timeline for RCR Project Plan WEHI

4.5.2 SYNTHETIC GENOMICS INC. (SGI) USAID approved the study design for Study W4 (Replicon Study in Rats); the study design is shown in Table 4.5-5. SGI will prepare monocistronic and polycistronic RNA replicons encoding the RCR proteins. These replicons serve as delivery platforms for in vivo expression of RCR proteins in rats. Procurement with SGI was completed, and a kickoff meeting was held on March 27, 2019. However, on May 9, 2019, SGI informed Leidos that their RNA platform would no longer be available due to acquisition of the company. On May 15, 2019, Leidos proposed an alternate RNA strategy to replace the SGI platform. However, on June 3, 2019, USAID notified Leidos that the alternate RNA approach would not be funded for the RCR project.

4.5.3 PRECISION ANTIBODY The procurement process was completed with Precision Antibody in Q2. Precision Antibody will develop an antibody reagent that specifically recognize the RCR complex. The antibody reagent would be used to detect complex formation in cultures transfected with RNA replicons. Although Study W4 (RNA study) will no longer be conducted, USAID has agreed to continue with the RCR antibody development. In the first phase of this project, Precision Antibody will evaluate RCR stability in the adjuvant used in monoclonal antibody development. The goal of the first phase is to ensure that the immunogen is in a complex formation when administered to mice. RCR stability evaluation, which will begin in Q3 (April 2019), will be performed using immunoprecipitation followed by Western Blotting and/or Size exclusion chromatography. If the RCR complex is stable in adjuvant, Precision Antibody will use the RCR as immunogen to generate monoclonal antibodies using Precision’s established workflow. If the RCR complex is not stable in the first adjuvant, another adjuvant will be tested. If the RCR complex is not stable in the second adjuvant, a decision will be made whether or not to continue with monoclonal antibody development.

MVDP Quarterly Report 39 Leidos Proprietary the plan were the study designs for four in-life studies (Studies O1-O4) as well as the description of two reagent/assay development activities (i.e., quantitative ELISA and VLPs decorated with RCR recombinant proteins). USAID provided edits and comments regarding the project plan on December 21, 2018 of Q1 FY2019. Leidos circulated the updated project plan to USAID on February 5, 2019; USAID approved the project plan on February 13, 2019. A kick-off meeting was held on March 4, 2019. An overview of the development plan for this project is provided in Table 4.5-7.

Table 4.5-7. Overview of the RCR Complex Vaccine Development Project Plan-Oxford

4.5.4.1 Dose-Ranging Study (Study O1) On November 30, 2018 of Q1 FY2019, USAID approved the design of Study O1. In Study O1, the immunogenicity of RH5, CyRPA and RIPR proteins as well as the ability of antibodies induced to elicit parasite growth inhibition will be evaluated individually in rats (Table 4.5-8). Leidos expects initiation of Study O1 during Q4 FY2019.

MVDP Quarterly Report 40 Leidos Proprietary

Table 4.5-8. Study O1 – Dose-Ranging Study

Study Day Cohort Formulation R # rats, strain -2 0 28 42 56 70 1 RH5.1 (2 µg) + Matrix-M IM 6, Wistar √ √ √

2 RH5.1 (200 ng) + Matrix-M IM 6, Wistar √ √ √

3 RH5.1 (20 ng) + Matrix-M IM 6, Wistar √ √ √ 4 CyRPA (2 µg) + Matrix-M IM 6, Wistar √ √ √ - bleed

5 RIPR (2 µg) + Matrix-M IM 6, Wistar Pre √ √ √ Testbleed

6 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √ TerminalBleed 7 Negative Control IM 3, Wistar ** **Terminal Bleed

Table 4.5-9. Study O2 – Individual Proteins and Double/Triple Protein Mixtures

(Dose TBD)

RIPR + CyRPA + Matrix-M (Dose 6 IM 6, Wistar √ √ √ TBD)

RH5.1 + CyRPA + RIPR + Matrix-M - bleed

7 (Dose TBD) IM 6, Wistar Pre √ √ √ Testbleed

Equivalent Molar Ratio TerminalBleed RH5.1 + CyRPA + RIPR + Matrix-M 8 (Dose TBD) IM 6, Wistar √ √ √ Equivalent Protein Concentration Reconstituted RCR + Matrix-M 9 IM 6, Wistar √ √ √ (Dose TBD) 10 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √

MVDP Quarterly Report 41 Leidos Proprietary

4.5.4.4 VLP Development USAID approved the development of SpyCatcher-HBsAg VLPs decorated with single antigens or the RCR complex on October 4, 2018 of Q1 FY2019. VLP development began in Q3 FY2019 with the production of SpyCatcher-HBsAg VLPs and initiation of cloning efforts for RH5-SpyTag, CyRPA-SpyTag, and RIPR-SpyTag. SpyCatcher-HBsAg VLPs were characterized by dynamic light scattering (DLS), transmission election microscopy (TEM), SDS-PAGE, and Western Blot (Figure 4.5-6). Analysis by DLS (Figure 4.5-6A) and TEM (Figure 4.5-6B) yielded expected results for VLP diameter (~26 nm) and VLP morphology. SDS-PAGE analysis revealed a single protein band of expected molecular weight (MW) at 37 kDa while Western Blot analysis using an anti-HBsAg antibody identified multiple bands in the VLP preparation that are consistent with protein monomers and dimers (Figure 4.5-6C).

Figure 4.5-6. Characterization of SpyCatcher-HBsAg VLPs

CyRPA-SpyTag, in which the tag is located at the C-terminus of CyRPA, was produced and subsequently used for the formation of the RCR complex. Note that in this preparation, RH5 and RIPR do not contain SpyTag. Analysis by size exclusion chromatography indicated that the RCR (CyRPA-SpyTag) complex eluted at a volume similar to the RCR complex in which all proteins were without SpyTag (RCR; Figure 4.5-7A). This result was consistent with complex formation. Preliminary results suggest that the RCR (CyRPA-SpyTag) complex can be conjugated onto the SpyCatcher-HBsAg VLPs (Figure 4.5-7B). In this experiment, the RCR (CyRPA-SpyTag) complex was incubated with SpyCatcher-HBsAg VLPs overnight at 4°C in Tris Buffered Saline (20mM Tris-HCl, 150mM NaCl, pH 7.4). Four different molar ratios of RCR:HBsAg were evaluated (0.5:1, 1:1, 1.5:1, and 2:1). As shown in Figure 4.5.7B, all molar ratios resulted in the formation of the RCR-HBsAg VLPs (yellow rectangle). However, using a

MVDP Quarterly Report 42 Leidos Proprietary molar ratio of 1:1 or higher resulted in the absence of unconjugated HBsAg VLPs (no red rectangle). Optimization of the conjugation reaction and cloning efforts for the remaining SpyTag proteins are ongoing at the time of this writing.

Figure 4.5-7. Characterization of RCR (CyRPA-SpyTag) (A) and Preliminary Conjugation of RCR-SpyTag Complex to SpyCatcher-HBsAg VLPs (B)

4.5.4.7 Updated Schedule: RCR Complex Project Oxford The initial project schedule provided by Oxford (included in the Project Plan) had a Study O1 start date in February 2019 and completion date in May 2020. Approval of the Project Plan was received from USAID on February 13, 2019. Of note is that due to the number of rats involved in the planned (USAID-approved) studies for this project, it is necessary for Oxford to subcontract the in life portions. Oxford finalized their subcontract in March 2019. Further, study approval by Novavax is needed prior to provision of Matrix-M. Oxford received study approval from Novavax in May 2019 and a signed MTA in June 2019. Based on this updated timeframe, Leidos requested a revised schedule from Oxford, and is awaiting receipt of the updated project schedule from Oxford at the time of this writing. Oxford communicated that Study O1 has an expected start date in July 2019 and scheduling options for Study O2 and Study O3 are being discussed. The project schedule shown in Table 4.5-10 was previously provided by Oxford (Q2 FY2019) and will be updated as more information is available. Note that the project schedule submitted in Q2 FY2019 has a Study O1 start date in April 2019 and a completion date

MVDP Quarterly Report 43 Leidos Proprietary

in August 2020, extending beyond the MVDP POP end date. Leidos recommends holding an IPT meeting in Q1 FY2020 to determine the project stopping point.

Table 4.5-10. Project Timeline Received from Oxford

4.6. RH5.1 HUMAN MAB IDENTIFICATION AND DEVELOPMENT PROJECT: VIN KOTRAIAH/CECILLE BROWNE The RH5.1 human mAb identification and development project is new for FY2019. Procurement efforts were started in Q1 FY2019 and are anticipated to be finalized in Q2 FY2019. Leidos (working with Oxford) provided the synopsis for this project to USAID in August 2018 and included this project in the FY2019 Annual Work Plan (submitted to USAID on September 1, 2018). In response to their review of the FY2019 annual work plan, USAID indicated their interest in funding portions of this proposal. On December 5, 2018, Leidos submitted to USAID a draft Project Plan on RH5.1 Human mAb Isolation and Development to USAID. USAID reviewed the plan and provided their comments on December 14, 2018. Leidos updated the plan based on feedback from USAID and submitted a final plan on December 19, 2018. USAID approved elements in the plan relating to mAb development, screening and characterization to be performed in the Draper lab on January 29, 2019. A decision on the R5.016 immunogen work was deferred. A kick-off meeting was held on March 4, 2019 where Oxford presented a slide deck titled “High-throughput cloning of RH5-specific mAbs” that contained the VAC063 data as well as their initial list of suggested VAC063 samples to use in this project. This presentation was circulated to USAID and an IPT meeting was held on March 18, 2019 to discuss the VAC063 data and to finalize sample selections. Additional sample selection updates were received from Oxford and circulated to USAID on March 22, 2019. Initiation of the technical work is pending USAID approval of sample selection, anticipated in early Q3 FY2019. As anticipated, a priority list of 20 samples was approved by USAID on April 1, 2019 (Table 4.6-2) and progress to date is described below. An overview of the development plan for this project is provided in Table 4.6-1 followed by a brief summary of the proposed project.

Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project

Phases Milestones Activities • Single B cell isolation (memory B cells (mBC) or plasmablasts) B Cell Isolation and • Reverse transcription and paired heavy and light chain nested PCR Cloning • DNA purification and cloning into heavy and light chain vectors B Cell Isolation and • Transfection of heavy and light chain vectors into HEK293 cells mAb Expression and mAb Development • Harvest of supernatant and screening by ELISA Purification • Affinity purification of antibodies from supernatant • Sequencing of heavy and light chain variable regions mAb Sequencing • Determination of sequence maturation and germline alleles ELISA Titer and • Determination of ELISA titers mAb Screening Protein Mapping • Protein region mapping using a panel of RH5 variants

MVDP Quarterly Report 44 Leidos Proprietary

Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project

Phases Milestones Activities GIA • Growth Inhibition Assay (using pLDH method) Epitope Similarity • Determination of similarity of B cell epitopes recognized by Screen antibodies using competition assay and SPR/BLI mAb Downselection • Monoclonal antibody down-selection mAb Affinity • Determination of antibody affinity by SPR mAb mAb Inhibition of • Assessment of inhibitory activity of antibodies on RCR, RH5-P113 Characterization Complex Formation and RH5-Basigin complexes mAb Downselection • Selection of monoclonal antibodies for structural studies Computational • Identification of sequence variants of the immunogen that are assessments stable and have the right conformation R5.016 Immunogen Production of select • Epitope grafting and production of selected immunogens Design (optional) immunogen designs Immunogen • SPR screening for binding to R5.016 mAb downselection Production of • Production and QC of downselected immunogen R5.016 Immunogen immunogen Production Coupling of (optional) • Conjugation of downselected immunogens to VLPs immunogen to VLP Formulation of • Selection and procurement of adjuvant immunogen:VLP • Formulation of immunogen R5.016 Immunogen • Rat immunizations with RH5.1 comparator Rat immunization Testing (optional) • Collection of sera Immunogenicity and • Humoral response and GIA activity with R5.016 mAb comparator GIA assessments • Recombinant antibody expression and purification Antibody production • Fab fragment generation New mAb • Screening for Fab:RH5 co-crystals Structure Crystal screening • Cryoprotection of crystals Determination • X-ray diffraction studies (optional) Crystal structure • Model building and refinement determination • Epitope delineation

MVDP Quarterly Report 45 Leidos Proprietary perform a sample selection inclusive of the RH5.1 clinical trial GIA data. Oxford conducted the same in March 2019, once the RH5.1 clinical study GIA data were available for analysis. Approval of sample selection by USAID is anticipated in early Q3 FY2019. Based on this updated timeframe, Leidos requested a revised schedule from Oxford, shown in Figure 4.6-1. Note that the updated project schedule begins in April 2019 and a completion date in September 2020, extending beyond the MVDP POP end date. Leidos recommends holding an IPT meeting in Q1 FY2020 to determine the project stopping point.

Figure 4.6-1. Project Timeline Received from Oxford The priority list of 20 samples approved by USAID on April 1, 2019 is shown in Table 4.6-2. Note that the first 20 samples were approved with the Oxford group aiming to deliver roughly 10 RH5-specific mAbs from each of these 20 samples and characterize them. In the “Group” column of Table 4.6-2, the samples are color coded by the VAC063 Group number to which they belong. Six samples drawn from Group 7, 10 drawn from Group 5 and 4 from Group 3 constitute the total of 20 approved samples. In the “Volunteer” column, the volunteer IDs are color coded by the selection criterion. The color yellow indicates that the volunteer was selected on the basis of In Vivo Growth Inhibition (IVGI) only. The green colored cells indicate that the volunteers were selected on the basis of IVGI and ELISA, Avidity or GIA. The blue colored cells indicate that the volunteers were selected on the basis of ELISA, Avidity or GIA. Note that Oxford’s slide summary with the ELISA, Avidity, GIA and IVGI data for all the VAC063 volunteers was provided to USAID in Q2 FY2019. On June 17, 2019, Oxford notified Leidos that volunteer 01-809 (01-032) from Group 7 with a priority number of 7 (Table 4.6-2) had withdrawn consent and that Oxford could no longer use this volunteer’s samples. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement. The consent withdrawal and proposed replacement sample were communicated to USAID in the bi-weekly meeting on June 19, 2019.

MVDP Quarterly Report 46 Leidos Proprietary

MVDP Quarterly Report 47 Leidos Proprietary

Table 4.6-3 shows the number of RH5-specific mAbs that have been isolated and cloned to date (“RH5-specific mAbs cloned”). A total of 18 mAbs have been isolated from three patient samples; all of these mAbs were isolated prior to the start of the USAID-funded project during test runs of the overall process. Of the three patient samples, only the patient ID “B” sample is present in the priority approved list of samples for the mAb identification and development project; the other two patient samples are not on the priority approved list. Sixteen of the 18 mAbs have been purified and characterized in ELISA and GIA assays; 2 mAbs could not be produced in sufficient quantities for characterization studies. Of note is that sorting of B cells from six additional samples from the priority list was completed in Q3 FY2019 as shown in Table 4.6-3; mAb cloning for these is ongoing. Table 4.6-3. Summary of progress in Q3 FY2019. The blank cells under the RH5-specific mAbs cloned column indicate that the cloning is pending.

The current set of 16 mAbs were characterized in ELISA using four different RH5-based plate antigens (Figure 4.6-2). All four antigens have a C-tag for purification and proper folding of RH5.1 and RH5.2 following purification has been verified using dot blots and mAbs previously characterized as recognizing conformational epitopes. Briefly, conformational mAbs were used to develop dot blots of denatured (boiled) protein, normal protein and “no protein” for each plate antigen. Only the natively expressed protein was recognized by the mAbs indicating proper folding of the recombinant plate antigen. Proper folding of the Bundle antigen has been confirmed by the Higgins Lab at Oxford and further details on the confirmation are awaited.

MVDP Quarterly Report 48 Leidos Proprietary the epitope of the R5.016 mAb isolated from the VAC057 trial (described in Alanine et al., Cell 2019) on a non- RH5 protein scaffold was also used as plate antigen. For the ELISA assessments, cut-off thresholds were used for determining binding versus non-binding by the mAbs, particularly regarding how “weak” binders are identified above background. Cut-off OD values were designated depending on the background typically experienced. Two measurements of background were used – control mAbs on PBS-coated wells and casein (the blocking buffer/dilutent used for all samples) on antigen-coated wells. The background values for the former never exceeded 0.09 OD and the latter OD value on RH5.1coated wells never exceeded a value of 0.1. Therefore, a value below an OD of 0.1 was regarded 'at or below background' and therefore negative for binding. The ELISA data for the 16 mAbs along with control mAbs are shown in Figure 4.6-3. Also shown on each graph are OD values for control mAbs, R5.016, h4BA7 and LB12. R5.016 is known to bind a conformational epitope in the bundle region, h4BA7 is a linear epitope in the intrinsic loop, and LB12 is known to bind a linear epitope in the N-terminus region. All of these control mAbs worked as expected except LB12, which was slightly positive for SV3 (i.e., RH5 delta NL) and strongly positive for Bundle. Oxford is currently evaluating the aliquot of LB12 used in these experiments. All mAbs with the exception of BC8 appear to bind conformational epitopes on RH5.

Figure 4.6-3. ELISA data for purified mAbs from patient IDs A, B and C. Also shown on each graph are OD data for control mAbs (R5.016, h4BA7 and LB12). GIA assays have been carried out by the LDH method and the sixteen mAbs were initially screened at 2 mg/ml final concentration. The 7 (of the 9) mAbs that showed high levels of growth inhibition at 2 mg/ml were next tested for dose-response. Both of these data sets are shown in Figure 4.6-4. The

MVDP Quarterly Report 49 Leidos Proprietary mAb BD5 from the fractional dose Group 3 volunteer (Priority number 19) showed nearly 80% inhibition even at the lowest concentration tested (<0.1 mg/mL). Based on the ELISA data (Figure 4.6- 3) the mAb BD5 epitope seems to overlap that of R5.016.

Figure 4.6-4. GIA data for the sixteen purified mAbs from patient IDs A, B and C at 2 mg/ml (left panel) and for select mAbs in dose titration (right panel) In addition to the work proposed by Oxford, an immune repertoire survey of RH5.1 vaccinees was also proposed (summarized in the section below). The final decision on this work is pending analysis of the clinical study GIA data.

4.6.1 IMMUNE REPERTOIRE SURVEY OF SELECT VAC063 VACCINEES In Q4 FY2018, USAID requested “project synopses” for several of the proposed projects including the VAC063 Immune Repertoire Survey Project. Leidos (working with the University of Texas Austin and Oxford) provided the synopsis for this project to USAID in August 2018 and included this project in the FY2019 Annual Work Plan (submitted to USAID on September 1, 2018). In their response to the proposed annual work plan, USAID indicated that a decision regarding funding for this project will not be made until after receipt and review of the GIA data from the RH5.1/AS01 clinical study. A brief summary of the proposed project is provided below. This project aims to use novel sequencing technologies (Boutz et al., 2014; DeKosky et al., 2016; Lavinder et al., 2015; Lavinder et al., 2014; Saphire et al., 2018; Williams et al., 2017) to identify the B cell and IgG immune repertoires for select RH5.1 vaccinees from the VAC063 clinical study and would involve collaboration between the Draper lab at Oxford and Drs. Georgiou and Ippolito at the University of Texas Austin. The humoral response to vaccination involves the generation of short-lived plasmablasts that peak approximately 8 days after immunization and the generation of broadly diverse memory B cells and long-lived plasma cells that secrete high-affinity mAbs in the germinal centers of the lymph nodes. The three iterative germinal center processes of somatic hypermutation of germline variable region DNA sequences, clonal expansion and selection of high affinity B cell receptor variants are involved in the evolution of the humoral response to vaccination. The data generated through this project will help us understand the evolution of the humoral response in vaccinees in a cohort over time as well as before and after primary and/or secondary CHMI. In addition, the evolution of the humoral response can be compared between dose cohorts and between different immunization regimens. Through this approach, we may for example, identify clusters of antibody or BCR sequences that encode highly functional mAbs but are present in very low frequency in the vaccinees. Such information together with structural information on how these mAbs recognize the cryptic epitopes on RH5.1 could mean that the RH5.1 immunogen has the potential to be engineered in the future, to elicit greater expansion of these highly functional mAbs and thereby better protective efficacy. In discussions with USAID, they indicated that the decision to fund this project would not be made until after review of the VAC063 GIA data. There are no Q1 FY2019 updates to report for this section. Preliminary sample

MVDP Quarterly Report 50 Leidos Proprietary selections for this project also were discussed in the IPT meeting on March 18, 2019. In further discussions, USAID tasked Leidos with following up with Drs. Georgiou and Ippolito regarding the sample selection and the use of pre-immune samples in their work. Additional sample selection updates were received from Oxford and circulated to USAID on March 22, 2019. A priority list of 20 samples was approved by USAID on April 1, 2019. On May 1, 2019, Leidos met with UTA and Oxford to discuss the clinical study data and the Oxford sample selection. During this call, Leidos noted the importance of scoping the UTA project so that it could be completed within the prime contract POP. Based on this and the factors included below, UTA indicated that it would be best to proceed with assessment of the five fractional dose group volunteer samples in the approved priority list of 20 samples. Oxford concurred with this selection. The factors considered are as follows:

• A sufficient number of samples should be tested to obtain a complete picture of the anti-RH5 antibody responses for the specific question(s) asked.

• Sample selection should facilitate publication of the data.

• Based on timeframe, with receipt of the sample in June, the number of samples screened should be seven or less.

• As the highest levels of immunogenicity (ELISA and GIA) are seen in samples from the fractional dose group, UTA noted that they anticipate this group being the richest source of antibodies.

• Each sample selected should have 2 vials of cells available for shipment to UTA (Oxford later confirmed that there are sufficient cells for the five selected fractional dose group volunteers).

• Assessment of the challenge group samples is complicated by a number of factors, the biggest of which is that none of the volunteers had sterile protection. Further, volunteers in this group were vaccinated a different number of times (3 or 4) and had a different number of CHMI challenges (1 or 2). UTA noted that it would be quite challenging to dissect such a sample set in order to provide a complete picture and that this would not be feasible during the POP timeframe. The above information was communicated to USAID during the May 15, 2019 biweekly meeting and via email follow-up. On May 21, 2019, USAID communicated via email approval to move ahead with the UTA assessment of the 5 fractional dose samples. During the UTA procurement process, based on concerns regarding cost, USAID changed the procurement method from advanced notification to consent to subcontract (letter from USAID received June 9, 2019). Leidos provided the consent package to USAID on June 28, 2019 and noted that, based on schedule, consent would need to be received no later than July 19, 2019 in order for the project to be completed within the MVDP contract POP. Further, Leidos informed USAID of UTA’s confirmation that the informal cost per sample budget estimate provided by UTA during the project proposal phase was not properly burdened.

MVDP Quarterly Report 51

Leidos Proprietary technical work on this project was completed in Q2 FY2019. A draft of the PD1 Blockade Inhibitor Project Final Report was provided to USAID on April 11, 2019 and USAID provided feedback on May 7, 2019. Leidos circulated an updated version of the project report to USAID on May 18, 2019, which USAID approved on May 29, 2019. Leidos circulated the final version (PDF) of the final project report on June 10, 2019. A draft of the PD1 manuscript was provided to USAID on May 28, 2019 and USAID provided feedback on June 5, 2019. Leidos circulated the updated manuscript on June 10, 2019, which was submitted for USAID clearance (clearance received June 19, 2019). The manuscript has been submitted to PLOS ONE. The PD1 blockers used in these studies include, but are not limited to, commercially available PD1 or PDL1 mAb and several PD1 peptide blockers identified by Leidos Life Sciences. These PD1 peptide blockers (~20 amino acids) bind to both human and mouse PD1 and have shown efficacy in other experimental models. The three PD1 peptide blockers currently being tested in these studies are: I. PD1 peptide combo that consists of four separate PD1 peptide blockers; II. Single PD1 peptide blocker, LD01, that is microbiome-derived; and III. Single PD1 peptide that is a derivative of LD01, named LD02.

Table 4.8-1. Overview of the PD1 Blockade Adjuvant Project Plan

Phase Milestone Task

PD1 Blockade 1. Assess the effect of PD1 blockers on Immunogenicity and efficacy testing with Studies vaccine efficacy liver and blood stage antigen 2. Assess PD1 expression and T Adjuvant reg Determine whether PD1 blockers modify generation following immunization with or Formulation Studies PD1 expression and/or T without PD1 blockers regs

MVDP Quarterly Report 53 Leidos Proprietary

Table 4.8-2. Cohorts for AdPyCS + PD1 Blockade Immunogenicity Study

Cohort # Test Sample 1 AdPyCS alone 2 AdPyCS + PD1 Peptide Combo 3 AdPyCS + Ova Peptide 4 AdPyCS + LD01 Peptide 5 AdPyCS + LD02 Peptide 6 AdPyCS + anti-PD1 mAb 7 AdPyCS + anti-PDL1 mAb

MVDP Quarterly Report 54 Leidos Proprietary

blockade BALB/c mice (n = 4) were immunized with 60 ng RH5.1 protein in 250 μg Alhydrogel. All immunizations are administered IM into both hind limbs. At days 1, 3, 6 and 8 post-immunization mice were treated IP with 200 µg of peptide(s) (LD01, LD02 or combo) or anti-PD1 mAb (Table 4.8-4). As shown in Table 4.8-4 peptide(s) or anti-PD1 mAb treatment occurs either after each immunization, only after the first immunization or after the second and third immunization to identify the optimal dosing schedule. At days 27, 55 and 63 post-immunization mice were bled to assess Ab levels via ELISA and GIA respectively. At day 63 post-immunization T cell responses were assessed by measuring the number of splenic RH5.1-specific, IFN-γ secreting T cells (spot-forming units =SFU) using the ELISPOT assay after stimulation with pooled (n = 7) RH5 peptides (10 µg/mL (Table 4.8-5)) or RH5.1 protein (5 µg/ml). The study was completed in Q1 FY2018 and T cell and humoral immunity assessments were completed by Q4 FY2018. Results of this study are detailed in the FY2018 USAID MVDP Annual Report.

Table 4.8-4. Groups for RH5.1/Alum + PD1 Blockade Immunogenicity Study

MVDP Quarterly Report 55 Leidos Proprietary

Table 4.8-5. PfRH5 Specific Peptides Used to Stimulate T Cells

Epitope Sequence of Peptide Position in PfRH5

A7 TYDKVKSKCNDIKNDLIATI T216-I235

C6 NIANSIDILQEKEGHLDFVI N156-I175

C9 NLNKKMGSYIYIDTIKFIHK N396-K415

D9 YIDTIKFIHKEMKHIFNRIE Y406-E425

F6 KHLSYNSIYHKSSTYGKCIA K186-A205

F10 NDVPIKMEYFQTYKKNKPLT N506-T525

G10 DVPIKMEYFQTYKKNKPLTQ D507-Q526

MVDP Quarterly Report 56

Leidos Proprietary

5. ELEMENT 3 ACTIVITIES: AMY NOE/JESSICA SMITH

5.2.1 SCG MEETING READ AHEAD MATERIALS At the end of Q2 FY2019, Leidos provide 2019 SCG read-ahead materials to USAID for the following projects:

• CSP Vaccine Development Project

• Liver Stage Vaccine Development Project

• Blood Stage Epitope Vaccine Development Project

• RH5.1 Clinical Study

• RH5.1 Human mAb Identification and Development Project

• RCR Complex Vaccine Development Project

MVDP Quarterly Report 58 Leidos Proprietary

5.2.2 SCG MEETING PRESENTATIONS Per USAID request, during Q3 FY2019, three Leidos MVDP project presentations were prepared for the 2019 SCG meeting as follows:

• RH5.1 Clinical Study

• RH5.1 Human mAb Identification and Development Project

6. ELEMENT 4 ACTIVITIES

7. LEIDOS POINTS OF CONTACT

MVDP Quarterly Report 59 Leidos Proprietary

7.1. PROGRAM MANAGER Amy Noe, Ph.D., MBA Leidos Life Sciences 5202 Presidents Court Suite 110 Frederick, MD 21703-8398 Phone: 858-826-6105 Mobile: 858-201-9176

7.2. ACTING OPERATIONS MANAGER Jessica L. Smith Leidos Life Sciences 5202 Presidents Court Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0475

7.3. CONTRACTS MANAGER Casey Koontz Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-529-0435 Fax: 301-846-0794

8. APPENDIX 1: LITERATURE CITED

MVDP Quarterly Report 60 Leidos Proprietary

Brune, K.D., Howarth, M., 2018. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front Immunol 9, 1432. Brune, K.D., Leneghan, D.B., Brian, I.J., Ishizuka, A.S., Bachmann, M.F., Draper, S.J., Biswas, S., Howarth, M., 2016. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234. Crompton, P.D., Kayala, M.A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G.E., Molina, D.M., Burk, C.R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D.L., Liang, X., Doumbo, O.K., Miller, L.H., Doolan, D.L., Baldi, P., Felgner, P.L., Pierce, S.K., 2010. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A 107, 6958-6963. DeKosky, B.J., Lungu, O.I., Park, D., Johnson, E.L., Charab, W., Chrysostomou, C., Kuroda, D., Ellington, A.D., Ippolito, G.C., Gray, J.J., Georgiou, G., 2016. Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires. Proc Natl Acad Sci U S A 113, E2636-2645. Dent, A.E., Nakajima, R., Liang, L., Baum, E., Moormann, A.M., Sumba, P.O., Vulule, J., Babineau, D., Randall, A., Davies, D.H., Felgner, P.L., Kazura, J.W., 2015. Plasmodium falciparum Protein Microarray Antibody Profiles Correlate With Protection From Symptomatic Malaria in Kenya. J Infect Dis 212, 1429-1438. Gilbert, S.C., 2013. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 31, 4241-4246. Goepfert, P.A., Elizaga, M.L., Seaton, K., Tomaras, G.D., Montefiori, D.C., Sato, A., Hural, J., DeRosa, S.C., Kalams, S.A., McElrath, M.J., Keefer, M.C., Baden, L.R., Lama, J.R., Sanchez, J., Mulligan, M.J., Buchbinder, S.P., Hammer, S.M., Koblin, B.A., Pensiero, M., Butler, C., Moss, B., Robinson, H.L., Group, H.S., National Institutes of, A., Infectious Diseases, H.I.V.V.T.N., 2014. Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis 210, 99-110. Hekele, A., Bertholet, S., Archer, J., Gibson, D.G., Palladino, G., Brito, L.A., Otten, G.R., Brazzoli, M., Buccato, S., Bonci, A., Casini, D., Maione, D., Qi, Z.Q., Gill, J.E., Caiazza, N.C., Urano, J., Hubby, B., Gao, G.F., Shu, Y., De Gregorio, E., Mandl, C.W., Mason, P.W., Settembre, E.C., Ulmer, J.B., Craig Venter, J., Dormitzer, P.R., Rappuoli, R., Geall, A.J., 2013. Rapidly produced SAM((R)) vaccine against H7N9 influenza is immunogenic in mice. Emerg Microbes Infect 2, e52. Lavinder, J.J., Horton, A.P., Georgiou, G., Ippolito, G.C., 2015. Next-generation sequencing and protein mass spectrometry for the comprehensive analysis of human cellular and serum antibody repertoires. Curr Opin Chem Biol 24, 112-120. Lavinder, J.J., Wine, Y., Giesecke, C., Ippolito, G.C., Horton, A.P., Lungu, O.I., Hoi, K.H., DeKosky, B.J., Murrin, E.M., Wirth, M.M., Ellington, A.D., Dorner, T., Marcotte, E.M., Boutz, D.R., Georgiou, G., 2014. Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc Natl Acad Sci U S A 111, 2259-2264. Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., Yan, S.F., Williamson, K.C., Holder, A.A., Carucci, D.J., Yates, J.R., 3rd, Winzeler, E.A., 2004. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res 14, 2308-2318. Le Roch, K.G., Zhou, Y., Blair, P.L., Grainger, M., Moch, J.K., Haynes, J.D., De La Vega, P., Holder, A.A., Batalov, S., Carucci, D.J., Winzeler, E.A., 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-1508. Lindner, S.E., Swearingen, K.E., Harupa, A., Vaughan, A.M., Sinnis, P., Moritz, R.L., Kappe, S.H., 2013. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol Cell Proteomics 12, 1127-1143. Pascolo, S., 2008. Vaccination with messenger RNA (mRNA). Handb Exp Pharmacol, 221-235.

MVDP Quarterly Report 61 Leidos Proprietary

MVDP Quarterly Report 62