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COVID-19 Antigen

COVID-19 Antigen

1 Recombinant protein , a proven approach against coronavirus pandemics

Jafar Amani

2 Introduction

• In December 2019, a cluster of patients with pneumonia surfaced in Wuhan, China.

• SARS-CoV-2 is a positive-strand RNA that belongs to the group of Betacoronaviruses.

• The of SARS-CoV-2 is approximately 29,700 nucleotides long and shares 79.5% sequence identity with SARS-CoV.

• The 3end of the genome encodes 4 major structural proteins, including the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein

3 4 Introduction

• SARS-CoV-2 binds to the receptor angiotensin converting 2 (ACE2) on host cell for the virus entry and subsequent pathogenesis. • resulting in severe respiratory illness with symptoms of fever, cough, and shortness of breath, and severe cases can be fatal.

5 6 SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) has accumulated multiple mutations during its global circulation. Recently, three SARS-CoV-2 lineages, B.1.1.7 (501Y.V1),B.1.351 (501Y.V2) and B.1.1.28.1 (P.1), have emerged in the United Kingdom, South Africa and Brazil, respectively. 7 Structural features of coronavirus SARS-CoV-2 spike protein: Targets for

• Fig. 1. Structural features of the SARS-CoV-2 spike (S) protein. (A) Ribbon diagram of the homotrimeric S, adopted from [19], (with permission from Elsevier Inc.). (B) Side view of the prefusion structure of S, with a single RBD in open (“up”) conformation (green), adopted from [18], (with permission from Science.org). (C) Top view of the prefusion structure of S, with two single RBDs in closed (“down”) conformation (white and grey) and one single RBD in open (“up”) conformation (green), adopted from [18], (with permission from Science.org). (D) Single monomer of S, with the RBD in closed (“down”) conformation (green), adopted from [18], (with permission from Science.org). (E) Single monomer of S, with the RBD in open (“up”) conformation (green), adopted from [18], (with permission from Science.org). (For interpretation of the references to colour in this figure legend, the reader is referred to • 8 9 SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications Notable non-synonymous mutations and deletions (D) in S, envelope (E), ORF8, nucleocapsid (N) proteins of SARS-CoV-2 501Y.V1, 501Y.V2 (*) and P.1 (#) variants. Co-occurring mutations other than D614G (S protein) and P323L (ORF1ab) are shown here. Protein names are shown in parenthesis. Table adapted from Portelli et al. and the Public Health England report. References for modulation in hACE2 affinity or resistance (shown as superscripts) are provided in supplementary information.

10 MD Simulation Analysis of S Protein with ACE2 Receptors

Binding interactions and energy of Spike proteins with ACE-2. (A) The number of hydrogen bonds between the wild type and mutant (N501Y and N440K) spike protein and ACE2 during the simulation. (B) Wild type spike–ACE2 interactions in the average structure extracted from MD simulations, N501 (circled) making hydrophobic contact (hydrogen bonds are shown with green dots and non-polar interactions with magenta and brick semicircle). (C) N501Y–ACE2 interactions in the average structure. (D) Molecular mechanics energies combined with the generalized Born and surface area continuum solvation (MM/GBSA) binding free energy of spike proteins with ACE2. 11 Different Types of SARS-CoV-2 Vaccines under Development

12 DRAFT landscape of COVID-19 candidate vaccines – 14 May 2021

13 Subunit Vaccines candidate • Full-length S-protein based vaccines • RBD-based vaccines • Multi-epitope vaccines

Protein production technologies • Escherichia coli • • Mammalian cell culture expression systems • Insect cells

14 Subunit Vaccines • the University of Queensland is developing a subunit • vaccine based on the “molecular clamp” technology. • Clover Biopharmaceuticals Inc. revealed that • they are developing a vaccine candidate against SARS- CoV-2 using the “Trimer-Tag” technology, • and the trimeric S protein candidate was produced via a mammalian cell expression • system. Novavax, Inc. announced that they had produced multiple nanoparticle vaccine candidates • based on S protein, and now is assessing efficacy in animal models to identify an optimal vaccine candidate for human testing. • Besides, Johnson & Johnson, , and Chongqing Zhifei Biological Products Co., Ltd. also started subunit vaccine development against SARS-CoV- 2.

15 . Spike Protein (S Protein)

• S protein is currently the most promising antigen formulation for SARS-CoV-2 vaccine research. • First, it is surface exposure and thus is able to be directly recognized by host . • Second, it mediates the interaction with host cell by binding to the receptor ACE2, which is essential for subsequent virus entry to target cells and causing subsequent pathogenicity. Finally, the homologue proteins were already used for vaccine development against SARS- CoV and MERS-CoV, and were proved to be effective. 16 . Spike Protein (S Protein) • The monomer of S protein from SARS-CoV-2 contains 1273 amino acids, with a molecular weight of about 140 kDa. The S protein contains two subunits (S1 and S2). The S1 subunit can be further defined with two domains termed the N-terminal domain (NTD) and the C-terminal domain (CTD). The receptor binding domain (RBD) located in the CTD. • S2 subunit contains the basic elements required for membrane fusion, including an internal membrane fusion peptide (FP), So far, the potential fragments of S protein for use as in vaccine development include the full-length S protein, the RBD domain, the S1 subunit, NTD, and FP.

17 Subunit COVID-19 vaccine (Spike Protein) SARS-CoV-2 rS/Matrix M1-Adjuvant (Full length recombinant SARS CoV-2 Phase glycoprotein nanoparticle vaccine 2 Day 0 + 21 IM Novavax 3 adjuvanted with Matrix M)

SARS-CoV-2 vaccine formulation 1 with adjuvant 1 (S protein (baculovirus Phase 2 Day 0 + 21 IM + GSK production) 1/2

SCB-2019 + AS03 or CpG 1018 adjuvant plus Alum adjuvant (Native like Clover Biopharmaceuticals Phase 2 Day 0 + 21 IM Trimeric subunit Spike Protein vaccine) Inc./GSK/Dynavax 2/3

Medigen Vaccine Biologics + MVC-COV1901 (S-2P protein + CpG Dynavax + National Institute Phase 2 Day 0 + 28 IM 1018) of Allergy and Infectious 2 (NIAID) COVAX-19® Recombinant spike Phase 1 Day 0 IM Vaxine Pty Ltd. protein + adjuvant 1 IM , recombinant spike Day 0 + 21 Razi Vaccine and Serum Phase 3 and protein +51 Research Institute 1 IN

18 RBD

• Since the RBD of S protein directly interacts with the ACE2 receptor on host cells, RBD induced specific may block this recognition and thus effectively prevent the invasion of the virus. As a matter of fact, most of SARS-CoV-2 subunit vaccines currently under development use RBD as the antigen. Moreover, the RBD domain was also used in the development of SARS-CoV and MERS-CoV vaccines.

19 Subunit COVID-19 vaccine (RBD Protein)

Day 0 + 28 or Anhui Zhifei Longcom Biopharmaceutical Recombinant SARS-CoV-2 vaccine 2-3 Day 0 + 28 + IM + Institute of Microbiology, Chinese Phase 3 (CHO Cell) 56 Academy of Sciences Phase KBP-COVID-19 (RBD-based) 2 Day 0 + 21 IM Kentucky Bioprocessing Inc. 1/2 FINLAY-FR anti-SARS-CoV-2 Vaccine 2 Day 0 + 28 IM Instituto Finlay de Vacunas Phase 2 (RBD + adjuvant) RBD (baculovirus production expressed in Sf9 cells) 2 Day 0 + 28 IM West China Hospital + Sichuan University Phase 2 Recombinant SARS-CoV-2 vaccine (Sf9 Cell) AdimrSC-2f (recombinant RBD +/- ND ND ND Adimmune Corporation Phase 1 Aluminium) Day 0 + 14 + Center for and Phase CIGB-669 (RBD+AgnHB) 3 28 or Day 0 IN Biotechnology (CIGB) 1/2 +28 + 56 Day 0 + 14 + CIGB-66 (RBD+aluminium Center for Genetic Engineering and Phase 3 28 or Day 0 IM hydroxide) Biotechnology (CIGB) 1/2 +28 + 56 University Medical Center Groningen + Phase SARS-CoV-2-RBD-Fc fusion protein SC or IM Akston Biosciences Inc. 1/2

20 NTD

• Similar to RBD, the N-terminal domains (NTD) of S protein from several coronaviruses were • reported to show carbohydrate receptor- binding activity. For example, the NTD of spike protein form transmissible gastroenteritis virus (TGEV) was reported to bind sialic acid via NTD. The carbohydrate-binding properties of IBV M41 strain are also related to the NTD of the S protein. Thus, this domain is also a candidate antigen for vaccine development.

21 S1 Subunit

• The S1 subunit, which contains both RBD and NTD described above, is mainly involved in the S • protein binding to the host receptor. It is also widely used in vaccine development. • Wang et al. reported that MERS-CoV S1 protein formulated with MF59 adjuvant protected hDPP4 transgenic mice against lethal virus challenge, and the protection correlated well with the neutralizing antibody titer. • Adney et al. confirmed that immunization with adjuvanted S1 protein reduced and delayed virus • shedding in the upper respiratory tract of dromedary camels and complete protection was observed in alpaca against MERS-CoV challenge.

22 FP

• The FP domain of the S2 subunit is involved in the membrane fusion of the virus, which is also a key step in viral pathogenicity [32]. Therefore, it may also serve as a vaccine candidate antigen. At present, Tianjin University has constructed an RBD-FP fusion protein, and high titer of antibodies was detected in mice immunized with this fusion protein, and the effcacy is under evaluation.

23 Nucleocapsid Protein (N Protein)

• The N protein is the most abundant protein in coronavirus, and it is normally highly conserved, • with a molecular weight of about 50 kDa. N protein has multiple functions including formation of nucleocapsids, signal transduction virus budding, RNA replication, and mRNA transcription. • This protein was reported to be highly antigenic, 89% of patients who developed SARS, produced antibodies to this antigen

24 Membrane Protein (M Protein)

• M protein is a trans-membrane glycoprotein with a molecular weight of about 25 kDa and • is involved in virus assembly, and this protein is the most abundant protein on the surface of • SARS-CoV. It was reported that immunization with the full length of M protein is able to elicit efficient neutralizing antibodies in SARS patients

25 Envelope Protein (E Protein)

• Compared with S, N, and M protein, E protein is not suitable for use as an immunogen. For one • reason, it consists of 76–109 amino acids in • different coronaviruses with channel activity, thus the immunogenicity is limited. Studies have shown that SARS-CoV E protein is an important virulence factor, and the secretion of inflammatory factors IL-1, TNF, and IL-6 are significantly reduced after knocking out E protein

26 Synthetic Peptide or Epitope Vaccine • These vaccines contain only certain fragments of intact antigens and are usually prepared by chemical synthesis techniques. They are easier in preparation and quality control. • Generex Biotechnology announced that they are working with third-party groups to generate peptide vaccines against pandemic using the patented NuGenerex Immuno-Oncology • Ii-Key technology that uses synthetic peptides in mimic essential protein regions from a virus that is • chemically linked to the 4-amino acid Ii-Key to ensure robust immune system activation.

27 Multi-epitope vaccines

IMP CoVac-1 (SARS-CoV-2 Phase HLA-DR peptides) 1 Day 0 SC University Hospital Tuebingen 1 UB-612 (Multitope peptide based S1-RBD-protein based Phase 2 Day 0 + 28 IM COVAXX + United Biomedical Inc vaccine) 2/3

EpiVacCorona (EpiVacCorona 2 Day 0 + 21 IM Federal Budgetary Research Phase vaccine based on peptide Institution State Research Center 1/2 antigens for the prevention of and Biotechnology of COVID-19) ""Russia

28 Adjuvant • In addition to live attenuated vaccines and live vector vaccines, adjuvants are required to enhance the immune response in the development of other types of vaccines. • (1) classic aluminum • (2) MF59, MF59 is an oil-in-water emulsion composed of Tween 80, sorbitol trioleate, and squalene, and it has already been used in flu vaccines in Europe and the United States. • (3) Adjuvant system (AS) series adjuvants, which are a series of adjuvants developed by GlaxoSmithKline (GSK), including AS01, AS02, AS03, and AS04. • DL-α-tocopherol, squalene oil, Tween80.

29 Vaccine delivery

• Parenteral vaccination • Mucosal vaccination • Outlook for alternative vaccine delivery systems

30 How the Novavax Vaccine Works

• The Maryland-based company Novavax has developed a protein-based coronavirus vaccine called NVX-CoV2373. In March the company announced an efficacy rate of 96 percent against the original coronavirus, 86 percent against the B.1.1.7 variant and 49 percent against the B.1.351 variant. • May Novavax launches clinical trials for their vaccine. • July The U.S. government awards Novavax $1.6 billion to support the vaccine’s clinical trials and manufacturing. • August Novavax launched a Phase 2 trial on 2,900 people in South Africa. • September Novavax launches a Phase 3 trial with up to 15,000 volunteers in the United Kingdom. The trial is expected to deliver results in early 2021.

31 How the Anhui Zhifei Longcom Vaccine Works • In December, Longcom began enrollment of a Phase III randomized, double- blind, placebo-controlled for 29,000 participants, including 750 participants between 18-59 and 250 participants 60 and older in China and 21,000 participants between 18-59 and 7,000 participants 60 and older outside China.[16][17] • In December, Malaysia's MyEG announced it would conduct Phase III trials. If the trials were successful, MyEG would be the sole distributor of ZF2001 in Malaysia for 3 years.[4] • In December, Uzbekistan began a year-long Phase III trial of ZF2001 with 5,000 volunteers between 18 and 59.[18][19] • In December, Ecuador's Minister of Health, Juan Carlos Zevallos announced Phase III trials would involve between 5,000 and 8,000 volunteers.[20] • In February, Pakistan's Drug Regulatory Authority (DRAP) approved Phase III trials with approximately 10,000 participants to be conducted at UHS Lahore, National Defense Hospital, and Agha Khan Hospital.[21] • Discussions to begin Phase III trials are also underway in Indonesia • No efficacy data has been made available from Phase III trials for Zhifei Longcom's ZF2001 vaccine, which requires three doses when used on its own.

32 Vaccination strategies to combat novel corona virus SARS-CoV-2

33 Immuno-informatics approach for B-cell and T-cell epitope based design against novel COVID-19 virus • Protein sequence retrieval Variability analysis of spike surface glycoprotein Prediction of antigenicity of spike surface glycoprotein B-cell epitope prediction Surface accessible regions prediction Antigenicity prediction of selected B-cell epitopes • Prediction of T cell epitopes and conservancy analysis The physiochemical and solubility properties analysis Assembled vaccine tertiary configuration projection Refinement and validation of putative vaccine Molecular dynamics (MD) simulation Codon optimization and in silico vaccine expression 34 Designing a conserved peptide-based subunit vaccine against SARS-CoV-2 using immunoinformatics approach

35 Multiepitope Subunit Vaccine Design against COVID-19 Based on the Spike Protein of SARS-CoV-2: An In Silico Analysis

36 Linear B-cell Epitopes Prediction Results

37 Surface accessibility of the SARS-CoV-2

38 Cytotoxic T lymphocytes (CTL) epitopes prediction

39 Helper T lymphocytes (HTL) epitopes prediction

40 Schematic Presentation of the final Multi-epitope Vaccine

41 Graphical Representation of the Secondary Structure Features.

42 Vaccine 3D Structure Modeling

43 Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralizing antibodies in COVID-19 patients • Patient serum and plasma fractions. Linear peptide library. Peptide-based ELISA. Peptides S14P5 and S21P2 ELISA for 41 COVID-19 patients. Peptide affinity depletion of pooled sera. Cell lines and cell culture. SARS-CoV-2 and SARS-CoV pseudotyped lentivirus production. Pseudotyped lentivirus neutralization assay. Live SARS-CoV-2 neutralisation assay in BSL3. Data visualization and statistical analysis. 44 COVID-19 patient sera recognize two linear epitopes in SARS-CoV-2 spike protein

45 Antibodies against S14P5 and S21P2 can neutralize SARSCoV-2

46 How Long Will a Vaccine Really Take?

47 48 49

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