Attenuated PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection Kirsten E. Lykea,1, Andrew S. Ishizukab,1, Andrea A. Berrya, Sumana Chakravartyc, Adam DeZureb, Mary E. Enamab, Eric R. Jamesc, Peter F. Billingsleyc, Anusha Gunasekerac, Anita Manojc, Minglin Lic,d, Adam J. Rubenc, Tao Lic, Abraham G. Eappenc, Richard E. Staffordc,d, Natasha KCc,d, Tooba Murshedkarc, Floreliz H. Mendozab, Ingelise J. Gordonb, Kathryn L. Zephirb, LaSonji A. Holmanb, Sarah H. Plummerb, Cynthia S. Hendelb, Laura Novikb, Pamela J. M. Costnerb, Jamie G. Saundersb, Nina M. Berkowitzb, Barbara J. Flynnb, Martha C. Nasone, Lindsay S. Garverf, Matthew B. Laurensa, Christopher V. Plowea, Thomas L. Richiec, Barney S. Grahamb, Mario Roedererb, B. Kim Lee Simc,d, Julie E. Ledgerwoodb, Stephen L. Hoffmanc,2, and Robert A. Sederb,2,3 aCenter for Vaccine Development and Division of Malaria Research, Institute for Global Health, University of Maryland School of Medicine, Baltimore,MD 21201; bVaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; cSanaria Inc., Rockville, MD 20850; dProtein Potential LLC, Rockville, MD 20850; eBiostatistics Research Branch, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and fEntomology Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910 Edited by Louis H. Miller, NIH, Rockville, MD, and approved January 19, 2017 (received for review September 27, 2016) A live-attenuated malaria vaccine, Plasmodium falciparum sporo- schedule showed a 26% and 36% reduction in clinical malaria zoite vaccine (PfSPZ Vaccine), confers sterile protection against among 6–12-wk-olds and 5–17-mo-olds, respectively, through 3–4y controlled human malaria infection (CHMI) with Plasmodium falci- of follow-up (7). Therefore, alternative vaccine approaches are parum (Pf) parasites homologous to the vaccine strain up to 14 mo needed that confer durable and sterile protection against homolo- after final vaccination. No injectable malaria vaccine has demon- gous and heterologous strains. strated long-term protection against CHMI using Pf parasites het- In contrast to recombinant subunit vaccine approaches, at- erologous to the vaccine strain. Here, we conducted an open-label tenuated PfSPZ immunization consistently achieves high-level × 5 trial with PfSPZ Vaccine at a dose of 9.0 10 PfSPZ administered i.v. (>80%) sterile protection against CHMI with homologous Pf three times at 8-wk intervals to 15 malaria-naive adults. After CHMI parasites (8–11). In addition, short-term protection (<8 wk) with homologous Pf parasites 19 wk after final immunization, nine – against a heterologous CHMI with the Pf7G8 clone was dem- (64%) of 14 (95% CI, 35 87%) vaccinated volunteers remained with- > out parasitemia compared with none of six nonvaccinated controls onstrated in five subjects after exposure to 1,000 irradiated (P = 0.012). Of the nine nonparasitemic subjects, six underwent re- PfSPZ-infected mosquitoes (10, 12). Therefore, high-level peat CHMI with heterologous Pf7G8 parasites 33 wk after final im- munization. Five (83%) of six (95% CI, 36–99%) remained without Significance parasitemia compared with none of six nonvaccinated controls. PfSPZ-specific T-cell and antibody responses were detected in all A highly effective malaria vaccine capable of long-term pro- vaccine recipients. Cytokine production by T cells from vaccinated tection against genetically diverse strains is urgently needed. subjects after in vitro stimulation with homologous (NF54) or het- Here, we demonstrate that a three-dose regimen of a live at- erologous (7G8) PfSPZ were highly correlated. Interestingly, PfSPZ- tenuated whole-parasite malaria vaccine conferred durable specific T-cell responses in the blood peaked after the first immuni- sterile protection through 33 weeks in ∼50% of subjects against MEDICAL SCIENCES zation and were not enhanced by subsequent immunizations. a controlled human malaria infection strain that is heterologous Collectively, these data suggest durable protection against homologous to the vaccine strain. Prior studies by others and us have shown and heterologous Pf parasites can be achieved with PfSPZ Vaccine. that T cells are critical to mediating sterile protection after live- Ongoing studies will determine whether protective efficacy can be attenuated malaria vaccination. Here, we provide evidence that enhanced by additional alterations in the vaccine dose and number this Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine) of immunizations. induces antigen-specific IFN-γ-producing CD8 and CD4 T cells that recognize both the homologous and the heterologous Pf strain. plasmodium | live-attenuated vaccine | T-cell immunology | malaria | sporozoite Author contributions: K.E.L., A.S.I., S.C., M.C.N., T.L.R., B.S.G., M.R., B.K.L.S., J.E.L., S.L.H., and R.A.S. designed research; K.E.L., A.S.I., A.A.B., S.C., A.D., M.E.E., E.R.J., P.F.B., A.G., A.M., M.L., A.J.R., T.L., A.G.E., R.E.S., N.K., T.M., F.H.M., I.J.G., K.L.Z., L.A.H., S.H.P., C.S.H., L.N., P.J.M.C., alaria caused by Plasmodium falciparum (Pf) is a major J.G.S., N.M.B., B.J.F., M.C.N., L.S.G., M.B.L., C.V.P., T.L.R., B.S.G., B.K.L.S., J.E.L., S.L.H., and Mcause of morbidity and mortality, particularly in children in R.A.S. performed research; K.E.L., A.S.I., B.K.L.S., S.L.H., and R.A.S. contributed new sub-Saharan Africa (1, 2). Travelers, military personnel, and reagents/analytic tools; K.E.L., A.S.I., S.C., A.D., M.E.E., A.J.R., M.C.N., T.L.R., M.R., B.K.L.S., international health care workers are also at risk (3, 4). The ideal J.E.L., S.L.H., and R.A.S. analyzed data; and K.E.L., A.S.I., S.L.H., and R.A.S. wrote the paper. vaccine would confer on individuals high-level, sterile protection Conflict of interest statement: S.C., E.R.J., P.F.B., A.G., A.M., M.L., A.J.R., T.L., A.G.E., R.E.S., N.K., T.M., T.L.R., B.K.L.S., and S.L.H. are salaried employees of Sanaria Inc., the developer against infection with Pf and would facilitate elimination efforts and owner of PfSPZ Vaccine and the investigational new drug (IND) application sponsor by interrupting parasite transmission (5, 6). of the clinical trials. S.L.H. and B.K.L.S. have a financial interest in Sanaria Inc. All other The most extensively studied candidate malaria vaccine, RTS,S/ authors declare no conflict of interest. AS01, is composed of a truncated form of the main sporozoite This article is a PNAS Direct Submission. (SPZ) surface protein (circumsporozoite protein, PfCSP), and an 1K.E.L. and A.S.I. contributed equally to this work. immune adjuvant (AS01). To our knowledge, this vaccine has never 2S.L.H. and R.A.S. contributed equally to this work. been tested using controlled human malaria infection (CHMI) with 3To whom correspondence may be addressed. Email: [email protected]. a heterologous Pf strain of parasites. However, a phase 3 efficacy This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. trial in Africa using a four-dose regimen on a 0-, 1-, 2-, and 20-mo 1073/pnas.1615324114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1615324114 PNAS | March 7, 2017 | vol. 114 | no. 10 | 2711–2716 durable sterile protection against heterologous CHMI has not 12 d for the controls (P < 0.0001, controls vs. all vaccine recip- been achieved with any injectable malaria vaccine (10, 13). ients by log-rank; SI Appendix, Table S4). Results Vaccine Efficacy, Heterologous CHMI. To assess the durability of Study Design. We recently showed that four i.v. immunizations vaccine efficacy against heterologous CHMI, six of the nine with PfSPZ Vaccine at a dose of 2.7 × 105 PfSPZ conferred vaccinated subjects who were not parasitemic after homologous ∼55% sterile protection after homologous CHMI up to 14 mo CHMI underwent CHMI by the bite of mosquitoes infected with after the final immunization (14). On the basis of the favorable 7G8, a clone of Brazilian origin (22), ∼33 wk after final vacci- safety profile and dose-dependent durable efficacy against ho- nation. Five (83%) of six vaccine recipients (95% CI, 36–99%) mologous CHMI (11, 14), we continued PfSPZ Vaccine dose and none of the six nonvaccinated controls remained without B escalation to 9.0 × 105 PfSPZ per dose. A three-dose regimen at parasitemia after heterologous CHMI (Fig. 1 ). Microsatellite 8-wk intervals was selected on the basis of data showing that an analysis of blood-stage parasites from vaccinated and non- 8-wk interval between immunizations resulted in enhanced im- vaccinated individuals confirmed that the parasites detected af- munogenicity in nonhuman primates (11). ter heterologous CHMI were genetically distinct from the 3D7 reference clone (SI Appendix, Fig. S4). Study Population. Thirty-one volunteers were enrolled: 15 vaccine recipients, 12 CHMI controls, and four back-up controls (SI Ap- Antibody Responses. Antibody responses against PfCSP after im- pendix,Fig.S1). Baseline demographic characteristics are shown in munization with PfSPZ Vaccine administered at lower doses SI Appendix, Table S1. Fifteen volunteers (100%) completed all have been shown to be a sensitive measure of vaccine immuno- genicity, have functional activity in vitro and in vivo, and may be three scheduled vaccinations. Fourteen volunteers underwent useful as an immune correlate of protection (14).