Attenuated PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection

Abstract

A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 105 PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35-87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls (P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36-99%) remained without parasitemia compared with none of six nonvaccinated controls. PfSPZ-specific T-cell and antibody responses were detected in all vaccine recipients. Cytokine production by T cells from vaccinated subjects after in vitro stimulation with homologous (NF54) or heterologous (7G8) PfSPZ were highly correlated. Interestingly, PfSPZ-specific T-cell responses in the blood peaked after the first immunization and were not enhanced by subsequent immunizations. Collectively, these data suggest durable protection against homologous and heterologous Pf parasites can be achieved with PfSPZ Vaccine. Ongoing studies will determine whether protective efficacy can be enhanced by additional alterations in the vaccine dose and number of immunizations.

Keywords: T-cell immunology; live-attenuated vaccine; malaria; plasmodium; sporozoite.

Conflict of interest statement

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 and owner of PfSPZ Vaccine and the investigational new drug (IND) application sponsor of the clinical trials. S.L.H. and B.K.L.S. have a financial interest in Sanaria Inc. All other authors declare no conflict of interest.

Figures

Fig. 1.

Efficacy against homologous and heterologous CHMI. Kaplan–Meier curves showing the percentage of volunteers remaining without parasitemia after CHMI with homologous Pf3D7 (A) or heterologous Pf7G8 (B). Six controls were enrolled for each CHMI. In A, nine (64%) of 14 vaccinated volunteers (black, solid line) and none of six controls (red, dashed line) remained without parasitemia. In B, five (83%) of six vaccinated volunteers (black, solid line) and none of six controls (red, dashed line) remained without parasitemia. Only vaccinated volunteers who remained without parasitemia after CHMI with the homologous strain, Pf3D7, underwent repeat CHMI with the heterologous strain, Pf7G8.

Fig. 2.

PfSPZ-specific antibody, CD8, and CD4 responses. (A) Antibody responses to PfCSP 2 wk after each vaccination and at the time of homologous (3D7) and heterologous (7G8) CHMI. Subjects who were parasitemic (red, dashed line and red squares) or not parasitemic (black, solid line and black circles) are shown. (B) Antibody responses to whole PfSPZ (NF54) compared with PfCSP responses. Spearman’s r was calculated using data from the three points combined: n = 15 for study week 18 (black circles), n = 14 for study week 35 (red squares), n = 6 for study week 49 (blue triangles). (C) Frequency of PfSPZ-specific cytokine-producing memory CD8 T cells. Results are the percentage of memory CD8 T cells producing IFN-γ, IL-2, and/or TNF-α after PfSPZ stimulation minus the percentage of cells after stimulation with vaccine diluent (1% human serum albumin in media). (D) Frequency of PfSPZ-specific cytokine-producing memory CD4 T cells. Results are calculated as in C. (E) Representative flow cytometry gating of the T-cell activation markers HLA-DR and CD38 over time showing memory CD4 T cells from a vaccinated subject without additional in vitro antigen stimulation. (F) CD4 T-cell activation in vivo. Results are the percentage of memory CD4 T cells expressing HLA-DR and CD38, as measured on PBMCs without antigen stimulation. (G) Correlation of CD4 T-cell activation in vivo and frequency of PfSPZ-specific memory CD4 T cells. The y axis shows the change in percentage of memory CD4 T cells expressing HLA-DR and CD38 from prevaccine to 2 wk after vaccination #1. The x axis shows the percentage of memory CD4 T cells expressing IFN-γ, IL-2, and/or TNF-α determined by intracellular cytokine staining after ex vivo stimulation with PfSPZ. Black circles denote the frequency 2 wk after vaccination #1; gray diamonds denote the frequency prevaccination. Spearman’s r was calculated using postvaccination data. For A, C, D, and F, n = 14 for study week 0 through 35 and n = 6 at week 49. Arrowhead designates PfSPZ Vaccine administration, arrows designate time of CHMI with 3D7 or 7G8. For weeks 0–35, data are stratified by outcome after homologous CHMI at study week 35. Data at week 49 are stratified by outcome after heterologous CHMI at study week 49. Individual points represent one subject. Line is geometric mean (A) or mean (C, D, F).

Fig. 3.

PfSPZ-specific γδ T-cell responses. (A) Flow cytometry gating showing γδ T-cell activation in vivo. PBMCs were stained as in Fig. 2E. (B) γδ T-cell activation in vivo. Results are the percentage of memory γδ T cells expressing HLA-DR and CD38 as measured on PBMCs without antigen stimulation. (C) Frequency of Vδ2+ T cells after immunization. Results are expressed as fold-change from the prevaccine frequency. For each graph, n = 14 for study week 0 through 35 (time of homologous CHMI) and n = 6 at week 49 (time of heterologous CHMI). Arrowhead designates PfSPZ Vaccine administration, arrows designate time of CHMI with 3D7 or 7G8. For B and C, data points are stratified as Fig. 2A, and lines are mean (B) and geometric mean (C).

Fig. 4.

T-cell responses to PfSPZ NF54 and 7G8. (A) Memory CD8 T-cell cytokine production after in vitro PfSPZ 7G8 antigen stimulation compared with PfSPZ NF54. Results are the percentage of memory CD8 T cells producing IFN-γ, IL-2, and/or TNF-α after stimulation with PfSPZ 7G8 (y axis) or PfSPZ NF54 (x axis) after subtracting the percentage of cytokine-producing cells in PBMCs incubated with the vaccine diluent (1% human serum albumin in media). (B) Percentage of memory CD8 T cells expressing the activation marker 4-1BB after PfSPZ antigen stimulation as in A. (C) Memory CD4 T-cell cytokine responses as in A. (D) Percentage of memory γδ T cells expressing 4-1BB after PfSPZ antigen stimulation as in B. n = 14 for each point. Each symbol represents an individual volunteer. Blue circles are samples 2 wk after vaccination #1, and gray squares are prevaccine samples. Blue line is linear regression on postvaccination sample percentages. P value is by Spearman’s correlation on postvaccination sample percentages.