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

Study Design.

We recently showed that four i.v. immunizations with PfSPZ Vaccine at a dose of 2.7 × 10⁵ PfSPZ conferred ∼55% sterile protection after homologous CHMI up to 14 mo after the final immunization (14). On the basis of the favorable safety profile and dose-dependent durable efficacy against homologous CHMI (11, 14), we continued PfSPZ Vaccine dose escalation to 9.0 × 10⁵ PfSPZ per dose. A three-dose regimen at 8-wk intervals was selected on the basis of data showing that an 8-wk interval between immunizations resulted in enhanced immunogenicity in nonhuman primates (11).

Study Population.

Thirty-one volunteers were enrolled: 15 vaccine recipients, 12 CHMI controls, and four back-up controls (SI Appendix, Fig. S1). Baseline demographic characteristics are shown in SI Appendix, Table S1. Fifteen volunteers (100%) completed all three scheduled vaccinations. Fourteen volunteers underwent CHMI with homologous Pf parasites; one vaccine recipient withdrew before CHMI to attend medical school.

Vaccine Safety.

Vaccinations were well tolerated. Solicited local and systemic adverse events (AEs) were generally graded as none or mild (SI Appendix, Table S2). After the third immunization, one subject had a temperature of 39.3 °C (graded as severe), which coincided with an acute viral illness. All unsolicited AEs reported during the vaccination period were classified as unrelated to the vaccine. Alanine aminotransferase levels measured 14 d after vaccination were not elevated in any vaccine recipient (SI Appendix, Fig. S2). There were no serious AEs attributed to vaccination.

Vaccine Efficacy, Homologous CHMI.

Vaccine efficacy was first assessed by CHMI with the Pf clone 3D7 by mosquito bite 19 wk after final immunization. The vaccine is composed of the NF54 strain, which is likely of African origin (15). 3D7 is a clone from the NF54 strain, and thus is homologous to the vaccine (16). 3D7 has been used to evaluate efficacy in the majority of malaria vaccine trials using CHMI (10, 17⇓⇓⇓–21). Nine (64%) of 14 vaccine recipients (95% CI, 35–87%) and none of the six nonvaccinated controls remained without parasitemia after homologous CHMI (P = 0.012) (Fig. 1A and SI Appendix, Table S3). In the five vaccinated volunteers who became parasitemic, the time to first positive malaria PCR ranged from 13 to 15 d compared with 9 to 12 d for the controls (P < 0.0001, controls vs. all vaccine recipients by log-rank; SI Appendix, Table S4).

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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.

Vaccine Efficacy, Heterologous CHMI.

To assess the durability of vaccine efficacy against heterologous CHMI, six of the nine vaccinated subjects who were not parasitemic after homologous CHMI underwent CHMI by the bite of mosquitoes infected with 7G8, a clone of Brazilian origin (22), ∼33 wk after final vaccination. Five (83%) of six vaccine recipients (95% CI, 36–99%) and none of the six nonvaccinated controls remained without parasitemia after heterologous CHMI (Fig. 1B). Microsatellite analysis of blood-stage parasites from vaccinated and nonvaccinated individuals confirmed that the parasites detected after heterologous CHMI were genetically distinct from the 3D7 reference clone (SI Appendix, Fig. S4).

Antibody Responses.

Antibody responses against PfCSP after immunization with PfSPZ Vaccine administered at lower doses have been shown to be a sensitive measure of vaccine immunogenicity, have functional activity in vitro and in vivo, and may be useful as an immune correlate of protection (14). After the first vaccination, low-level PfCSP IgG responses were detected in 13/14 subjects [Fig. 2A; geometric mean (GM), 213.5]. Such responses were substantially boosted ∼40-fold after the second vaccination (14/14 subjects with PfCSP responses; GM, 9,336), with modest boosting after the third vaccination (GM, 13,066). There was a trend toward higher antibody responses among subjects who remained without parasitemia after homologous CHMI [GM, 15,858 (not parasitemic) vs. 8,717 (parasitemic)]. This comparison did not reach the threshold for statistical significance (P = 0.23) with this sample size (nine not parasitemic vs. five parasitemic). We also measured antibody responses against whole PfSPZ by the automated immunofluorescence assay and showed that they were highly concordant with PfCSP antibody levels (Spearman’s r = 0.8787; P < 0.0001; Fig. 2B and SI Appendix, Tables S5 and S6). Finally, we measured the ability of serum to inhibit SPZ invasion of hepatocytes in vitro as a functional assessment of antibodies. Inhibitory activity increased significantly after the three immunizations compared with the preimmunization serum (P = 0.0012; SI Appendix, Fig. S4). These data showed that at doses of 9.0 × 10⁵ PfSPZ, malaria-specific antibody responses were induced in all subjects, but did not easily discriminate between protected and unprotected subjects (SI Appendix, Table S7).

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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).

CD8 and CD4 T-Cell Responses.

CD8 T cells are the primary cellular effectors that are critical for mediating protection after whole-SPZ vaccination in mice and nonhuman primates (23⇓–25). Here, after PfSPZ vaccination, the frequency of PfSPZ-specific cytokine (IFN-γ, IL-2, or TNF-α) producing memory CD8 T-cell responses peaked after the first immunization [0.38 ± 0.12% (mean ± SEM); P = 0.002 compared with prevaccine; Fig. 2C], as assessed by intracellular cytokine staining (ICS) after in vitro stimulation with PfSPZ. Of note, IFN-γ⁺ CD8 T-cell responses declined markedly after the second immunization and were not different from prevaccine (background) levels 2 wk after the third immunization (P = 0.12). The frequency of cytokine-producing PfSPZ-specific memory CD4 T cells also peaked after the first immunization [1.55 ± 0.38% (mean ± SEM); Fig. 2D]. Such responses declined to 0.59 ± 0.08% and 0.51 ± 0.07% after the second and third immunizations, respectively. CD8 T-cell responses were detected in 11/14 volunteers, and CD4 T-cell responses were detected in 14/14 volunteers. The magnitude of the total PfSPZ-specific memory T-cell cytokine responses did not significantly differ among vaccinated subjects who did or did not develop parasitemia after homologous CHMI at the assessed points (SI Appendix, Table S7).

Analysis of activation markers HLA-DR and CD38 on T cells without ex vivo antigen stimulation has been used as a sensitive measure of T-cell activation in vivo after immunization (Fig. 2E) (26, 27). The frequency of HLA-DR⁺CD38⁺ memory CD4 T cells increased by 1.69 ± 0.42 percentage points (mean ± SEM; P < 0.001; Fig. 2F) 2 wk after first immunization compared with baseline (prevaccine). The mean increase was similar to the level of memory CD4 T cells that were determined to be PfSPZ-specific by the ICS assay (Fig. 2D; i.e., 1.55%), and individual responses were highly correlated (Spearman’s r = 0.8462; P = 0.0003; Fig. 2G), suggesting the ICS assay used to assess the frequency of PfSPZ-specific T-cell responses after in vitro stimulation with PfSPZ accurately reflects the absolute magnitude of in vivo CD4 T-cell activation by the first PfSPZ vaccination. Interestingly, although circulating PfSPZ-specific CD4 T cells were detected at >0.5% by in vitro PfSPZ stimulation after the second and third vaccination, the frequency of HLA-DR⁺CD38⁺ memory CD4 T cells 2 wk after second and third immunizations were only 0.17 ± 0.15 percentage points (mean ± SEM) and 0.25 ± 0.18 percentage points, respectively, above the prevaccination frequencies. These findings indicate that the second and third vaccinations resulted in a dramatic reduction in in vivo activation of circulating PfSPZ-specific CD4 T cells compared with the first vaccination.

γδ T-Cell Responses.

Prior studies have shown that γδ T cells expand after immunization with PfSPZ Vaccine, and the frequency of the Vδ2⁺ subfamily of γδ T cells is a potential immune correlate of protection (11, 14). Here, we extend these findings and show that γδ T cells have a high level of in vivo activation after vaccination, as measured by HLA-DR⁺CD38⁺ coexpression (Fig. 3A). Activation peaked at a mean of 34% (compared with 6.4% at prevaccination) 2 wk after second immunization (Fig. 3B). The frequency of circulating Vδ2⁺ T cells increased after each vaccination (Fig. 3C). The frequency was 2.8-fold higher than prevaccination, and this increase persisted through follow-up (2.5-fold increase at the time of CHMI with 3D7).

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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).

T-Cell Responses Against 7G8.

Because we observed protection against homologous and heterologous CHMI in subjects that received only the homologous NF54 vaccine strain, we assessed the PfSPZ-specific T-cell cytokine producing responses against the heterologous 7G8 strain and compared them with responses against the homologous NF54 vaccine strain. There was a significant correlation in the magnitude of CD8 T-cell cytokine responses to NF54 and 7G8 PfSPZ (Spearman r = 0.8052; P = 0.0005; Fig. 4A) 2 wk after the first immunization, when such responses were measurable at detectable frequencies. CD8 T-cell responses were also assessed by expression of the TNF-receptor superfamily member 4-1BB (a marker of T-cell activation also known as CD137). This marker has been used to identify antigen-specific CD8 T cells after ex vivo antigen stimulation (28). Using this alternative assessment, CD8 T-cell responses were also highly correlated (Spearman’s r = 0.9033; P < 0.0001; Fig. 4B). CD4 T-cell cytokine responses were significantly correlated between the two strains (Fig. 4C). Finally, γδ T-cell activation by the two strains (as measured by 4-1BB) was also highly correlated (Fig. 4D). Expression of 4-1BB in the γδ T-cell compartment in response to PfSPZ was evident in the prevaccine sample, as expected, as this lineage of cells is prevalent before vaccination, albeit at lower absolute frequencies than found after vaccination.

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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.