Virus-like particle vaccines targeting a key epitope in circumsporozoite protein provide sterilizing immunity against malaria

Abstract

Vaccines that target the pre-erythrocytic stage of the malaria lifecycle have the potential to provide sterilizing immunity but must elicit sustained, high-titer antibody responses to completely prevent infection. Most pre-erythrocytic vaccines target circumsporozoite protein (CSP), the major surface antigen on Plasmodium falciparum sporozoites. Antibodies targeting distinct epitopes within the central repeat region of CSP have the potential to provide protection from infection, but we have focused on developing vaccines that target a highly vulnerable CSP epitope that is targeted by the potent monoclonal antibody L9. In a previous study, we produced a pre-erythrocytic vaccine displaying a synthetic peptide representing the L9 epitope on Qβ bacteriophage virus-like particles (VLPs). This vaccine elicited strong anti-CSP antibody responses that protected mice from malaria challenge. Here, we asked whether the structural context of the L9 epitope influences the quality of antibody responses. We compared the immunogenicity and protective efficacy of Qβ L9 VLPs to recombinant VLPs that display the L9 peptide in a structure that is hypothesized to mimic its native conformation. Recombinant MS2 bacteriophage VLPs displaying various lengths of the L9 epitope were produced and immunogenicity and protective efficacy were evaluated in mice. Our results demonstrate that MS2 L9 VLPs, particularly those displaying longer L9 peptides and in combination with a potent novel adjuvant, elicit strong and durable antibody responses that lower malaria liver burden and prevent infection. We also compared the efficacy of L9-targeted vaccines to the licensed vaccine, RTS,S/AS01_E (Mosquirix™, GSK). Immunization with Qβ L9 VLPs, MS2 L9 VLPs, and RTS,S/AS01_E provided significant protection from liver-stage infection in a mouse model. Interestingly, immunization with a combination vaccine consisting of MS2 L9 and Qβ L9 VLPs, each presenting the L9 epitope in a distinct structural context, elicited sterilizing immunity in the highest percentage of mice.

Fig. 1. Characterization of MS2 L9 VLPs.

a Structure of the MS2 coat protein dimer (left) and the MS2 VLP (right). 90 coat protein dimers self-assemble into a VLP. The location of the AB-loop is highlighted in red on both structures. Images of the MS2 coat protein dimer and the MS2 VLP were generated by the authors using online tools available through the RCSB protein data bank. b PfCSP domains and the sequence of the junctional and minor repeat regions. The sequence of the three inserted L9 sequences and the location of the core NPNV epitopes (underlined) are shown. c SDS-PAGE analysis of wildtype MS2 VLPs and MS2 L9 VLPs displaying the 8, 15, and 27 amino acid L9 epitopes. The unmodified gel is shown in Supplementary Fig. 2. d Transmission electron microscopy (TEM) images of recombinant MS2 L9 VLPs. The scale bar (in white) represents 100 nm. e Binding of L9 mAb to MS2 L9 VLPs or wildtype MS2 VLPs, as measured by ELISA.

Fig. 2. MS2 L9 VLPs elicit strong anti-CSP antibody responses.

a Comparison of the immunogenicity of MS2 L9 VLPs displaying 8, 15, and 27 amino acid L9 epitopes. Groups of Balb/c mice (n = 5) were immunized intramuscularly with 5 µg of VLPs at weeks 0 and 3. Anti-CSP IgG end-point dilution titers were calculated by ELISA using sera collected one week after the second immunization. Results show antibody titers in individual mice, lines represent the geometric mean titer from each group. Statistical comparisons were performed using a one-way ANOVA with post-hoc analysis. b Mice immunized with MS2 L9(15aa) and MS2 L9(27aa) received a third immunization, given 7 weeks after the prime. Sera were obtained one week following the third immunization. Statistical comparisons between titers following the second dose and the third dose were performed using an unpaired t-test. c Anti-CSP antibody titers in mice immunized twice with unadjuvanted MS2 L9(15aa) VLPs compared to mice that received two doses MS2 L9(15aa) plus Cquim-MA adjuvant and mice that received a Qβ L9 prime (plus Cquim-MA) followed by an MS2 L9(15aa) boost (also with Cquim-MA). Sera were obtained one week following the second immunization. Statistical comparisons were performed using a one-way ANOVA with post-hoc analysis (Tukey’s test).

Fig. 3. Longevity of antibody responses.

a Groups of Balb/c mice (n = 5) were immunized intramuscularly with 5 µg of VLPs at weeks 0, 3, and 7 and were measured following each immunization and at 38–47 weeks post-prime. Anti-CSP IgG titers were calculated by end-point dilution ELISA. Geometric means plus SEM are shown at each timepoint. b Mice that received vaccine plus Cquim-MA adjuvant received two immunizations, at weeks 0 and 3.

Fig. 4. Both Qβ L9 and MS2 L9(15aa) VLPs plus Cquim-MA elicit strong antibody responses and protect against malaria infection.

a Experimental timeline. C57BL/6 mice were immunized three times at weeks 0, 3, and 6, followed by a challenge with five Pb-PfCSP-Luc-infected mosquitoes at week 8. Liver luminescence was measured 42 h after the mosquito challenge, and blood smears were taken starting on day 3 post-infection. b Anti-CSP antibody levels in serum obtained following the third vaccination, prior to challenge. Horizontal lines represent the mean anti-CSP antibody concentration for each group. Groups are compared by 2-tailed unpaired t test. c Parasite liver burden measured via luminescence. Horizontal lines indicate geometric mean luminescence for each group. Statistical comparisons were performed using a one-way ANOVA in which experimental groups were compared to every other group. Multiple comparisons were corrected using the Bonferroni adjustment. d Protection from blood-stage infection, as measured by percent of blood parasite-free mice post-challenge. A log-rank test (correcting for multiple comparisons by controlling the False Discovery Rate using the two-stage step-up method of Benjamini, Krieger and Ykutieli) was used to statistically compare vaccinated groups to naïve, infected mice.

Fig. 5. A combination vaccine targeting the L9 epitope provides the strongest protection from malaria infection.

a Anti-CSP antibody levels in serum obtained following the third vaccination. Horizontal lines represent the mean anti-CSP antibody concentration for each group. No significant differences were observed between groups (p > 0.05, unpaired t-test). b Parasite liver burden measured via luminescence. Horizontal lines indicate geometric mean luminescence for each group. Statistical comparisons were performed using a one-way ANOVA in which experimental groups were compared to every other group. Multiple comparisons were corrected using the Bonferroni adjustment. c Protection from blood-stage infection, as measured by percent of blood parasite-free mice post-challenge. A log-rank test (correcting for multiple comparisons by controlling the False Discovery Rate using the two-stage step-up method of Benjamini, Krieger and Yekutieli) was used to statistically compare vaccinated groups to naïve, infected mice.