Malaria bivalent viral vectored vaccine protects against Plasmodium falciparum and vivax and blocks parasite transmission

Abstract

Malaria remains a major infectious disease, with Plasmodium falciparum and Plasmodium vivax often co-endemic, requiring a dual-target vaccine for adequate control. We previously developed monovalent vaccines against P. falciparum or P. vivax using vaccinia virus LC16m8Δ (m8Δ) and adeno-associated virus type 1 (AAV1). Here, we demonstrate the efficacy of a novel bivalent malaria vaccine against P. falciparum and P. vivax. The m8Δ vaccine harbors two gene cassettes encoding Pfs25-PfCSP and Pvs25-PvCSP fusion proteins, while the AAV1 vaccine includes two recombinant AAV1s carrying one of these cassettes as a mixture. Heterologous m8Δ-prime and AAV1-boost immunization provided 70% protection against both PfCSP/Pb and PvCSP/Pb transgenic sporozoites. Moreover, a membrane feeding assay using P. vivax isolates from infected patients in the Brazilian Amazon showed 90% transmission-blocking efficacy. The bivalent vaccine outperformed monovalent combinations, maintaining immune responses for over 7 months, and shows promise for malaria control and elimination.

Fig. 1. Construction of the recombinant bivalent vaccine.

The a m8Δ-Pf(s25-CSP) and b m8Δ-Pv(s25-CSP) monovalent vaccines. c The m8Δ-Pf/Pv(s25-CSP) bivalent vaccine. The Pv(s25-CSP-VK210/247) gene cassette was introduced into the m8∆-Pf genome to generate m8∆-Pf/Pv. The mPH5 and P7.5 promoters drive the expression of the pvs25-pvcsp fusion gene and pfs25-PfCSP fusion gene cassette in m8∆. The d AAV1-Pf(s25-CSP) and e AAV1-Pv(s25-CSP-VK210/247) monovalent vaccines. The CMV promoter drives the expression of the pvs25-PvCSP and pfs25-PfCSP fusion gene cassettes in AAV (b). HA hemagglutinin gene, A46R A46R region gene, S mouse IgGκ signal peptide sequence, P7.5 7.5 promoter, mPH5 modified H5 promoter, pCMV CMV immediate early promoter, F FLAG epitope tag, G6S GGGGGS hinge sequence, G VSV-G TM, WPRE woodchuck hepatitis virus posttranscriptional regulatory element.

Fig. 2. Evaluation of the antibody titer.

BALB/c mice (n = 10 per group) were immunized with a monovalent vaccine (m8Δ/AAV1-Pf or m8Δ/AAV1-Pv), the bivalent vaccine (m8Δ/AAV1-Pf/Pv), or the mixed vaccine (m8Δ/AAV1-Pf + m8Δ/AAV1-Pv). a–d Sera were collected 1 day before and 4 weeks after the boost immunization. Antibody titers against PfCSP (a) Pfs25 (b) PvCSP-VK210 (c) Pvs25 (d) and PvCSP-VK247 (e) are shown. Each data point represents a single mouse, and horizontal lines indicate the median of the antibody titers ± interquartile range. In (a) and (c), closed circles represent protected mice, while open circles represent infected mice. P values were calculated by Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig. 3. Evaluation of long-term antibody titers.

Bivalent vaccine (m8Δ/AAV1-Pf/Pv) immunized BALB/c mice (n = 10 per group), sera were collected weekly for up to 224 days. Antibody titers against PfCSP (a) Pfs25 (b) PvCSP-VK210 (c) and Pvs25 (d) after the last immunization with AAV1-boost are shown. The data points indicate the median of the antibody titers ± interquartile range.

Fig. 4. Protective efficacy against sporozoite challenge.

BALB/c mice were immunized with the bivalent vaccine (m8Δ/AAV1-Pf/Pv) or the mixed vaccine (m8Δ/AAV1-Pf + m8Δ/AAV1-Pv). a Experiment I: Mice were challenged with PfCSP/Pb sporozoites 28 days after immunization with the bivalent vaccine (n = 10), the mixed vaccine (n = 10), or PBS (n = 10). b The surviving mice in (a) (bivalent vaccine group; n = 4, mixed vaccine group; n = 7) were rechallenged with PvCSP-VK210/Pb sporozoites at 56 days (28 days after initial challenge) and compared with naïve mice (n = 10). c Experiment II: Mice were challenged with PvCSP-VK210/Pb sporozoites 35 days after immunization with the bivalent vaccine (n = 10), the mixed vaccine (n = 10), or PBS (n = 10). d The surviving mice in (c) (bivalent vaccine group; n = 5, mixed vaccine group; n = 7) were rechallenged with PfCSP/Pb sporozoites at 63 days (28 days after initial challenge) and compared with naïve mice (n = 10). In all experiments, parasitemia was monitored daily from day 4 after the challenge up to day 14. The p-values were calculated by log-rank (Mantel–Cox) tests comparing the vaccine groups with the control group (PBS or naive) or between the vaccine groups. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Fig. 5. Transmission-blocking efficacy of the bivalent vaccine against P. falciparum, as determined using a DFA.

Transmission-blocking efficacy against P. falciparum in mice immunized with m8Δ/AAV1-Pf/Pv bivalent vaccine at day 28 (n = 4) after the last immunization. Each data point represents the oocyst number from a single blood-fed mosquito, and horizontal lines indicate the mean number (blue dot, naïve mouse; red dot, immunized mouse). The mean ± standard deviation, % of infected mosquitoes, number of mosquitoes, % parasitemia, and % gametocytemia are summarized below each mouse ID.

Fig. 6. Transmission-blocking efficacy of the bivalent vaccine against P. vivax, as determined using a DMFA (short-term regimen).

BALB/c mice (n = 5) were immunized with the m8Δ/AAV1-Pf/Pv bivalent vaccine, and sera were collected 28 days after the final immunization. Pooled sera were tested using the DMFA methodology with six P. vivax isolates (isolate nos. 1–6). Oocyst intensity per midgut is shown. Each data point represents the oocyst number from a single blood-fed mosquito (blue dot, control; red dot, immune sera of different dilutions). The black scale represents the serum dilution: 1:50—highest dilution; 1:5—lowest dilution; 1:10—intermediate dilution. The p values were calculated using the Kruskal–Wallis test followed by Dunn’s multiple comparisons (*p < 0.05; **p < 0.01; ***; p < 0.001; ****p < 0.0001).

Fig. 7. Transmission-blocking activity of the bivalent vaccine against P. vivax, as determined using a DMFA (long-term regimen).

Oocyst intensity per midgut at day 232 post-boost using sera from immunized BALB/c mice (n = 4), tested against four P. vivax isolates. Red dots represent immune sera; blue dots represent control sera. Each point reflects a single mosquito midgut.