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. 2024 Apr 30:15:1372584.
doi: 10.3389/fimmu.2024.1372584. eCollection 2024.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study

Yutaro Yamamoto  1 , Camila Fabbri  2   3 , Daiki Okuhara  1 , Rina Takagi  1 , Yuna Kawabata  1 , Takuto Katayama  1 , Mitsuhiro Iyori  4 , Ammar A Hasyim  1 , Akihiko Sakamoto  1 , Hiroaki Mizukami  5 , Hisatoshi Shida  6 , Stefanie Lopes  2   3 , Shigeto Yoshida  1
Affiliations

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study

Yutaro Yamamoto et al. Front Immunol. .

Abstract

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Keywords: LC16m8Δ; Plasmodium vivax; PvCSP; Pvs25; adeno-associated virus; malaria; vaccine.

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Conflict of interest statement

Authors SY, HS, HM, and MI are credited as inventors of patents concerning viral-vectored malaria vaccines 2022-24221. HS is also credited as an inventor on a pending patent related to LC16m8Δ WO 2005/054451 A1. However, neither of these products has been brought to market. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Construction of the m8Δ/AAV1 vaccine. (A) The gene cassette encoding the Pvs25-PvCSP fusion protein is shown; the chimeric pvcsp gene encoding amino acids 19–373 of PvCSP VK210 (i.e., lacking the N-terminal signal peptide and C-terminal glycosylphosphatidylinositol anchor sequences) and the three repeat sequence units (a, GDRADGQPA; b, GDRAAGQPA; c, GAGNQPGAN) of PvCSP VK247 are fused to the C-terminus of Pvs25. (B) The gene cassette was introduced into the AAV1 and m8Δ genomes to generate AAV1-Pv(s25-CSP-VK210/247) and m8Δ-Pv(P7.5-s25-CSP-VK210/247)-HA, respectively. Expression of the pvcsp-pvs25 fusion gene cassette was driven by the CMV promoter in AAV1 and by the P7.5 promoter in m8Δ. HA, hemagglutinin gene; S, gp64 signal sequence; P7.5, 7.5 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. (C–E) Analysis of Pvs25-PvCSP fusion protein expression in HEK293T cells transduced with AAV1-Pv(s25-CSP-VK210/247) (MOI = 105) or m8Δ-Pv(P7.5-s25-CSP-VK210/247)-HA (MOI = 5). Cells were lysed and loaded onto a 10% SDS-PAGE gel and immunoblotted with anti-PvCSP VK210 mAb 2F2 (C), anti-PvCSP VK247 mAb 2E10E9 (D), or anti-Pvs25 mAb N1-1H10 (E). (F, G) Localization of the Pvs25-PvCSP fusion protein in mammalian cells after transduction with AAV1-Pv(s25-CSP-VK210/247) in HEK293T cells (F) or with m8Δ-Pv(P7.5-s25-CSP-VK210/247)-HA in RK13 cells (G). (F) After 24 h, the cells were fixed with paraformaldehyde and blocked with 10% normal goat serum. Then, the cells were incubated with R-Phycoerythrin LK23-conjugated anti-PvCSP VK210 mAb 2F2 (red), Fluorescein LK01-conjugated anti-PvCSP VK247 mAb 2E10E9 (green), and HiLyte Flour™ 647 LK13-conjugated anti-Pvs25 mAb N1-1H10 (light blue). Cell nuclei were visualized with DAPI (blue). Original magnification, 100×. Scale bars = 200 µm. (G) After 24 h, the infected cells formed plaques and were directly immunostained with R-Phycoerythrin LK23-conjugated anti-PvCSP VK210 mAb 2F2 (red), Fluorescein LK01-conjugated anti-PvCSP VK247 mAb 2E10E9 (green), and HiLyte Flour™ 647 LK13-conjugated anti-Pvs25 mAb N1-1H10 (light blue). Original magnification, 400×. Scale bar = 20 μm.
Figure 2
Figure 2
Durability of antibody responses. BALB/c mice (n = 20) were immunized with the m8Δ/AAV1-Pv(s25-CSP-VK210/247) vaccine. (A–C) Sera were collected 1 day before the boost immunization (shown as “prime”) and 4 weeks after the boost immunization (shown as “boost”). Antibody titers against PvCSP-VK210 (A), PvCSP-VK247 (B), and Pvs25 (C) are shown. Each datapoint represents a single mouse, and horizontal lines indicate the median of antibody titers ± interquartile range. Differences between prime and boost were calculated by the Mann–Whitney U-test. ****p < 0.0001; ns, not significant. (D, E) BALB/c mice (n = 5) were immunized and sera were collected weekly up to 248 days. Antibody titers against PvCSP-VK210 (D) and Pvs25 (E) are shown. The data points indicate the median of antibody titers ± interquartile range.
Figure 3
Figure 3
Protective efficacy against sporozoite challenge. BALB/c mice were immunized with the m8Δ/AAV1-Pv(s25-CSP-VK210/247) vaccine. (A, B) Experiment I: Mice were challenged with PvCSP-VK210/Pb sporozoites 28 days after immunization with m8Δ/AAV1 (n = 10) or PBS (n = 10) (A). The surviving mice in (A) (n = 10) were rechallenged at 63 days (35 days after initial challenge) and compared with naïve mice (n = 10) (B). (C, D) Experiment II: Mice were challenged with PvCSP-VK210/Pb sporozoites 242 days after immunization with m8Δ/AAV1 (n = 5) or PBS (n = 5) (C). The surviving mice in (C) (n = 3) were rechallenged at 277 days (35 days after initial challenge) and compared with naïve mice (n = 10) (D). Data of naïve control mice in (B) and (D) were identical in the rechallenge experiments (B, D) conducted on the same day. (E) Experiment III: Mice were challenged with PvCSP-VK247/Pb sporozoites 28 days after immunization with m8Δ/AAV1 (n = 10) or PBS (n = 10). In all experiments, parasitemia was monitored daily from day 4 after challenge up to day 14. P-values were calculated by log-rank (Mantel-Cox) tests versus the control group (PBS or naive). ****p < 0.0001, **p < 0.01.
Figure 4
Figure 4
Transmission-blocking activity for P. vivax using DMFA. BALB/c mice (n = 5) were immunized with the m8Δ/AAV1-Pv(s25-CSP-VK210/247) vaccine and sera were collected 28 days after the final immunization. Pooled sera were tested using a direct membrane feeding assay (DMFA) involving four P. vivax isolates from Brazilian patients (Isolate Nos. 1–4). 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). Horizontal lines indicate the mean number. The mean ± standard deviation, % of infected mosquitoes and number of mosquitoes are summarized below each P. vivax isolate. P-values were calculated using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test for oocyst intensity of the immunized sample versus the control. *p < 0.05, ***p < 0.001, ****p < 0.0001.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was partially supported by a Grant-in-Aid for Young Scientists (B) (JSPS KAKENHI grant number 21K16317), a Fostering Joint International Research grant (A) (JSPS KAKENHI grant number 21KK0295) to YY, a Grant-in-Aid for Scientific Research (B) (JSPS KAKENHI grant number 19H03458) to SY, JSPS Bilateral Joint Research Projects (grant number JPJSBP120205704) to SY, and a Grant-in-Aid for Scientific Research (C) (JSPS KAKENHI grant numbers 18K06655 and 21K06559) to MI. The research was also supported by the Global Health Innovative Technology Fund (grant number GHIT T2021-256). The funding sources played no role in study design, the collection, analysis, or interpretation of data, or publication.