In a recent study published in NPJ vaccines, researchers evaluated the immunogenicity of two vaccine candidates, Pfs25 and PfCSP, based on the messenger ribonucleic acid (mRNA)-lipid nanoparticle (LNP) platform.
Study: mRNA-LNP expressing PfCSP and Pfs25 vaccine candidates targeting infection and transmission of Plasmodium falciparum. Image Credit: Chinnapong/Shutterstock
Background
Malaria, caused by the parasite Plasmodium falciparum, is a fatal disease claiming close to 627,000 lives per year worldwide. There is an urgent need to develop vaccines for the eradication of malaria.
The malaria-causal parasite has a complex life cycle presenting multiple stages, each of which female anopheline mosquitoes could ingest during blood feeding, leading to P. falciparum transmission. For instance, if an infected Anopheles mosquito starts the malaria infection by injecting P. falciparum sporozoites into the host, which invades its hepatocytes resulting in a blood-stage infection.
Thus, malaria vaccine development has focused on antigens expressed during various stages of its life cycle. Any transmission-blocking response (TBR) by a vaccine (TBV) intervenes with both sexual- and mosquito stages is crucial in malaria eradication efforts.
About the study
In the present study, researchers assessed the antibody responses to Pfs25 and PfCSP separately and in combination using a mouse model. First, they vaccinated female Balb/c mice with three, 10, and 30 μg doses of Pfs25 mRNA-LNP and evaluated the elicited antibody responses. Likewise, they assessed the antibody responses elicited by the PfCSP mRNA-LNP vaccine in mice at similar dosages.
Next, the researchers vaccinated a group of mice with a combination of both vaccines (10 μg of each vaccine) to explore the immunogenicity of co-immunization with antigens targeting different life cycle stages of P. falciparum. They also compared the antigen-specific antibody titers due to co-immunization with antibody titers in mice vaccinated with only Pfs25 or PfCSP.
Further, the team performed mosquito standard membrane feeding assays (SMFA) to evaluate immunoglobulin G (IgG) in serum from final bleeds from each mouse. To this end, they evaluated mice vaccinated with 3 μg and 10 μg Pfs25 mRNA-LNP thrice and also compared mice that received 30 μg Pfs25 only with recipients of (Pfs25+PfCSP) mRNA-LNP combination post-four vaccinations.
SMFA also helped the researchers evaluate the functional efficacy of the antibodies elicited by lower doses of the Pfs25 mRNA-LNP vaccine. They assessed purified IgG at one, 0.5, 0.25, and 0.125 mg/ml of final concentrations. IgG purified from pre-vaccination sera served as a negative control in this experiment. They also characterized the antibody isotype (IgG1/IgG2a ratio) and avidity in the sera used for the SMFA.
Initially, all three concentrations of purified IgG isolated from all the groups that received 3 μg, 10 μg, and 30 μg of Pfs25 and Pfs25+PfCSP revealed >94% TRA. In subsequent experiments, they evaluated lower concentrations of IgG (0.25, 0.125, 0.0625, 0.03125 mg/ml). They used enzyme-linked immunosorbent assay (ELISA) to evaluate Pfs25-specific antibody avidity and its isotypes in pooled serum of terminal bleeds of each mouse group.
Finally, the researchers evaluated the protection conferred by PfCSP in an in vivo model using sporozoites of P. berghei, a transgenic organism expressing PfCSP and PbPfCSP-green fluorescent protein-luciferase (GFPLuc). During the first challenge (Ch1), they inoculated mice with ~2000 PbPfCSP-GFPLuc sporozoites four weeks post-third vaccination and evaluated their liver stage parasite burden 42 to 44 hours later.
Results
The PfCSP mRNA LNP attacked circumsporozoite proteins, and Pfs25 targeted a transmission-blocking vaccine (TBV) antigen. From doses ranging between 0.1 μg and 30 μg, Pfs25 mRNA-LNPs elicited antibody responses when administered in a three-dose vaccination regimen four weeks apart. After priming, the antibody responses were modest but increased substantially post-boosting.
The researchers also noted decreased germinal center B cells in the spleen following one Pfs25 dose, which increased markedly post-boosting. In both mice strains used in the study, Balb/c, and C57Bl/6, Pfs25 triggered a robust cluster of differentiation (CD)4 and CD8 T cell responses. Overall, antibodies to Pfs25 mediated the transmission-blocking response. However, how elevation in these T cells might be impacting TRA and TBA warrants more work.
SMFAs revealed strong transmission reversing and blocking activities (TRA and TBA) amid low IgG concentrations by all three doses of Pfs25 mRNA-LNP. Pfs25 doses as low as 0.1 μg elicited substantial TRA, which enhanced the effect of higher vaccine doses.
Vaccination with subsequently higher doses of Pfs25 mRNA-LNP provided strong and functional TBA (>90%) at 0.125 mg/ml IgG concentration. Although TRA and TBA activities of 10μg Pfs25 and 1 μg Pfs25 were comparable, post-boosting, the 10 μg Pfs25 mRNA-LNPs induced almost 2.5 higher antibody titers than the one μg dose. Thus, in the case of Pfs25 mRNA-LNP, a booster dose invariably invoked stronger functional activity.
The parasite challenge did not modulate antibody avidity and isotypes markedly. However, interestingly, mice vaccinated with 30 μg vaccine(highest dose tested) had superior antibody titers with less effective TRA. Also, the researchers had no clue if the active infection caused antibodies to lose potency in SMFA.
On the contrary, PfCSP mRNA-LNP elicited modest antibody responses following one vaccination only and increasingly strong responses after each following dose(s) or boosting. Four doses made immunized mice completely protected against parasite challenges. The co-immunization of Pfs25 and PfCSP induced similar antibody responses and functional activity as a single antigen mRNA-LNP.
Thus, providing evidence of the feasibility of combining multiple mRNA-LNP platform-based vaccines with no adverse consequences. Although PfCSP alone could not confer complete protection, in combination with Pfs25 mRNA-LNP, its efficacy showed considerable enhancements over multiple generations.
Conclusions
The current study showed that the mRNA-LNP platform-based two vaccines, Pfs25 and PfCSP, targeting the malarial antigens, conferred adequate immune protection. The former vaccine type elicited potent neutralizing activity as low-dose vaccination and short prime-boost regimes. PfCSP, on the other hand, required multiple booster vaccination or a heterologous protein boost to provide complete protection in mice.
Nevertheless, a vaccine targeting sporozoite will prevent the development of gametocytes of the parasite in an infected person; TBV would prevent the sexual reproduction of the gametocytes. The combination of both vaccines targeting the infection and sexual stages could effectively interrupt P. falciparum transmission, which is critical for malaria eradication.
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