A mosquito AgTRIO mRNA vaccine contributes to immunity against malaria

Abstract

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. To prevent malaria, vaccination is the most effective strategy and there is an urgent need for new strategies to enhance current pathogen-based vaccines. Active or passive immunization against a mosquito saliva protein, AgTRIO, contributes to protection against Plasmodium infection of mice. In this study, we generated an AgTRIO mRNA-lipid nanoparticle (LNP) and assessed its potential usefulness as a vaccine against malaria. Immunization of mice with an AgTRIO mRNA-LNP generated a robust humoral response, including AgTRIO IgG2a isotype antibodies that have been associated with protection. AgTRIO mRNA-LNP immunized mice exposed to Plasmodium berghei-infected mosquitoes had markedly reduced initial Plasmodium hepatic infection levels and increased survival compared to control mice. In addition, as the humoral response to AgTRIO waned over 6 months, additional mosquito bites boosted the AgTRIO IgG titers, including IgG1 and IgG2a isotypes, which offers a unique advantage compared to pathogen-based vaccines. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

Fig. 1. AgTRIO mRNA-LNP immunization generates a robust IgG response against recombinant AgTRIO and salivary gland extracts.

a Experiment scheme showing groups of C57BL/6 female mice injected with 10 μg of AgTRIO mRNA-LNP or control mRNA (Luc mRNA-LNP), and boosted twice, at two-week intervals. b Two weeks after each immunization, mice were bled. 1:2,500 and 1:12,500 dilutions of sera were examined for AgTRIO-specific IgG antibodies by ELISA using recombinant AgTRIO as the antigen. c 1:2,500 dilution of sera collected after the final were examined for AgTRIO-specific IgG antibodies against salivary gland extracts by ELISA. (Median ± IQR, p < 0.05 using the Mann Whitney U-test) d 1:2,500 dilution of sera were used to determine AgTRIO-specific IgG1, IgG2a and IgG2b. 1:500 dilution of sera were examined for AgTRIO specific IgG3 antibodies.

Fig. 2. Immunization with AgTRIO mRNA-LNP reduces the initial Plasmodium berghei infection level and offers protection in mice.

C57BL/6 mice were immunized with 10 µg of AgTRIO mRNA-LNP or control mRNA (Luc) three times. 3 weeks after last boost, the immunized mice were exposed to 3 P. berghei-infected mosquitoes. a After 40 h, livers were dissected, and the Plasmodium infection level was determined by RT-PCR. (Median ± IQR, p < 0.05 using the Mann Whitney U-test). b In a separate experiment with mice vaccinated in the same way and exposed to infected mosquito bites, the parasitemia levels were determined at day 5 post infection. (Median ± IQR, p < 0.05 using the Mann Whitney U-test). c Kaplan–Meier survival curves were used to present the percent of uninfected mice. Infection was determined as 0.01% of parasitemia by flow cytometry. p = 0.036 by Log rank test between the two groups.

Fig. 3. Mosquito bites can maintain AgTRIO-specific IgG, including IgG2a antibodies, after AgTRIO mRNA-LNP immunization.

A group of mice was injected with 10 μg of AgTRIO mRNA-LNP or control mRNA (Gluc mRNA-LNP) and boosted twice over 4 weeks. Sera were collected at 10, 18, and 25 weeks after the beginning of the experiments. a 1:2,500 dilution of sera were examined for total AgTRIO-specific IgG antibodies by ELISA. b 1:2,500 dilution of sera were used to determine AgTRIO specific IgG1, IgG2a, and IgG2b. 1:500 dilution of sera were examined for AgTRIO specific IgG3 antibodies. (Median ± IQR, p < 0.05 using the Wilcoxon matched-pairs signed rank test). Red bar: exposed to 10 mosquitoes weekly after 18 weeks for three times. Blue bar: non-exposure.