Development of a bivalent conjugate vaccine candidate against malaria transmission and typhoid fever

Abstract

Immune responses to poorly immunogenic antigens, such as polysaccharides, can be enhanced by conjugation to carriers. Our previous studies indicate that conjugation to Vi polysaccharide of Salmonella Typhi may also enhance immunogenicity of some protein carriers. We therefore explored the possibility of generating a bivalent vaccine against Plasmodium falciparum malaria and typhoid fever, which are co-endemic in many parts of the world, by conjugating Vi polysaccharide, an approved antigen in typhoid vaccine, to Pfs25, a malaria transmission blocking vaccine antigen in clinical trials. Vi-Pfs25 conjugates induced strong immune responses against both Vi and Pfs25 in mice, whereas the unconjugated antigens are poorly immunogenic. Functional assays of immune sera revealed potent transmission blocking activity mediated by anti-Pfs25 antibody and serum bactericidal activity due to anti-Vi antibody. Pfs25 conjugation to Vi modified the IgG isotype distribution of antisera, inducing a Th2 polarized immune response against Vi antigen. This conjugate may be further developed as a bivalent vaccine to concurrently target malaria and typhoid fever.

Keywords: Conjugate vaccine; Malaria; Pfs25; Transmission blocking vaccine; Typhoid fever; Vi capsular polysaccharide.

Fig. 1.

Anti-Pfs25 antibody titers and transmission blocking activity of immune sera. (A) Anti-Pfs25 ELISA antibody titers on Days 42 and 83 for immune sera from mice vaccinated with unconjugated Pfs25, Vi conjugates of Pfs25, and EPA-Pfs25 formulated in PBS. (B) Anti-Pfs25 antibody titers on Days 42 and 91 for immune sera from mice vaccinated with Vi conjugates of Pfs25 and EPA-Pfs25 formulated in Alhydrogel®. ELISA data were analyzed with Prism software (GraphPad Software, Inc., La Jolla, CA) and statistical differences between groups were measured using a Kruskal-Wallis analysis followed by a Dunn multiple comparator test for comparing three or more groups. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001. C) Standard Membrane Feeding Assay results showing the number of oocysts developed in each mosquito fed on P. falciparum gametocytes mixed with immune sera obtained from mice immunized with PBS or Alhydrogel formulations of various Vi-Pfs25 conjugates, EPA-Pfs25 and Pfs25. Immune sera from Day 42 was used for the assay and mice immunized with Vi was used as control. Table shows percentage reduction in mean oocyst count compared to Vi control sera.

Fig. 2.

Anti-Vi antibody titer of conjugates and serum bactericidal activity (SBA) of conjugates. Anti-VI ELISA antibodies obtained for immune sera from mice vaccinated with Vi conjugates of Pfs25, unconjugated Vi and EPA-Pfs25 formulated in PBS or Alhydrogel®. ELISA for sera obtained on (A) day 42 sera, and (B) on days 83 (for PBS formulations) and 91 (for Alhydrogel® formulations) was performed using Vi as coating antigen and sera diluted 500-fold. (C) Serum bactericidal activity (SBA) assay titer observed for sera from mice immunized with Vi-Pfs25 conjugates, unconjugated Vi and EPA-Pfs25 formulated in PBS or Alhydrogel®. Mice were vaccinated on days 0 and 28 and sera obtained on Day 83 (PBS formulations) or Day 91 (Alhydrogel® formulations) were analyzed for bactericidal activity. Data were analyzed with Prism software (GraphPad Software, Inc., La Jolla, CA) and statistical differences between groups were measured using a Kruskal-Wallis analysis followed by a Dunn multiple comparator test for comparing three or more groups. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001.

Fig. 3.

Percentage contribution of IgG subclasses to total IgG against (A) Pfs25 and (B) Vi. Percentage contribution from IgG1, IgG2a, IgG2b and IgG3 to total IgG response against (A) Pfs25 and (B) Vi was determined from isotype specific ELISA titers obtained using pooled sera from each group of mice. Day 83 sera were analyzed for groups immunized with PBS formulations and day 91 sera were analyzed for groups received Alhydrogel® formulations of various conjugates and antigens. Percentage contribution is expressed as the percentage of subclass specific ELISA titer to the sum of all subclass titers.