Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Abstract

The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-microg dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants.

Fig. 1.

Conjugation enhanced antibody responses of Pfs25H in mice. Seven groups of mice, 10 in each group, were immunized twice i.p. on days 0 and 28 with the indicated vaccines. Pfs25H-OMPC indicates the conjugate vaccine. Anti-Pfs25 antibody responses were evaluated in sera collected 2 weeks after the second immunization. The histogram bars indicate geometric mean antibody levels in these animal groups, with dots indicating the antibody level of each animal within each group.

Fig. 2.

Conjugation process did not affect the functional epitopes for TBA. The mouse (A), rabbit (B), and rhesus (C) antisera obtained from animals immunized with Pfs25H-OMPC or Pfs25H/ISA51 were compared for their TBAs relative to the ELISA titers in membrane feeding assays. Mouse and rabbit sera used in the assays were obtained 2 weeks after the second immunization. Rhesus sera used in the assay were obtained on day 611, 2 weeks after the booster dose on day 596 (see also Fig. 3 and Table 1). Antisera and sexual-stage parasites were fed to mosquitoes together with the blood meals containing parasites, and the oocyst numbers were counted as an indication of the infectivity. The negative control samples were preimmune sera from the same species. Lines are the nonlinear regression fit of the data to a simple hyperbolic equation: % inhibition = 100·[C]/(IC₅₀ + [C]), where [C] is the anti-Pfs25 ELISA units and IC₅₀ is the anti-Pfs25 ELISA units giving 50% inhibition of oocyst number. Filled circles and solid lines indicate antisera induced by Pfs25H-OMPC conjugate, and open circles and dashed lines indicate antisera induced by Pfs25H/ISA51.

Fig. 3.

Pfs25H-OMPC induced sustained antibody responses in rhesus monkeys. Two groups of monkeys were given two immunizations of 4 or 40 μg of Pfs25H conjugated to OMPC (diamond and square, respectively) on days 0 and 70. To compare the responses induced by the water-in-oil formulation, a third group of monkeys was given 40 μg of Pfs25H/ISA51 (circles) on days 0 and 70. On day 596, a dose of 40 μg of nonconjugated Pfs25H adsorbed to MAA was given to all three groups of monkeys. The antibody levels were assayed in sera by ELISA throughout the study. The data are expressed as the geometric mean ×/÷ geometric standard error. Arrows indicate the days of immunization.