The C-terminal 42 kDa fragments of the P. falciparum Merozoite Surface Protein 1, MSP1-42 is a leading malaria vaccine candidate. MSP1-33, the N-terminal processed fragment of MSP1-42, is rich in T cell epitopes and it is hypothesized that they enhance antibody response toward MSP1-19. Here, we gave in vivo evidence that T cell epitope regions of MSP1-33 provide functional help in inducing anti-MSP1-19 antibodies. Eleven truncated MSP1-33 segments were expressed in tandem with MSP1-19, and immunogenicity was evaluated in Swiss Webster mice and New Zealand White rabbits. Analyses of anti-MSP1-19 antibody responses revealed striking differences in these segments' helper function despite that they all possess T cell epitopes. Only a few fragments induced a generalized response (100%) in outbred mice. These were comparable to or surpassed the responses observed with the full length MSP1-42. In rabbits, only a subset of truncated antigens induced potent parasite growth inhibitory antibodies. Notably, two constructs were more efficacious than MSP1-42, with one containing only conserved T cell epitopes. Moreover, another T cell epitope region induced high titers of non-inhibitory antibodies and they interfered with the inhibitory activities of anti-MSP1-42 antibodies. In mice, this region also induced a skewed TH2 cellular response. This is the first demonstration that T cell epitope regions of MSP1-33 positively or negatively influenced antibody responses. Differential recognition of these regions by humans may play critical roles in vaccine induced and/or natural immunity to MSP1-42. This study provides the rational basis to re-engineer more efficacious MSP1-42 vaccines by selective inclusion and exclusion of MSP1-33 specific T cell epitopes.

Many adjuvants are known to enhance expression of co-stimulatory and adhesion molecules secondarily to the activation of immune cells. Whether interactions via these molecules are obligatory in adjuvants' ability to potentiation vaccine immunogenicity is less clear. We investigated the ability of eight adjuvant formulations to potentiate the immunogenicity of a malaria vaccine in mice deficient in the prominent co-stimulatory molecules, CD80 and CD86; and the adhesion ligand, ICAM-1. While no adjuvants could bypass co-stimulatory requirements, more formulations exhibited dependency for CD86 than for CD80. In CD80 or CD86 KO mice, formulations with the saponin derivative, QS21 could efficiently default to the other B7 molecule. This effect was dominant over other adjuvant constituents. The requirement for ICAM-1 could be readily bypassed using adjuvant formulations containing immunomodulators; whereas this was not the case with emulsion-type adjuvants in which reduction in adjuvanticity was associated with decreases in antigen-specific IFN-gamma responses. These studies may help to guide the formulation of vaccine adjuvants to maintain effectiveness in hosts with altered immunological environment that often result from infections.

Infections and chronic diseases can alter the host's immunological balance or result in immunodeficiencies. We hypothesize that this may also affect the performance of vaccine adjuvants. Accordingly, the potency and adjuvanticity of eight adjuvant formulations based on Montanide ISA720, MF59, monophosphoryl lipid A (MPL), QS21 (saponin derivative), MPL-SE (stable emulsion of a MPL derivative), and MPL-AF (MPL in aqueous formulation) were studied in immune gene knockout mice, IFN-γ −/−, IL-4 −/−, and STAT6 −/−, using the P. falciparum MSP1 vaccine, P30P2MSP1-19 as a model immunogen. The adjuvants showed preferential requirements for the immune mediators to induce immune responses to MSP1-19, and the effects were formulationspecific. While emulsion-type adjuvants were highly effective in mice, their potency was more readily suppressed by immune knockouts; and additions of immunomodulators were required to restore efficacy. Formulated adjuvants had characteristics distinct from their individual components, and multi-components formulations were not necessarily superior. We conclude that perturbation of immune environments will have measurable impact on adjuvant's potency. Evaluation of adjuvants in immune knockout models may be a supplementary approach to measure and compare adjuvants' efficacy, and to further unveil their distinct biological activities.

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The efficacy of vaccine adjuvants can be influenced by the immunological environment of the host, depending on the mechanism(s) by which they exert their immunopotentiating activities. Interleukin-6 is a pleiotropic cytokine that has a broad range of biological activities on immune and non-immune cells. We investigated the role of IL-6 on the ability of nine adjuvant formulations to induce antibody responses to the P. falciparum MSP1-19 malaria vaccine, using IL-6 -/-(KO) mice. Results showed that some adjuvants, ie. MPL-SE, CFA/IFA, ISA720/QS21/MPL, depended on IL-6 for their efficacy, while others exhibited increased potency in its absence. The efficacy of adjuvants in the IL-6 KO environment cannot be solely attributed to their ability to stimulate antigen-specific cellular responses, suggesting that other biological activities of IL-6 are also important. The results further suggest that two adjuvants utilized dissimilar pathways to potentiate the same type of immune response.

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1 42 ) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1 42 was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 g/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1 42 were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1 19 proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1 19 . Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1 42 . Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1 42 at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1 42 vaccine for human use.

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