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Abstract Background Establishing antibody correlates of protection against malaria in human field studies and clinical trials requires, amongst others, an accurate estimation of antibody levels. For polymorphic antigens such as apical membrane antigen 1 (AMA1), this may be confounded by the occurrence of a large number of allelic variants in nature. Methods To test this hypothesis, plasma antibody levels in an age-stratified cohort of naturally exposed children from a malaria-endemic area in Southern Ghana were determined by indirect ELISA. Titres against four single PfAMA1 alleles were compared with those against three different allele mixtures presumed to have a wider repertoire of epitope specificities. Associations of antibody levels with the incidence of clinical malaria as well as with previous exposure to parasites were also examined. Results Antibody titres against PfAMA1 alleles generally increased with age/exposure while antibody specificity for PfAMA1 variants decreased, implying that younger children (≤ 5 years) elicit a more strain-specific antibody response compared to older children. Antibody titre measurements against the FVO and 3D7 AMA1 alleles gave the best titre estimates as these varied least in pair-wise comparisons with titres against all PfAMA1 allele mixtures. There was no association between antibody levels against any capture antigen and either clinical malaria incidence or parasite density. Conclusions The current data shows that levels of naturally acquired antigen-specific antibodies, especially in infants and young children, are dependent on the antigenic allele used for measurement. This may be relevant to the interpretation of antibody titre data from measurements against single PfAMA1 alleles, especially in studies involving infants and young children who have experienced fewer infections.

Abstract Background Increasing the breadth of the functional antibody response through immunization with Plasmodium falciparum apical membrane antigen 1 (PfAMA1) multi-allele vaccine formulations has been demonstrated in several rodent and rabbit studies. This study assesses the safety and immunogenicity of three PfAMA1 Diversity-Covering (DiCo) vaccine candidates formulated as an equimolar mixture (DiCo mix) in CoVaccine HT™ or Montanide ISA 51, as well as that of a PfAMA1-MSP119 fusion protein formulated in Montanide ISA 51. Methods Vaccine safety in rhesus macaques was monitored by animal behaviour observation and assessment of organ and systemic functions through clinical chemistry and haematology measurements. The immunogenicity of vaccine formulations was assessed by enzyme-linked immunosorbent assays and in vitro parasite growth inhibition assays with three culture-adapted P. falciparum strains. Results These data show that both adjuvants were well tolerated with only transient changes in a few of the chemical and haematological parameters measured. DiCo mix formulated in CoVaccine HT™ proved immunologically and functionally superior to the same candidate formulated in Montanide ISA 51. Immunological data from the fusion protein candidate was however difficult to interpret as four out of six immunized animals were non-responsive for unknown reasons. Conclusions The study highlights the safety and immunological benefits of DiCo mix as a potential human vaccine against blood stage malaria, especially when formulated in CoVaccine HT™, and adds to the accumulating data on the specificity broadening effects of DiCo mix.

Abstract Background A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp142) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof. Methods Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition. Results After vaccination, PkAMA1 and PkMSP119 antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1. Conclusions This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity in vitro and protection, either after vaccination or after challenge.

Abstract Background Antibodies to key Plasmodium falciparum surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achieve strain-transcending immunity. Such antibody responses against Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail. Methods A standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different PfAMA1 alleles in sequence. The in vitro parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies. Results A mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different PfAMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations. Conclusions These findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design.

Introduction: Polymorphism in vaccine antigens poses major challenges to vaccinologists. ThePlasmodium falciparumApical Membrane Antigen 1 (AMA1) poses such a challenge. We found that immunization with a mixture of three variants yielded functional antibody levels to all variants comparable to levels induced by monovalent immunization. The mechanism behind the observed broadening was shown to be an increase in the fraction of cross-reactive antibodies, most likely because strain-specific epitopes are present at lower frequency relative to conserved epitopes. Areas covered: We hereby introduce the Epitope Dilution Phenomenon (EDiP) as a practical strategy for the induction of broad, cross-variant antibody responses against polymorphic antigens and discuss the utility and applicability of this phenomenon for the development of vaccines against polymorphic antigens of pathogens like Influenza, HIV, Dengue andPlasmodium. Expert commentary: EDiP can be used to broaden antibody responses by immunizing with a mixture of at least 3 antigenic variants, where the variants included can differ, yet yield broadened responses. [ABSTRACT FROM PUBLISHER]

The absence of pathological hallmarks of progressive multiple sclerosis (MS) in commonly used rodent models of experimental autoimmune encephalomyelitis (EAE) hinders the development of adequate treatments for progressive disease. Work reviewed here shows that such hallmarks are present in the EAE model in marmoset monkeys (Callithrix jacchus). The minimal requirement for induction of progressive MS pathology is immunization with a synthetic peptide representing residues 34–56 from human myelin oligodendrocyte glycoprotein (MOG) formulated with a mineral oil [incomplete Freund’s adjuvant (IFA)]. Pathological aspects include demyelination of cortical gray matter with microglia activation, oxidative stress, and redistribution of iron. When the peptide is formulated in complete Freund’s adjuvant, which contains mycobacteria that relay strong activation signals to myeloid cells, oxidative damage pathways are strongly boosted leading to more intensive pathology. The proven absence of immune potentiating danger signals in the MOG34–56/IFA formulation implies that a narrow population of antigen-experienced T cells present in the monkey’s immune repertoire is activated. This novel pathway involves the interplay of lymphocryptovirus-infected B cells with MHC class Ib/Caja-E restricted CD8+ CD56+ cytotoxic T lymphocytes. [ABSTRACT FROM AUTHOR]