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Adjuvants have long been critical components of vaccines, but the exact mechanisms of their action and precisely how they alter or enhance vaccine-induced immune responses are often unclear. In this study, we used broad immunoprofiling of antibody, cellular, and cytokine responses, combined with data integration and machine learning to gain insight into the impact of different adjuvant formulations on vaccine-induced immune responses. A Self-Assembling Protein Nanoparticles (SAPN) presenting the malarial circumsporozoite protein (CSP) was used as a model vaccine, adjuvanted with three different liposomal formulations: liposome plus Alum (ALFA), liposome plus QS21 (ALFQ), and both (ALFQA). Using a computational approach to integrate the immunoprofiling data, we identified distinct vaccine-induced immune responses and developed a multivariate model that could predict the adjuvant condition from immune response data alone with 92% accuracy (p = 0.003). The data integration also revealed that commonly used readouts (i.e. serology, frequency of T cells producing IFN-γ, IL2, TNFα) missed important differences between adjuvants. In summary, broad immune-profiling in combination with machine learning methods enabled the reliable and clear definition of immune signatures for different adjuvant formulations, providing a means for quantitatively characterizing the complex roles that adjuvants can play in vaccine-induced immunity. The approach described here provides a powerful tool for identifying potential immune correlates of protection, a prerequisite for the rational pairing of vaccines candidates and adjuvants. more...

Background Whole parasite vaccines provide a unique opportunity for dissecting immune mechanisms and identify antigens that are targeted by immune responses which have the potential to mediate sterile protection against malaria infections. The radiation attenuated sporozoite (PfSPZ) vaccine has been considered the gold standard for malaria vaccines because of its unparalleled efficacy. The immunogenicity of this and other vaccines continues to be evaluated by using recombinant proteins or peptides of known sporozoite antigens. This approach, however, has significant limitations by relying solely on a limited number of known pathogen-associated immune epitopes. Using the full range of antigens expressed by the sporozoite will enable the comprehensive immune-profiling of humoral immune responses induced by whole parasite vaccines. To address this challenge, a novel ELISA based on sporozoites was developed. Results The SPZ-ELISA method described in this report can be performed with either freshly dissected sporozoites or with cryopreserved sporozoite lysates. The use of a fixative for reproducible coating is not required. The SPZ-ELISA was first validated using monoclonal antibodies specific for CSP and TRAP and then used for the characterization of immune sera from radiation attenuated sporozoite vaccinees. Conclusion Applying this simple and highly reproducible approach to assess immune responses induced by malaria vaccines, both recombinant and whole parasite vaccines, (1) will help in the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine, (2) will facilitate and accelerate the identification of immune correlates of protection, and (3) can also be a valuable assessment tool for antigen discovery as well as down-selection of vaccine formulations and, thereby, guide vaccine design. Electronic supplementary material The online version of this article (10.1186/s12936-017-2129-9) contains supplementary material, which is available to authorized users. more...

To date, no immune correlates for blood stage-specific immunity against Plasmodium falciparum malaria parasites have been identified. Growth and/or invasion inhibition assays using sera from Phase 2a/b trials will aid in determining whether correlations with protective immunity can be established for these assays. A major constraint in the ability to evaluate functional antibody activities from populations in endemic areas is the relatively limited availability of sufficient sample quantity. For this reason, we developed a miniaturized and high-throughput method to measure growth inhibitory activity by quantification of parasite lactate dehydrogenase (pLDH) in a 384-microtiter plate format. This culture method can be extended beyond the pLDH-based readout to other techniques commonly used to determine growth/invasion inhibition. more...

Antibodies are thought to be the primary immune effectors in the defense against erythrocytic stage Plasmodium falciparum. Thus, malaria vaccines directed to blood stages of infection are evaluated based on their ability to induce antibodies with anti-parasite activity. Such antibodies may have different effector functions (e.g., inhibition of invasion or inhibition of parasite growth/development) depending on the target antigen. We evaluated four methods with regards to their ability to differentiate between invasion and/or growth inhibitory activities of antibodies specific for two distinct blood stage antigens: AMA1 and MSP142. We conclude that antibodies induced by these vaccine candidates have different modes of action that vary not only by the antigen, but also by the strain of parasite being tested. Analysis based on parasitemia and viability was essential for defining the full range of anti-parasite activities in immune sera. more...

The immunogenicity of recombinant protein or anti-viral DNA vaccines can be significantly improved by the addition of tandem copies of the complement fragment C3d. We sought to determine if the efficacy of a circumsporozoite protein (CSP)-based DNA vaccine delivered to mouse skin by gene gun was improved by using this strategy. Instead, we found that C3d suppressed the protective immunity against Plasmodium berghei malaria infection and deviated immunity, most notably by suppressing the induction of antibodies specific for the CSP C-terminal flanking sequence and by suppressing the induction of CSP-specific IL-4-producing spleen cells. We further showed that C3d bound to the C-terminal flanking sequence of the CSP, which may explain the immune deviation observed in CS/C3d chimeric antigen. We have thus identified C3d-mediated epitope masking and shifting of both the humoral and cellular immune responses as a potential novel escape mechanism, which plasmodia may use to divert the induction of protective immunity. more...

Background MSP1 is the major surface protein on merozoites and a prime candidate for a blood stage malaria vaccine. Preclinical and seroepidemiological studies have implicated antibodies to MSP1 in protection against blood stage parasitaemia and/or reduced parasite densities, respectively. Malaria endemic areas have multiple strains of Plasmodium falciparum circulating at any given time, giving rise to complex immune responses, an issue which is generally not addressed in clinical trials conducted in non-endemic areas. A lack of understanding of the effect of pre-existing immunity to heterologous parasite strains may significantly contribute to vaccine failure in the field. The purpose of this study was to model the effect of pre-existing immunity to MSP142 on the immunogenicity of blood-stage malaria vaccines based on alternative MSP1 alleles. Methods Inbred and outbred mice were immunized with various recombinant P. falciparum MSP142 proteins that represent the two major alleles of MSP142, MAD20 (3D7) and Wellcome (K1, FVO). Humoral immune responses were analysed by ELISA and LuminexTM, and functional activity of induced MSP142-specific antibodies was assessed by growth inhibition assays. T-cell responses were characterized using ex vivo ELISpot assays. Results Analysis of the immune responses induced by various immunization regimens demonstrated a strong allele-specific response at the T cell level in both inbred and outbred mice. The success of heterologous regimens depended on the degree of homology of the N-terminal p33 portion of the MSP142, likely due to the fact that most T cell epitopes reside in this part of the molecule. Analysis of humoral immune responses revealed a marked cross-reactivity between the alleles. Functional analyses showed that some of the heterologous regimens induced antibodies with improved growth inhibitory activities. Conclusion The development of a more broadly efficacious MSP1 based vaccine may be hindered by clonally imprinted p33 responses mainly restricted at the T cell level. In this study, the homology of the p33 sequence between the clonally imprinted response and the vaccine allele determines the magnitude of vaccine induced responses. more...

Natural immunity against malaria is acquired after repeated infections for an extended period of time resulting in a state of immunological non-responsiveness against the malaria parasite. This state ultimately prevents the onset of severe disease such as cerebral malaria thereby reducing the risk of death from malaria. Individuals with acquired natural immunity to malaria still harbor parasites (albeit in low densities) in the blood, and therefore natural immunity against malaria is not sterile. For this reason, natural immunity to malaria cannot be compared to immunity achieved against other diseases where the immune response neutralizes and eliminates the pathogen. The second hallmark of natural immunity to malaria is that protection wanes once a “protected” individual leaves the malaria-endemic area indicating that protection depends on continued antigen exposure. Immunity to malaria is stageand species-specific and distinct immune mechanisms confer protection against the different developmental stages of the parasite. In the case of blood stage infection, passive transfer experiments with purified human immunoglobulins derived from immune individuals living in malaria-endemic areas have demonstrated that antibodies can mediate protection (Cohen et al., 1961; Butcher et al., 1970; BouharounTayoun et al., 1990). Mechanistic studies revealed that the effect of blood stage-specific antibodies on the asexual erythrocytic parasite depends on their antigen-specificity; antibodies can bind to merozoites, opsonize and target them towards phagocytic cells of the host (Groux and Gysin, 1990), or prevent invasion of new erythrocytes (Perkins, 1991). Once infected, antibodies against asexual blood stage antigens such as Pf332 or MSP-1 inhibit the intra-erythrocytic development of Plasmodium falciparum (Ahlborg et al., 1996; Siddique et al., 1998; Bergmann-Leitner et al., 2009). Antibodies directed to antigens expressed by sexual erythrocytic stages (gametocytes) have been shown to prevent transmission of malaria by blocking either the infection of the mosquito or the development in the mosquito (Lavazec and Bourqouin, 2008). As will be outlined in this chapter, it becomes increasingly clear that a blood stage vaccine may never be able to induce sterile protection, but can prevent mortality. Why bother developing blood stage vaccines? Extensive studies characterizing more...

Background Antibodies are the main effector molecules in the defense against blood stages of the malaria parasite Plasmodium falciparum. Understanding the mechanisms by which vaccine-induced anti-blood stage antibodies work in protecting against malaria is essential for vaccine design and testing. Methods The effects of MSP-1p42-specific antibodies on the development of blood stage parasites were studied using microscopy, flow cytometry and the pLDH assay. To determine allele-specific effects, if present, allele-specific antibodies and the various parasite clones representative of these alleles of MSP-1 were employed. Results The mode of action of anti-MSP-1p42 antibodies differs among the parasite clones tested: anti-MSP-1p42 sera act mainly through invasion-inhibitory mechanisms against FVO parasites, by either preventing schizonts from rupturing or agglutinating merozoites upon their release. The same antibodies do not prevent the rupture of 3D7 schizonts; instead they agglutinate merozoites and arrest the development of young parasites at the early trophozoite stage, thus acting through both invasion- and growth inhibitory mechanisms. The second key finding is that antibodies have access to the intra-erythrocytic parasite, as evidenced by the labeling of developing merozoites with fluorochrome-conjugated anti-MSP-1p42 antibodies. Access to the parasite through this route likely allows antibodies to exert their inhibitory activities on the maturing schizonts leading to their inability to rupture and be released as infectious merozoites. Conclusion The identification of various modes of action by which anti-MSP-1 antibodies function against the parasite during erythrocytic development emphasizes the importance of functional assays for evaluating malaria vaccines and may also open new avenues for immunotherapy and vaccine development. more...

Vaccination with Plasmodium falciparum MSP1(42)/complete Freund's adjuvant (FA) followed by MSP1(42)/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP1(42) expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP1(42) in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = -0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = -0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP1(42) vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component. more...

Background Antibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested in vitro. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies. Methods To address this concern, various immunoglobulin (Ig) purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation) were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution) and for functional activity by in vitro parasite growth inhibition assay (GIA). Results This comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies. Conclusion Consequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection. more...

Antibodies are thought to be the primary immune effectors in the defense against erythrocytic stage Plasmodium falciparum. Thus, malaria vaccines directed to blood stages of infection are evaluated based on their ability to induce antibodies with anti-parasite activity. Such antibodies may have different effector functions (e.g., inhibition of invasion or inhibition of parasite growth/development) depending on the target antigen. We evaluated four methods with regards to their ability to differentiate between invasion and/or growth inhibitory activities of antibodies specific for two distinct blood stage antigens: AMA1 and MSP1(42). We conclude that antibodies induced by these vaccine candidates have different modes of action that vary not only by the antigen, but also by the strain of parasite being tested. Analysis based on parasitemia and viability was essential for defining the full range of anti-parasite activities in immune sera. more...