Asexual Stage Vaccine Research Articles

B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.

Evidence that seasonal malaria chemoprevention with SPAQ influences blood and pre-erythrocytic stage antibody responses of Plasmodium falciparum infections in Niger

BackgroundIn endemic areas, children develop slowly and naturally anti-Plasmodium antibodies and become semi-immune. Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine + amodiaquine (SPAQ) is a new strategy to reduce malaria morbidity in West African young children. However, SMC may impact on the natural acquisition of anti-Plasmodium immunity. This paper evaluates the effect of SMC with SPAQ on antibody concentration in young children from Niger.MethodsThis research was conducted in areas benefitting from SMC since 2014 (Zinder district), without SMC (Dosso district), and with 1 year of SMC since 2016 (Gaya district). To assess the relationship between SMC and Plasmodium falciparum IgG antibody responses, the total antibody concentrations against two P. falciparum asexual stage vaccine candidate antigens, circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2), in children aged 3 to 59 months across the three areas were compared. Antibody concentrations are quantified using an enzyme-linked immunosorbent assay on the elution extracted from positive and negative malaria Rapid Diagnostic Test cassettes.ResultsThe analysis concerns two hundred and twenty-nine children aged from 3 to 59 months: 71 in Zinder, 77 in Dosso, and 81 in Gaya. In Zinder (CSP = 17.5 µg/ml and GLURP-R2 = 14.3 µg/ml) median antibody concentration observed are higher than in Gaya (CSP = 7.7 µg/ml and GLURP-R2 = 6.5 µg/ml) and Dosso (CSP = 4.5 µg/ml and GLURP-R2 = 3.6 µg/ml) (p < 0.0001).ConclusionThe research reveals some evidences which show that seasonal malaria chemoprevention with SPAQ has an effect on blood stage antibody responses and pre-erythrocytic stage of P. falciparum infections in Niger. Increased antibody titres with increased SMC/SPAQ implementation. This contradicts hypothesis that SMC/SPAQ could reduce immunity to erythrocyte and liver-stage antigens. Further studies are necessary to provide better understanding of the SMC effect on malaria immunity.

Malaria vaccines for eradication

In achieving the goal of malaria eradication, vaccines could play a crucial role. Current attempts to develop malaria vaccines are primarily focused on Plamosdium falciparum and are directed towards reducing morbidity and mortality. This is the case of the RTS,S vaccine candidate, the most developed at the moment and currently undergoing the final analysis of a multicentric, international Phase III clinical trial. Continued support for these efforts is essential, but if malaria vaccines are to be used as part of a repertoire of tools for malaria eradication, they will need to have an impact on malaria transmission, contributing to lower the reproductive rate to less than one. The consultative group on vaccines of the Malaria Eradication Research Agenda (malERA) initiative identified key features of vaccines especially suited for malaria eradication. The group introduced the concept of “Vaccines that Interrupt Malaria Transmission” (VIMTs), broadening the classical definition of Transmission Blocking Vaccines (TBVs) -that target sexual and mosquito stages of the parasite-, in order to also include any other parasite life stages that may interrupt transmission of malaria. This would include pre-erythrocytic and asexual stage vaccines to reduce prevalence and densities of sexual forms of the parasite, as well as vaccines against mosquito gut antigens impeding parasite development in the vector. Moreover, malaria eradication would necessarily need to target not only P. falciparum but also P. vivax, for which therapeutic vaccines against hypnozoites or preventive vaccines with effect against multiple parasite stages could have enormous impact. Development of VIMTs require the advancement of basic understanding of interactions of the malaria parasite with humans and vectors, as well as the development of tools to measuring infectivity at the individual level and assessing transmission within a given population. VIMTs may have primarily an effect at the population level and could be crucial not only for eradication, but also in areas where malaria control is still the main issue to be addressed. In order to accelerate the actual implementation of such vaccines, it is also important to address regulatory issues and to develop appropriate delivery platforms.

Important advances in malaria vaccine research

The Drug Development and Therapeutics (DDT) is an open access peer-reviewed journal committed to publishing high-quality articles in the field of Drug synthesis and development and Therapeutics from industry, Clinicians and Academia.

A Research Agenda for Malaria Eradication: Vaccines

Vaccines could be a crucial component of efforts to eradicate malaria. Current attempts to develop malaria vaccines are primarily focused on Plasmodium falciparum and are directed towards reducing morbidity and mortality. Continued support for these efforts is essential, but if malaria vaccines are to be used as part of a repertoire of tools for elimination or eradication of malaria, they will need to have an impact on malaria transmission. We introduce the concept of "vaccines that interrupt malaria transmission" (VIMT), which includes not only "classical" transmission-blocking vaccines that target the sexual and mosquito stages but also pre-erythrocytic and asexual stage vaccines that have an effect on transmission. VIMT may also include vaccines that target the vector to disrupt parasite development in the mosquito. Importantly, if eradication is to be achieved, malaria vaccine development efforts will need to target other malaria parasite species, especially Plasmodium vivax, where novel therapeutic vaccines against hypnozoites or preventive vaccines with effect against multiple stages could have enormous impact. A target product profile (TPP) for VIMT is proposed and a research agenda to address current knowledge gaps and develop tools necessary for design and development of VIMT is presented.

Genetic diversity and the ensuing strain specific immunity to asexual erythrocytic stage vaccine candidate antigens, MSP-1, AMA-1 and DBP, of Plasmodium vivax in Sri Lanka

Characterization of genetic diversity among locally prevalent parasite strains and the ensuing strain-specific immunity will be particularly important in a specific geographical setting for development of vaccine constructs and for planning future vaccination strategies. The present study characterized the prevailing genetic diversity of three major asexual stage putative vaccine antigens of Plasmodium vivax, 42 kDa fragment of the Merozoite Surface Protein -1 (MSP-142), Apical Membrane Antigen-1 domain II (AMA-1 DII) and the Duffy Binding Protein region II (PvDBPII), in patients infected with P. vivax malaria from two endemic areas, Anuradhapura and Kataragama, in Sri Lanka, where unstable malaria prevails with low transmission, and from a malaria non-endemic area, Colombo, where the patients contracted the infections while visiting areas endemic to vivax malaria. Based on nucleotide sequences, extensive allelic polymorphism was evident in the N-terminal region of MSP-142(Pvmsp-133), Pvama- 1dII and PvdbpII, due to recombination, mutations, natural selection and genetic differentiation. Nucleotide sequences of Pvmsp-142(N=95), Pvama-1dII (64) and PvdbpII (100), generated 27, 21 and 33 amino acid (a.a) haplotypes, respectively. Clinical isolates with amino acid haplotypes available for all three antigens (N=24) revealed uniquely different haplotype combinations in each isolate. Conversely, PvMSP-119, domain II loop of PvAMA-1 and the six a.a. in the critical binding domain of PvDBPII responsible for direct binding with the Duffy antigen on human RBC, showed 100% conservation of the amino acid sequences. In order to evaluate whether sequence differences among each antigen was indicative of strain-specific immunity, amino acid sequences of PvMSP-142, PvAMA-1DII and PvDBPII were aligned with the homologous total (IgM + IgG) antibody responses assayed by ELISA against the recombinant protein of each antigen, and the seven (P01-P07) PvAMA-1DII peptides. Though Anti-PvMSP-119 and anti-P04 antibody prevalence precludes strain-specific immune responses against the highly conserved PvMSP-119 and the P04 peptide on domain II loop of PvAMA-1, a protective immune response was clearly elicited to these two regions by the marked isotype switch from IgM to IgG with increasing exposure in areas of endemicity. Further, the presence of strain transcending (cross reactive) PvDBPII specific functionally active binding inhibitory antibodies were demonstrated among locally exposed P. vivax patients. Thus this study may support the inclusion of PvMSP-119, P04 region of PvAMA-1 domain II and region II of PvDBP as veritable vaccine components for a multi component cocktail asexual stage vaccine against P. vivax malaria in Sri Lanka.

Vaccinating with the genome: a Sisyphean task?

Human trials of subunit vaccines against the asexual blood stage of malaria are yielding disappointing results, supporting the premise that a single recombinant protein will not be particularly efficacious and that additional proteins must be added. The genome sequence of Plasmodium falciparum offers a large number of additional candidates, but which should be chosen? Various criteria have been suggested to rank the additional candidates, but in the absence of even a partially effective asexual-stage vaccine, the criteria remain unvalidated. These issues are discussed here, together with some suggestions as to how the development of an asexual-stage vaccine could be progressed.

Allele Specificity of Naturally Acquired Antibody Responses against Plasmodium falciparum Apical Membrane Antigen 1

Antibody responses against proteins located on the surface or in the apical organelles of merozoites are presumed to be important components of naturally acquired protective immune responses against the malaria parasite Plasmodium falciparum. However, many merozoite antigens are highly polymorphic, and antibodies induced against one particular allelic form might not be effective in controlling growth of parasites expressing alternative forms. The apical membrane antigen 1 (AMA1) is a polymorphic merozoite protein that is a target of naturally acquired invasion-inhibitory antibodies and is a leading asexual-stage vaccine candidate. We characterized the antibody responses against AMA1 in 262 individuals from Papua New Guinea exposed to malaria by using different allelic forms of the full AMA1 ectodomain and some individual subdomains. The majority of individuals had very high levels of antibodies against AMA1. The prevalence and titer of these antibodies increased with age. Although antibodies against conserved regions of the molecule were predominant in the majority of individuals, most plasma samples also contained antibodies directed against polymorphic regions of the antigen. In a few individuals, predominantly from younger age groups, the majority of antibodies against AMA1 were directed against polymorphic epitopes. The D10 allelic form of AMA1 apparently contains most if not all of the epitopes present in the other allelic forms tested, which might argue for its inclusion in future AMA1-based vaccines to be tested. Some important epitopes in AMA1 involved residues located in domain II or III but depended on more than one domain.

The alanine-rich heptad repeats are intact in the processed form of Plasmodium falciparum MSP3

The potential of Plasmodium falciparum merozoite surface protein 3 as a component of an asexual-stage malaria vaccine is currently being assessed. The precursor form of MSP3 undergoes cleavage during schizogony to generate a mature processed form. It is unknown if this cleavage event is necessary for MSP3 function, but it may be an important consideration for assessing and developing MSP3 as an asexual-stage vaccine candidate. We have therefore determined the cleavage site in MSP3 by sequencing the N-terminus of the processed form of MSP3, which was isolated from parasite material. The position of the cleavage site indicates that the processed form of MSP3 retains the three blocks of alanine-rich heptad repeats, which are predicted to provide the structural framework for an intramolecular coiled-coil. The cleavage-site motif has many features in common with the published cleavage sites of MSP1 30, MSP6 36, and MSP7 22, which are all located on the merozoite surface and are implicated in the erythrocyte invasion process. The common cellular location and similar cleavage-site motifs suggest that these merozoite proteins may be cleaved by the same or related proteases.

TESTING VACCINES IN HUMAN EXPERIMENTAL MALARIA: STATISTICAL ANALYSIS OF PARASITEMIA MEASURED BY A QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION

Clinical trials are an essential step in evaluation of safety and efficacy of malaria vaccines, and human experimental malaria infections have been used for evaluation of protective immunity of Plasmodium falciparum malaria. In this study, a quantitative real-time polymerase chain reaction was used to measure P. falciparum malaria parasitemia in non-immune volunteers who had been experimentally infected by mosquito bites. Based on a remarkably small variation in the kinetics of parasitemia, a statistical model was developed that provides detailed estimates of pre-patent periods and parasite multiplication of blood stages. Using this model, we could predict results on vaccine efficacy for 1) pre-erythrocytic vaccines in the asymptomatic incubation period and 2) asexual stage vaccines after a limited number of multiplication cycles. The model shows that stage-specific vaccines even with limited efficacy can be highly efficacious when used in combination. This P. falciparum challenge method significantly adds to the potential to evaluate efficacy of candidate malaria vaccines before going into field trials.

Geographical structure of diversity and differences between symptomatic and asymptomatic infections for Plasmodium falciparum vaccine candidate AMA1.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is a prime malaria vaccine candidate. Antigenic diversity within parasite populations is one of the main factors potentially limiting the efficacy of any asexual-stage vaccine, including one based on AMA1. The DNA coding for the most variable region of this antigen, domain I, was sequenced in 168 samples from the Wosera region of Papua New Guinea, including samples from symptomatic and asymptomatic infections. Neutrality tests applied to these sequences provided strong evidence of selective pressure operating on the sequence of ama1 domain I, consistent with AMA1 being a target of protective immunity. Similarly, a peculiar pattern of geographical diversity and the particular substitutions found were suggestive of strong constraints acting on the evolution of AMA1 at the population level, probably as a result of immune pressure. In addition, a strong imbalance between symptomatic and asymptomatic infections was detected in the frequency of particular residues at certain polymorphic positions, pointing to AMA1 as being one of the determinants of the morbidity associated with a particular strain. The information yielded by this study has implications for the design and assessment of AMA1-based vaccines and provides additional data supporting the importance of AMA1 as a malaria vaccine candidate.

Chronic Malaria Infection and Vaccines

Pierre Druilhe and Jean-Louis Pérignon, in a recent article in Parasitology Today[ 1 Druilhe P. Pérignon J-L. Parasitol. Today. 1997; 13: 353-357 Abstract Full Text PDF PubMed Scopus (56) Google Scholar ], discussed how chronic infections of Plasmodium falciparum are maintained in partially immune individuals. They reasoned that a mechanism must operate that is triggered by the parasites themselves, and argued that this is a monocyte-mediated, antibody-dependent merozoite-triggered process. Whether or not the mechanism which they propose is accurate in detail, they are surely correct in pointing out that natural immunity to asexual stage parasites must not only be very potent, because it is able to reduce parasite densities to levels several orders of magnitude lower than those in non-immune individuals, but also that it exhibits an incomplete or partially effective (non-sterilizing) antiparasite action[ 1 Druilhe P. Pérignon J-L. Parasitol. Today. 1997; 13: 353-357 Abstract Full Text PDF PubMed Scopus (56) Google Scholar ]. These observations have profound implications for asexual stage vaccine development. As Druilhe and Pérignon point out, if antibody acts by a direct mechanism, it is difficult to imagine how antibody effective enough to remove 99.9% of the parasite population would leave the other 0.1% alive[ 1 Druilhe P. Pérignon J-L. Parasitol. Today. 1997; 13: 353-357 Abstract Full Text PDF PubMed Scopus (56) Google Scholar ]. It follows that a vaccine designed to stimulate such a direct antibody response would tend to eliminate infections completely, rather than promote the chronic infections characteristic of premunition. This idea can be, and indeed has been, tested experimentally in humans. Beck et al.[ 2 Beck H-P. et al. J. Infect. Dis. 1997; 175: 921-926 Crossref PubMed Scopus (142) Google Scholar ] used PCR-RFLP typing of the MSP-2 locus of P. falciparum to determine the number of distinct parasite genotypes in recipients of the SPf66 vaccine, during the trial of the vaccine in Tanzanian children[ 3 Alonso P.L. et al. Lancet. 1994; 344: 1175-1181 Abstract PubMed Scopus (292) Google Scholar ]. They found that the vaccine reduced the average number of concurrent infections per individual from 5.0 to 3.9 (Ref. [ 2 Beck H-P. et al. J. Infect. Dis. 1997; 175: 921-926 Crossref PubMed Scopus (142) Google Scholar ]). There was no difference in the incidence of infection between vaccine and placebo recipients, and hence the difference in multiplicity of infection could only be because SPf66 recipients eliminate infections more rapidly than do controls.

Attenuated parasites and the development of an erythrocytic-stage vaccine

Asexual-stage vaccine candidates may be identified by recognition of antibodies to them in the sera of naturally immune individuals. Once identified, recombinant molecules expressing the relevant antigen can be produced, although such recombinant molecules only seem to be effective at inducing immunity if mixed with adjuvant. More information is needed about the role of antibodies in immunity to malaria. In an animal model, that of Plasmodium berghei in mice, a fairly effective immune response can be produced by inoculating mice with live, proliferating parasites and then keeping the infections subclinical, by drug treatment, for 5 weeks. However, the resultant ‘immune1 individuals carry small numbers of parasites despite the presence of antibody (premunition). Repeated challenge infections are limited by the hosts’ immune responses but sterile immunity is not observed, The immunity that is established only fades in the absence of the parasite, In contrast, inoculation with preparations of disrupted parasites, which do not seem to contain all the antigenic information of the living parasites, do not lead to an immune response which limits parasitaemias. These observations may be the result of antigenic variation. Immunity induced in mice by live, attenuated parasites (but not by extracts) protects against challenge with virulent parasites. Immunity against the attenuated but not the virulent parasite can be transferred by inoculating naive mice with spleen cells from the immunized mice. The mice given spleen cells can then be protected against the virulent parasite by challenge with proliferating, attenuated parasites (but not with extracts). Immune B cells from the immune donor and non-immune CD4 cells in the naive recipient are essential for transfer of immunity.

A vaccine candidate from the sexual stage of human malaria that contains EGF-like domains.

Malaria vaccines are being developed against different stages in the parasite's life cycle, each increasing the opportunity to control malaria in its diverse settings. Sporozoite vaccines are designed to prevent mosquito-induced infection; first generation recombinant or synthetic peptide vaccines have been tested in humans. Asexual erythrocytic stage vaccines, developed to prevent or reduce the severity of disease, have been tested in animals and in humans. A third strategy is to produce sexual stage vaccines that would induce antibodies which would prevent infection of mosquitoes when ingested in a bloodmeal containing sexual stage parasites. Although not directly protective, the sexual stage vaccine combined with a sporozoite or asexual stage vaccine (protective component) could prolong the useful life of the protective component by reducing transmission of resistant vaccine-induced mutants. In areas of low endemnicity, the sexual stage vaccine could reduce transmission below the critical threshold required to maintain the infected population, thereby assisting in the control or eradication of malaria. Transmission of Plasmodium falciparum, the major human malaria, can be blocked by monoclonal antibodies against three sexual stage-specific antigens. We have cloned the gene encoding the surface protein of relative molecular mass Mr 25,000 (25K; Pfs25), expressed on zygotes and ookinetes of P. falciparum. The deduced amino-acid sequence consists of a signal sequence, a hydrophobic C-terminus, and four tandem epidermal growth factor EGF-like domains.