Plasmodium Falciparum Apical Membrane Antigen Research Articles

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Genetic diversity and natural selection of apical membrane antigen-1 (ama-1) in Cameroonian Plasmodium falciparum isolates

Antigenic variation associated with genetic diversity in global Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major impediment to designing an effective malaria vaccine. Here, we report the first study on genetic diversity and natural selection of the Pfama-1 gene in P. falciparum isolates from Cameroon. A total of 328 P. falciparum positive samples collected during 2016 and 2019 from five localities of Cameroon were analysed. The ectodomain coding fragment of Pfama-1 gene was amplified for polymorphism profiling and natural selection analysis. A total of 108 distinct haplotypes were found in 203 P. falciparum isolates with considerable nucleotide diversity (π = 0.016) and haplotype diversity (Hd = 0.976). Most amino acid substitutions detected were scattered in ectodomain-I and few specific mutations viz P145L, K148Q, K462I, L463F, N471K, S482L, E537G, K546R and I547F were seen only in Cameroonian isolates. A tendency of natural selection towards positive diversifying selection was observed (Taj-D = 2.058). Five positively selected codon sites (P145L, S283L, Q308E/K, P330S and I547F) were identified, which overlapped with predicted B-cell epitopes and red blood cell (RBC) binding sites, suggesting their potential implication in host immune pressure and parasite-RBC binding complex modulation. The Cameroonian P. falciparum populations indicated a moderate level of genetic differentiation when compared with global sequences, with few exceptions from Vietnam and Venezuela. Our findings provide baseline data on existing Pfama-1 gene polymorphisms in Cameroonian field isolates, which will be useful information for malaria vaccine design.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Tentative indicators of malaria in archaeological skeletal samples, a pilot study testing different methods

ObjectiveThis study attempts to integrate multiple methods to investigate the presence of malaria in human skeletal samples from an archaeological context. Materials33 well preserved human remains originating from a 17th-century archaeological site in southeastern Romania. MethodsThe human bone samples were analyzed using rapid diagnostic tests for malaria antigens and PCR amplification of Plasmodium falciparum apical membrane antigen 1. A preliminary test was performed to identify and briefly characterize the presence of hemozoin using a combination of TEM imaging and diffraction. ResultsThe rapid diagnostic tests indicated that more than half of the examined samples were positive for Plasmodium antigens, but no traces of the parasites’ genetic material were detected despite repeated attempts. The TEM images indicated that hemozoin might be a promising diagnostic marker of malaria in ancient bones. ConclusionsThe indisputable identification of malaria in the analyzed archaeological population was not possible as none of the applied methodological strategies turned out to be straightforward. SignificanceThis study reinforces the intricacy and limitations of unequivocally identifying malaria in past populations and sets the stage for future studies on such life-threatening infectious disease in a geographical space, which is currently underrepresented in the bioarchaeological literature. LimitationsThe low sample size and the lack of consistency across all assays hindered understanding the role of malaria in the studied population. Suggestions for further researchFurther thorough multidisciplinary approaches on malaria detection in ancient settlements would be appropriate to inform our knowledge of its origins, frequency, and pathogen changes over centuries.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial.

Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 ( ama1), and multidrug resistance gene 1 ( mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Molecular Assessment of Domain I of Apical Membrane Antigen I Gene in Plasmodium falciparum: Implications in Plasmodium Invasion, Taxonomy, Vaccine Development, and Drug Discovery.

Given its global morbidity and mortality rates, malaria continues to be a major public health concern. Despite significant progress in the fight against malaria, efforts to control and eradicate the disease globally are in jeopardy due to lack of a universal vaccine. The conserved short peptide sequences found in Domain I of Plasmodium falciparum apical membrane antigen 1 (PfAMA1), which are exposed on the parasite cell surface and in charge of Plasmodium falciparum invasion of host cells, make PfAMA1 a promising vaccine candidate antigen. The precise amino acids that make up these conserved short peptides are still unknown, and it is still difficult to pinpoint the molecular processes by which PfAMA1 interacts with the human host cell during invasion. The creation of a universal malaria vaccine based on the AMA1 antigen is challenging due to these knowledge limitations. This study used genome mining techniques to look for these particular short peptides in PfAMA1. Thirty individuals with Plasmodium falciparum malaria had blood samples taken using Whatman's filter papers. DNA from the parasite was taken out using the Chelex technique. Domain I of the Plasmodium falciparum AMA1 gene was amplified using nested polymerase chain reactions, and the amplified products were removed, purified, and sequenced. The DNA sequence generated was converted into the matching amino acid sequence using bioinformatic techniques. These amino acid sequences were utilized to search for antigenic epitopes, therapeutic targets, and conserved short peptides in Domain I of PfAMA1. The results of this investigation shed important light on the molecular mechanisms behind Plasmodium invasion of host cells, a potential PfAMA1 vaccine antigen sequence, and prospective malaria treatment options in the future. Our work offers fresh information on malaria medication and vaccine research that has not been previously discussed.

Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon deep sequencing (TADS) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, TADS Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterize the complexity of infection (COI) and drug-resistance genotypes, respectively. P. falciparum positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Of the 30 participants, 9 presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. The COI was comparable between ama1 and msp1, msp2 and glurp, however, overall ama1 detected more haplotypes. Based on ama1, a stable number of haplotypes were detected throughout treatment up until day 3. Additionally, a recrudescent infection was identified with an ama1 haplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 haplotypes and parasitaemia, we identified a fast (&lt;1h) and slow (&gt;5h) clearing haplotype. As expected, only two mdr1 haplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. This study highlights TADS as a sensitive tool for tracking parasite haplotypes throughout treatment and can detect variation in haplotype clearance estimates. TADS can also identify slow clearing haplotypes, a potential early sign of selection during treatment. Consequently, TADS has the capability of improving the discriminatory power to accurately distinguish recrudescences from reinfections.

Genetic diversity of Plasmodium falciparum AMA-1 antigen from the Northeast Indian state of Tripura and comparison with global sequences: implications for vaccine development

BackgroundMalaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen.MethodsPatients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique.ResultsAmong 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation.ConclusionsBesides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite.

Retrospective study of toxoplasmosis prevalence in pregnant women in Benin and its relation with malaria.

Globally distributed with variable prevalence depending on geography, toxoplasmosis is a zoonosis caused by an obligate intracellular protozoan parasite, Toxoplasma gondii. This disease is usually benign but poses a risk for immunocompromised people and for newborns of mothers with a primary infection during pregnancy because of the risk of congenital toxoplasmosis (CT). CT can cause severe damage to fetuses-newborns. To our knowledge, no study has been conducted in sub-Saharan Africa on toxoplasmosis seroprevalence, seroconversion and CT in a large longitudinal cohort and furthermore, no observation has been made of potential relationships with malaria. We performed a retrospective toxoplasmosis serological study using available samples from a large cohort of 1,037 pregnant women who were enrolled in a malaria follow-up during the 2008-2010 period in a rural area in Benin. We also used some existing data to investigate potential relationships between the maternal toxoplasmosis serological status and recorded malaria infections. Toxoplasmosis seroprevalence, seroconversion and CT rates were 52.6%, 3.4% and 0.2%, respectively, reflecting the population situation of toxoplasmosis, without targeted medical intervention. The education level influences the toxoplasmosis serological status of women, with women with little or no formal education have greater immunity than others. Surprisingly, toxoplasmosis seropositive pregnant women tended to present lower malaria infection during pregnancy (number) or at delivery (presence) and to have lower IgG levels to Plasmodium falciparum Apical Membrane Antigen 1, compared to toxoplasmosis seronegative women. The high toxoplasmosis seroprevalence indicates that prevention against this parasite remains important to deploy and must be accessible and understandable to and for all individuals (educated and non-educated). A potential protective role against malaria conferred by a preexisting toxoplasmosis infection needs to be explored more precisely to examine the environmental, parasitic and/or immune aspects.

Comparison of the impact of allelic polymorphisms in PfAMA1 on the induction of T Cell responses in high and low malaria endemic communities in Ghana

BackgroundMalaria eradication requires a combined effort involving all available control tools, and these efforts would be complemented by an effective vaccine. The antigen targets of immune responses may show polymorphisms that can undermine their recognition by immune effectors and hence render vaccines based on antigens from a single parasite variant ineffective against other variants. This study compared the influence of allelic polymorphisms in Plasmodium falciparum apical membrane antigen 1 (PfAMA1) peptide sequences from three strains of P. falciparum (3D7, 7G8 and FVO) on their function as immunodominant targets of T cell responses in high and low malaria transmission communities in Ghana.MethodsPeripheral blood mononuclear cells (PBMCs) from 10 subjects from a high transmission area (Obom) and 10 subjects from a low transmission area (Legon) were tested against 15 predicted CD8 + T cell minimal epitopes within the PfAMA1 antigen of multiple parasite strains using IFN-γ ELISpot assay. The peptides were also tested in similar assays against CD8 + enriched PBMC fractions from the same subjects in an effort to characterize the responding T cell subsets.ResultsIn assays using unfractionated PBMCs, two subjects from the high transmission area, Obom, responded positively to four (26.7%) of the 15 tested peptides. None of the Legon subject PBMCs yielded positive peptide responses using unfractionated PBMCs. In assays with CD8 + enriched PBMCs, three subjects from Obom made positive recall responses to six (40%) of the 15 tested peptides, while only one subject from Legon made a positive recall response to a single peptide. Overall, 5 of the 20 study subjects who had positive peptide-specific IFN-γ recall responses were from the high transmission area, Obom. Furthermore, while subjects from Obom responded to peptides in PfAMA1 from multiple parasite strains, one subject from Legon responded to a peptide from 3D7 strain only.ConclusionsThe current data demonstrate the possibility of a real effect of PfAMA1 polymorphisms on the induction of T cell responses in malaria exposed subjects, and this effect may be more pronounced in communities with higher parasite exposure.

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development.

BackgroundBovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized.ResultsOur results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups.ConclusionOur results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

Accelerated phase Ia/b evaluation of the malaria vaccine candidate PfAMA1 DiCo demonstrates broadening of humoral immune responses

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. PfAMA1’s polymorphic nature impacts vaccine-induced protection. To address polymorphism, three Diversity Covering (DiCo) protein sequences were designed and tested in a staggered phase Ia/b trial. A cohort of malaria-naive adults received PfAMA1-DiCo adjuvanted with Alhydrogel® or GLA-SE and a cohort of malaria-exposed adults received placebo or GLA-SE adjuvanted PfAMA1 DiCo at weeks 0, 4 and 26. IgG and GIA levels measured 4 weeks after the third vaccination are similar in malaria-naive volunteers and placebo-immunised malaria-exposed adults, and have a similar breadth. Vaccination of malaria-exposed adults results in significant antibody level increases to the DiCo variants, but not to naturally occurring PfAMA1 variants. Moreover, GIA levels do not increase following vaccination. Future research will need to focus on stronger adjuvants and/or adapted vaccination regimens, to induce potentially protective responses in the target group of the vaccine.

Memory B-Cell Responses Against Merozoite Antigens After Acute Plasmodium falciparum Malaria, Assessed Over One Year Using a Novel Multiplexed FluoroSpot Assay.

Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-119, MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria.

Secreted heat shock protein gp96-Ig vaccine induces malaria specific intrahepatic CD8 T cell responses

Abstract A highly-efficacious and durable malaria vaccine is an essential tool for global malaria eradication. One promising strategy to develop such a vaccine is to induce robust CD8+ T cell-mediated protective immunity against malaria liver stage parasites. We developed and tested a novel malaria vaccine platform based on the secreted form of the heat shock protein gp96 immunoglobulin, (gp96-Ig) to induce malaria antigen-specific liver resident memory CD8+ T cells. Gp96-Ig has ideal properties as an antigen carrier and vaccine adjuvant to activate antigen presenting cells (APCs) and effectively chaperone antigens for cross presentation via MHC I to CD8+ T lymphocytes. Our study shows that vaccination of mice and non-human primate (NHP) animal models with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum (Pf) vaccine candidate antigens, circumsporozoite protein (CSP) and apical membrane antigen 1 (AMA1), induce liver infiltrating, antigen-specific, cytotoxic memory CD8+ T cell responses. The majority of the CSP and AMA specific intrahepatic CD8+ T cells expressed CD69 and CXCR3, hallmarks of tissue resident memory T cells (TRM). Also, we found an increased frequency of intrahepatic CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses. Our findings are strongly supportive of a novel gp96-Ig-based malaria vaccine as a unique systemic and liver-homing, liver-stage antigen-specific CD8+ cytotoxic T lymphocyte (CTL) vaccine strategy.

Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

HIV infection drives IgM and IgG3 subclass bias in Plasmodium falciparum-specific and total immunoglobulin concentration in Western Kenya

BackgroundHIV infection is associated with more frequent and severe episodes of malaria and may be the result of altered malaria-specific B cell responses. However, it is poorly understood how HIV and the associated lymphopenia and immune activation affect malaria-specific antibody responses.MethodsHIV infected and uninfected adults were recruited from Bondo subcounty hospital in Western Kenya at the time of HIV testing (antiretroviral and co-trimoxazole prophylaxis naïve). Total and Plasmodium falciparum apical membrane antigen-1 (AMA1) and glutamate rich protein-R0 (GLURP-R0) specific IgM, IgG and IgG subclass concentrations was measured in 129 and 52 of recruited HIV-infected and uninfected individuals, respectively. In addition, HIV-1 viral load (VL), CD4+ T cell count, and C-reactive protein (CRP) concentration was quantified in study participants. Antibody levels were compared based on HIV status and the associations of antibody concentration with HIV-1 VL, CD4+ count, and CRP levels was measured using Spearman correlation testing.ResultsAmong study participants, concentrations of IgM, IgG1 and IgG3 antibodies to AMA1 and GLURP-R0 were higher in HIV infected individuals compared to uninfected individuals (all p < 0.001). The IgG3 to IgG1 ratio to both AMA1 and GLURP-R0 was also significantly higher in HIV-infected individuals (p = 0.02). In HIV-infected participants, HIV-1 VL and CRP were weakly correlated with AMA1 and GLURP-R0 specific IgM and IgG1 concentrations and total (not antigen specific) IgM, IgG, IgG1, and IgG3 concentrations (all p < 0.05), suggesting that these changes are related in part to viral load and inflammation.ConclusionsOverall, HIV infection leads to a total and malaria antigen-specific immunoglobulin production bias towards higher levels of IgM, IgG1, and IgG3, and HIV-1 viraemia and systemic inflammation are weakly correlated with these changes. Further assessments of antibody affinity and function and correlation with risk of clinical malaria, will help to better define the effects of HIV infection on clinical and biological immunity to malaria.

Identification of adjuvants for clinical trials performed with Plasmodium falciparum AMA1 in rabbits

BackgroundIn this study, seven adjuvants were compared for use with Plasmodium falciparum DiCo-Apical Membrane Antigen 1 (Pf-DiCo-AMA1), with the aim to identify an ideal adjuvant which yields high antibody titres and potentially broadens the responses in clinical trials. The following adjuvant formulations were evaluated: SE, SE-GLA, Liposomes, Liposomes-GLA, CoVaccine HT™, ImSaVac-P and ImSaVac-P o/w. The study was performed in rabbits, which were immunized with FVO-AMA1 in combination with one of the seven adjuvants. Antibody levels (humoral responses) and functional activity of the antibodies induced against malaria vaccine candidate AMA1 were evaluated. Thus, in this study the ideal adjuvant is expected to induce high functional antibody levels, a long-lived response, and a broad cross-strain activity.ResultsAMA1 formulated in all adjuvants was immunogenic. However, the magnitude of the immune responses differed between the seven adjuvants. The highest IgG levels were observed for the CoVaccine HT™ group, this was statistically significant for all four AMA1 variants versus all other adjuvant groups. No differences were observed in the breadth of the humoral response, i.e., increased recognition of AMA1 variants. Also, Growth Inhibition Activity (GIA) for both Plasmodium falciparum strains (FCR3 – homologous to FVO AMA1 protein and NF54 – heterologous to FVO AMA1 protein) were significantly higher in the CoVaccine HT™ group as compared to the other adjuvant groups.ConclusionsIn brief, all seven vaccine – adjuvant formulations were immunogenic. The magnitude of the immune responses differed between the seven adjuvants. No statistically significant differences were observed in the breadth of the humoral response, nor in longevity of the response. Nevertheless, AMA1 formulated in CoVaccine HT™ appeared as the best adjuvant for use in clinical trials.

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Immunoglobulin G subclass and antibody avidity responses in Malian children immunized with Plasmodium falciparum apical membrane antigen 1 vaccine candidate FMP2.1/AS02A

BackgroundA malaria vaccine based on Plasmodium falciparum apical membrane antigen 1 (AMA1) elicited strain specific efficacy in Malian children that waned in the second season after vaccination despite sustained AMA1 antibody titers. With the goal of identifying a humoral correlate of vaccine-induced protection, pre- and post-vaccination sera from children vaccinated with the AMA1 vaccine and from a control group that received a rabies vaccine were tested for AMA1-specific immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3, and IgG4) and for antibody avidity.MethodsSamples from a previously completed Phase 2 AMA1 vaccine trial in children residing in Mali, West Africa were used to determine AMA1-specific IgG subclass antibody titers and avidity by ELISA. Cox proportional hazards models were used to assess correlation between IgG subclass antibody titers and risk of time to first or only clinical malaria episode and risk of multiple episodes. Asexual P. falciparum parasite density measured for each child as area under the curve were used to assess correlation between IgG subclass antibody titers and parasite burden.ResultsAMA1 vaccination did not elicit a change in antibody avidity; however, AMA1 vaccinees had a robust IgG subclass response that persisted over the malaria transmission season. AMA1-specific IgG subclass responses were not associated with decreased risk of subsequent clinical malaria. For the AMA1 vaccine group, IgG3 levels at study day 90 correlated with high parasite burden during days 90–240. In the control group, AMA1-specific IgG subclass rise and persistence over the malaria season was modest and correlated with age. In the control group, titers of several IgG subclasses at days 90 and 240 correlated with parasite burden over the first 90 study days, and IgG3 at day 240 correlated with parasite burden during days 90–240.ConclusionsNeither IgG subclass nor avidity was associated with the modest, strain-specific efficacy elicited by this blood stage malaria vaccine. Although a correlate of protection was not identified, correlations between subclass titers and age, and correlations between IgG subclass titers and parasite burden, defined by area under the curve parasitaemia levels, were observed, which expand knowledge about IgG subclass responses. IgG3, known to have the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing malaria exposure when examining antibody responses to AMA1.