Blood-stage Antigens Research Articles

IMMUNOGENICITY OF THE MEROZOITE SURFACE PROTEIN-1 (MSP-1) OF HUMAN PLASMODIUM SP.

Malaria is a major cause of mortality and morbidity globally. Great efforts have been made in the prevention and the elimination of malaria, especially in controlling the malaria vector, the mosquito. Another promising approach would be the development of malaria vaccines. Malaria vaccine studies can be focused on the pre-erythrocytic-stage antigens and the blood-stage antigens, and on the transmission blocking agents targeting the malaria gametocytes. The blood-stage antigens are the leading candidates in malaria vaccine development, as the blood-stage parasites are responsible for causing symptomatic malaria. Human acquired immunity largely targets on blood-stage antigens. This review focuses on one of the most extensively studied blood-stage antigen, the merozoite surface protein-1 (MSP-1), specifically on its evaluation and immunogenicity in rodents and primate models, and its safety and immunogenicity in human clinical trials.

Placental Malaria: Decreased Transfer of Maternal Antibodies Directed to Plasmodium falciparum and Impact on the Incidence of Febrile Infections in Infants.

The efficacy of mother-to-child placental transfer of antibodies specific to malaria blood stage antigens was investigated in the context of placental malaria infection, taking into account IgG specificity and maternal hypergammaglobulinemia. The impact of the resulting maternal antibody transfer on infections in infants up to the age of 6 months was also explored. This study showed that i) placental malaria was associated with a reduced placental transfer of total and specific IgG, ii) antibody placental transfer varied according to IgG specificity and iii) cord blood malaria IgG levels were similar in infants born to mothers with or without placental malaria. The number of malaria infections was negatively associated with maternal age, whereas it was not associated with the transfer of any malaria-specific IgG from the mother to the fetus. These results suggest that i) malaria-specific IgG may serve as a marker of maternal exposure but not as a useful marker of infant protection from malaria and ii) increasing maternal age contributes to diminishing febrile infections diagnosed in infants, perhaps by means of the transmission of an effective antibody response.

Contrasting Patterns of Serologic and Functional Antibody Dynamics to Plasmodium falciparum Antigens in a Kenyan Birth Cohort.

IgG antibodies to Plasmodium falciparum are transferred from the maternal to fetal circulation during pregnancy, wane after birth, and are subsequently acquired in response to natural infection. We examined the dynamics of malaria antibody responses of 84 Kenyan infants from birth to 36 months of age by (i) serology, (ii) variant surface antigen (VSA) assay, (iii) growth inhibitory activity (GIA), and (iv) invasion inhibition assays (IIA) specific for merozoite surface protein 1 (MSP1) and sialic acid-dependent invasion pathway. Maternal antibodies in each of these four categories were detected in cord blood and decreased to their lowest level by approximately 6 months of age. Serologic antibodies to 3 preerythrocytic and 10 blood-stage antigens subsequently increased, reaching peak prevalence by 36 months. In contrast, antibodies measured by VSA, GIA, and IIA remained low even up to 36 months. Infants sensitized to P. falciparum in utero, defined by cord blood lymphocyte recall responses to malaria antigens, acquired antimalarial antibodies at the same rate as those who were not sensitized in utero, indicating that fetal exposure to malaria antigens did not affect subsequent infant antimalarial responses. Infants with detectable serologic antibodies at 12 months of age had an increased risk of P. falciparum infection during the subsequent 24 months. We conclude that serologic measures of antimalarial antibodies in children 36 months of age or younger represent biomarkers of malaria exposure rather than protection and that functional antibodies develop after 36 months of age in this population.

Analysis of antibody profiles in symptomatic malaria in three sentinel sites of Ivory Coast by using multiplex, fluorescent, magnetic, bead-based serological assay (MAGPIX™).

BackgroundAdvances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead-based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast.MethodsRecombinant proteins or peptides derived from liver or blood stage antigens of Plasmodiumfalciparum (CSP, LSA141, LSA3, SALSA, PF13-DBL1α1, GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour-coded microsphere (Luminex™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed.ResultsHighly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97 % (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite-derived recombinant proteins or peptides. Prevalence of responders to LSA141 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post-treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50 % of the parasite antigens tested.ConclusionThe multiplex MBA-Magpix technology assay provides an accurate high throughput monitoring of parasite-specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection. Additionally, they can be used as an indicator in the implementation of malaria prevention and local control strategies.

Progress with viral vectored malaria vaccines: A multi-stage approach involving “unnatural immunity”

Viral vectors used in heterologous prime-boost regimens are one of very few vaccination approaches that have yielded significant protection against controlled human malaria infections. Recently, protection induced by chimpanzee adenovirus priming and modified vaccinia Ankara boosting using the ME-TRAP insert has been correlated with the induction of potent CD8+ T cell responses. This regimen has progressed to field studies where efficacy against infection has now been reported. The same vectors have been used pre-clinically to identify preferred protective antigens for use in vaccines against the pre-erythrocytic, blood-stage and mosquito stages of malaria and this work is reviewed here for the first time. Such antigen screening has led to the prioritization of the PfRH5 blood-stage antigen, which showed efficacy against heterologous strain challenge in non-human primates, and vectors encoding this antigen are in clinical trials. This, along with the high transmission-blocking activity of some sexual-stage antigens, illustrates well the capacity of such vectors to induce high titre protective antibodies in addition to potent T cell responses. All of the protective responses induced by these vectors exceed the levels of the same immune responses induced by natural exposure supporting the view that, for subunit vaccines to achieve even partial efficacy in humans, “unnatural immunity” comprising immune responses of very high magnitude will need to be induced.

Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1

Malaria is still a grave public health problem in tropical areas of the world. The greater genetic diversity of Plasmodium vivax at geographic sites with less control over infection evidences the importance of genetic studies of these parasites. The present genetic study compares P. vivax in Nicaragua, which is still in the control phase, with this species in several other countries. In Nicaragua, P. vivax causes over 80% of malaria cases, most occurring in two remote northern regions. Plasmodium asexual blood-stage antigens, implicated in reticulocyte invasion, are possible molecular markers for analyzing parasite population genetics and for developing vaccines. The aim of this work was to investigate the genetic structure of P. vivax based on the 42kDa merozoite surface protein-1 (PvMSP-142), which may represent a sensitive marker for evaluating malaria transmission control. From blood samples of patients with P. vivax, we amplified PvMSP-142, obtained the nucleotide sequences, and compared them to homologous sequences of parasites from other geographic sites, retrieved from the GenBank. The 92 nucleotide sequences of P. vivax resulted in the resolution of eight haplotypes, six exclusive to Nicaragua. The great nucleotide diversity (π=0.020), the minimal recombination events (Rm=11), and the dN–dS values were similar to other control phase countries. FST values between parasites were low (0.069) for Nicaragua versus Brazil but higher for Nicaragua versus other regions (0.134–0.482). The haplotype network revealed five lineages: two were very frequent in Nicaragua and closely related to American parasites; three have been detected in multiple geographic sites around the world. These results suggest that P. vivax in Nicaragua is a differentiated and genetically diverse population (mainly due to mutation, positive balancing selection and recombination) and that PvMSP-142 may be a sensitive marker for evaluating sustained reduction in malaria transmission and for developing vaccines.

Genetic determinants of anti-malarial acquired immunity in a large multi-centre study

BackgroundMany studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels.MethodsSera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels.ResultsMalaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2.ConclusionAlthough the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0833-x) contains supplementary material, which is available to authorized users.

Natural acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein (PvDBP-II) equally block erythrocyte binding of homologous and heterologous expressed PvDBP-II on the surface of COS-7 cells.

The binding domain of Plasmodium vivax Duffy binding protein (PvDBP-II) is a promising blood-stage vaccine candidate for vivax malaria. For the development of a successful vivax malaria vaccine based on DBP-II, the antigenic diversity and also naturally occurring functional antibodies to different PvDBP-II variant types in the various populations must be determined. However, similar to other blood-stage antigens, allelic variation within the PvDBP-II is a fundamental challenge for the development of a broadly efficient vaccine. The present study was performed to define whether the polymorphisms in PvDBP-II influence the nature of functional inhibitory activity of naturally acquired or induced anti-DBP-II antibodies in mice. In this investigation, five genetically distinct variants of PvDBP-II were transiently expressed on the COS-7 cell surface. Erythrocyte-binding inhibition assay (EBIA) was performed using human sera infected with corresponding and non-corresponding P. vivax variants as well as by the use of mice sera immunized with different expressed recombinant PvDBP-IIs. EBIA results showed that the inhibitory percentage varied between 50 and 63 % by using sera from infected individuals, and in case of mouse antisera, inhibition was in the range of 76-86 %. Interestingly, no significant difference was detected in red blood cell binding inhibition when different PvDBP-II variants on the COS-7 cell surfaces were incubated with heterologous and homologous sera infected with PvDBP-II variants. This suggests that the detected polymorphisms in all five forms of PvDBP-II may not affect functional activity of anti-DBP-II antibodies. In conclusion, our results revealed that there are functional cross-reactive antibody responses to heterologous PvDBP-II variants that might provide a broader inhibitory response against all, or at least the majority of strains compared to single allele of this protein that should be considered in development of PvDBP-II-based vaccine.

The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines.

Multicomponent vaccines targeting different stages of Plasmodium falciparum represent a promising, holistic concept towards better malaria vaccines. Additionally, an effective vaccine candidate should demonstrate cross-strain specificity because many antigens are polymorphic, which can reduce vaccine efficacy. A cocktail of recombinant fusion proteins (VAMAX-Mix) featuring three diversity-covering variants of the blood-stage antigen PfAMA1, each combined with the conserved sexual-stage antigen Pfs25 and one of the pre-erythrocytic-stage antigens PfCSP_TSR or PfCelTOS, or the additional blood-stage antigen PfMSP1_19, was produced in Pichia pastoris and used to immunize rabbits. The immune sera and purified IgG were used to perform various assays determining antigen specific titers and in vitro efficacy against different parasite stages and strains. In functional in vitro assays we observed robust inhibition of blood-stage (up to 90%), and sexual-stage parasites (up to 100%) and biased inhibition of pre-erythrocytic parasites (0-40%). Cross-strain blood-stage efficacy was observed in erythrocyte invasion assays using four different P. falciparum strains. The quantification of antigen-specific IgGs allowed the determination of specific IC50 values. The significant difference in antigen-specific IC50 requirements, the direct correlation between antigen-specific IgG and the relative quantitative representation of antigens within the cocktail, provide valuable implementations for future multi-stage, multi-component vaccine designs.

Malaria invasion ligand RH5 and its prime candidacy in blood-stage malaria vaccine design

With drug resistance to available therapeutics continuing to develop against Plasmodium falciparum malaria, the development of an effective vaccine candidate remains a major research goal. Successful interruption of invasion of parasites into erythrocytes during the blood stage of infection will prevent the severe clinical symptoms and complications associated with malaria. Previously studied blood stage antigens have highlighted the hurdles that are inherent to this life-cycle stage, namely that highly immunogenic antigens are also globally diverse, resulting in protection only against the vaccine strain, or that naturally acquired immunity to blood stage antigens do not always correlate with actual protection. The blood stage antigen reticulocyte binding homolog RH5 is essential for parasite viability, has globally limited diversity, and is associated with protection from disease. Here we summarize available information on this invasion ligand and recent findings that highlight its candidacy for inclusion in a blood-stage malaria vaccine.

The effect of daily co-trimoxazole prophylaxis on natural development of antibody-mediated immunity against P. falciparum malaria infection in HIV-exposed uninfected Malawian children.

Background and ObjectivesCo-trimoxazole prophylaxis, currently recommended in HIV-exposed, uninfected (HEU) children as protection against opportunistic infections, also has some anti-malarial efficacy. We determined whether daily co-trimoxazole prophylaxis affects the natural development of antibody-mediated immunity to blood-stage Plasmodium falciparum malaria infection.MethodsUsing an enzyme-linked immunosorbent assay, we measured antibodies to 8Plasmodium falciparum antigens (AMA-1, MSP-119, MSP-3, PfSE, EBA-175RII, GLURP R0, GLURP R2 and CSP) in serum samples from 33 HEU children and 31 HIV-unexposed, uninfected (HUU) children, collected at 6, 12 and 18 months of age.ResultsCompared to HIV-uninfected children, HEU children had significantly lower levels of specific IgG against AMA-1 at 6 months (p = 0.001), MSP-119 at 12 months (p = 0.041) and PfSE at 6 months (p = 0.038), 12 months (p = 0.0012) and 18 months (p = 0.0097). No differences in the IgG antibody responses against the rest of the antigens were observed between the two groups at all time points. The breadth of specificity of IgG response was reduced in HEU children compared to HUU children during the follow up period.ConclusionsCo-trimoxazole prophylaxis seems to reduce IgG antibody responses to P. falciparum blood stage antigens, which could be as a result of a reduction in exposure of those children under this regime. Although antibody responses were regarded as markers of exposure in this study, further studies are required to establish whether these responses are correlated in any way to clinical immunity to malaria.

RTS,S Vaccination Is Associated With Serologic Evidence of Decreased Exposure to Plasmodium falciparum Liver- and Blood-Stage Parasites*[S

The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator vaccine (n = 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together, these data provide insight into the mechanism by which RTS,S protects from malaria.

IgG responses to candidate malaria vaccine antigens in the urban area of Dakar (Senegal): evolution according to age and parasitemia in patients with mild symptoms

Malaria remains a major problem in African countries despite substantial decreases in morbidity and mortality due to sustained control programs. Studies for the evaluation of qualitative or quantitative Ab responses to key targets of anti-plasmodium immunity were mostly done in rural endemic setting compared to urban area. In a cohort of 200 patients with mild malaria and living in Dakar, we analyze total and subclasses IgG responses to a panel of P. falciparum blood stage antigens: MSP1p19, MSP3, EB200, GST-5 and R23. A mean age of 15 yrs (4 to 56 yrs) and parasitemia between 0.1 to 17% were found. Levels of IgG anti-MSP3 were higher in patients with low parasitemia (≤1%) and appear negatively correlated to parasite densities (Rho =. 0.54; p= 0.021). This correlation is more significant in children (≤ 15 yrs). In addition, an increase of IgG responses against MSP1p19 is highly observed in adults having a parasitemia less than 1%. In those patients, we find that IgG1 subclasses were predominant (p <0.01). Our study shows an association between Ab responses and parasitemia. This association is dependant to IgG anti-MSP3 in children and IgG anti-MSP1p19 in adults living in urban area.

Effect of antenatal parasitic infections on anti-vaccine IgG levels in children: a prospective birth cohort study in Kenya.

BackgroundParasitic infections are prevalent among pregnant women in sub-Saharan Africa. We investigated whether prenatal exposure to malaria and/or helminths affects the pattern of infant immune responses to standard vaccinations against Haemophilus influenzae (Hib), diphtheria (DT), hepatitis B (Hep B) and tetanus toxoid (TT).Methods and Findings450 Kenyan women were tested for malaria, schistosomiasis, lymphatic filariasis (LF), and intestinal helminths during pregnancy. After three standard vaccinations at 6, 10 and 14 weeks, their newborns were followed biannually to age 36 months and tested for absolute levels of IgG against Hib, DT, Hep B, and TT at each time point. Newborns’ cord blood (CB) lymphocyte responses to malaria blood-stage antigens, soluble Schistosoma haematobium worm antigen (SWAP), and filaria antigen (BMA) were also assessed. Three immunophenotype categories were compared: i) tolerant (those having Plasmodium-, Schistosoma-, or Wuchereria-infected mothers but lacking respective Th1/Th2-type recall responses at birth to malaria antigens, SWAP, or BMA); ii) sensitized (those with infected/uninfected mothers and detectable Th1/Th2-type CB recall response to respective parasite antigen); or iii) unexposed (no evidence of maternal infection or CB recall response).Overall, 78.9% of mothers were infected with LF (44.7%), schistosomiasis (32.4%), malaria (27.6%) or hookworm (33.8%). Antenatal maternal malaria, LF, and hookworm were independently associated with significantly lower Hib-specific IgG. Presence of multiple maternal infections was associated with lower infant IgG levels against Hib and DT antigens post-vaccination. Post-vaccination IgG levels were also significantly associated with immunophenotype: malaria-tolerized infants had reduced response to DT, whereas filaria-tolerized infants showed reduced response to Hib.ConclusionsThere is an impaired ability to develop IgG antibody responses to key protective antigens of Hib and diphtheria in infants of mothers infected with malaria and/or helminths during pregnancy. These findings highlight the importance of control and prevention of parasitic infections among pregnant women.

Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity.

Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethal Plasmodium yoelii yoelii 17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses of P. yoelii yoelii 17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.

Genetic diversity in MSP-1 gene of Plasmodium falciparum and its association with malaria severity, parasite density, and host factors of asymptomatic and symptomatic patients in Papua, Indonesia

Background: Genetic diversity of Plasmodium falciparum represents a major issue in understanding the several aspects of malaria infection and diseases. The merozoite surface protein (MSP)-1 gene of P. falciparum codes for a major asexual blood-stage antigen, presently proposed as a major malarial vaccine candidate. Objective: To determine the length of the DNA bands and genetic diversity of the MSP-1 gene of P. falciparum, which is associated with malaria severity and host factors in population of the asymptomatic and symptomatic patients in Papua, Indonesia. Materials and Methods: A total of 152 isolates from the blood samples of the patients infected with P. falciparum were analyzed by microscopic examination. DNA was extracted from MSP-1 gene and nested PCR amplification carried out. The active case detection was conducted in Koya and Skow villages, whereas passive detection was obtained from general hospitals. Result: The main allele of the gene MSP-1, which related to the severity of malaria, the parasite density, and host factors of the population with a single infection, was found to MAD20 that was higher in mild malarial cases. The family alleles K1, MAD20, and RO33 of the MSP-1 gene of P. falciparum were found to be in large numbers in severe malaria and light malaria of the symptomatic patients. In the asymptomatic patients, it was found that the alleles of MSP-1 (K1, MAD20, and RO33) were more numerous when the parasite density was less than 10,000/μL of blood. Conclusion: There was no significant correlation between the genetic diversity of the MSP-1 gene of P. falciparum with malaria severity, parasite density, age group, and gender of asymptomatic patients, but there was a significant correlation with the age groups and gender in the symptomatic patients. The K1, MAD20, and RO33 alleles showed a higher density in asymptomatic patients, but K1 and RO33 alleles were higher at severe and mild malaria and MAD20 was higher at mild malaria cases. Therefore, MSP-1 gene allele could be used as a genetic marker to determine the genetic diversity of P. falciparum and as a candidate molecular vaccine.

IFNγ Responses to Pre-erythrocytic and Blood-stage Malaria Antigens Exhibit Differential Associations With Past Exposure and Subsequent Protection.

The malaria-specific T-cell response is believed to be important for protective immunity. Antimalarial chemoprevention may affect this response by altering exposure to malaria antigens. We performed interferon γ (IFNγ) ELISpot assays to assess the cellular immune response to blood-stage and pre-erythrocytic antigens longitudinally from 1 to 3 years of age in 196 children enrolled in a randomized trial of antimalarial chemoprevention in Tororo, Uganda, an area of high transmission intensity. IFNγ responses to blood-stage antigens, particularly MSP1, were frequently detected, strongly associated with recent malaria exposure, and lower in those adherent to chemoprevention compared to nonadherent children and those randomized to no chemoprevention. IFNγ responses to pre-erythrocytic antigens were infrequent and similar between children randomized to chemoprevention or no chemoprevention. Responses to blood-stage antigens were not associated with subsequent protection from malaria (aHR 0.96, P = .83), but responses to pre-erythrocytic antigens were associated with protection after adjusting for prior malaria exposure (aHR 0.52, P = .009). In this high transmission setting, IFNγ responses to blood-stage antigens were common and associated with recent exposure to malaria but not protection from subsequent malaria. Responses to pre-erythrocytic antigens were uncommon, not associated with exposure but were associated with protection from subsequent malaria.

PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen

BackgroundErythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes.ResultsPfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes.MethodsThe expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies.ConclusionThis study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-490) contains supplementary material, which is available to authorized users.

Effect of transmission intensity and age on subclass antibody responses to Plasmodium falciparum pre-erythrocytic and blood-stage antigens

Cytophilic immunoglobulin (IgG) subclass responses (IgG1 and IgG3) to Plasmodium falciparum antigens have been associated with protection from malaria, yet the relative importance of transmission intensity and age in generation of subclass responses to pre-erythrocytic and blood-stage antigens have not been clearly defined. We analyzed IgG subclass responses to the pre-erythrocytic antigens CSP, LSA-1, and TRAP and the blood-stage antigens AMA-1, EBA-175, and MSP-1 in asymptomatic residents age 2 years or older in stable (n=116) and unstable (n=96) transmission areas in Western Kenya. In the area of stable malaria transmission, a high prevalence of cytophilic (IgG1 and IgG3) antibodies to each antigen was seen in all age groups. Prevalence and levels of cytophilic antibodies to pre-erythrocytic and blood-stage P. falciparum antigens increased with age in the unstable transmission area, yet IgG1 and IgG3 responses to most antigens for all ages in the unstable transmission area were less prevalent and lower in magnitude than even the youngest age group from the stable transmission area. The dominance of cytophilic responses over non-cytophilic (IgG2 and IgG4) was more pronounced in the stable transmission area, and the ratio of IgG3 over IgG1 generally increased with age. In the unstable transmission area, the ratio of cytophilic to non-cytophilic antibodies did not increase with age, and tended to be IgG3-biased for pre-erythrocytic antigens yet IgG1-biased for blood-stage antigens. The differences between areas could not be attributed to active parasitemia status, as there were minimal differences in antibody responses between those positive and negative for Plasmodium infection by microscopy in the stable transmission area. Individuals in areas of unstable transmission have low cytophilic to non-cytophilic IgG subclass ratios and low IgG3:IgG1 ratios to P. falciparum antigens. These imbalances could contribute to the persistent risk of clinical malaria in these areas and serve as population-level, age-specific biomarkers of transmission.

Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits.

One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P. falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2 × 10(6) were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC₅₀ values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.