BackgroundThe antibody response generated during malaria infections is of particular interest, since the production of specific IgG antibodies is required for acquisition of clinical immunity. However, variations in antibody responses could result from genetic polymorphism of the HLA class II genes. Given the increasing focus on the development of subunit vaccines, studies of the influence of class II alleles on the immune response in ethnically diverse populations is important, prior to the implementation of vaccine trials.Methods and FindingsIn this study, we evaluated the influence of HLA-DRB1* and -DQB1* allelic groups on the naturally acquired humoral response from Brazilian Amazon individuals (n = 276) against P. vivax Merozoite Surface Protein-1 (MSP-1), MSP-3α and MSP-9 recombinant proteins. Our results provide information concerning these three P. vivax antigens, relevant for their role as immunogenic surface proteins and vaccine candidates. Firstly, the studied population was heterogeneous presenting 13 HLA-DRB1* and 5 DQB1* allelic groups with a higher frequency of HLA-DRB1*04 and HLA-DQB1*03. The proteins studied were broadly immunogenic in a naturally exposed population with high frequency of IgG antibodies against PvMSP1-19 (86.7%), PvMSP-3 (77%) and PvMSP-9 (76%). Moreover, HLA-DRB1*04 and HLA-DQB1*03 alleles were associated with a higher frequency of IgG immune responses against five out of nine antigens tested, while HLA-DRB1*01 was associated with a high frequency of non-responders to repetitive regions of PvMSP-9, and the DRB1*16 allelic group with the low frequency of responders to PvMSP3 full length recombinant protein.ConclusionsHLA-DRB1*04 alleles were associated with high frequency of antibody responses to five out of nine recombinant proteins tested in Rondonia State, Brazil. These features could increase the success rate of future clinical trials based on these vaccine candidates.

Haematological and cytokine alterations in malaria are a broad and controversial subject in the literature. However, few studies have simultaneously evaluated various cytokines in a single patient group during the acute and convalescent phases of infection. The aim of this study was to sequentially characterise alterations in haematological patters and circulating plasma cytokine and chemokine levels in patients infected with Plasmodium vivax or Plasmodium falciparum from a Brazilian endemic area during the acute and convalescent phases of infection. During the acute phase, thrombocytopaenia, eosinopaenia, lymphopaenia and an increased number of band cells were observed in the majority of the patients. During the convalescent phase, the haematologic parameters returned to normal. During the acute phase, P. vivax and P. falciparum patients had significantly higher interleukin (IL)-6, IL-8, IL-17, interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein-1β and granulocyte-colony stimulating factor levels than controls and maintained high levels during the convalescent phase. IL-10 was detected at high concentrations during the acute phase, but returned to normal levels during the convalescent phase. Plasma IL-10 concentration was positively correlated with parasitaemia in P. vivax and P. falciparum-infected patients. The same was true for the TNF-α concentration in P. falciparum-infected patients. Finally, the haematological and cytokine profiles were similar between uncomplicated P. falciparum and P. vivax infections.

The Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3α) is considered as a potential vaccine candidates. However, the detailed investigations of the type of immune responses induced in naturally exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to PvMSP-3α in 282 individuals with different levels of exposure to malaria infections residents in Brazilian Amazon. PvMSP3 specific antibodies (IgA, IgG and IgG subclass) to five recombinant proteins and the epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immunogenic in naturally exposed populations, where 78% of studied individuals present IgG immune response against the full-length recombinant protein (PVMSP3-FL) and IgG subclass profile was similar to all five recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the two blocks of repeats, confirming the high immunogenicity of this region. In conclusion, PvMSP-3α is immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggest that this mechanisms could also occur in P. vivax.

Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines.

Plasmodium falciparum‐specific antibodies tend to be short‐lived, but their cognate memory B cells (MBCs) circulate in the peripheral blood of exposed subjects for several months or years after the last infection. However, the time course of antigen‐specific antibodies and B‐cell responses to the relatively neglected parasite Plasmodium vivax remains largely unexplored. Here, we showed that uncomplicated vivax malaria elicits short‐lived antibodies but long‐lived MBC responses to a major blood‐stage P vivax antigen, apical membrane protein 1 (PvAMA‐1), in subjects exposed to declining malaria transmission in the Amazon Basin of Brazil. We found that atypical (CD19+CD10−CD21−CD27−) MBCs, which appear to share a common precursor with classical MBCs but are unable to differentiate into antibody‐secreting cells, significantly outnumbered classical MBCs by 5:1 in the peripheral blood of adult subjects currently or recently infected with P vivax and by 3:1 in healthy residents in the same endemic communities. We concluded that malaria can drive classical MBCs to differentiate into functionally impaired MBCs not only in subjects repeatedly exposed to P falciparum, but also in subjects living in areas with low levels of P vivax transmission in the Amazon, leading to an impaired B‐cell memory that may affect both naturally acquired and vaccine‐induced immunity.

In Brazil, malaria is prevalent in the Amazon region and these regions coincide with high prevalence of intestinal parasites but few studies explore the interaction between malaria and other parasites. Therefore, the present study evaluates changes in cytokine, chemokine, C-reactive protein, and nitric oxide (NO) concentrations in 264 individuals, comparing plasma from infected individuals with concurrent malaria and intestinal parasites to individuals with either malaria infection alone and uninfected. In the studied population 24% of the individuals were infected with Plasmodium and 18% coinfected with intestinal parasites. Protozoan parasites comprised the bulk of the intestinal parasites infections and subjects infected with intestinal parasites were more likely to have malaria. The use of principal component analysis and cluster analysis associated increased levels of IL-6, TNF-α, IL-10, and CRP and low levels of IL-17A predominantly with individuals with malaria alone and coinfected individuals. In contrast, low levels of almost all inflammatory mediators were associated predominantly with individuals uninfected while increased levels of IL-17A were associated predominantly with individuals with intestinal parasites only. In conclusion, our data suggest that, in our population, the infection with intestinal parasites (mainly protozoan) does not modify the pattern of cytokine production in individuals infected with P. falciparum and P. vivax.

The haematological changes and release of soluble mediators, particularly C-reactive protein (CRP) and nitric oxide (NO), during uncomplicated malaria have not been well studied, especially in Brazilian areas in which the disease is endemic. Therefore, the present study examined these factors in acute (day 0) and convalescent phase (day 15) patients infected with Plasmodium falciparum and Plasmodium vivax malaria in the Brazilian Amazon. Haematologic parameters were measured using automated cell counting, CRP levels were measured with ELISA and NO plasma levels were measured by the Griess reaction. Our data indicate that individuals with uncomplicated P. vivax and P. falciparum infection presented similar inflammatory profiles with respect to white blood cells, with high band cell production and a considerable degree of thrombocytopaenia during the acute phase of infection. Higher CRP levels were detected in acute P. vivax infection than in acute P. falciparum infection, while higher NO was detected in patients with acute and convalescent P. falciparum infections. Although changes in these mediators cannot predict malaria infection, the haematological aspects associated with malaria infection, especially the roles of platelets and band cells, need to be investigated further.

BackgroundCytokines play an important role in human immune responses to malaria and variation in their production may influence the course of infection and determine the outcome of the disease. The differential production of cytokines has been linked to single nucleotide polymorphisms in gene promoter regions, signal sequences, and gene introns. Although some polymorphisms play significant roles in susceptibility to malaria, gene polymorphism studies in Brazil are scarce.MethodsA population of 267 individuals from Brazilian Amazon exposed to malaria was genotyped for five single nucleotide polymorphisms (SNPs), IFNG + 874 T/A, IL10A-1082G/A, IL10A-592A/C, IL10A-819 T/C and NOS2A-954G/C. Specific DNA fragments were amplified by polymerase chain reaction, allowing the detection of the polymorphism genotypes. The polymorphisms IL10A-592A/C and IL10A-819 T/C were estimated by a single analysis due to the complete linkage disequilibrium between the two SNPs with D’ = 0.99. Plasma was used to measure the levels of IFN-γ and IL-10 cytokines by Luminex and nitrogen radicals by Griess reaction.ResultsNo differences were observed in genotype and allelic frequency of IFNG + 874 T/A and NOS2A-954G/C between positive and negative subjects for malaria infection. Interesting, the genotype NOS2A-954C/C was not identified in the study population. Significant differences were found in IL10A-592A/C and IL10A-819 T/C genotypes distribution, carriers of IL10A -592A/-819 T alleles (genotypes AA/TT + AC/TC) were more frequent among subjects with malaria than in negative subjects that presented a higher frequency of the variant C allele (p < 0.0001). The presence of the allele C was associated with low producer of IL-10 and low parasitaemia. In addition, the GTA haplotypes formed from combinations of investigated polymorphisms in IL10A were significantly associated with malaria (+) and the CCA haplotype with malaria (−) groups. The IL10A-1082G/A polymorphism showed high frequency of heterozygous AG genotype in the population, but it was not possible to infer any association of the polymorphism because their distribution was not in Hardy Weinberg equilibrium.ConclusionThis study shows that the IL10A-592A/C and IL10A-819 T/C polymorphisms were associated with malaria and decreased IL-10 levels and low parasite density suggesting that this polymorphism influence IL-10 levels and may influence in the susceptibility to clinical malaria.