Daiana de Souza Perce-da-Silva

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.

Intestinal Parasites Coinfection Does Not Alter Plasma Cytokines Profile Elicited in Acute Malaria in Subjects from Endemic Area of Brazil

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.

Killer cell immunoglobulin-like receptor (KIR) gene diversity in a population naturally exposed to malaria in Porto Velho, Northern Brazil

Killer cell immunoglobulin-like receptors (KIR) are expressed mainly in natural killer cells and specifically recognize human leukocyte antigen (HLA) class I molecules. The repertoire of KIR genes and KIR-HLA pairs is known to play a key role in the susceptibilities to and the outcomes of several diseases, including malaria. The aim of this study was to investigate the distribution of KIR genes, KIR genotypes and KIR-HLA pair combinations in a population naturally exposed to malaria from Brazilian Amazon. All 16 KIR genes investigated were present in the studied population. Overall, 46 KIR genotypes were defined. The two most common genotypes in the Porto Velho communities, genotypes 1 and 2, were present at similar frequencies as in the Americas. Principal component analysis based on the frequencies of the KIR genes placed the Porto Velho population closer to the Venezuela Mestizos, USA California hispanic and Brazil Paraná Mixed in terms of KIR gene frequencies. This analysis highlights the multi-ethnic profile of the Porto Velho population. Most of the individuals were found to have at least one inhibitory KIR-HLA pair. Seventy-five KIR-HLA pair combinations were identified. The KIR-2DL2/3_HLA-C1, KIR3DL1_HLA-Bw4 and KIR2DL1_HLA-C2 pairs were the most common. There was no association between KIR genes, KIR genotypes or KIR-HLA pair combinations and malaria susceptibility in the studied population. This is the first report on the distribution of KIR and known HLA ligands in the Porto Velho population. Taken together, these results should provide baseline information that will be relevant to population evolutionary history, malaria and other diseases studies in populations of the Brazilian Amazon.

Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites: Naturally Acquired Humoral Immune Response and B-Cell Epitope Mapping in Brazilian Amazon Inhabitants.

The cell-traversal protein for ookinetes and sporozoites (CelTOS), a highly conserved antigen involved in sporozoite motility, plays an important role in the traversal of host cells during the preerythrocytic stage of Plasmodium species. Recently, it has been considered an alternative target when designing novel antimalarial vaccines against Plasmodium falciparum. However, the potential of Plasmodium vivax CelTOS as a vaccine target is yet to be explored. This study evaluated the naturally acquired immune response against a recombinant P. vivax CelTOS (PvCelTOS) (IgG and IgG subclass) in 528 individuals from Brazilian Amazon, as well as the screening of B-cell epitopes in silico and peptide assays to associate the breadth of antibody responses of those individuals with exposition and/or protection correlates. We show that PvCelTOS is naturally immunogenic in Amazon inhabitants with 94 individuals (17.8%) showing specific IgG antibodies against the recombinant protein. Among responders, the IgG reactivity indexes (RIs) presented a direct correlation with the number of previous malaria episodes (p = 0.003; r = 0.315) and inverse correlation with the time elapsed from the last malaria episode (p = 0.031; r = −0.258). Interestingly, high responders to PvCelTOS (RI > 2) presented higher number of previous malaria episodes, frequency of recent malaria episodes, and ratio of cytophilic/non-cytophilic antibodies than low responders (RI < 2) and non-responders (RI < 1). Moreover, a high prevalence of the cytophilic antibody IgG1 over all other IgG subclasses (p < 0.0001) was observed. B-cell epitope mapping revealed five immunogenic regions in PvCelTOS, but no associations between the specific IgG response to peptides and exposure/protection parameters were found. However, the epitope (PvCelTOSI136-E143) was validated as a main linear B-cell epitope, as 92% of IgG responders to PvCelTOS were also responders to this peptide sequence. This study describes for the first time the natural immunogenicity of PvCelTOS in Amazon individuals and identifies immunogenic regions in a full-length protein. The IgG magnitude was mainly composed of cytophilic antibodies (IgG1) and associated with recent malaria episodes. The data presented in this paper add further evidence to consider PvCelTOS as a vaccine candidate.

Malaria-Cutaneous Leishmaniasis Co-infection: Influence on Disease Outcomes and Immune Response

Malaria and Cutaneous Leishmaniasis (CL) are co-endemic throughout large regions in tropical countries and co-infection may impact the evolution of host-parasite interactions. In the present study, we evaluate Malaria/Leishmaniasis disease outcome, Th1/Th2 cytokine levels and the CD4 and CD8 T-cell profiles in a co-infection murine model (BALB/c) of Plasmodium yoelii 17XNL (Py) and Leishmania amazonensis (La) or L. braziliensis (Lb). Malaria parasitaemia was assessed through blood strains stained with Giemsa. Leishmania lesions were monitored with a digital caliper and parasite loads determined by limiting-dilution assay. Serum levels of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10, and IL-17 were determined using multiplexed bead assay and expression of CD3, CD4, and CD8 T-cells markers were determined by Flow Cytometry in the thymus, spleens and lymph nodes. Parasitaemia in Lb+Py co-infected group was lower than in Py single-infected group, suggesting a protective effect of Lb co-infection in Malaria progression. In contrast, La+Py co-infection increased parasitaemia, patent infection and induced mortality in non-lethal Malaria infection. Regarding Leishmaniasis, Lb+Py co-infected group presented smaller lesions and less ulceration than Lb single-infected animals. In contrast, La+Py co-infected group presented only a transitory delay on the development of lesions when compared to La single-infected mice. Decreased levels of IFN-γ, TNF, IL-6, and IL-10 were observed in the serum of co-infected groups, demonstrating a modulation of Malaria immune response by Leishmania co-infections. We observed an intense thymic atrophy in Py single-infected and co-infected groups, which recovered earlier in co-infected animals. The CD4 and CD8 T cell profiles in thymus, spleens and lymph nodes did not differ between Py single and co-infected groups, except for a decrease in CD4+CD8+ T cells which also increased faster in co-infected mice. Our results suggest that Py and Leishmania co-infection may change disease outcome. Interestingly Malaria outcome can be altered according to the Leishmania specie involved. Alternatively Malaria infection reduced the severity or delayed the onset of leishmanial lesions. These alterations in Malaria and CL development seem to be closely related with changes in the immune response as demonstrated by alteration in serum cytokine levels and thymus/spleens T cell phenotypes dynamics during infection.

Evaluation of allelic forms of the erythrocyte binding antigen 175 (EBA-175) in Plasmodium falciparum field isolates from Brazilian endemic area

BackgroundThe Plasmodium falciparum Erythrocyte Binding Antigen-175 (EBA-175) is an antigen considered to be one of the leading malaria vaccine candidates. EBA-175 mediates sialic acid-dependent binding to glycophorin A on the erythrocytes playing a crucial role during invasion of the P. falciparum in the host cell. Dimorphic allele segments, termed C-fragment and F-fragment, have been found in high endemicity malaria areas and associations between the dimorphism and severe malaria have been described. In this study, the genetic dimorphism of EBA-175 was evaluated in P. falciparum field isolates from Brazilian malaria endemic area.MethodsThe study was carried out in rural villages situated near Porto Velho, Rondonia State in the Brazilian Amazon in three time points between 1993 and 2008. The allelic dimorphism of the EBA-175 was analysed by Nested PCR.ResultsThe classical allelic dimorphism of the EBA-175 was identified in the studied area. Overall, C-fragment was amplified in a higher frequency than F-fragment. The same was observed in the three time points where C-fragment was observed in a higher frequency than F-fragment. Single infections (one fragment amplified) were more frequent than mixed infection (two fragments amplified).ConclusionsThese findings confirm the dimorphism of EBA175, since only the two types of fragments were amplified, C-fragment and F-fragment. Also, the results show the remarkable predominance of CAMP allele in the studied area. The comparative analysis in three time points indicates that the allelic dimorphism of the EBA-175 is stable over time.

Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon

BackgroundBrazil has seen a great decline in malaria and the country is moving towards elimination. However, for eventual elimination, the control program needs efficient tools in order to monitor malaria exposure and transmission. In this study, we aimed to evaluate whether seroprevalence to the circumsporozoite protein (CSP) is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium species in the Brazilian Amazon.MethodsCross-sectional surveys were conducted in a rural area of Porto Velho, Rondônia state. Parasite infection was detected by microscopy and polymerase chain reaction. Antibodies to the sporozoite CSP repeats of Plasmodium vivax, P. falciparum, and P. malariae (PvCS, PfCS, and PmCS) were detected using the enzyme-linked immunosorbent assay technique. Human leukocyte antigen (HLA)-DRB1 and DQB1 genes were typed using Luminex® xMAP® technology.ResultsThe prevalence of immunoglobulin G against P. vivax CSP peptide (62%) was higher than P. falciparum (49%) and P. malariae (46%) CSP peptide. Most of the studied individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although the majority of the population was not infected at the time of the survey, 74.3% of parasite-negative individuals had antibodies to at least one of the CSPs. Importantly, among individuals carrying the haplotypes DRB1*04~DQB1*03, there was a significantly higher frequency of PfCS responders, and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders. In contrast, HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P. vivax and P. falciparum CSP repeats, and the haplotype DRB1*01~DQB1*05 was also associated with non-responders, including non-responders to P. malariae.ConclusionsOur results show that in low transmission settings, naturally acquired antibody responses against the CSP repeats of P. vivax, P. falciparum, and P. malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II molecules play an important role in antibody response and require further study with a larger sample size. It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.

Synthetic Antigens Derived from Plasmodium falciparum Sporozoite, Liver, and Blood Stages: Naturally Acquired Immune Response and Human Leukocyte Antigen Associations in Individuals Living in a Brazilian Endemic Area

Peptide vaccine strategies using Plasmodium-derived antigens have emerged as an attractive approach against malaria. However, relatively few studies have been conducted with malaria-exposed populations from non-African countries. Herein, the seroepidemiological profile against Plasmodium falciparum of naturally exposed individuals from a Brazilian malaria-endemic area against synthetic peptides derived from vaccine candidates circumsporozoite protein (CSP), liver stage antigen-1 (LSA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein-3 (MSP-3) was investigated. Moreover, human leukocyte antigen (HLA)-DRB1* and HLA-DQB1* were evaluated to characterize genetic modulation of humoral responsiveness to these antigens. The study was performed using blood samples from 187 individuals living in rural malaria-endemic villages situated near Porto Velho, Rondônia State. Specific IgG and IgM antibodies and IgG subclasses were detected by enzyme-linked immunosorbent assay, and HLA-DRB1* and HLA-DQB1* low-resolution typing was performed by PCR-SSP. All four synthetic peptides were broadly recognized by naturally acquired antibodies. Regarding the IgG subclass profile, only CSP induced IgG1 and IgG3 antibodies, which is an important fact given that the acquisition of protective immunity appears to be associated with the cytophilicity of IgG1 and IgG3 antibodies. HLA-DRB1*11 and HLA-DQB1*7 had the lowest odds of responding to EBA-175. Our results showed that CSP, LSA-1, EBA, and MSP-3 are immunogenic in natural conditions of exposure and that anti-EBA antibody responses appear to be modulated by HLA class II antigens.