The influence of intestinal parasites on Plasmodium vivax-specific antibody responses to MSP-119 and AMA-1 in rural populations of the Brazilian Amazon

Abstract\ud \ud Background\ud Polyparasitism is a common condition in humans but its impact on the host immune system and clinical diseases is still poorly understood. There are few studies of the prevalence and the effect of malaria-intestinal parasite co-infections in the immune response to malaria vaccine candidates. The present study determines whether the presence of malaria and intestinal parasites co-infection is associated with impaired IgG responses to Plasmodium vivax AMA-1 and MSP-119 in a rural population of the Brazilian Amazon.\ud \ud \ud Methods\ud A cross-sectional survey was performed in a rural area of Rondonia State and 279 individuals were included in the present study. At recruitment, whole blood was collected and Plasmodium and intestinal parasites were detected by microscopy and molecular tests. Blood cell count and haemoglobin were also tested and antibody response specific to P. vivax AMA-1 and MSP-119 was measured in plasma by ELISA. The participants were grouped according to their infection status: singly infected with Plasmodium (M); co-infected with Plasmodium and intestinal parasites (CI); singly infected with intestinal parasites (IP) and negative (N) for both malaria and intestinal parasites.\ud \ud \ud Results\ud The prevalence of intestinal parasites was significantly higher in individuals with malaria and protozoan infections were more prevalent. IgG antibodies to PvAMA-1 and/or PvMSP-119 were detected in 74 % of the population. The prevalence of specific IgG was similar for both proteins in all four groups and among the groups the lowest prevalence was in IP group. The cytophilic sub-classes IgG1 and IgG3 were predominant in all groups for PvAMA-1 and IgG1, IgG3 and IgG4 for PvMSP-119. In the case of non-cytophilic antibodies to PvAMA-1, IgG2 was significantly higher in IP and N group when compared to M and CI while IgG4 was higher in IP group.\ud \ud \ud Conclusions\ud The presence of intestinal parasites, mainly protozoans, in malaria co-infected individuals does not seem to alter the antibody immune responses to P. vivax AMA-1 and MSP-119. However, IgG response to both AMA1 and MSP1 were lower in individuals with intestinal parasites.The authors are in debt to the individuals who participated in this study, the\ud Secretary of Health and Laboratory Central (LACEN) of Rondonia, the local\ud malaria control team in Joana D´Arc settlement for their logistic support\ud and the Institute Oswaldo Cruz (Fiocruz) for overall support. This work was\ud supported by PRONEX Malaria network funded by the Brazilian Ministry of\ud Science and Technology (MCT), Conselho Nacional de Desenvolvimento\ud Cientifico e Tecnologico (CNPq, Brazil) and Fundação de Amparo à Pesquisa\ud do Estado do Rio de Janeiro (FAPERJ, Brazil). PROEP, Instituto Oswaldo Cruz\ud (FIOCRUZ, Brazil). JOF is recipient of a Research Productivity Fellowship from\ud CNPq, JCSA is recipient of a fellowship from Instituto Oswaldo Cruz and VAR,\ud MM from CNPq

Cells and mediators of inflammation (C-reactive protein, nitric oxide, platelets and neutrophils) in the acute and convalescent phases of uncomplicated Plasmodium vivax and Plasmodium falciparum infection

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

Plasmodium vivax cell traversal protein for Ookinetes and Sporozoites (PvCelTOS) gene sequence and potential epitopes are highly conserved among isolates from different regions of Brazilian Amazon

This work was supported by Brazilian National Research Council–CNPq/PAPES, (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Programa de Apoio e Pesquisa Estratégica em Saúde) Fiocruz. JdCLJ is recipient of a FAPERJ APQ1 (E-26/210.653/2015), Jovem Cientista do Nosso Estado (E26/203.255/2016).Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Clinical Immunology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Computational Modeling Group. Fortaleza, CE, Brazil.University of Campinas. Department of Genetics, Evolution and Bioagents. Laboratory of Tropical Diseases - Prof. Luiz Jacintho da Silva. Campinas, SP, BrazilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Imunogenética da Malária. Ananindeua, PA, Brasil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Malaria Research. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Clinical Immunology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.The Plasmodium vivax Cell-traversal protein for ookinetes and sporozoites (PvCelTOS) plays an important role in the traversal of host cells. Although essential to PvCelTOS progress as a vaccine candidate, its genetic diversity remains uncharted. Therefore, we investigated the PvCelTOS genetic polymorphism in 119 field isolates from five different regions of Brazilian Amazon (Manaus, Novo Repartimento, Porto Velho, Plácido de Castro and Oiapoque). Moreover, we also evaluated the potential impact of non-synonymous mutations found in the predicted structure and epitopes of PvCelTOS. The field isolates showed high similarity (99.3% of bp) with the reference Sal-1 strain, presenting only four Single-Nucleotide Polymorphisms (SNP) at positions 24A, 28A, 109A and 352C. The frequency of synonymous C109A (82%) was higher than all others (p<0.0001). However, the non-synonymous G28A and G352C were observed in 9.2% and 11.7% isolates. The great majority of the isolates (79.8%) revealed complete amino acid sequence homology with Sal-1, 10.9% presented complete homology with Brazil I and two undescribed PvCelTOS sequences were observed in 9.2% field isolates. Concerning the prediction analysis, the N-terminal substitution (Gly10Ser) was predicted to be within a B-cell epitope (PvCelTOS Accession Nos. AB194053.1) and exposed at the protein surface, while the Val118Leu substitution was not a predicted epitope. Therefore, our data suggest that although G28A SNP might interfere in potential B-cell epitopes at PvCelTOS N-terminal region the gene sequence is highly conserved among the isolates from different geographic regions, which is an important feature to be taken into account when evaluating its potential as a vaccine candidate

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

Plasmodium vivax Cell Traversal Protein for Ookinetes and Sporozoites (PvCelTOS) gene sequence and potential epitopes are highly conserved among isolates from different regions of Brazilian Amazon

The Plasmodium vivax Cell-traversal protein for ookinetes and sporozoites (PvCelTOS) plays an important role in the traversal of host cells. Although essential to PvCelTOS progress as a vaccine candidate, its genetic diversity remains uncharted. Therefore, we investigated the PvCelTOS genetic polymorphism in 119 field isolates from five different regions of Brazilian Amazon (Manaus, Novo Repartimento, Porto Velho, Pla A cido de Castro and Oiapoque). Moreover, we also evaluated the potential impact of non-synonymous mutations found in the predicted structure and epitopes of PvCelTOS. The field isolates showed high similarity (99.3% of bp) with the reference Sal-1 strain, presenting only four Single-Nucleotide Polymorphisms (SNP) at positions 24A, 28A, 109A and 352C. The frequency of synonymous C109A (82%) was higher than all others (p<0.0001). However, the non-synonymous G28A and G352C were observed in 9.2% and 11.7% isolates. The great majority of the isolates (79.8%) revealed complete amino acid sequence homology with Sal-1, 10.9% presented complete homology with Brazil I and two undescribed PvCelTOS sequences were observed in 9.2% field isolates. Concerning the prediction analysis, the N-terminal substitution (Gly10Ser) was predicted to be within a B-cell epitope (PvCelTOS Accession Nos. AB194053.1) and exposed at the protein surface, while the Val118Leu substitution was not a predicted epitope. Therefore, our data suggest that although G28A SNP might interfere in potential B-cell epitopes at PvCelTOS N-terminal region the gene sequence is highly conserved among the isolates from different geographic regions, which is an important feature to be taken into account when evaluating its potential as a vaccine candidate112CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO CARLOS CHAGAS FILHO DE AMPARO À PESQUISA DO ESTADO DO RIO DE JANEIRO - FAPERJsem informaçãoE-26/210.653/201

Genetic Diversity of Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) in Field Isolates from Five Different Areas of the Brazilian Amazon

The Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) has an important role in erythrocyte invasion and has been considered a target for vivax malaria vaccine development. Nonetheless, its genetic diversity remains uncharted in Brazilian malaria-endemic areas. Therefore, we investigated the pvcyrpa genetic polymorphism in 98 field isolates from the Brazilian Amazon and its impact on the antigenicity of predicted B-cell epitopes. Genetic diversity parameters, population genetic analysis, neutrality test and the median-joining network were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. One synonymous and 26 non-synonymous substitutions defined fifty haplotypes. The nucleotide diversity and Tajima&rsquo;s D values varied across the coding gene. The exon-1 sequence had greater diversity than those of exon-2. Concerning the prediction analysis, seven sequences were predicted as linear B cell epitopes, the majority contained in conformational epitopes. Moreover, important amino acid polymorphism was detected in regions predicted to contain residues participating in B-cell epitopes. Our data suggest that the pvcyrpa gene presents a moderate polymorphism in the studied isolates and such polymorphisms alter amino acid sequences contained in potential B cell epitopes, an important observation considering the antigen potentiality as a vaccine candidate to cover distinct P. vivax endemic areas worldwide

Extensive genetic diversity of Plasmodium vivax dbp-II in Rio de Janeiro Atlantic Forest and Brazilian Amazon Basin: evidence of positive selection

Submitted by Fábio Marques ([email protected]) on 2020-03-20T19:58:45Z No. of bitstreams: 1 Extensive Genetic_Patricia_Brasil_etal_INI_2020.pdf: 1629547 bytes, checksum: b147ad8b01b1a4dd498b8eb6e699ea99 (MD5)Approved for entry into archive by Regina Costa ([email protected]) on 2020-03-24T00:40:21Z (GMT) No. of bitstreams: 1 Extensive Genetic_Patricia_Brasil_etal_INI_2020.pdf: 1629547 bytes, checksum: b147ad8b01b1a4dd498b8eb6e699ea99 (MD5)Made available in DSpace on 2020-03-24T00:40:21Z (GMT). No. of bitstreams: 1 Extensive Genetic_Patricia_Brasil_etal_INI_2020.pdf: 1629547 bytes, checksum: b147ad8b01b1a4dd498b8eb6e699ea99 (MD5) Previous issue date: 2020Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz, Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil / Centro Universitário Serra dos Órgãos. Teresópolis, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microrganismos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Background: Plasmodium vivax is the most widespread human malaria parasite outside Africa and is the predominant parasite in the Americas. Increasing reports of P. vivax disease severity, together with the emergence of drugresistant strains, underscore the urgency of the development of vaccines against P. vivax. Polymorphisms on DBP-IIgene could act as an immune evasion mechanism and, consequently, limited the vaccine efcacy. This study aimed to investigate the pvdbp-II genetic diversity in two Brazilian regions with diferent epidemiological patterns: the unstable transmission area in the Atlantic Forest (AF) of Rio de Janeiro and; the fxed malaria-endemic area in Brazilian Amazon (BA). Methods: 216 Brazilian P. vivax infected blood samples, diagnosed by microscopic examination and PCR, were investigated. The region fanking pvdbp-II was amplifed by PCR and sequenced. Genetic polymorphisms of pvdbp-II were estimated based on the number of segregating sites and nucleotide and haplotype diversities; the degree of diferentiation between-regions was evaluated applying Wright’s statistics. Natural selection was calculated using the rate of nonsynonymous per synonymous substitutions with the Z-test, and the evolutionary distance was estimated based on the reconstructed tree. Results: 79 samples from AF and 137 from BA were successfully sequenced. The analyses showed 28 polymorphic sites distributed in 21 codons, with only 5% of the samples Salvador 1 type. The highest rates of polymorphic sites were found in B- and T cell epitopes. Unexpectedly, the nucleotide diversity in pvdbp-II was higher in AF (0.01) than in BA (0.008). Among the 28 SNPs detected, 18 are shared between P. vivax isolates from AF and BA regions, but 8 SNPs were exclusively detected in AF—I322S, K371N, E385Q, E385T, K386T, K411N, I419L and I419R—and 2 (N375D and I419M) arose exclusively in BA. These fndings could suggest the potential of these geographical clusters as population-specifc-signatures that may be useful to track the origin of infections. The sample size should be increased in order to confrm this possibility

Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection's role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions

Geographical map showing the five study sites and the respective Annual Parasitic Incidence (API) (SIVEP-Malaria).

<p>Geographical map showing the five study sites and the respective Annual Parasitic Incidence (API) (SIVEP-Malaria).</p