Polymorphisms in Toll-Like receptors genes and their associations with immunological parameters in Plasmodium vivax malaria in the Brazil-French Guiana Border.

BACKGROUND: The innate immune response plays an important role during malaria. Toll-like receptors (TLR) are capable of recognizing pathogen molecules. We aimed to evaluate five polymorphisms in TLR-4, TLR-6, and TLR-9 genes and their association with cytokine levels and clinical parameters in malaria from the Brazil-French Guiana border. METHODS: A case-control study was conducted in Amapá, Brazil. P. vivax patients and individuals not infected were evaluated. Genotyping of five SNPs was carried out by qPCR. Circulating cytokines were measured by CBA. The MSP-119 IgG antibodies were performed by ELISA. RESULTS: An association between TLR4 A299G with parasitemia was observed. There was an increase for IFN-ɤ, TNF-ɑ, IL-6, and IL-10 in the TLR-4 A299G and T3911, TLR-6 S249P, and TLR-9 1486C/T, SNPs for the studied malarial groups. There were significant findings for the TLR-4 variants A299G and T3911, TLR-9 1237C/T, and 1486C/T. For the reactivity of MSP-119 antibodies levels, no significant results were found in malaria, and control groups. CONCLUSIONS: The profile of the immune response observed by polymorphisms in TLRs genes does not seem to be standard for all types of malaria infection around the world. This can depend on the human population and the species of Plasmodium.

Analysis of anti-Plasmodium IgG profiles among Fulani nomadic pastoralists in northern Senegal to assess malaria exposure.

BACKGROUND: Northern Senegal is a zone of very low malaria transmission, with an annual incidence of < 5/1000 inhabitants. This area, where the Senegal National Malaria Control Programme has initiated elimination activities, hosts Fulani, nomadic, pastoralists that spend the dry season in the south where malaria incidence is higher (150-450/1000 inhabitants) and return to the north with the first rains. Previous research demonstrated parasite prevalence of < 1% in this Fulani population upon return from the south, similar to that documented in the north in cross-sectional surveys. METHODS: A modified snowball sampling survey of nomadic pastoralists was conducted in five districts in northern Senegal during September and October 2014. Demographic information and dried blood spots were collected. Multiplex bead-based assays were used to assess antibody responses to merozoite surface protein (MSP-119) antigen of the four primary Plasmodium species, as well as circumsporozoite protein (CSP) and liver stage antigen (LSA-1) of Plasmodium falciparum. RESULTS: In the five study districts, 1472 individuals were enrolled, with a median age of 22 years (range 1 to 80 years). Thirty-two percent of subjects were under 14 years and 57% were male. The overall seroprevalence of P. falciparum MSP-119, CSP and LSA-1 antibodies were 45, 12 and 5%, respectively. Plasmodium falciparum MSP-119 antibody responses increased significantly with age in all study areas, and were significantly higher among males. The highest seroprevalence to P. falciparum antigens was observed in the Kanel district (63%) and the lowest observed in Podor (28%). Low seroprevalence was observed for non-falciparum species in all the study sites: 0.4, 0.7 and 1.8%, respectively, for Plasmodium ovale, Plasmodium vivax and Plasmodium malariae MSP-1. Antibody responses to P. vivax were observed in all study sites except Kanel. CONCLUSION: Prevalence of P. falciparum MSP-119 antibodies and increases by study participant age provided data for low levels of exposure among this transient nomadic population. In addition, antibody responses to P. falciparum short half-life markers (CSP and LSA-1) and non-falciparum species were low. Further investigations are needed to understand the exposure of the Fulani population to P. vivax.

Specificity of the IgG antibody response to Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale MSP119 subunit proteins in multiplexed serologic assays.

BACKGROUND: Multiplex bead assays (MBA) that measure IgG antibodies to the carboxy-terminal 19-kDa sub-unit of the merozoite surface protein 1 (MSP119) are currently used to determine malaria seroprevalence in human populations living in areas with both stable and unstable transmission. However, the species specificities of the IgG antibody responses to the malaria MSP119 antigens have not been extensively characterized using MBA. METHODS: Recombinant Plasmodium falciparum (3D7), Plasmodium malariae (China I), Plasmodium ovale (Nigeria I), and Plasmodium vivax (Belem) MSP119 proteins were covalently coupled to beads for MBA. Threshold cut-off values for the assays were estimated using sera from US citizens with no history of foreign travel and by receiver operator characteristic curve analysis using diagnostic samples. Banked sera from experimentally infected chimpanzees, sera from humans from low transmission regions of Haiti and Cambodia (N = 12), and elutions from blood spots from humans selected from a high transmission region of Mozambique (N = 20) were used to develop an antigen competition MBA for antibody cross-reactivity studies. A sub-set of samples was further characterized using antibody capture/elution MBA, IgG subclass determination, and antibody avidity measurement. RESULTS: Total IgG antibody responses in experimentally infected chimpanzees were species specific and could be completely suppressed by homologous competitor protein at a concentration of 10 µg/ml. Eleven of 12 samples from the low transmission regions and 12 of 20 samples from the high transmission area had antibody responses that were completely species specific. For 7 additional samples, the P. falciparum MSP119 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays. A pan-malaria MSP119 cross-reactive antibody response was observed in elutions of blood spots from two 20-30 years old Mozambique donors. The antibody response from one of these two donors had low avidity and skewed almost entirely to the IgG3 subclass. CONCLUSIONS: Even when P. falciparum, P. malariae, P. ovale, and P. vivax are co-endemic in a high transmission setting, most antibody responses to MSP119 antigens are species-specific and are likely indicative of previous infection history. True pan-malaria cross-reactive responses were found to occur rarely.

Seroepidemiology of Plasmodium species infections in Zimbabwean population.

BACKGROUND: Individuals living in malaria-endemic regions may be exposed to more than one Plasmodium species; there is paucity of data on the distribution of the different species of Plasmodium in affected populations, in part due to the diagnostic method of microscopy, which cannot easily differentiate between the species. Sero-epidemiological data can overcome some of the shortcomings of microscopy. METHODS: The specificity of IgG antibodies to recombinant merozoite surface protein 1 (MSP-119) derived from four human Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale) was investigated using competition enzyme-linked immunosorbent assay. Subsequently, these antigens were used to determine the exposure prevalence to the different Plasmodium species in serum samples of participants. One-hundred individuals, aged five-18 years, from each of the three Plasmodium meso-endemic Zimbabwean villages (Burma Valley, Mutoko, Chiredzi) were recruited in the study. RESULTS: The study demonstrated that the host serum reactivity to MSP-119 antigens was species-specific and that no cross-reactivity occurred. The overall prevalence of antibody response to MSP-119 antigens was 61 % in Burma Valley, 31 % in Mutoko and 32 % in Chiredzi. Single species IgG responses to MSP-119 were most frequent against P. falciparum, followed by P. malariae and P. ovale, with responses to P. vivax being the least prevalent. Interestingly, 78-87 and 50 % of sera with IgG responses to P. malariae and P. ovale MSP-119, respectively, also had IgG specific response for P. falciparum MSP-119 antigens, indicating that exposure to these species is a common occurrence in these populations. Single species IgG responses to the non-falciparum species were at a very low frequency, ranging between 0 and 13 % for P. malariae. CONCLUSIONS: There is evidence of a higher exposure to the non-falciparum parasite species than previously reported in Zimbabwe. The recombinant MSP-119 antigens could be used as additional diagnostic tools in antibody assays for the detection of exposure to the different Plasmodium species. The results also introduce an interesting concept of the co-infection of non-falciparum Plasmodium almost always with P. falciparum, which requires further validation and mechanistic studies.

Antimalarial activity of cupredoxins: the interaction of Plasmodium merozoite surface protein 119 (MSP119) and rusticyanin.

The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.

Acquired antibody responses against merozoite surface protein-119 antigen during Plasmodium falciparum and P.vivax infections in South Indian city of Mangaluru.

Merozoite surface protein-1 (MSP-1) of malaria parasites has been extensively studied as a malaria vaccine candidate and the antibody response to this protein is an important indicator of protective immunity to malaria. Mangaluru city and its surrounding areas in southwestern India are endemic to malaria with Plasmodium vivax being the most widespread and prevalent species although P. falciparum also frequently infects. However, no information is available on the level of protective immunity in this population. In this regard, a prospective hospital-based study was performed in malarial patients to assess antibody responses against the 19-kDa C-terminal portion of P. vivax and P. falciparum MSP-1 (MSP-119). Serum samples from 51 healthy endemic controls and 267 infected individuals were collected and anti-MSP-119 antibody levels were analyzed by ELISA. The possible association between the antibody responses and morbidity parameters such as malarial anemia and thrombocytopenia was investigated. Among the 267 infected cases, 144 had P. vivax and 123 had P. falciparum infections. Significant levels of anti-MSP-119 antibody were observed both in P. vivax (123/144; 85.4%) and P. falciparum (108/123; 87.9%) infected individuals. In both type of infections, the major antibody isotypes were IgG1 and IgG3. The IgG levels were found to be increased in patients with severe anemia and thrombocytopenia. The antibody levels were also higher in infected individuals who had several previous infections, although antibodies produced during previous infections were short lived. The predominance of cytophilic anti-MSP-119 IgG1 and IgG3 antibodies suggests the possibility of a dual role of Pv MSP-119 and Pf MSP-119 during malarial immunity and pathogenesis.

Diversity and natural selection of Merozoite surface Protein-1 in three species of human malaria parasites: Contribution from South-East Asian isolates.

The present study aimed to examine the genetic diversity of human malaria parasites (i.e., P. falciparum, P. vivax and P. knowlesi) in Malaysia and southern Thailand targeting the 19-kDa C-terminal region of Merozoite Surface Protein-1 (MSP-119). This region is essential for the recognition and invasion of erythrocytes and it is considered one of the leading candidates for asexual blood stage vaccines. However, the genetic data of MSP-119 among human malaria parasites in Malaysia is limited and there is also a need to update the current sequence diversity of this gene region among the Thailand isolates. In this study, genomic DNA was extracted from 384 microscopy-positive blood samples collected from patients who attended the hospitals or clinics in Malaysia and malaria clinics in Thailand from the year 2008 to 2016. The MSP-119 was amplified using PCR followed by bidirectional sequencing. DNA sequences identified in the present study were subjected to Median-joining network analysis with sequences of MSP-119 obtained from GenBank. DNA sequence analysis revealed that PfMSP-119 of Malaysian and Thailand isolates was not genetically conserved as high number of haplotypes were detected and positive selection was prevalent in PfMSP-119, hence questioning its suitability to be used as a vaccine candidate. A novel haplotype (Q/TNG/L) was also detected in Thailand P. falciparum isolate. In contrast, PvMSP-119 was highly conserved, however for the first time, a non-synonymous substitution (A1657S) was reported among Malaysian isolates. As for PkMSP-119, the presence of purifying selection and low nucleotide diversity indicated that it might be a potential vaccine target for P. knowlesi.

Variation of antibody responses to Plasmodium falciparum MSP1-19 antigen with parasitaemia and IL4vntr polymorphism in Khartoum state, Sudan.

A hospital-based cross-sectional study was conducted at Khartoum state to investigate the variation of antibody responses to Plasmodium falciparum 19-kDa C-terminal region of merozoite surface protein 1 antigen and the variation of human IL4 polymorphism with parasitaemia. Measurements of natural acquisition of anti-Plasmodium falciparum MSP1-19 IgG, IgG1 and IgG3 antibodies were performed using ELISA. Molecular characterization of IL4vntr polymorphism was achieved. We were able to detect a statistically significant negative correlation between parasitaemia and different age groups (r = - 0.262 and p value = 0.043) and with anti-P.fMSP1-19 IgG1 (r = - 0.418, p value = 0.047). Anti-P.fMSP1-19 IgG showed a significant difference among age groups (p < 0.001). Only anti-P.fMSP1-19 IgG showed a significant association with general appearance (p value < 0.001). The mean for total anti-P.fMSP1-19 IgG3 was statistically significantly higher in females compared to males (p value < 0.001). There was no significant difference in the distribution of human IL4 vntr genotypic and allelic frequencies between cases and control group as well as among different clinical manifestation.We concluded that IgG1 levels to MSP1-19 were found to be negatively correlated with parasitaemia and anti-PfMSP1-19 IgG was significantly increased in ill and severely ill with age considered as a cofactor. Further studies are needed to ascertain the role of IgG and IgG1 in protection and to investigate the IgG and subclasses' response against other antigenic markers. These findings are valuable for advancing vaccine development by providing evidence supporting merozoite antigens as targets of protective immunity in humans.

Protective Immunity in Mice Immunized With P. vivax MSP119-Based Formulations and Challenged With P. berghei Expressing PvMSP119.

The lack of continuous in vitro cultures has been an obstacle delaying pre-clinical testing of Plasmodium vivax vaccine formulations based on known antigens. In this study, we generated a model to test available formulations based on the P. vivax MSP119 antigen. The Plasmodium berghei strains ANKA and NK65 were modified to express PvMSP119 instead of the endogenous PbMSP119. The hybrid parasites were used to challenge C57BL/6 or BALB/c mice immunized with PvMSP119-based vaccine formulations. The PvMSP119 was correctly expressed in the P. berghei hybrid mutant lines as confirmed by immunofluorescence using anti-PvMSP119 monoclonal antibodies and by Western blot. Replacement of the PbMSP119 by the PvMSP119 had no impact on asexual growth in vivo. High titers of specific antibodies to PvMSP119 were not sufficient to control initial parasitemia in the immunized mice, but late parasitemia control and a balanced inflammatory process protected these mice from dying, suggesting that an established immune response to PvMSP119 in this model can help immunity mounted later during infection.

Malaria transmission and individual variability of the naturally acquired IgG antibody against the Plasmodium vivax blood-stage antigen in an endemic area in Brazil.

Plasmodium vivax remains an important cause of malaria in South America and Asia, and analyses of the antibody immune response are being used to identify biomarker of parasite exposure. The IgG antibody naturally acquired predominantly occurs against targets on blood-stage parasites, including C-terminal of the merozoite surface protein 1 (MSP1-19). Epidemiological and immunological evidence has been showed that antibodies to malaria parasite antigens are lost in the absence of ongoing exposure. We describe the IgG antibody response in individuals living in an unstable malaria transmission area in Pará state, Amazon region, Brazil, where an epidemic of P. vivax malaria was recorded and monitored over time. As indicated by epidemiological data, the number of P. vivax-caused malaria cases decreased by approximately 90% after three years and the prevalence of IgG positive to PvMSP1-19 decreased significantly over time, in 2010 (93.4%), 2012 (78.3%), and 2013 (85.1%). Acquisition and decay of the IgG antibody against P. vivax MSP1-19 showed variability among individuals living in areas with recent circulating parasites, where the malaria epidemic was being monitored until transmission had been completely controlled. We also found that previous malaria episodes were associated with an increased in the IgG positivity . Our results showed epidemiological, spatial, temporal and individual variability. The understanding on dynamics of antibodies may have implications for the design of serosurveillance tools for monitoring parasite circulation, especially in a context with spatial and temporal changes in P. vivax malaria transmission.

Magnetic Nanovectors for the Development of DNA Blood-Stage Malaria Vaccines.

DNA vaccines offer cost, flexibility, and stability advantages, but administered alone have limited immunogenicity. Previously, we identified optimal configurations of magnetic vectors comprising superparamagnetic iron oxide nanoparticles (SPIONs), polyethylenimine (PEI), and hyaluronic acid (HA) to deliver malaria DNA encoding Plasmodium yoelii (Py) merozoite surface protein MSP119 (SPIONs/PEI/DNA + HA gene complex) to dendritic cells and transfect them with high efficiency in vitro. Herein, we evaluate their immunogenicity in vivo by administering these potential vaccine complexes into BALB/c mice. The complexes induced antibodies against PyMSP119, with higher responses induced intraperitoneally than intramuscularly, and antibody levels further enhanced by applying an external magnetic field. The predominant IgG subclasses induced were IgG2a followed by IgG1 and IgG2b. The complexes further elicited high levels of interferon gamma (IFN-γ), and moderate levels of interleukin (IL)-4 and IL-17 antigen-specific splenocytes, indicating induction of T helper 1 (Th1), Th2, and Th17 cell mediated immunity. The ability of such DNA/nanoparticle complexes to induce cytophilic antibodies together with broad spectrum cellular immunity may benefit malaria vaccines.

Transdermal immunization of P. falciparum surface antigen (MSP-119) via elastic liposomes confers robust immunogenicity.

As transdermal immunization results in poor immunogenicity, which is attributed to poor permeability of antigens through the skin, we believed ultradeformable lipid vesicles (elastic liposome) might address the challenges encountered during transdermal immunization. The elastic liposome, versatile carrier, proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. Our recently published article (1) is suggestive of improved immunogenicity of carboxyl-terminal 19 kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously via elastic liposomes ( Fig. 1 ).

Elastic liposome-mediated transdermal immunization enhanced the immunogenicity of P. falciparum surface antigen, MSP-119.

Transdermal immunization results in poor immunogenicity, which can be attributed to poor permeability of antigens through the skin. Therefore, elastic liposome, ultradeformable lipid vesicles, may overcome the challenges faced during transdermal immunization. This versatile carrier proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. The present results are suggestive of improved immunogenicity of carboxyl-terminal 19 kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously through elastic liposomes. The prepared elastic liposomes were characterized with respect to vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, stability and in vitro release. Humoral and cell-mediated immune (CMI) response elicited by topically applied PfMSP-119-loaded elastic liposomes, intramuscularly administered alum-adsorbed PfMSP-119 solution, and topically applied PfMSP-119-loaded conventional liposomes were compared and normalized with vehicle control. Results suggest greater transcutaneous immunization via elastic liposomes, and induced robust and perdurable IgG-specific antibody and cytophilic isotype responses. We report to have achieved sizeable CMI activating factor (IFNγ), a crucial player in conferring resistance to asexual blood stage malaria, responses with elastic liposomes when compared with other formulations. The fluorescence microscopy and histopathology results are suggestive of prominent skin permeation and biodistribution, and demonstrate efficient delivery of malaria antigen via elastic liposomes to immunocompetent Langerhans cells (LC) and lymphatics. In conclusion, elastic liposomal formulation provided greater entrapment efficiency, enhanced penetration and heightened and long-lasting immune response. Moreover, effective immunoadjuvant property of this carrier justifies its potential for improved vaccine delivery, and opens new avenues to explore further on the development of malaria vaccine.

Ficolin-A Enhances Inhibition of the C-Terminal 19 kDa Region of Merozoite Surface Protein-1 of Plasmodium berghei Using Test In Vivo.

BACKGROUND: Malaria remains a serious public health problem with significant morbidity and mortality. This study was conducted to identify whether ficolin-A could play an active role of against malaria infection. METHODS: The function of ficolin-A was analyzed in mouse model. The open reading frame of ficolin-A was cloned from the liver of new born C57BL/6 mice by RT-PCR and then inserted into the expression vector of eukaryon to construct pVAX1-ficolin-A plasmid. Meanwhile, the open reading frame of the 19-kDa fragment of merozoite surface protein-1 of Plasmodium berghei (MSP119) was cloned and then the expression vector of eukaryon, pVAX1- MSP119 was constructed. Both recombinant vectors were used in the mouse model of infection by Plasmodium berghei. RESULTS: pVAX1-ficolin-A alone could not significantly suppress parasite density and prolong survival time of infection mice; however, when injected pVAX1-ficolin-A and pVAX1-MSP119 together, the percent of invasion by Plasmodium was decreased (from 43.78% to 22.23% at 10 day after infection, compared to vector) and the survival time was prolonged significantly in the infection mouse model (P=0.01). CONCLUSION: Ficolin-A can enhance the immunoprotection of MSP119, it implies ficolin-A may be used as immunoenhancer in the study of vaccine defending malaria.

Absence of asymptomatic malaria infection in endemic area of bashagard district, hormozgan province, iran.

BACKGROUND: A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran. METHODS: Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques. RESULTS: Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1(19)-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1(19), using ELISA. CONCLUSION: This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.

Plataforma sorológica pela citometria de fluxo para detecção de anticorpos contra antígenos sanguíneos de Plasmodium vivax

A malária causada pelo P. vivax atinge milhões de pessoas no mundo sendo responsável por grande parte da morbidade da malária humana. Considerando que as medidas de controle atualmente disponíveis apresentam limitações, faz-se necessário intensificar esforços para o desenvolvimento de metodologias alternativas que possam efetivamente contribuir para o controle e eliminação da doença em longo-prazo. Para isso, torna-se necessário avaliar a resposta imune contra os principais antígenos candidatos à vacina, e marcadores de exposição, considerando o novo perfil epidemiológico de transmissão reduzida da malária no Brasil e no mundo. Neste contexto, o objetivo do presente trabalho foi de desenvolver plataformas sorológicas pela citometria de fluxo para avaliar anticorpos contra as formas sanguíneas do P. vivax que possam ser indicativos de imunidade adquirida (Parte I) ou de infeção aguda ou recente (Parte II). Para identificar anticorpos relacionados a imunidade adquirida, o presente trabalho concentrou-se na resposta de anticorpos que bloqueiam a principal via utilizada pelo P. vivax na invasão dos reticulócitos, mediada pela região II da Duffy binding protein (DBPII) e seu receptor DARC presente na superfície dos eritrócitos. Apesar dos anticorpos capazes de bloquear a interação DBPII-DARC (binding inhibitory antibodies, BIAbs) estarem associados a proteção clínica, poucos estudos têm investigado estes anticorpos funcionais. Parte da dificuldade se deve a complexidade dos ensaios funcionais que avaliam a interação DBPII-DARC. Baseado na hipótese de que uma resposta de anticorpos anti-DBPII contra diferentes variantes alélicas da proteína poderia ser indicativa de BIAbs, padronizou-se um ensaio multiplex para a detecção da resposta de anticorpos contra diferentes construções de DBPII, incluindo epítopos conservados (DEKnull-2) e polimórficos de variantes nativas prevalentes na Amazônia (Sal-1 e Brz-1). O desenho do estudo incluiu 245 amostras plasmáticas de indivíduos com histórico de longa exposição á malária na Amazônia. As amostras foram caracterizadas pela presença de BIAbs através do ensaio funcional de referência que utiliza células COS-7 expressando a DBPII (padrão-ouro). Os resultados mostraram que a plataforma de citometria aqui desenvolvida permitiu, com elevada acurácia, identificar indivíduos com anticorpos BIAbs. De relevância, o ensaio poderá ser adaptado para outras áreas endêmicas, sendo necessário a inclusão de variantes alélicas prevalentes na área a ser estudada. Em uma segunda parte do projeto, buscou-se identificar anticorpos associados a presença de infecção aguda ou recente por P. vivax. A hipótese do trabalho foi a de que a resposta de anticorpos contra antígenos sanguíneos altamente imunogênicos seria indicativa de infecção aguda, e/ou exposição recente ao P. vivax, particularmente, para indivíduos sem história de exposição prévia à malária. Para tal, padronizou-se um ensaio sorológico pela citometria de fluxo para determinar a resposta de anticorpos contra uma proteína quimérica composta pelos antígenos AMA-1 e MSP1-19 (AMA1/MSP1-19). A população de estudo foi constituída de indivíduos primoinfectados pelo P. vivax, residentes em Minas Gerais (área não endêmica) ou na Amazônia (RO e AM). Em conjunto, os resultados da resposta de anticorpos IgM e IgG mostraram que a proteína quimérica foi altamente imunogênica nas populações de estudo (frequência de resposta variando de 57-76% para IgM e 78% para IgG). No ensaio de multiplex, a acurácia da proteína quimérica foi superior as proteínas individuais, com especificidade de 90-95% e sensibilidade 60-64%. De interesse, nas amostras dos indivíduos residentes fora da área de transmissão, ensaios de cinética demonstram que anticorpos IgG (mas não IgM) permitiram identificar um "booster" na resposta em indivíduos que apresentaram episódios de recaídas. Em resumo, os resultados demonstram o potencial da plataforma multiplex na avaliação da resposta de anticorpos em primoinfecção pelo P. vivax. Ensaios futuros fazem-se necessários para aumentar a sensibilidade do ensaio visando o potencial uso em bancos de sangue localizado fora da área endêmica e/ou para identificar indivíduos com infecção aguda ou recente em surtos de transmissão por P. vivax.

Avaliação em modelos animais de uma vacina para malária utilizando como vetor de expressão o vírus de febre amarela vacinal 17D

O desenvolvimento de uma vacina para malária é considerado atualmente uma prioridade em saúde pública pelo impacto socioeconômico e morbidade da doença com 250 milhões de novos casos por ano. A vacina de febre amarela é considerada uma das vacinas mais bem sucedidas por sua imunogenicidade duradora obtida após uma única dose. Frente a elucidação das respostas polivalentes dirigidas ao vírus vacinal 17D, a utilização do mesmo como vetor de expressão para antígenos heterólogos têm sido encorajada. Tendo em vista que febre amarela e malária compartilham grandes zonas endêmicas nos continentes americano e africano, a construção de uma vacina para malária baseada no vetor de febre amarela 17D se tornou uma abordagem interessante. Foram construídos dois vírus recombinantes contendo a proteína heteróloga MSP-119de P. falciparum (FA17D/MSP-119fal) e P. vivax (FA17D/MSP-119vivax) entre os genes E/NS1 de FA. Esta proteína é constituída de um fragmento de 19 kDa obtido após o processamento proteolítico da proteína de superfície do merozoíta 1 (MSP-1) durante a invasão do eritrócito e é descrita como alvo de anticorpos protetores em animais e pessoas imunes. Os vírus recombinantes foram caracterizados in vitro quanto à capacidade proliferativa em células Vero, estabilidade genética e expressão da proteína heteróloga por microscopia confocal de imunofluorescência e western blotting. A imunização de camundongos BALB/c e primatas não-humanos da espécie Saimiri sciureus foi usada para avaliar as construções quanto à imunogenicidade. Ambos os vírus foram capazes de induzir a produção de anticorpos neutralizantes contra a FA, porém em menores títulos do que os induzidos pelo vírus vacinal 17DD. A indução de anticorpos específicos para a proteína heteróloga após a imunização com os diferentes vírus recombinantes, também foi demonstrada e resultou em baixos títulos de IgG.em ambos os modelos. Os anticorpos induzidos no modelo com macacos Saimiri reconheceram a proteína nativa do parasita em hemácias infectadas por P. falciparum. No entanto, o desafio realizado neste modelo de primata não-esplenectomizado após a imunização com FA17D/MSP-119fal não gerou resultados conclusivos. Estes resultados sugerem a necessidade de aprimoramento da plataforma de expressão em busca de maior imunogenicidade.