RESUMO
Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.
Assuntos
Epitopos de Linfócito B/imunologia , Imunogenicidade da Vacina , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Peptídeos/síntese química , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Malária Vivax/prevenção & controle , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Plasmodium vivax , Proteínas de Protozoários/genética , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
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.
Assuntos
Anticorpos Antiprotozoários/imunologia , Epitopos de Linfócito B/imunologia , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Peptídeos/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/genética , Simulação por Computador , Epitopos de Linfócito B/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Proteínas de Membrana/genética , Camundongos , Peptídeos/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genéticaRESUMO
Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host's metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.
Assuntos
Interações Hospedeiro-Parasita , Malária/parasitologia , Metabolômica , Plasmodium/química , Animais , Biologia Computacional , Humanos , Espectrometria de Massas , Plasmodium/metabolismo , Plasmodium/patogenicidadeRESUMO
Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.
Assuntos
Animais , Humanos , Interações Hospedeiro-Parasita , Metabolômica , Malária/parasitologia , Plasmodium/química , Biologia Computacional , Espectrometria de Massas , Plasmodium/metabolismo , Plasmodium/patogenicidadeRESUMO
A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3ß of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3ß (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3ß, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3ß elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.
Assuntos
Antígenos de Protozoários/uso terapêutico , Vacinas Antimaláricas/uso terapêutico , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/uso terapêutico , Animais , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/sangue , Malária Vivax/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêuticoRESUMO
BACKGROUND: The 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 FINDINGS: In this study, we evaluated the influence of HLA-DRB1* and -DQB1* allelic groups on the naturally acquired humoral response from Brazilian Amazon individuals (nâ=â276) 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. CONCLUSIONS: HLA-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.
Assuntos
Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Imunoglobulina G/imunologia , Malária Vivax/epidemiologia , Malária Vivax/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Protozoários/imunologia , Brasil/epidemiologia , Criança , Etnicidade/genética , Frequência do Gene , Humanos , Entrevistas como Assunto , Proteínas de Membrana/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Pessoa de Meia-Idade , Prevalência , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologiaRESUMO
An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-119 and PvMSP-3α359â798. Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-119 was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3α359â798. Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3α), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3α359â798 during natural infection.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Idoso , Brasil , Criança , Feminino , Humanos , Imunoglobulina G/sangue , Vacinas Antimaláricas/administração & dosagem , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Latin America contributes 1-1.2 million clinical malaria cases to the global malaria burden of about 300 million per year. In 21 malaria endemic countries, the population at risk in this region represents less than 10% of the total population exposed worldwide. Factors such as rapid deforestation, inadequate agricultural practices, climate change, political instability, and both increasing parasite drug resistance and vector resistance to insecticides contribute to malaria transmission. Recently, several malaria endemic countries have experienced a significant reduction in numbers of malaria cases. This is most likely due to actions taken by National Malaria Control Programs (NMCP) with the support from international funding agencies. We describe here the research strategies and activities to be undertaken by the Centro Latino Americano de Investigación en Malaria (CLAIM), a new research center established for the non-Amazonian region of Latin America by the National Institute of Allergy and Infectious Diseases (NIAID). Throughout a network of countries in the region, initially including Colombia, Guatemala, Panama, and Peru, CLAIM will address major gaps in our understanding of changing malaria epidemiology, vector biology and control, and clinical malaria mainly due to Plasmodium vivax. In close partnership with NMCPs, CLAIM seeks to conduct research on how and why malaria is decreasing in many countries of the region as a basis for developing and implementing new strategies that will accelerate malaria elimination.
Assuntos
Erradicação de Doenças/métodos , Erradicação de Doenças/organização & administração , Projetos de Pesquisa Epidemiológica , Malária/prevenção & controle , Animais , Atenção à Saúde/organização & administração , Resistência a Medicamentos , Variação Genética , Humanos , Imidazóis/farmacologia , Insetos Vetores/parasitologia , Insetos Vetores/fisiologia , Cooperação Internacional , América Latina/epidemiologia , Malária/epidemiologia , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Programas Nacionais de Saúde/organização & administração , Niacina/análogos & derivados , Niacina/farmacologia , Plasmodium/efeitos dos fármacos , Plasmodium/genética , Plasmodium/imunologia , Plasmodium/patogenicidade , Fatores SocioeconômicosRESUMO
Plasmodium vivax Merozoite Surface Protein-3alpha and 3beta are members of a family of related merozoite surface proteins that contain a central alanine-rich domain with heptad repeats that is predicted to form alpha-helical secondary and coiled-coil tertiary structures. Seven recombinant proteins representing different regions of MSP-3alpha and MSP-3beta of P. vivax were generated to investigate their structure. Circular dichroism spectra analysis revealed that some proteins are folded with a high degree of alpha-helices as secondary structure, whereas other products contain a high content of random coil. Using size exclusion chromatography, we found that the two smaller fragments of the MSP-3alpha, named CC4 and CC5, predicted to form coiled-coil (CC) structures, eluted at volumes corresponding to molecular weights larger than their monomeric masses. This result suggests that both proteins are oligomeric molecules. Analytical ultracentrifugation experiments showed that the CC5 oligomers are elongated molecules. Together, these data may help to understand important aspects of P. vivax biology.
Assuntos
Antígenos de Protozoários/química , Biofísica/métodos , Plasmodium vivax/química , Proteínas de Protozoários/química , Alanina/química , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Dicroísmo Circular , Plasmídeos , Plasmodium vivax/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Naturally acquired antibody reactivity to two major Plasmodium vivax vaccine candidates was investigated in 294 donors from three malaria-endemic communities of Rondônia state, Brazil. Antibody recognition of recombinantly expressed antigens covering five different regions of P. vivax reticulocyte binding protein 1 (PvRBP1) and region II of P. vivax Duffy binding protein (PvDBP-RII) were compared. Positive IgG responses to these antigens were significantly related to the level of malaria exposure in terms of past infections and years of residence in the endemic area when corrected for age. The highest prevalence of anti-PvRBP1 total IgG antibodies corresponded to the amino acid regions denoted PvRBP1(431-748) (41%) and PvRBP1(733-1407) (47%). Approximately one-fifth of positively responding sera had titers of at least 1:1,600. Total IgG responses to PvDBP-RII were more prevalent (67%), of greater magnitude, and acquired more rapidly than those to individual PvRBP1 antigens. Responses to both PvRBP1 and PvDBP-RII were biased toward the cytophilic subclasses IgG1 and IgG3. These data provide the first insights on acquired antibody responses to PvRBP1 and a comparative view with PvDBP-RII that may prove valuable for understanding protective immune responses to these two vaccine candidates as they are evaluated as components of multitarget blood-stage vaccines.
Assuntos
Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Malária Vivax/epidemiologia , Proteínas de Membrana/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Brasil/epidemiologia , Criança , Sistema do Grupo Sanguíneo Duffy/metabolismo , Feminino , Humanos , Imunoglobulina G/classificação , Vacinas Antimaláricas , Malária Vivax/imunologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Plasmodium vivax/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas Recombinantes/imunologiaRESUMO
Merozoite surface protein-9 of Plasmodium vivax (PvMSP-9) is highly conserved and present in several malaria species. Here, we present the immunogenic properties of two recombinant glutathione S-transferase (GST) fusion proteins comprising the N-terminus (PvMSP-9-Nt) and the second block of tandem repeats (PvMSP-9-RepII) of PvMSP9. These recombinants proteins were used to immunize BALB/c mice. The specificity and subtyping of the antibodies and the cellular immune responses were evaluated by enzyme-linked immunosorbent assay (ELISA) and ELISPOT, respectively, using the recombinant proteins as antigens. Our results demonstrate that both the N-terminal and the tandem repeat regions of MSP9 are immunogenic in mice. The ELISA antibody titers elicited by PvMSP-9-Nt were significantly higher (1:819,200) than the antibody titers elicited by PvMSP-9-RII (1:409,600). Analysis of IgG subclasses showed that both recombinant proteins induce similar antibody patterns where IgG1, IgG2a and IgG2b were most predominant. Moreover, all sera from mice immunized with either PvMSP-9-Nt or PvMSP-9-RII, which were positive by ELISA showed reactivity with P. vivax, P. cynomolgi, P. knowlesi and P. coatneyi schizonts by immunofluorescence assays (IFA). Similar results were observed in western immunoblot analyses using parasite extracts. Furthermore, immunization of mice with the PvMSP-9-Nt upon stimulation with PvMSP-9-Nt secreted IFN-gamma and IL-5. We have also used the two PvMSP-9 recombinant constructs to show that individuals exposed to P. vivax infections in an endemic area of Brazil had IgG antibodies reactive with the recombinant proteins.
Assuntos
Escherichia coli/metabolismo , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Animais , Formação de Anticorpos/imunologia , Western Blotting , Brasil , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Imunofluorescência , Humanos , Imunidade Celular/imunologia , Interferon gama/análise , Interferon gama/biossíntese , Interleucina-5/análise , Interleucina-5/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Vacinas Sintéticas/imunologiaRESUMO
Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1* alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.