Influence of HLA-DRB1 and HLA-DQB1 Alleles on IgG Antibody Response to the P. vivax MSP-1, MSP-3α and MSP-9 in Individuals from Brazilian Endemic Area

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 (n = 276) against P. vivax Merozoite Surface Protein-1 (MSP-1), MSP-3 alpha 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.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Yerkes National Primate Research Center BaseNational Center for Research Resources of the National Institutes of HealthNIHCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Inst Oswaldo Cruz, Lab Immunoparasitol, BR-20001 Rio de Janeiro, BrazilOswaldo Cruz Fdn Fiocruz, Ctr Technol Dev Hlth CDTS, Rio de Janeiro, BrazilInst Oswaldo Cruz, Lab Simulideos & Oncocercose, BR-20001 Rio de Janeiro, BrazilEmory Univ, Emory Vaccine Ctr, Atlanta, GA 30322 USAUniv Estado Rio de Janeiro, Histocompatibil & Cryopreservat Lab, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol CTCMol, Escola Paulista Med, São Paulo, BrazilEmory Univ, Sch Med, Div Infect Dis, Atlanta, GA USACDC Natl Ctr Infect Dis, Div Parasit Dis, Atlanta, GA USAUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol CTCMol, Escola Paulista Med, São Paulo, BrazilFAPESP: 2009/15132-4Yerkes National Primate Research Center Base: RR00165NIH: RO1 AI0555994Web of Scienc

Plasmodium vivax merozoite surface protein-3 (PvMSP3): expression of an 11 member multigene family in blood-stage parasites.

Three members of the Plasmodium vivax merozoite surface protein-3 (PvMSP3) family (PvMSP3-α, PvMSP3-β and PvMSP3-γ) were initially characterized and later shown to be part of a larger highly diverse family, encoded by a cluster of genes arranged head-to-tail in chromosome 10. PvMSP3-α and PvMSP3-β have become genetic markers in epidemiological studies, and are being evaluated as vaccine candidates. This research investigates the gene and protein expression of the entire family and pertinent implications.A 60 kb multigene locus from chromosome 10 in P. vivax (Salvador 1 strain) was studied to classify the number of pvmsp3 genes present, and compare their transcription, translation and protein localization patterns during blood-stage development. Eleven pvmsp3 paralogs encode an N-terminal NLRNG signature motif, a central domain containing repeated variable heptad sequences, and conserved hydrophilic C-terminal features. One additional ORF in the locus lacks these features and was excluded as a member of the family. Transcripts representing all eleven pvmsp3 genes were detected in trophozoite- and schizont-stage RNA. Quantitative immunoblots using schizont-stage extracts and antibodies specific for each PvMSP3 protein demonstrated that all but PvMSP3.11 could be detected. Homologs were also detected by immunoblot in the closely related simian species, P. cynomolgi and P. knowlesi. Immunofluorescence assays confirmed that eight of the PvMSP3s are present in mature schizonts. Uniquely, PvMSP3.7 was expressed exclusively at the apical end of merozoites.Specific proteins were detected representing the expression of 10 out of 11 genes confirmed as members of the pvmsp3 family. Eight PvMSP3s were visualized surrounding merozoites. In contrast, PvMSP3.7 was detected at the apical end of the merozoites. Pvmsp3.11 transcripts were present, though no corresponding protein was detected. PvMSP3 functions remain unknown. The ten expressed PvMSP3s are predicted to have unique and complementary functions in merozoite biology

Naturally acquired immune responses to P. vivax merozoite surface protein 3α and merozoite surface protein 9 are associated with reduced risk of P. vivax malaria in young Papua New Guinean children

Plasmodium vivax is the most geographically widespread human malaria parasite. Cohort studies in Papua New Guinea have identified a rapid onset of immunity against vivax-malaria in children living in highly endemic areas. Although numerous P. vivax merozoite antigens are targets of naturally acquired antibodies, the role of many of these antibodies in protective immunity is yet unknown.; In a cohort of children aged 1-3 years, antibodies to different regions of Merozoite Surface Protein 3α (PvMSP3α) and Merozoite Surface Protein 9 (PvMSP9) were measured and related to prospective risk of P. vivax malaria during 16 months of active follow-up. Overall, there was a low prevalence of antibodies to PvMSP3α and PvMSP9 proteins (9-65%). Antibodies to the PvMSP3α N-terminal, Block I and Block II regions increased significantly with age while antibodies to the PvMSP3α Block I and PvMSP9 N-terminal regions were positively associated with concurrent P. vivax infection. Independent of exposure (defined as the number of genetically distinct blood-stage infection acquired over time (molFOB)) and age, antibodies specific to both PvMSP3α Block II (adjusted incidence ratio (aIRR) = 0.59, p = 0.011) and PvMSP9 N-terminus (aIRR = 0.68, p = 0.035) were associated with protection against clinical P. vivax malaria. This protection was most pronounced against high-density infections. For PvMSP3α Block II, the effect was stronger with higher levels of antibodies.; These results indicate that PvMSP3α Block II and PvMSP9 N-terminus should be further investigated for their potential as P. vivax vaccine antigens. Controlling for molFOB assures that the observed associations are not confounded by individual differences in exposure

In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

Submitted by sandra infurna ([email protected]) on 2016-05-31T17:35:39Z No. of bitstreams: 1 dalma_banic_etal_IOC_2016.PDF: 1194916 bytes, checksum: b3b7e7ecb81de1803e07ac4391fc1c17 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-06-02T12:56:12Z (GMT) No. of bitstreams: 1 dalma_banic_etal_IOC_2016.PDF: 1194916 bytes, checksum: b3b7e7ecb81de1803e07ac4391fc1c17 (MD5)Made available in DSpace on 2016-06-02T12:56:12Z (GMT). No. of bitstreams: 1 dalma_banic_etal_IOC_2016.PDF: 1194916 bytes, checksum: b3b7e7ecb81de1803e07ac4391fc1c17 (MD5) Previous issue date: 2016Made available in DSpace on 2016-06-03T12:34:16Z (GMT). No. of bitstreams: 2 dalma_banic_etal_IOC_2016.PDF: 1194916 bytes, checksum: b3b7e7ecb81de1803e07ac4391fc1c17 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Grupo de Modelagem Computacional. Fortaleza, CE, Brasil.Emory University. Yerkes National Primate Research Center. Emory Vaccine Center. Atlanta, GA, USA.Emory University. Yerkes National Primate Research Center. Emory Vaccine Center. Atlanta, GA, USA.Emory University. Environmental Health and Safety Office. Atlanta, GA, USA.Fundação Nacional de Saúde. Departamento de Entomologia. Laboratório central. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Referência Nacional de Simulídeos e Oncocercose. Rio de Janeiro, RJ, Brasil.Emory University. Yerkes National Primate Research Center. Emory Vaccine Center. Atlanta, GA, USA / Emory University. Emory University School of Medicine. Department of Medicine. Division of Infectious Diseases. Atlanta, GA, USA.Emory University. Yerkes National Primate Research Center. Emory Vaccine Center. Atlanta, GA, USA / Emory University. Emory University School of Medicine. Department of Medicine. Division of Infectious Diseases. Atlanta, GA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.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

Association between antibodies to PvMSP3α(Block II) and PvMSP9(N-terminal) proteins and risk of <i>P. vivax</i> malaria.

<p>Data are plotted as exposure and age adjusted incidence rate ratios (aIRR(exp)) ± 95% confidence intervals for febrile episodes with different levels of concurrent <i>P. vivax</i> parasitaemia.</p

Optical density values (OD) as a measurement of total IgG to different PvMSP3α and PvMSP9 proteins.

a<p>The cut-off for positivity was determined as the mean+3 standard deviations of negative control plasma samples (Australian residents) included in each assay.</p>b<p>Responders defined as individuals whose plasma OD value was above the cut-off for positivity for a given antigen.</p

IgG positivity to different PvMSP3α and PvMSP9 proteins.

<p>(a) Cumulative IgG positivity for different PvMSP3α proteins. Data are plotted as the percentage of 183 individuals who are antibody positive for 0–4 of the proteins tested. (b) Associations between age and IgG positivity to PvMSP3α and PvMSP9 proteins. Children were divided into two age groups (<21 mths: n = 96, ≥21 mths: n = 87) to examine associations with age. P values≤0.05 were considered significant and are shown. (c) Associations between <i>P. vivax</i> infection status (post-PCR LDR-FMA positive: n = 90, negative: n = 93) and IgG positivity to PvMSP3α and PvMSP9 proteins. As indicated, the presence of <i>P. vivax</i> was determined by a semi-quantitative post-PCR ligase detection reaction-fluorescent microsphere assay (LDR-FMA). P values≤0.05 were considered significant and are shown.</p

Association between antibody positivity and protection against subsequent <i>P. vivax</i> malaria (density>500/µl).

a<p>Adjustment for season (month, year), spatial variation (village or residence) and individual differences in exposure (as measured by molecular force of blood-stage infection (_molFOB).</p>b<p>Adjustment for age of child, season (month, year), spatial variation (village or residence) and individual differences in exposure (as measured by molecular force of blood-stage infection (_molFOB).</p>c<p>Adjustment as performed for age-adjusted ^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002498#nt104" target="_blank">b</a>}. Multivariate analyses of antibodies univariately associated with protection.</p

Three-dimensional structure prediction of the PvMSP9 RIRII domain.

<p>(A) 3D model of the PvMSP9 domain constructed using the Robetta algorithm. Red structures depict alpha helices, while the disordered region is represented in green. A small beta sheet was found between residues 850 and 858. (B) B-factor as calculated by GROMACS after 10ns simulation. The thicker segments represent the most flexible regions, while the thinnest represent the most rigid. (C) Electrostatic surface of the disordered region, showing a predominantly negative segment in red. (D) Same region show in B, without the electrostatic surface, showing the region 793–866 highlighted in green. The color scale was set from 5 kT/e (red) to 5 kT/e (blue), as calculated by APBS.</p