"Genetic Diversity and Selection in Three Plasmodium vivax Merozoite Surface Protein 7 (Pvmsp-7) Genes in a Colombian Population"

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7 genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.

Low genetic diversity and functional constraint in loci encoding Plasmodium vivax P12 and P38 proteins in the Colombian population

Background Plasmodium vivax is one of the five species causing malaria in human beings, affecting around 391 million people annually. The development of an anti-malarial vaccine has been proposed as an alternative for controlling this disease. However, its development has been hampered by allele-specific responses produced by the high genetic diversity shown by some parasite antigens. Evaluating these antigens’ genetic diversity is thus essential when designing a completely effective vaccine. Methods The gene sequences of Plasmodium vivax p12 (pv12) and p38 (pv38), obtained from field isolates in Colombia, were used for evaluating haplotype polymorphism and distribution by population genetics analysis. The evolutionary forces generating the variation pattern so observed were also determined. Results Both pv12 and pv38 were shown to have low genetic diversity. The neutral model for pv12 could not be discarded, whilst polymorphism in pv38 was maintained by balanced selection restricted to the gene’s 5′ region. Both encoded proteins seemed to have functional/structural constraints due to the presence of s48/45 domains, which were seen to be highly conserved.

Characterizing PvARP, a novel Plasmodium vivax antigen

Background Plasmodium vivax continues to be the most widely distributed malarial parasite species in tropical and sub-tropical areas, causing high morbidity indices around the world. Better understanding of the proteins used by the parasite during the invasion of red blood cells is required to obtain an effective vaccine against this disease. This study describes characterizing the P. vivax asparagine-rich protein (PvARP) and examines its antigenicity in natural infection. Methods The target gene in the study was selected according to a previous in silico analysis using profile hidden Markov models which identified P. vivax proteins that play a possible role in invasion. Transcription of the arp gene in the P. vivax VCG-1 strain was here evaluated by RT-PCR. Specific human antibodies against PvARP were used to confirm protein expression by Western blot as well as its subcellular localization by immunofluorescence. Recognition of recombinant PvARP by sera from P. vivax-infected individuals was evaluated by ELISA. Results VCG-1 strain PvARP is a 281-residue-long molecule, which is encoded by a single exon and has an N-terminal secretion signal, as well as a tandem repeat region. This protein is expressed in mature schizonts and is located on the surface of merozoites, having an apparent accumulation towards their apical pole. Sera from P. vivax-infected patients recognized the recombinant, thereby suggesting that this protein is targeted by the immune response during infection.

A sequence in subdomain 2 of DBL1 alpha of plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1 alpha previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1 alpha-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1 alpha antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface.

Genetic diversity and selection in three plasmodium vivax merozoite surface protein 7 (pvmsp-7) genes in a colombian population

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7 genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.

3D Analysis of the TCR/pMHCII Complex Formation in Monkeys Vaccinated with the First Peptide Inducing Sterilizing Immunity against Human Malaria

T-cell receptor gene rearrangements were studied in Aotus monkeys developing high antibody titers and sterilizing immunity against the Plasmodium falciparum malaria parasite upon vaccination with the modified synthetic peptide 24112, which was identified in the Merozoite Surface Protein 2 (MSP-2) and is known to bind to HLA-DR beta 1*0403 molecules with high capacity. Spectratyping analysis showed a preferential usage of V beta 12 and V beta 6 TCR gene families in 67% of HLA-DR beta 1*0403-like genotyped monkeys. Docking of peptide 24112 into the HLA-DR beta 1*0401-HA peptide-HA1.7TCR complex containing the VDJ rearrangements identified in fully protected monkeys showed a different structural signature compared to nonprotected monkeys. These striking results show the exquisite specificity of the TCR/pMHCII complex formation needed for inducing sterilizing immunity and provide important hints for a logical and rational methodology to develop multiepitopic, minimal subunit-based synthetic vaccines against infectious diseases, among them malaria.

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

Background: Multi-drug resistance and severe/ complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. Methods: 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. Results: 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 +/- 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 +/- 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. Conclusions: The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Identification of plasmodium vivax proteins with potential role in invasion using sequence redundancy reduction and profile hidden markov models

Background: This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results: A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions: The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.

Diário da Constituinte [gravação de vídeo] : [programa n. 616 ]

A estabilidade dos servidores é a nova polêmica da constituinte. A votação da emenda que concede a anistia aos microempresários é adiada. O texto que chegou ontem para ser votado na sessão estendia a anistia aos médios empresários, cooperativas agrícolas e entidades filantrópicas, segundo alguns líderes partidários. Os defensores da anistia se reuniram com o presidente da constituinte e decidirem que a emenda será votada na próxima quarta, independente de acordo. Na reunião dos líderes partidários recomeçou uma tentativa de se chegar a um acordo. A dificuldade está em saber quem ficará dentro e quem ficará fora da anistia. Foram discutidos outros pontos das disposições transitórias. A discussão agora é em torno do artigo que trata da estabilidade dos servidores públicos com cinco anos de serviço. Essa emenda beneficia 150 mil funcionários da administração direta e está dividindo os constituintes. Eles se preocupam no estabelecimento de critérios seletivos para o ingresso no serviço. Essa questão poderá ser votada ainda nesta sessão. Foi colocada em votação uma emenda que tentava acabar com o direito de transmissão de imóvel mediante o pagamento de uma quantia anual. A emenda acabou sendo recusada. Também foi rejeitada uma emenda propondo o cancelamento das atividades nucleares da Marinha. Durante a sessão de hoje foi divulgado o novo texto da emenda da anistia. Pela proposta serão anistiados os micros e pequenos empresários que contraíram dívidas entre 28 de fevereiro de 1986 e 31 de dezembro de 1987. A receita anual máxima para ser considerados microempresários é de cinco mil OTNS.