Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

"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.

Effect of the human papillomavirus (HPV) quadrivalent vaccine in a subgroup of women with cervical and vulvar disease: Retrospective pooled analysis of trial data

Objectives To determine the effect of human papillomavirus (HPV) quadrivalent vaccine on the risk of developing subsequent disease after an excisional procedure for cervical intraepithelial neoplasia or diagnosis of genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia. Design Retrospective analysis of data from two international, double blind, placebo controlled, randomised efficacy trials of quadrivalent HPV vaccine (protocol 013 (FUTURE I) and protocol 015 (FUTURE II)). Setting Primary care centres and university or hospital associated health centres in 24 countries and territories around the world. Participants Among 17 622 women aged 15–26 years who underwent 1:1 randomisation to vaccine or placebo, 2054 received cervical surgery or were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia. Intervention Three doses of quadrivalent HPV vaccine or placebo at day 1, month 2, and month 6. Main outcome measures Incidence of HPV related disease from 60 days after treatment or diagnosis, expressed as the number of women with an end point per 100 person years at risk. Results A total of 587 vaccine and 763 placebo recipients underwent cervical surgery. The incidence of any subsequent HPV related disease was 6.6 and 12.2 in vaccine and placebo recipients respectively (46.2% reduction (95% confidence interval 22.5% to 63.2%) with vaccination). Vaccination was associated with a significant reduction in risk of any subsequent high grade disease of the cervix by 64.9% (20.1% to 86.3%). A total of 229 vaccine recipients and 475 placebo recipients were diagnosed with genital warts, vulvar intraepithelial neoplasia, or vaginal intraepithelial neoplasia, and the incidence of any subsequent HPV related disease was 20.1 and 31.0 in vaccine and placebo recipients respectively (35.2% reduction (13.8% to 51.8%)). Conclusions Previous vaccination with quadrivalent HPV vaccine among women who had surgical treatment for HPV related disease significantly reduced the incidence of subsequent HPV related disease, including high grade disease.

Four year efficacy of prophylactic human papillomavirus quadrivalent vaccine against low grade cervical, vulvar, and vaginal intraepithelial neoplasia and anogenital warts: randomised controlled trial

Objectives: To evaluate the prophylactic efficacy of the human papillomavirus (HPV) quadrivalent vaccine in preventing low grade cervical, vulvar, and vaginal intraepithelial neoplasias and anogenital warts (condyloma acuminata). Design: Data from two international, double blind, placebo controlled, randomised efficacy trials of quadrivalent HPV vaccine (protocol 013 (FUTURE I) and protocol 015 (FUTURE II)). The trials were to be 4 years in length, and the results reported are from final study data of 42 months' follow-up. Setting: Primary care centres and university or hospital associated health centres in 24 countries and territories around the world. Participants: 17 622 women aged 16-26 years enrolled between December 2001 and May 2003. Major exclusion criteria were lifetime number of sexual partners (>4), history of abnormal cervical smear test results, and pregnancy. Intervention: Three doses of quadrivalent HPV vaccine (for serotypes 6, 11, 16, and 18) or placebo at day 1, month 2, and month 6. Main outcome measures: Vaccine efficacy against cervical, vulvar, and vaginal intraepithelial neoplasia grade I and condyloma in a per protocol susceptible population that included subjects who received all three vaccine doses, tested negative for the relevant vaccine HPV types at day 1 and remained negative through month 7, and had no major protocol violations. Intention to treat, generally HPV naive, and unrestricted susceptible populations were also studied. Results: In the per protocol susceptible population, vaccine efficacy against lesions related to the HPV types in the vaccine was 96% for cervical intraepithelial neoplasia grade I (95% confidence interval 91% to 98%), 100% for both vulvar and vaginal intraepithelial neoplasia grade I (95% CIs 74% to 100%, 64% to 100% respectively), and 99% for condyloma (96% to 100%). Vaccine efficacy against any lesion (regardless of HPV type) in the generally naive population was 30% (17% to 41%), 75% (22% to 94%), and 48% (10% to 71%) for cervical, vulvar, and vaginal intraepithelial neoplasia grade I, respectively, and 83% (74% to 89%) for condyloma. Conclusions: Quadrivalent HPV vaccine provided sustained protection against low grade lesions attributable to vaccine HPV types (6, 11, 16, and 18) and a substantial reduction in the burden of these diseases through 42 months of follow-up. Trial registrations: NCT00092521 and NCT00092534.

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.

Development of designed site-directed pseudopeptide-peptido-mimetic immunogens as novel minimal subunit-vaccine candidates for malaria

Synthetic vaccines constitute the most promising tools for controlling and preventing infectious diseases. When synthetic immunogens are designed from the pathogen native sequences, these are normally poorly immunogenic and do not induce protection, as demonstrated in our research. After attempting many synthetic strategies for improving the immunogenicity properties of these sequences, the approach consisting of identifying high binding motifs present in those, and then performing specific changes on amino-acids belonging to such motifs, has proven to be a workable strategy. In addition, other strategies consisting of chemically introducing non-natural constraints to the backbone topology of the molecule and modifying the a-carbon asymmetry are becoming valuable tools to be considered in this pursuit. Non-natural structural constraints to the peptide backbone can be achieved by introducing peptide bond isosters such as reduced amides, partially retro or retro-inverso modifications or even including urea motifs. The second can be obtained by strategically replacing L-amino-acids with their enantiomeric forms for obtaining both structurally site-directed designed immunogens as potential vaccine candidates and their Ig structural molecular images, both having immunotherapeutic effects for preventing and controlling malaria.