Plasmodium vivax vaccine candidate MSP1 displays conserved B-cell epitope despite high genetic diversity.

The polymorphic nature of merozoite surface protein 1(MSP1) raises doubts whether it may serve as a vaccine target against Plasmodium vivax malaria. This study analyses the impact of genetic variability on the epitope organization of different Pvmsp1 blocks. Ten blood samples collected from P. vivax infected malaria patients from West Bengal, India were used to analyze sequence and antigenic diversities of block 2 region of Pvmsp1. An additional 48 block 2 sequences from other countries were also analyzed. Global genetic framework of Pvmsp1 block 2 was represented by 12 indel clusters & 33 haplotypes (haplotype diversiy = 0.965 ± 0.024). Parasite sequences pertaining to other Pvmsp1 modules, namely block 6 and 10 displayed 14 & 29 (haplotype diversiy = 0.975 ± 0.003) and 22 & 30 indel clusters and haplotypes (haplotype diversiy = 0.947 ± 0.004), respectively. In spite of this remarkable genetic diversity, a small number of conserved epitopes were detected in all three PvMSP1 blocks. This novel finding substantiates that MSP1 could serve as a promising vaccine candidate against vivax malaria.

Population genetic and biophysical evidences reveal that purifying selection shapes the genetic landscape of Plasmodium falciparum RH ligands in Chhattisgarh and West Bengal, India.

BACKGROUND: Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Plasmodium falciparum malaria. Present study explores the possible evolutionary drivers shaping the genetic organization of Pfrhs in Indian parasite population. It simultaneously evaluates a putative gain-of-function variant of PfRH5, a keystone member of PfRH family. METHODS: Receptor binding regions of Pfrh1, Pfrh2a/b, Pfrh4 and whole Pfrh5 were amplified using blood samples of P. falciparum malaria patients from Chhattisgarh and West Bengal and sequenced. Assembled sequences were analysed using MEGA7 and DnaSPv6. Binding affinities of recombinant PfRH5 proteins with basigin (BSG) were compared using in silico (CHARMM and AUTODOCK) and in vitro (Circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry) methods. RESULTS: Pfrh1 (0.5), Pfrh2a/b (0.875), Pfrh4 (0.667) and Pfrh5 (0.778) sequence changes corresponded to low frequency (< 0.05) variants which resulted in an overall negative Tajima's D. Since mismatch distribution of none of the Pfrh loci corroborated with the model of demographic expansion, a possible role of natural selection formulating Pfrh sequence diversity was investigated. Among the 5 members, Pfrh5 displayed very high dN/dS (5.7) ratio. Nevertheless, the model of selective sweep due to presence of any advantageous substitutions could not be invoked as polymorphic nonsynonymous sites (17/18) for Pfrh5 exceeded significantly over the divergent (62/86) ones (p = 0.0436). The majority of extant PfRH5 sequences (52/83) differed from the reference Pf3D7 allele by a single amino acid mismatch (C203Y). This non-conservative alteration was predicted to lower the total interaction energy of that PfRH5variant with BSG, compared to PfRH53D7. Biophysical evidences validated the proposition that PfRH5variant formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5variant was also found to be higher (Kavariant = 3.63E6 ± 2.02E6 M-1 and Ka3D7 = 1.31E6 ± 1.21E6 M-1). CONCLUSIONS: Together, the study indicates that the genetic architecture of Pfrhs is principally shaped by purifying selection. The most abundant and ubiquitous PfRH5 variant harbouring 203Y, exhibits a greater affinity for BSG compared to PfRH53D7 possessing 203C allele. The study underscores the importance of selecting the functional allele that best represents circulating strains in natural parasite populations as vaccine targets.

Diversity analysis of MSP1 identifies conserved epitope organization in block 2 amidst high sequence variability in Indian Plasmodium falciparum isolates.

BACKGROUND: Despite its immunogenicity, the polymorphic nature of merozoite surface protein 1, an important vaccine candidate for Plasmodium falciparum malaria, remains a concern. This study analyses the impact of genetic variability and parasite population structure on epitope organization of different MSP1 segments. METHODS: Altogether 98 blood samples collected from P. falciparum infected mild and severe malaria patients of Chhattisgarh and West Bengal were used to sequence regions encoding block 2 and MSP1-19 of msp1. Sequences were analysed using MEGA7, DnaSPv5, Arlequin3.5 and BepiPred. RESULTS: All three major MSP1 block 2 allele families namely K1, MAD20 and RO33 were detected in the samples and they together resulted in 41 indel variants. Chhattisgarh samples displayed an average MOI of 2.07 ± 1.59 which was higher in mild malaria and in age group < 18 years. Ultra-structure of block 2 alleles revealed that mutation and repeat expansion were two major mechanisms responsible for allelic variability of K1 and MAD20. Regions flanking block 2 were highly variable in Chhattisgarh with average mismatch differences (k) ranging from 1.198 to 5.156 for three families. In contrast, region encompassing MSP1-19 exhibited limited heterogeneity (kChhattisgarh = 1.45, kWest Bengal = 1.363). Of the 50 possible B cell linear epitopes predicted from block 2 variants, 94.9% (131 of 138) of the parasites could be represented by three conserved antigens. CONCLUSIONS: Present data indicates that natural selection and transmission intensity jointly play a role in controlling allelic diversity of MSP1 in Indian parasite isolates. Despite remarkable genetic variability, a limited number of predominant and conserved epitopes are present in Indian parasite isolates reinstating the importance of MSP1 as a promising malaria vaccine candidate.

Effectiveness of early clinical exposure in learning respiratory physiology among the newly entrant MBBS students.

INTRODUCTION: Early Clinical Exposure has been conceptualized to orient medical students towards actual clinical scenario and help them correlate their theoretical knowledge with real life situations in early years of MBBS courses. In the present study we explored the outcome of early clinical exposure in the context of basic science topics (Physiology) in fresh MBBS entrants and compared their performance with a conventionally taught control group. METHODS: One hundred fifty voluntary students of 1st year MBBS (2015-16) batch consisted the sample of this study. They were divided into two groups through the simple random method (using computer generated random number table with roll numbers of the students). They were evaluated by MCQ (Multiple Choice Question) and OSCE (Objective Structured Clinical Examination) before and after being taught a basic Physiology topic (respiratory system) theoretically. The study group underwent clinical exposure before the post-test while the control group did not. Performance of the students was compared between the two groups by unpaired student's t-test whereas marks of pre and post-test within the same group were compared by paired Student's t-test. Everywhere p<0.05 was considered to be significant. RESULTS: The marks of each group in the pre and post-tests differed significantly (P<0.05 in each case). Post-test marks were significantly greater in each group though the level of improvement was strikingly higher in the study group (p=0.01). Though there was no significant difference in pre-test marks of both groups (P=0.73), post-test marks were significantly higher in the study group (P=0.04). Among the exposed students, majority (92%) opined that ECE was a better technique being practically oriented and more interesting while some (8%) found it to be more time and energy-consuming, suitable for selective portions of basic science topics. CONCLUSION: Early clinical exposure may be an effective technique to supplement the traditional theoretical teaching and improve the performance of fresh medical entrants in Physiology. It has better acceptability by the students and may be considered for inclusion in the existing pre-clinical curriculum with proper allocation of time and manpower.