Prediction of an immunogenic peptide ensemble and multi-subunit vaccine for Visceral leishmaniasis using bioinformatics approaches.

Visceral Leishmaniasis (VL) is a neglected tropical disease of public health importance in the Indian subcontinent. Despite consistent elimination initiatives, the disease has not yet been eliminated and there is an increased risk of resurgence from active VL reservoirs including asymptomatic, post kala azar dermatitis leishmaniasis (PKDL) and HIV-VL co-infected individuals. To achieve complete elimination and sustain it in the long term, a prophylactic vaccine, which can elicit long lasting immunity, is desirable. In this study, we employed immunoinformatic tools to design a multi-subunit epitope vaccine for the Indian population by targeting antigenic secretory proteins screened from the Leishmania donovani proteome. Out of 8014 proteins, 277 secretory proteins were screened for their cellular location and proteomic evidence. Through NCBI BlastP, unique fragments of the proteins were cropped, and their antigenicity was evaluated. B-cell, HTL and CTL epitopes as well as IFN-ɣ, IL-17, and IL-10 inducers were predicted, manually mapped to the fragments and common regions were tabulated forming a peptide ensemble. The ensemble was evaluated for Class I MHC immunogenicity and toxicity. Further, immunogenic peptides were randomly selected and used to design vaccine constructs. Eight vaccine constructs were generated by linking random peptides with GS linkers. Synthetic TLR-4 agonist, RS09 was used as an adjuvant and linked with the constructs using EAAK linkers. The predicted population coverage of the constructs was ∼99.8 % in the Indian as well as South Asian populations. The most antigenic, nontoxic, non-allergic construct was chosen for the prediction of secondary and tertiary structures. The 3D structures were refined and analyzed using Ramachandran plot and Z-scores. The construct was docked with TLR-4 receptor. Molecular dynamic simulation was performed to check for the stability of the docked complex. Comparative in silico immune simulation studies showed that the predicted construct elicited humoral and cell-mediated immunity in human host comparable to that elicited by Leish-F3, which is a promising vaccine candidate for human VL.

Publisher Correction: High resolution structural and functional analysis of a hemopexin motif protein from Dolichos.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High resolution structural and functional analysis of a hemopexin motif protein from Dolichos.

It is increasingly evident that seed proteins exhibit specific functions in plant physiology. However, many proteins remain yet to be functionally characterized. We have screened the seed proteome of Dolichos which lead to identification and purification of a protein, DC25. The protein was monomeric and highly thermostable in extreme conditions of pH and salt. It was crystallized and structure determined at 1.28 Å resolution using x-ray crystallography. The high-resolution structure of the protein revealed a four-bladed ß-propeller hemopexin-type fold containing pseudo four-fold molecular symmetry at the central channel. While the structure exhibited homology with 2S albumins, variations in the loops connecting the outermost strands and the differences in surface-charge distribution may be relevant for distinct functions. Comparative study of the protein with other seed hemopexins revealed the presence of four conserved water molecules in between the blades which cross-link them and maintain the tertiary structure. The protein exhibited intrinsic peroxidase activity, which could be inhibited by binding of a heme analog. The identification of redox-sensitive cysteine and inhibition of peroxidase activity by iodoacetamide facilitated characterization of the possible active site. The determined peroxidase activity of DC25 may be responsible for rescuing germinating seeds from oxidative stress.

Cross-clade antibody reactivity may attenuate the ability of influenza virus to evade the immune response.

Vaccines developed against influenza lose efficacy primarily due to the ability of the virus to generate variants that escape recognition of the immune system. Frequent accumulation of mutations in the virus surface proteins is believed to be responsible for immune evasion. Surprisingly, despite the high mutation rate, the appearance of new viral strains through antigenic drift is slow. This delay in the emergence of new strains has been explained by several different hypotheses over the past decade. In the present study, we have probed the antibody response against multiple clades of influenza neutralizing epitope in the context of antigenic drift. Both, the serum IgG and the monoclonal antibodies raised against the epitope showed strong predisposition against different variants even with non-conservative mutations. The physiologically relevant binding with hemagglutinin protein and its variants revealed multi-reactive recognition potential of human single-chain variable fragments (scFvs). Differential scope for antibody cross-reactivity was evident among different clades that could counterbalance the effect of antigenic drift. Our findings reveal that the majority of epitope variants, which could manifest as single or double amino acid substitutions, would not escape immune surveillance. However, mutations beneficial for the virus do appear causing effective antigenic changes. It is suggested that inherent antibody promiscuity could reduce the deleterious effects of natural mutations on antigen recognition and may be responsible for the delay in the appearance of new antigenic variants of the fast-mutating viruses.