Reverse vaccinology and immunoinformatics approach to design a chimeric epitope vaccine against Orientia tsutsugamushi.

Scrub typhus is a vector-borne infectious disease caused by Orientia tsutsugamushi and it is reportedly associated with up to 20 % of hospitalized cases of febrile illnesses. The major challenge of vaccine development is the lack of identified antigens that can induce both heterotypic and homotypic immunity including the production of antibodies, cytotoxic T lymphocyte, and helper T lymphocytes. We employed a comprehensive immunoinformatic prediction algorithm to identify immunogenic epitopes of the 56-kDa type-specific cell membrane surface antigen and surface cell antigen A of O. tsutsugamushi to select potential candidates for developing vaccines and diagnostic assays. We identified 35 linear and 29 continuous immunogenic B-cell epitopes and 51 and 27 strong-binding T-cell epitopes of major histocompatibility complex class I and class II molecules, respectively, in the conserved and variable regions of the 56-kDa type-specific surface antigen. The predicted B- and T-cell epitopes were used to develop immunogenic multi-epitope candidate vaccines and showed to elicit a broad-range of immune protection. A stable interactions between the multi-epitope vaccines and the host fibronectin protein were observed using docking and simulation methods. Molecular dynamics simulation studies demonstrated that the multi-epitope vaccine constructs and fibronectin docked models were stable during simulation time. Furthermore, the multi-epitope vaccine exhibited properties such as antigenicity, non-allergenicity and ability to induce interferon gamma production and had strong associations with their respective human leukocyte antigen alleles of world-wide population coverage. A correlation of immune simulations and the in-silico predicted immunogenic potential of multi-epitope vaccines implicate for further investigations to accelerate designing of epitope-based vaccine candidates and chimeric antigens for development of serological diagnostic assays for scrub typhus.

Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India.

Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases.

Generation of a novel chimeric PALFn antigen of Bacillus anthracis and its immunological characterization in mouse model.

Bacillus anthracis chimeric molecule PALFn, comprising the immunodominant domains of protective antigen (PA) and lethal factor (LF), has been developed in the past and has been shown to confer enhanced protection against anthrax in mouse model when challenged with anthrax lethal toxin (LeTx). However, the immunological correlates for this chimeric antigen, both in terms of humoral as well as cell-mediated immune responses, have not been described in detail. To address this gap, we have determined the immunological responses both at humoral as well as cellular levels for the protection conferred by the novel chimeric antigen PALFn constructed in our laboratory in comparison to PA antigen. The biological functionality of the chimeric antigen was ascertained by the trypsin digestion assay. The trypsin cleavage activated the functionality of PALFn and rendered it to interact and bind with the LF molecule. Similarly, the LFn component in the chimera could independently interact and bind to the trypsin-activated wild-type PA. Further, it was observed that the PALFn-immunized mice sera could readily react to both PA and LF antigens while PA-immunized mice sera showed reaction to PA and PALFn alone and not to the individual LF antigen. The in vitro toxin neutralizing ability of PALFn antisera on macrophage cell line J774.1 was robust but with 1.3-fold lesser titer than PA-immunized antisera. PALFn-immunized mouse splenocytes showed a significant lymphocyte proliferation when stimulated with PALFn. There was a remarkable increase in the level of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin 10 (IL-10), interferon-γ (IFN- γ), and tumor necrosis factor α (TNFα) from PALFn- and PA-stimulated splenocytes. In addition, there was a significant increase in antigen-specific CD4+ and CD8+ T-cell counts from both PALFn- and PA-immunized mouse splenocytes. The results clearly demonstrate the ability of chimeric molecule PALFn in eliciting robust humoral and cell-mediated immune responses in mouse model that is parallel to the wild-type PA but has additional anti-LF antibody response. Considering the enhanced protection offered by the chimera PALFn, we can conclude that it can be a better alternative to the wild-type PA-based recombinant vaccine against anthrax.