The recent progress in immunoinformatics provided the basis for an accelerated development of target-specific peptide vaccines as an alternative to the traditional vaccine concept. However, there is still limited information on whether the in silico predicted immunoreactive epitopes correspond to those obtained from the actual experiments. Here, humoral and cellular immune responses to two major Yersinia pestis protective antigens, F1 and LcrV, were studied in human donors immunized with the live plague vaccine (LPV) based on the attenuated Y. pestis strain EV line NIIEG. The F1 antigen provided modest specific cellular (mixed T helper 1 (Th1)/Th2 type) and humoral immune responses in vaccinees irrespective of the amount of annual vaccinations and duration of the post-vaccination period. The probing of the F1 overlapping peptide library with the F1-positive sera revealed the presence of seven linear B cell epitopes, which were all also predicted by in silico assay. The immunoinformatics study evaluated their antigenicity, toxicity, and allergenic properties. The epitope TSQDGNNH was mostly recognized by the sera from recently vaccinated donors rather than antibodies from those immunized decades ago, suggesting the usefulness of this peptide for differentiation between recent and long-term vaccinations. The in silico analysis predicted nine linear LcrV-specific B-cell epitopes; however, weak antibody and cellular immune responses prevented their experimental evaluation, indicating that LcrV is a poor marker of successful vaccination. No specific Th17 immune response to either F1 or LcrV was detected, and there were no detectable serum levels of F1-specific immunoglobulin A (IgA) in vaccinees. Overall, the general approach validated in the LPV model could be valuable for the rational design of vaccines against other neglected and novel emerging infections with high pandemic potency.

To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis. PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination. The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen. Yersinia pestis, the causative agent of plague, has been recognized as one of the most devastating pathogen experienced by mankind. It remains endemic in many parts of the world, and is considered emerging pathogen. A live attenuated Y. pestis strain EV line NIIEG has been used for decades in the former Soviet Union for human vaccination and has proven effective against all forms of plague. We began characterizing the Y. pestis-specific antibody and T cell-mediated immune responses in people immunized with live plague vaccine. The long term goal of our research is to understand the protective mechanisms underlying immunity to plague in humans and to discover novel protective antigens for their incorporation into a subunit vaccine. Here, we describe our study on immune responses in vaccinees to one of the essential virulence factors of Y. pestis, namely Pla antigen. The results of the study shed light on the development of the optimal markers to assess the correlation with vaccine-induced protection.

Omptins represent a family of proteases commonly found in various Gram-negative pathogens. These proteins play an important role in host–pathogen interaction and have been recognized as key virulence factors, highlighting the possibility of developing an omptin-based broad-spectrum vaccine. The prototypical omptin, His-tagged recombinant Pla, was used as a model target antigen. In total, 46 linear and 24 conformational epitopes for the omptin family were predicted by the use of ElliPro service. Among these we selected highly conserved, antigenic, non-allergenic, and immunogenic B-cell epitopes. Five epitopes (2, 6, 8, 10, and 11 corresponding to Pla regions 52–60, 146–150, 231–234, 286–295, and 306–311, respectively) could be the first choice for the development of the new generation of target-peptide-based vaccine against plague. The partial residues of omptin epitopes 6, 8, and 10 (regions 136–145, 227–230, and 274–285) could be promising targets for the multi-pathogen vaccine against a group of enterobacterial infections. The comparative analysis and 3D modeling of amino acid sequences of several omptin family proteases, such as Pla (Yersinia pestis), PgtE (Salmonella enterica), SopA (Shigella flexneri), OmpT, and OmpP (Escherichia coli), confirmed their high cross-homology with respect to the identified epitope clusters and possible involvement of individual epitopes in host–pathogen interaction.

Live plague vaccines have saved thousands of human lives in the 20th century and have continued to be used in Russia and other countries of the former Soviet Union for prophylaxis of plague. A live attenuated Y. pestis EV strain line NIIEG is used for the routine annual vaccination of plague workers, as well as people from the groups at risk. This vaccination can offer immunity against both bubonic and pneumonic plague. However, serologic markers of the human response to vaccination with EV NIIEG are poorly investigated. It is not clear whether other antigens, in addition to the established capsular antigen F1 and lipopolysaccharide, can elicit specific and long-lasting antibody responses in vaccinees. In this study, a humoral immunity to a panel of recombinant Y. pestis-specific antigens, such as F1, Pla, LcrV, YopM and YscF, was examined in volunteers, who received multiple annual immunizations with EV NIIEG during the period of 5 to 30 years. To evaluate a long-term immune response to these antigens, we chose a cohort of donors, who had their last immunization 4-30 years ago. The immunoblotting technique revealed that sera of 14 out of 17 donors contained antibodies to at least one of the tested antigens. As expected, the occurrence of anti-F1 antibodies was detected in a large group of vaccinees (57%). In contrast, the presence of specific antibodies to either LcrV or YscF was less pronounced (26% and 36%, respectively), and only two donors possessed anti-YopM (10%). Surprisingly, we found that sera of the vast majority of volunteers (82%) gave positive reaction with the outer membrane protease Pla, specific to Y. pestis. This analysis will provide insights into the correlates of immunity elicited in humans by live plague vaccine, aid in the search for markers of exposure to plague, and help to develop new diagnostic protocols

Polyoxidonium (PO) has been recognized as an effective, safe immunomodulator with a marked immuno-stimulating activity suitable for the complex treatment of both acute and chronic infections in humans. The PO contains high-polymeric units of 100 kDa based on both N-oxide 1,4- ethylene piperazine and (N-carboxyethyl-) 1,4 ethylene piperazine bromide. Recently, PO has been licensed for human use in the new Russian trivalent polymeric subunit flu vaccine “Grippol.” This will open a possibility of using the PO for the development of vaccines against other infectious diseases. Here we studied the potency of the PO in eliciting the humoral immune response to specific antigens of Chlamydia spp. (C1 and C2) and Yersinia pestis (Pla, LcrV, and YopM) in comparison with the Freund's complete (FCA) and Titermax (TMC) adjuvants. The Balb/c mice were primed with a single intraperitoneal injection of the 10-20μg of antigen mixed 1:1 with either adjuvant and boosted 3 times with the same antigen alone. We found at least 8- to 16-fold and 4- to 8-fold increases in IgG titers to Chlamydia and Yersinia antigens, respectively, after injections of the antigens with the PO in relation to the use of the either FCA or TMC. The PO was found to be absolutely safe for animal health, since there were no visible adverse reactions at the inoculation site characteristic of those seen with the other adjuvants, in particular FCA. Thus, the PO could be the adjuvant of choice to elicit a strong immune response to the major antigens of Chlamydia and Yersinia, suggesting the possibility of its use in developing bi- and tri-valent subunit vaccines with enhanced immunity. The exact mechanism of priming the immune response by the PO is under investigation