Search

The aim of the work was to study the effect of the azoximer bromide immunoadjuvant (polyoxidonium, PO) on certain molecular-genetic and proteomic properties of Yersinia pestis EV NIIEG strain, when added to the culture medium. Materials and methods. Y. pestis EV NIIEG was grown at 28 °C for 48 hours on LB agar pH 7.2 (Miller), with and without PO (EV+PO). Whole-genome sequencing of EV and EV+PO strains was performed on the Ion S5 XG generation II platform. Whole-genome SNP analysis and search for marker SNPs were conducted in the Wombac 2.0 program. Mass-spectra of Y. pestis EV extracts and EV+PO cells were recorded using a Microflex LT mass spectrometer. Protective properties of the test cultures were evaluated by the integral ImD50 index in BALB/c mice when infected with Y. pestis 231(708). Results and discussion. Comparative analysis has not revealed deletions, insertions and single nucleotide polymorphisms in the structure of Y. pestis EV+PO strain genome leading to a violation of the production of pathogenicity, immunogenicity and ensuring the vital activity factors of the plague pathogen. The MALDI-TOF mass spectrometry has shown that Y. pestis EV+PO strains changed the intensity of 22 % of the total number of peaks in the range of 2000–20000 Da. Most of the altered peaks in the UniProtKB protein bank belong to uncharacterized proteins and metabolic proteins. At the same time, the ImD50 was 2–3.3 times lower in cultures grown with the addition of PO than in Y. pestis EV. Thus, the addition of PO to Y. pestis EV NIIEG culture medium does not cause changes in the genes of pathogenicity and vital activity support factors of plague pathogen, but modulates its protein profile, which is accompanied by an increase in the protective potential of the EV vaccine strain.

ABSTRACT Outer membrane vesicles (OMVs) from Gram-negative bacteria can be used as a vaccine platform to deliver heterologous antigens. Here, the major protective antigens of Yersinia pestis, F1 and LcrV, were fused either with the leader sequence or the transmembrane domain of the outer membrane protein A (OmpA), resulting in chimeric proteins OmpA-ls-F1V and OmpA46-159-F1V, respectively. We show that OmpA-ls-F1V and OmpA46-159-F1V can be successfully delivered into the lumen and membrane of the OMVs of Escherichia coli, respectively. Mutation of ompA but not tolR in E. coli enhanced the delivery efficiency of OmpA-ls-F1V into OMVs. The OmpA-ls-F1V protein comprises up to 20% of the total protein in OMVs derived from the ompA mutant (OMVdA-ALS-F1V), a proportion significantly higher than the 1% observed for OmpA46-159-F1V in OMVs produced by an ompA mutant that expresses OmpA46-159-F1V, referred to as OMVdA-LATM5-F1V. Intramuscular (i.m.) immunization of mice with OMVdA-ALS-F1V induced significantly higher levels of serum anti-LcrV and anti-F1 IgG, and provided higher efficacy in protection against subcutaneous (s.c.) Y. pestis infection compared to OMVdA-LATM5-F1V and the purified recombinant F1V (rF1V) protein adsorbed to aluminum hydroxide. The three-dose i.m. immunization with OMVdA-ALS-F1V, administered at 14-day intervals, provides complete protection to mice against s.c. infection with 130 LD50 of Y. pestis 201 and conferred 80% against intranasal (i.n.) challenge with 11.4 LD50 of Y. pestis 201. Taken together, our findings indicate that the engineered OMVs containing F1V fused with the leader sequence of OmpA provide significantly higher protection than rF1V against both s.c. and i.n. infection of Y. pestis and more balanced Th1/Th2 responses.IMPORTANCEThe two major protective antigens of Y. pestis, LcrV and F1, have demonstrated the ability to elicit systemic and local mucosal immune responses as subunit vaccines. However, these vaccines have failed to provide adequate protection against pneumonic plague in African green monkeys. Here, Y. pestis F1 and LcrV antigens were successfully incorporated into the lumen and the surface of the outer membrane vesicles (OMVs) of E. coli by fusion either with the leader sequence or the transmembrane domain of OmpA. We compared the humoral immune response elicited by these OMV formulations and their protective efficacy in mice against Y. pestis. Our results demonstrate that the plague OMV vaccine candidates can induce robust protective immunity against both s.c. and i.n. Y. pestis infections, surpassing the effectiveness of rF1V. In addition, immunization with OMVs generated a relatively balanced Th1/Th2 immune response compared to rF1V immunization. These findings underscore the potential of OMVs-based plague vaccines for further development.

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the coapplication of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy. [ABSTRACT FROM AUTHOR]

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.

Outer membrane vesicles (OMVs) from Gram-negative bacteria can be used as a vaccine platform to deliver heterologous antigens. Here, the major protective antigens of Yersinia pestis, F1 and LcrV, were fused either with the leader sequence or the transmembrane domain of the outer membrane protein A (OmpA), resulting in chimeric proteins OmpA-ls-F1V and OmpA 46-159 -F1V, respectively. We show that OmpA-ls-F1V and OmpA 46-159 -F1V can be successfully delivered into the lumen and membrane of the OMVs of Escherichia coli, respectively. Mutation of ompA but not tolR in E. coli enhanced the delivery efficiency of OmpA-ls-F1V into OMVs. The OmpA-ls-F1V protein comprises up to 20% of the total protein in OMVs derived from the ompA mutant (OMV dA -ALS-F1V), a proportion significantly higher than the 1% observed for OmpA 46-159 -F1V in OMVs produced by an ompA mutant that expresses OmpA46-159-F1V, referred to as OMV dA -LATM5-F1V. Intramuscular ( i.m .) immunization of mice with OMV dA -ALS-F1V induced significantly higher levels of serum anti-LcrV and anti-F1 IgG, and provided higher efficacy in protection against subcutaneous ( s.c. ) Y. pestis infection compared to OMV dA -LATM5-F1V and the purified recombinant F1V (rF1V) protein adsorbed to aluminum hydroxide. The three-dose i.m . immunization with OMV dA -ALS-F1V, administered at 14-day intervals, provides complete protection to mice against s.c. infection with 130 LD 50 of Y. pestis 201 and conferred 80% against intranasal ( i.n .) challenge with 11.4 LD 50 of Y. pestis 201. Taken together, our findings indicate that the engineered OMVs containing F1V fused with the leader sequence of OmpA provide significantly higher protection than rF1V against both s.c . and i.n . infection of Y. pestis and more balanced Th1/Th2 responses.IMPORTANCEThe two major protective antigens of Y. pestis , LcrV and F1, have demonstrated the ability to elicit systemic and local mucosal immune responses as subunit vaccines. However, these vaccines have failed to provide adequate protection against pneumonic plague in African green monkeys. Here, Y. pestis F1 and LcrV antigens were successfully incorporated into the lumen and the surface of the outer membrane vesicles (OMVs) of E. coli by fusion either with the leader sequence or the transmembrane domain of OmpA. We compared the humoral immune response elicited by these OMV formulations and their protective efficacy in mice against Y. pestis . Our results demonstrate that the plague OMV vaccine candidates can induce robust protective immunity against both s.c . and i.n. Y. pestis infections, surpassing the effectiveness of rF1V. In addition, immunization with OMVs generated a relatively balanced Th1/Th2 immune response compared to rF1V immunization. These findings underscore the potential of OMVs-based plague vaccines for further development., Competing Interests: The authors declare no conflict of interest.

A new Yersinia pseudotuberculosis mutant strain, YptbS46, carrying the lpxE insertion and pmrF-J deletion is constructed and shown to exclusively produce monophosphoryl lipid A (MPLA) having adjuvant properties. Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, are designated OMV 46 -LcrV, which contained MPLA and high amounts of LcrV (Low Calcium response V) and displayed low activation of Toll-like receptor 4 (TLR4). Intramuscular prime-boost immunization with 30 µg of of OMV 46 -LcrV exhibited substantially reduced reactogenicity than the parent OMV 44 -LcrV and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV 46 -LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which are correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV 46 -LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV 46 -LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. The studies strongly demonstrate the feasibility of OMV 46 -LcrV as a new type of plague vaccine candidate., (© 2023 Wiley‐VCH GmbH.)

Vaccine strains Yersinia pestis EV NIIEG at a dose of 10 3 CFU and Francisella tularensis 15 NIIEG at a dose of 10 2 CFU induced changes in the concentration of cyclic nucleotides in the thymus and spleen of white mice. Antigen-induced changes in the cAMP/cGMP ratio in immunocompetent organs had a phase or oscillatory character, which seems to be related to the regulation of postvaccination immunoreactivity in the body. Synthetic organoselenium compound 974zh stimulated an increase in the amplitude of cAMP/cGMP oscillations, indicating its stimulating effect on the immunogenic properties of vaccine strains at doses an order of magnitude below the standard doses., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)