Plague Vaccine Research Articles

Study of allergic reaction to somatic antigen made from a live plague vaccine of the EV strain

Study of allergic reaction to somatic antigen made from a live plague vaccine of the EV strain

Delivery of Yersinia pestis antigens via Escherichia coli outer membrane vesicles offered improved protection against plague.

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.

Sex differences in immune protection in mice conferred by heterologous vaccines for pneumonic plague.

Yersinia pestis is the etiological agent of plague, which can manifest as bubonic, septicemic, and/or pneumonic disease. Plague is a severe and rapidly progressing illness that can only be successfully treated with antibiotics initiated early after infection. There are no FDA-approved vaccines for plague, and some vaccine candidates may be less effective against pneumonic plague than bubonic plague. Y. pestis is not known to impact males and females differently in mechanisms of pathogenesis or severity of infection. However, one previous study reported sex-biased vaccine effectiveness after intranasal Y. pestis challenge. As part of developing a safe and effective vaccine, it is essential that potential sex differences are characterized. In this study we evaluated novel vaccines in male and female BALB/c mice using a heterologous prime-boost approach and monitored survival, bacterial load in organs, and immunological correlates. Our vaccine strategy consisted of two subcutaneous immunizations, followed by challenge with aerosolized virulent nonencapsulated Y. pestis. Mice were immunized with a combination of live Y. pestis pgm- pPst-Δcaf1, live Y. pestis pgm- pPst-Δcaf1/ΔyopD, or recombinant F1-V (rF1-V) combined with adjuvants. The most effective vaccine regimen was initial priming with rF1-V, followed by boost with either of the live attenuated strains. However, this and other strategies were more protective in female mice. Males had higher bacterial burden and differing patterns of cytokine expression and serum antibody titers. Male mice did not demonstrate synergy between vaccination and antibiotic treatment as repeatedly observed in female mice. This study provides new knowledge about heterologous vaccine strategies, sex differences in plague-vaccine efficacy, and the immunological factors that differ between male and female mice.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Co-formulation of the rF1V plague vaccine with depot-formulated cytokines enhances immunogenicity and efficacy to elicit protective responses against aerosol challenge in mice.

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.

A novel sORF gene mutant strain of Yersinia pestis vaccine EV76 offers enhanced safety and improved protection against plague.

We recently identified two virulence-associated small open reading frames (sORF) of Yersinia pestis, named yp1 and yp2, and null mutants of each individual genes were highly attenuated in virulence. Plague vaccine strain EV76 is known for strong reactogenicity, making it not suitable for use in humans. To improve the immune safety of EV76, three mutant strains of EV76, Δyp1, Δyp2, and Δyp1&yp2 were constructed and their virulence attenuation, immunogenicity, and protective efficacy in mice were evaluated. All mutant strains were attenuated by the subcutaneous (s.c.) route and exhibited more rapid clearance in tissues than the parental strain EV76. Under iron overload conditions, only the mice infected with EV76Δyp1 survived, accompanied by less draining lymph nodes damage than those infected by EV76. Analysis of cytokines secreted by splenocytes of immunized mice found that EV76Δyp2 induced higher secretion of multiple cytokines including TNF-α, IL-2, and IL-12p70 than EV76. On day 42, EV76Δyp2 or EV76Δyp1&yp2 immunized mice exhibited similar protective efficacy as EV76 when exposed to Y. pestis 201, both via s.c. or intranasal (i.n.) routes of administration. Moreover, when exposed to 200-400 LD50 Y. pestis strain 201Δcaf1 (non-encapsulated Y. pestis), EV76Δyp2 or EV76Δyp1&yp2 are able to afford about 50% protection to i.n. challenges, significantly better than the protection afforded by EV76. On 120 day, mice immunized with EV76Δyp2 or EV76Δyp1&yp2 cleared the i.n. challenge of Y. pestis 201-lux as quickly as those immunized with EV76, demonstrating 90-100% protection. Our results demonstrated that deletion of the yp2 gene is an effective strategy to attenuate virulence of Y. pestis EV76 while improving immunogenicity. Furthermore, EV76Δyp2 is a promising candidate for conferring protection against the pneumonic and bubonic forms of plague.

Possibility of Using Disposable Polymeric Containers in the Production Technology of Live Plague Vaccine

The aim of the work was to assess the possibility of using disposable polymeric containers in the production technology of the live plague vaccine.Materials and methods. We deployed the vaccine strain Yersinia pestis ЕV NIIEG for the work. A standard disposable 10 L Flexboy type polymeric container manufactured by Sartorius Stedim Biotech, equipped with Sartopore 2 filter capsules, was used for submerged cultivation of plague microbe inoculum. This method was compared to the regulated technology for obtaining seed cultures using glass bottles with a volume of 20 liters. The control of the produced seed cultures of the vaccine strain EV was performed in accordance with FS.3.3.1.0022.15. The cultivation of the seed culture in a disposable polymeric container was carried out in a liquid nutrient medium at a temperature of 26 to 28°C with continuous barbotage and mechanical agitation with a platform oscillation frequency of 80 to 90 per minute. For aeration, compressed air with a pressure of 0.3 to 0.4 kgf/cm2 was used. The volumetric flow rate of sterile air supplied for aeration ranged from 0.9 to 1.0 l/min.Results and discussion. The use of disposable polymeric containers made it possible to reduce the duration of the technological stage of obtaining a seed culture by 1.7 times and increase the yield of live microbes per unit volume of the nutrient medium by 2.8 times, as compared to the regulated production technology. Thus, the possibility and prospects of using disposable polymeric containers in the production technology of live plague vaccine at the stage of preparation of sowing cultures is evidenced.

Prospects for using flow cytometry in the quality control of live plague vaccines

Scientific relevance. The number of live bacteria is a quality parameter controlled at all stages of live plague vaccine production. Currently, live microbial cell counting uses a bacteriological method. However, flow cytometry has the potential to increase analytical accuracy and reduce testing time.Aim. This study aimed at testing the applicability of flow cytometry to assessing the quality of live plague vaccines.Materials and methods. The study quantified live microbial cells in 5 experimental batches of live plague vaccine as part of their quality control using the bacteriological method according to the State Pharmacopoeia of the Russian Federation (FS.3.3.1.0022.15). Cytofluorometry of the samples used the SynaptoGreen fluorescent dye.Results. The study quantified live microbial cells in live plague vaccine samples using the bacteriological method and flow cytometry. The results obtained by the bacteriological method ranged from 27.8±2.2 to 56.5±3.1% with an average of 39.8±5.4%. The results obtained by flow cytometry ranged from 29.2±1.2 to 59.1±2.1% with an average of 41.7±5.5%. The statistical analysis showed no significant difference between the results of vaccine quality control by both methods, as well as a high coefficient of determination.Conclusions. The results show that flow cytometry is an appropriate method for the quantification of live microbial cells as part of the quality control of plague vaccines. Being quick, easy, and highly informative, flow cytometry is preferable to traditional methods.

Prairie dog responses to vector control and vaccination during an initial Yersinia pestis invasion

We evaluated the invasion of plague bacteria Yersinia pestis into a population of black-tailed prairie dogs (Cynomys ludovicianus; BTPDs) in South Dakota. We aimed to ascertain if Y. pestis invaded slowly or rapidly, and to determine if vector (flea) control or vaccination of BTPDs assisted in increasing survival rates. We sampled BTPDs in 2007 (before Y. pestis documentation), 2008 (year of confirmed invasion), and 2009 (after invasion). We estimated annual BTPD re-encounter rates on three 9-ha plots treated annually with deltamethrin dust for flea control and three 9-ha plots lacking dust. In 2007 and 2008, approximately half the adult BTPDs live-trapped were injected subcutaneously with either an experimental plague vaccine (F1–V fusion protein) or placebo formulation; the remaining individuals were not inoculated. From 2007 to 2009, we sampled 1559 BTPDs on 2542 occasions. During 2007–2008, the prevalence and intensity of fleas on BTPDs were 69–97% lower on the dusted vs. no dust plots. From 2007 to 2008, the annual re-encounter rate of non-inoculated BTPDs was 150% higher on the dusted vs. no dust plots. During the same interval on the dusted plots, the re-encounter rate was 55% higher for vaccinated adult female BTPDs vs. nonvaccinated adult females, but the annual re-encounter rate was 19% lower for vaccinated adult males. By late August 2008, BTPDs were nearly extirpated from the no dust plots. During 2007–2008 and 2008–2009 on the dusted plots, which persisted, the BTPD re-encounter rate was 41% higher for vaccinated vs. non-vaccinated adult females but 35% lower for vaccinated adult males. Yersinia pestis erupted with vigor as it invaded. Flea control enhanced BTPD survival but did not offer full protection. Flea control and F1–V vaccination seemed to have additive, positive effects on adult females. Annual re-encounter rates were reduced for vaccinated adult males; additional experimentation is needed to further evaluate this trend.

Pneumonic Plague Protection Induced by a Monophosphoryl Lipid A Decorated Yersinia Outer-Membrane-Vesicle Vaccine.

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 OMV46-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 30µg of of OMV46-LcrV exhibited substantially reduced reactogenicity than the parent OMV44-LcrV and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV46-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, OMV46-LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV46-LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. The studies strongly demonstrate the feasibility of OMV46-LcrV as a new type of plague vaccine candidate.

A self-amplifying RNA vaccine provides protection in a murine model of bubonic plague.

Mice were immunized with a combination of self-amplifying (sa) RNA constructs for the F1 and V antigens of Yersinia pestis at a dose level of 1 μg or 5 μg or with the respective protein sub-units as a reference vaccine. The immunization of outbred OF1 mice on day 0 and day 28 with the lowest dose used (1 μg) of each of the saRNA constructs in lipid nanoparticles protected 5/7 mice against subsequent sub-cutaneous challenge on day 56 with 180 cfu (2.8 MLD) of a 2021 clinical isolate of Y. pestis termed 10-21/S whilst 5/7 mice were protected against 1800cfu (28MLD) of the same bacteria on day 56. By comparison, only 1/8 or 1/7 negative control mice immunized with 10 μg of irrelevant haemagglutin RNA in lipid nanoparticles (LNP) survived the challenge with 2.8 MLD or 28 MLD Y. pestis 10-21/S, respectively. BALB/c mice were also immunized with the same saRNA constructs and responded with the secretion of specific IgG to F1 and V, neutralizing antibodies for the V antigen and developed a recall response to both F1 and V. These data represent the first report of an RNA vaccine approach using self-amplifying technology and encoding both of the essential virulence antigens, providing efficacy against Y. pestis. This saRNA vaccine for plague has the potential for further development, particularly since its amplifying nature can induce immunity with less boosting. It is also amenable to rapid manufacture with simpler downstream processing than protein sub-units, enabling rapid deployment and surge manufacture during disease outbreaks.

Plague: Symptoms, Transmission, Diagnosis, Prevention and Treatment

Plague is a contagious disease caused by the zoonotic bacteria, (Yersinia pestis) but transmitted by fleas. It is found on rodents and their fleas. The most common ways for humans to contract plague are through handling a plague-infected animal or by getting bitten by a rodent flea that is carrying the pathogen. Septicemic, pneumonic, and bubonic plague are the three most prevalent types. The bubonic plague typically takes 2 to 8 days to incubate. Patients experience weakness, one or more enlarged, painful lymph nodes, headache, chills, and fever (called buboes). The bite of an infected flea results in plague, which is the most widespread type of plague. Pneumonic plague, a more severe form of bubonic plague, can progress and disseminate to the lungs. Septicemic plague incubation period isn’t exactly known but, it most likely happens within days of exposure and is accompanied by high temperature, chills, intense exhaustion, stomach discomfort, shock, and maybe, bleeding into the skin and other organs. Pneumonic plague often only takes 1 to 3 days to incubate and is accompanied by a fever, headache, weakness, rapidly progressing pneumonia, shortness of breath, chest pain, coughing up occasionally bloody or watery mucus, and other symptoms. Generally, fever, chills, headaches, and nausea are some of the general plague symptoms. There is also the case of lymph nodes enlargement, coughing, chest pain, and hemoptysis. Plague can be transmitted through; bites by fleas, contact with contaminated fluid or tissue, and infectious droplets. The symptoms of plague are typically non-specific; hence laboratory investigation is necessary for a certain diagnosis. Y. pestis infection can be detected using a microscope and by bacteria culture. The sample (buboes) can be gotten from blood, mucus (sputum), or an aspirate taken from swollen lymph nodes. Polymerase chain reaction (PCR), can also be employed to diagnose plague (F1 capsule antigen). It is also possible to identify plague by using blood tests that look for antibodies to Y. pestis. Plague can be prevented by eliminating rodent’s habitats around living houses, workplace, and leisure locations, and elimination of bushes, rock piles, garbage, cluttered firewood, and potential rodent food sources such as pet and wild animal food. Plague vaccines are only recommended for high-risk categories such as laboratory professionals who are constantly exposed to the risk of contamination, and health care workers. All forms of plague respond to antibiotic treatment if they are diagnosed early. Those suspected to have made contact with infected patients or animals should be isolated and observed. Plague remains one of the most infectious diseases worldwide. Although as at the time of this review, there is no recent case of plague in Nigeria, or in Africa, effort is still required to create robust awareness among the population on the subject of plague. The focus of this review is to educate and create more awareness among the citizens on plague as a highly infectious disease.

Protection of Khaki Campbell Ducks against Duck Plague Using an Inactivated Duck Plague Vaccine

Duck plague (DP) or duck viral enteritis is a fatal viral disease of ducks that causes huge economic losses in the duck industry. The present study was performed to determine the immune response and protective efficacy of an inactivated DP vaccine prepared from a local virulent DP virus. A virulent DP virus was obtained from the laboratory repository of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh (Bangladesh). The DP virus (EID50 105.3/ml) was inactivated using 0.04% formalin. The alum (40 g/L) was added to the inactivated DP virus as an adjuvant. A total of 60 Khaki Campbell male ducks aged 17 weeks were randomly divided into three groups. Ducks of groups A (n = 20) and B (n = 20) were vaccinated intramuscularly in the breast muscle with 1 ml of inactivated DP vaccine and a live attenuated DP vaccine, respectively. Ducks of group C (n = 20) were kept as unvaccinated control. Booster vaccination was administered at 2 weeks after primary vaccination. Antibody titers of vaccinated ducks were measured at 7, 14, 21, and 28 days post-vaccination (DPV) using a passive haemagglutination (PHA) test. Ducks of both vaccinated and unvaccinated groups were challenged with 1 ml virulent DP virus (EID50 104.3/ml) at 28 DPV. Clinical signs, morbidity and mortality, and gross pathological lesions of vaccinated and control ducks were observed for 10 days post-challenge to evaluate the protective efficacy of inactivated DP vaccine. The mean PHA antibody titers of vaccinated ducks of group A at 7, 14, 21, and 28 DPV were 5 ± 0.43, 26 ± 1.71, 43 ± 3.4, and 54 ± 3.28, respectively. Ducks in group B had mean serum PHA antibody titers of 21 ± 1.71, 41 ± 3.28, 52 ± 3.41, and 84 ± 7.25 at 7, 14, 21, and 28 DPV, respectively. No mortality or gross pathological lesions were observed in vaccinated ducks after they were subjected to a challenge infection. Additionally, no significant difference was observed between groups A and B in terms of the challenge infection. The mortality rate of the control group of ducks was 70%. Hemorrhage in the trachea and intestine and necrotic foci in the liver were seen in unvaccinated control ducks (group C). Experimentally developed inactivated DP vaccine induced a protective serum antibody titer and conferred 100% protection against virulent challenge infection up to 10 days observation period.

Increased expression of tyrosine protein phosphatase (CD45) on the surface of human blood granulocytes under the influence of the plague microbe vaccine strain <I>Yersinia pestis</I> EV NIIEG <I>ex vivo</I> and <I>in vivo</I>

Tyrosine protein phosphatase (common leukocyte antigen CD45) regulates FcᵧR-mediated cell signaling and secretory function of neutrophilic granulocytes (NG) when interacting with antigen-antibody immune complexes. The aim of the work is to study changes in the expression of CD45 on the surface of human granulocytes in ex vivo modeling of bacteremia by live cells of the plague microbe vaccine strain Yersinia pestis EV NIIEG and to evaluate the priming effect of the live plague vaccine (LPV) in vivo in terms of this parameter. The expression density of CD45 on NG was determined by flow cytometry in conventional units of fluorescence intensity (MFI) after staining the cells with the CD45-FITC labeled mouse antibody reagent (Backman Coulter, USA) during immunophenotyping of blood leukocytes according to the Lyse/No Wash protocol. In donors not previously vaccinated against plague (group 1), the value of the indicator was assessed before and 30 min, 2 h, 6 h after the addition of Y pestis EV cells to whole blood at a dose of 108 mc/ml, as well as 1 month and 6 months after the primary anti-plague vaccination. In individuals who had previously been repeatedly vaccinated with LPV in the territory of the natural plague focus (group 2), CD45 expression on blood granulocytes was determined one year after the last annual vaccination, and then 1 month and 6 months after revaccination. Getting into human blood, living cells of the vaccine strain Y pestis EV of the plague microbe induced a change in the NC phenotype already after 30 minutes, associated with a 3.5-fold increase in the surface expression of CD45, which remained at an elevated level for 6 hours. The studied indicator depended ex vivo on the degree of resistance of plague microbes to phagocytosis and killing of NG. Plague vaccination had a similar stimulating effect on human peripheral blood NG in vivo. Under the influence of HPV, CD45 expression increased on blood NG in groups 1 and 2 one month after vaccination, and the changes persisted in volunteers for 6 months. The experimental data obtained in the work may reflect the result of NG priming with lipopolysaccharide and other Y. pestis antigens. The registered functional activation of NG by expression of tyrosine protein phosphatase probably indicates the formation of “immune memory” at the level of innate immunity cells under the influence of LPV, the functioning of which explains the development of a faster and more intense antigen-specific immune response to repeated introduction of the plague vaccine into the body.

Evaluation of the applicability of immunochromatography to the identification of live plague vaccines and the tularaemia allergen (Tularin)

The regulatory standards require that the identification of live plague vaccines and the liquid tularaemia allergen (Tularin) should be performed by immunofluorescence. A major drawback of the recommended method is its labour intensive nature. However, immunochromatography represents an alternative method that offers a number of advantages, including rapid testing and easy result interpretation. The aim of the study was to assess the applicability of immunochromatography to the identification of live plague vaccines and the liquid tularaemia allergen (Tularin).Materials and methods. The authors performed identification tests using samples of the pharmacopoeia standard for live plague vaccines, three commercial batches of a live plague vaccine, and two batches of the liquid tularaemia allergen (Tularin). These samples were tested using immunochromatographic assay (ICA) reagent kits for rapid detection and identification of Yersinia pestis (ICA System for Y. pestis) and Francisella tularensis (ICA System for F. tularensis) manufactured by the State Scientific Center for Applied Microbiology and Biotechnology.Results. The findings show that immunochromatography is an effective, rapid, and species-specific method to confirm the presence of Y. pestis in a sample of a live plague vaccine or F. tularensis in a sample of the liquid tularaemia allergen (Tularin). To perform identification tests by immunochromatography, the authors recommend diluting live plague vaccine samples to a concentration of 109 bacterial cells/mL and using undiluted samples of the liquid tularaemia allergen (Tularin).Conclusions. The study results may support the inclusion of ICA into the regulatory standards for live plague vaccines and the liquid tularaemia allergen (Tularin) as an alternative identification method.

Polyclonal Antibodies Derived from Transchromosomic Bovines Vaccinated with the Recombinant F1-V Vaccine Increase Bacterial Opsonization In Vitro and Protect Mice from Pneumonic Plague.

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human.

Direct Quantification of Protein Antigens in Subunit Plague and Rickettsial Vaccine Preparations

The aim of the work was to put forward the methods for direct quantitative determination of the content of Yersinia pestis and Rickettsia raoultii protein antigens in preparations and various prototypes of subunit vaccines. Materials and methods. Y. pestis LcrV and Caf1 antigens enclosed in the substance of the molecular microencapsulated plague vaccine (MMPV) and separately, in microcrystals of amino acids co-precipitated with plague proteins were used as model antigens. R. raoultii Adr2, OmpB24, and YbgF antigens were adsorbed on the prototype substance of the rickettsia vaccine. The release of plague antigens from MMPV microcapsules was carried out through successive treatment of the latter with organic solvents, methylene chloride and methanol, respectively; the carrier microcrystals were dissolved in 0.1 M sodium citrate buffer at pH 6.0. The antigen content in the prototype substance of the rickettsial vaccine was determined by measuring the amount of proteins not bound to the alumogel. Quantitative parameters characterizing the content of antigens in the substances and prototypes of vaccine preparations were calculated by processing digital images of polyacrylamide gels obtained by electrophoresis of protein antigen fractions extracted from carriers. Results and discussion. Methods for direct extraction and subsequent quantitative analysis of Y. pestis LcrV and Caf1 antigens from subunit vaccine preparations based on amino acid microcrystals and polylactide microcapsules that do not cause protein degradation have been studied. A different nature of the binding of LcrV and Caf1 in the substances of microcrystals has been established, while the proportion of antigens released from microcrystals has been quantified only in case of their complete dissolution. It was found that at low concentrations of LcrV and Caf1 proteins extracted from microcrystals, it is necessary to concentrate the extracts with subsequent removal of salts for their reliable visualization. It has been confirmed that 10 μg of plague antigens and proteins of R. raoultii in a dose volume of 200 μl of suspension is sufficient for quantitative analysis using electrophoretic method. The prospects of other physicochemical methods alternative to direct extraction of antigens for evaluating the composition and quality of vaccine preparations are discussed.

Lung Resident Memory T Cells Activated by Oral Vaccination Afford Comprehensive Protection against Pneumonic Yersinia pestis Infection.

A growing body of evidence has shown that resident memory T (TRM) cells formed in tissue after mucosal infection or vaccination are crucial for counteracting reinfection by pathogens. However, whether lung TRM cells activated by oral immunization with Yptb1(pYA5199) play a protective role against pneumonic plague remains unclear. In this study, we demonstrated that lung CD4+ and CD8+ TRM cells significantly accumulated in the lungs of orally Yptb1(pYA5199)-vaccinated mice and dramatically expanded with elevated IL-17A, IFN-γ, and/or TNF-α production after pulmonary Yersinia pestis infection and afforded significant protection. Short-term or long-term treatment of immunized mice with FTY720 did not affect lung TRM cell formation and expansion or protection against pneumonic plague. Moreover, the intratracheal transfer of both lung CD4+ and CD8+ TRM cells conferred comprehensive protection against pneumonic plague in naive recipient mice. Lung TRM cell-mediated protection was dramatically abolished by the neutralization of both IFN-γ and IL-17A. Our findings reveal that lung TRM cells can be activated via oral Yptb1(pYA5199) vaccination, and that IL-17A and IFN-γ production play an essential role in adaptive immunity against pulmonary Y. pestis infection. This study highlights an important new target for developing an effective pneumonic plague vaccine.

Direct Detection of Antibodies to <i>Yersinia pestis</i> Using Glass Microstructural Waveguides as an Express Method for Assessing Seroconversion in Individuals Vaccinated against Plague

Relevance. As part of ensuring sanitary and epidemiological well­-being, in the territory of natural plague foci of the Russian Federation, according to epidemiological indications, the population is vaccinated against this infection. The lack of a unified scheme for evaluating the effectiveness of vaccination dictates the need to develop universal express methods that allow screening studies of anti­-plague immunity, including in the field.Aims. To evaluate the effectiveness of the express method for the direct detection of antibodies to Yersinia pestis in biological material using microstructural glass waveguides with a hollow core (MGW HC) as immunosensors in studying the dynamics of seroconversion in people vaccinated with the plague live people.Materials and methods. In the study, blood serum samples were taken from 30 individuals vaccinated according to epidemic indications with the live plague vaccine (PLV) and 30 volunteers who were not vaccinated and did not have a history of contact with the plague agent.Results. An assessment was made of the possibility of using the method of direct detection of antibodies using MGW HC at different time intervals according to the receipt of blood sera in the framework of monitoring vaccinated individuals from among the inhabitants of the Caspian sandy natural plague focus. The presence of specific antibodies to the F1 protein in the blood serum of vaccinated volunteers was revealed both with the help of ELISA and with the use of MGW HC. The speed of the method of direct detection of antibodies (maximum 2 minutes per test), the absence of the need to use species­specific secondary antibodies, enzymes and substrates and additional costs for laborious sample preparation were noted.Conclusions. The method of direct detection of antibodies using glass microstructural waveguides is promising for introduction into the list of express methods for assessing the immunological effectiveness of anti­plague vaccination.

Determination of Conformational and Functional Stability of Potential Plague Vaccine Candidate in Formulation.

Generally, protein-based vaccines are available in liquid form and are highly susceptible to instability under elevated temperature changes including freezing conditions. There is a need to create a convenient formulation of protein/peptides that can be stored at ambient conditions without loss of activity or production of adverse effects. The efficiency of naturally occurring biocompatible polymer dextran in improving the shelf-life and biological activity of a highly thermally unstable plague vaccine candidate protein called Low Calcium Response V antigen (LcrV), which can be stored at room temperature (30 ± 2 °C), has been evaluated. To determine the preferential interactions with molecular-level insight into solvent-protein interactions, analytical techniques such asspectroscopy, particle size distribution, gel electrophoresis, microscopy, and thermal analysis have been performed along with the evaluation of humoral immune response, invivo. The analytical methods demonstrate the structural stability of the LcrV protein by expressing its interaction with the excipients in the formulation. The invivo studies elicited the biological activity of the formulated antigen with a significantly higher humoral immune response (p-value = 0.047) when compared to the native, adjuvanted antigen. We propose dextran as a potential biopolymer with its co-excipient sodium chloride (NaCl) to provide protein compactness, i.e., prevent protein unfolding by molecular crowding or masking mechanism using preferential hydrophobic interaction for up to three weeks at room temperature (30 ± 2 °C).