Nanodisc assembly from bacterial total lipid extracts.

Nanodiscs are discoidal lipoproteins that have often been used as vehicles to study membrane proteins in their native configuration. Nanodiscs have been primarily made from synthetic lipids. However, nanodiscs also offer a format by which native lipids can be studied in their natural configuration. Here, we present a method to synthesize nanodiscs from bacterial total lipid extracts using the biothreat agent, Yersinia pestis, as a proof-of-concept. The creation of nanoparticles entirely composed of bacterial lipids supports membrane characterization and vaccine antigen discovery without the inherent safety concerns associated with live bacterial cells of this Tier 1 select agent pathogen.

Symbiosis of a lytic bacteriophage and Yersinia pestis and characteristics of plague in Marmota himalayana.

Surveillance for animal plague was conducted in the Marmota himalayana plague focus of the Qinghai-Tibet Plateau from 2020 to 2023. A 22.89% positive rate of serum F1 antibody was detected in live-caught marmots, alongside a 43.40% incidence of Yersinia pestis isolation from marmot carcasses. Marmot carcasses infected with plague exhibited a significantly higher spleen-somatic index (P < 0.05). Twenty-one Y. pestis-specific phages were isolated, among which one Y. pestis lytic phage (AKS2022HT87GU_phi) was isolated from the bone marrow of a marmot carcass (no. AKS2022HT87) and was found to be symbiotic with Y. pestis. Microscopy revealed the coexistence of lysed and non-lysed colonies of Y. pestis AKS2022HT87. Genome-wide analysis showed that certain strains of the Y. pestis AKS2022HT87 carried phage DNA fragments consistent with phage AKS2022HT87GU_phi. The rare symbiotic relationship between a lytic phage and Y. pestis observed in vitro was highlighted in this study, laying the basis for further exploring the relationship between Y. pestis and its bacteriophages.IMPORTANCEBacteriophages and host bacteria commonly coexist in vivo or in soil environments through complex and interdependent microbial interactions. However, recapitulating this symbiotic state remains challenging in vitro due to limited medium nutrients. In this work, the natural symbiosis between Yersinia pestis and specific phages has been discovered in a Marmota himalayana specimen. Epidemiological analysis presented the characteristics of the Y. pestis and specific phages in the area with a strong plague epidemic. Crucially, comparative genomics has been conducted to analyze the genetic changes in both the Y. pestis and phages over different periods, revealing the dynamic and evolving nature of their symbiosis. These are the critical steps to study the mechanism of the symbiosis.

Differential pathogenicity and lethality of bubonic plague (1720-1945) by sex, age and place.

COVID-19 brought back to the attention of the scientific community that males are more susceptible to infectious diseases. What is clear for other infections-that sex and gender differences influence both risk of infection and mortality-is not yet fully elucidated for plague, particularly bubonic plague, although this knowledge can help find specific defences against a disease for which a vaccine is not yet available. To address this question, we analysed data on plague from hospitals in different parts of the world since the early eighteenth century, which provide demographic information on individual patients, diagnosis and course of the disease in the pre-antibiotic era. Assuming that the two sexes were equally represented, we observe a worldwide prevalence of male cases hospitalized at any age, a result which seems better explained by gender-biased (thus cultural) behaviours than biological sex-related factors. Conversely, case fatality rates differ among countries and geographic macro-areas, while globally, lethality appears slightly prevalent in young females and older adults (regardless of sex). Logistic regression models confirm that the main risk factor for bubonic plague death was the geographical location of the cases and being older than 50 years, whereas sex only showcased a slight trend.

Distinct Mechanisms of Type 3 Secretion System Recognition Control LTB4 Synthesis in Neutrophils versus Macrophages.

Leukotriene B4 (LTB4) is critical for initiating the inflammatory cascade in response to infection. However, Yersinia pestis colonizes the host by inhibiting the timely synthesis of LTB4 and inflammation. Here, we show that the bacterial type 3 secretion system (T3SS) is the primary pathogen associated molecular pattern (PAMP) responsible for LTB4 production by leukocytes in response to Yersinia and Salmonella, but synthesis is inhibited by the Yop effectors during Yersinia interactions. Moreover, we unexpectedly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require two distinct host signaling pathways. We show that the SKAP2/PLC signaling pathway is essential for LTB4 production by neutrophils but not macrophages. Instead, phagocytosis and the NLRP3/CASP1 inflammasome are needed for LTB4 synthesis by macrophages. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered a second unrelated PAMP-mediated signal independently activates the MAP kinase pathway needed for LTB4 synthesis. Together, these data demonstrate significant differences in the signaling pathways required by macrophages and neutrophils to quickly respond to bacterial infections.

Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Recombinant YopE and LcrV vaccine candidates protect mice against plague and yersiniosis.

No licensed vaccine exists for the lethal plague and yersiniosis. Therefore, a combination of recombinant YopE and LcrV antigens of Yersinia pestis was evaluated for its vaccine potential in a mouse model. YopE and LcrV in formulation with alum imparted a robust humoral immune response, with isotyping profiles leaning towards the IgG1 and IgG2b subclasses. It was also observed that a significantly enhanced expression of IFN-γ, TNF-α, IL-6, IL-2, and IL-1ß from the splenic cells of vaccinated mice, as well as YopE and LcrV-explicit IFN-γ eliciting T-cells. The cocktail of YopE + LcrV formulation conferred complete protection against 100 LD50Y. pestis infection, while individually, LcrV and YopE provided 80 % and 60 % protection, respectively. Similarly, the YopE + LcrV vaccinated animal group had significantly lower colony forming unit (CFU) counts in the spleen and blood compared to the groups administered with YopE or LcrV alone when challenged with Yersinia pseudotuberculosis and Yersinia enterocolitica. Histopathologic evidence reinforces these results, indicating the YopE + LcrV formulation provided superior protection against acute lung injury as early as day 3 post-challenge. In conclusion, the alum-adjuvanted YopE + LcrV is a promising vaccine formulation, eliciting a robust antibody response including a milieu of pro-inflammatory cytokines and T-cell effector functions that contribute to the protective immunity against Yersinia infections. YopE and LcrV, conserved across all three human-pathogenic Yersinia species, provide cross-protection. Therefore, our current vaccine (YopE + LcrV) targets all three pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. However, the efficacy should be tested in other higher mammalian models.

Antimicrobial therapies administrated during the Third Plague Pandemic in Europe.

Plague raged in Europe for over 1400 years and was responsible for three major pandemics. Today, plague still poses a serious threat to global public health and surveillance is imperative. Plague is still present in natural reservoirs on several continents, including Africa, Asia and the Americas, and sometimes causes local cases and epidemics. The Third Plague Pandemic caused millions of deaths worldwide, including in Europe. Plague arrived in Europe in the autumn of 1896 mostly through maritime trade routes, where it spread with several epidemic events until 1945, when, in the port city of Taranto, the last known outbreak was recorded. In this paper, we present an overview of the natural history and pathogenicity of Yersinia pestis, the bacterium responsible for plague, its spread from Asia to Europe during the Third Pandemic, and the therapies used to treat and prevent the disease in Europe, with particular focus on the case of Taranto. In Taranto, the Pasteur Institute's antiserum antimicrobial therapy, and vaccination were used to treat and stop the advance of the bacterium, with mixed results.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Phylogenetic Relationships and Evolution of the Genus Eganvirus (186-Type) Yersinia pestis Bacteriophages.

Plague is an endemic infectious disease caused by Yersinia pestis. In this study, we isolated fourteen phages with similar sequence arrangements to phage 186; these phages exhibited different lytic abilities in Enterobacteriaceae strains. To illustrate the phylogenetic relationships and evolutionary relationships between previously designated 186-type phages, we analysed the complete sequences and important genes of the phages, including whole-genome average nucleotide identity (ANI) and collinearity comparison, evolutionary analysis of four conserved structural genes (V, T, R, and Q genes), and analysis of the regulatory genes (cI, apl, and cII) and integrase gene (int). Phylogenetic analysis revealed that thirteen of the newly isolated phages belong to the genus Eganvirus and one belongs to the genus Felsduovirus in the family Peduoviridae, and these Eganvirus phages can be roughly clustered into three subgroups. The topological relationships exhibited by the whole-genome and structural genes seemed similar and stable, while the regulatory genes presented different topological relationships with the structural genes, and these results indicated that there was some homologous recombination in the regulatory genes. These newly isolated 186-type phages were mostly isolated from dogs, suggesting that the resistance of Canidae to Y. pestis infection may be related to the wide distribution of phages with lytic capability.

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.

Dynamics of Changes in the cAMP/cGMP Concentration Ratio in the Thymus and Spleen of Laboratory Mice during Vaccination against Plague and Tularemia against the Background of Immunomodulation.

Vaccine strains Yersinia pestis EV NIIEG at a dose of 103 CFU and Francisella tularensis 15 NIIEG at a dose of 102 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.

Decrypting the multi-genome data for chimeric vaccine designing against the antibiotic resistant Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a gram-negative bacterium that can be fatal if not treated properly. Three types of plague are currently known: bubonic, septicemic, and pneumonic plague, among which the fatality rate of septicemic and pneumonic plague is very high. Bubonic plague can be treated, but only if antibiotics are used at the initial stage of the infection. But unfortunately, Y. pestis has also shown resistance to certain antibiotics such as kanamycin, minocycline, tetracycline, streptomycin, sulfonamides, spectinomycin, and chloramphenicol. Despite tremendous progress in vaccine development against Y. pestis, there is no proper FDA-approved vaccine available to protect people from its infections. Therefore, effective broad-spectrum vaccine development against Y. pestis is indispensable. In this study, vaccinomics-assisted immunoinformatics techniques were used to find possible vaccine candidates by utilizing the core proteome prepared from 58 complete genomes of Y. pestis. Human non-homologous, pathogen-essential, virulent, and extracellular and membrane proteins are potential vaccine targets. Two antigenic proteins were prioritized for the prediction of lead epitopes by utilizing reverse vaccinology approaches. Four vaccine designs were formulated using the selected B- and T-cell epitopes coupled with appropriate linkers and adjuvant sequences capable of inducing potent immune responses. The HLA allele population coverage of the T-cell epitopes selected for vaccine construction was also analyzed. The V2 constructs were top-ranked and selected for further analysis on the basis of immunological, physicochemical, and immune-receptor docking interactions and scores. Docking and molecular dynamic simulations confirmed the stability of construct V2 interactions with the host immune receptors. Immune simulation analysis anticipated the strong immune profile of the prioritized construct. In silico restriction cloning ensured the feasible cloning ability of the V2 construct in the expression system of E. coli strain K12. It is anticipated that the designed vaccine construct may be safe, effective, and able to elicit strong immune responses against Y. pestis infections and may, therefore, merit investigation using in vitro and in vivo assays.

High genetic diversity of the himalayan marmot relative to plague outbreaks in the Qinghai-Tibet Plateau, China.

Plague, as an ancient zoonotic disease caused by Yersinia pestis, has brought great disasters. The natural plague focus of Marmota himalayana in the Qinghai-Tibet Plateau is the largest, which has been constantly active and the leading source of human plague in China for decades. Understanding the population genetics of M. himalayana and relating that information to the biogeographic distribution of Yersinia pestis and plague outbreaks are greatly beneficial for the knowledge of plague spillover and arecrucial for pandemic prevention. In the present research, we assessed the population genetics of M. himalayana. We carried out a comparative study of plague outbreaks and the population genetics of M. himalayana on the Qinghai-Tibet Plateau. We found that M. himalayana populations are divided into two main clusters located in the south and north of the Qinghai-Tibet Plateau. Fourteen DFR genomovars of Y. pestis were found and exhibited a significant region-specific distribution. Additionally, the increased genetic diversity of plague hosts is positively associated with human plague outbreaks. This insight gained can improve our understanding of biodiversity for pathogen spillover and provide municipally directed targets for One Health surveillance development, which will be an informative next step toward increased monitoring of M. himalayana dynamics.

Lyophilization for bacteria preservation: a promising approach for Yersinia pestis strains from an unique collection in Brazil (Fiocruz-CYP).

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

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.

The Natural and Clinical History of Plague: From the Ancient Pandemics to Modern Insights.

The human pathogen Yersinia pestis is responsible for bubonic, septicemic, and pneumonic plague. A deeply comprehensive overview of its historical context, bacteriological characteristics, genomic analysis based on ancient DNA (aDNA) and modern strains, and its impact on historical and actual human populations, is explored. The results from multiple studies have been synthesized to investigate the origins of plague, its transmission, and effects on different populations. Additionally, molecular interactions of Y. pestis, from its evolutionary origins to its adaptation to flea-born transmission, and its impact on human and wild populations are considered. The characteristic combinations of aDNA patterns, which plays a decisive role in the reconstruction and analysis of ancient genomes, are reviewed. Bioinformatics is fundamental in identifying specific Y. pestis lineages, and automated pipelines are among the valuable tools in implementing such studies. Plague, which remains among human history's most lethal infectious diseases, but also other zoonotic diseases, requires the continuous investigation of plague topics. This can be achieved by improving molecular and genetic screening of animal populations, identifying ecological and social determinants of outbreaks, increasing interdisciplinary collaborations among scientists and public healthcare providers, and continued research into the characterization, diagnosis, and treatment of these diseases.

The ecological and etiological investigation of ticks and rodents in China: results from an ongoing surveillance study in Zhejiang Province.

Objectives: This study aimed to analyze the population density of vector ticks and reservoir hosts rodents, and to investigate the relevant pathogen infection in Zhejiang Province, China. Methods: In this surveillance study, the data of ticks density were collected with the tick picking method on animal body surface and the drag-flag method, while the rodent density with the night trapping method. The samples of ticks were examined for the severe fever with thrombocytopenia syndrome virus (SFTSV), and blood serum and organs from rodents were subjected for SFTSV, hantavirus, Leptospira, Orientia tsutsugamushi (O. tsutsugamushi) and Yersinia pestis (Y. pestis) screening in the laboratory. Results: From 2017 to 2022 in Zhejiang Province, 16,230 parasitic ticks were found in 1848 positive animals, with the density of parasitic ticks of 1.29 ticks per host animal, and a total of 5,201 questing ticks were captured from 1,140,910 meters of vegetation distance with the questing tick density of 0.46 ticks/flag·100 m. Haemaphysalis longicornis (H. longicornis) was the major species. A total of 2,187,739 mousetraps were distributed and 12,705 rodents were trapped, with the density of 0.58 per 100 trap-nights. Rattus norvegicus was the major species. For SFTSV screening, two groups nymphal ticks of H. longicornis were tested to be positive. For the rodents samples, the Leptospira had a positive rate of 12.28% (197/1604), the hantavirus was 1.00% (16/1604), and the O. tsutsugamushi was 0.15% (2/1332). No positive results were found with SFTSV and Y. pestis in the rodents samples. Conclusion: Findings from this study indicated that the ticks and rodents were widely distributed in Zhejiang Province. Particularly, the positive detection of SFTSV, Leptospira, hantavirus and O. tsutsugamushi in ticks or rodents from this area suggested that more attention should be paid to the possibilities of relevant vector-borne diseases occurrence.

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism.

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.