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The ongoing coronavirus disease pandemic has fostered major advances in vaccination technologies; however, there are urgent needs of mucosal immune responses and single-dose, non-invasive administration. Here, we develop a SARS-CoV-2 vaccine for single-dose, dry-powder aerosol inhalation that induces potent systemic and mucosal immune responses. Our vaccine encapsulates proteinaceous cholera toxin B subunit-assembled nanoparticles displaying the SARS-CoV-2 RBD antigen (R-CNP) within microcapsules of optimal aerodynamic size, and such unique nano-micro coupled structure supports efficient alveoli delivery, sustained R-CNP release, and antigen presenting cell uptake, which are favorable for invocation of immune responses. Moreover, our vaccine successfully induces robust serological IgG and secretory IgA production, collectively conferring effective protection from SARS-CoV-2 challenge (including pseudovirus and the authentic virus) in mice, hamsters, and non-human primates. Finally, we also demonstrate a “mosaic iteration” of our vaccine that co-displays ancestral and Omicron’s antigens, thus extending the breadth of antibody response against co-circulating strains and transmission of Omicron variant. These findings support our inhalable vaccine as a promising candidate to prevent SARS-CoV-2 infection, disease, and transmission.

Choroidal neovascularization induced by age-related macular degeneration and retinal neovascularization induced by diabetic retinopathy-two leading causes of blindness-are often treated using antibodies targeting vascular endothelial growth factor (VEGF). Here we report a strong association between inflammation and high VEGF expression in aqueous humour samples from patients with choroidal or retinal neovascularization, and show that intravitreally injected exosomes derived from regulatory T cells and conjugated with an anti-VEGF antibody via a peptide linker that is cleavable by matrix metalloproteinases markedly suppressed ocular neovascularization in mouse and non-human primate models of choroidal neovascularization. The engineered exosomes, which selectively accumulate in the neovascularization lesions, could be adapted for other combination therapies of therapeutic antibodies and anti-inflammatory cargo.

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is generally known to be the most poisonous of all biological toxins. In this study, we evaluate the protection conferred by intratracheal (i.t.) inoculation immunization with recombinant Hc subunit (AHc) vaccines against aerosolized BoNT/A intoxication. Three AHc vaccine formulations, i.e., conventional liquid, dry powder produced by spray freeze drying, and AHc dry powder reconstituted in water are prepared, and mice are immunized via i.t. inoculation or subcutaneous (s.c.) injection. Compared with s.c.-AHc-immunized mice, i.t.-AHc-immunized mice exhibit a slightly stronger protection against a challenge with 30,000× LD50 aerosolized BoNT/A. Of note, only i.t.-AHc induces a significantly higher level of toxin-neutralizing mucosal secretory IgA (SIgA) production in the bronchoalveolar lavage of mice. In conclusion, our study demonstrates that the immune protection conferred by the three formulations of AHc is comparable, while i.t. immunization of AHc is superior to s.c. immunization against aerosolized BoNT/A intoxication.

Botulinum neurotoxin type A (BoNT/A) is traditional medicine and well known for its therapeutic use as an anesthetic and in cosmetic applications that work through the inhibition of acetylcholine exocytosis in neuronal cells. BoNT/A also has the potential to function as a biological weapon due to its high mortality rate and ease of dispersal. Emerging evidence suggests that BoNT/A exhibits biological effects on nonneuronal cells. In cytology experiments, BoNT/A induces global gene expression alterations. However, pulmonary effects from exposure to aerosolized BoNT/A have not been evaluated. This study investigated the global transcriptional profile of lung tissues after botulism inhalation. A mice model of inhaled botulism was established using intratracheal exposure to aerosolized BoNT/A and described through histological examination and flow cytometry. Transcriptomic analysis revealed that genes related to acute inflammatory responses were upregulated at 12-h postexposure. Increased expression of multiple anti-inflammatory marker genes and decreased expression of pro-inflammatory marker genes were observed at 48- to 72-h postexposure, underscoring a transcriptional shift toward a pro-reparative phenotype. Histological examination and cell proportions analysis mirrored these expression patterns. Accordingly, the orchestration of a quick phenotype transition prompted by BoNT/A may have the potential for promoting the resolution of the inflammatory lung. To our knowledge, this study represents the first research to investigate the pulmonary transcriptional responses of aerosolized BoNT/A exposure; the results may provide new insights in elucidating the molecular mechanism for pulmonary inhaled botulism and highlight the potential therapeutic application of BoNT/A in mitigating inflammatory conditions.

In this study, a ricin toxin (RT)-induced pulmonary intoxication model was established in mice by intratracheal-delivered RT at a dose of 2× LD50. Based on this model, the histopathological evaluation of the lungs at 24 h and 48 h post-exposure was executed, and the genome-wide transcriptome of the lungs at 4, 12, 24 and 48 h post-exposure was analyzed. Histopathological analysis showed that a large number of neutrophils infiltrated the lungs at 24 h post-exposure, and slight pulmonary edema and perivascular-peribronchiolar edema appeared in the lungs at 48 h. Transcriptome analysis showed that the expression of a large number of genes related to leukocyte migration and chemotaxis consistently increased in the lungs upon exposure to RT, and the expression of genes that participate in acute phase immune and/or inflammatory response, also increased within 12 h of exposure to RT, which could be confirmed by the measurement of cytokines, such as IL-1β, TNF-α and IL-6, in bronchoalveolar lavage fluid. While the expression of genes related to cellular components of the extracellular matrix and cell membrane integrity consistently decreased in the lungs, and the expression of genes related to antioxidant activity also decreased within the first 12 h. There are 17 differentially expressed genes (DEGs) that participate in ribotoxic stress response, endoplasmic reticulum stress response or immune response in the lungs at 4 h post-exposure. The expression of these DEGs was upregulated, and the number of these DEGs accounted for about 59 % of all DEGs at 4 h. The 17 DEGs may play an important role in the occurrence and development of inflammation. Notably, Atf3, Egr1, Gdf15 and Osm, which are poorly studied, may be important targets for the subsequent research of RT-induced pulmonary intoxication. This study provides new information and insights for RT-induced pulmonary intoxication, and it can provide a reference for the subsequent study of the toxicological mechanism and therapeutic approaches for RT-induced pulmonary intoxication.

Background: Docosahexaenoic acid-acylated phloridzin (PZ-DHA), a novel polyphenol fatty acid ester derivative, is synthesized through an acylation reaction of phloridzin (PZ) and docosahexaenoic acid (DHA). PZ-DHA is more stable than DHA and exhibits higher cellular uptake and bioavailability than PZ. Objective: The study aims to investigate the effects of PZ-DHA on insulin resistance in the skeletal muscle and the related mechanisms; we used palmitic acid (PA)-treated C2C12 myotubes as an insulin resistance model. Results: We found that PZ-DHA increased the activity of AMP-activated protein kinase (AMPK) and improved glucose uptake and mitochondrial function in an AMPK-dependent manner in untreated C2C12 myotubes. PZ-DHA treatment of the myotubes reversed PA-induced insulin resistance; this was indicated by increases in glucose uptake and the expression of membrane glucose transporter 4 (Glut4) and phosphorylated Akt. Moreover, PZ-DHA treatment reversed PA-induced inflammation and oxidative stress. These effects of PZ-DHA were mediated by AMPK. Furthermore, the increase in AMPK activity, improvement in insulin resistance, and decrease in inflammatory and oxidative responses after PZ-DHA treatment diminished upon co-treatment with a liver kinase B1 (LKB1) inhibitor, suggesting that PZ-DHA improved AMPK activity by regulating its upstream kinase, LKB1. Conclusion: The effects of PZ-DHA on insulin resistance in C2C12 myotubes may be mediated by the LKB1- AMPK signaling pathway. Hence, PZ-DHA is a promising therapeutic agent for insulin resistance in type 2 diabetes.

The high level noise caused by intense acoustic weapons and blasting is a common source of acute acoustic trauma faced by some special environmental personnel. Studies have shown that high level noise can cause auditory and non-auditory effects. However, there are few reports on the biological effects, especially the non-auditory effects of acute high level noise exposure in simulated special working environments, and the great differences between experimental animals and human beings make it difficult to extrapolate from research conclusions. In this study, macaque monkeys were used to detect the effects of acute high level noise exposure on hearing, cognition, and cardiovascular function. Auditory brainstem response, auditory P300, and electrocardiogram (ECG) of macaque monkeys were measured. Results showed that acute high level noise exposure caused permanent hearing threshold shifts, partial hearing loss which couldn’t recover to normal levels in the detection period, pathological changes in T wave and QRS complexes, and large fluctuations in cognitive ability after exposure, which finally recovered to normal. These alterations may be a combination of effects caused by stress-induced neuroendocrine dysfunction and mechanical damage of auditory organs. To elaborate the exact mechanism, further studies are still needed. Meanwhile, positive measures should be taken to reduce the incidence of acute high level noise injury.

Choroidal neovascularization induced by age-related macular degeneration and retinal neovascularization induced by diabetic retinopathy-two leading causes of blindness-are often treated using antibodies targeting vascular endothelial growth factor (VEGF). Here we report a strong association between inflammation and high VEGF expression in aqueous humour samples from patients with choroidal or retinal neovascularization, and show that intravitreally injected exosomes derived from regulatory T cells and conjugated with an anti-VEGF antibody via a peptide linker that is cleavable by matrix metalloproteinases markedly suppressed ocular neovascularization in mouse and non-human primate models of choroidal neovascularization. The engineered exosomes, which selectively accumulate in the neovascularization lesions, could be adapted for other combination therapies of therapeutic antibodies and anti-inflammatory cargo.

Background As a natural host of simian immunodeficiency virus (SIV), African green monkeys (AGM) do not develop AIDS. AGM has recently been shown to rapidly activate and maintain evolutionarily conserved regenerative wound healing mechanisms in mucosal tissue in case of SIV acute infection, suggesting a role of mucosal integrity in AIDS resistance.However, little is known about the underlying mechanisms, especially of which the gut microbiome as well as the metabolites participate in. This study aims to characterize the profiles of gut microbiome and metabolites of AGM with chronic SIV infection (AGM_P), AGM without SIV infection (AGM_N), Cynomolgus macaque (CM), Rhesus macaque (RM). Results Compared with CM and RM, significant decreases in the abundances of Streptococcus, Alistipes, Treponema, Bacteoides, Methanobrevibacter, Methanobrevibacter (P Clostridium, Eubacterium, Blautia, Roseburia, Faecalibacterium, Dialister (P Streptococcus and Roseburia were found in AGM_P. Quantitative RT-PCR analysis validated the increased abundances of butyrate-producing Faecalibacterium prausnitzii and Eubacteria in AGM_N (P

Recent years have seen enormous advances in nanovaccines for both prophylactic and therapeutic applications, but most of these technologies employ chemical or hybrid semi-biosynthetic production methods. Thus, production of nanovaccines has to date failed to exploit biology-only processes like complex sequential post-translational biochemical modifications and scalability, limiting the realization of the initial promise for offering major performance advantages and improved therapeutic outcomes over conventional vaccines. A Nano-B5 platform for in vivo production of fully protein-based, self-assembling, stable nanovaccines bearing diverse antigens including peptides and polysaccharides is presented here. Combined with the self-assembly capacities of pentamer domains from the bacterial AB5 toxin and unnatural trimer peptides, diverse nanovaccine structures can be produced in common Escherichia coli strains and in attenuated pathogenic strains. Notably, the chassis of these nanovaccines functions as an immunostimulant. After showing excellent lymph node targeting and immunoresponse elicitation and safety performance in both mouse and monkey models, the strong prophylactic effects of these nanovaccines against infection, as well as their efficient therapeutic effects against tumors are further demonstrated. Thus, the Nano-B5 platform can efficiently combine diverse modular components and antigen cargos to efficiently generate a potentially very large diversity of nanovaccine structures using many bacterial species.

Persistent infection with human papillomavirus (HPV) is a key factor in the development of precancerous lesions and invasive cervical cancer. Prophylactic vaccines to immunize against HPV are an effective approach to reducing HPV related disease burden. In this study, we investigated the immunogenicity and dosage effect of a trivalent HPV 16/18/58 vaccine (3vHPV) produced in Escherichia coli (E.coli), with Gardasil quadrivalent vaccine (4vHPV, Merck & Co.) as a positive control. Sera collected from rhesus macaques vaccinated with three dosage formulations of 3vHPV (termed low-, mid-, and high-dosage formulations, respectively), and the 4vHPV vaccine were analyzed by both Pseudovirus-Based Neutralization Assay (PBNA) and Enzyme-Linked Immunosorbent Assay (ELISA). Strong immune responses against HPV 16/18/58 were successfully elicited, and dosage-dependence was observed, with likely occurrence of immune interference between different L1-VLP antigens. HPV 16/18 specific neutralizing antibody (nAb) and total immunoglobulin G (IgG) antibody responses in rhesus macaques receiving 3vHPV at the three dosages tested were generally non-inferior to those observed in rhesus macaques receiving 4vHPV throughout the study period. Particularly, HPV 18 nAb titers induced by the mid-dosage formulation that contained the same amounts of HPV 16/18 L1-VLPs as Gardasil 4vHPV were between 7.3 to 12.7-fold higher compared to the positive control arm from weeks 24â64. The durability of antibody responses specific to HPV 16/18 elicited by 3vHPV vaccines was also shown to be non-inferior to that associated with Gardasil 4vHPV. Keywords: Human papillomavirus, HPV 16/18/58, GMTs, Trivalent, Immunogenicity

The purpose of this study was to characterize mechanisms of plasmid-mediated antimicrobial resistance in Shigella boydii. S. boydii strain 2246 with resistance to ciprofloxacin, ceftriaxone and azithromycin was isolated from a human case of watery diarrhea in a Chinese public hospital. Resistance in strain 2246 to ceftriaxone and azithromycin was attributable to the presence of blaCTX-M-14, and erm(B) and mph(A), respectively, which were co-located on a multidrug-resistant (MDR) plasmid p2246-CTXM. p2246-CTXM represented a novel IncFII-type MDR plasmid with a very complex chimera structure. Its master backbone was genetically closely related to the R100 plasmid, but p2246-CTXM had evolved to integrate additional R100-unrelated backbone regions as well as massive exogenous mobile elements that carried multiple resistance determinants. In p2246-CTXM, erm(B) together with its leading peptide gene erm(C), mph(A) together with its regulatory genes mrx and mphR(A), and blaCTX-M-14 were captured by three different mobile elements Tn6295, the IS26-mph(A)-mrx-mphR(A)-IS6100 unit, and a truncated ISEcp1-blaCTX-M-14-IS903D-iroN transposition unit, respectively, all of which were harbored in a large Tn3-family transposon Tn6285. p2246-CTXM still carried additional resistance determinants mer (mercury resistance), aacA4 (aminoglycoside resistance), cmlA1 (chloramphenicol resistance), and qacED1 (quaternary ammonium compound resistance). This is the first report of identifying a clinical S. boydii strain simultaneously resistant to ciprofloxacin, ceftriaxone, and azithromycin, and determining the complete sequence of a resistance plasmid from S. boydii.

Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.

The subunit vaccine SV1 (20 μg F1 + 10 μg rV270) has been identified as the optimal formulation in mice, which provided a good protection against plague in mice, guinea pigs and rabbits. To compare SV1 and SV2 (200 μg F1 + 100 μg rV270) with live attenuated vaccine EV76, antibody responses, protective efficacy, cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-10 and IL-12) production, CD4/CD8 ratio and CD69+ T-cell activation marker were determined in sera of the immunized Chinese-origin rhesus macaques, Macaca mulatta. The immunized animals with SV1 or SV2 developed higher anti-rV270 IgG titre, while those immunized with EV76 elicited a negligible IgG to V antigen, indicating that subunit vaccine (SV) had an advantage over EV76 in terms of the indispensable role of anti-V antibody against Yersinia pestis. There was no significant antibody titre difference between SV1 and SV2, suggesting that the immune response may have been saturated at the dose level of SV1. There were no statistical changes for CD4/CD8 ratios, IL-4 and CD69 levels between the three-vaccine immunized groups. However, a significant higher level of IL-12 was observed in the EV76 immunized animals, indicating that EV76 had an advantage over SV in respect of cellular immunity. Complete protection was observed for the immunized animals with SV and EV76, revealing that SV has a similar protective efficacy with EV76 against 6 × 106 CFU of Y. pestis challenge by subcutaneous route in Chinese-origin rhesus macaques.

In this study, a new subunit vaccine that comprised native F1 and recombinant rV270 was evaluated for protective efficacy using mouse, guinea pig and rabbit models in comparison with the live attenuated vaccine EV76. Complete protection against challenging with 10 6 colony-forming units (CFU) of virulent Yersinia pestis strain 141 was observed for mice immunized with the subunit vaccines and EV76 vaccine. In contrast, the subunit vaccine recipes VII (F1-20 μg + rV270-10 μg) and IX (F1-40 μg + rV270-20 μg) and EV76 vaccine provided 86%, 79% and 93% protection against the same level of challenge in guinea pigs and 100%, 83% and 100% protection in rabbits, respectively. The immunized mice with the vaccines had significantly higher IgG titres than the guinea pigs and rabbits, and the immunized guinea pigs developed significantly higher IgG titres than the rabbits, but the anti-F1 response in guinea pigs was more variable than in the mice and rabbits, indicating that guinea pig is not an ideal model for evaluating protective efficacy of plague subunit vaccine, instead the rabbits could be used as an alternative model. All the immunized animals with EV76 developed a negligible IgG titre to rV270 antigen. Furthermore, analysis of IgG subclasses in the immunized animals showed a strong response for IgG1, whereas those receiving EV76 immunization demonstrated predominant production of IgG1 and IgG2a isotypes. The subunit vaccine and EV76 vaccine are able to provide protection for animals against Y. pestis challenge, but the subunit vaccines have obvious advantages over EV76 in terms of safety of use.

Despite the importance of pneumonic plague caused by Yersinia pestis, a few is known about the interaction between Y. pestis and its host at the molecular level during the pneumonic plague development. In this study, we employed an intranasally challenged plague model in mice for investigating the kinetics of the disease progression by transcriptional profiling of Y. pestis and mice using qRT-PCR and microarray, respectively. The increasing transcription of important virulence genes of Y. pestis and of mice genes involving in immune and inflammatory defensive responses, and responses to stimuli, presents an overview of interaction between Y. pestis and mice during development of pneumonic plague. The early and persisting up-regulation of caf 1, psa A and lcr V in vivo indicated their role in resisting the host innate immune responses. The up-regulation of fur, ybt A and hms H in vivo reflected the ability of Y. pestis for acquiring iron. The transcription regulators, including pho P, oxy R and omp R, were up-regulated during plague development, suggesting their roles in interaction between Y. pestis and mice. Many genes encoding cytokines, such as IL2, IL-1B, CXCL2, CXCL5, CCL20, CD14 and TNFRSF13B, were up-regulated during the infection, confirming the report that they are important mediators to activate host responses to invading pathogens. The up-regulation of some genes encoding important virulent factors of Y. pestis and expression alterations of some genes encoding cytokines in the host reflect the interaction between the pathogen and the host, which will help us better understand plague pathogenesis. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The catalase or catalase-peroxidase activity commonly exists in many pathogens and plays an important role in resisting the oxidative burst of phagocytes helping the pathogen persistently colonize in the host. Yersinia pestis is a facultative pathogen and the causative agent of plague. KatY has been identified as a thermosensing antigen with modest catalase activity in this pathogen. Here Y. pestis KatA and KatY were experimentally confirmed as a monofunctional catalase and bifunctional catalase-peroxidase, respectively. Their expression induced by H2O2 was proven to be mediated by the oxidative regulator, OxyR. Expression of KatA changed with growth phases and was crucial to its traditional physiological role in protecting Y. pestis cells against toxicity of exogenous H2O2. KatY was regulated by temperature and H2O2, two major elements of phagolysosomal microenvironments. Consistent with the above results, gene expression of katY increased significantly during intracellular growth of Y. pestis compared with that in vitro growth. However, a DeltakatY mutant was fully virulent to mice, suggesting that KatY is not required for Y. pestis virulence.

Raoultella ornithinolytica YNKP001 and Leclercia adecarboxylata P10164, which harbor conjugative plasmids pYNKP001-NDM and pP10164-NDM, respectively, were isolated from two different Chinese patients, and their complete nucleotide sequences were determined. Production of NDM-1 enzyme by these plasmids accounts for the carbapenem resistance of these two strains. This is the first report of bla NDM in L. adecarboxylata and third report of this gene in R. ornithinolytica. pYNKP001-NDM is very similar to the IncN2 NDM-1-encoding plasmids pTR3, pNDM-ECS01, and p271A, whereas pP10164-NDM is similar to the IncFIIY bla NDM-1-carrying plasmid pKOX_NDM1. The bla NDM-1 genes of pYNKP001-NDM and pP10164-NDM are embedded in Tn125-like elements, which represent two distinct truncated versions of the NDM-1-encoding Tn125 prototype observed in pNDM-BJ01. Flanking of these two Tn125-like elements by miniature inverted repeat element (MITE) or its remnant indicates that MITE facilitates transposition and mobilization of bla NDM-1 gene contexts.

Objectives The objective of this study was to characterize the molecular mechanism of coproduction of KPC-2 and NDM-1 in Citrobacter freundii. Methods C. freundii strain 112298 was isolated from a human case of septic shock in a Chinese teaching hospital. The major carbapenemase and ESBL genes were detected by PCR. The MIC values were determined by using VITEK 2 and antimicrobial susceptibility was judged by CLSI standards. The resistance plasmid was transferred into Escherichia coli by electroporation, followed by plasmid DNA isolation from the electroporant, and then fully sequenced and compared with closely related plasmids. Results Strain 112298 produces KPC-2 and NDM-1, encoded by the novel non-typeable plasmid p112298-KPC and an IncX3-type plasmid p112298-NDM, respectively. In p112298-KPC, a Tn1722-based blaKPC-2-carrying transposon is associated with several additional resistance modules, constituting a single MDR region. Assembly of these resistance modules is likely mediated by homologous recombination between five copies of IS26 elements at different sites within the MDR region. p112298-NDM is a very close relation of pNDM-HN380. blaNDM-1 in p112298-NDM is carried by a Tn125 variant, which differs from the prototype Tn125 as observed in pNDM-BJ01 by disruption of an upstream copy of ISAba125 by IS5 and absence of a downstream copy of ISAba125. Conclusions Production of KPC-2 and NDM-1 by p112298-KPC and p112298-NDM, respectively, makes C. freundii 112298 highly resistant to carbapenems and, moreover, these two plasmids still harbour genes for resistance to cephalosporins, chloramphenicol, chromate, fosfomycin, quaternary ammonium, rifampicin and sulphonamides.

Vibrio parahaemolyticus, a leading cause of seafood-associated diarrhea and gastroenteritis, harbors three major virulence gene loci T3SS1, Vp-PAI (T3SS1+tdh2) and T6SS2. As showing in this study, the nucleoid-associated DNA-binding regulator H-NS binds to multiple promoter-proximal regions in each of the above three loci to repress their transcription, and moreover H-NS inhibits the cytotoxicitiy, enterotoxicity, hemolytic activity, and mouse lethality of V. parahaemolyticus. H-NS appears to act as a major repressor of the virulence of this pathogen. Date presented here would promote us to gain a deeper understanding of H-NS-mediated silencing of horizontally acquired virulence loci in V. parahaemolyticus.

Our previous study has demonstrated that Yersinia pestis Microtus 201 is a low virulent strain to the Chinese-origin rhesus macaques, Macaca mulatta, and can protect it against high dose of virulent Y. pestis challenge by subcutaneous route. To investigate whether the Y. pestis Microtus 201 can be used as a live attenuated vaccine candidate, in this study its intravenous virulence was determined and compared with the live attenuated vaccine strain EV in the Chinese-origin rhesus macaque model. The results showed that the Chinese-origin rhesus macaques can survive intravenous infection with approximately 10(9) CFU of the Y. pestis Microtus 201, but all the animals succumbed to 10(10) CFU of intravenous infection. By contrast, all the animals survive intravenous infection with 10(10) CFU of the vaccine EV. Post-mortem examination showed multiple areas of severe abscess in the lungs of the dead animals infected with 10(10) CFU of the Y. pestis Microtus 201, whereas histopathology observation, microbiological examination and immunohistochemistry staining showed that the Y. pestis Microtus 201 also invaded hearts, livers, spleens, kidneys and lymph nodes and caused different degrees of pathological changes in these organs. These results indicated that the Y. pestis Microtus 201 is indeed low virulent to monkeys, but it is more virulent than the vaccine EV when administered by intravenous route. The Y. pestis Microtus 201 mainly attack the lungs when administered by intravenous infection, which may be the leading cause of animal death.

Yersinia pestis biovar Microtus is considered to be a virulent to larger mammals, including guinea pigs, rabbits and humans. It may be used as live attenuated plague vaccine candidates in terms of its low virulence. However, the Microtus strain’s protection against plague has yet to be demonstrated in larger mammals. In this study, we evaluated the protective efficacy of the Microtus strain 201 as a live attenuated plague vaccine candidate. Our results show that this strain is highly attenuated by subcutaneous route, elicits an F1-specific antibody titer similar to the EV and provides a protective efficacy similar to the EV against bubonic plague in Chinese-origin rhesus macaques. The Microtus strain 201 could induce elevated secretion of both Th1-associated cytokines (IFN-γ, IL-2 and TNF-α) and Th2-associated cytokines (IL-4, IL-5, and IL-6), as well as chemokines MCP-1 and IL-8. However, the protected animals developed skin ulcer at challenge site with different severity in most of the immunized and some of the EV-immunized monkeys. Generally, the Microtus strain 201 represented a good plague vaccine candidate based on its ability to generate strong humoral and cell-mediated immune responses as well as its good protection against high dose of subcutaneous virulent Y. pestis challenge.

Yersinia pestis 201 contains 4 plasmids pPCP1, pMT1, pCD1 and pCRY, but little is known about the effects of these plasmids on the dissemination of Y. pestis. We developed a plasmid-based luxCDABE bioreporter in Y. pestis 201, Y. pestis 201-pCD1(+), Y. pestis 201-pMT1(+), Y. pestis 201-pPCP1(+), Y. pestis 201-pCRY(+), Y. pestis 201-p(-) and Yersinia pseudotuberculosis Pa36060 strains, and investigated their dissemination by bioluminescence imaging during primary septicemic plague in a mouse model. These strains mainly colonized the livers and spleens shortly after intravenous inoculation. Y. pestis 201-pMT1(+) appeared to have a stronger ability to survive in the livers, spleens and blood, and to be more virulent than other plasmid-deficient strains. Y. pestis 201-pPCP1(+) appeared to have a stronger ability to colonize lungs than other plasmid-deficient strains. Pa36060 has the strongest ability to colonize intestines and lungs. Y. pestis 201 has the strongest ability to survive in blood, and the strongest virulence. These results indicated that the plasmid pMT1 was an important determinant in the colonization of livers, spleens and blood, whereas the plasmid pPCP1 appeared to correlate with the colonization in lungs. The resistance to killing in mouse blood seemed to be the critical factor causing animal death.

Many Brucella species are isolated from nonpreferred hosts, and these bacteria may show genetic differences from isolates from the preferred hosts. Here, we report the draft genome sequence of Brucella abortus BCB027, a novel strain isolated from a domestic deer.

Brucellosis is an important zoonotic disease of almost worldwide distribution. One significant immune phenomenon of this disease is the ability of the pathogen to hide and survive in the host, establishing long lasting chronic infections. Brucella was found to have the ability to actively modulate the host immune response in order to establish chronic infections, but the mechanism by which the pathogen achieves this remains largely unknown. In our screening for protective antigens of Brucella abortus, 3 proteins (BAB1_0597, BAB1_0917, and BAB2_0431) were found to induce significantly higher levels of gamma interferon (IFNγ) in splenocytes of PBS immunized mice than those immunized with S19. This finding strongly implied that these three proteins inhibit the production of IFNγ. Previous studies have shown that LPS, PrpA, and Btp1/TcpB are three important immunomodulatory molecules with the capacity to interfere with host immune response. They have been shown to have the ability to inhibit the secretion of IFNγ, or to increase the production of IL-10. Due to the role of these proteins in virulence and immunomodulation, they likely offer significant potential as live, attenuated Brucella vaccine candidates. Understanding the mechanisms by which these proteins modulate the host immune responses will deepen our knowledge of Brucella virulence and provide important information on the development of new vaccines against Brucellosis.

Brucellosis is an important zoonotic disease caused primarily by the bacterial pathogens Brucella melitensis and B. abortus. The pathogens cause debilitating febrile illness that can progress into a long-lasting disease with severe complications in humans. Understanding the mechanisms by which the host immune system responds to the infection will provide important information on the pathogenesis and development of differential diagnostic assays. In this study, a protein microarray was used to evaluate the antibody responses of brucellosis patients at different infection stages. A total of 107 outer membrane proteins, surface-exposed or secreted proteins, and known or putative virulence-associated proteins of B. melitensis were successfully expressed in Escherichia coli and used to fabricate the protein microarray. Then, 99 serum samples from acute, chronic, primary infection, or relapse brucellosis patients were probed with the protein microarray. Antibodies to 66 of the proteins were detected at least in one serum sample. Among the antigens, the combination of BMEII0318, BMEII0513, BMEI0748, and BMEII1116 could be used as serodiagnostic antigens for brucellosis. Patients at different infection stages show distinct antibody profiles. The numbers of antibodies in the relapse patients were superior to those in the primary infection patients, and the response magnitude of antibodies in the chronic infection patients was higher than those in the acute brucellosis patients. The sustained and differential antibody profiles of patients at different infection stages have implications for the development of new serological methods for the accurate diagnosis of human brucellosis, and contribute to a more detailed understanding of the pathogenesis of chronic brucellosis.

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 µg F1 and 10 µg rV270) and SV2 (200 µg F1 and 100 µg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×10(6) CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague.

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.

Objective LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co2+ affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. Results Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Background Yersinia pestis is the causative agent of plague, which is transmitted primarily between fleas and mammals and is spread to humans through the bite of an infected flea or contact with afflicted animals. Hfq is proposed to be a global post-transcriptional regulator that acts by mediating interactions between many regulatory small RNAs (sRNAs) and their mRNA targets. Sequence comparisons revealed that Y. pestis appears to produce a functional homologue of E. coli Hfq. Methodology and Principal Findings Phenotype comparisons using in vitro assays demonstrated that Y. pestis Hfq was involved in resistance to H2O2, heat and polymyxin B and contributed to growth under nutrient-limiting conditions. The role of Hfq in Y. pestis virulence was also assessed using macrophage and mouse infection models, and the gene expression affected by Hfq was determined using microarray-based transcriptome and real time PCR analysis. The macrophage infection assay showed that the Y. pestis hfq deletion strain did not have any significant difference in its ability to associate with J774A.1 macrophage cells. However, hfq deletion appeared to significantly impair the ability of Y. pestis to resist phagocytosis and survive within macrophages at the initial stage of infection. Furthermore, the hfq deletion strain was highly attenuated in mice after subcutaneous or intravenous injection. Transcriptome analysis supported the results concerning the attenuated phenotype of the hfq mutant and showed that the deletion of the hfq gene resulted in significant alterations in mRNA abundance of 243 genes in more than 13 functional classes, about 23% of which are known or hypothesized to be involved in stress resistance and virulence. Conclusions and Significance Our results indicate that Hfq is a key regulator involved in Y. pestis stress resistance, intracellular survival and pathogenesis. It appears that Hfq acts by controlling the expression of many virulence- and stress-associated genes, probably in conjunction with small noncoding RNAs.

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH) 3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10 4 CFU of Y. pestis virulent strain 141.

Brucella canis is considered a rare cause of human brucellosis because of difficulties in presumptive diagnosis and underestimation of the incidence. Here, we report the draft genome sequence of a Brucella canis isolate, BCB018, isolated from a human patient, providing precious resources for comparative genomics analysis of Brucella field strains.

Live attenuated vaccines play essential roles in the prevention of brucellosis. Here, we report the draft genome sequences of three vaccine strains, Brucella melitensis M5-10, B. suis S2-30, and B. abortus 104M. Primary genome sequence analysis identified mutations, deletions, and insertions which have implications for attenuation and signatures for differential diagnosis.