The ΔfbpAΔsapM candidate vaccine derived from Mycobacterium tuberculosis H37Rv is markedly immunogenic in macrophages and induces robust immunity to tuberculosis in mice.

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.

Characterization of two affinity matured Anti-Yersinia pestis F1 human antibodies with medical countermeasure potential.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 µg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

Ag85B with c-di-AMP as mucosal adjuvant showed immunotherapeutic effects on persistent Mycobacterium tuberculosis infection in mice.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality by a single infectious agent in the world. M. tuberculosis infection could also result in clinical chronic infection, known as latent TB infection (LTBI). Compared to the current limited treatment, several subunit vaccines showed immunotherapeutic effects and were included in clinical trials. In this study, a subunit vaccine of Ag85B with a novel mucosal adjuvant c-di-AMP (Ag85B:c-di-AMP) was delivered intranasally to a persistent M. tuberculosis H37Ra infection mouse model, which also presented the asymptomatic characteristics of LTBI. Compared with Ag85B immunization, Ag85B:c-di-AMP vaccination induced stronger humoral immune responses, significantly higher CD4+ T cells recruitment, enhanced Th1/Th2/Th17 profile response in the lung, decreased pathological lesions of the lung, and reduced M. tuberculosis load in mice. Taken together, Ag85B:c-di-AMP mucosal route immunization provided an immunotherapeutic effect on persistent M. tuberculosis H37Ra infection, and c-di-AMP, as a promising potential mucosal adjuvant, could be further used in therapeutic or prophylactic vaccine strategies for persistent M. tuberculosis infection as well as LTBI.

An integrated comparative genomics, subtractive proteomics and immunoinformatics framework for the rational design of a Pan-Salmonella multi-epitope vaccine.

Salmonella infections pose a significant global public health concern due to the substantial expenses associated with monitoring, preventing, and treating the infection. In this study, we explored the core proteome of Salmonella to design a multi-epitope vaccine through Subtractive Proteomics and immunoinformatics approaches. A total of 2395 core proteins were curated from 30 different isolates of Salmonella (strain NZ CP014051 was taken as reference). Utilizing the subtractive proteomics approach on the Salmonella core proteome, Curlin major subunit A (CsgA) was selected as the vaccine candidate. csgA is a conserved gene that is related to biofilm formation. Immunodominant B and T cell epitopes from CsgA were predicted using numerous immunoinformatics tools. T lymphocyte epitopes had adequate population coverage and their corresponding MHC alleles showed significant binding scores after peptide-protein based molecular docking. Afterward, a multi-epitope vaccine was constructed with peptide linkers and Human Beta Defensin-2 (as an adjuvant). The vaccine could be highly antigenic, non-toxic, non-allergic, and have suitable physicochemical properties. Additionally, Molecular Dynamics Simulation and Immune Simulation demonstrated that the vaccine can bind with Toll Like Receptor 4 and elicit a robust immune response. Using in vitro, in vivo, and clinical trials, our findings could yield a Pan-Salmonella vaccine that might provide protection against various Salmonella species.

Immunogenic profiling of Mycobacterium tuberculosis Rv1513 reveals its ability to switch on Th1 based immunity.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.

Antibody responses to immunoevasion proteins BBK32 and OspE constitute part of the serological footprint in neuroborreliosis but are insufficient to prevent the disease.

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is the most common tickborne disease. Its neuronal form, neuroborreliosis, comprises 3 to 38% of borreliosis cases in Europe. Borrelia outer surface proteins and virulence factors, OspE and BBK32, have been previously reported to help cause infection by promoting attachment to human host epithelial cells and evading complement attack. We assessed the serological responses to BBK32 and OspE in 19 individuals diagnosed with neuroborreliosis to see whether antibodies that could both target the bacteria and neutralize the virulence mechanisms on the microbial surface emerge. Results evaluate levels of total protein, IgG and the chemokine CXCL13, a determinant for B-cell recruitment during neuroinflammation, in patients' cerebrospinal fluid samples. Antibody levels against BBK32 and OspE correlated with those against VlsE, a well-characterized diagnostic serological marker of the disease. A dual serological profile of the patients was observed. K-means clustering split the cohort into two discrete groups presenting distinct serological and CNS responses. One group contained young patients with low levels of anti-BBK32 and OspE antibodies. The other group showed stronger responses, possibly following prolonged infections or reinfections. Additionally, we assessed anti-ganglioside antibodies that could cause autoimmunity or complement dysregulation but observed that they did not correlate with neuroborreliosis in our patient cohort. The dual nature of antibody responses against the virulence factors BBK32 and OspE in neuroborreliosis patients may suggest the necessity of repeated exposures for efficient immune responses. Better protection could be achieved if the virulence factors were formulated into vaccines.

[Development of an ELISA method for detection of antibodies against Mycoplasma hyopneumoniae based on Mhp336 protein].

This study aims to establish an ELISA method with high specificity for the detection of antibodies against Mycoplasma hyopneumoniae. Firstly, we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen. Then, we optimized the ELISA parameters, including antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, colorimetric reaction time, and cut-off value. Afterwards, reproducibility experiments were conducted, and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined. Finally, 226 porcine serum samples were detected using the method established in this study, a commercial ELISA kit for M. hyopneumoniae antibody detection, and a convalescent serum ELISA kit for M. hyopneumoniae antibody detection. The detection results of the three methods were compared to evaluate the sensitivity and specificity of the ELISA method established in this study. For this method, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, and colorimetric reaction time were 0.05 µg/mL, PBS containing 2.5% skim milk, 1 h, 1:500, 0.5 h, 1:10 000, 1 h, and 5 min, respectively. Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332. The coefficients of variation of both intra-batch and inter-batch kits were below 7%. The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M. hyopneumoniae antibody detection in terms of sensitivity and specificity. We successfully established an ELISA method for detecting the antibodies against M. hyopneumoniae based on Mhp336 protein. This method demonstrated high sensitivity and specificity, serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.

Genetic characterization and estimated 4CMenB vaccine strain coverage of 284 Neisseria meningitidis isolates causing invasive meningococcal disease in Argentina in 2010-2014.

Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.

The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.

Evaluation of vaccine candidates against Rhodococcus equi in BALB/c mice infection model: cellular and humoral immune responses.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that mainly causes fatal lung and extrapulmonary abscesses in foals and immunocompromised individuals. To date, no commercial vaccine against R. equi exists. We previously screened all potential vaccine candidates from the complete genome of R. equi using a reverse vaccinology approach. Five of these candidates, namely ABC transporter substrate-binding protein (ABC transporter), penicillin-binding protein 2 (PBD2), NlpC/P60 family protein (NlpC/P60), esterase family protein (Esterase), and M23 family metallopeptidase (M23) were selected for the evaluation of immunogenicity and immunoprotective effects in BALB/c mice model challenged with R. equi. The results showed that all five vaccine candidate-immunized mice experienced a significant increase in spleen antigen-specific IFN-γ- and TNF-α-positive CD4 + and CD8 + T lymphocytes and generated robust Th1- and Th2-type immune responses and antibody responses. Two weeks after the R. equi challenge, immunization with the five vaccine candidates reduced the bacterial load in the lungs and improved the pathological damage to the lungs and livers compared with those in the control group. NlpC/P60, Esterase, and M23 were more effective than the ABC transporter and PBD2 in inducing protective immunity against R. equi challenge in mice. In addition, these vaccine candidates have the potential to induce T lymphocyte memory immune responses in mice. In summary, these antigens are effective candidates for the development of protective vaccines against R. equi. The R. equi antigen library has been expanded and provides new ideas for the development of multivalent vaccines.

Role of the Pseudomonas plecoglossicida fliL gene in immune response of infected hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

Pseudomonas plecoglossicida, a gram-negative bacterium, is the main pathogen of visceral white-point disease in marine fish, responsible for substantial economic losses in the aquaculture industry. The FliL protein, involved in torque production of the bacterial flagella motor, is essential for the pathogenicity of a variety of bacteria. In the current study, the fliL gene deletion strain (ΔfliL), fliL gene complement strain (C-ΔfliL), and wild-type strain (NZBD9) were compared to explore the influence of the fliL gene on P. plecoglossicida pathogenicity and its role in host immune response. Results showed that fliL gene deletion increased the survival rate (50%) and reduced white spot disease progression in the hybrid groupers. Moreover, compared to the NZBD9 strain, the ΔfliL strain was consistently associated with lower bacterial loads in the grouper spleen, head kidney, liver, and intestine, coupled with reduced tissue damage. Transcriptomic analysis identified 2 238 differentially expressed genes (DEGs) in the spleens of fish infected with the ΔfliL strain compared to the NZBD9 strain. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the DEGs were significantly enriched in seven immune system-associated pathways and three signaling molecule and interaction pathways. Upon infection with the ΔfliL strain, the toll-like receptor (TLR) signaling pathway was activated in the hybrid groupers, leading to the activation of transcription factors (NF-κB and AP1) and cytokines. The expression levels of proinflammatory cytokine-related genes IL-1ß, IL-12B, and IL-6 and chemokine-related genes CXCL9, CXCL10, and CCL4 were significantly up-regulated. In conclusion, the fliL gene markedly influenced the pathogenicity of P. plecoglossicida infection in the hybrid groupers. Notably, deletion of fliL gene in P. plecoglossicida induced a robust immune response in the groupers, promoting defense against and elimination of pathogens via an inflammatory response involving multiple cytokines.

Acetylated bacterial proteins as potent antigens inducing an anti-modified protein antibody response.

OBJECTIVE: Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA). METHODS: E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA. RESULTS: Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins. CONCLUSIONS: Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Fit-for-purpose heterodivalent single-domain antibody for gastrointestinal targeting of toxin B from Clostridium difficile.

Single-domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that display extraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross-inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesses high stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications.

Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Selenium nanoparticles enhance mucosal immunity against Mycobacterium bovis infection.

Selenium nanoparticles (SeNPs) enhance the immune response as adjuvants, increasing the efficacy of viral vaccines, including those for COVID-19. However, the efficiency of mucosal SeNPs in boosting vaccine-induced protective immunity against tuberculosis remains unclear. Therefore, this study aims to investigate whether the combination of SeNPs with the AH antigen (Ag85A-HspX) can boost respiratory mucosal immunity and thereby enhance the protective effects against tuberculosis. We synthesized SeNPs and assessed their impact on the immune response and protection against Mycobacterium bovis (M. bovis) as a mucosal adjuvant in mice, administered intranasally at a dose of 20 µg. SeNPs outperformed polyinosinic-polycytidylic acid (Poly IC) in stimulating the maturation of bone marrow-derived dendritic cells (BMDCs), which enhanced antigen presentation. SeNPs significantly activated and proliferated tissue-resident memory T cells (TRMs) and effector CD4+ T cells in the lungs. The vaccines elicited specific antibody responses in the respiratory tract and stimulated systemic Th1 and Th17 immune responses. Immunization with AH and SeNPs led to higher levels of mucosal secretory IgA in bronchoalveolar lavage fluid (BALF) and secretory IL-17 in splenocytes. Moreover, SeNPs immunized mice showed reduced M. bovis infection loads and inflammatory lesions in the lungs post-challenge. Notably, immunization with AH and SeNPs significantly reduced bacterial load in the lungs, achieving the lowest levels compared to all other tested groups. This study calls for pre-clinical investigation of AHB-SeNPs as an anti-bovine tuberculosis vaccine and for exploring its human vaccine potential, which is anticipated to aid in the development of innovative vaccines or adjuvants.

The effector-triggered immunity landscape of tomato against Pseudomonas syringae.

Tomato (Solanum lycopersicum) is one of the world's most important food crops, and as such, its production needs to be protected from infectious diseases that can significantly reduce yield and quality. Here, we survey the effector-triggered immunity (ETI) landscape of tomato against the bacterial pathogen Pseudomonas syringae. We perform comprehensive ETI screens in five cultivated tomato varieties and two wild relatives, as well as an immunodiversity screen on a collection of 149 tomato varieties that includes both wild and cultivated varieties. The screens reveal a tomato ETI landscape that is more limited than what was previously found in the model plant Arabidopsis thaliana. We also demonstrate that ETI eliciting effectors can protect tomato against P. syringae infection when the effector is delivered by a non-virulent strain either prior to or simultaneously with a virulent strain. Overall, our findings provide a snapshot of the ETI landscape of tomatoes and demonstrate that ETI can be used as a biocontrol treatment to protect crop plants.

Expression and field evaluation of new Mycobacterium bovis antigens.

Bovine tuberculosis (bTB) represents a threat to livestock production. Mycobacterium bovis is the main causative agent of bTB and a pathogen capable of infecting wildlife and humans. Eradication programs based on surveillance in slaughterhouses with mandatory testing and culling of reactive cattle have failed to eradicate bTB in many regions worldwide. Therefore, developing effective tools to control this disease is crucial. Using a computational tool, we identified proteins in the M. bovis proteome that carry predictive binding peptides to BoLADRB3.2 and selected Mb0309, Mb1090, Mb1810 and Mb3810 from all the identified proteins. The expression of these proteins in a baculovirus-insect cell expression system was successful only for Mb0309 and Mb3810. In parallel, we expressed the ESAT-6 family proteins EsxG and EsxH in this system. Among the recombinant proteins, Mb0309 and EsxG exhibited moderate performance in distinguishing between cattle that test positive and negative to bTB using the official test, the intradermal tuberculin test (IDT), when used to stimulate interferon-gamma production in blood samples from cattle. However, when combined as a protein cocktail, Mb0309 and EsxG were reactive in 50 % of positive cattle. Further assessments in cattle that evade the IDT (false negative) and cattle infected with Mycobacterium avium paratuberculosis are necessary to determine the potential utility of this cocktail as an additional tool to assist the accurate diagnosis of bTB.

Deletion of an immune evasion gene, steD, from a live Salmonella enterica serovar Typhimurium vaccine improves vaccine responses in aged mice.

Introduction: Non-typhoidal Salmonella (NTS) generally causes self-limiting gastroenteritis. However, older adults (≥65 years) can experience more severe outcomes from NTS infection. We have previously shown that a live attenuated S. Typhimurium vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), was immunogenic in adult but not aged mice. Here we describe modification of CVD 1926 through deletion of steD, a Salmonella effector responsible for host immune escape, which we hypothesized would increase immunogenicity in aged mice. Methods: Mel Juso and/or mutuDC cells were infected with S. Typhimurium I77, CVD 1926, and their respective steD mutants, and the MHC-II levels were evaluated. Aged (18-month-old) C57BL/6 mice received two doses of PBS, CVD 1926, or CVD 1926 ΔsteD perorally (109 CFU) and the number of FliC-specific CD4+ T cells were determined. Lastly, aged C57BL/6 mice received three doses of PBS, CVD 1926, or CVD 1926 ΔsteD perorally (109 CFU) and then were challenged perorally with wild-type S. Typhimurium SL1344 (108 CFU). These animals were also evaluated for antibody responses. Results: MHC-II induction was higher in cells treated with steD mutants, compared to their respective parental strains. Compared to PBS-vaccinated mice, CVD 1926 ΔsteD elicited significantly more FliC-specific CD4+ T cells in the Peyer's Patches. There were no significant differences in FliC-specific CD4+ T cells in the Peyer's patches or spleen of CVD 1926- versus PBS-immunized mice. CVD 1926 and CVD 1926 ΔsteD induced similar serum and fecal anti-core and O polysaccharide antibody titers after three doses. After two immunizations, the proportion of seroconverters for CVD 1926 ΔsteD was 83% (10/12) compared to 42% (5/12) for CVD 1926. Compared to PBS-immunized mice, mice immunized with CVD 1926 ΔsteD had significantly lower S. Typhimurium counts in the spleen, cecum, and small intestine upon challenge. In contrast, there were no differences in bacterial loads in the tissues of PBS-vaccinated and CVD 1926-immunized animals. Conclusion: These data suggest that the steD deletion enhanced the immunogenicity of our live attenuated S. Typhimurium vaccine. Deletion of immune evasion genes could be a potential strategy to improve the immunogenicity of live attenuated vaccines in older adults.

The extracellular serine protease from Staphylococcus epidermidis elicits a type 2-biased immune response in atopic dermatitis patients.

Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis). Objective: To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp). Methods: To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry. Results: We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients' T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33. Conclusion: The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

Immunoinformatics-Based Designing of Novel and Potent Multi-Epitope PSA D15 and Cag11 Immunogens for Helicobacter pylori Immunodiagnostic Assay Development.

Helicobacter pylori (H. pylori) strain is the most genetically diverse pathogenic bacterium and now alarming serious human health concern ranging from chronic gastritis to gastric cancer and human death all over the world. Currently, the majority of commercially available diagnostic assays for H. pylori is a challenging task due to the heterogeneity of virulence factors in various geographical regions. In this concern, designing of universal multi-epitope immunogenic biomarker targeted for all H. pylori strains would be crucial to successfully immunodiagnosis assay and vaccine development for H. pylori infection. Hence, the present study aimed to explore the potential immunogenic epitopes of PSA D15 and Cag11 proteins of H. pylori, using immunoinformatics web tools in order to design novel immune-reactive multi-epitope antigens for enhanced immunodiagnosis in humans. Through an in silico immunoinformatics approach, high-ranked B-cell, MHC-I, and MHC-II epitopes of PSA D15 and Cag11 proteins were predicted, screened, and selected. Subsequently, a novel multi-epitope PSA D15 and Cag11 antigens were designed by fused the high-ranked B-cell, MHC-I, and MHC-II epitopes and 50S ribosomal protein L7/L12 adjuvant using linkers. The antigenicity, solubility, physicochemical properties, secondary and tertiary structures, 3D model refinement, and validations were carried. Furthermore, the designed multi-epitope antigens were subjected to codon adaptation and in silico cloning, immune response simulation, and molecular docking with receptor molecules. A novel, stable multi-epitope PSA D15 and Cag11 H. pylori antigens were developed and immune simulation of the designed antigens showed desirable levels of immunological response. Molecular docking of designed antigens with immune receptors (B-cell, MHC-I, MHC-II, and TLR-2/4) revealed robust interactions and stable binding affinity to the receptors. The codon optimized and in silico cloned showed that the designed antigens were successfully expressed (CAI value of 0.95 for PSA D15 and 1.0 for Cag11) after inserted into pET-32ba (+) plasmid of the E. coli K12 strain. In conclusion, this study revealed that the designed multi-epitope antigens have a huge immunological potential candidate biomarker and useful in developing immunodiagnostic assays and vaccines for H. pylori infection.