Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Outer membrane vesicles derived from Bordetella pertussis are potent adjuvant that drive Th1-biased response.

For several years, we have been committed to exploring the potential of Bordetella pertussis-derived outer membrane vesicles (OMVBp) as a promising third-generation vaccine against the reemerging pertussis disease. The results of our preclinical trials not only confirm its protective capacity against B. pertussis infection but also set the stage for forthcoming human clinical trials. This study delves into the examination of OMVBp as an adjuvant. To accomplish this objective, we implemented a two-dose murine schedule to evaluate the specific immune response induced by formulations containing OMVBp combined with 3 heterologous immunogens: Tetanus toxoid (T), Diphtheria toxoid (D), and the SARS-CoV-2 Spike protein (S). The specific levels of IgG, IgG1, and IgG2a triggered by the different tested formulations were evaluated using ELISA in dose-response assays for OMVBp and the immunogens at varying levels. These assays demonstrated that OMVBp exhibits adjuvant properties even at the low concentration employed (1.5 µg of protein per dose). As this effect was notably enhanced at medium (3 µg) and high concentrations (6 µg), we chose the medium concentration to determine the minimum immunogen dose at which the OMV adjuvant properties are significantly evident. These assays demonstrated that OMVBp exhibits adjuvant properties even at the lowest concentration tested for each immunogen. In the presence of OMVBp, specific IgG levels detected for the lowest amount of antigen tested increased by 2.5 to 10 fold compared to those found in animals immunized with formulations containing adjuvant-free antigens (p<0.0001). When assessing the adjuvant properties of OMVBp compared to the widely recognized adjuvant alum, we detected similar levels of specific IgG against D, T and S for both adjuvants. Experiments with OMVs derived from E. coli (OMVE.coli) reaffirmed that the adjuvant properties of OMVs extend across different bacterial species. Nonetheless, it's crucial to highlight that OMVBp notably skewed the immune response towards a Th1 profile (p<0.05). These collective findings emphasize the dual role of OMVBp as both an adjuvant and modulator of the immune response, positioning it favorably for incorporation into combined vaccine formulations.

A conserved 3D pattern in a Streptococcus pyogenes M protein immunogen elicits M-type crossreactivity.

Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220 M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens because of type specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was shown to elicit M-type crossreactivity. The basis for this crossreactivity is unknown but may be due in part to antibody recognition of a 3D pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit crossreactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S. pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum-recognized M proteins displayed natively on the strep A surface and promoted the opsonophagocytic killing of strep A strains expressing these M proteins. As C4BP binding is a conserved virulence trait of strep A, we propose that targeting the 3D pattern may prove advantageous in vaccine design.

Outer surface protein E (OspE) mediates Borrelia burgdorferi sensu stricto strain-specific complement evasion in the eastern fence lizard, Sceloporus undulatus.

In North America, Lyme disease is primarily caused by the spirochetal bacterium Borrelia burgdorferi sensu stricto (Bb), which is transmitted between multiple vertebrate hosts and ixodid ticks, and is a model commonly used to study host-pathogen interactions. While Bb is consistently observed in its mammalian and avian reservoirs, the bacterium is rarely isolated from North American reptiles. Two closely related lizard species, the eastern fence lizard (Sceloporus undulatus) and the western fence lizard (Sceloporus occidentalis), are examples of reptiles parasitized by Ixodes ticks. Vertebrates are known to generate complement as an innate defense mechanism, which can be activated before Bb disseminate to distal tissues. Complement from western fence lizards has proven lethal against one Bb strain, implying the role of complement in making those lizards unable to serve as hosts to Bb. However, Bb DNA is occasionally identified in distal tissues of field-collected eastern fence lizards, suggesting some Bb strains may overcome complement-mediated clearance in these lizards. These findings raise questions regarding the role of complement and its impact on Bb interactions with North American lizards. In this study, we found Bb seropositivity in a small population of wild-caught eastern fence lizards and observed Bb strain-specific survivability in lizard sera. We also found that a Bb outer surface protein, OspE, from Bb strains viable in sera, promotes lizard serum survivability and binds to a complement inhibitor, factor H, from eastern fence lizards. Our data thus identify bacterial and host determinants of eastern fence lizard complement evasion, providing insights into the role of complement influencing Bb interactions with North American lizards.

Bioinformatics design of recombinant chimeric protein containing SipD and LptD immunogens and evaluation of its immunogenicity against Salmonella Typhimurium.

The growing emergence of resistant bacteria is the current global concern for the humans and animals. Vaccination could be the desirable method to preventing such infectious diseases. Safe and effective vaccines are urgently needed to manage and prevent Salmonella contamination. Subunit vaccines are safe approaches for the protection against Salmonella spp. The bioinformatics methods were performed to determine the gene structure. Gene cassette (rLPSI) was ordered in pET28a (+), and cloned into E.coli BL21 (DE3), and the recombinant protein was expressed using IPTG (1 mM). Mice were immunized by subcutaneous administration of recombinant protein. Then, the mice were challenged by oral administration of 100LD50 of live S. Typhimurium. The Codon adaptation index of the chimeric gene was multiplied by 0.92. Validation results showed that >90% of residues lie in the desired or extra allowed area of the Ramachandran plot. The recombinant protein (65.9 kDa) was expressed in E.coli. Antibody titers in vaccinated mice were significantly different from those in the control groups. Recombinant protein immunization of the mice provided 90% and 70% protection against 10LD50 and 100LD50 of S. Typhimurium, respectively. In general, the results showed the high efficiency of rLPSI chimeric protein as a protective antigen against S. Typhimurium infection. The rLPSI chimeric protein could be an effective recombinant vaccine candidate against S. Typhimurium infection, but more research is needed.

Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model.

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

FHUSPA2/10 is a bactericidal monoclonal antibody targeting multiple repeated sequences of Moraxella catarrhalis UspA2.

Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.

VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force.

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.

A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

Antibodies binding diverse pertactin epitopes protect mice from Bordetella pertussis infection.

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness.

Immunogenic characteristics of the outer membrane phosphoporin as a vaccine candidate against Klebsiella pneumoniae.

In recent years, Klebsiella pneumoniae (KP) has caused disease outbreaks in different animals, resulting in serious economic losses and biosafety concerns. Considering the broad antibiotic resistance of KP, vaccines are the most effective tools against infection. However, there is still no KP vaccine available in the veterinary field. Our results indicate that the highly conserved outer membrane phosphoporin (PhoE) of KP is immunogenic in mice and elicits high titers of antibodies that were shown to be specific for PhoE by immunoblotting. Immunization with PhoE also induced robust cell-mediated immunity and elicited the secretion of high levels of IFN-γ and IL-4, suggesting the induction of mixed Th1 and Th2 responses. Sera from PhoE-immunized mice induced significantly higher complement-mediated lysis of KP cells than did sera from the PBS control mice. Finally, mice immunized with PhoE were significantly protected against KP challenge, with better survival and a reduced visceral bacterial load. Our data underscore the great potential of PhoE as a novel candidate antigen for a vaccine against KP infection.

Shigella Outer Membrane Vesicles as Promising Targets for Vaccination.

The clinical symptoms of shigellosis, a gastrointestinal infection caused by Shigella spp. range from watery diarrhea to fulminant dysentery. Endemic infections, particularly among children in developing countries, represent the majority of clinical cases. The situation is aggravated due to the high mortality rate of shigellosis, the rapid dissemination of multi-resistant Shigella strains and the induction of only serotype-specific immunity. Thus, infection prevention due to vaccination, encompassing as many of the circulating serotypes as possible, has become a topic of interest. However, vaccines have turned out to be ineffective so far. Outer membrane vesicles (OMVs) are promising novel targets for vaccination. OMVs are constitutively secreted by Gram-negative bacteria including Shigella during growth. They are composed of soluble luminal portions and an insoluble membrane and can contain toxins, bioactive periplasmic and cytoplasmic (lipo-) proteins, (phospho-) lipids, nucleic acids and/or lipopolysaccharides. Thus, OMVs play an important role in bacterial cell-cell communication, growth, survival and pathogenesis. Furthermore, they modulate the secretion and transport of biomolecules, the stress response, antibiotic resistance and immune responses of the host. Thus, OMVs serve as novel secretion machinery. Here, we discuss the current literature and highlight the properties of OMVs as potent vaccine candidates because of their immunomodulatory, antigenic and adjuvant properties.

HomA and HomB, outer membrane proteins of Helicobacter pylori down-regulate activation-induced cytidine deaminase (AID) and Ig switch germline transcription and thereby affect class switch recombination (CSR) of Ig genes in human B-cells.

H. pylori is one of the major causes of chronic gastritis, peptic ulcer disease (PUD), gastric mucosa-associated lymphoid tissue lymphoma (MALT) and gastric carcinoma. H. pylori toxin VacA is responsible for host cell apoptosis, whereas CagA is known to aberrantly induce expression of activation-induced cytidine deaminase (AID) in gastric epithelial cells that causes mutations in oncogenes and tumour suppressor genes, leading to the transformation of normal cells into cancerous cells. Although, a significant amount of research has been conducted to understand the role of bacterial factors modulating deregulated host cell pathways, the interaction between H. pylori and immune cells of the marginal zone and its consequences are still not well understood. HomB and HomA, outer membrane proteins (OMPs) from H. pylori, which assist in the adhesion of bacteria to host cells, are found to be associated with H. pylori virulent strains and promote inflammation. Interestingly, we observed that the interaction of HomB/HomA OMPs with B-cells transiently downregulates AID expression and Ig switch germline transcription. Downregulation of AID leads to impairment of class switch recombination (CSR), resulting in significantly reduced switching to IgG and IgA antibodies. Besides, we examined the immune-suppressive response of B-cells and observed that the cells stimulated with HomA/B show upregulation in the levels of IL10, IL35, as well as PDL1, a T-cell inhibition marker. Our study suggests the potential role of OMPs in immune response modulation strategies used by the pathogen to evade the immune response. These results provide a better understanding of H. pylori pathogenesis and assist in identifying novel targets for therapy.

A brief history of OspA vaccines including their impact on diagnostic testing for Lyme disease.

The only United States Food and Drug Administration approved vaccine preparation to prevent Lyme disease consisted of a single recombinant outer surface protein A (OspA), which was marketed for use from late 1998 until early 2002, with no vaccine currently available for humans for nearly 20 years. OspA vaccines generate an antibody-mediated, transmission blocking immunity, that prevents Borrelia burgdorferi from being transmitted during a tick bite. Although this OspA vaccine was safe and effective, it likely would have required booster doses to maintain immunity, and vaccination regularly caused false positive results on first-tier serologic testing for Lyme disease, when a whole cell-based enzyme immunoassay was used. Clinical trials are in progress to test a new multivalent OspA vaccine designed to prevent Lyme disease in both the United States and Europe.

Two Potential Syphilis Vaccine Candidates Inhibit Dissemination of Treponema pallidum.

Syphilis, caused by the spirochete Treponema pallidum subspecies pallidum, continues to be a major public health problem worldwide. Recent increases in the number of syphilis cases, in addition to the lack of an efficient vaccine against T. pallidum for humans, highlights an urgent need for the design and development of an efficacious syphilis vaccine. Here, we assess the vaccine potential of the adhesion protein Tp0136 and the outer membrane protein Tp0663. Rabbits were subcutaneously immunized with recombinant proteins Tp0136, Tp0663, or control PBS. Immunization with Tp0136 or Tp0663 generated a strong humoral immune response with high titers of IgG, as assessed by ELISA. Moreover, animals immunized with Tp0136 or Tp0663 exhibited attenuated lesion development, increased cellular infiltration at the lesion sites, and inhibition of treponemal dissemination to distant organs compared to the unimmunized animals. These findings indicate that Tp0136 and Tp0663 are promising syphilis vaccine candidates. Furthermore, these results provide novel and important information for not only understanding the pathogenic mechanisms of spirochetes, but also the development of spirochete-specific subunit vaccines.

Immunoinformatics-Based Designing of a Multi-Epitope Chimeric Vaccine From Multi-Domain Outer Surface Antigens of Leptospira.

Accurate information on antigenic epitopes within a multi-domain antigen would provide insights into vaccine design and immunotherapy. The multi-domain outer surface Leptospira immunoglobulin-like (Lig) proteins LigA and LigB, consisting of 12-13 homologous bacterial Ig (Big)-like domains, are potential antigens of Leptospira interrogans. Currently, no effective vaccine is available against pathogenic Leptospira. Both the humoral immunity and cell-mediated immunity of the host play critical roles in defending against Leptospira infection. Here, we used immunoinformatics approaches to evaluate antigenic B-cell lymphocyte (BCL) and cytotoxic T-lymphocyte (CTL) epitopes from Lig proteins. Based on certain crucial parameters, potential epitopes that can stimulate both types of adaptive immune responses were selected to design a chimeric vaccine construct. Additionally, an adjuvant, the mycobacterial heparin-binding hemagglutinin adhesin (HBHA), was incorporated into the final multi-epitope vaccine construct with a suitable linker. The final construct was further scored for its antigenicity, allergenicity, and physicochemical parameters. A three-dimensional (3D) modeled construct of the vaccine was implied to interact with Toll-like receptor 4 (TLR4) using molecular docking. The stability of the vaccine construct with TLR4 was predicted with molecular dynamics simulation. Our results demonstrate the application of immunoinformatics and structure biology strategies to develop an epitope-specific chimeric vaccine from multi-domain proteins. The current findings will be useful for future experimental validation to ratify the immunogenicity of the chimera.

BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis.

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

Persistent Anti-Borrelia IgM Antibodies without Lyme Borreliosis in the Clinical and Immunological Context.

The aim of the study was to investigate the etiology of persistent IgM antibodies against Borrelia burgdorferi sensu lato (sl) and to analyze their association with nonspecific symptoms. The study group comprised individuals with persistent IgM antibodies in the absence of IgG. The relation between ELISA values and time elapsed since past erythema migrans (EM) was analyzed. Previous antibiotic treatments were assessed. The association between persistent IgM and nonspecific symptoms was evaluated statistically. Specificity of IgM antibodies for outer surface protein C (OspC) of B. burgdorferi sl was examined by immunoblotting. Further, we investigated the cross-reactivity with Borrelia-unrelated proteins. Fifty-nine patients (46 women; 78%) were included in the study group. The mean IgM-ELISA values did not change significantly during follow-up (median 6.2 months). The mean ELISA value in the study group was dependent on time elapsed since past EM. Nonspecific symptoms improved significantly more often in patients with lower IgM ELISA results. Persistent IgM antibodies were specific for the C-terminal PKKP motif of OspC. Cross-reacting C-terminal PKKP antigens from both human and prokaryotic origins were identified. We demonstrate that the C-terminal PKKP motif plays a main role for the reactivity of persistent Borrelia IgM toward OspC. However, cross-reactivity to other eukaryotic and/or prokaryotic antigens may hamper the specificity of OspC in the serological diagnosis of Lyme borreliosis. Lack of improvement of nonspecific symptoms was associated with higher IgM ELISA values. IMPORTANCE The reactivity of human IgM with the outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is frequently used to detect Borrelia specific IgM in commercial immunoassays, and such antibodies usually occur in the early phase of the infection. We identified a group of individuals with persistent Borrelia IgM without symptoms of Lyme borreliosis. We used their sera to demonstrate that the C-terminal epitope of OspC binds the IgM. Strikingly, we found that the same epitope occurs also in certain proteins of human and environmental origin; the latter include other bacteria and food plants. Our experimental data show that these Borrelia-unrelated proteins cross-react with the OpsC-specific IgM. This knowledge is important for the development of serologic assays for Lyme borreliosis and provides a cross-reactive explanation for the persistence of Borrelia-IgM.

Immunoinformatic identification of the epitope-based vaccine candidates from Maltoporin, FepA and OmpW of Shigella Spp, with molecular docking confirmation.

Shigella is a bacterial pathogen that causes shigellosis, fatal bacillary dysentery, responsible for a higher level of mortality worldwide. We adopted a number of computational approaches to predict potential epitope-based vaccine candidates of immunogenic proteins of Shigella spp. We selected three cell surface proteins of the bacterium according to their antigenicity using the VaxiJen server, including, FepA, Maltoporin, and OmpW. The sequence analyses by the IEDB server resulted in three 15-mer peptides of the core epitope, FTAEHTQSV, FLVNQTLTL, and MRAGSATVR from FepA, Maltoporin, and OmpW, respectively, as the most potential epitopes that have an affinity with both cytotoxic and helper T-cells. Moreover, the epitopes showed 73.76%, 99.0%, and 93.07% world population coverage, along with 100% conservancy among the Shigella subspecies. The molecular docking simulation studies were performed to verify the interactions between the peptides and the respective HLAs. Docking analyses showed that the Epitope-MHC complexes had a higher level of global energy score dictating strong binding. We have also predicted B-cell epitopes from the sequences of these three proteins. In vivo study of the proposed epitope might contribute to the development of a functional and efficient vaccine, which might be an effective way to elude dysentery from the world.

Identification and Evaluation of Recombinant Outer Membrane Proteins as Vaccine Candidates Against Klebsiella pneumoniae.

Klebsiella pneumoniae found in the normal flora of the human oral and intestinal tract mainly causes hospital-acquired infections but can also cause community-acquired infections. To date, most clinical trials of vaccines against K. pneumoniae have ended in failure. Furthermore, no single conserved protein has been identified as an antigen candidate to accelerate vaccine development. In this study, we identified five outer membrane proteins of K. pneumoniae, namely, Kpn_Omp001, Kpn_Omp002, Kpn_Omp003, Kpn_Omp004, and Kpn_Omp005, by using reliable second-generation proteomics and bioinformatics. Mice vaccinated with these five KOMPs elicited significantly higher antigen-specific IgG, IgG1, and IgG2a. However, only Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 were able to induce a protective immune response with two K. pneumoniae infection models. These protective effects were accompanied by the involvement of different immune responses induced by KOMPs, which included KOMPs-specific IFN-γ-, IL4-, and IL17A-mediated immune responses. These findings indicate that Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 are three potential Th1, Th2, and Th17 candidate antigens, which could be developed into multivalent and serotype-independent vaccines against K. pneumoniae infection.