Development of a Tag/Catcher-mediated capsid virus-like particle vaccine presenting the conserved Neisseria gonorrhoeae SliC antigen that blocks human lysozyme.

Virus-like particles (VLPs) are promising nanotools for the development of subunit vaccines due to high immunogenicity and safety. Herein, we explored the versatile and effective Tag/Catcher-AP205 capsid VLP (cVLP) vaccine platform to address the urgent need for the development of an effective and safe vaccine against gonorrhea. The benefits of this clinically validated cVLP platform include its ability to facilitate unidirectional, high-density display of complex/full-length antigens through an effective split-protein Tag/Catcher conjugation system. To assess this modular approach for making cVLP vaccines, we used a conserved surface lipoprotein, SliC, that contributes to the Neisseria gonorrhoeae defense against human lysozyme, as a model antigen. This protein was genetically fused at the N- or C-terminus to the small peptide Tag enabling their conjugation to AP205 cVLP, displaying the complementary Catcher. We determined that SliC with the N-terminal SpyTag, N-SliC, retained lysozyme-blocking activity and could be displayed at high density on cVLPs without causing aggregation. In mice, the N-SliC-VLP vaccines, adjuvanted with AddaVax or CpG, induced significantly higher antibody titers compared to controls. In contrast, similar vaccine formulations containing monomeric SliC were non-immunogenic. Accordingly, sera from N-SliC-VLP-immunized mice also had significantly higher human complement-dependent serum bactericidal activity. Furthermore, the N-SliC-VLP vaccines administered subcutaneously with an intranasal boost elicited systemic and vaginal IgG and IgA, whereas subcutaneous delivery alone failed to induce vaginal IgA. The N-SliC-VLP with CpG (10 µg/dose) induced the most significant increase in total serum IgG and IgG3 titers, vaginal IgG and IgA, and bactericidal antibodies.

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.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages.

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

New Rapid Helicobacter Pylori Blood Test Based on Dual Detection of FliD and CagA Antibodies for On-Site Testing.

Helicobacter pylori is the most prevalent bacterial infection, affecting half of the world's population, with a high morbidity and mortality rate.1,2 Several invasive and noninvasive testing procedures are available, and their selective use serves the specific needs of diverse clinical scenarios. For gastric cancer prevention, mass screening is necessary and requires a noninvasive, rapid, accurate and cost-effective test. For this purpose H pylori serology currently seems to be the preferred noninvasive diagnostic method. Population-based testing and treatment for H pylori is cost effective in high-risk countries, but less effective in low- and medium-risk countries.3,4 Many serologic tests are available on the market, with inconsistent performance often being observed. Therefore, international guidelines recommend considering only serologic tests with high accuracy that have been validated in the respective local populations. To date, no rapid point-of-care test (POCT) has reached a sufficient degree of accuracy.

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.

Twice the tolerance.

A gut microbiota-derived antigen elicits distinct subsets of regulatory T cells to suppress inflammation in mice.

Identification of the Bartonella autotransporter CFA as a protective antigen and hypervariable target of neutralizing antibodies in mice.

The bacterial genus Bartonella comprises numerous emerging pathogens that cause a broad spectrum of disease manifestations in humans. The targets and mechanisms of the anti-Bartonella immune defense are ill-defined and bacterial immune evasion strategies remain elusive. We found that experimentally infected mice resolved Bartonella infection by mounting antibody responses that neutralized the bacteria, preventing their attachment to erythrocytes and suppressing bacteremia independent of complement or Fc receptors. Bartonella-neutralizing antibody responses were rapidly induced and depended on CD40 signaling but not on affinity maturation. We cloned neutralizing monoclonal antibodies (mAbs) and by mass spectrometry identified the bacterial autotransporter CFA (CAMP-like factor autotransporter) as a neutralizing antibody target. Vaccination against CFA suppressed Bartonella bacteremia, validating CFA as a protective antigen. We mapped Bartonella-neutralizing mAb binding to a domain in CFA that we found is hypervariable in both human and mouse pathogenic strains, indicating mutational antibody evasion at the Bartonella subspecies level. These insights into Bartonella immunity and immune evasion provide a conceptual framework for vaccine development, identifying important challenges in this endeavor.

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.

Remodeling Yersinia pseudotuberculosis to generate a highly immunogenic outer membrane vesicle vaccine against pneumonic plague.

SignificanceYersinia pestis, the etiologic agent of plague, has been responsible for high mortality in several epidemics throughout human history. This plague bacillus has been used as a biological weapon during human history and is currently one of the deadliest biological threats. Currently, no licensed plague vaccines are available in the Western world. Since an array of immunogens are enclosed in outer membrane vesicles (OMVs), immune responses elicited by OMVs against a diverse range of antigens may reduce the likelihood of antigen circumvention. Therefore, self-adjuvanting OMVs from a remodeled Yersinia pseudotuberculosis strain as a type of plague vaccine could diversify prophylactic choices and solve current vaccine limitations.

A multi-epitope fusion antigen candidate vaccine for Enterotoxigenic Escherichia coli is protective against strain B7A colonization in a rabbit model.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's and travelers' diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.

Immune signature of acute pharyngitis in a Streptococcus pyogenes human challenge trial.

Streptococcus pyogenes causes at least 750 million infections and more than 500,000 deaths each year. No vaccine is currently available for S. pyogenes and the use of human challenge models offer unique and exciting opportunities to interrogate the immune response to infectious diseases. Here, we use high-dimensional flow cytometric analysis and multiplex cytokine and chemokine assays to study serial blood and saliva samples collected during the early immune response in human participants following challenge with S. pyogenes. We find an immune signature of experimental human pharyngitis characterised by: 1) elevation of serum IL-1Ra, IL-6, IFN-γ, IP-10 and IL-18; 2) increases in peripheral blood innate dendritic cell and monocyte populations; 3) reduced circulation of B cells and CD4+ T cell subsets (Th1, Th17, Treg, TFH) during the acute phase; and 4) activation of unconventional T cell subsets, γδTCR + Vδ2+ T cells and MAIT cells. These findings demonstrate that S. pyogenes infection generates a robust early immune response, which may be important for host protection. Together, these data will help advance research to establish correlates of immune protection and focus the evaluation of vaccines.

Aerosolized Intratracheal Inoculation of Recombinant Protective Antigen (rPA) Vaccine Provides Protection Against Inhalational Anthrax in B10.D2-Hc0 Mice.

Anthrax caused by Bacillus anthracis is a fatal zoonotic disease with a high lethality and poor prognosis. Inhalational anthrax is the most severe of the three forms of anthrax. The currently licensed commercial human anthrax vaccines require a complex immunization procedure for efficacy and have side effects that limit its use in emergent situations. Thus, development of a better anthrax vaccine is necessary. In this study, we evaluate the potency and efficacy of aerosolized intratracheal (i.t.) inoculation with recombinant protective antigen (rPA) subunit vaccines against aerosolized B. anthracis Pasteur II spores (an attenuated strain) challenge in a B10.D2-Hc0 mouse (deficient in complement component C5) model. Immunization of rPA in liquid, powder or powder reconstituted formulations via i.t. route conferred 100% protection against a 20× LD50 aerosolized Pasteur II spore challenge in mice, compared with only 50% of subcutaneous (s.c.) injection with liquid rPA. Consistently, i.t. inoculation of rPA vaccines induced a higher lethal toxin (LeTx) neutralizing antibody titer, a stronger lung mucosal immune response and a greater cellular immune response than s.c. injection. Our results demonstrate that immunization with rPA dry powder vaccine via i.t. route may provide a stable and effective strategy to improve currently available anthrax vaccines and B10.D2-Hc0 mice challenged with B. anthracis attenuated strains might be an alternative model for anthrax vaccine candidate screening.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals a Novel Binding Region of a Neutralizing Fully Human Monoclonal Antibody to Anthrax Protective Antigen.

Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5-10 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody-antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.

Prospective serological and molecular cross-sectional study focusing on Bartonella and other blood-borne organisms in cats from Catalonia (Spain).

BACKGROUND: There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. METHODS: We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.). RESULTS: From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp. CONCLUSIONS: Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats.

The Mycobacterium tuberculosis PE_PGRS Protein Family Acts as an Immunological Decoy to Subvert Host Immune Response.

Mycobacterium tuberculosis (M.tb) is a successful pathogen that can reside within the alveolar macrophages of the host and can survive in a latent stage. The pathogen has evolved and developed multiple strategies to resist the host immune responses. M.tb escapes from host macrophage through evasion or subversion of immune effector functions. M.tb genome codes for PE/PPE/PE_PGRS proteins, which are intrinsically disordered, redundant and antigenic in nature. These proteins perform multiple functions that intensify the virulence competence of M.tb majorly by modulating immune responses, thereby affecting immune mediated clearance of the pathogen. The highly repetitive, redundant and antigenic nature of PE/PPE/PE_PGRS proteins provide a critical edge over other M.tb proteins in terms of imparting a higher level of virulence and also as a decoy molecule that masks the effect of effector molecules, thereby modulating immuno-surveillance. An understanding of how these proteins subvert the host immunological machinery may add to the current knowledge about M.tb virulence and pathogenesis. This can help in redirecting our strategies for tackling M.tb infections.

Vaccination of mice with hybrid protein containing Exotoxin S and PcrV with adjuvants alum and MPL protects Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa as a common pathogen causing urinary tract infections (UTIs) has been resistant to different antibiotics and developing an effective vaccine can be an alternative strategy. In the present study, the immunogenicity and protection efficacy of formulations composed of a hybrid protein composed of P. aeruginosa V-antigen (PcrV) and exoenzyme S (ExoS) with alum and MPL were evaluated. The hybrid protein could increase the specific systemic and mucosal immune responses, as well as cellular responses as compared with control groups. Combining of alum or MPL adjuvant with the hybrid protein significantly improved the levels of IgG1, serum IgA, mucosal IgG, and IL-17 as compared to the ExoS.PcrV alone. After bladder challenge with a P. aeruginosa strain, the bacterial loads of bladder and kidneys were significantly decreased in mice received ExoS.PcrV admixed with alum and ExoS.PcrV admixed with MPL than controls. The present study indicated that immunization of mice with a hybrid protein composed of ExoS and PcrV could induce multifactorial immune responses and opsonize the bacteria and decrease the viable bacterial cells. Because P. aeruginosa have caused therapeutic challenges worldwide, our study proposed ExoS.PcrV + alum and ExoS.PcrV + MPL as promising candidates for the prevention of infections caused by P. aeruginosa.

Identification of vaccine and drug targets in Shigella dysenteriae sd197 using reverse vaccinology approach.

Shigellosis is characterized as diarrheal disease that causes a high mortality rate especially in children, elderly and immunocompromised patients. More recently, the World Health Organization advised safe vaccine designing against shigellosis due to the emergence of Shigella dysenteriae resistant strains. Therefore, the aim of this study is to identify novel drug targets as well as the design of the potential vaccine candidates and chimeric vaccine models against Shigella dysenteriae. A computational based Reverse Vaccinology along with subtractive genomics analysis is one of the robust approaches used for the prioritization of drug targets and vaccine candidates through direct screening of genome sequence assemblies. Herein, a successfully designed peptide-based novel highly antigenic chimeric vaccine candidate against Shigella dysenteriae sd197 strain is proposed. The study resulted in six epitopes from outer membrane WP_000188255.1 (Fe (3+) dicitrate transport protein FecA) that ultimately leads to the construction of twelve vaccine models. Moreover, V9 construct was found to be highly immunogenic, non-toxic, non-allergenic, highly antigenic, and most stable in terms of molecular docking and simulation studies against six HLAs and TLRS/MD complex. So far, this protein and multiepitope have never been characterized as vaccine targets against Shigella dysenteriae. The current study proposed that V9 could be a significant vaccine candidate against shigellosis and to ascertain that further experiments may be applied by the scientific community focused on shigellosis.

Bacterial coloration immunofluorescence strip for ultrasensitive rapid detection of bacterial antibodies and targeted antibody-secreting hybridomas.

The indirect enzyme-linked immunosorbent assay (ELISA) is the gold standard method for monoclonal antibody (McAb) detection and plays a unique role in the preparation of bacterial antibodies. To solve the laborious issues associated with indirect ELISA, a novel bacterial coloration immunofluorescence strip (BCIFS) for antibody detection using colored bacteria instead of a labeled antibody as the antigen and tracer simultaneously and goat anti-mouse IgG as the test line was developed. The affinity range survey of BCIFS indicated that hybridoma cell cultures of E. coli O157:H7 (D3, E7) and Vibrio parahemolyticus (H7, C9) were detected, which complied with the results of indirect ELISA. Compared with the traditional indirect ELISA, the BCIFS sensitivity for E7 cell cultures, ascites, and purified antibodies was at least 4-fold more sensitive, and the BCIFS cross-reactivity for E7 cell cultures was almost consistent with that of indirect ELISA. In addition, the BCIFS isotypes for E. coli O157:H7 cell cultures and Vibrio parahemolyticus were IgG2a and IgG1, respectively, which were identical to the indirect ELISA. Furthermore, the BCIFS method was confirmed by McAb preparation, effective antibody use, and targeted antibody-secreted hybridoma preparation and screening, which showed excellent performance and substitution of the indirect ELISA method. Combined with methylcellulose semisolid medium, BCIFS offers a novel, easy to operate, rapid preparation method for antigen-specific hybridomas. This is the first report using BCIFS instead of indirect ELISA for bacterial antibody detection and application in different samples, which demonstrates a rapid and powerful tool for antibody engineering.

Characterization of Pathogenic CD8+ T cells in Chlamydia-Infected OT1 Mice.

Chlamydia trachomatis is a leading infectious cause of infertility in women due to its induction of lasting pathology such as hydrosalpinx. Chlamydia muridarum induces mouse hydrosalpinx because C. muridarum can both invade tubal epithelia directly (as a first hit) and induce lymphocytes to promote hydrosalpinx indirectly (as a second hit). In the current study, a critical role of CD8+ T cells in chlamydial induction of hydrosalpinx was validated in both wild type C57BL/6J mice and OT1 transgenic mice. OT1 mice failed to develop hydrosalpinx partially due to the failure of their lymphocytes to recognize chlamydial antigens. CD8+ T cells from naive C57BL/6J mice rescued the ability of recipient OT1 mice to develop hydrosalpinx when naive CD8+ T cells were transferred at the time of infection with Chlamydia. However, when the transfer was delayed for 2 weeks or longer after the Chlamydia infection, naive CD8+ T cells no longer promoted hydrosalpinx. Nevertheless, CD8+ T cells from mice immunized against Chlamydia still promoted significant hydrosalpinx in the recipient OT1 mice even when the transfer was delayed for 3 weeks. Thus, CD8+ T cells must be primed within 2 weeks after Chlamydia infection to be pathogenic, but, once primed, they can promote hydrosalpinx for >3 weeks. However, Chlamydia-primed CD4+ T cells failed to promote chlamydial induction of pathology in OT1 mice. This study optimized an OT1 mouse-based model for revealing the pathogenic mechanisms of Chlamydia-specific CD8+ T cells.