Murine Genital Tract Infection Model Research Articles

Irradiated whole cell Chlamydia vaccine confers significant protection in a murine genital tract challenge model

Chlamydia trachomatis infections are the most common bacterial STIs globally and can lead to serious morbidity if untreated. Development of a killed, whole-cell vaccine has been stymied by coincident epitope destruction during inactivation. Here, we present a prototype Chlamydia vaccine composed of elementary bodies (EBs) from the related mouse pathogen, Chlamydia muridarum (Cm). EBs inactivated by gamma rays (Ir-Cm) in the presence of the antioxidant Mn2+-Decapeptide (DEHGTAVMLK) Phosphate (MDP) are protected from epitope damage but not DNA damage. Cm EBs gamma-inactivated with MDP retain their structure and provide significant protection in a murine genital tract infection model. Mice vaccinated with Ir-Cm (+MDP) exhibited elevated levels of Cm-specific IgG and IgA antibodies, reduced bacterial burdens, accelerated clearance, and distinctive cytokine responses compared to unvaccinated controls and animals vaccinated with EBs irradiated without MDP. Preserving EB epitopes with MDP during gamma inactivation offers the potential for a polyvalent, whole-cell vaccine against C. trachomatis.

Meningococcal Detoxified Outer Membrane Vesicle Vaccines Enhance Gonococcal Clearance in a Murine Infection Model.

Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea. We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets. Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB. Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Gonococcal Adaptation to Palmitic Acid Through farAB Expression and FadD Activity Mutations Increases In Vivo Fitness in a Murine Genital Tract Infection Model.

Neisseria gonorrhoeae is a bacterial pathogen that colonizes mucosal epithelia that are rich in antimicrobial molecules such as long-chain fatty acids. Here we studied the mechanisms involved in palmitic acid resistance and their impact on in vivo biological fitness in a murine genital tract infection model. A stable palmitic acid-resistant derivative was obtained by serial passage with incremental palmitic acid concentrations. This derivative outcompeted its parent strain for colonization and survival in the murine infection model. Subsequent whole-genome sequencing resulted in the identification of the 3 resistance-related SNPs ihfAC5T, fadDC772T, and farAG-52T (promoter) that were verified for resistance against palmitic acid. Subsequent characterization of the associated resistance determinants showed that ihfAC5T and farAG-52T induced gene expression of the FarAB efflux pump, whereas fadDC772T increased the maximum enzyme activity of the FadD long-chain fatty acid-coenzyme A ligase. Our results highlight the mechanisms involved in gonococcal adaptation to the murine host environment.

Pharmacokinetic Data Are Predictive of In Vivo Efficacy for Cefixime and Ceftriaxone against Susceptible and Resistant Neisseria gonorrhoeae Strains in the Gonorrhea Mouse Model.

There is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESCs) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fTMIC]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESCr) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, with in vivo efficacy against an ESCs strain requiring doses that yielded an fTMIC in excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESCr strain and were useful in designing effective dosing regimens.

Vaccination with the polymorphic membrane protein A reduces Chlamydia muridarum induced genital tract pathology

Chlamydia trachomatis serovars D-K are one of the most frequent causes of sexually transmitted infections of the female genital tract, with possible complications such as hydrosalpinx, pelvic inflammatory disease, extra-uterine gravidity or infertility. We used the murine genital tract infection model with C. muridarum for vaccination studies and found that more than 70% of the infected mice suffered from uterus dilatations and/or hydrosalpinx. Systemic consequences of the vaginal infection were apparent by splenomegaly ten to fifteen days post infection. While cultivable microorganisms were detectable for the first 23days post infection, the first lesions of the genital tract developed at day 15, however, many lesions occurred later in the absence of cultivable bacteria. Lesions were not accompanied by pro-inflammatory cytokines such as IFNɣ, TNF and IL-6, since these cytokines were almost undetectable in the genital tract 43days post infection. To prevent genital tract lesions, we vaccinated mice with the polymorphic membrane protein (Pmp) A in combination with CpG-ODN 1826 as adjuvant. The vaccine lowered the chlamydial burden and the differences were significant at day 10 post infection but not later. More importantly the vaccine decreased the rate and severity of genital tract lesions. Interestingly, control vaccination with the protein ovalbumin plus CpG-ODN 1826 enhanced significantly the severity but not the rate of pathologic lesions, which was presumably caused by the activation of innate immune responses by the adjuvant in the absence of a C. muridarum-specific adaptive immune response. In summary, vaccination with recombinant PmpA plus CpG-ODN 1826 significantly reduced C. muridarum-induced tissue damage, however, CpG-ODN 1826 may aggravate C. muridarum-induced tissue injuries in the absence of a protective antigen.

Mutational Analysis of the Chlamydia muridarum Plasticity Zone.

Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed--and some may produce functional proteins--but are dispensable for infection of the murine genital tract.

Vaccines against gonorrhea: Current status and future challenges

Gonorrhea occurs at high incidence throughout the world and significantly impacts reproductive health and the spread of human immunodeficiency virus. Current control measures are inadequate and seriously threatened by the rapid emergence of antibiotic resistance. Progress on gonorrhea vaccines has been slow; however, recent advances justify significant effort in this area. Conserved vaccine antigens have been identified that elicit bactericidal antibodies and, or play key roles in pathogenesis that could be targeted by a vaccine-induced response. A murine genital tract infection model is available for systematic testing of antigens, immunization routes and adjuvants, and transgenic mice exist to relieve some host restrictions. Furthermore, mechanisms by which Neisseria gonorrhoeae avoids inducing a protective adaptive response are being elucidated using human cells and the mouse model. Induction of a Th1 response in mice clears infection and induces a memory response, which suggests Th1-inducing adjuvants may be key in vaccine-induced protection. Continued research in this area should include human testing and clinical studies to confirm or negate findings from experimental systems and to define protective host factors.

Chlamydia muridarum T Cell Antigens and Adjuvants That Induce Protective Immunity in Mice

Major impediments to a Chlamydia vaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model against Chlamydia infection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for a Chlamydia vaccine, we evaluated 10 new Chlamydia T cell antigens discovered via immunoproteomics in addition to the 3 antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6'-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720-CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4(+) T cells coexpressing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), and reduced infection by more than 3 logs. Using DDA-MPL as an adjuvant, we found that 7 of 13 Chlamydia T cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly defined Chlamydia vaccine.

Protective efficacy of major outer membrane protein-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection.

The protective efficacy of immunoglobulin A (IgA) and IgG monoclonal antibodies (MAbs) specific for the major outer membrane protein of Chlamydia trachomatis MoPn was evaluated in a murine genital tract infection model. MAbs were delivered into serum and vaginal secretions of naive mice by using the backpack hybridoma tumor system, and protective efficacy was assessed over the first 8 days following challenge by quantitative determination of chlamydial recovery from cervicovaginal swabs, histopathological evaluation of genital tract tissue, and immunohistochemical detection of chlamydial inclusions. IgA and IgG significantly reduced the incidence of infection following vaginal challenge with 5 50% infectious doses, but such protection was overwhelmed by 10- and 100-fold higher challenge doses. Both MAbs also consistently reduced vaginal shedding from infected animals with all three challenge doses compared with the negative control MAb, although the magnitude of this effect was marginal. Blinded pathological evaluation of genital tract tissues at 8 days postinfection showed a significant reduction in the severity of the inflammatory infiltrate in oviduct tissue of infected IgA- and IgG-treated animals. Immunohistochemical detection of chlamydial inclusions revealed a marked reduction in the chlamydial burden of the oviduct epithelium; this finding is consistent with the reduced pathological changes observed in this tissue. These studies indicate that the presence of IgA or IgG MAbs specific to major outer membrane proteins has a marginal effect in preventing chlamydial colonization and shedding from the genital tract but has a more pronounced effect on ascending chlamydial infection and accompanying upper genital tract pathology.