Immunogenicity and Protective Capacity of a Virus-like Particle Vaccine against Chlamydia trachomatis Type 3 Secretion System Tip Protein, CT584

Abstract

An effective vaccine against Chlamydia trachomatis is urgently needed as infection rates continue to rise and C. trachomatis causes reproductive morbidity. An obligate intracellular pathogen, C. trachomatis employs a type 3 secretion system (T3SS) for host cell entry. The tip of the injectosome is composed of the protein CT584, which represents a potential target for neutralization with vaccine-induced antibody. Here, we investigate the immunogenicity and efficacy of a vaccine made of CT584 epitopes coupled to a bacteriophage virus-like particle (VLP), a novel platform for Chlamydia vaccines modeled on the success of HPV vaccines. Female mice were immunized intramuscularly, challenged transcervically with C. trachomatis, and assessed for systemic and local antibody responses and bacterial burden in the upper genital tract. Immunization resulted in a 3-log increase in epitope-specific IgG in serum and uterine homogenates and in the detection of epitope-specific IgG in uterine lavage at low levels. By contrast, sera from women infected with C. trachomatis and virgin controls had similarly low titers to CT584 epitopes, suggesting these epitopes are not systemically immunogenic during natural infection but can be rendered immunogenic by the VLP platform. C. trachomatis burden in the upper genital tract of mice varied after active immunization, yet passive protection was achieved when immune sera were pre-incubated with C. trachomatis prior to inoculation into the genital tract. These data demonstrate the potential for antibody against the T3SS to contribute to protection against C. trachomatis and the value of VLPs as a novel platform for C. trachomatis vaccines.

Keywords: Chlamydia trachomatis; type 3 secretion system; virus-like particle vaccines.

Figure 1

Development of virus-like particle vaccines against C. trachomatis T3SS tip protein CT584. (A) Linear B-cell epitopes of C. trachomatis CT584 were identified by the Immune Epitope Database and used in VLP vaccines. Bolded residues were added for chemical conjugation to the VLP composed of the capsid from the bacteriophage Qβ. (B,C) Localization of vaccine epitopes 70–77 (pink) and 154–164 (cyan) in the CT584 hexamer from the top (B) and side (C) view. Structure retrieved from NCBI PDB #4MLK.

Figure 2

Urogenital tract infection with C. trachomatis in women does not elicit robust antibody responses to Qβ-CT584 vaccine epitopes. Virgin and C. trachomatis (Ct)-infected women were evaluated for serum IgG to CT584 epitopes 70-77 and 154-164, as well as to the immunodominant C. trachomatis epitope MOMP VD4. Genital tract infection with C. trachomatis resulted in an average 5.45 fold increase in MOMP VD4 titers (*** p < 0.001, data adapted from [12]) compared to an average 1.58 fold and 1.56 fold increase to CT584 epitopes 70–77 and 154–164, respectively.

Figure 3

Immunogenicity and protective capacity of single epitope and mixed epitope vaccines against C. trachomatis T3SS tip protein CT584. (A) Immunization with Qβ VLPs conjugated to CT584 epitopes 70–77 (Qβ-70), 154–164 (Qβ-154) or a mixture of both (Qβ-CT584) induced high titer IgG specific to epitopes 70–77 (left) and 154–164 (right) as measured by ELISA. End point dilution was defined as the reciprocal of the highest sample dilution with an OD450 reading twice that of blank wells. Horizontal lines represent means +/− standard error of the mean (SEM), dotted lines represent lower limit of detection. (B) Bacterial burden in the upper genital tract of female mice immunized with either Qβ-CT584, Qβ-70, Qβ-154 or the negative control Qβ alone was measured by qPCR. Immunization with Qβ-CT584 resulted in a 92% reduction in C. trachomatis load, ** p = 0.0079 by Mann Whitney U test.

Figure 4

Immunogenicity and protective capacity of vaccine Qβ-CT584. A second, statistically-powered vaccine trial (n = 20/group) was performed to assess the protective capacity and immunogenicity of Qβ-CT584. (A) Bacterial burden in the upper genital tract of female mice immunized with either Qβ-CT584 or the negative control Qβ alone was measured by qPCR. (B) Immunization with Qβ-CT584 induced high titer IgG specific to vaccine epitopes CT584 70-77 (p < 0.0001) and CT584 154-164 (p < 0.0001) as measured by ELISA. End point dilution was defined as the reciprocal of the highest sample dilution with an OD450 reading twice that of blank wells. Horizontal lines represent means +/− SEM.

Figure 5

Vaccine epitope-specific antibody is detectable at low levels in uterine lavage. Serum (A) and lavage of the lumen of the uterus (B,C) was collected from mice immunized with either Qβ-CT584 or the negative control Qβ alone. (A,B) IgG specific to vaccine epitopes CT584 70–77 and CT584 154–164 was measured by ELISA. End point dilution was defined as the reciprocal of the highest sample dilution with an OD450 reading twice that of blank wells. Antigen-specific IgG was detectable in uterine lavage (B) but at lower levels than in serum (A). (C) The amount of total IgG in uterine lavage was comparable between the two vaccination groups.

Figure 6

Pre-incubation of C. trachomatis with immune serum from Qβ-CT584-vaccinated mice reduces infection in the upper genital tract. C. trachomatis were pre-opsonized in vitro with heat-inactivated serum pooled from mice immunized with Qβ-CT584 or Qβ alone, immediately prior to transcervical inoculation into the upper genital tract of naïve female mice. Bacterial burden in the upper genital tract was measured by qPCR. Data are presented from independent pre-opsonization experiments using immune serum from two independent vaccine trials, Trial 1 ((A), p = 0.0159) and Trial 2 ((B), p = 0.095). Pre-incubation with Qβ-CT584 serum resulted in a 83–84% reduction in bacterial burden compared to pre-incubation with negative control Qβ serum. Horizontal lines represent means +/− SEM.