Use of a Guinea pig-specific transcriptome array for evaluation of protective immunity against genital chlamydial infection following intranasal vaccination in Guinea pigs

Abstract

Guinea pigs have been used as a second animal model to validate putative anti-chlamydial vaccine candidates tested in mice. However, the lack of guinea pig-specific reagents has limited the utility of this animal model in Chlamydia sp. vaccine studies. Using a novel guinea pig-specific transcriptome array, we determined correlates of protection in guinea pigs vaccinated with Chlamydia caviae (C. caviae) via the intranasal route, previously reported by us and others to provide robust antigen specific immunity against subsequent intravaginal challenge. C. caviae vaccinated guinea pigs resolved genital infection by day 3 post challenge. In contrast, mock vaccinated animals continued to shed viable Chlamydia up to day 18 post challenge. Importantly, at day 80 post challenge, vaccinated guinea pigs experienced significantly reduced genital pathology - a sequelae of genital chlamydial infections, in comparison to mock vaccinated guinea pigs. Sera from vaccinated guinea pigs displayed antigen specific IgG responses and increased IgG1 and IgG2 titers capable of neutralizing GPIC in vitro. Th1-cellular/inflammatory immune genes and Th2-humoral associated genes were also found to be elevated in vaccinated guinea pigs at day 3 post-challenge and correlated with early clearance of the bacterium. Overall, this study provides the first evidence of guinea pig-specific genes involved in anti-chlamydial vaccination and illustrates the enhancement of the utility of this animal model in chlamydial pathogenesis.

Figure 1. Vaccination of Guinea pigs with C. caviae Protects Against Genital Chlamydial Infection.

Groups (5 per group) of guinea pigs were immunized i.n. with 1×10⁵ IFU C. caviae or treated with PBS as mock vaccination controls. All animals were rested for 30 days and challenged i.vag. with 1×10⁵ IFU C. caviae. Chlamydial shedding was monitored every third day after challenge for a month, and are presented as mean ± SD for each group at each time point.

Figure 2. Intranasal Vaccination with C. caviae Induced Robust Humoral Responses.

Groups (5 per group) of guinea pigs were immunized i.n. with 1×10⁵ IFU C. caviae or treated with PBS as mock vaccination controls. All animals were rested for 30 days and challenged i.vag. with 1×10⁵ IFU C. caviae. Serum antibody titer (total antibody, IgG1 and IgG2a) to UV-inactivated C. caviae was determined at day 30 post vaccination (A) or at day 15 after challenge (B). Total Ig, IgG1 and IgG2a antibody titers were significantly (p<0.05) elevated in C. caviae EB vaccinated guinea pigs compared to mock vaccinated animals prior to challenge (A), but not after challenge (B).

Figure 3. C. caviae Immune Serum and Chlamydial Infectivity.

Sera obtained from guinea pigs (n = 5 per group) 30 days post vaccination with C. caviae or mock (PBS) or 15 days after challenge (+ challenge) were heat-inactivated (HK) at 56°C for 30 min or left untreated and diluted 1∶25 in DMEM before addition into 96 well plate. Sera from each animal was incubated with C. caviae (2×10⁴ IFU) for 1 hr in a shaking incubator, then sera/bacterial mixtures were added to HeLa cells (0.01 MOI) for bacterial enumeration. Bacterial numbers for each group were presented as a box-and-whisker plot. * p<0.05.

Figure 4. Effect of C. caviae Vaccination on Histopathological Lesions in the Genital tract from Guinea pigs following Chlamydial Challenge.

The genital tracts from each humanely euthanized guinea pig were removed at day 80 post C. caviae challenge fixed and embedded and then sectioned, and analyzed microscopically after H&E staining. (A) Representative photomicrographs of histological sections from uterine tissues are shown for each group of challenged guinea pigs with C. caviae EB vaccination (EB, n = 3) or mock vaccinated (Mock, n = 3). The superimposed images are magnifications of the regions of the indicated boxes to show details of inflammatory cell infiltration (c and f) and hemorrhage (a and d). Original magnification of the images (b and e) is ×200, while a, c, d and f are ×400. The light blue dash lines mark superficial layer exfoliation of the endometrial epithelium of the uterus (b), whereas the light blue solid lines indicate the intact endometrial epithelium of the uterus (e). Histopathological injury scores were calculated from five distinct morphological parameters (inflammatory cell infiltration, superficial layer exfoliation, edema, congestion and hemorrhage) in the uterus (B). Scores were calculated by examination of 5 consecutive sections (2 mm-interval) in every animal. Graphs expressed as mean ± SD, and compared using paired t- test. The asterisk indicates statistically significant differences (* p<0.05) between the C. caviae group and the mock group for the respective parameter.

Figure 5. Comparative Heatmap Depiction of Differential Gene Expression using RT-PCR array screening.

Three groups of guinea pigs were used for the comparative study: non vaccinated and non challenged (naïve), mock vaccinated but challenged (PBS/C), and C. caviae EB vaccinated and challenged (EB/C) groups. Each group contained three animals. The tissues (upper and lower genital tracts, U and L, respectively) from the respective groups of animals were collected at days 3 and 9 after challenge. Red shading indicates an increase in expression, while blue shading indicates suppression of expression, of the gene indicated on the right side of the panel. Lighter shades including white indicate similar levels of expression. Functional gene clustering is indicated by the brackets on the left showing 3 major groups consisting of innate, Th2 and Th1/inflammatory related genes.

Figure 6. Quantitative PCR Assessment of Bacterial Genomic Burdens in Lower and Upper Genital Tracts from C. caviae Mock or (PBS) Vaccinated Guinea Pigs.

Groups of three animals were euthanized on days 3 and 9 after C. caviae i.vag. challenge and tissues representing the lower or upper genital tract were aseptically collected. DNA was subjected to qPCR for GAPDH (host target used for normalization) and the single copy C. caviae tryptophan synthase gene (quantification of bacterial load). The average bacterial burdens for each tissue are depicted as grey (mock-vaccinated) or black (vaccinated) bars for each tissue and time point. LGT: lower genital tract. UGT: upper genital tract. * p<0.05, ** p<0.01.