Chlamydial Protease-Like Activity Factor Mediated Protection Against C. trachomatis In Guinea Pigs

Abstract

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared to day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high level 4 weeks post challenge. Upregulation of antigen specific IFN-γ gene expression was observed in rCPAF/CpG vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared to CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

Introduction

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted diseases (STD) worldwide¹. When left untreated, it leads to chronic inflammatory conditions including pelvic inflammatory disorder, ectopic pregnancies and infertility^1–3. Given the asymptomatic nature of infection in a high proportion of affected individuals, and the intracellular persistence of the organism, recurring infections and chronic disease lead to significant health care costs^4–6. Although the infection can be treated with antibiotics⁷, preventive intervention by vaccination is considered the most effective measure to control chlamydial STD.

Currently, there is no licensed vaccine against Chlamydia spp.; however, our laboratory has extensively demonstrated that immunization with recombinant chlamydial protease-like activity factor (rCPAF) is highly effective in protection against subsequent challenge in the mouse model^8–16. Specifically, intranasal (i.n.) vaccination with rCPAF, with murine recombinant IL-12 or CpG deoxynucleotides as adjuvant, protects against subsequent genital chlamydial challenge and reduces the incidence of hydrosalpinx development in the upper reproductive tract^{10, 11}. Moreover, rCPAF vaccination preserved fertility in mice repeatedly challenged with Chlamydia¹⁵. Protection by rCPAF vaccination has been found to be mediated via IFN-γ secreting antigen specific CD4⁺ T cells and antibodies^{11, 12}.

Although the mouse model is widely used for Chlamydia studies^17–19; the availability of other models, such as guinea pigs, pigs, minipigs, sheep, cattle, and macaques^20–23, to study chlamydial pathogenesis and vaccine strategies is highly beneficial. Guinea pigs serve as a translation model between mice and human Chlamydia studies as they have a reproductive physiology and estrous cycle (15 to 17 days) similar to that of humans^{20, 24}. Additionally, certain guinea pig strains are outbred (as used in our study) and may add to understanding the genetic basis of differences in anti-chlamydial immunity in human cohorts. To date, guinea pigs have been used to evaluate the protective efficacy of recombinant major outer membrane protein (rMOMP) against genital chlamydial infection, where reduction in genital pathology following Chlamydia challenge is associated with systematic and mucosal antibody production^25–27. Furthermore, our recent report on the regulation of guinea pig-specific genes in vaccinated animals using a 96-gene transcriptome array has advanced our understanding of guinea pig immune responses to vaccination and infection²⁸. In that report, we found that intranasal inoculation/vaccination with Chlamydia caviae elementary bodies induced robust neutralizing antibodies and regulated Th1 and Th2 related cytokine/chemokine genes, including IFN-γ²⁸ which has been shown to play a major role in the control of chlamydial infection in mice^{8, 29, 30}.

While C. caviae is the naturally infecting strain in guinea pigs and has been extensively used to study chlamydial infection in the guinea pig model^{26, 28}, de Jonge et al., has developed the human C. trachomatis serovars D and E infection model in guinea pigs and reported development of upper genital tract pathology comparable to human disease³¹. The rCPAF vaccine extensively characterized by our group in the mouse model is derived from the human C. trachomatis L2 serovar and shares a 99% and 82% identical amino acid sequence to the human CT-D serovar and mouse C. muridarum strain, respectively³². However, this rCPAF has only 54% identity to C. caviae CPAF protein, therefore, we utilized the newly developed guinea pig CT-D infection model³¹ to examine the efficacy of rCPAF as a vaccine in this second animal model.

Results

Resolution of genital C. trachomatis challenge in guinea pigs immunized with rCPAF

Using a vaccination regimen similar to the one used for mice studies^{10, 11, 16}, we evaluated the protective efficacy of rCPAF in guinea pigs. In this study, we used CpG-10109 and a vaccination dosage based on other guinea pig reports^{25, 33, 34} and pilot studies in our laboratory. As shown in Fig. 1a, CpG (mock) immunized guinea pigs displayed higher levels of chlamydial shedding initially which then progressively decreased with complete clearing of the infection (no detectable bacterial shedding) by day 33. In contrast, guinea pigs i.n. inoculated with 10⁵ IFUs live CT-D (serves as positive control for immunization) exhibited significant reduction of bacterial shedding on days 3 and 6 post-challenge with complete clearance on day 9. Immunization with rCPAF+CpG resulted in a significant reduction in chlamydial shedding compared to those receiving CpG alone as early as day 3 post-challenge. The bacterial shedding profile displayed a significant reduction in CT-D and rCPAF+CpG immunized guinea pigs when compared to CpG-mock immunized animals (Figure 1b). Also as shown in figure 1c, the area under the curve showed a significant reduction in bacterial burdens in CT-D and rCPAF+CpG immunized guinea pigs compared to CpG-mock immunized guinea pigs (Figure 1c). Furthermore, 40% of the rCPAF immunized guinea pigs resolved infection on day 21 and all animals cleared infection by day 24; whereas, all CpG-mock immunized animals still exhibited chlamydial shedding on day 27 and ultimately resolved the infection by day 33 (Fig. 1d).

Figure 1.

Vaccination enhanced chlamydial clearance from the guinea pigs genital tract. Groups (n =5) of guinea pigs were immunized i.n. with rCPAF/CpG or CpG alone (mock) and boosted twice at two-week intervals. Another group (n = 5) of guinea pigs received one i.n. dose of live C. trachomatis serovar D EBs (CT-D; 1×10⁵ IFUs). One month after the final immunization, guinea pigs were challenged i.vag. with 10⁵ IFU CT-D. Chlamydial shedding was monitored every third day post challenge until day 36 and presented as mean ± SD for each group at each time point. * Significant reductions (p < 0.05; one-way ANOVA) in bacterial shedding by (a) bar graph and (b) overall area and (c) area under the curve between the indicated group and CpG-immunized (mock) guinea pigs are shown. (d) The number of guinea pigs shedding Chlamydia after genital challenge for each immunization group is summarized. (c) ** Significant reduction (p=0.002), ANOVA with Tukey B) between groups; (d) ** Significant reduction (p=0.006 and p=0.0001, respectively, Fisher’s exact test) in number of rCPAF+CpG and CT-D immunized animals, compared to CpG immunized animals, shedding chlamydia across all time-points evaluated. Results are representative of two independent experiments.

rCPAF/CpG vaccination induced antibody production in guinea pigs

Using cyclophosphamide treatment to preferentially suppress humoral, but not cell-mediated, immunity, Rank et.al, have demonstrated that a humoral response is essential for guinea pigs to resolve primary³⁵ and secondary³⁶ C. caviae genital infections. To assess humoral responses induced by vaccination and challenge, we collected sera from immunized animals 2 weeks prior to challenge and 4 weeks post-challenge and measured antibody reactivity. Prior to bacterial challenge, CpG vaccinated guinea pigs produced minimal serum antibody against rCPAF and EBs (UV-inactivated CT-D) as shown in Fig. 2a. In contrast, rCPAF/CpG and CT-D vaccinated guinea pigs induced high levels of serum antibodies against rCPAF and CT-D, respectively. All animals, including CpG vaccinated guinea pigs, mounted anti- CT-D humoral responses to i.vag. CT-D challenge (Fig. 2b). Among the 3 study groups, CT-D i.n. vaccinated guinea pigs produced the highest level of anti-CT-D antibody pre and post challenge. Serum anti-CT-D antibody production also increased in CpG and rCPAF/CpG vaccinated animals after CT-D challenge (Fig. 2b).

Figure 2.

Vaccination induced antigen specific serum IgG responses in guinea pigs. Groups (n=5–6) of guinea pigs were immunized i.n. with CpG alone (mock), rCPAF/CpG or CT-D (1×10⁵ IFUs). Guinea pigs were rested for 4 (CpG, rCPAF/CpG) or 8 (CT-D) weeks before challenging i.vag. with 1×10⁵ IFU of CT-D. Total serum IgG reacting with rCPAF (a) and UV-inactivated CT-D (b) was determined 2 weeks prior to (pre-challenge) and 4 weeks after (post-challenge) challenge. * p < 0.05, ** p < 0.01 (One-Way ANOVA) comparison between indicated groups. Results are presented as average ± standard deviation of endpoint titer of each group. Results are representative of two independent experiments.

rCPAF/CpG vaccination induced cellular IFN-γ response in guinea pigs

We have previously demonstrated that immunization with rCPAF induced a robust cell-mediated immune response with IFN-γ production and led to protection against genital chlamydial disease in mice^{10, 11}. To evaluate the cell-mediated response by vaccination in guinea pigs, we collected 3 spleens from each study group one day prior to challenge. Splenocytes were stimulated in vitro with antigens rCPAF and UV-inactivated CT-D (UV-CT-D) or unstimulated (with medium) for 24 hrs and the gene expression of IFN-γ was assessed by quantitative real time PCR. As shown in Fig 3, significantly higher transcript levels of IFN-γ were observed in splenocytes from the rCPAF/CpG group stimulated with rCPAF and in CT-D immunized animals stimulated with UV-CT-D compared to their respective medium stimulations. IFN-γ gene transcript levels corresponded to elevated IFN-γ secretion in supernatants of splenocytes from rCPAF/CpG or CT-D immunized guinea pigs stimulated with CPAF or CT-D compared to CpG vaccinated guinea pigs (Supplementary Figure 1). Additionally, TNF-α - a cytokine associated with its role in multifactorial Ag-specific T-cell mediated protection^37–40, displayed increased/ higher transcript levels (although not statistically significant) in splenocytes from the rCPAF/CpG group stimulated with rCPAF and in CT-D immunized animals stimulated with UV-CT-D compared to their respective medium stimulations (Supplementary Figure 2). Taken together, these results indicate that both rCPAF/CpG and CT-D vaccination mounted modest increases (given the small numbers of animals/ group) in Ag-specific Th₁ cytokines such as IFN-γ which contribute to the cell-mediated immune response.

Figure 3.

Vaccination induced antigen specific IFN-γ gene expression. Guinea pigs (n=3 per group) were euthanized one day before challenge and splenocytes were stimulated with 0.5µg of rCPAF or UV-inactivated CT-D (1×10⁵ IFUs), or unstimulated (media alone), for 24 hrs. IFN-γ gene expression was then measured by qRT-PCR analysis and normalized to respective GAPDH expression for each sample. Subsequently, within each vaccination group, IFN-γ expression by rCPAF and CT-D (UV) stimulation was calculated and presented as a relative value to medium mock stimulation. Significant increase in IFN-γ expression between stimuli was indicated * p < 0.05, ** p < 0.01 (One-Way ANOVA). Results are representative of two independent experiments.

rCPAF/CpG vaccination reduced upper genital tract pathology

Chlamydial infections result in upper genital tract pathology and reproductive sequelae such as tubal damage and infertility^{10, 11}. In order to evaluate the efficacy of vaccination against development of reproductive tract pathology, tissue sections were obtained from healthy and diseased guinea pigs (Figure 4a) at day 65 post challenge and the severity of pathology was scored using a comprehensive system reported previously²⁸. Following CT-D challenge, CpG vaccinated guinea pigs developed moderate to severe congestion, hemorrhage, and edema accompanied by severe infiltration of inflammatory cells (lymphocytes and neutrophils) in uterine tissues (Fig. 4b). In contrast, the rCPAF/CpG vaccinated animals had moderate cellular inflammation and edema, and exhibited a reduction in congestion and hemorrhage. Minimal pathology was developed in CT-D vaccinated guinea pigs and was comparable to mock vaccinated healthy naive animals. The difference in pathological severity among CpG, rCPAF/CpG, and CT-D vaccinated guinea pigs (8.8, 4.8, and 2.4, respectively, sum of 4 evaluated parameters in Fig. 4b for each group) was evident and comparable to similarly vaccinated/challenged mice (5.6, 2.0, and 1.5, respectively, combination of mouse oviduct dilatation and cellular infiltration scores, in our previous report⁴¹).

Figure 4.

Vaccination reduced histopathological lesions in indicated genital tracts following chlamydial challenge. Histopathology was assessed in naïve (uninfected/ healthy) and Chlamydia challenged CpG-, rCPAF/CpG-, CT-D-vaccinated guinea pigs. The genital tract of each guinea pig was removed at day 65 post CT-D challenge, sectioned, H&E stained, and analyzed microscopically (original magnification of the images is 100× or 200×). Histopathological injury in the genital tract of representative healthy and diseased guinea pigs was scored (a) and graphically represented for the respective group as a whole (b), for four distinct parameters (inflammatory cell infiltration, congestion, hemorrhage and edema). Obviously, the individual micrographs shown (a) represent the types of pathology observed in different regions of the genital tract of healthy and diseased animals, whereas the graph (b) summarizes these types of observations for all sections of all regions of all animals examined. The asterisk indicates significant reductions (* p <0.05; Kruskal-Wallis test with Dunns post hoc test) between CT-D or rCPAF/CpG immunized groups in comparison to CpG group for the respective parameters.

Discussion

We have previously demonstrated that vaccination with rCPAF protects against genital chlamydial challenge by robust induction of cellular and humoral immune responses in the murine model^{10, 11, 15, 16}. We now have demonstrated the protective efficacy of rCPAF in a second animal model, the guinea pig. Guinea pigs immunized with rCPAF/CpG exhibited lower levels of chlamydial shedding, a shortened duration of infection, and reduced upper genital tract pathology compared to CpG (mock) immunized animals. This protection was correlated with robust antibody production and cellular IFN-γ induction in an antigen specific manner. Also in line with our previous findings, we found a limited role for rCPAF specific antibodies in the CT-D immunization regimen⁴². Collectively, these results are consistent with the protective efficacy of rCPAF observed in the mouse model of chlamydial infection and further validate rCPAF role as a potential candidate to be used for the development of a licensed human Chlamydia vaccine.

In the current study, animals that received live i.n. C. trachomatis (CT-D) immunization exhibited rapid bacterial clearance and significant reduction in upper genital pathology following a secondary intravaginal chlamydial challenge. These results are consistent with those from the mouse model^{41, 43} and guinea pig vaccination studies^{25, 31} wherein, robust protection against genital chlamydial challenge is induced by live chlamydial EB immunization. Hydrosalpinx (fluid-filled oviduct dilatations) are a characteristic feature of pathological sequelae following chlamydial infections in their respective hosts. Hydrosalpinx was not a common feature of the guinea pig model; however, histopathology was observed in the uterine tissue sections of the previously estradiol-treated, CpG immunized and CT-D challenged animals. Estradiol, not progesterone, treatment prior to chlamydial challenge in guinea pigs has been shown to increase the intensity and duration of C. caviae infection⁴⁴ and is required to establish a sustained CT-D infection³¹. Although genital tract pathology induced by estradiol injection has been documented in guinea pigs⁴⁵, the estradiol treatment following our study regimen (two 5 mg injections 1-week apart) in the absence of chlamydial infection did not cause visible inflammation 68 days after the 2^nd injection (data not shown) suggesting that hormonal treatment alone contributed minimally to upper genital pathology in our model. Furthermore, similar to other animal models²⁰, CT-D challenge in guinea pigs resulted in milder genital tract pathology and uterine inflammation than that seen with the natural pathogen C. caviae, as demonstrated by our group and others^{28, 46}. In this context, Chlamydiae have been shown to exhibit host tropism due to their adaptation to the restrictive influence of IFN-γ in respective hosts⁴⁷.

However, despite the reduced severity of pathological outcomes following CT-D infection in the guinea pig, this model produces sufficient bacterial shedding and pathology to evaluate the effects of experimental vaccines. To this end, studies by us and others using live or UV-EBs for vaccination have demonstrated significantly accelerated chlamydial clearance within 7 days post challenge^{41, 48} compared to single antigens including rCPAF/CpG and MOMP which result in protection 7–10 days later⁴⁹. A correlation between early protection with neutralizing antibody induced by the live-EB or UV-EB regimen may occur, whereas given that CPAF is a RB-specific protein, anti-CPAF antibodies do not neutralize chlamydial infectivity, as demonstrated using B-cell deficient mice⁴²¹³. We have previously characterized the efficacy of rCPAF cloned from C. trachomatis genome⁴⁹ and further evaluated the effectiveness of this vaccine candidate (with only 54% amino acid identity to C. caviae) against a CT-D challenge in this study.

Consistent with our previous findings in mouse studies, vaccination with rCPAF (derived from CT) in guinea pigs resulted in reduced chlamydial shedding and genital tract pathology, correlating with CPAF-specific immune responses. CPAF is a dominant antigen expressed in CT positive individuals^{50, 51}. We have previously demonstrated that rCPAF immunization protects against a subsequent genital challenge in humanized HLA-DR4 transgenic mice⁹, and that it provides cross-serovar protection⁴⁹. The vaccination regimen conferred by rCPAF plus adjuvants (CpG or IL-12) exhibited greater protection that that observed by rCPAF vaccination alone¹³.

Taken together, previous reports and this current study extend the importance of rCPAF as a vaccine candidate and highlight its use as a protective molecule in a second animal model against a human serovar of CT. Despite new and evolving information on cellular targets of CPAF^{52, 53}, its role as a putative anti-Chlamydia vaccine candidate holds continued promise^{13, 54, 55}. With rapid progression in genetic manipulation of Chlamydia⁵⁶, there also is enthusiasm for the development of a safe and more effective live attenuated vaccine. Additionally, recent studies have highlighted anti-chlamydial immune responses via protective tissue resident memory T cell subsets in vaccinated mice⁴⁸, and B cells enhancing Ag-specific CD4⁺ T cell priming upon infection^{56, 57}.

In the context of a future Chlamydia vaccine and a possible role for CPAF, there is no consensus as to whether the desired outcome is a reduction in infectivity/transmission or a reduction in the clinically relevant chronic pathologies. Along with the reduction in bacterial shedding (around 2^nd week), we have previously demonstrated superior protection against upper reproductive pathologies with the rCPAF regimen¹¹, including protection against infertility induced following repeated chlamydial challenge¹⁵. Our findings, with regard to efficacy of rCPAF immunization, are in accordance with other reports^{58, 59}. Additionally, the concept that a reduction in shedding does not always correlate to protection against pathology is supported by O’Meara et al⁶⁰.

In summary, the guinea pig CT-D challenge model may be a useful for the evaluation of new and previously identified vaccine candidates. Vaccination with single protein antigens (such as CPAF and MOMP) may not be sufficient to generate desired protective efficacy in humans^{1, 4}. To this end, effective vaccination strategies using multiple antigens, formulations and routes of delivery are critical⁶¹. Importantly, rCPAF with its ability to preserve fertility in mice¹⁵, and reduce infection severity in guinea pigs has the potential to serve as an ideal antigen to be formulated into multivalent vaccines or be overexpressed in an attenuated live vaccine platform to prophylactically control human Chlamydia infection.

Methods

Bacteria

Chlamydia trachomatis serovar D (from the Zhong Lab, UT Health Sciences Center, San Antonio, TX) was grown on confluent HeLa cell monolayers. Infected HeLa cells were mechanically dislodged using glass beads. The disrupted cells were then vortexed with glass beads in a falcon tube for 5 min with 30 seconds intervals on ice followed by centrifugation for 10 min at 1200 rpm 4°C. The supernatant was collected and spun at of 27000 × g for 1 hr at 4°C to obtain a bacterial pellet that was further purified on Renografin gradient as described earlier²⁸. Purified elementary bodies were aliquoted and stored at −80°C in sucrose-phosphate-glutamate (SPG) buffer until use.

Guinea pigs

Dunkin Hartley strain guinea pigs (350g–450g) were purchased from Charles River Laboratories (Massachusetts, USA) and were housed in the AAALAC-accredited University of Texas at San Antonio Vivarium. Food and water were supplied ad libitum and all experimental studies were completed humanely and followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (IS0146) was approved for conducting this study by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas at San Antonio.

Immunization and challenge

Groups of guinea pigs (n=5) were immunized i.n. on day 0 with 150µg rCPAF (derived from human C. trachomatis serovar L2 with a 99% homology to serovar D³² and expressed as fusion proteins in Escherichia coli as described previously⁹) and 25µg of CpG nucleotides (CpG-10109^{25, 62}), or CpG alone (mock), in 100µl of sterile phosphate buffered saline (PBS). The dose of rCPAF was based on other reported protein vaccination studies using the guinea pig infection model^{25, 33, 34}. Booster i.n. immunizations were provided on days 14 and 28 with 100µg rCPAF and 25µg of CpG. One month following the last booster, guinea pigs were challenged i.vag. with 1×10⁵ IFU of CT-D resuspended in 50µl SPG buffer. Another group of guinea pigs was immunized i.n. with 1×10⁵ IFUs of live CT-D once and rested for 8 weeks before challenge. To achieve a sustained CT-D infection in guinea pigs, all animals received a subcutaneous injection of 5mg β-estradiol (Sigma) in 100µl sesame oil (Sigma) on days −10 and −3 prior to challenge as described by de Jonge et.al.³¹. All guinea pigs were anesthetized with 3% isoflurane before immunization and challenge procedures. Following challenge, vaginal swabs were collected at a 3-day interval for 36 days from all groups of guinea pigs and plated onto HeLa cell monolayers to determine the chlamydial burden as described previously²⁸.

Assay of humoral immune responses

Guinea pigs were bled from the lateral saphenous leg vein 15 days after the last booster (2 weeks prior to challenge) and 4 weeks post-challenge as described⁶³. To measure antibody reactivity, microtiter plates were coated with 1×10⁵ IFUs of UV-inactivated CT-D or 0.5µg rCPAF and incubated at 4°C overnight. ELISA was performed using serial 2-fold diluted sera (starting with1:100) as described²⁸ to assess antibody reactivity. Following serial dilution of serum samples, plates were incubated for 2 h, followed by incubation with goat anti-guinea pig total IgG conjugated to horseradish peroxidase (HRP) (ABD Serotec). Tetramethylbenzidine substrate was added and the absorbance quantified at 630 nm using a µQuant ELISA plate reader (BioTek Instruments, Winooski, VT). Endpoint titers of each serum for specific antigen(s) was determined by selecting the highest dilution factor at which the sample O.D. was greater than the average O.D. of 6 CpG mock immunized pre-challenge sera, plus 2.2 standard deviations (to achieve 95% confidence as suggested by Frey et.al.⁶⁴), and O.D. value greater than 0.1.

Cytokine PCR and ELISA

Groups of guinea pigs (n=3) were immunized i.n. with rCPAF/CpG or live CT-D as described above and euthanized one day prior to challenge to collect spleens aseptically for antigen-specific splenocyte stimulation assay. Single splenocyte cell suspensions were prepared (1×10⁶ cells/well in 100µl DMEM plus 10% FBS) and stimulated with 0.5µg rCPAF, UV-inactivated CT-D (1×10⁵ IFUs) or left unstimulated with media alone in a 96 well microtiter plate for 24 hrs. Following stimulation, cells were collected to assess IFN-γ gene expression. Messenger RNA was isolated from stimulated splenocytes as per manufacturer’s instructions (RNeasy mini kit, Qiagen) and quantified using a Nanodrop spectrophotometer (Thermo Scientific NanoDropTM 1000). RNA (0.5µg) was used to generate cDNA using the Verso cDNA Synthesis (Thermo Scientific). The guinea pig IFN-γ (Forward 5’-CCATCAAGGAACAAATTATTAC and Reverse 5’-TGACCGAAATTTGAATCAG), TNF-α (Forward 5’- GGAAGAGCAGTTCTCCAG and Reverse 5’-GCTTGTCATTATCGTTTTGAG), and GAPDH (Forward 5’-CTCGTCATCAATGGAAAG, Reverse 5’- GTGGATTCCACTACATAC) gene specific primers were used to amplify gene transcripts from the synthesized cDNA. qRT-PCR was conducted under a previously optimized condition using the CFX96 Touch™ Real-Time PCR Detection System (Bio-rad)²⁸ and IFN-γ and TNF-α mRNA levels were normalized to GAPDH mRNA and expressed as relative level to medium mock-stimulated samples using the comparative cycle threshold method⁶⁵. Supernatants from each condition were collected and stored at −80°C until further use. Guinea pig IFN-γ was assessed by a quantitative competitive immunoassay (NeoScientific) according to manufacturers’ instructions. In brief, 100ul of experimental supernatants or standards were co-incubated in wells with an IFN-γ conjugate. Binding of IFN-γ HRP was visualized by production of colorimetric reaction products that were quantitatively measured by absorbance using a µQuant ELISA plate reader (BioTek Instruments, Winooski, VT).

Genital tract pathology

Guinea pigs were euthanized and genital tract tissues were collected in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histopathology imaging and pathological scoring were performed using a Carl Zeiss microscope as described previously²⁸. Pathology scores for each group was represented as mean and SD of each guinea pig in the respective group.

Animals and Statistical analyses

For experiments, female guinea pigs were age- matched and numbers/ group was selected based on previous findings^{11, 28}. All result datasets from experiments were included for analyses and were not excluded from the study. No randomization or blinding was done. GraphPad Prism 5 (La Jolla, CA) was used to perform all statistical tests. P < 0.05 was considered statistically significant. One way ANOVA or Kruskal-Wallis test with Dunns post hoc test was used for determining the protective efficacy of rCPAF-vaccination compared to other treatment groups. Appropriate statistical tests are indicated in legends of respective figures. All data are representative of two independent experiments and each experiment was analyzed independently.