Introduction

Chlamydiae is an obligate intracellular of Gram-negative bacteria and has a biphasic development cycle. The elementary bodies (EBs) exhibit infectivity without metabolic activity, and the reticulate bodies (RBs) are metabolically active without infectivity. EBs infect nearby host cells and transform into RBs to complete one life cycle [1]. Bacteria of the genus Chlamydiae are composed of nine species, and four of these bacteria infect humans: C. trachomatis, C. abortus, C. pneumoniae and C. psittaci. C. psittaci has received less research attention than the other three species.

C. psittaci is a zoonotic pathogen that causes psittacosis or ornithosis, which results in appreciable economic damage to pet birds and poultry. C. psittaci also infects humans and leads to life-threatening atypical pneumonia via the spread of infection from birds to humans [2]. Psittacosis is a rare, but potentially severe, disease in humans. No or merely flu-like symptoms, such as headache, fever, listlessness, and dry cough, appear early in the infection. However, the infection may further affect other systems and produce a fatal outcome without timely and effective antibiotic therapy [3].

Chlamydia causes a wide range of infections, such as respiratory, genital, and ocular infections, which result in considerable public health threats. Several antibiotics effectively treat chlamydial infection, but the current medical opinion is that the most effective strategy is prevention and protection of humans against chlamydial using an efficacious vaccination [4,5,6]. An effective vaccine should produce a persistent protective immune response via specific serotype and pathological immune stimulation. Unfortunately, no human vaccine has been developed despite significant advances and considerable efforts.

Plasmids may play a key role in chlamydial pathogenesis, and these structures widely exist in Chlamydiae species [7]. The plasmids of C. trachomatis and C. muridarum have been highly researched. Plasmid-deficient C. trachomatis and C. muridarum fail to induce severe pathology, which suggests that the plasmids are vital virulence factors in chlamydial infection [8,9,10]. Most C. psittaci strains also harbor a small highly conserved cryptic plasmid (~ 7.5 kb) [11, 12]. The plasmid of C. psittaci encodes eight open reading frames (ORFs) designated CPSIT_p1-8, and CPSIT_p7 encodes the protein Pgp3. Pgp3 is the only plasmid protein secreted into the host cell cytosol [13], and it is a major virulence factor for the induction of hydrosalpinx for C. muridarum [14]. Notably, human antibodies recognize Pgp3 as an important immunodominant antigen in C. trachomatis infection [15]. Immunization with Pgp3 protein induces protective immunity against C. trachomatis infection in a mouse model [16]. However, the role of CPSIT_p7 (Pgp3) in C. psittaci is not clear. Research demonstrated that the CPSIT_p8 (Pgp4) protein of C. psittaci induced significant protective immunity in BALB/c mice [17]. Pgp4 regulates the expression of pgp3, but Pgp3 does not affect the function of Pgp4. Therefore, we hypothesized that CPSIT_p7 would exhibit protective effects against C. psittaci infection and may be a candidate vaccine for C. psittaci.

The present study investigated the efficacy of immunization with the plasmid-encoded protein of C. psittaci CPSIT_p7 (Pgp3) in protecting against C. psittaci infection in BALB/c mice using recombinant CPSIT_p7 proteins. The results demonstrated that three immunizations with recombinant CPSIT_p7 proteins induced strong immune responses and protection against C. psittaci infection. The protective effects of the CPSIT_p7-specific immune response relied on CD4+ T lymphocytes.

Materials and methods

Cultivation of C. psittaci

C. psittaci 6BC EBs (a gift from G.M. Zhong, USA) were cultured on HeLa 229 monolayer cells, purified from infected cells using density gradient centrifugation, and stored at − 80 °C. The number of C. psittaci EBs was counted using indirect immunofluorescence (IFA) [17]. Serial dilutions of the infectious EBs were inoculated onto HeLa 229 monolayer cells after culturing for 36 h, and the infected cells were fixed and stained using an FITC-labeled monoclonal anti-Chlamydia LPS antibody (Meridian Diagnostics, Inc., Cincinnati, OH) and anti-mouse IgG (Sigma, St. Louis, MO, USA). The titer is expressed as inclusion-forming units/ml (IFU/ml).

Preparation of recombinant proteins CPSIT_p7

The open reading frames of CPSIT_p7 gene containing 795 bp DNA fragments (GenBank:NC_015217) were amplified using polymerase chain reaction (PCR) from the genomic DNA of C. psittaci 6BC and inserted into the pET28a vector with CPSIT_p7 primer pairs (5′-CGC-GGATCC(BamHI)-ATGGGTAATTCTGGTTTTTAC-3′, 5′-CCC-AAGCTT(HindIII)-TTAACCATTTGTTTGTTGTTTT-3′) (Fig. 1a). The constructed DNA sequence was used to verify inclusion. The vectors were inserted into Escherichia coli BL21 strains and grown in Luria-Bertani medium (kanamycin 60 μg/ml). The his-tagged proteins were expressed from E. coli BL21 strains and induced using 0.6 M isopropyl-β-d-thiogalactoside (IPTG) for 4 h at 30 °C in LB medium (Fig. 1b). Bacterial suspensions were collected, washed with phosphate-buffered saline (PBS), and lysed using a lysis buffer (10 mM imidazole, 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 20% glycerol, and 1% Triton X-100) and lysozymes (final concentration, 2~4 g/ml). The resulting CPSIT_p7 proteins were purified using a Ni-NTA affinity resin (QIAGEN) (Fig. 1c). Fusion proteins were treated filtered through a polymyxin B cartridge (Sigma, St. Louis, MO, USA) to remove the endotoxins, and concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA).

Fig. 1
figure 1

Preparation of recombinant protein CPSIT_p7. a Amplification of the CPSIT_p7 (795 bp) gene from C. psittaci 6BC genomic DNA. b Recombinant proteins CPSIT_p7 were expressed from E.coli BL21 strains containing a pET28a-CPSIT_p7 plasmid and induced with (+) or without (−) IPTG. c CPSIT_p7 proteins purified using Ni-NTA affinity resin. d Western blotting analysis was used to identify the CPSIT_p7 proteins and production of CPSIT_p7-specific antibodies in immunized-CPSIT_p7 mice. Ten microliters of purified CPSIT_p7 was loaded onto a 12% SDS-PAGE gel, and the gel was blotted onto a PVDF membrane using anti-His monoclonal antibody sera, e sera of BALB/c mice immunized with CPSIT_p7, and f sera of control group mice

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Western blotting

Purified recombinant proteins CPSIT_p7 were diluted (4:1) in 5× sample loading buffer and boiled for 10 min. Protein samples (10 μl) were separated using 12% SDS polyacrylamide gel (SDS-PAGE) electrophoresis. Separated proteins were blotted onto polyvinylidene fluoride (PVDF) membranes and blocked with Tris-buffered saline (TBS) containing 0.05% Tween 20 (Sigma, St. Louis, MO, USA) (TBST) and 5% skim milk at room temperature (RT) for 3 h [2]. The PVDF membrane was probed using an anti-His-tag mouse monoclonal antibody (Fig. 1f) (1:500 dilution in TBST containing 5% skim milk) or a pool of sera collected from CPSIT_p7 (Fig. 1e) or sera of control group mice (Fig. 1f). The membrane was washed with TBST and incubated with HRP-conjugated goat anti-mouse IgG (Abcam, UK) for 1 h at 37 °C. The color was developed using ECL Prime Western Blotting Detection Reagent (Auragene).

Mice and immunization

Three groups (n = 3) of 30 pathogen-free 6-week-old female BALB/c mice (Hunan SJA Laboratory Animal Co. Ltd.) were immunized three times at 2-week intervals via intraperitoneal injection (i.p.) with 40 μg purified recombinant CPSIT_p7 proteins diluted in PBS. PBS and Freund’s adjuvant (FA, Sigma, St. Louis, MO, USA) were used as the negative control and blank control, respectively. The first immunization of CPSIT_p7 proteins was emulsified in an equivalent volume of Freund’s complete adjuvant (FCA) in a volume of 0.1 ml and subsequently in Freund’s incomplete adjuvant (FIA). Clinical manifestations and body weight were observed and recorded during immunization. Blood samples of the 30 mice in each group were harvested via tail bleeding 2 weeks after the first two immunizations, and 15 mice from each group were euthanized 2 weeks after the last immunization. Blood was harvested via retro-orbital bleeding, and spleens were removed using aseptic surgery. Serum samples were recovered via centrifugation (4000 rpm, 8 min) and stored at − 20 °C for the detection of antibody production and neutralization of C. psittaci. Twelve spleens from each group were homogenized in RPMI 1640 (Hyclone, Logan, Utah, USA) containing 10% fetal calf serum (FBS; Invitrogen, USA) for the detection of cytokine level and adoptive transfer experiments 2 weeks after the last immunization. All of the animal experiments complied with the University of South China Guide for the Use of Laboratory Animals.

Mouse challenge and culturing of C. psittaci from the lungs

Immunized and control mice were anesthetized with ether and infected intranasally (i.n.) with 30 μl of 5 × 105 IFUs C. psittaci in a sucrose-phosphate-glutamic acid (SPG) buffer 2 weeks after the last immunization to evaluate the protective efficiencies [18, 19]. All mice were weighed before and 3, 7, and 10 days after challenge. The remaining 15 mice in each group were euthanized, and ten infected lungs from each group were dissected and homogenized in Hanks’ solution (Hyclone, Logan, Utah, USA) 10 days after infection, and one-half of the lung homogenates were re-suspended in SPG buffer for cytokine level determinations. The other homogenates were re-suspended in SPG buffer containing gentamicin (10 μg/ml), vancomycin (100 μg/ml), and streptomycin (100 μg/ml) for C. psittaci titer measurements. Lung homogenates from each mouse were centrifuged (20 min, 4000 rpm), and serial dilutions of the supernatants were inoculated onto HeLa 229 cell monolayers. The number of inclusion was counting using a fluorescence microscopy assay. The lungs of five mice from each group were fixed in 10% formaldehyde and embedded in paraffin for histopathological evaluation with hematoxylin-eosin (H&E) and an UltraSensitive™SP (Rabbit) IHC Kit (Maixin, China).

Splenic cell proliferation assay

Three spleens from each group were harvested, and splenic cells from each mouse were re-suspended in RPMI-1640 (1% antibiotics) to evaluate the CPSIT_p7-specific cellular immune response. Cells were diluted to 1 × 105 cells/ml and seeded onto culture plates. Each well received 10 μg/ml of puried-CPSIT_p7 for re-stimulation, and the plate was incubated at 37 °C with 5% CO2 for 48 h. Lymphocyte proliferative capacity was estimated using a Cell Counting Kit-8 (Dojindo, Japan).

Cytokine determinations from the spleen and lung

Splenic cells were cultured for 48 h and centrifuged (4 °C, 20 min, 4000 rpm). The supernatants were harvested for determination of cytokines. The supernatants of lung homogenates were collected from C. psittaci-infected mice after immunization and centrifuged as described above. Cytokine concentrations were measured using the Ready-SET-Go! Kits (eBioscience, USA). The limits of the IL-4, IL-6, IL-10, IL-2, IL-12, IL-17, and interferon-γ (IFN-γ) measurements were 500 pg/ml, 500 pg/ml, 4000 pg/ml, 200 pg/ml, 2000 pg/ml, 700 pg/ml, and 2000 pg/ml, respectively. Duplicate clarified supernatants were prepared according to the manufacturer’s guidelines.

In vivo neutralization of mice

Three groups (n = 3) of five mice were infected intranasally with 5 × 105 IFUs of C. psittaci. The infected mice were injected with 200 μl of sera obtained from mice immunized with PBS, FA, or CPSIT_p7 24 h after infection. All mice were euthanized 10 days after sera injections, and C. psittaci titers were measured as described above.

In vitro neutralization of C. psittaci

Sera obtained from mice immunized with CPSIT_p7, FA, or PBS were mixed with 5 × 105 IFUs of C. psittaci and incubated for 1 h at RT. Three groups (n = 3) of five mice were treated intranasally with C. psittaci, which were pre-treated with immunized mice sera in vitro. All mice were euthanized 10 days after infection, and the C. psittaci titers were measured as described above.

Spleen cell negative selection and adoptive transfer

All spleen cells were collected individually from CPSIT_p7-, FA-, or PBS-immunized mice 2 weeks after the last immunization via lysis of the erythrocytes, washed twice with PBS, and re-suspended in PBS (pH 7.0). CD4+ or CD8+ T cells were depleted from the spleen cells using micro-beads coated with CD4 (L3T4) or CD8a (Ly-2) antibodies (Miltenyi Biotec, Germany) and a magnetic cell sorting system (Miltenyi Biotec, Germany) for the negative selective transfer. FACS analysis evaluated the efficiency of cell sorting after staining with anti-CD4-TC and anti-CD8-rPE antibodies. The remaining CD4+ or CD8+ cells were < 1%. Approximately 1 × 106 depleted cells were inoculated into the tail vein of native mice. Mice were infected with 5 × 105 IFUs of C. psittaci 24 h after transfer. Lung tissues of the mice were isolated and homogenized in SPG on day 10 post-infection. C. psittaci titers in lungs were measured as described above.

Statistical analysis

Statistical comparisons of the data between groups for C. psittaci burden and cytokine levels after infection were computed using SPSS 18.0 software and one-way analysis of variance (ANOVA) with the Student-Newman-Keuls test. Differences were considered statistically significant at P < 0.05.

Results

Evaluation of CPSIT_p7-specfic humoral and cellular immune responses

Sera obtained from immunized mice 2 weeks after the last immunization were assessed using Western blotting to investigate the ability of CPSIT_p7 proteins to induce the humoral immune response. No adverse reactions or weight changes were observed between CPSIT_p7-immunized and control groups during immunization (data not shown). Three immunizations with CPSIT_p7 proteins (Fig. 1e) induced the production of protein-specific antibodies, and the control sera did not react with CPSIT_p7 proteins (Fig. 1f). Splenic cells collected from PBS-, FA-, or CPSIT_p7-immunized mice were stimulated in vitro for 48 h with 10 μg/ml CPSIT_p7 proteins to determine cell proliferative responses and cytokine levels in spleen cell supernatants. The stimulation index (SI) of the CPSIT_p7-immunized group was significantly higher than the negative control groups (PBS- or FA-immunized mice) (Fig. 1s). Spleen cell supernatants were collected after stimulation with CPSIT_p7 proteins to determine the expressions of IL-2, IL-4, IL-6, or IFN-γ (Fig. 2) using ELISA. IL-4 and IL-10 levels were not significantly different between the CPSIT_p7-immunized and control mice, but IL-2 and IFN-γ levels of the CPSIT_p7-immunized group were significantly higher than control mice.

Fig. 2
figure 2

Cytokine recall responses after immunization. The production of IL-2, IL-4, IL-10, and IFN-γ in splenic cell culture supernatants was detected using an ELISA kit according to the manufacturer’s instructions. Means ± standard deviation of the data from 10 mouse lungs in three independent experiments. **P < 0.01. ***P < 0.001

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Effect of the protective immunization of the CPSIT_p7 proteins against C. psittaci infection

Mice were infected i.n. with C. psittaci and sacrificed 10 days after infection to examine whether the CPSIT_p7-specific immune response protected the mice against C. psittaci infection 2 weeks after the final immunization. Mice were weighed during the infection, and no significant differences between the CPSIT_p7-immunized and control groups were observed (data not shown). The protective efficacy of CPSIT_p7 was measured using the C. psittaci burden in the lungs of infected mice (Fig. 3). The CPSIT_p7-immunized mice exhibited a significantly lower chlamydial load compared to PBS- or FA-immunized mice.

Fig. 3
figure 3

The C. psittaci burden in the lungs of immunized mice after C. psittaci infection. Mice were infected with 5 × 105 IFUs of C. psittaci 2 weeks after the last immunization and sacrificed 10 days later. Lung homogenates were prepared and inoculated on HeLa cells, and the C. psittaci inclusions were counted using IFA. Means ± standard deviations of C. psittaci titers (IFU/lung) from 10 mice in three independent experiments are shown.*P = 0.028. **P = 0.008

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Serious pathological damages were observed in PBS-immunized (Fig. 4a(1)) and FA-immunized (Fig. 4a(2)) mice, and inflammatory infiltrates were significantly reduced in H&E-stained lung sections stained of C. psittaci-infected mice immunized with CPSIT_p7 proteins (Fig. 4a(3)). The alveolar structure was clearly recognized, and fewer pathological features were observed, such as alveolar interstitial thickening, alveolar hemorrhage, and inflammatory cell infiltration, compared to control animals (Fig. 4a). Streptavidin-peroxidase (S-P) immunohistochemistry revealed that CPSIT_p7-immunized mice (Fig. 4b(3)) exhibited a remarkably lower chlamydial load (brown granule) (black arrow) compared to control animals (Fig. 4b(1, 2)).

Fig. 4
figure 4

Pathological lesion and C. psittaci burden of lung tissues from PBS-immunized (1), FA-immunized (2), or CPSIT_p7-immunized (3) mice after C. psittaci infection. a Ten days after C. psittaci infection, the lungs of immunized mice were stained with H&E. b The S-P immunohistochemistry kit with a rabbit anti-C. psittaci 6BC was the first antibody, and it was used to test C. psittaci inclusion. Brown granules (black arrow) express the C. psittaci inclusion

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Potential vaccine candidates must induce a protective immune response without stimulation of pathological immunity during infection. Therefore, we measured the production of IL-4, IL-6, IL-10, IL-12, IL-17, and IFN-γ in the lungs of immunized mice using ELISA (Fig. 5). The concentrations of IFN-γ in the lungs of CPSIT_p7-immunized mice were significantly lower than control mice, and there were no significant differences in the levels of other cytokines between the three groups.

Fig. 5
figure 5

Concentration of inflammatory cytokines in the lungs of immunized mice after C. psittaci infection. The supernatants of lung homogenates were tested using IL-4, IL-6, IL-10, IL-12, IL-17, or IFN-γ ELISA kits. Means ± standard deviations of the data from 10 mouse lungs in three independent experiments. ***P < 0.001

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Effect of serum neutralization and spleen cells of immunized mice on the outcome of C. psittaci infection

Immunization with CPSIT_p7 proteins induced specific humoral immune responses. Therefore, we further evaluated the capacity of these antibodies to protect against C. psittaci infection. Mice were intranasally infected with C. psittaci, and sera harvested from CPSIT_p7-, FA-, or PBS-immunized mice 2 weeks after the final immunization were intraperitoneally injected in infected mice. Mice were sacrificed 10 days after sera injection, and the Chlamydiae collected from lungs were measured. The C. psittaci burden was not significantly different between CPSIT_p7-treated immune sera and control sera (Fig. S2). Native mice were injected with C. psittaci-treated immune sera and sacrificed after 10 days to examine the in vitro neutralization of C. psittaci. Similarly, the chlamydial load of experimental group was not reduced compared with the control groups (Fig. S2). These results revealed that the sera of the CPSIT_p7-immunized mice did not contain antibodies to protect against C. psittaci.

The CPSIT_p7-specific antibodies did not exhibit neutralizing activity to C. psittaci infection. Therefore, we further investigated the protective efficiency of the CPSIT_p7-specific cellular immune responses. Spleen cells harvested from PBS-, FA- or CPSIT_p7-immunized mice were depleted of CD4+ or CD8+ cells and transferred to native mice. The native mice were infected with C. psittaci after 24 h and euthanized after 10 days. The adoptive transfer of specific spleen cells depleted of CD4+ cells produced no significant difference in C. psittaci burden between the three groups. The adoptive transfer of CPSIT_p7-specific spleen cells depleted of CD8+ cells significantly reduced the C. psittaci burden in the lungs of mice compared to control groups (Fig. 6).

Fig. 6
figure 6

C. psittaci burden in the lungs of mice after adoptive transfer. Spleen cells of groups of 10 mice were collected individually from PBS-, FA, or CPAIT-P7-immunized mice, depleted of CD4+ or CD8+ cells, and transferred to native mice. The native mice were infected with C. psittaci 24 h after transfer and euthanized 10 days later. Lung tissues of the mice were collected, and the C. psittaci titers were detected using IFA assay. **P = 0.002. ***P < 0.001

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Discussion

C. psittaci is an important zoonotic pathogen that infects humans and results in severe atypical pneumonia [2]. C. psittaci contains a highly conserved plasmid that is an essential virulence factor in chlamydial infection, similar to C. trachomatis and C. muridarum. The function of the plasmids of C. trachomatis and C. muridarum was the subject of considerable research, but little is known about the C. psittaci plasmid. Pgp3 is a plasmid-encoded protein of chlamydia. Analysis of the Smart BLAST database from NCBI demonstrated 70% sequence identity between CPSIT_p7 (C. psittaci plasmid-encode) and pORF5 (C. trachomatis plasmid-encode) and 71% between CPSIT_p7 and TCA04 (C. muridarum plasmid-encode), all of which encode plasmid protein Pgp3. The function and immunological role of Pgp3 are well-defined in C. trachomatis and C. muridarum, but not in C. psittaci.

The current research evaluated the immune responses and protective efficacy of BALB/c mice immunized with CPSIT_p7 against C. psittaci infection to aid in the development of candidate vaccines for the prevention and control of C. psittaci infection. We examined whether immunization of BALB/c mice with CPSIT_p7 induced an immune response that was sufficient to protect against C. psittaci infection in mice.

The sera of immunized mice were used to detect specific antibodies using Western blotting, and the results confirmed that multiple immunization with CPSIT_p7 proteins induced the production of CPSIT_p7-specific antibodies in mice. We evaluated the cellular immune response of protein-immunized mice. Previous studies demonstrated that immunity against chlamydia infection was primarily dependent on the contribution of Th1 cells [20,21,22,23], and CD4+ Th1-related IFN-γ played a key role in providing protective immunity against chlamydial infection [24, 25]. The levels of IFN-γ and IL-2 in CPSIT_p7-immunized mice were significantly increased in our experiments, which are CD4+ Th1-related cytokines. We found that T-cell proliferative responses of mice immunized with CPSIT_p7 were significantly increased compared to control mice. These results indicated that the CPSIT_p7 proteins induced a strong CD4+ T cell response.

The present study discovered that immunization with CPSIT_p7 induced a significant reduction in chlamydial burden and inflammatory infiltrates. Donati et al. demonstrated that immunization with Pgp3 DNA induced resistance to re-infection of C. trachomatis in mice [26]. Immunization with the pORF5 DNA vaccine induced protective immunity against genital C. trachomatis infection in a murine model [27]. Recombinant Pgp3 protein-immunized mice exhibited a significantly lower C. muridarum burden [16]. These results suggest that the role of Pgp3 of C. psittaci is similar to C. trachomatis and C. muridarum. The very low level of INF-γ in our experiments supports the decrease in inflammatory infiltrates in the lungs of CPSIT_p7-immunized mice. Earlier studies revealed that IFN-γ was responsible for the clearance of chlamydial infection in female mice [28], which supports CPSIT_p7 as a vaccine candidate to induce this immune response.

We designed in vitro and in vivo neutralization experiments and adoptive transfer experiments of CD4+/CD8+ T cells to further examine the potential mechanisms of CPSIT_p7 protection against C. psittaci. The number of recoverable chlamydia was not significantly different between CPSIT_p7-immunized sera and control sera for in vivo and in vitro neutralization experiments. These data suggest that the specific-CPSIT_p7 antibodies exert no protective effect against C. psittaci infection. Similar results were observed in previous experiments [16, 26]. These results demonstrated that CPSIT_p7-immunized mice elicited a powerful Th1 immune response, which suggests that the specific-CPSIT_p7 antibodies may assist Th1 immune responses by strengthening antigen uptake and presentation [29].

The adoptive transfer of CPSIT_p7-specific CD4+ T cells significantly reduced the chlamydial load in the present experiment, but not the transfer of negatively selected CD8+ T cells. Pgp3-specific (C. muridarum encoded) and MOMP-specific (outer membrane protein of C. muridarum) CD4+ cells also significantly reduced the amount of recoverable Chlamydiae after infection [16, 30]. Our results support these findings. MOMP induced efficient protective immunity against C. muridarum genital infection in C3H/HeN [31,32,33,34]. CD4+ T cells are important for protection against intracellular zoonotic bacterial pathogens, which are involved in the production of macrophage-activating factors and assist B cells to enhance antibody production [35,36,37]. In contrast, Morrison et al. found that CD4+ T cells were not necessary to protect against murine Chlamydia trachomatis genital infection [38]. Some studies indicated that CD8+ cells provided a very restricted or no protective effect, which is similar to our study results [39, 40]. However, the significance of the reduction of the chlamydial load in lungs is not certain, but the reduction of the chlamydial load reduced the transmission of chlamydial infection.

In conclusion, vaccination with CPSIT_p7 induced a strong Th1-type immunity response and elicited protective effects against C. psittaci infection. CPSIT_p7-specific CD4+ T cells reduced the chlamydial load in the lungs of native mice after infection challenge. These results provide important directions for future studies of vaccine development against C. psittaci infection.