Design,Optimization Of Chlamydia Psittaci Multi-epitopes Fusion Vaccine And The Protective Immunity Induced By Multi-epitopes Fusion Vaccine

Background and objective:Chlamydia psittaci is a widely distributed and strict obligate intracellular pathogen with multiple hosts and may cause different infectious diseases in different hosts,humans,birds,livestock and some mammals are susceptible hosts of Cps.In recent years,Cps infectious diseases are increasing year by year in the world,and the prevalence and incidence of Cps in humans and animals has reached their highest levels worldwide in a decade.Thus,effective prevention and control of Cps infection is a great significance for protecting public health and promoting the development of animal husbandry.As the best way to prevent chlamydia infection,vaccination has been paid more attention in humans.Although chlamydia has made some achievements in attenuated whole bacteria vaccine and subunit vaccine for many years,there is still no clinically available anti-infection vaccine for chlamydia due to the limitations of high toxic,side effects and weak efficacy of the above vaccines.Multi-epitope fusion vaccine is a research focus for the past few years,due to the characteristics of well-targeted immunity,less allergenic components and high stability,it has more advantages than traditional vaccines.However,there are still few reports on multi-epitope fusion vaccine of chlamydia.In this study,we predict and identify the dominant epitopes of Cps MOMP and CPSIT_p6 by bioinformatics prediction,and construct a new multi-epitope fusion antigen of Cps based on above dominant epitopes to evaluate its immunogenicity and protection against chlamydia infection.And then,the optimal immunization strategy was designed and a new vaccine adjuvant was constructed to further optimize the multi-epitope fusion vaccine of Cps.Overall,our study will provide new insight into the design and development of chlamydia vaccine,and it is of great significance for the prevention and control of chlamydiaMethods:1.The amino acid sequences of Cps MOMP and CPSIT_p6 proteins were obtained from Genbank.The secondary structure and the hydrophilicity,flexibility and accessibility of above proteins were predicted by DNAStar and IEBD,respectively.The potential T-cell sites was evaluated by SYFPEITHI software and the antigenic index was calculated to verify the prediction of the dominant epitopes Finally,the dominant epitopes of MOMP and CPSIT_p6 proteins were comprehensively predicted based on the above analysis2.The dominant epitope peptides of MOMP and CPSIT_p6 proteins were constructed for intraperitoneal immunization of BALB/c mice After immunized three times with peptides,the serum IgG and IgA antibodies were detected to assess humoral immune response.Splenocyte samples of immunized mice were collected to determine the effect of peptides on the proliferation of splenocytes,and the levels of IFN-y and IL-10 secreted by peptides stimulated-splenocytes were detected by ELISA.3.The multi-epitope fusion antigen based on MOMP and CPSIT_p6 were constructed and BALB/c mice were immunized three times with the multi-epitope antigen.Blood samples were collected to determine the levels of serum IgG,IgM,IgA and IgG subclasses antibodies.Splenocyte samples of immunized mice were collected to detected the levels of IFN-y,IL-2,IL-4 and IL-10 in the supernatants of splenocyte.2 w after the final immunization,mice were challenged intranasally with Cps.On day 10,infected-lung tissues were isolated and partial homogenized for Cps burden determination.And other lung tissues were stained with H&E and S-P immunohistochemistry to evaluate the protective efficiencies.4.After intraperitoneally immunizied with the multiple-epitope fusion antigen for three times,splenocyte samples were collected to depleted CD4+or CD8+cells by magnetic beads separation,and then adoptively transferred to the new native BALB/c mice.The BALB/c mice were challenged intranasally with Cps 1 d later.Lungs were collected on day 7 post-infection to measured the Cps titers.5.BALB/c mice were simultaneous immunized with intramuscular and intranasal for three times.2 w after the final immunization,blood,nasal washes and vaginal washes were collected from mice to detect the levels of serum antibodies and secretory IgA(sIgA).The levels of splenocyte supernatant or intracellular cytokines were detected by ELISA and flow cytometry.2 w after the final immunization,BALB/c mice were challenged intranasally with Cps,and the infected-lung tissues were isolated and partial homogenized for cytokine levels,Cps burden determinations and pathological examination.Finally,qRT-PCR were used to assess the Cps burdens in different tissues.6.CNPs/HBc-144 was constructed based on CNPs and HBc-144 adjuvants,the morphological structure and stability of CNPs/HBc-144 were detected by physical-structural characterization analysis.And then,CNPs/HBc-144 was mixed with fusion antigen and immunized continuously for 7 days to detected the weight changes of mice.After immunization for 7 days,the mice were sacrificed to collected the lung tissues and injected-muscle tissues for H&E staining.7.BALB/c mice were immunized four times with CNPs/HBc-144-Ags at 2-week intervals.Blood samples,nasal washes and vaginal washes were collected from imunnized BALB/c mice to detect the levels of serum IgG,IgA and sIgA.The levels of CD4+,CD8+cells and the secretion of CD44/CD62,intracellular cytokines were detected by flow cytometry,and the levels of supernatant cytokines were detected by ELISA.The infected lung tissues were collected for inflammatory cytokines level and pathological damage determinations,and the Cps titer in the lung was further examined by IFA.Finally,the Cps load in heart,liver,spleen and other tissues was detected by qRT-PCR.Results:1.Based on the predictive analysis by DNAStar and IEBD,the potential epitopes of MOMP and CPSIT_p6 were identified at positions 24-32,86-100,262-272,328-340 and 15-25,109-119,173-181,280-290 Moreover,MOMP24-32,MOMP262-272 and CPSIT_p6109-119,CPSIT_p6173-181 were predicted to contained potential T-cell epitopes and with high antigen index.2.The peptides of MOMP24-32,MOMP262-272,CPSIT_p6109-119,CPSIT_p6173-181 can induce high levels of serum IgG and IgA in immunized mice.In addition,the peptides of MOMP and CPSIT_p6 can induce a significant proliferation of splenocytes and the secretion of the Th1 cytokine IFN-? in the supernatant of splenocytes.3.After immunized with multi-epitopes fusion antigen,serum specific antibodies contained IgG,IgM,IgA and IgG subclasses were significantly increased compared with negative control group,and the levels of cytokines IFN-? and IL-2 were also significantly increased in fusion antigen immunizations.In addition,after Cps infection,the Cps titer and the secretion levels of IFN-? and IL-6 in the lung were significantly lower in the multi-epitopes antigen group than the negative controls.Remarkably reduced infiltration and milder lesions were also detected in multi-epitopes antigen immunized mice.4.The adoptive transfer of antigen-specific splenocytes depleted of CD8+cells significantly reduced the Cps burden in the lungs of immunized BALB/c mice.Nevertheless,the adoptive transfer of splenocytes depleted of CD4+cells from the multi-epitopes antigen group did not cause a remarkable reduction in the Cps load compared with the adoptive transfer of those from the PBS and FA groups.5.The levels of serum IgG and sIgA were significantly higher in simultaneous intramuscular and intranasal(SIM)vaccinated group than control groups.Immunization with fusion antigen via the SIM route generated an antigen-specific proliferation response in the splenocytes,and the secretion of IFN-??IL-2?TNF-? and IL-17 A in the splenocytes of SIM group was significantly increased compared to the individual immunizations.Moreover,the results revealed a decreased chlamydial burden and IFN-?,TNF-?,IL-6 levels in the lungs of the SIM group compared to control group,and the Cps burden in lung,liver and spleen of the mice immunized by SIM route was significantly lower than that of the control-immunized mice.6.After physical-structural characterization analysis,the images of CNPs/HBc-144-Ags revealed the appearance of nanoparticles to be obviously more spherical with nanorange sizes,and all nanoparticles appeared to be in similar sizes.CNPs/HBc-144-Ags showed 181.6±41.2 nm average particle size,+6.62±0.23 mV zeta potential and 0.391±0.083 dispersion index.The mice showed a stable body weight after continuous immunization with CNPs/HBc-144-Ags for 7 days.In addition,pathological examination showed that there were no severe inflammatory infiltration in the muscle and lung tissues.7.The levels of serum IgG and IgA induced by CNPs/HBc-144-Ags were increased significantly with the immunization times,but there was no significant difference compared with CNPs-Ags and HBc-144-Ags group,while the sIgA induced by CNPs/HBc-144-Ags was significantly increased than the control immunization.The levels of IFN-?,IL-2 and IL-17A in the supernatant of splenocytes were increased significantly in the CNPs/HBc-144-Ags immunized mice,and the levels of intracellular IFN-? and CD44/CD62 were also significantly higher than the control group.Vaccinations with CNPs/HBc-144-Ags gave rise to an effectively lower Cps load,reduced inflammatory pathological damage in lung tissue,and effectively reduce the content of Cps in liver,spleen and other tissues.Conclusions:1.MOMP24-32?MOMP262-272?CPSIT_p6109-119?CPSIT_p6173-181 could induce specific humoral and cellular immune responses with good immunogenicity,which were the dominant epitopes of Cps MOMP and CPSIT_p6.2.The multi-epitope antigens based on Cps MOMP and CPSIT_p6 can produce robust humoral and cellular immune responses against Cps lung infection.And CD4+cells are crucial for the cellular immune responses induced by the multi-epitope antigens3.Simultaneous intramuscular and intranasal immunizations of multi-epitope fusion antigens can induce stronger humoral,mucosal immunity and cell-mediated immunity,and effectively enhance the protective role against lung infection and inhibit Cps disseminating to other tissues in infected BALB/c mice4.CNPs/HBc-144 novel vaccine adjuvants can singnificantly improve the mucosal immunity and cell immunity induced by multi-epitope antigens,and promote the secretion of memory T cells to forms a memory immune response against Cps infection in vivo.