Vaccines against Chlamydia trachomatis: Immunogenicity analysis via peptide pool
January 21, 2025
Dr. Lilian Schoefer
Research updates
Research updates
Scientists have investigated two novel recombinant vaccines based on the modified vaccinia virus Ankara (MVA). These vaccines express a chimeric protein containing several antigens from the most common serovars of Chlamydia trachomatis.
To analyze the immune responses elicited by these vaccines, the researchers developed the peptide pool spCTH522, which encompasses the entire chimeric protein. peptides&elephants synthesized the consecutive 15-mer peptides, which overlapped by 11 amino acids. In addition to its capacity to generate individual peptide pools, peptides&elephants has a large peptide shop from which the scientists selected more peptides.
To analyze the immune responses elicited by these vaccines, the researchers developed the peptide pool spCTH522, which encompasses the entire chimeric protein. peptides&elephants synthesized the consecutive 15-mer peptides, which overlapped by 11 amino acids. In addition to its capacity to generate individual peptide pools, peptides&elephants has a large peptide shop from which the scientists selected more peptides.
Chlamydia trachomatis: most common cause of STDs
Chlamydia trachomatis is the leading pathogen responsible for sexually transmitted diseases (STDs), with infection rates continuing to rise. Despite often being asymptomatic, chlamydia infection can have severe consequences, including sterility, if left untreated. Furthermore, Chlamydia trachomatis is associated with an increased risk of co-infection with other pathogens such as the human immunodeficiency virus (HIV). Unfortunately, a commercial vaccine against this pathogen is currently not available.
Novel vaccines based on the modified vaccinia virus Ankara
Giuseppe Andreacchio and colleagues from the Institute of Virology at the University Hospital Düsseldorf in Germany have now investigated two novel recombinant vaccines based on the modified vaccinia virus Ankara. [1] These vaccines express the proteins spCTH522 and CTH522:B7, which are designed to elicit an immune response against Chlamydia trachomatis. The chimeric protein CTH522 is composed of antigenic regions from the most common serovars of Chlamydia trachomatis:
- Amino acid sequence 56–349 of the major outer membrane protein (MOMP) of serovar D
- Amino acid sequence 282–349 of the external VD4 domain of serovar E
- Amino acid sequence 283–351 of the external VD4 domain of serovar F
- Amino acid sequence 283–351 of the external VD4 domain of serovar G
Immune response depends on mouse model
The researchers observed a significant difference in the immune responses between two mouse models: C57BL/6J mice and an HLA-transgenic mouse model.
In C57BL/6J mice, both vaccines elicited CD4+ T cell responses, but antigen-specific systemic CD8+ T cells were not detected. Notably, the membrane-anchored CTH522:B7 vaccine induced robust IgG2b and IgG2c antibody reactions. In an HLA-transgenic mouse model, however, the two recombinant vaccines induced Th1-directed CD4+ T cells and multifunctional CD8+ T cells. Once again, only the membrane-anchored vaccine CTH522:B7 generated antibody reactions.
To validate their results, the scientists used ovalbumin-specific peptides (OVA 257–264 and OVA 323–339) and peptides specific for the modified vaccinia virus Ankara (B8 20−27 and B5 46–60) as negative and positive controls, respectively.
Transgenic mouse model for HLA-restricted T cell responses
Since the researchers wanted to focus on the development of a vaccine that specifically targets CD8+ T cell responses, they turned their attention to the HLA-transgenic mouse model. The A2.DR1 mice used are knock-out mice for the major histocompatibility complexes (MHCs) I and II and transgenic for HLA-A*02:01/Db and HLA-DR1 as the sole sources of MHC-I and MHC-II. This genetic background allows the scientists to study T cell responses in a HLA-restricted manner.
The researchers immunized the HLA-transgenic mice intraperitoneally with either the modified wild-type vaccinia virus Ankara or the antigen-expressing variants spCTH522 (MVA-spCTH522) or CTH522:B7 (MVA-CTH522:B7). After 7 days, they removed the animals' spleens to examine the splenocytes.
Analysis of immune response using peptide pools and specific peptides
The scientists crushed the spleen on a 70 µm cell strainer to release the cells, then incubated them with lysis buffer for one minute at room temperature to remove the erythrocytes. They then passed the cells through another 70 µm cell strainer and counted them using a Neubauer cell counting chamber. They pipetted 200 µL of 2 × 106 cells into each well of a 96-well plate and incubated them for 4–5 hours with various peptide preparations:
- the entire spCTH522 peptide pool
- subsets of the spCTH522 peptide pool
- the specific peptides MOMP282–290 and MOMP200–209 or
- control peptides
All peptide preparations were added at a concentration of 5 µg/mL in the presence of 1 µg/mL brefeldin A.
Two HLA-A*02:01-restricted epitopes within the CTH522 protein sequence
Neither MVA-spCTH522 nor MVA-CTH522:B7 induced strong CD4+ T cell responses that were specific for the spCTH522 peptide pool (Figure). However, using the peptide pool, the researchers were able to identify two HLA-A*02:01-restricted epitopes within the CTH522 protein sequence that trigger CD8+ T cell responses (Figure). One epitope was the 9-mer NMFTPYIGV, which corresponds to MOMP282–290 and has previously been described as a dominant CD8+ T cell-specific epitope in PBMCs of Chlamydia trachomatis-positive patients. [2] The other epitope was new: the 10-mer ALWECGCATL, which corresponds to MOMP200–209.
When the HLA-transgenic mice received a booster dose of MVA-spCTH522 or MVA-CTH522:B7 after 28 days, this increased the magnitude of the T cell responses.
New epitope and insights for improved vaccine development
With MOMP200–209, the scientists were able to identify a new HLA-A*02:01-restricted epitope within the CTH522 protein sequence. Their results also contribute to understanding how vaccine development can be improved for targeted immune responses. Their studies show the importance of both antigen localization and the selection of suitable animal models to study the immune response of Chlamydia trachomatis vaccine candidates. Whether the immune responses observed in the mouse models correlate with those required for protection against chlamydia in humans remains to be determined.
Literature:
- Andreacchio, Giuseppe et al. “Viral Vector-Based Chlamydia trachomatis Vaccines Encoding CTH522 Induce Distinct Immune Responses in C57BL/6J and HLA Transgenic Mice.” Vaccines vol. 12,8 944. 22 Aug. 2024, doi:10.3390/vaccines12080944
- Kim, S K et al. “Induction of HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein of Chlamydia trachomatis in human genital tract infections.” Journal of immunology (Baltimore, Md. : 1950) vol. 162,11 (1999): 6855-66.
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