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Cell-free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development.

ABSTRACT: Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (<1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

SUBMITTER: Gilmore SF

PROVIDER: S-EPMC9236854 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chlamydia pecorum is responsible for causing ocular infection and disease which can lead to blindness in koalas (Phascolarctos cinereus). Antibiotics are the current treatment for chlamydial infection and disease in koalas, however, they can be detrimental for the koala's gastrointestinal tract microbiota and in severe cases, can lead to dysbiosis and death. In this study, we evaluated the therapeutic effects provided by a recombinant chlamydial major outer membrane protein (MOMP) vaccine on ocular disease in koalas. Koalas with ocular disease (unilateral or bilateral) were vaccinated and assessed for six weeks, evaluating any changes to the conjunctival tissue and discharge. Samples were collected pre- and post-vaccination to evaluate both humoral and cell-mediated immune responses. We further assessed the infecting C. pecorum genotype, host MHC class II alleles and presence of koala retrovirus type (KoRV-B). Our results clearly showed an improvement in the clinical ocular disease state of all seven koalas, post-vaccination. We observed increases in ocular mucosal IgA antibodies to whole C. pecorum elementary bodies, post-vaccination. We found that systemic cell-mediated immune responses to interferon-?, interleukin-6 and interleukin-17A were not significantly predictive of ocular disease in koalas. Interestingly, one koala did not have as positive a clinical response (in one eye primarily) and this koala was infected with a C. pecorum genotype (E') that was not used as part of the vaccine formula (MOMP genotypes A, F and G). The predominant MHC class II alleles identified were DAb*19, DAb*21 and DBb*05, with no two koalas identified with the same genetic sequence. Additionally, KoRV-B, which is associated with chlamydial disease outcome, was identified in two (29%) ocular diseased koalas, which still produced vaccine-induced immune responses and clinical ocular improvements post-vaccination. Our findings show promise for the use of a recombinant chlamydial MOMP vaccine for the therapeutic treatment of ocular disease in koalas.

Project description:Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To determine what type of adjuvant can better enhance the immunogenicity of a Chlamydia vaccine, we formulated the recombinant major outer membrane protein (Ct-rMOMP) with several ligands for Toll-like receptors (TLR) and the nucleotide-binding oligomerization domain (NOD) including Pam(2)CSK(4) (TLR2/TLR6), Poly (I:C) (TLR3), monophosphoryl lipid A (TLR4), flagellin (TLR5), imiquimod R837 (TLR7), imidazoquinoline R848 (TRL7/8), CpG-1826 (TLR9), M-Tri-(DAP) (NOD1/NOD2) and muramyldipeptide (NOD2). Groups of female BALB/c mice were immunized intramuscularly (i.m.) three times with the Ct-rMOMP and each one of those adjuvants. Four weeks after the last immunization the mice were challenged intranasally (i.n.) with 10(4)C. trachomatis mouse pneumonitis (MoPn) inclusion forming units (IFU). As negative antigen control, mice were immunized with the Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) and the same adjuvants. As a positive vaccine control, mice were inoculated i.n. with 10(4)IFU of MoPn. The humoral and cell mediated immune responses were determined the day before the challenge. Following the challenge the mice were weighed daily and, at 10 days post-challenge (p.c.), they were euthanized, their lungs weighted and the number of IFU in the lungs counted. As determined by the IgG2a/IgG1 ratio in the sera, mice immunized with Ct-rMOMP+Pam(2)CSK(4) showed a strong Th2 biased humoral immune response. Furthermore, these mice developed a robust cellular immune response with high Chlamydia-specific T cell proliferation and levels of IFN-γ production. In addition, based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, the mice immunized with Ct-rMOMP+Pam(2)CSK(4), were better protected against the i.n. challenge than any group of mice immunized with Ct-rMOMP and the other adjuvants. In conclusion, Pam(2)CSK(4) should be evaluated as a candidate adjuvant for a C. trachomatis vaccine.

Project description:BackgroundChlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes.ResultsC. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) beta-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands.ConclusionsC. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with beta-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins.

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Cell-free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development.

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