Bacterial carbonic anhydrase as a candidate vaccine target against Helicobacter pylori

György Miklós Buzás ^* György MIklós Buzás ^*

• Ferencváros Health Center, Budapest, Hungary

The lack of an ideal eradication therapy prompted researchers to seek alternative ways to prevent and treat H. pylori-related diseases. Many vaccine types have been developed against H. pylori (inactivated whole-cell vaccines, urease, outer membrane proteins, heat shock proteins, lipopolysaccharide, cytotoxin-associated gene A, flagellar sheath protein, DNA vaccines, recombinant Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae and measles virus vaccine), but carbonic anhydrase (CA) was omitted (Zhang et al., 2022). Just a few made it to human trials, therefore new avenues of research must be explored. Although the worldwide prevalence of H. pylori decreased from 58.2% to 43.1% between 198058.2% to 43.1% between and 202258.2% to 43.1% between (Li et al., 2024)), and efficient vaccination against H. pylori infection should be a possibility to prevent gastric cancer and other, less life-threatening conditions associated with this infection (Ilic et al., 2022).Carbonic anhydrase (CA) (EC 4.2.1.1) was discovered in 1933. Its role in gastric acid secretion was demonstrated in 1939. The enzyme inhibitor acetazolamide was synthesized in 1950. CAs have a molecular mass of about 35,000-45,000 kDa and are highly conserved, with significant structural homology but also organ specificity. The spatial structure and protein folding as well as the zinc-containing active centre and mechanism of action were characterised (Supuran, 2024). CA inhibitors were used in the 1980's to treat peptic ulcers, obtaining high healing (>90%) and low relapse rates (<10%). At that time, this was considered the result of strong acid inhibition. Later, the low relapse rate was interpreted as a possible effect of H. pylori eradication (Buzás and Supuran, 2015).Bacterial H. pylori CA (HpCA) has two forms, HpCAά and HpCAβ, localised on the membrane, in periplasma and cytoplasm, respectively. The membrane-bound CA is either anchored to the cell surface or enclosed in the outer-membrane vesicles (Li et al., 2024). The three dimensional structure resembles those of human CAs, the amino acid sequence presents differences in the active catalytic site and other segments of the protein chain (Supuran, 2017). Both HpCAs are involved in the acid acclimation of H. pylori. Inhibitors of HpCAsuch as ethoxzolamide -kills the bacteria in vitro at low concentrations, whereby the enzyme is essential for the survival of the bacterium (Supuran, 2024). Monoclonal antibodies against human and bacterial soluble and membrane-bound CAs were recently generated to identify enzymatic isoforms (Stravinskiene, 2019).Autoantibodies against CA isoenzymes occur in patients with rheumatoid arthritis, systemic lupus erythematosus, polymyositis, systemic sclerosis, Sjögren's syndrome, autoimmune liver disease, diabetes mellitus and endometriosis (D'Cruz et al., 1996, Liu et al., 2012). In genetically predisposed patients, H. pylori can trigger autoimmune pancreatitis via molecular mimicry. In silico protein analysis showed homology between pancreatic CA II isoform and HpCA, and the homologous segments contained the binding motif of a HLA molecule (Guarneri et al.,2005). Moreover, human CA IX can serve as a tumour marker in renal cell carcinoma which can be identified by monoclonal antibodies. A complex of recombinant heat shock protein and CA IX -based vaccine was even evaluated in targeting renal cell carcinoma (Combe et al., 2015). Association of H. pylori with immune thrombocytopenic purpura, Hashimoto's disease, rheumatoid arthritis, autoimmune hepatitis, chronic urticaria is rather due to pro-inflammatory cytokines and virulence factors than to antibodies against CA (Wang et al., 2022).Recently, several attemps have been made to identify epitopes of H. pylori virulence factors. Firstly, Chinese authors constructed a multivalent epitope-based vaccine with selected antigens (urease, lipopolysaccharide 20, Hp adhesin A and CagL). The specificity, immunogenicity and antibody production were verified in BALB/mice and the multiepitope vaccine proved to be more effective than the anti-urease vaccine (Guo et al., 2017). Another Chinese research group prepared antibodies against cytotoxin-associated gene A, vacuolating cytotoxin-associated gene A and urease A and B gene (Du et al., 2023). An international group determined the crystal structure of H. pylori adhesin A, which plays an important role in cell adhesion of the bacterium and induces TNF-alpha production. The results could contribute to a further vaccine preparation against this important virulence factor (Martini et al., 2024). A Bangladesh research group identified outer-membrane proteins from H. pylori, which were scrutinized to identify cytotoxic and helper T lymhpocytes and B cell epitopes and developed a non-allergic, immunogenic vaccine. The non-toxic, soluble preparation exhibited binding to toll-like receptor 4. In silico testing and immune simulation revealed that it is able to initiate an immune response in humans. The authors suppose that is has the potential to induce robust immunity against H. pylori (Tamanna et al., 2023). Mexican authors used baculovirus carrying Thp1 transgene coding for epitopes from urease B, CagL, Cag7, gamma-glutamyl transpeptidase and CA and a multiepitope recombinant baculovirus Th1 protein was produced and inoculated in mice. A strong IgG-response was obtained after intranasal, intragastric, intramuscular and combined administration which persisted in sera after 125 days and IgA antibodies were found in faces after 82 days. However, none of the above studies excepting those using baculovirus employed CA as target (Montiel-Martinez et al., 2023). Finally, an Iranian research group created a multi-epitope vaccine using lipid nanoparticles, targeting five H. pylori proteins (urease, CagA, HopE, BabA and SabA), but CA was omitted. The designed product was non-toxic and non-allergic and needs additional testing to establish its immunogenicity and safety (Jebali A. et al., 2024).Knowing the role of human CA in acid secretion and that of HpCA in acid acclimation of the bacterium, and the recent results in the field of HpCA immunology and vaccinological research, I propose identification of specific HpCA epitopes as single or multiple structures and generating specific antibodies to avoid cross-reaction with other CAs. It is noteworthy, that antibodies against other H. pylori proteins (CagA, VacA, urease, GGT, heat-shock proteins, etc) were not developed further into efficient human vaccines. After selecting the specific antibodies against bacterial CA, a new vaccine should be prepared, using the mRNA method as designed by the Nobel laureate Katalin Karikó (Karikó et al., 2008, Pardi et al., 2020). CAs are vital for survival in all H. pylori strains, a vaccine against the enzyme can have an advantage compared to preparations targeting other virulence factors which are not present in all strains and not essential for survival. After adequate laboratory, animal and human testing, such preparations could be used as a vaccine against H. pylori infection, hopefully with more success than before. Some mRNA-based vaccines have already been prepared against Clostridioides difficile (Alameh et al., 2024), Listeria monocytogenes (Mayer et al., 2022) and Pseudomonas aedruginosa (Wang et al., 2023). Why than H. pylori must be an exception? A successful vaccine can change the worldwide epidemiology and clinical,outcomes of H. pylori infection.