Development of mucosal adjuvant based on a mutant immunoglobulin binding domain of protein A from Staphylococcus aureus

Background. The development of efficient and safe adjuvants for mucosal vaccines remains one of the key challenges in modern immunology, as traditional adjuvants, such as aluminum salts, have been shown to be insufficient in stimulating local immunity, particularly the production of secretory immunoglobulin A (IgA). Therefore, the development of novel, potent and cost-effective adjuvants presents a significant opportunity.

The aim. Development of a recombinant A4Z protein based on the Z-domain of Staphylococcus aureus protein A and investigation of its potential use as a mucosal adjuvant in a subunit vaccine model.

Materials and methods. A synthetic gene encoding the A4Z protein was constructed using genetic engineering methods. The protein consists of four repeats of the mutant Z-domain of protein A (with N3A, N6D, N23T amino acid substitutions to reduce toxicity) and a C-terminal hexahistidine tag. The protein was expressed in the E. coli BL21(DE3) system. Purification was performed using a two-step chromatography process: affinity chromatography and ion-exchange chromatography. Acute toxicity was assessed in CD-1 mice following intraperitoneal administration of doses up to 2 g/kg. The adjuvant properties were studied in Balb/c mice via intranasal immunization with a conjugate of the A4Z protein and the SARS-CoV-2 spike protein’s RBD domain. Immunogenicity was evaluated by measuring specific IgG antibody titers using ELISA.

Results. The A4Z protein was shown to be virtually nontoxic (hazard class 4), causing no animal mortality or significant toxic effects even at a dose of 2 g/kg. Intranasal administration of the A4Z conjugate with RBD induced high levels of specific IgG antibodies, the titers of which were not statistically different from those obtained with intramuscular immunization and significantly (p < 0.05) exceeded the titers in the control groups (RBD without adjuvant or in combination with chitosan).

Conclusions. These findings highlight the potential of A4Z as a promising mucosal adjuvant capable of enhancing humoral immunity. Further research should focus on investigating its effects on the local IgA response and cellular immunity.

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