Suitability of Polymyxin B as a Mucosal Adjuvant for Intranasal Influenza and COVID-19 Vaccines

Abstract

Polymyxin B (PMB) is an antibiotic that exhibits mucosal adjuvanticity for ovalbumin (OVA), which enhances the immune response in the mucosal compartments of mice. Frequent breakthrough infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants indicate that the IgA antibody levels elicited by the mRNA vaccines in the mucosal tissues were insufficient for the prophylaxis of this infection. It remains unknown whether PMB exhibits mucosal adjuvanticity for antigens other than OVA. This study investigated the adjuvanticity of PMB for the virus proteins, hemagglutinin (HA) of influenza A virus, and the S1 subunit and S protein of SARS-CoV-2. BALB/c mice immunized either intranasally or subcutaneously with these antigens alone or in combination with PMB were examined, and the antigen-specific antibodies were quantified. PMB substantially increased the production of antigen-specific IgA antibodies in mucosal secretions and IgG antibodies in plasma, indicating its adjuvanticity for both HA and S proteins. This study also revealed that the PMB-virus antigen complex diameter is crucial for the induction of mucosal immunity. No detrimental effects were observed on the nasal mucosa or olfactory bulb. These findings highlight the potential of PMB as a safe candidate for intranasal vaccination to induce mucosal IgA antibodies for prophylaxis against mucosally transmitted infections.

Keywords: S protein; S1 subunit; SARS-CoV-2; hemagglutinin; influenza A virus; mucosal adjuvant; polymyxin B.

Figure 1

Quantity of HA-specific Ab in mice immunized with HA split. Mice were subjected to intranasal (IN) or subcutaneous (SC) immunization with 1 μg of HA split (4–6 mice per experimental group). Mucosal secretions (TBL: tracheal–bronchial lavages; NW: nasal washes; saliva; FE: fecal extract; VW: vaginal wash) and plasma were collected. HA-specific IgA and IgG Abs in mucosal secretions (1:2) or plasma (1:10,000) diluted in 1% BSA-PBS were quantified using an ELISA system described in the Materials and Methods section. Each dot represents a single mouse. The horizontal dotted line indicates the detection limit. Significant differences in OVA-specific Ab titers between experimental groups are indicated by asterisks: ** p < 0.005.

Figure 2

Particle diameter of PMB-virus antigen complex. Influenza HA split (1 μg), SARS-CoV-2 S1 subunit (1 μg), or SARS-CoV-2 S protein (1 μg) was mixed with PMB (500 μg) in saline. Particle diameters of the complexes were measured using a Zetasizer Nano ZS system. X- and y-axes indicate particle diameter and intensity, respectively.

Figure 3

Amount of S-specific Ab in mice immunized with 1 μg of S protein. Mice were subjected to intranasal (IN) or subcutaneous (SC) immunization with 1 μg of the SARS-CoV-2 S protein (4–6 mice per experimental group). The S-specific Abs in the mucosal secretions (1:2) or plasma (1:10,000 or 1:100) diluted in 1% BSA-PBS were quantified using ELISA. ** p < 0.005.

Figure 4

Histopathology of the nasal mucosa of immunized mice. Nasal mucosa (pseudostratified ciliated epithelium and olfactory epithelium) and olfactory bulbs of immunized mice in the S (IN) (a,d,g), PMB + S (IN) (b,e,h), or S (SC) (c,f,i) groups were examined using hematoxylin-eosin (HE) staining. Bar; 20 μm.