The COVID-19 pandemic caused by SARS-CoV-2 highlighted the urgent need for effective vaccines. While current vaccines significantly reduce mortality, their effectiveness in preventing infection is limited. Breakthrough infections and the emergence of variants of concern (VOCs) like Delta and Omicron underscore the need for improved vaccine strategies. One promising approach is mucosal vaccination, which aims to enhance immunity within the respiratory tract, thereby creating a barrier against infection and minimizing viral spread. Existing vaccines, primarily delivered intramuscularly, predominantly induce systemic IgG and T-cell responses, neglecting the crucial mucosal immune system. Mucosal IgA antibodies and resident memory B and T cells in the respiratory tract are critical for preventing infection and reducing viral shedding. Previous studies using live-attenuated viruses or viral vector vaccines showed the potential of mucosal vaccination, but these approaches have limitations including safety concerns and anti-vector immunity. Protein-based vaccines offer a safer and cost-effective alternative but require effective adjuvants to boost their immunogenicity. Toll-like receptor 2 (TLR2), expressed by respiratory epithelium and immune cells, is a promising target for adjuvant development. The TLR2 ligand Pam₂Cys has proven effective as a mucosal adjuvant in previous studies, prompting this investigation into its use in a SARS-CoV-2 subunit vaccine.

The literature extensively documents the limitations of current SARS-CoV-2 vaccines in preventing infection and transmission. Studies show waning immunity post-vaccination or infection, coupled with the emergence of VOCs, resulting in breakthrough infections even in highly vaccinated populations. The impact of VOCs on vaccine effectiveness is significant, with reduced control of viral burden observed for variants like Delta. Issues of vaccine equity further complicate the global response. Mucosal immunity is recognized as a crucial component of effective protection against respiratory viruses. Pre-clinical studies demonstrate the exciting potential of mucosal vaccination for various respiratory pathogens in generating lower respiratory tract immunity. Intranasal vaccination, in particular, elicits IgA antibodies and resident memory cells crucial in preventing infection and reducing viral shedding. However, most previous mucosal vaccines utilized live-attenuated viruses or viral vectors, raising safety concerns and limitations in booster immunizations. Protein-based vaccines, coupled with safe and effective mucosal adjuvants, offer a promising solution, especially in addressing the need for broad population coverage including vulnerable groups. The role of TLR2 in effective host defense against infections and its potential as a target for mucosal adjuvants is well-established, with prior research showing the efficacy of Pam₂Cys in pulmonary immunization against Mycobacterium tuberculosis.

This study used a subunit vaccine consisting of recombinant trimeric SARS-CoV-2 Spike protein and the TLR2-stimulating adjuvant Pam₂Cys (Pam₂Cys Spike). C57BL/6 mice received three immunizations, either subcutaneously (s.c.) or intranasally (i.n.), two weeks apart. Immune responses were assessed one and eight weeks post-final vaccination using various techniques. Antibody responses (IgG isotypes, IgA, neutralizing antibodies) were measured using ELISA and pseudovirus/live virus neutralization assays. T-cell responses (CD4+, CD8+) were evaluated by ex vivo recall stimulation, intracellular cytokine staining, and flow cytometry. To determine the contribution of TLR2 expression in different compartments (respiratory epithelium vs. immune cells), irradiated wild-type and TLR2-deficient mice were reconstituted with bone marrow from either wild-type or TLR2-deficient donors, creating four groups: KO/KO, KO/WT, WT/KO, and WT/WT. Early innate responses (24h post-vaccination) and adaptive immune responses (one week post-final vaccination) were then analyzed by flow cytometry. Protective efficacy was evaluated in K18-hACE2 mice challenged with SARS-CoV-2, assessing weight loss, clinical scores, viral load, and lung inflammation using histology. Statistical significance was determined using appropriate tests (Mann-Whitney, Kruskal-Wallis, ANOVA). The study also assessed the durability of protection in a separate cohort of mice.

Both s.c. and i.n. vaccination with Pam₂Cys Spike induced potent anti-Spike IgG responses in serum and BALF. However, i.n. vaccination uniquely generated high titres of anti-Spike IgA, significantly elevated serum and airway nAb titres, and increased lung CD4+ T-cell responses compared to s.c. vaccination. Analysis of TLR2-deficient chimeric mice revealed that TLR2 expression in either respiratory epithelium or hematopoietic cells facilitates early innate responses to mucosal vaccination. However, TLR2 on hematopoietic cells was essential for optimal lung-localized, antigen-specific adaptive responses. In K18-hACE2 mice, Pam₂Cys Spike vaccination provided complete protection against SARS-CoV-2 challenge, with sterilizing lung immunity. Intranasal Pam₂Cys alone showed a short-term, non-specific protective effect. Longitudinal studies demonstrated that sterilizing immunity, induced by Pam₂Cys Spike, was maintained at least six weeks post-vaccination.

This study demonstrates that mucosal vaccination with a Pam₂Cys-adjuvanted SARS-CoV-2 Spike protein vaccine elicits superior immune responses compared to parenteral vaccination. The increased IgA production and elevated nAb titres in serum and airways, coupled with enhanced lung CD4+ T-cell responses, contribute to the observed superior protection against SARS-CoV-2 challenge. The crucial role of TLR2 on hematopoietic cells in generating optimal antigen-specific responses suggests a key mechanism underlying the vaccine's efficacy. The short-term, non-specific protective effect of intranasal Pam₂Cys alone highlights the potential of TLR2 agonists as adjuvants for enhancing innate immunity. The findings strongly support mucosal vaccination as a strategy to enhance protection against SARS-CoV-2 and possibly other respiratory viruses. The generation of robust mucosal immunity, including IgA and resident memory cells, could significantly reduce the risk of infection and transmission, addressing the limitations of currently available vaccines.

This study provides compelling evidence for the efficacy of a mucosal TLR2-activating protein-based vaccine against SARS-CoV-2 in mice. The vaccine induced potent pulmonary immunity, characterized by high IgA and nAb levels, robust T cell responses and sterilizing immunity. The importance of TLR2 expression on hematopoietic cells for optimal adaptive immunity was highlighted. These findings support the development of mucosal vaccines as a strategy to enhance protection against SARS-CoV-2 and reduce transmission. Future research should focus on evaluating the durability of protection in the upper respiratory tract and exploring the potential of a prime-boost strategy combining parenteral and mucosal vaccination.

The study was conducted in mice, and the results may not fully translate to humans. The use of a specific mouse model (K18-hACE2) might limit the generalizability of the findings. Long-term durability of protection and the effectiveness against emerging variants beyond Delta need further investigation. The short-term non-specific effect observed with Pam₂Cys alone warrants more detailed mechanistic studies.