1 Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared in Wuhan, China, in late of 2019, causing coronavirus disease 2019 (COVID-19) and infecting approximately 560 million individuals worldwide based on July 2022 data, and death of approximately 6.5 million individuals.1 The seriousness of the global situation called for urgent development of COVID-19 vaccine and several vaccines were approved at unprecedented speed and used clinically throughout the world.2-4
The approved vaccines are delivered by intramuscular (IM) injection and induce SARS-CoV-2 specific IgG in serum and showed effectiveness in prevention of severe infection and death.5 However, vaccines administrated intramuscularly are, in general, known to be minimally effective in inducing mucosal secretory IgA (S-IgA), which plays an important role in boosting mucosal immunity, and preventing respiratory and gastrointestinal tract infections and viral transmission.6 Recent reports have described intramuscular administration of COVID-19 mRNA vaccine (e.g., Comirnaty)-induced SARS-CoV-2 specific IgA at mucosal sites.7, 8 However, the effect of mucosal IgA induced by mRNA vaccine is presumably limited in providing protection against virus infection through mucosal sites.9 Thus, although the first-generation COVID-19 vaccines produced limited benefits, such as reduction of severe infection and death in pandemic emergency situation, it is necessary to develop the next generation COVID-19 vaccines that can induce effective mucosal S-IgA, in addition to systemic IgG and IgA, to achieve protection against infection and prevention of transmission of the virus.
Mucosal vaccines that are directly administered onto mucosal surfaces, such as the nasal cavity and gastrointestinal tract, can induce mucosal S-IgA.10 Since the mucosa is equipped with exclusion systems, namely the cilia, mucus and various proteases, against foreign substances including vaccine antigens,11 the use of vaccines that contain antigen alone is unlikely to produce protective immunity. Thus, for successful mucosal vaccination, live virus vaccines and mucosal adjuvants that enhance the vaccine effects, have been developed worldwide.12 However, the live attenuated vaccines, such as Flumist®, have problems related to their efficacy and safety and thus their worldwide use is limited.13
To overcome these problems, we reported previously the use of an antigen delivery type adjuvant,14 pulmonary surfactant (PS)-based compound, surfacten®, as an effective and safe intranasal mucosal adjuvant for influenza ether-split hemagglutinin vaccine (HAv) and that it enhanced the production of both HAv-specific IgG in serum and S-IgA in the respiratory mucosae of mice and swine.15, 16 Based on these results, we then successfully developed synthetic surfactant (SSF), which mimicked human PS, consisting of three major lipids [1,2-dipalmitoyl-phosphatidylcholine (DPPC), phosphatidylglycerol (PG) and palmitic acid (PA)] plus the K6L16 peptide [which resembles surfactant protein C (SP-C)], as a potent and safe synthetic mucosal adjuvant.17, 18 In addition, to increase the effectiveness of the vaccine, we prepared the antigen and SSF complex by lyophilization, thus allowing the lyophilized powder to be suspended in carboxy vinyl polymer (CVP) to increase the viscosity of the antigen and SSF complex solution in order to prolong antigen uptake on the mucosal surface.18 The final vaccine solution was termed antigen-SF-10. In a series of studies, we reported that intranasal application of antigen-SF-10 enhanced the absorption of antigens to nasal antigen presenting cells and induced systemic as well as respiratory humoral immunity in mice and cynomolgus monkeys18-20 and cell-mediated immunity in mice.21
The present study is an extension to the above work and was designed to determine the outcome of mucosal application of recombinant S1 spike protein of SARS-CoV-2, as an antigen combined with SF-10 (S1-SF-10) on respiratory and systemic immunity against SARS-CoV-2 infection. For this purpose, S1-SF-10 vaccine was inoculated intratracheally (IT) through nasal cavity to the lower respiratory tract (S1-SF-10-IT) of mice, the site of viral infection and proliferation. To test the effectiveness of a new administration method of intratracheal vaccination, we analyzed the levels of induced S1-specific antibodies, the antibody secreting cells and S1-responsive Th cytokine secreting cells in lung lymphocytes and splenocytes. We also compared the level of protective immunity induced by S1-SF-10-IT and intramuscular injection of S1 with an already reported potent adjuvant AS03 (S1-AS03-IM) against SARS-CoV-2 infection using the S1/angiotensin converting enzyme 2 (ACE2) binding inhibition assay.