A live attenuated influenza virus-vectored intranasal COVID-19 vaccine provides rapid, prolonged, and broad protection against SARS-CoV-2 infection

Remarkable progress has been made in developing intramuscular vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they are limited with respect to eliciting local immunity in the respiratory tract, which is the primary infection site for SARS-CoV-2. To overcome the limitations of intramuscular vaccines, we constructed a nasal vaccine candidate based on an influenza vector by inserting a gene encoding the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, named CA4-dNS1-nCoV-RBD (dNS1-RBD). A preclinical study showed that in hamsters challenged 1 day and 7 days after single-dose vaccination or 6 months after booster vaccination, dNS1-RBD largely mitigated lung pathology, with no loss of body weight, caused by either the prototype-like strain or beta variant of SARS-CoV-2. Lasted data showed that the animals could be well protected against beta variant challenge 9 months after vaccination. Notably, the weight loss and lung pathological changes of hamsters could still be significantly reduced when the hamster was vaccinated 24 h after challenge. Moreover, such cellular immunity is relatively unimpaired for the most concerning SARS-CoV-2 variants. The protective immune mechanism of dNS1-RBD could be attributed to the innate immune response in the nasal epithelium, local RBD-specific T cell response in the lung, and RBD-specific IgA and IgG response. Thus, this study demonstrates that the intranasally delivered dNS1-RBD vaccine candidate may offer an important addition to fight against the ongoing COVID-19 pandemic, compensating limitations of current intramuscular vaccines, particularly at the start of an outbreak.

Riboflavin as a Mucosal Adjuvant for Nasal Influenza Vaccine

Intranasal immunization with whole inactivated virus (WIV) is an important strategy used for influenza prevention and control. However, a powerful mucosal adjuvant is required to improve nasal vaccine efficacy. Riboflavin, as a food additive with the advantages of being safe and low-cost, widely exists in living organisms. In this paper, the mucosal adjuvant function of riboflavin was studied. After intranasal immunization with H1N1 WIV plus riboflavin in mice, we found that the mucosal immunity based on the secretory IgA (sIgA) levels in the nasal cavity, trachea, and lung were strongly enhanced compared with H1N1 WIV alone. Meanwhile, the IgG, IgG1, and IgG2a levels in serum also showed a high upregulation and a decreased ratio of IgG1/IgG2a, which implied a bias in the cellular immune response. Moreover, riboflavin strongly improved the protection level of H1N1 inactivated vaccine from a lethal influenza challenge. Furthermore, riboflavin was found to possess the capacity to induce dendritic cell (DC) phenotypic (MHCII, CD40, CD80, and CD86) and functional maturation, including cytokine secretion (TNF-α, IL-1β, IL-12p70, and IL-10) and the proliferation of allogeneic T cells. Lastly, we found that the DC maturation induced by riboflavin was dependent on the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which plays an important role in immune regulation. Therefore, riboflavin is expected to be developed as an alternative mucosal adjuvant for influenza nasal vaccine application.

Nasal Immunization With Small Molecule Mast Cell Activators Enhance Immunity to Co-Administered Subunit Immunogens

Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, small molecule mast cell activators may be a more cost-efficient adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. To identify novel mast cell activating compounds that could be evaluated for mucosal vaccine adjuvant activity, we employed high-throughput screening to assess over 55,000 small molecules for mast cell degranulation activity. Fifteen mast cell activating compounds were down-selected to five compounds based on in vitro immune activation activities including cytokine production and cellular cytotoxicity, synthesis feasibility, and selection for functional diversity. These small molecule mast cell activators were evaluated for in vivo adjuvant activity and induction of protective immunity against West Nile Virus infection in BALB/c mice when combined with West Nile Virus envelope domain III (EDIII) protein in a nasal vaccine. We found that three of the five mast cell activators, ST101036, ST048871, and R529877, evoked high levels of EDIII-specific antibody and conferred comparable levels of protection against WNV challenge. The level of protection provided by these small molecule mast cell activators was comparable to the protection evoked by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Thus, novel small molecule mast cell activators identified by high throughput screening are as efficacious as previously described mast cell activators when used as nasal vaccine adjuvants and represent next-generation mast cell activators for evaluation in mucosal vaccine studies.

Mucosal vaccines: wisdom from now and then

The oral and nasal cavities are covered by the mucosal epithelium that starts at the beginning of the aero-digestive tract. These mucosal surfaces are continuously exposed to environmental antigens including pathogens and allergens and are thus equipped with a mucosal immune system that mediates initial recognition of pathogenicity and initiates pathogen-specific immune responses. At the dawn of our scientific effort to explore the mucosal immune system, dental science was one of the major driving forces as it provided insights into the importance of mucosal immunity and its application for the control of oral infectious diseases. The development of mucosal vaccines for the prevention of dental caries was thus part of a novel approach that contributed to building the scientific foundations of the mucosal immune system. Since then, mucosal immunology and vaccines have gone on a scientific journey to become one of the major entities within the discipline of immunology. Here, we introduce our past and current efforts and future directions for the development of mucosal vaccines, specifically a rice-based oral vaccine (MucoRice) and a nanogel-based nasal vaccine, with the aim of preventing and controlling gastrointestinal and respiratory infectious diseases using the interdisciplinary fusion of mucosal immunology with agricultural science and biomaterial engineering, respectively.

Lipopolysaccharide Derived From the Lymphoid-Resident Commensal Bacteria Alcaligenes faecalis Functions as an Effective Nasal Adjuvant to Augment IgA Antibody and Th17 Cell Responses

Alcaligenes spp., including A. faecalis, is a gram-negative facultative bacterium uniquely residing inside the Peyer’s patches. We previously showed that A. faecalis-derived lipopolysaccharides (Alcaligenes LPS) acts as a weak agonist of toll-like receptor 4 to activate dendritic cells and shows adjuvant activity by enhancing IgG and Th17 responses to systemic vaccination. Here, we examined the efficacy of Alcaligenes LPS as a nasal vaccine adjuvant. Nasal immunization with ovalbumin (OVA) plus Alcaligenes LPS induced follicular T helper cells and germinal center formation in the nasopharynx-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs), and consequently enhanced OVA-specific IgA and IgG responses in the respiratory tract and serum. In addition, nasal immunization with OVA plus Alcaligenes LPS induced OVA-specific T cells producing IL-17 and/or IL-10, whereas nasal immunization with OVA plus cholera toxin (CT) induced OVA-specific T cells producing IFN-γ and IL-17, which are recognized as pathogenic type of Th17 cells. In addition, CT, but not Alcaligenes LPS, promoted the production of TNF-α and IL-5 by T cells. Nasal immunization with OVA plus CT, but not Alcaligenes LPS, led to increased numbers of neutrophils and eosinophils in the nasal cavity. Together, these findings indicate that the benign nature of Alcaligenes LPS is an effective nasal vaccine adjuvant that induces antigen-specific mucosal and systemic immune responses without activation of inflammatory cascade after nasal administration.

ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever

The African swine fever virus (ASFV) is currently causing a pandemic affecting wild and domestic swine from Western Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in overwhelming economic losses to the swine industry. We recently developed a recombinant vaccine candidate, ASFVG-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV strain Georgia (ASFV-G). ASFV-G-ΔI177L has been proven safe and highly efficacious in challenge studies using parental ASFV-G. Here, we present data demonstrating that ASFV-G-ΔI177L can be administered by the oronasal (ON) route to achieve a similar efficacy to that of intramuscular (IM) administration. Animals receiving ON ASFV-G-ΔI177L were completely protected against virulent ASFV-G challenge. As previously described, similar results were obtained when ASFV-G-ΔI177L was given intramuscularly. Interestingly, viremias induced in animals inoculated oronasally were lower than those measured in IM-inoculated animals. ASFV-specific antibody responses, mediated by IgG1, IgG2 and IgM, do not differ in animals inoculated by the ON route from that had IM inoculations. Therefore, the ASFV-G-ΔI177L vaccine candidate can be administered oronasally, a critical attribute for potential vaccination of wild swine populations.

Polymeric Caffeic Acid Acts as a Nasal Vaccine Formulation against Streptococcus pneumoniae Infections in Mice

Infectious diseases are the second leading cause of death worldwide, highlighting the importance of the development of a novel and improved strategy for fighting pathogenic microbes. Streptococcus pneumoniae is a highly pathogenic bacteria that causes pneumonia with high mortality rates, especially in children and elderly individuals. To solve these issues, a mucosal vaccine system would be the best solution for the prevention and treatment of these diseases. We have recently reported that enzymatically polymerized caffeic acid (pCA) acts as a mucosal adjuvant when co-administered with antigenic proteins via the nasal route. Moreover, the sources of caffeic acid and horseradish peroxidase are ingredients found commonly in coffee beans and horseradish, respectively. In this study, we aimed to develop a pneumococcal nasal vaccine comprising pneumococcal surface protein A (PspA) and pCA as the mucosal adjuvant. Intranasal immunization with PspA and pCA induced the production of PspA-specific antibody responses in the mucosal and systemic compartments. Furthermore, the protective effects were tested in a murine model of S. pneumoniae infection. Intranasal vaccination conferred antigen-dependent protective immunity against a lethal infection of S. pneumoniae. In conclusion, pCA is useful as a serotype-independent universal nasal pneumococcal vaccine formulation.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.