Intranasal immunization with recombinant Vaccinia virus encoding trimeric SARS-CoV-2 spike receptor-binding domain induces neutralizing antibody

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

Intranasal immunization with recombinant Vaccinia virus Tiantan harboring Zaire Ebola virus gp elicited systemic and mucosal neutralizing antibody in mice

SummaryMucosal vaccines are considered to be a promising strategy for preventing upper airway infections. As immunization routes to induce mucosal immune responses, oral, intranasal and sublingual immunizations have been proposed for clinical application. Recently, it was reported that transcutaneous immunization (TC) is also capable of inducing mucosal as well as systemic immune responses. However, it remains unclear which route is most effective for inducing mucosal immune responses in upper respiratory airways. In this study, the mucosal immune responses against phosphorylcholine (PC), a structural component of a wide variety of Gram‐positive and ‐negative bacteria, were investigated after intranasal, sublingual, or TC. Female BALB/c mice were immunized intranasally, sublingually, or transcutaneously with PC and cholera toxin. For the TC, the dorsal region was shaved and a cotton patch soaked with the antigen was attached. Nasal wash, saliva and serum samples were collected at 7 days after the final immunization, and examined for their PC‐specific antibody activities using ELISA. Phosphorylcholine‐specific antibodies in serum and PC‐specific IgA in nasal washes and saliva were detected in mice after intranasal and sublingual immunization with PC. Although salivary IgA was higher following intranasal immunization, nasal wash IgA was significantly higher after sublingual immunization. Furthermore, significantly increased IgA in vaginal washes and remarkably decreased serum IgE production were observed after sublingual immunization. TC increased PC‐specific IgA in faecal samples, but not in saliva. The PC antigen can induce mucosal as well as systemic immune responses when administered via intranasal, sublingual and transcutaneous routes. Sublingual immunization is superior to intranasal immunization in terms of safety for inducing mucosal immune responses. Although TC might be an alternative way to induce mucosal immune responses, further investigation is needed for its application as a vaccine route to prevent upper airway infection.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

(1) Background: The COVID-19 pandemic highlights the critical necessity for the development of mucosal vaccines. (2) Objective: In this study, we aimed to develop mucosal vaccines based on the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. (3) Methods: We engineered the RBD of the Spike protein by incorporating ten lysine residues (K10), thereby enhancing its positive charge under physiological conditions. (4) Results: Although this modification did not directly augment the immunogenicity of the antigen, its combination with the mucosal adjuvant cholera toxin B subunit (CTB) and administration via the pulmonary route in BALB/c mice resulted in the induction of robust neutralizing antibody titers. Antigen-specific antibody responses were observed in both serum and bronchoalveolar lavage fluid. Importantly, serum IgG antibody titers remained above 104 six months following third immunization, suggesting the establishment of sustained long-term immunity. Additionally, the incorporation of five lysine residues (K5) into the RBD, in conjunction with CTB, significantly increased serum IgG and IgA antibody titers. (5) Conclusions: Adding poly-lysine to RBD and combining it with CTB can stimulate robust mucosal and humoral immune responses in mice. These findings offer valuable insights for the design of subunit mucosal vaccines.

Intranasal immunization with the recombinant measles virus encoding the spike protein of SARS-CoV-2 confers protective immunity against COVID-19 in hamsters

Intranasal immunization with liposome-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal, cellular and humoral immune responses

Fibroblast-stimulating lipopeptide-1 as a potential mucosal adjuvant enhances mucosal and systemic immune responses to enterovirus 71 vaccine

<b>Introduction:</b> The mRNA COVID-19 BNT162b2 vaccine has successfully reduced the burden of coronavirus disease 2019 (COVID-19). The serum IgG and IgA responses to the vaccine have been extensively reported. Recent studies suggest that mRNA vaccine induces IgG and IgA production (or secretion) from the upper respiratory tract mucosa. <b>Aim:</b> To evaluate whether BNT162b2 vaccine provides lower airways protection by inducing IgG and IgA neutralizing antibodies from the lung. <b>Methods:</b> Prospective observational study in patients undergoing medically indicated bronchoscopy and bronchoalveolar lavage (BAL). Serum, saliva and BAL fluids were collected from the participants in order to assess immunoglobulins targeting the Receptor Binding Domain (RBD) of SARS-CoV-2. The functional neutralization is measured in cultured cells, using SARS-CoV-2 spike pseudotyped reporter particles. <b>Results:</b> Serum, saliva and BAL fluid were obtained from 68 individuals (6 unvaccinated, 7 vaccinated with 2 doses, 34 vaccinated with 3 doses, 5 vaccinated with 4 doses, 7 recovered, 6 recovered and vaccinated (1 doses at least)). We have adjusted custom enzyme linked immunoassay to quantify molar equivalents of anti-RBD IgG and IgA in serum, saliva and BAL fluids. We detected and characterized anti-RBD IgG and IgA in serum, saliva and BAL of vaccinated and recovered individuals. <b>Conclusion:</b> BNT162b2 mRNA COVID-19 vaccine induces IgG and IgA responses against SARS-CoV-2 in the lungs

SARS-CoV-2 Vaccines: The Mucosal Immunity Imperative

The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape.

As the COVID-19 pandemic has progressed, new variants of the virus SARS-CoV-2 have emerged that are more infectious than the original form. The variants known as Alpha, Beta and Gamma have mutations in a protein on the virus’s surface that is vital for attaching to cells and infecting them. This protein, called Spike, carries out its role by binding to ACE2, a protein on the surface of human cells. Mutations on Spike are found on the region where it binds to ACE2. The interaction between these two proteins appears to be important to the behaviour of SARS-CoV-2, but the impact of individual mutations in Spike is unknown. In addition, some people have different variants of ACE2 with mutations in the region that interacts with Spike, but it is not known whether this affects these people’s risk of contracting COVID-19. To answer these questions, Barton et al. measured the precise effect of mutations in Spike and ACE2 on the strength of the interaction between the two proteins. The experiments showed that three of the five common Spike mutations in the Alpha, Beta and Gamma SARS-CoV-2 variants strengthened binding to ACE2. The two mutations that weakened binding were only found together with other mutations that strengthened binding. This meant that the Spike proteins in all three of these SARS-CoV-2 variants bind to ACE2 more strongly than the original form. The experiments also showed that two common variants of ACE2 also increased the strength of binding to Spike. Interestingly, one of these ACE2 variants reversed the effect of a specific SARS-CoV-2 mutation, suggesting that carriers would be resistant to SARS-CoV-2 variants with this mutation. Identifying the precise effects of Spike mutations on ACE2 binding helps understand why new variants of SARS-CoV-2 spread more rapidly. This could help to identify concerning new variants before they spread widely and inform the response by health authorities. The finding that two common ACE2 variants bind more strongly to Spike suggests that people with these mutations could be more susceptible to SARS-CoV-2.

Induction of neutralizing antibodies and mucosal IgA through intranasal immunization with the receptor binding domain of SARS-CoV-2 spike protein fused with the type IIb E. coli heat-labile enterotoxin A subunit

Host defenses against Streptococcus pneumoniae depend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) of Escherichia coli is a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans.

The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. To determine whether an immune response could be elicited by intranasal delivery of recombinant lactobacilli, a Lactobacillus plantarum strain of human origin (NCIMB8826) was selected as the expression host. Cytoplasmic production of the 47-kDa fragment C of tetanus toxin (TTFC) was achieved at different levels depending on the plasmid construct. All recombinant strains proved to be immunogenic by the intranasal route in mice and able to elicit very high systemic immunoglobulin G (IgG1, IgG2b, and IgG2a) responses which correlated to the antigen dose. No significant differences in enzyme-linked immunosorbent assay IgG titers were observed when mice were immunized with live or mitomycin C-treated recombinant lactobacilli. Nevertheless, protection against the lethal effect of tetanus toxin was obtained only with the strains producing the highest dose of antigen and was greater following immunization with live bacteria. Significant TTFC-specific mucosal IgA responses were measured in bronchoalveolar lavage fluids, and antigen-specific T-cell responses were detected in cervical lymph nodes, both responses being higher in mice receiving a double dose of bacteria (at a 24-h interval) at each administration. These results demonstrate that recombinant lactobacilli can induce specific humoral (protective) and mucosal antibodies and cellular immune response against protective antigens upon nasal administration.