The novel coronavirus disease (COVID-19) and its infamous "Variants" of the etiological agent termed Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has proven to be a global health concern. The three antibodies, IgA, IgM, and IgG, perform their dedicated role as main workhorses of the host adaptive immune system in virus neutralization. Immunoglobulin-A (IgA), also known as "Mucosal Immunoglobulin", has been under keen interest throughout the viral infection cycle. Its importance lies because IgA is predominant mucosal antibody and SARS family viruses primarily infect the mucosal surfaces of human respiratory tract. Therefore, IgA can be considered a diagnostic and prognostic marker and an active infection biomarker for SARS CoV-2 infection. Along with molecular analyses, serological tests, including IgA detection tests, are gaining ground in application as an early detectable marker and as a minimally invasive detection strategy. In the current review, it was emphasized the role of IgA response in diagnosis, host defense strategies, treatment, and prevention of SARS-CoV-2 infection. The data analysis was performed through almost 100 published peer-reviewed research reports and comprehended the importance of IgA in antiviral immunity against SARS-CoV-2 and other related respiratory viruses. Taken together, it is concluded that secretory IgA- Abs can serve as a promising detection tool for respiratory viral diagnosis and treatment parallel to IgG-based therapeutics and diagnostics. Vaccine candidates that target and trigger mucosal immune response may also be employed in future dimensions of research against other respiratory viruses.

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. In this study, we demonstrate the efficacy of Bordetella colonization factor A (BcfA), a novel bacteria-derived protein adjuvant, in SARS-CoV-2 spike-based prime-pull immunizations. We show that i.m. priming of mice with an aluminum hydroxide- and BcfA-adjuvanted spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17-polarized CD4+ tissue-resident memory T cells and neutralizing Abs. Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 (MA10) and reduced viral replication in the respiratory tract. Histopathology showed a strong leukocyte and polymorphonuclear cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. Importantly, neutralizing Abs and tissue-resident memory T cells were maintained until 3 mo postbooster. Viral load in the nose of mice challenged with the MA10 virus at this time point was significantly reduced compared with naive challenged mice and mice immunized with an aluminum hydroxide-adjuvanted vaccine. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, provide sustained protection against SARS-CoV-2 infection.

Antinucleocapsid (anti-N) immunoglobulin G antibody responses were lower in plasma and oral fluid after severe acute respiratory syndrome coronavirus 2 infection in vaccinated patients compared with patients infected before vaccination or infected without vaccination. This raises questions about the long-term use of anti-N antibodies as a marker for natural infection for surveillance.

Based on mucosal immunization to promote both mucosal and systemic immune responses, next-generation coronavirus disease 2019 (COVID-19) vaccines would be administered intranasally or orally. The goal of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines is to provide adequate immune protection and avoid severe disease and death. Mucosal vaccine candidates for COVID-19 including vector vaccines, recombinant subunit vaccines and live attenuated vaccines are under development. Furthermore, subunit protein vac-cines and virus-vectored vaccines have made substantial progress in preclinical and clinical settings, resulting in SARS-CoV-2 intranasal vaccines based on the previously successfully used nasal vaccines. Additional to their ability to trigger stable, protective immune responses at the sites of pathogenic infection, the development of 'specific' mucosal vaccines targeting coronavirus antigens could be an excellent option for preventing future pandemics. However, their efficacy and safety should be confirmed.

As SARS-CoV-2 will likely continue to circulate, low-impact methods become more relevant to monitor antibody-mediated immunity. Saliva sampling could provide a non-invasive method with reduced impact on children. Studies reporting on the differences between systemic and mucosal humoral immunity to SARS-CoV-2 are inconsistent in adults and scarce in children. These differences may be further unraveled by exploring associations to demographic and clinical variables.

To evaluate the use of saliva antibody assays, we performed a cross-sectional cohort study by collecting serum and saliva of 223 children attending medical services in the Netherlands (irrespective of SARS-CoV-2 exposure, symptoms or vaccination) from May to October 2021. With a Luminex and a Wantai assay, we measured prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD) and nucleocapsid-specific IgG and IgA in serum and saliva and explored associations with demographic variables.

The S-specific IgG prevalence was higher in serum 39% (95% CI 32 - 45%) than in saliva 30% (95% CI 24 - 36%) (P ≤ 0.003). Twenty-seven percent (55/205) of children were S-specific IgG positive in serum and saliva, 12% (25/205) were only positive in serum and 3% (6/205) only in saliva. Vaccinated children showed a higher concordance between serum and saliva than infected children. Odds for saliva S-specific IgG positivity were higher in girls compared to boys (aOR 2.63, P = 0.012). Moreover, immunocompromised children showed lower odds for S- and RBD-specific IgG in both serum and saliva compared to healthy children (aOR 0.23 - 0.25, P ≤ 0.050).

We showed that saliva-based antibody assays can be useful for identifying SARS-CoV-2 humoral immunity in a non-invasive manner, and that IgG prevalence may be affected by sex and immunocompromisation. Differences between infection and vaccination, between sexes and between immunocompromised and healthy children should be further investigated and considered when choosing systemic or mucosal antibody measurement.

SARS-CoV-2 is primarily an airborne infection of the upper respiratory tract, which on reaching the lungs causes the severe acute respiratory disease, COVID-19. Its first contact with the immune system, likely through the nasal passages and Waldeyer's ring of tonsils and adenoids, induces mucosal immune responses revealed by the production of secretory IgA (SIgA) antibodies in saliva, nasal fluid, tears, and other secretions within 4 days of infection. Evidence is accumulating that these responses might limit the virus to the upper respiratory tract resulting in asymptomatic infection or only mild disease. The injectable systemic vaccines that have been successfully developed to prevent serious disease and its consequences do not induce antibodies in mucosal secretions of naïve subjects, but they may recall SIgA antibody responses in secretions of previously infected subjects, thereby helping to explain enhanced resistance to repeated (breakthrough) infection. While many intranasally administered COVID vaccines have been found to induce potentially protective immune responses in experimental animals such as mice, few have demonstrated similar success in humans. Intranasal vaccines should have advantage over injectable vaccines in inducing SIgA antibodies in upper respiratory and oral secretions that would not only prevent initial acquisition of the virus, but also suppress community spread via aerosols and droplets generated from these secretions.

Despite the remarkable efficacy of currently approved COVID-19 vaccines, there are several opportunities for continued vaccine development against SARS-CoV-2 and future lethal respiratory viruses. In particular, restricted vaccine access and hesitancy have limited immunization rates. In addition, current vaccines are unable to prevent breakthrough infections, leading to prolonged virus circulation. To improve access, a subunit vaccine with enhanced thermostability was designed to eliminate the need for an ultra-cold chain. The exclusion of infectious and genetic materials from this vaccine may also help reduce vaccine hesitancy. In an effort to prevent breakthrough infections, intranasal immunization to induce mucosal immunity was explored. A prototype vaccine comprised of receptor-binding domain (RBD) polypeptides formulated with additional immunoadjuvants in a chitosan (CS) solution induced high levels of RBD-specific antibodies in laboratory mice after 1 or 2 immunizations. Antibody responses were durable with high titers persisting for at least five months following subcutaneous vaccination. Serum anti-RBD antibodies contained both IgG1 and IgG2a isotypes suggesting that the vaccine induced a mixed Th1/Th2 response. RBD vaccination without CS formulation resulted in minimal anti-RBD responses. The addition of CpG oligonucleotides to the CS plus RBD vaccine formulation increased antibody titers more effectively than interleukin-12 (IL-12). Importantly, generated antibodies were cross-reactive against RBD mutants associated with SARS-CoV-2 variants of concern, including alpha, beta and delta variants, and inhibited binding of RBD to its cognate receptor angiotensin converting enzyme 2 (ACE2). With respect to stability, vaccines did not lose activity when stored at either room temperature (21-22°C) or 4°C for at least one month. When delivered intranasally, vaccines induced RBD-specific mucosal IgA antibodies, which may protect against breakthrough infections in the upper respiratory tract. Altogether, data indicate that the designed vaccine platform is versatile, adaptable and capable of overcoming key constraints of current COVID-19 vaccines.

The development and deployment of vaccines against COVID-19 demonstrated major successes in providing immunity and preventing severe disease and death. Yet SARS-CoV-2 evolves and vaccine-induced protection wanes, meaning progress in vaccination strategies is of upmost importance. New vaccines directed at emerging viral strains are being developed while vaccination schemes with booster doses and combinations of different platform-based vaccines are being tested in trials and real-world settings. Despite these diverse approaches, COVID-19 vaccines are only delivered intramuscularly, whereas the nasal mucosa is the primary site of infection with SARS-CoV-2. Preclinical mucosal vaccines with intranasal or oral administration demonstrate promising results regarding mucosal IgA generation and tissue-resident lymphocyte responses against SARS-CoV-2. By mounting an improved local humoral and cell-mediated response, mucosal vaccination could be a safe and effective way to prevent infection, block transmission and contribute to reduce SARS-CoV-2 spread. However, questions and limitations remain: how effectively and reproducibly will vaccines penetrate mucosal barriers? Will vaccine-induced mucosal IgA responses provide sustained protection against infection?

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Despite global vaccination efforts, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and spread globally. Relatively high vaccination rates have been achieved in most regions of the United States and several countries worldwide. However, access to vaccines in low- and mid-income countries (LMICs) is still suboptimal. Second generation vaccines that are universally affordable and induce systemic and mucosal immunity are needed. Here we performed an extended safety and immunogenicity analysis of a second-generation SARS-CoV-2 vaccine consisting of a live Newcastle disease virus vector expressing a pre-fusion stabilized version of the spike protein (NDV-HXP-S) administered intranasally (IN), intramuscularly (IM), or IN followed by IM in Sprague Dawley rats. Local reactogenicity, systemic toxicity, and post-mortem histopathology were assessed after the vaccine administration, with no indication of severe local or systemic reactions. Immunogenicity studies showed that the three vaccination regimens tested elicited high antibody titers against the wild type SARS-CoV-2 spike protein and the NDV vector. Moreover, high antibody titers were induced against the spike of B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants of concern (VOCs). Importantly, robust levels of serum antibodies with neutralizing activity against the authentic SARS-CoV-2 USA-WA1/2020 isolate were detected after the boost. Overall, our study expands the pre-clinical safety and immunogenicity characterization of NDV-HXP-S and reinforces previous findings in other animal models about its high immunogenicity. Clinical testing of this vaccination approach is ongoing in different countries including Thailand, Vietnam, Brazil and Mexico.