key: cord-1020838-yinnu3ke authors: Tiboni, Mattia; Casettari, Luca; Illum, Lisbeth title: Nasal vaccination against SARS-CoV-2: synergistic or alternative to intramuscular vaccines? date: 2021-05-06 journal: Int J Pharm DOI: 10.1016/j.ijpharm.2021.120686 sha: e033b04a16c08426f86c2e202f160c904cf1b801 doc_id: 1020838 cord_uid: yinnu3ke It is striking that all marketed SARS-CoV-2 vaccines are developed for intramuscular administration designed to produce humoral and cell mediated immune responses, preventing viremia and the COVID-19 syndrome. They have a high degree of efficacy in humans (70-95%) depending on the type of vaccine. However, little protection is provided against viral replication and shedding in the upper airways due to the lack of a local sIgA immune response, indicating a risk of transmission of virus from vaccinated individuals. A range of novel nasal COVID-19 vaccines are in development and preclinical results in non-human primates have shown a promising prevention of replication and shedding of virus due to the induction of mucosal immune response (sIgA) in upper and lower respiratory tracts as well as robust systemic and humoral immune responses. Whether these results will translate to humans remains to be clarified. An IM prime followed by an IN booster vaccination would likely result in a better well-rounded immune response, including prevention (or strong reduction) in viral replication in the upper and lower respiratory tracts. Many human pathogens enter the human organism via a mucosal site such as the gastrointestinal mucosa (e.g., poliovirus, Vibrio cholerae, HIV-1), genital mucosa (e.g., human papilloma virus (HPV), HIV-1) and respiratory mucosa (e.g., influenza virus, Mycobacterium tuberculosis, coronavirus, adenovirus, rhinovirus, respiratory syncytial virus (RSV) (Belyakov and Ahlers, 2009) . Some mucosal pathogens can spread to systemic sites by entering the blood circulation, whereas others only develop the disease at a local site such as for HIV-1. The innate mucosal immune system present in humans has evolved to protect humans from invading pathogens, by specifically recognizing and eliminating harmful species. The innate mucosal immune system comprises a variety of recognition receptor molecules (e.g., TLRs, NOD-like receptors), which after activation can effectively recognize invading pathogens and generate an immune response that prevents or limits pathogen entry and neutralises any adverse reactions such as tissue damage. Furthermore, it regulates the adaptive response in cases of severe infection and also helps generate a memory response (Aich and Dwivedy, 2011; Belyakov and Ahlers, 2009) . A comprehensive review (Poland et al., 2020) In humans, the airways are highly prone to the risk of viral infections which can be the cause of seasonal epidemics or even pandemics and thereby pose a severe health risk to the world's population, especially those with underlying medical conditions or those of certain ethnicities. For example, one of the most widespread viral infections is caused by the influenza virus which exists as four types, A, B, C and D. It is, however, the influenza virus A and B that are the cause of seasonal epidemics every year and only influenza A virus is known to cause flu pandemics. Pandemics generally occur when a variant influenza A virus emerges that is highly infective and with the ability to efficiently transmit between people (Rose et al., 2012) . called the "A(H1N1)pdm09 virus", or in common terms "2009 H1N1", has since continued to circulate in the population and has undergone relatively limited genetic changes and changes to their antigenic properties that affect immunity over time. The COVID-19 pandemic, that started in Wuhan, China in the end of 2019, was caused by the transmission of "severe acute respiratory syndrome coronavirus 2" the so-called SARS-CoV-2 virus. SARS-CoV-2 is a member of the coronavirus family which can cause common colds and the more fatal Middle East respiratory syndrome (MERS). The SARS-CoV-2 is a positivesense single-stranded RNA (+ssRNA) virus with a single linear RNA segment. The genome of CoV is the largest RNA genome (26.4-31.7 kilobases) of all known RNA viruses (Woo et al., 2009 ). Each virion is from 50 to 200 nm in diameter and comprises four different structural proteins, namely S (spike), E (envelope), M (membrane) and N (nucleocapsid), where the N protein surrounds the RNA genome and the S, E and M proteins form the viral envelope ( Figure 1 ). The S protein (a glycoprotein) forms homo trimeric spikes on the virion and is responsible for the ability of the virus to attach to and fuse with the membrane of the host cell, engaging the cell surface receptor angiotensin-converting enzyme 2 (ACE2), and thereby allowing it cell entry ("Coronaviruses -a general introduction"; Letko et al., 2020; Wu et al., 2020) . SARS-CoV-2 is efficiently transmitted from person to person and therefore rapidly spread across all continents. The transmission of the virus occurs via respiratory droplets from cough and sneezes, from speaking and also at least indoors with air flow, suggesting that the virus may be airborne ("239 Experts With One Big Claim: The Coronavirus Is Airborne -The New York Times", "Talking is worse than coughing for spreading COVID-19 indoors | Live Science"). It has been shown that the nasal epithelium has the highest concentration of ACE2 and the lowest is found in the alveoli (Hou et al., 2020) . Hence, it is to be expected that the replication of the virions mostly takes place in nasal mucosa (Sims et al., 2005) and furthermore in the salivary gland ducts that also are rich in the expression of ACE2 (Liu et al., 2011) . The SARS-CoV-2 has a high mutation rate because of the error prone RdRp (RNA-dependent RNA polymerases) of the virus which is responsible for the duplication of genetic information. Hence, the virus is prone to create variants of the virus, of which the most prominent at present are a) the UK (or Kent) variant known as B.1.1.7, which show several mutations and especially one in the S protein that causes the virus to bind more tightly to the ACE2 receptor; b) the South African variant known as B.1.351, also with mutations in the S protein and c) the Danish variant appearing in minks and mink farmers with four changes in the spike protein which makes the virus moderately resistant to neutralizing antibodies, d) the Brazilian virus, known as P1, that is feared to be more contagious than the original virus and very recently the Indian variant that appears to have two mutations ("Science Brief: Emerging SARS-CoV-2 Variants | CDC", "WHO | SARS-CoV-2 mink-associated variant strain -Denmark"). In order to combat such viral infections, developed countries at least, have immunization programmes for yearly vaccination, for example against influenza, with most emphasis on vaccination of the older part of the population. This is also reflected in the current situation with the COVID-19 pandemic where at least the developed countries presently are competing to vaccinate as quickly as possible their most vulnerable subjects. For example, the UK has managed to vaccinate more than 30 million people over a period of 4 months (Jan-April 2021) which has taken planning, co-ordination and investment of a magnitude only previously seen 7 location, are named the e.g., nasopharynx-associated lymphoid tissue (NALT), the bronchusassociated lymphoid tissue (BALT) and the gut-associated lymphoid tissue (GALT) (Brandtzaeg et al., 2008) . Therefore, mucosal immunity often is best induced by administration of vaccines by a mucosal route since mucosal immunisation generally, if an optimal vaccine formulation is developed, will result in both a mucosal and a systemic immune response (Borges et al., 2010) . Of the various routes of mucosal administration, the nasal and the oral routes are the most acceptable and accessible, but due to the hostile gastrointestinal environment, where the antigen can potentially be degraded or denaturated, and the dilution by intestinal content requiring high doses of antigenic material and specialised vaccine formulations, the nasal route is preferential to the oral. In humans the nasal lymphoid tissue is situated in the oropharynx and described as a ring of tissues (Waldeyer's ring), comprising the nasopharyngeal adenoids (or tonsils), the paired tubal tonsils and the paired palatine and lingual tonsils ( Figure 2 ). The adenoids are similar to the Peyer's patches in the intestines in that they contain aggregates of lymphoid tissue. The NALT is strategically placed in the nasopharynx and oropharynx areas so that they can be exposed not only to airborne antigens but also alimentary antigens. Furthermore, the epithelial surface of the NALT invaginates into valleys, the so-called crypts that increases the area for antigen interaction and for retainment. M-like cells (or microfold cells) are located in these crypts (Brandtzaeg, 2011; Cesta, 2006) . It should also be noted that the epithelial cells are covered with mucus that acts as a barrier to invasion of pathogens and cilia that through the mucociliary clearance mechanism may quickly transport the pathogens down the esophagus. Antigens reaching the nasal mucosa can be transported to the NALT. Soluble antigens can penetrate between epithelial cells and reach the antigen-presenting cells (APC) such as macrophages and dendritic cells whereas particulate antigens are transported across the epithelium via M-like cells (or microfold M-cells) that are present in the epithelial cell layer overlying the NALT. The APC process and present the antigen to the T cells e.g., CD4+ T cells in the lymphoid tissue that can then induce IgA-committed B-cell development in the lymphoid follicle. The B-cells migrate from the NALT to the regional cervical lymph nodes via the efferent lymphatics and then the antigen specific CD4+ cells and IgA+ B cells migrate to the nasal passage through the thoracic duct and the blood circulation. The IgA+ B cells then, in the presence of cytokines (e.g., IL-5 and IL-6 produced by T helper cells), differentiate into IgA producing plasma cells that create dimeric forms of IgA which subsequently become secretory IgA by binding to polymeric Ig receptors present on the epithelial mucosal cells. This secretory IgA is then released into the nasal mucosal surface. Specific neutralising IgG (antibodies) are also present within the mucosal tissues derived from local plasma cells or from blood by diffusion from local fenestrated epithelia ( Figure 3 ) (Kiyono and Fukuyama, 2004) . Hence, as has been reported by some researchers, after an appropriate antigen stimulation of the NALT, both a potent humoral and cellular immune response is normally elicited both at a mucosal and systemic level (Rose et al., 2012; . The antigens reaching the NALT are met with two different defence mechanisms involving antibodies namely the production of secretory IgA which helps in preventing further viral infection and IgG antibodies which can neutralize viruses that are generated in the mucosa. As indicated above, secretory IgA is an important effector molecule for protecting the mucosal surface, however, the contribution of the cellular immune system in this defence should not be underestimated. A cell-mediated immune response has a strategic advantage, as opposed to an antibody-mediated immune response, in that T cells can recognize peptides from the core proteins of for example influenza virus and that the core proteins are normally expressed and presented earlier during infection than proteins that are targeted for neutralising antibodies, as for example is the case for hemagglutinin and neuraminidase of influenza virus . Two mechanisms are involved in the killing of infected cells that entail specific cytotoxic T lymphocytes (CTLs) or antibody-dependent cell-mediated cytotoxicity (ADCC), a collaboration between natural killer (NK) cells and antibodies. It should be noted that vaccination by a mucosal route such as the nasal can induce generalized mucosal immune responses, not only at the nasal mucosa but also at distant mucosal effector sites (Belyakov and Ahlers, 2009 ). The SARS-CoV-2 S protein binds primarily to the ACE2 receptors to mediate viral entry, in the upper and lower respiratory tracts. The mature S protein is a trimeric class I fusion protein located on the surface of the virion. It possesses two fragments, the S1 containing the receptor binding domain (RBD) and the S2 containing the fusion peptide. Different studies with monoclonal antibodies have demonstrated that infected humans develop robust neutralizing antibodies against the S protein and in particular against the S1 fragment with the receptorbinding domain (RBD) of the SARS-CoV-2 Hansen et al., 2020; Ju et al., 2020) . In early studies for SARS-CoV-2 vaccines, the N protein was also evaluated for effectiveness but, using in vivo models, N-based vaccines resulted in no protection. Furthermore, they showed an exacerbation of the infection due to increased pulmonary eosinophilic infiltration (Deming et al., 2006) . M and E proteins are of less interest as vaccine targets due to lower immunogenicity (Du et al., 2008) . Advances in virology, molecular biology and immunology have created many alternatives to traditional vaccine approaches. More than 100 vaccine candidates against the SARS-CoV-2 virus are currently in development ("Vaccines -COVID19 Vaccine Tracker"), based on several different platforms ( Figure 4 ). These platforms can be divided into "traditional" approaches (i.e., live attenuated or inactivated virus vaccines) and "innovative approaches" such as RNA or DNA vaccines and recombinant viral-vectored vaccines. Live attenuated vaccines derive directly from the pathogenic viruses that still possess the ability to infect cells and replicate but are treated in order to cause no or only very mild disease. The attenuation can be completed by growing the virus at unfavourable conditions such as at nonoptimal temperature or by rational modification of the virus genome (e.g., codon deoptimization, removal of genes responsible for counteracting innate immune recognition (Broadbent et al., 2016; Talon et al., 2000) ). However, these techniques are time-consuming and technically challenging, resulting in a difficult and long development. Being nearly identical to the natural virus causing the infection, a live attenuated virus usually creates a strong and long-lasting humoral and cell-mediated immune response after a prime/boost vaccination regimen. Moreover, since the virus is replicating after the vaccination, the immune response is targeting both structural and non-structural viral proteins, widening the humoral and cellular immune responses without the use of adjuvants since these vaccines already contain naturally occurring adjuvants (Lee and Nguyen, 2015) . This type of vaccine can be given intranasally to induce a mucosal immune response such as in the case of the quadrivalent influenza vaccine against A(H1N1), A(H3N2) and two influenza B viruses available in the market with the brand name FluMist Quadrivalent ("FluMist Quadrivalent | FDA"). It is easily administered as 0.2 mL suspension supplied in a single-dose pre-filled intranasal spray device to be divided approximately one-half into each nostril. In inactivated viral vaccines the whole disease-causing virus or a part of it (where the genetic material has been wrecked) is usually present. Compared to live attenuated viral vaccines, they are considered safer and more stable and although their genetic material has been destroyed, they still contain many antigenic proteins and hence, as in the case for coronaviruses (e.g. SARS-CoV-2), the immune responses are likely to target many different proteins such as the S but also M, E, and N. Inactivated vaccines only stimulate antibody-mediated responses, which can be weaker and less long-lived, as compared to live attenuated vaccines, and hence, inactivated vaccines are often administered alongside adjuvants and also booster doses may be required. The vaccine production requires biosafety level 3 facilities in which the virus is grown in a cell culture (usually Vero cells) followed by the inactivation. The productivity of the virus in cell culture could affect the final production yield (Yadav et al., 2021) . This type of vaccine has proven to be safe and effective in the prevention of diseases like polio and influenza (https://www.who.int/vaccine_safety/initiative/tech_support/Part-2.pdf -accessed March 22, 2021). Viral vector-based vaccines (in the form of a modified harmless version of an alternative virus) use a modified virus (the vector) to deliver the genetic code (RNA or DNA) for an antigen, (e.g., in the case of COVID-19 the S protein) into human cells which then will produce the antigen. Infecting the cells and instructing them to produce the antigen, this type of vaccine mimic a natural viral infection in order to generate the requested immune response (Rollier et al., 2011) . This mechanism induces a strong cellular immune response by T cells as well the production of antibodies by B cells. The viral vectors are grown in cell lines and their production is quick and easy (Sebastian and Lambe, 2018) . Viral vectors can be replicating and non-replicating. Replicating viral vectors possess the ability to replicate and thus they can produce new viral particles providing a continuous source of vaccine antigens for prolonged periods. This results in a stronger immune response with a single dose compared to the non-replicating viral vectors. Replicating viral vectors are selected so that the virus cannot cause a disease whilst infecting the host. They typically derive from attenuated viruses engineered to express the specific antigen protein such as the S protein for COVID-19 vaccine. On the other hand, non-replicating viral vectors do not retain the ability to make new viral particles because the key viral genes for the replication have been previously removed. The most common approaches of this vaccine type are based on an adenovirus delivered intramuscularly. As an advantage of viral vectored vaccines, their production does not require the use of live pathogen viruses, the vectors can be easily produced in large quantities showing a good stimulation of both B and T cell responses in vivo (Zhu et al., 2020a) . As a disadvantage, pre-existing vector immunity can neutralize the vaccine efficacy. However, this problem can be easily avoided by using vectors that are rare in humans (Mercado et al., 2020) , derived from animals (Folegatti et al., 2020) or viruses that do not generate much immunity. Moreover, as vector immunity can be problematic during the second dose in a prime-boost regimen, the use of two different viral vectors during the two doses can help avoiding this problem. Nevertheless, in this case, vaccine antigen can only be produced as long as the initial vaccine remains in infected cells, resulting in a generally weaker immune response. Booster doses are likely to be required. An example of a viral vector vaccine is the recombinant, replication-competent rVSV-ZEBOV vaccine against Ebola (Marzi et al., 2011) Protein subunit vaccines (also called acellular vaccines) do not contain any whole virus, but instead purified antigenic fragments such as isolated proteins (e.g., the S protein on the SARS-CoV-2 virus) specifically selected because of their capacity to stimulate the immune system. Many different antigens can be selected to develop acellular vaccine such as specific isolated proteins from viral or bacterial pathogens, chains of sugar molecules (polysaccharides) found in the cell walls of some bacteria or a carrier protein binding a polysaccharide chain in order to boost the immune response. Acellular vaccines are generally considered very safe since they cannot cause the disease. The immune response usually is not as robust as for live attenuated vaccines, hence, booster doses are most often required. A possible disadvantage of this type of vaccine is that isolated proteins could be denatured and thus bind to different antibodies than the protein of the pathogen. In the case of SARS-CoV-2, the antigenic proteins used are the S protein or the RBD. The advantage of this type of vaccine is that live virus is not handled. Commonly used protein subunit vaccines are the acellular pertussis (aP) vaccines that contain the inactivated pertussis toxin detoxified either by treatment with a chemical or by using molecular genetic techniques Nucleic acid-based vaccines follow a different strategy compared to the other vaccines. Instead of directly providing the protein antigen to the body, they deliver the genetic code of the antigen to the cells in the body instructing the cells to produce the antigen that then will stimulate an immune response. This type of vaccines is quick and easy to develop and are the most promising vaccines for the future. They are divided in RNA-and DNA-based vaccines. RNA vaccines use messenger RNA (mRNA) or self-replicating RNA normally formulated in a particulate carrier such as a lipidic bilayer membrane (liposome). This formulation protects the mRNA when first enters the body and helps cell internalization (Pardi et al., 2015) . Higher doses are required for mRNA than for self-replicating RNA, which amplifies itself. When the mRNA is inside the cells, it can be translated into the antigen protein by ribosomes to start the stimulation of the immune response. Then the mRNA is naturally broken down and removed by the body. A main advantage of this technology is that the vaccine can be produced completely without the use of cell cultures, however, the long-term storage stability is challenging since it requires frozen storage. RNA-based vaccines are usually administered by injection and are therefore unlikely to induce strong mucosal immunity (Pardi et al., 2018) . Being more stable than mRNA/RNA, DNA do not require to be formulated in particulate carriers. They are based on plasmid DNA that can be produced at large scale in bacteria. The DNA contains mammalian expression promoters and the specific gene that encodes for the antigen (e.g., the spike protein) produced after the uptake in the cells of the vaccinated person. To be delivered, they usually need delivery strategies such as electroporation that help the DNA cellular uptake. Both these technologies based on nucleic acids are the latest frontier of vaccination and up till now two different mRNA vaccines have been approved for human use (i.e., Moderna and Pfizer/BioNTech (Baden et al., 2021; Polack et al., 2020) ) meanwhile the most advanced DNA vaccine so far is the INO-4800 from Inovio that has entered Phase 2/3 clinical trials ("Safety, Immunogenicity, and Efficacy of INO-4800 for COVID-19 in Healthy Seronegative Adults at High Risk of SARS-CoV-2 Exposure -Full Text View -ClinicalTrials.gov"). Many vaccine formulations contain an adjuvant or adjuvants combinations that enhance the immune response to the vaccination. The word "adjuvant" means "to help/aid", and initially adjuvants were used only to increase the immunogenic potential of purified antigens. Not all the types of vaccines need an adjuvant such as the live attenuated virus that possess naturally occurring adjuvants. In recent years, by knowing and understanding the immunology of vaccination, the role of adjuvants has expanded (Pasquale et al., 2015) . The first adjuvants authorized (nearly 70 years ago) for human use were aluminium salts (e.g., aluminium hydroxide, aluminium phosphate, aluminium potassium sulphate (alum)). They are still the most widely used because of their wide-spectrum ability to strengthen immune responses and their safety. They act primarily to increase antibody production with an immune mechanism that remains incompletely understood (Lee and Nguyen, 2015) . Newer adjuvants have been developed to target specific components of the body's immune response such as the tall-like receptors (TLR) that, when triggered, stimulate the production of pro-inflammatory cytokines/chemokines and type I interferons that increase the host's ability to eliminate the pathogen. Adaptive immunity is developed immediately after the innate immune response so that the protection against disease is stronger and lasts longer (Steinhagen et al., 2011) . So far, at the time of writing this review, ten SARS-CoV-2 vaccines have been fully approved or approved under Emergency Use Authorisation (EUA) (or similar) by the regulatory authorities and distributed for use in various countries such as EU, UK, Russia, USA, India and China. The marketed injectable vaccines are listed in Table 1 . Vaccine Stability Data at Standard Freezer Temperature to the U.S. FDA Nasdaq:BNTX"). The vaccine, code-named BTN 162b2, is administered IM as a series of two doses ( The AstraZeneca/Oxford Jenner Institute COVID-19 vaccine was approved the 30 th December 2020 as a conditional marketing authorisation (CMA) by the MHRA in the UK and as a CMA in the EU by EMA the 29 th January 2021 for active immunisation to prevent coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 in individuals 18 years of age and older. Approval in the USA is pending. The vaccine (ChAdOx1-S) is supplied as a ready-made aqueous suspension for IM injection. Each multidose vial contain 8 x 0.5 mL doses with not less than 2.5 x 10 8 infectious units and can be stored for six months at 2 o C to 8 o C and when opened for no more than 48h at the same temperature. The vaccination regimen is two separate doses of 0.5 mL each with an interval of 4-12 weeks between doses. The AstraZeneca COVID-19 vaccine works by delivering the genetic code of the SARS-CoV-2 spike protein to the body's cells, that will produce the antigen (i.e., the S-glycoproteins). It is a monovalent vaccine comprising a single recombinant replication-deficient chimpanzee adenovirus vector encoding the full-length SARS-CoV-2 spike glycoprotein gene (DNA), where the immunogen in the vaccine is expressed in the trimeric pre-fusion conformation. After administration, the S glycoprotein is expressed locally and able to stimulate the production of neutralising antibody (humoral response) and cellular immune responses. The conditional approval of the COVID-19 vaccine was based on a range of preclinical and phase 1, 2 and 3 clinical studies evaluation safety and efficacy of the vaccine of which some results are described here. A recently reported phase 1/2 clinical study in 5,258 healthy volunteers of age 18-55 years were administered either ChAdOx1 nCoV-19 at a dose of 5 x 10 10 viral particles or the meningitis vaccine control (MenACWY) as a single IM injection whereas ten participants also received a booster dose 28 days after the first ChAdOx1 nCoV-19 dose. There were no serious adverse events related to ChAdOx1 nCoV-19. It was found that the vaccine induced a spike-specific T-cell responses that peaked on day 14, whereas a potent anti-spike IgG response rose by day 28 and were augmented following a second dose. The trial did not show to what extent both CD4+ and CD8+ T cell subsets were activated (Folegatti et al., 2020) . Vaccine efficacy was found to be 62.6% in subjects receiving two recommended doses with any dose interval between 3 -23 weeks with no cases of COVID-19 hospitalisation in subjects who received two doses of the COVID-19 vaccine as compared to eight in the control. A single blind, randomised, controlled phase 2/3 clinical in healthy volunteers of 18 years and older were divided in age groups of 18-55 years, 56-69 years and 70 years and older. In a lowdose cohort subjects received either IM ChAdOx1 nCoV-19 (2.2 x 10 10 virus particles) or a control vaccine (MENACWY) using a complicated block randomisation and stratified by age and dose group and study site. Secondly, subjects were recruited to the standard dose cohort (3.5 x 10 10 virus particles) and a similar randomisation procedure. The specific aim of the study was to assess the safety and humoral and cellular immunogenicity of single-dose and doubledose regimen in subjects older than 55 years. In subjects who received two doses of vaccine the median anti-spike SARS-CoV-2 IgG response were similar in all age groups at 28 days after the booster dose. By 14 days after the booster dose, 99% of the boosted subjects had neutralising antibody responses. The T-cell responses peaked at 14 days after a single standard dose. It was also concluded that the ChAdOx1 nCoV-19 vaccine was better tolerated in older subjects than in younger but had a similar immunogenicity across all age groups (Ramasamy et al., 2020) . Recently, Voysey et al. (2021) published an interim analysis of four randomised controlled trials (phase 1/2/3) pooling results from studies COV001 (UK), COV002 (UK), COV003 (Brazil) and COV005 (South Africa). Pooling all results, the mean efficacy was 70.4%. But remarkably, in subjects who received a low dose (LD) followed by a standard dose (SD) the efficacy was 90.0%. There were ten subjects hospitalised due to COVID-19 but these were all in the control group. The duration of the protection was not determined. On the 22 nd March AstraZeneca announced that a US phase 3 trial (two doses 4 weeks apart) showed a statistically significant vaccine efficacy of 79% at preventing symptomatic COVID-19 and 100% efficacy at preventing severe disease and hospitalisation. Notably in subjects aged 65 years and over the vaccine efficacy was found to be 80%. The study was based on 32,449 subjects, with a 2:1 randomisation of vaccine to placebo and accruing 141 symptomatic cases of COVID-19 ("AZD1222 US Phase III trial met primary efficacy endpoint in preventing COVID-19 at interim analysis"). It should be noted that in an earlier study in non-human primates, although the rhesus macaques showed a reduced viral load in the bronchoalveolar lavage (BAL) fluid after IM vaccination there was no difference in nasal viral shedding between vaccinated and control SARS-CoV-2 infected macaques (van Doremalen et al., 2020). The Oxford Vaccine Group published a study (yet to be peer reviewed) in Lancet on February to 85% and 94% drop in risk of coronavirus hospital admissions in Scotland, study shows | UK News | Sky News"). The In an expansion of the Phase 1, dose-escalating, open-label clinical of the mRNA-1273 vaccine described above, 40 older subjects (56-70 or more than 70 years of age) were recruited and received two doses of either 25 µg or 100 µg 28 days apart. Interestingly, by day 57 the anti-S-2P geometric mean titre was higher among subjects of more than 70 years than of subjects between 56-70 years of age. It was also confirmed that the 100 µg dose of vaccine induced higher binding and neutralising antibody titres than the 25 µg dose, supporting the use of the 100 µg dose in the Phase 3 study . In a further "correspondence paper" the authors reported that serum neutralizing antibodies continued to be detected (with a slight expected decline in titres of binding and neutralising antibodies) in all participants at day 119 and that, although correlates of protection against SARS-CoV-2 infection in humans have not been established, the mRNA-1273 had the potential to provide durable humoral immunity (Widge et al., 2021 The storage at 2°C to 8°C) (to be reconstituted in 1.0 mL of sterile water for injection before use) for delivery to distant regions of Russia (Logunov et al., 2020) . (https://roszdravnadzor.gov.ru/i/upload/files/Новости/Файлы/28.12.2020/инструкцияпо применению ЛС.pdf -accessed March 22, 2021). The two-component vaccine was evaluated for safety and immunogenicity in two separate open, non-randomised phase 1/2 clinical studies in 76 healthy subjects, planned to be aged 18-60 years of age (although the authors declared that the "volunteers were fairly young"). In the first stage of the study (36 subjects) the subjects were given either; a single dose of rAd26-S or rAd5-S (either frozen or lyophilised) and assessed for safety for 28 days. In Stage 2 of the studies 40 subjects were given a prime dose of rAd26-S and on day 21 a booster dose of the rAd5-S. Both vaccine formulations were safe and well tolerated and most adverse effects were mild, and no serious adverse events were found. All subjects in both studies were, according to the authors, found to have seroconverted at day 21 showing RBD-specific (neutralising) IgGs with titres observed equal to or higher than those seen in patients recovered from COVID-19. Furthermore, T cell responses (CD4+ and CD8+) were detected in all subject at day 28 (Logunov et al., 2020 ). An interim analysis of a controlled phase 3 clinical trial, initiated September 7th 2020, evaluating the safety and efficacy of the rAd26-S or rAd5-S heterologous vaccine, was published February 2nd, 2021 (Logunov et al., 2021) . The study was randomised, double-blind and placebo controlled and took place at 25 hospitals or polyclinics in Moscow. The primary outcome was the proportion of subjects confirmed with COVID-19 infection 21 days after receiving the first dose. Secondary outcomes were the severity of COVID-19 infections, changes in antibody levels against the spike protein S and N protein, changes in neutralising antibody titres and changes in antigen specific cellular immunity levels. 19,866 subjects received either two doses of vaccine or placebo and were included in the analysis. From day 21, 0.1% of the vaccination group subjects and 1.3% of the placebo group subjects, were found to have contracted COVID-19. The vaccine efficacy was calculated to be 91.6%. No serious side effects were considered to be associated with vaccination. RBD-specific IgG was detected in 98% of the samples with a seroconversion rate of 98.25%, whereas, the data for the placebo samples were 15% and 14.9%, respectively. In terms of neutralising antibodies, on day 42 after first vaccination, the GMT was 44.5 and the seroconversion was 95.83%, compared to 1.6 and 7.14%, respectively, in the placebo group. The cellular immune response was highest in the vaccine group (expressed as IFN-g secretion 28 days after the first vaccination . The tolerability profile of the vaccine in subjects aged 18 and older was good. Studies are ongoing to investigate a single dose regimen of vaccination (Logunov et al., 2021) . Warnings were published from the Paul-Ehrlich Institute in Germany, together with the WHO, on the 11th August 2020 against the limited transparency of the regulatory approval of the Sputnik V vaccine, when at that time no data from phase 2/3 clinical trials with thousands of subjects (or even interim data) had been released ("Paul-Ehrlich-Institut -Homepage - Institute,"). Another concern, in our opinion, is that the vaccine was approved for subjects over 18 but the mean age of the volunteers was between 25.3 years and 31.4 years which (as was also admitted by the authors) would (taking into account the standard deviations), mean very few if any volunteers were over 40 years of age. (https://cattiviscienziati.com/2020/09/07/noteof-concern/ -accessed March 22, 2021). The Experimental Covid-19 Vaccines -WSJ"). There also seems to be a second similarly produced vaccine developed in a collaboration between Sinopharm and Wuhan Institute of Biological Products. Studies with both of these vaccines are described below. As described above, the use of inactivated whole virus has been a standard method of development of vaccines against a range of viral infections such as influenza, polio and hepatitis and often need coadministration with an adjuvant in order to induce efficient immunogenicity (Murdin et al., 1996; Vellozzi et al., 2009 (Xia et al., 2020) , the present study did not find any noticeable changes in lymphocyte subsets or cytokines, indicating no cellular immunity was induced. It should be noted that a seroconversion rate of 100% was reached earlier in the 18-59 years age group compared to the group aged 60 and over and more over that the titres of neutralising antibodies were lower in the older group (Xia et al., 2021) . As far as we are aware, results from Phase 3 studies have not been published, but it has been reported by UAE that interim results showed that the BBIBP-CorV vaccine had an 86% efficacy rate, 99% seroconversion rate of neutralising antibody and 100% effectiveness in preventing moderate to severe cases of COVID-19. However, Sinopharm announced that its internal data showed an efficacy rate of 79% ("China Approves Sinopharm's Covid-19 Vaccine as it Moves to Inoculate Millions -The New York Times", "UAE: Ministry of Health announces 86 per cent vaccine efficacy | Health -Gulf News"). Zhang et al. reported results from a safety, tolerability and immunogenicity phase 1/2 clinical trial in healthy adults 18-59 years of age. The study was randomised, double blind and placebo controlled, and as for the Sinopharm studies, the clinical trial was separated in a phase 1 and a phase 2 study. 144 subjects were enrolled in the phase 1 study and separated into two vaccination regimen cohorts, i.e., vaccination at day 0 and 14 and vaccination at day 0 and 28. Also, within each of these cohorts, using block randomisation, the first 36 subjects were assigned to a low dose of CoronaVac (3 µg per 0.5 mL of alum diluent per dose) and the other 36 subjects to a high dose of CoronaVac (6 µg per 0.5 mL of alum diluent per dose). Furthermore, within each block, the subjects were given either two doses of CoronaVac or of placebo (aluminium hydroxide in phosphate buffered saline). For the phase 2 study 600 subjects were enrolled and separated into two vaccination regimen cohorts, i.e., vaccination at day 0 and 14 and vaccination at day 0 and 28, as for the phase 1 study. The subjects were randomly assigned (2:2:1) using block randomisation to receive two doses of either low-dose or high-dose CoronaVac vaccine or the placebo. No serious adverse effects were recorded for any of the subjects in the two studies. For the phase 1 part of the study, seroconversion for neutralising antibodies was seen in 83% in the 3 µg group, 79% in the 6 µg group and 4% in the placebo group. For the phase 2 study, the seroconversion for neutralising antibodies, was 92% in the 3 µg group, 98% in the 6 µg group and 3% in the placebo group at day 14 in the days 0-and 14-day dosing regimen, whereas at day 28, in the days 0 and 28 day dosing regimen, seroconversion was higher, with the respective results of 97%, 100% and 0%. Importantly, the induced humoral immune responses (neutralising antibodies) were significantly higher in the younger subjects (18-39 years of age) than in the older (40-59 years of age). The study did not assess whether the vaccine induced cellular immune responses (T cell responses) in the subjects . Zhang et al. (2021) states that three phase 3 studies are ongoing in Brazil, Indonesia and Turkey evaluating the low vaccine dose of 3 µg CoronaVac in 0.5 mL of diluent, with a 0-and 14-day vaccination regimen. Future phase 3 trials will also evaluate the 0-and 28-day dosing regimen. Further, the study in Brazil will also assess the T cell responses in the subjects. No formal scientific papers have been published describing the outcome of the various Phase 3 studies. However, in a press release on the 5 th February 2021, Sinovac announced Phase 3 results from its CoronaVac vaccine ("Sinovac Announces Phase III Results of Its COVID-19 Vaccine-SINOVAC -Supply Vaccines to Eliminate Human Diseases"). The Press release first states that Phase 3 trials started July 21, 2020 in Brazil, Turkey, Indonesia and Chile and that a total of 25,000 subjects have been enrolled across those four countries. All studies were randomised, double blind and placebo controlled and followed a vaccination regimen on days 0 and 14. The dose given was, as seen above, 3 µg CoronaVac in 0.5 mL of diluent including alum. The press release goes on to state that as of December 2020, 12,396 health workers of more than 18 years of age were enrolled, presumably in Brazil only (Palacios et al., 2020) . The vaccine efficacy against SARS-CoV-2 was 50.65% for all cases, but 83.7% for cases requiring medical treatment and 100% for hospitalized, severe and fatal cases. The press release then describes the outcome of the Turkish two stage study (first health workers and then those from the general population) as of December 23, 2020 with all subjects (7,371) ranging from 18 -59 years. The study found an efficacy for prevention of COVID-19 injection of 91.25%. In a separate press release ("Indonesia green lights China's Sinovac COVID-19 vaccine") data from the Indonesian trial showed a 65.3% efficacy, with no information given on whether this efficacy data was the combined overall result. ChiCTR2000040153) enrolling 29,000 volunteers. As mentioned above, many human pathogens such as influenza virus and SARS-CoV-2 enter the human body via the respiratory tract and hence, it is a natural progression to investigate and exploit the possibility of developing nasal vaccines to combat such infections. Nasal vaccines offer an attractive alternative to injectable vaccines in that it may be possible to use a lower dose than for IM/SC injection, the vaccine can be delivered to the appropriate site, namely the NALT, nasal vaccines do not necessarily require to be administered by a health-care person, and it is a better alternative for vaccination of children who generally are not keen on injections. Furthermore, nasal vaccines can be delivered in simple low-bioburden single/or bi-dose nasal devices, avoiding the need for a sterile environment during administration, which is of great benefit for vaccination programmes in third world countries. Also, dry powder nasal vaccines have been developed that can avoid the cold-chain production which is cost saving. The nasal epithelium, especially at Waldeyer's ring in the nasopharynx, encloses follicleassociated lymphoid tissue, the NALT, that is important for creating (local and disseminated) mucosal immune responses. As discussed further below, nasal vaccines have been shown to induce both humoral and cell mediated immune responses and furthermore both serum IgG and local nasal neutralizing mucosal IgA protecting against colonization by invading pathogens. Moreover, intranasal immunization has been reported to enable the induction of cross-reactive antibodies that could be indicative of cross-protection (Jang et al., 2012) . As further discussed below, after IM/SC injection of vaccines systemic replication of virus is prevented, but only limited mucosal protection in the form of IgG transudation to airways surfaces are induced. In order to induce the required immune response and provide long term immunity after nasal vaccination it is of essence to select an optimal delivery system for the specific nasal vaccine, since depending on the type of vaccine formulation e.g live-attenuated vaccines, inactivated viral vaccines, MRA/ DNA encoded particulate systems, subunit or purified antigens with or with/out the use of adjuvants, different immune responses may be induced. Furthermore, it is important that the nasal vaccine remains in the nasal cavity/nasopharynx at sufficient time to enable the vaccine to reach the NALT. This can generally be achieved as necessary with liquid or powder bio-adhesive vaccine formulations, that to some extent is able to overcome the mucociliary clearance system. The potential problem of toxicological effects of nasally applying vaccines will be discussed below. responses and mucosal antibody responses in the form of secretory immunoglobulin A (SIgA) (Hagenaars et al., 2008; Isho et al., 2020) . The upper respiratory tract, such as the nasal cavity, is suggested to mainly be protected by the SIgA, and the lower respiratory tract, by IgG (Spiekermann et al., 2002) . IM injected vaccine prevents systemic replication of the virus but induces only limited mucosal protection through IgG transudation to airway surfaces, such as in the lungs. It is the general perception that, whereas mucosal (e.g., nasal) vaccination results in high titres of protective secretory IgA antibodies at the mucosal site with lower systemic IgG antibodies and cell-mediated immunity, the opposite is the case for parenteral vaccination (Krammer, 2020; MacPherson et al., 2008; Su et al., 2016) . Matsuda et al. (2021) also state that there are many examples of a failure to protect against respiratory virus infections when using IM non-replicating vaccines, for example RSV, parainfluenza virus type 3, Ad4, rotavirus and measles vaccines. It is possible that IM vaccines against respiratory viruses induce disease-preventing or disease-attenuating immunity but does not lead to "sterilizing" immunity (Krammer, 2020) . (Schreckenberger et al., 2000) . significantly more effective than the IM vaccine in inducing a mucosal IgA response, which they further suggested, may prevent influenza at its early stages and contribute to the reduction of morbidity and complications in the elderly (Muszkat et al., 2003) . In a study published by Samdal et al. (2005) , Caledonia/20/99(h 1 N 1 )-like re-assortant IVR116) influenza vaccine, either in saline, mixed with formaldehyde inactivated Bordetella pertussis or in a thixotropic vehicle, were given to 3 groups of subjects for IN immunisation, as four doses, with one-week intervals. All vaccinated groups developed significant IgG and IgA antibody responses after four doses, and 6 weeks after the immunisation 80% of the subject reached hemagglutination inhibition titres of more than 40, which was considered to be protective. In addition, significant increases in CD4+ Tcell proliferation and cytotoxic T-cells were detected. However, no additive effect was found for the addition of B. pertussis or for the thixotropic formulation, that probably was added to evaluate the effect of a prolonged residence in the nasal cavity. Recently, Matsuda et al. (2021) reported on a study in subjects vaccinated with a replicationcompetent, Ad4-based vaccine carrying a full-length HA gene from the influenza AH5N1 virus (A/Vietnam/1194/2004) (Ad-4-H5-Vtn recombinant vaccine). The vaccine was given, either orally (10 10 vp), directly to the tonsils (10 3 -10 8 vp) or nasally (10 3 -10 8 vp). Viral shedding, from nose, mouth and rectum, together with H5 specific IgG and IgA antibodies and T cell responses, were detected. It was found that Ad-4-H5-Vtn DNA was shed from most subjects immunised in the upper respiratory tract. The vaccine induced increases in the H5, specific CD4+ and CD8+ T cells in the peripheral blood, as well as increases in IgG and IgA in nasal, cervical and rectal secretions and high levels of serum neutralising antibodies against H5 that remained stable for 26 weeks. The authors concluded that the Ad4 vaccine platform showed considerable promise for vaccines designed to stimulate B cell response to viral surface glycoproteins. Hence, as seen above, the literature does describe examples of studies where mucosal immune responses, to some extent, are induced after IM injection of a respiratory virus vaccine and that complete or partial protection against such a virus is attainable. However, it is also evident, that for some virus antigens mucosal strategies, including specific adjuvant formulations and a combination of antigens that activate multiple arms of the immune system, would be necessary in order to generate a robust up-front protective immunity. It has been suggested by Bleier et al. (2021) The amount of SARS-CoV-2 virus that is required for efficient human transmission is presently not known, however, it is known that the amount of virus found in the upper airways of subjects just after infection, is in the order of 10 6 RNA copies per nasal swab, which is close to the challenge doses given in the challenge studies discussed above. Presently, it is also unclear whether the detection of viral shedding in the upper airways in non-human primate translates directly to humans. Recently, there has been an intensive discussion about a conclude that at present it cannot be excluded that the IM vaccines have the potential to trigger ITP, albeit very rarely. The AstraZeneca vaccine is (as described below) presently in development for nasal vaccination but, as far as we are aware, no information has been published concerning potential serious side effects after using these vaccines for nasal administration. Relative few vaccines have been licensed for nasal application including Fluenz Tetra TM (EU)/ FluMist Quadravalent (US, Can) which are tetravalent cold-adapted live-attenuated influenza vaccines (LAIV) produced by Medimmune/AstraZeneca and Nasovac ® which is a similar trivalent influenza vaccine produced in India by CiplaMed in collaboration with the Serum Institute of India Ltd. For the latter vaccine a post marketing study reported that the vaccine was safe in that 90% of all events were non-serious and mild and 10% moderate in severity with no event lasting more than 4 days. No deaths, life threatening events, permanent disability or hospitalisation were reported (Kulkarni et al., 2013) . Similarly, the Fluenz Tetra TM /FluMist Quadravalent nasal vaccines were found to be safe (Lycke, 2012) . However, due to the involvement of eggs in the production process, LAIV have been reported to have particular allergic side effects in that it causes significant wheezing for up till 42 days after nasal administration. Hence, the vaccine is precluded for administration to asthmatic patients with unstable asthma (Vasu et al., 2008) . The addition of adjuvants to a vaccine can be necessary for the enhancement of the immune response especially for vaccines comprising purified antigens. As outlined above a range of adjuvants are available including alum, chitosan and also bacterial toxins such as cholera toxin (CT) or heat-labile enterotoxin (LT), or inactivated viral envelopes such as recombinant adenoviruses. Some side effects have been reported after the use of bacterial toxins in vaccines given nasally. It is therefore necessary to consider both the potential toxicity as well as the protective immunity conferred by the selected adjuvant. For example, an inactivated viral subunit influenza vaccine (Nasalflu, Berna Biotech) was adjuvanted with E. coli heat-labile toxin and found after intranasal vaccination to increase the risk of Bell's palsy. The licence for the vaccine was revoked and is no longer available (Mutsch et al., 2004) . It was suggested that the toxin may have been transported from the nasal cavity to the CNS in the same way as was shown for the adjuvant CT Fujihashi et al., 2002) . The SARS-CoV-2 virus enter cells by engaging the spike protein with an ACE2 receptor. The ACE2 receptors are present throughout the body especially with high levels in the small intestine, testis, kidneys, heart, thyroid, and adipose tissue, while blood, spleen, bone marrow, brain, blood vessels, and muscle had the lowest ACE2 expression levels and medium expression was found in the lungs, colon, liver, bladder, and adrenal gland (Li et al., 2020) . Hence theoretically it is possible for the SARS-CoV-2 virus to enter all of these tissue since the S-protein is attached to the surface of the virus and therefore available for interaction with the ACE2 receptors. However, at least for most of the nasal vaccines described below that is under development the carrier e.g. adenovirus and lentiviral vectors are encoded for the S protein and hence is not accessible for interaction with the ACE2 receptor before the S protein has entered a cell and is being produced. The exception is the nasal vaccine in development by University of Houston that comprise liposomes with surface adsorbed S protein It has been suggested that the SARS-CoV-2 virus can reach the brain by using the transneural route of entry into the olfactory epithelium (where ACE2 is expressed) and transsynaptic routes to spread further into the brain (Butowt and Bilinska, 2020). However, whether the recombinant vectors carrying the encoding for the S-protein are able to enter the olfactory tissue either by paracellular or transcellular transport and potentially result in adverse effects of the vaccine, has, as far as we are aware, not been investigated. Adenovirus has been used for nose to brain delivery of drugs, but it was not shown that the adenovirus itself actually entered the brain, only that the drug did (Ma et al., 2016) . Using the paracellular route to enter the olfactory tissue, through the tight junctions, particles should be less than 20 nm whereas using the transcellular route either into olfactory tissue cells or olfactory neural cells the particle size should ideally be less than 100 nm (Illum, 2007 (Illum, , 2015 . The adenovirus vectors used for many of the nasal Covid-19 vaccines under investigation are about 90 nm in diameter, the lentiviral vector about 80-100 nm (similar to the SARS-CoV-2 virus of about 100 nm) and the Newcastle Disease virus is between 150-400 nm. It should be noted that the endocytosis process is dependent not only on particle size but also particle characteristics such as charge and surface properties. Furthermore, there is a difference in deposition of the SARS-CoV-2 virus particles in the nose that has entered the nasal cavity by normal inhalation and vaccine particles that are sprayed with stronger force into the cavity. Therefore, whether a nasal spray will reach the olfactory region (2.5% of nasal surface area) positioned in the top of the nasal cavity, is highly unlikely unless a specialised nasal delivery device is used. Hence, it is difficult to predict whether the vaccine carriers could enter the olfactory epithelium, whether any toxic events would result from such entry and hence toxicological studies will need to be done in line with normal regulatory demands before the nasal SARS-CoV-2 vaccines are licensed for marketing. As discussed above, it is important that a COVID-19 vaccine should protect humans against a later SARS-CoV-2 viral infection by creating the necessary humoral and cell mediated responses, to include neutralising antibodies, not only in the blood, but also at the upper respiratory tract, such as the nasal mucosal membrane, together with the lower respiratory tract i.e., the lungs. Furthermore, it is also of importance that vaccinated subjects are not prone to asymptomatic nasal viral shedding and therefore potential transmission of disease to other subjects. Hence, there is presently a great interest in the development of nasal COVID-19 vaccines, although at the time of writing no mucosal vaccine has been approved by regulatory authorities. The following discussion only includes developments where at least preclinical studies have been published. It should be noted that many of the publications discussed below have been preliminarily published on-line in non-peer review publications such as "www.BioRxiv.org". However, taken together the papers still give a good overview and information of the potential benefits of nasal COVID-19 vaccines as compared to the IM vaccines. preclinical study in mice, tested the immunogenicity of AdCOVID™ after intranasal administration of one of three doses of vaccine 3.35 x 10 8 ifu (high-dose), 6 x 10 7 ifu (middose) or 6 x 10 6 ifu (low-dose) given in a volume of 50 mL, or a control in the form of buffer. The vaccine demonstrated a strong IgG serum neutralising activity, several fold higher than the titre recommended by the FDA, and a potent mucosal immunity with a 29-fold increase in mucosal IgA in the respiratory tract as measured in the BAL fluid. Furthermore, a potent stimulation of the cell mediated immunity, in the form of antigen specific CD8+ killer T cells, was found in the lungs as early as 10 days after vaccination. No nasal samples were collected for identification of secretory nasal IgA. The authors concluded that their AdCOVID™ vaccine generated both humoral and cellular responses at both systemic and mucosal sites, particularly within the lungs, which is a major site for infection and disease (King et al., 2020) . A Phase 1 clinical trial is ongoing which will evaluate the safety and immunogenicity of a single dose of AdCOVID™ in up to 180 healthy adult volunteers between 18 and 55 years of age. AdCOVID™ will be administered to subjects at one of three dose levels as a nasal spray. In addition to the primary study endpoint, the immunogenicity of AdCOVID™ will be evaluated by serum IgG binding and neutralizing antibody titres, mucosal IgA antibody levels from nasal samples, and T cell responses. The study was approved by the FDA on the 25 th February 2021 ("Altimmune Commences Enrollment in Phase 1 Clinical Trial of AdCOVID TM --a Needle-Free, Single-Dose Intranasal COVID-19 Vaccine Candidate -Altimmune"). Washington Codagenix Inc. has developed an intranasal vaccine against SARS-CoV-2 (COVI-VAC) based on a live attenuated whole virus platform, which uses "synthetic biology" to re-code the genes of viruses into a safe and stable vaccine. The Codagenix COVI-VAC "de-optimised" virus can be grown easily in cell culture. As far as the present authors are aware, results from preclinical studies have not been published and the information available is from a news review ("First patient dosed with intranasal COVID-19 vaccine candidate"). However, a phase 1 clinical study, to evaluate the safety and immune responses of intranasally administered COVI-VAC in 48 healthy young subjects (18-30 years of age), is presently ongoing in the UK. The subjects, divided into three groups, will receive either two doses of COVI-VAC, 28 days apart, two doses of placebo (saline) or one dose of COVI-VAC and 1 dose of placebo. The dose is administered by drops (no information of number of drops) into each nostril. Each subject will record any symptoms and oral temperature daily for 14 days. Blood samples and intranasal samples will be collected to assess the immune response. The study plan was approved by the MHRA on the 22 nd December 2020. The first subject was dosed on the 12 th January 2021 ("First patient dosed with intranasal COVID-19 vaccine candidate"). AstraZeneca/Oxford Jenner Inst. (who developed ChAdOx1 nCoV-19/AZD1222 for intramuscular injection as discussed above) have also evaluated the same vaccine administered nasally in hamsters and in non-human primates (van Doremalen et al., 2021) . After IM injection of the vaccine in rhesus macaques, the animals were protected against pneumonia but no reduction in sub-genomic and genomic viral shedding (RNA) from the nasal cavity was found, with the shedding being similar to that from control animals, indicating replicating virus in the upper respiratory tract. In the non-human primate studies, four rhesus macaques were vaccinated IN with a dose of 2.5 x 10 10 virus particles ChAdOx1 nCoV-19 in a prime/boost regimen and compared with four control animals. Blood, nasal swabs and BAL fluid samples were also collected throughout the studies. Animals were challenged with 10 6 SARS-Cov-2/human virus particles both intratracheally and nasally. Higher fractions of IgA to total Ig antibodies were found in the nasal swabs compared to BAL fluid and serum samples. S and RBD -specific IgG antibodies was found in serum and nasal swabs but not in BAL fluid at day seven after the prime vaccination (at -49 days post infection ~ DPI). Higher IgG titres were found after the booster vaccination (-28 DPI). SARS-CoV-2 specific IgA titres were low after the prime vaccination, but higher after the booster vaccination, and also detected in BAL fluid 7 days after the booster vaccination. Serum neutralising antibodies were found in vaccinated animals at titres similar to those found in previous studies after IM vaccination. After challenge, the nasal swabs in control animals contained genomic and sub-genomic RNA and infectious virus. Viral RNA was found in nasal swabs of vaccinated animals but at a lower level and in fewer animals. Genomic and sub-genomic RNA was detected in BAL fluid of all control animals. Genomic The Division of Biomedical and Life Sciences, Lancaster University has engineered a COVID-19 vaccine based on a live attenuated and vectored Newcastle Disease virus (NDV) encoding a human codon-optimised S glycoprotein gene of SARS-CoV-2, that is administered by the intranasal route. The NDV vaccine platform has been shown in preclinical models and in humans to be safe and effective against a range of other viruses including influenza. In a published study, Park et al. (Park et al., 2021) evaluated the immunogenicity and safety of the rNDV-S based live attenuated virus vaccine in mice and the protective efficacy in hamsters. Groups of 12 BALB/c mice were inoculated with 10 6 PFU of the test vaccine in a prime/booster regimen 7 days apart, rNDV-S, a wild type NDV (rNDV-WT) or with phosphate buffered saline. The rNDV-S induced robust systemic humoral (S protein specific IgG and anti-RBD specific IgG) and cell-mediated immune responses in the lungs and in serum in mice, where CD4+ T cell IFNg and NK T-cell TNF+ were significantly increased only for the rNDV-S vaccinated animals. The vaccine also appeared to be safe, since no clinical disease signs were observed throughout the experiments nor was any adverse pathology found in the tissues examined (Park et al., 2021) . In a further study, a total of 8 Syrian hamsters in each group were vaccinated IN with 1 x 10 6 PFU of rNDV-WT, rNDT-S or a mock control once or twice with two weeks interval. To assess protection efficacy of the rNDT-S vaccine, hamsters immunised (prime or boosted) were Institut Pasteur-TheraVectys Joint Laboratory published studies recently in two preclinical models, mice (with induced expression of the human SARS-CoV-2 receptor, hACE2) and hamsters. They evaluated a novel COVID-19 vaccine candidate based on a lentiviral vector eliciting neutralising antibodies against the S glycoprotein of SARS-CoV-2. The mice studies included prime/boost (1 x 10 7 /1 x 10 7 transduction units (TU)) intraperitoneal (IP) injections and prime/target (1 x 10 7 /3 x 10 7 TU) IP/IN administration of vaccine compared to control, together with challenge studies (0.3 x 10 5 TCID 50 of SARS-CoV-2). The prime/boost injection of the vaccine resulted in very high serum neutralising IgG against the S protein together with cellular immunity. Furthermore, partial protection was observed after the challenge test, with lung viral load significantly reduced for both prime/boost (10-fold) and prime/target (1000fold) in vaccinated animals, whereas, IgA was detectable in the upper respiratory tract only in the prime/target vaccinated animals. The authors concluded, from this part of the study, that local IgA in the upper respiratory tract is necessary for full protection against a challenge with SARS-CoV-2 virus. The study regimen was repeated in golden hamsters, which are naturally permissive to SARS-CoV-2 replication. Strong and comparable anti S IgG were detected in the sera of animals, from both the prime/boost and prime/target groups. Neutralising activity was found to be highest in the prime/target animals and comparable to those seen in COVID-19 cases in humans. After challenge with SARS-CoV-2 virus, the viral lung loads were significantly lower than in control for both vaccination groups and the prime/target vaccination strategy induced almost full protection. The authors concluded that the studies provided evidence of the substantial prophylactic effects of vaccination with the lentiviral based vaccine against SARS-CoV-2 and showed intranasal immunisation as a powerful means to combat COVID-19 infection (Ku et al., 2021) . In a recent study, the acute humoral responses to a SARS-CoV-2 virus infection, such as antibody secreting cells and the presence of virus specific neutralising antibodies in saliva, BAL fluid and serum, were measured in 159 patients with COVID-19 (Sterlin et al., 2021) . It was found that the early humoral immune responses to the viral infection were dominated by IgA antibodies, and that SARS-CoV-2 neutralisation was more closely correlated with IgA than IgM or IgG. One month after onset of the symptoms from a SARS-CoV-2 infection, the serum IgA concentrations decreased notably, whereas neutralising IgA in saliva were detectable for up to 73 days after onset of symptoms. It has also been shown that the dimeric form of IgA, found in the mucosa, is more potent against SARS-CoV-2 than both IgA and IgG monomers (Wang et al., 2021) . The authors concluded from the study, that IgA mediated mucosal immunity could be the most critical defence mechanism against SARS-CoV-2 and may reduce viral shedding and transmission of the virus from person to person (Sterlin et al., 2021) . Likewise, Butler et al. (2021) such as the nasal cavity, but also in the lower respiratory tract and prevented or provided a significant reduction in viral shedding and therefore, also transmission between animals. From the results in the preclinical studies on intranasal vaccines, it is likely that a similar protective efficacy seen in the IM COVID-129 vaccines in humans, will be found with the IN COVID-19 vaccine candidates. Results from the first clinical studies should be available in second quarter of 2021. However, whether these IN vaccines will also afford a strong prevention (or reduction) of viral replication in the nasal cavity and lungs and hence prevent transmission of virus by asymptomatic subjects, will only be clarified when viral titre endpoints are incorporated into vaccine clinical trials. It is likely that a combination of an IM prime vaccination and an IN-booster vaccination (IM/IN) would provide a viable alternative to the IM/IM prime/booster vaccines, with a better well-rounded humoral and cell mediated immune response. Presently, the longevity of the immune responses created by the vaccines is not known and hence a further development could be that (as is the case for flu vaccination) a yearly vaccination will be needed against SARS-CoV-2. Such a booster could be given as a IN vaccine. 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Hum. Vaccines Immunother Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial Mattia Tiboni: Conceptualization, Writing -original draft, Writing -review & editing Luca Casettari: Conceptualization, Writing -original draft, Writing -review & editing Lisbeth Illum: Conceptualization, Writing -original draft, Writing -review & editing ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests The authors acknowledge Fabio de Belvis for the design of the graphical abstract. Images created with Biorender.com.Funding: This research received no external funding. The authors declare no conflict of interest