key: cord-0925832-pmd78j61
authors: Afkhami, Sam; D’Agostino, Michael R.; Zhang, Ali; Stacey, Hannah D.; Marzok, Art; Kang, Alisha; Singh, Ramandeep; Bavananthasivam, Jegarubee; Ye, Gluke; Luo, Xiangqian; Wang, Fuan; Ang, Jann C.; Zganiacz, Anna; Sankar, Uma; Kazhdan, Natallia; Koenig, Joshua F.E.; Phelps, Allyssa; Gameiro, Steven F.; Tang, Shangguo; Jordana, Manel; Wan, Yonghong; Mossman, Karen L.; Jeyanathan, Mangalakumari; Gillgrass, Amy; Medina, Maria Fe C.; Smaill, Fiona; Lichty, Brian D.; Miller, Matthew S.; Xing, Zhou
title: Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2
date: 2022-02-09
journal: Cell
DOI: 10.1016/j.cell.2022.02.005
sha: 6b75b8170f204c8505e90f19561dfaca112b5056
doc_id: 925832
cord_uid: pmd78j61

The emerging SARS-CoV-2 variants of concern (VOC) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing Spike-1, Nucleocapsid and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

Since its outbreak in Wuhan China in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally infected 359M people and claimed 5.6M lives and counting. Besides general mitigation/infection control measures, the only effective way to control the pandemic of coronavirus disease 2019 is to establish herd immunity through vaccination (Fontanet and Cauchemez, 2020; Jeyanathan et al., 2020) . Thus, based on a pandemic vaccine paradigm (Lurie et al., 2020) , there have been at least 100 vaccines tested in clinical trials and another 180 under preclinical development. These efforts have led a growing number of firstgeneration COVID-19 vaccines to receive emergency use authorization in various countries.

Notably, several authorized vaccines are based on mRNA and adenoviral platforms to express the spike protein of the ancestral SARS-CoV-2 and elicit neutralizing antibody responses following 1-2 intramuscular injections .

The global rollout of COVID-19 vaccines has played a critical role in reducing viral transmission, hospitalizations and deaths. However, since September 2020 there have been five SARS-CoV-2 variants of concern (VOC) emerged which are B.1.1.7 (UK, Alpha), B.1.351 (South Africa, Beta), P.1 (Brazil, Gamma), B.1.617.2 (India, Delta) and B.1.1.529 (South Africa, Omicron) (Andreano and Rappuoli, 2021; Gupta, 2021) . While they all have multiple mutations in the spike protein, B.1.351, P.1 and B.1.1.529 harbor multiple mutations within the RBD that reduce their neutralization by antibodies present in convalescent or vaccine-induced sera Garcia-Beltran et al., 2021; Geers et al., 2021; Hoffmann et al., 2021b; Planas et al., 2021; Wang et al., 2021; Wilhelm et al., 2021) . Some B.1.617 sub-lineages also carry E484Q and L452R mutations that reduce antibody binding (Starr et al., 2021) . Of importance, several firstgeneration vaccines including ChAdOx1 nCoV-19 (AstraZeneca/Oxford) (Madhi et al., 2021) , Ad26.COV2.S (J&J) (Sadoff et al., 2021) , NVX-CoV2373 (Novavax) (Shinde et al., 2021) and

BNT162b2 (Pfizer-BioNTech) (Abu-Raddad et al., 2021) have demonstrated reduced effectiveness in protecting from mild to moderate COVID-19 caused by B.1.351 . Likewise, sera from those immunized with mRNA-1273 (Moderna) show reduced neutralization of B.1.351 (Shen et al., 2021) . Thus, the emerging VOC capable of escaping the immunity by first-generation vaccines constantly threaten to impede or disrupt the establishment and sustainability of vaccineinduced herd immunity (Aschwanden, 2021; Harvey et al., 2021) .

To meet the challenges from VOC and limited durability of first-generation vaccineinduced immunity, there is an urgent need to develop next-generation COVID-19 vaccine strategies (Callaway and Ledford, 2021; Gupta, 2021; Jeyanathan et al., 2020) . Although updating the spike antigen to specific VOC represents one such strategy (Callaway and Ledford, 2021; Gupta, 2021) , it is cumbersome and expensive, and requires selection of specific VOC sequence(s) which may result in inherently inaccurate prediction of antigenic drift, akin to current seasonal influenza vaccines. An alternative strategy is to develop recombinant viral-vectored multivalent vaccines amenable to respiratory mucosal immunization . Besides the spike antigen, such vaccines express additional conserved SARS-CoV-2 antigens to broaden T cell immunity. Since antigenic changes in conserved, internal viral proteins that are the primary focus of T cell responses are rare/improbable in SARS-CoV-2 viruses including VOC (Alter et al., 2021; Geers et al., 2021) , such multivalent vaccines do not require frequent updating while they can boost the spike-specific immunity induced by first-generation vaccines. Thus, these vaccines are expected to be effective against both ancestral and variants of SARS-CoV-2. Furthermore, adenoviral vectors delivered via the respiratory tract confer protection via eliciting mucosal tissueresident trained innate and adaptive immunity at the site of viral entry Teijaro and Farber, 2021; Xing et al., 2020; Yao et al., 2018) . Unfortunately, to-date there has been a paucity of next-generation COVID-19 vaccine strategies capable of robust and durable protection against the ancestral strain and variants of SARS-CoV-2.

Herein we have developed and evaluated a next-generation COVID-19 vaccine strategy in murine models. Our vaccine is built upon adenoviral vectors of human (Tri:HuAd) or chimpanzee (Tri:ChAd) origin, expressing three SARS-CoV-2 antigens (spike protein 1, full-length nucleocapsid protein, and truncated polymerase), and is suitable for respiratory mucosal delivery.

We show that single-dose intranasal, but not intramuscular, immunization, particularly with the Tri:ChAd vaccine, induces all-around respiratory mucosal immunity against both ancestral SARS-CoV-2 and B.1.1.7 and B.1.351 VOC. Our study thus indicates that respiratory mucosal delivery of multivalent viral-vectored COVID-19 vaccine is an effective next-generation COVID-19 vaccine strategy.

Currently approved recombinant first-generation COVID-19 vaccines only encode the spike (S) protein from the Wuhan-Hu-1 ancestral SARS-CoV-2 and were designed primarily to induce neutralizing antibodies following intramuscular injections, a strategy inadequate to combat VOC (Aschwanden, 2021; Harvey et al., 2021) . To develop next-generation adenoviral-vectored COVID-19 vaccines, we utilized human serotype 5 (Tri:HuAd) and chimpanzee serotype 68 (Tri:ChAd) adenoviral vectors. Our vaccines were constructed to include the full-length S1 domain of spike which contains the NTD and RBD and numerous T cell epitopes (Geers et al., 2021; Tarke et al., 2021) . The S1 was fused to the vesicular stomatitis virus G protein (VSVG) transmembrane/trimerization domain ( Figure 1A ) to anchor it to the membrane and facilitate its trimerization and exosomal targeting for enhanced antibody responses (Bliss et al., 2020; Kuate et al., 2007) . To broaden T cell immunity against additional viral antigens, the full-length Nucleocapsid (N) and truncated nsp12 (RNA-dependent RNA polymerase; RdRp) proteins were included in vaccine design as a single polyprotein downstream of a porcine teschovirus 2A sequence (P2A) ( Figure 1A) . N is the most abundant viral protein rich in T cell epitopes in humans including convalescent COVID-19 patients (Altmann and Boyton, 2020; Dai and Gao, 2021; Peng et al., 2020) , and the genetic vaccines expressing N were shown to induce protective immunity in preclinical COVID-19 models (Class et al., 2021; Hajnik et al., 2021; Matchett et al., 2021) . A region of RdRp was selected for maximal high-affinity T cell epitopes based on bioinformatic analysis. T cells specific for N and RdRp are also cross-reactive with other bat-derived coronaviruses (Alter et al., 2021; Altmann and Boyton, 2020; Geers et al., 2021; Tarke et al., 2021) .

Prior to viral rescue, the transgene cassette was verified to be in-frame by Sanger sequencing, with translation initiating at the TpA signal sequence. Following virus rescue, amplification and purification, A549 cells were transduced with Tri:HuAd or Tri:ChAd and transgene expression at the protein level was verified by Western blot for S1-VSVG and Nucleocapsid:RdRp fusion proteins of expected sizes ( Figure 1B) .

In anticipation of their further clinical evaluation, we assessed the safety of Tri:HuAd and Tri:ChAd during the acute phase following intramuscular (i.m.) or intranasal (i.n.) delivery in mice J o u r n a l P r e -p r o o f ( Figure S1A ). There was little change in body weight following single-dose i.m. or i.n. vaccination ( Figure S1B ). Regardless of immunization route or vaccine vector, there was no significant elevations in the lung or bronchoalveolar lavage fluid (BAL) in either neutrophils ( Figure S1C) or pro-inflammatory cytokines ( Figure S1D ), relative to naïve controls. Furthermore, there was no indications of hepatic or renal toxicity based on alkaline phosphatase/alanine aminotransferase and creatinine, respectively ( Figure S1E) . These data support an overall satisfactory safety profile of Tri:HuAd and Tri:ChAd COVID-19 vaccines.

Given the close relationship between spike-specific humoral responses and protective immunity (Huang et al., 2020; Khoury et al., 2021; Krammer, 2021) , we first examined the kinetics of spike-specific antibody responses following i.m. or i.n. immunization with a single-dose of Tri:HuAd or Tri:ChAd vaccine. Serum and BAL were collected from BALB/c mice 2-and 4weeks post-immunization ( Figure 1C ). IgG responses against spike and RBD were quantified by ELISA. While the control sera from i.m. or i.n. empty viral vector (HuAd:EV or ChAd:EV)-treated animals had little anti-spike/RBD IgG responses ( Figure S2A Since vaccine-associated enhanced respiratory disease (VAERD) is potentially associated with Th2-biased immune responses to certain viral infection and has also been experimentally observed post-inactivated SARS-CoV-1 vaccination (Bournazos et al., 2020; Jeyanathan et al., 2020) , we determined the ratio of S-specific IgG2a/IgG1 antibodies as a surrogate of the Th1/Th2 immune response. Regardless of vaccine route or vector, no Th2-skewing of antibody responses was seen at either timepoint ( Figure 1F ).

We next assessed the neutralizing capacity of serum antibodies 4-weeks post-immunization by a surrogate Virus Neutralization test (sVNT) (Tan et al., 2020) . Whereas immunization route had no significant effect on the neutralizing potential of serum antibodies in Tri:HuAd-vaccinated J o u r n a l P r e -p r o o f animals (i.m. 6.1%±0.2% vs. i.n. 11.92%±2.7%), i.n. Tri:ChAd generated antibody responses with markedly enhanced neutralizing potential (87.70%±2.3%) over that by i.m. route or by Tri:HuAd immunization ( Figure 1G ).

To assess humoral responses at the respiratory mucosa, BAL fluids collected 4-weeks postimmunization with either trivalent vaccine were assessed for S-specific IgG. As expected, we were only able to reliably detect S-specific antibodies in the airway following i.n., but not i.m., immunization ( Figure 1H ). Of note, airway S-specific IgG responses following Tri:ChAd immunization almost doubled that by Tri:HuAd.

We next assessed the durability of antibody responses at 8-weeks post-vaccination ( Figure   1I ). Overall, compared to 4-weeks data ( Figure 1D/E) , serum S-and RBD-specific IgG responses largely sustained following i.m. immunization and remained significantly higher following i.n.

immunization with either vaccine ( Figure 1J ). Once again, the serum neutralization profile determined by sVNT at 8-weeks ( Figure 1K ) was similar to that at 4-weeks ( Figure 1G) , showing i.n. Tri:ChAd to induce the highest titers of neutralizing antibodies. Given the robust neutralizing capacity exhibited by serum from i.n. Tri:ChAd mice, we next tested it in a microneutralization assay with live SARS-CoV-2. Congruent with the sVNT results, i.m. immunization with either vaccine afforded minimal neutralization against live SARS-CoV-2 ( Figure 1L ). In contrast, while i.n. immunization with either vaccine increased their respective neutralization capacities, i.n.

Tri:ChAd elicited superior neutralization capacity over Tri:HuAd counterpart ( Figure 1L ).

Compared to 4-weeks BAL data ( Figure 1H ), anti-S IgG from the BAL fluid was somewhat increased at 8-weeks following i.n. immunization with higher levels induced by Tri:ChAd vaccine while i.m. immunization with either vaccine failed to induce anti-S IgG in the airway ( Figure 1M ).

Moreover, significant amounts of anti-S IgA were detected only in the BAL of i.n. Tri:ChAd animals ( Figure 1M ).

To examine the relationship of vaccine vector and immunization route to detectable antigen-experienced memory B cells in systemic lymphoid and local lung tissues, we tetramerized biotinylated RBD conjugated to a fluorochrome and probed for RBD-specific B cells by FACS (Hartley et al., 2020; Rodda et al., 2021) . A decoy tetramer was included during staining to gateout vector-specific B cells ( Figure S3A ). While all immunizations induced a detectable population of RBD-specific B cells in the spleen, i.n. Tri:ChAd induced significantly higher levels than i.n.

Tri:HuAd ( Figure 1N ). In addition, only i.n. Tri:ChAd vaccine induced detectable RBD-specific B cells in the lung tissue ( Figure 1N ).

The above data indicate that single-dose intranasal immunization, particularly with Tri:ChAd vaccine, induces superior functional humoral responses both systemically and locally in the lung over the intramuscular route.

We next examined T cell responses with a focus on those within the airways. Besides antibodies, airway T cells play pivotal roles in immunity against coronaviruses Zhao et al., 2016) . BALB/c mice were immunized intramuscularly or intranasally with a single-dose of either trivalent COVID-19 vaccine. Antigen-specific T cells in mononuclear cells from the BAL harvested 2-and 4-weeks post-immunization were analyzed by FACS for intracellular IFNγ, TNFα, IL-2 and Granzyme B expression upon ex vivo stimulation with 15mer peptide pools for S1 (132 peptides), N (82 peptides) and RdRp (12 peptides). As expected, both CD8 + and CD4 + T cells from 4-weeks i.m. or i.n. empty vector (HuAd:EV or ChAd:EV)-treated animals did not respond to any of these peptide pools ( Figure S2D ). In agreement with our previous work (Jeyanathan et al., 2017; Lai et al., 2015; Santosuosso et al., 2005) , i.m. immunization failed to induce antigen-specific CD8 + T cells in the airways, irrespective of vaccine vector (Figure 2A /D/G). In contrast, i.n. immunization induced a significant number of IFNγ + CD8 + T cells specific for S1, N, or RdRp in the airways. Of interest, S1-specific T cells were dominant relative to those for N or RdRp. Compared to Tri:HuAd, i.n. Tri:ChAd vaccine induced greater airway CD8 + T cell responses to the three antigens, particularly at 2-weeks (Figure   2A /D/G). I.n., but not i.m., immunization also induced a similar profile of antigen-specific CD4 + T cell responses in the airways, but to a lesser degree than the CD8 + T cell response ( Figure S4A ).

Of importance, such CD4 + T cells were predominantly of Th1 phenotype based on their IFNγ vs.

IL-4 production capacity and ratios, upon spike-specific or polyclonal (αCD3/αCD28) stimulation ( Figure S4B ), in keeping with a Th1-skewed S-specific IgG2a antibody response ( Figure 1F ).

We further assessed the multifunctionality of CD8 + T cells with a focus on 4-weeks timepoint post-i.n. immunization when T cells entered the contraction/memory phase. The majority of S1-specific CD8 + T cells induced by either Tri:HuAd or Tri:ChAd vaccine were We also assessed systemic antigen-specific T cells in the spleen at 4-weeks postimmunization. In keeping with our previous findings (Santosuosso et al., 2005) , i.m., but not i.n., immunization with either vaccine induced robust systemic CD8 + T cell responses to S1, N, and RdRp ( Figure S4C , top panels). Once again, S1-specific T cell responses were dominant compared to those to N or RdRp. Antigen-specific CD4 + T cells were also induced in the spleen, but again to a lesser degree than CD8 + T cells ( Figure S4C , bottom panels).

The above data indicate that single-dose intranasal, but not intramuscular, immunization, particularly with Tri:ChAd vaccine, is able to induce multifunctional CD8 + T cells with cytotoxic potential within the respiratory tract.

Compelling evidence indicates a critical role of mucosal tissue-resident memory T cells (TRM) in host defense (Szabo et al., 2019) and i.n., but not i.m., vaccination can induce such T cells Teijaro and Farber, 2021) . To investigate whether Ad-vectored trivalent COVID-19 vaccine could induce respiratory mucosal TRM, we first established t-SNE maps based on pooled CD3 + /CD8 + /CD4mononuclear cells from lung tissues of all animals 8weeks post-i.m. or i.n. delivery of Tri:HuAd or Tri:ChAd vaccine ( Figure 3A , top panel). Upon overlaying these cells concatenated from i.m. and i.n. animals, two unique CD8 + T cell clusters (orange-yellow color) were identified to be associated only with i.n. immunization ( Figure 3A, bottom panel). We next generated heatmaps to overlay expression intensities for the surface markers CD44, CD69, CD103 and CD49a associated with mucosal TRM (Szabo et al., 2019) . This analysis shows the two unique clusters of T cells of CD8 + TRM phenotype induced by i.n. We next examined CD8 + TRM within the airways (BAL) 8-weeks post-immunization.

Given the lack of airway T cells following i.m. immunization (Figure 2A /D/G), we focused our analysis on BAL cells from i.n.-immunized animals. We found that regardless of vaccine vector, approximately 50% of antigen-experienced CD44 + CD8 + T cells in the BAL co-expressed TRM surface markers CD69, CD103, and CD49a ( Figure 3E ). Similar observations were made with antigen-experienced CD69 + CD11a + CD4 + TRM in the airways except that they were present in smaller frequencies compared to CD8 + TRM ( Figure S5C ). Potent induction of TRM in the lung by i.n. immunization, particularly with Tri:ChAd, was also observed at 4-weeks (Figure S5D/E/F).

In contrast, i.n. delivery of an empty vector (HuAd:EV or ChAd:EV) had a minimal effect on TRM induction in the lung or airways ( Figure S2E ).

We further determined the multi-functionality of long-term memory CD8 + T cells in the airways 8-weeks post-i.n. immunization. Compared to the 4-week timepoint (Figure 2A 

airway antigen-specific IFNγ + CD8 + T cells further contracted, irrespective of vaccine vector, with the majority being specific for S1 ( Figure 3F ). However, multi-cytokine expression analysis reveals that the memory CD8 + T cells induced by i.n. Tri:ChAd were more functional than those by Tri:HuAd based on co-expression of IFNγ, TNFα and/or IL-2 ( Figure 3G ). Of note, S1-specific memory T cells showed a greater breadth of multifunctionality than those specific to N or RdRp.

These data indicate that intranasal, but not intramuscular, COVID-19 immunization is able to induce durable multifunctional respiratory mucosal TRM responses. Furthermore, Tri:ChAd vaccine is more potent than Tri:HuAd platform.

Since alveolar macrophages (AM) as the main respiratory mucosal-resident innate immune cells have been shown to interact with SARS-CoV-2 (Grant et al., 2021) and other RNA viruses (Kumagai et al., 2007; Schneider et al., 2014) , they likely play a critical role in early innate immune control of SARS-CoV-2. Aside from producing robust adaptive immunity within the airways, we have shown that Ad-vectored i.n., but not i.m., TB immunization induces long-lasting airwayresident memory AM and trained innate immunity Xing et al., 2020; . To investigate whether Ad-vectored COVID-19 vaccines induces trained AM, mice were immunized intramuscularly or intranasally with Tri:HuAd or Tri:ChAd vaccine and the immune phenotype of airway (BAL) macrophages was analyzed 8-weeks post-immunization ( Figure 4A ). To better enable our flow cytometric characterization of CD45 + CD11b + CD11c + airway macrophages, t-SNE maps were first generated with the BAL cells pooled from all animals to visualize the overall differences between both vaccine vectors and routes ( Figure We next specifically determined the extent to which COVID-19 vaccine vectors and routes induced trained MHC II high airway macrophages. Whereas i.m. Tri:HuAd or Tri:ChAd vaccine resulted in hardly any MHC II high AM ( Figure 4C , left 2 panels), i.n. immunization, notably with Tri:ChAd, generated several distinct populations expressing high levels of MHC II or CD11b separate from the large AM cluster ( Figure 4C , right 2 panels). This is phenotypically consistent with an influx of interstitial macrophages (IM), and trained AM observed within the airway following i.n. vaccination with a HuAd-vectored TB vaccine . Thus, using a comprehensive gating strategy, we compared MHC II expression in both AM and IM in the airways. While in keeping with our previous findings with a TB vaccine Yao et al., 2018) , i.m. Tri:HuAd or Tri:ChAd COVID-19 vaccine was unable to induce trained AM or IM (Figure 4D ), i.n. immunization with either vaccine induced markedly increased MHC II high AM and IM ( Figure 4D ). Furthermore, in line with t-SNE analysis ( Figure 4C ), i.n.

Tri:ChAd induced significantly further increased MHC II MFI on both AM and IM over that by Tri:HuAd ( Figure 4D ).

Furthermore, we also examined whether i.n. immunization induced trained airway macrophages even earlier at 4-weeks with the empty vector (HuAd:EV or ChAd:EV) included as control ( Figure 4E ). Visualization of t-SNE plots of CD45 + CD11C + CD11b + BAL cells showed the absence of MHC II high macrophages in the airways of HuAd:EV or ChAd:EV animals, compared to i.n. Tri:HuAd-or Tri:ChAd animals ( Figure Tri:ChAd vaccine induced significantly further increased MHC II MFI on airway macrophages over that by i.n. Tri:HuAd ( Figure 4H) . The above data together suggest that single-dose respiratory mucosal immunization with Ad-vectored COVID-19 vaccines induces trained airway macrophages, besides its potent effects on inducing memory B and T cells in the lung.

Having demonstrated the robust immunogenicity by intranasal immunization with trivalent COVID-19 vaccines, we next assessed the protection by both vaccine vectors against a mouse- Mice were then infected with SARS-CoV-2 MA10 at 4-weeks post-immunization ( Figure 5D ). T cell depletion was carried out from day 25 post-immunization when B cells/antibody responses were fully developed and prior to viral challenge by 2-3 repeated intraperitoneal injections of a T cell depleting antibody cocktail . Unvaccinated wildtype mice rapidly succumbed to infection, reaching humane endpoint by 4-days ( Figure 5E ). Unvaccinated mice lacking either T cells or B cells showed similar weight loss kinetics, with 80% T cell dep. and 20% B cell KO mice reaching humane endpoint at the same time as unvaccinated wildtype controls ( Figure 5E ).

In contrast, i.n. Tri:ChAd vaccine protected wildtype, T cell dep., and B cell KO animals equally well as they did not show any weight losses throughout ( Figure 5F ). These data indicate that the superior immunogenicity of i.n. Tri:ChAd vaccine is capable of compensating for the lack of either T or B cells to retain protection against clinical disease.

Besides clinical outcomes, we examined the role of B and T cells in control of viral burden in the lung 4-days post-infection ( Figure 5D ). Compared to wildtype hosts, lack of T or B cells in unvaccinated animals had little effects on high lung viral burden ( Figure 5G ). In contrast, i.n.

Tri:ChAd vaccination of wildtype control animals led to complete viral clearance at 4-days ( Figure 5H) . However, lack of either T or B cells in i.n. Tri:ChAd-vaccinated animals resulted in significantly elevated lung viral titers (2-4 log) (Figure 5H) , despite no changes in morbidity/mortality ( Figure 5F ). In comparison, similar to i.n. Tri:ChAd vaccine, i.n. Tri:HuAd also well protected wildtype and B cell KO mice but vaccinated mice lacking T cells experienced a transient moderate weight loss with 20% of them succumbing to infection (Figure S7A/B) .

However, while similar to the 2-day data (Figure 5C ), i.n. Tri:HuAd vaccine at 4-days only moderately reduced viral loads in the lung of wildtype animals by approximately 2 logs, lack of J o u r n a l P r e -p r o o f either T or B cells in these animals also resulted in increased lung viral burden (~1 log) ( Figure   S7C ).

To further understand the mechanisms of i.n. Tri:ChAd-induced protection in either T celldepleted or B cell-deficient hosts, we determined the protection in i.n. Tri:ChAd-vaccinated B cell KO mice depleted of T cells. Since we have reported that T cell-help is required for vaccinemediated airway macrophage priming and trained innate immunity (TII) only at early (3-5 days), but not later, times post-i.n. Ad-vectored vaccination , this model also provided the opportunity to address the protective role of TII in the absence of both T and B cell immunity.

Thus, a continuous T cell depletion protocol initiated shortly after i.n. Tri:ChAd vaccination was employed in B cell KO mice to render deficiencies in TII, and T and B cells (Figure 5I The above data indicate that intranasal immunization provides superior protection against a mouse-adapted SARS-CoV-2 over intramuscular route; Tri:ChAd vaccine is more potent than Tri:HuAd; both humoral and T cell immunity are required for optimal protection by intranasal immunization; and vaccine-induced trained innate immunity contributes to protection primarily by improving clinical outcomes while it plays a minor role in control of viral burden.

We next assessed vaccine-induced protection against wildtype ancestral and VOC strains of SARS-CoV-2 in highly susceptible K18-hACE2 mouse model (Khoury et al., 2020) . Since our data indicate the superiority of i.n. Tri:ChAd vaccine in both immunogenicity and protection, we focused on evaluating the protection by i.n. Tri:ChAd immunization in K18-hACE2 models.

Since the bulk of our immunogenicity data were from BALB/c mice, we began by assessing vaccine immunogenicity in C57BL/6 K18-hACE2 mice at 4-weeks post-i.n. Tri:ChAd immunization ( Figure S7D) . Similar to the neutralization titers in BALB/c hosts (Figure 1L) , sera from i.n. Tri:ChAd-immunized C57BL/6 animals showed robust SARS-CoV-2 neutralization ( Figure S7E) . Similarly, airway antigen-specific CD8 + T cell responses in C57BL/6 mice were also comparable ( Figure S7F ) to those in BALB/c mice (Figure 2A Since SARS-CoV-2 infection of K18-hACE2 mice also causes viral dissemination to the brain (Zheng et al., 2021), the brain was included in the assay. Unvaccinated naïve mice had similarly high B.1.1.7 or B.1.351 viral burden in the lung ( Figure 6I ) and brain ( Figure 6J ). Of interest, while ChAd:EV mice had similarly high viral titers in the lung as in naïve mice (Figure 6I) , they showed moderately reduced viral burden in the brain ( Figure 6J) . Remarkably, i.n. Tri:ChAd immunized mice developed sterilizing immunity against both B.1.1.7 and B.1.351 variants in the lung ( Figure 6I ) and brain ( Figure 6J) .

ChAd-vectored COVID-19 vaccine induces robust mucosal immunity against lethal infection by not only ancestral SARS-CoV-2 but also immune-evasive VOC.

We next examined the protective role of the N and RdRp antigens included in Tri:ChAd vaccine design. To this end, we developed a bi-valent and a mono-valent ChAd vaccine only expressing the N/RdRp (Bi:ChAd) and the S1 (Mono:ChAd), respectively ( Figure 7A) . We first J o u r n a l P r e -p r o o f examined the protection by i.n. Bi:ChAd vaccine and compared it with Tri:ChAd in BALB/c mouse model (Figure 7B) . In contrast to severe clinical illness in unvaccinated animals, i.n.

Bi:ChAd protected mice as well as i.n. Tri:ChAd against infection ( Figure 7C) . On the other hand, while i.n. Tri:ChAd led to no detectable viruses, i.n. Bi:ChAd markedly reduced lung viral titers by 3 logs relative to unvaccinated controls ( Figure 7D) . Figure 7J) . Indeed, extensive gross pathology was seen in the lungs of Mono:ChAd animals whereas the Bi:ChAd lungs appeared nearly free of gross pathology as did the Tri:ChAd animals ( Figure 7K) . Furthermore, unvaccinated animals had high viral loads in the lung whereas i.n. Tri:ChAd significantly reduced viral loads by 3.5 logs ( Figure 7L ). In comparison, both i.n. Bi:ChAd and Mono:ChAd vaccines only moderately reduced viral load.

The above data indicate the protective superiority of ChAd-vectored vaccine when expressing the S1, N and RdRp antigens over its bi-valent and mono-valent counterparts. Inclusion of N/RdRp antigens in our tri-valent vaccine design offers additional protection via neutralizing antibody-independent T cell and trained innate immunity.

The effective global control of COVID-19 via immunization with first-generation vaccines has been threatened by the VOC and waning vaccine-induced antibody immunity (Goldberg et al., 2021; Harvey et al., 2021; Krause et al., 2021) . This situation calls for the development of not only J o u r n a l P r e -p r o o f next-generation/"universal" vaccines but also diversified vaccine strategies. In response, we have developed Ad-vectored next-generation trivalent COVID-19 vaccines expressing the original S1 antigen and highly conserved T cell antigens N and RdRp for respiratory mucosal route of delivery.

We show that respiratory mucosal immunization is superior to intramuscular immunization at inducing neutralizing antibodies, mucosal tissue-resident memory T cells and trained airway macrophages. We further show that the choice of Ad vector also is of importance, with chimpanzee-derived Ad68 platform (Tri:ChAd) outperforming the human Ad5 counterpart (Tri:HuAd) and that inclusion of both the spike and conserved internal T cell antigens in vaccine design is required for optimal protection against both ancestral SARS-CoV-2 and VOC. We also reveal that both B and T cells and trained innate immunity are required for robust respiratory mucosal immunity.

To our knowledge, our study is the first to have developed a multivalent next-generation vaccine strategy against both ancestral SARS-CoV-2 and emerging VOC in animal models.

Although a recent murine study shows the ability of a first-generation S-encoding mRNA vaccine (CVnCoV) to protect from B.1.351 infection (Hoffmann et al., 2021a) , this vaccine has led to disappointing efficacy results from clinical trials. It is widely accepted that the next-generation vaccine strategies ought to take into consideration both vaccine multivalency and route of delivery Teijaro and Farber, 2021) . While almost all first-generation genetic COVID-19 vaccines were designed for intramuscular delivery and to express only the S protein, there is a growing interest in studying their utility for respiratory mucosal delivery in preclinical models (Bricker et al., 2021; Cao et al., 2021; Hassan et al., 2020 Hassan et al., , 2021a Ku et al., 2021) .

However, most of these studies did not compare the intranasal with the intramuscular route of immunization, nor did they test their protection against VOC. The studies by scientists at Washington University in St. Louis (WUSTL) are the only ones to compare the intranasal delivery with intramuscular immunization (Bricker et al., 2021; Hassan et al., 2020) and this group also extended their study to show the ability of intranasal ChAd-S immunization to protect against a virus displaying B.1.351 spike protein (Hassan et al., 2021b) . By comparison, with the goal of developing next-generation COVID-19 vaccines, we have bioengineered two Ad-vectored trivalent vaccines (Tri:HuAd & Tri:ChAd) and extensively compared their immunogenicity and protection against ancestral and variant strains of SARS-CoV-2 following single-dose intramuscular or intranasal immunization. Our findings indicate the respiratory mucosal delivery J o u r n a l P r e -p r o o f of a trivalent ChAd-vectored vaccine to be the most effective next-generation COVID-19 vaccine strategy. Our study thus supports its further clinical development.

The superiority of i.n. COVID-19 immunization at inducing both protective humoral and mucosal T cell immunity over the i.m. route observed in our study is well aligned with the established paradigm associated with other vaccines (Belyakov and Ahlers, 2009; Neutra and Kozlowski, 2006) . It has also been observed in animal models of COVID-19 using a ChAd-vectored first-generation vaccine (Bricker et al., 2021; Hassan et al., 2020) . The high degree of tissue compartmentalization of immunity dictated by the route of immunization is not only limited to animal models. Inhaled aerosol MVA TB vaccine induced respiratory mucosal immunity in humans whereas intradermal injection of the same vaccine failed to do so (Satti et al., 2014) . We have also recently reported that inhaled aerosol, but not intramuscular delivery, of HuAd-vectored TB vaccine induces respiratory mucosal immunity in humans (Jeyanathan et al., 2021) . Given that all of the currently approved viral-vectored COVID-19 vaccines including ChAdOx1nCov-19 (AstraZeneca/Oxford), Ad26.COV2-S (J&J), Gam-COVID-Vac (Gamaleya) and Ad5-nCoV (CanSino) are intramuscularly administered, they are unlikely to induce protective respiratory mucosal immunity . Although aerosol delivery of Ad5-nCoV was tested in humans, it is unclear whether it induced respiratory mucosal immunity (Wu et al., 2021) . We have also recently shown that SARS-CoV-2-specific IgA enriched at mucosal surfaces like the lung can induce neutrophils to undergo NETosis capable of trapping and killing virus, thereby limiting spread . Thus, the well-recognized limitations of i.m. vaccine delivery, along with our current findings and those from others (Bricker et al., 2021; Hassan et al., 2020) , should bolster the global effort in developing respiratory mucosal-delivered nextgeneration COVID-19 vaccines. In this regard, there are at least two clinical trials testing inhaled aerosol ChAdOx1nCov-19 (Singh et al., 2021) or intranasally delivered ChAd-SARS-CoV-2-S (Clinical Trial NCT04751682).

Our study has also provided the evidence that both B and T cells are required for optimal protection. We find that the clinical outcomes/illness do not always corroborate with viral burden.

Indeed, while i.n. Tri:ChAd vaccine protected wildtype and B cell-and T cell-deficient mice in terms of clinical outcomes, lack of B or T cells led to partially impaired viral clearance in the lung.

Importantly, we observed that while lack of both B and T cells at the time of infection completely abolished the control of viral infection, the animals remained reasonably protected from clinical J o u r n a l P r e -p r o o f illness and lung pathology due to the presence of vaccine-induced trained innate immunity. Thus, the optimal protection in both clinical disease and viral clearance is accomplished when vaccines effectively elicited functional antibodies, T cells and trained innate immunity.

Globally, while waning vaccine-induced antibody immunity may account for recent increases in break-through infections by VOC (Wilhelm et al., 2021) , the first-generation vaccines have thus far largely protected against severe disease (Scott et al., 2021) , supporting the critical role of T cell immunity in controlling established infection in human lungs. In our study, besides using a T cell-depletion approach, the role of vaccine-induced T cell immunity is supported further by our observations from both BALB/c and K18-hACE2 models immunized i.n. with a bi-valent ChAd-vectored vaccine (Bi:ChAd) that only expresses T cell antigens N/RdRp. Intranasal Bi:ChAd vaccine protected against clinical illness as well as Tri:ChAd vaccine in BALB/c hosts and offered partial protection against viral load in the lung. Since K18-hACE2 animals are much more susceptible than BALB/c animals to SARS-CoV-2, due in part to early viral dissemination to the brain, i.n. Bi:ChAd vaccination was less protective against clinical illness but still significantly reduced viral burden both in the lung and brain. Furthermore, when Bi:ChAd and S1expressing Mono:ChAd vaccines were compared in our K18-hACE2/B.1.351 model, i.n. Bi:ChAd vaccine better protected against lung pathology than Mono:ChAd vaccine whereas both vaccines reduced viral loads in the lungs. These findings together support the relevance of broadening the breadth of T cell immunity in COVID-19 vaccine design.

Delivery of Ad-vectored vaccines via the respiratory mucosal route to humans helps to bypass pre-existing anti-vector immunity which is more prevalent in the circulation than in the lung. This is particularly relevant to the use of human Ad5 and Ad26 vectors . Indeed, we have seen little presence of pre-existing anti-Ad5 antibodies in human airways, contrast to the peripheral blood (Jeyanathan et al., 2021) . Nonetheless, ChAd-vectored vaccines have an advantage over Ad5 and Ad26 vectors in that humans have little pre-existing immunity against ChAd viruses. We previously found that not only intranasal ChAd-vectored vaccine was not impacted by anti-Ad5 immunity in murine lungs but it also triggered T cell responses to additional antigenic epitopes . Besides these potential advantages, our current study provides strong evidence that ChAd-vectored trivalent COVID-19 vaccine is also much more potent than its HuAd-vectored counterpart. Furthermore, since intramuscular injection of ChAdOx1nCov-19 (AstraZeneca/Oxford) and Ad26.COV2-S (J&J) has been associated with J o u r n a l P r e -p r o o f rare cases of VITT (vaccine-induced thrombotic thrombocytopenia) which is likely related to the activation of platelets and endothelium by accidental intravenous introduction of adenovirus (Nicolai et al., 2021; Tsilingiris et al., 2021) , respiratory mucosal delivery of Ad-vectored vaccines may avert this adverse outcome.

In summary, we have developed a next-generation COVID-19 vaccine strategy which is unique in both vaccine design and route of delivery. We show the superiority of a single-dose intranasal immunization with trivalent ChAd-vectored vaccine in inducing the tripartite respiratory mucosal immunity against both ancestral and variant strains of SARS-CoV-2.

We show respiratory mucosal Ad-vectored immunization to induce the tripartite mucosal immunity which includes trained innate immunity. Although our evidence supports the protective role of trained innate immunity against COVID-19, its potency may be underestimated given the limitations of murine models. Unlike infected humans, mice infected with relatively large viral inoculums do not have a pre-symptomatic period where trained innate immunity may play a critical role . Likewise, while we show strong evidence that intranasal ChAdvectored vaccine is more potent than HuAd-vectored counterpart, whether the same holds true in humans remains to be investigated. We have recently begun a phase 1 clinical trial (ClinicalTrials.gov: NCT05094609) to compare these two COVID-19 vaccines following inhaled aerosol delivery to mRNA-vaccinated humans.

Although our experimental evidence supports the superiority of intranasal immunization over the intramuscular route in inducing respiratory mucosal immunity, murine lungs differ from human lungs in that the latter are not "naïve" and are therefore potentially amenable to the translocation of circulating antibodies following intramuscular COVID-19 immunization. This may explain vaccine-mediated protection in humans at least within the first 6-8 months. In addition, our data showing the immunodominance of S1 over N and RdRp antigens should be interpreted with caution. This is particularly relevant to T cell responses. It is known that the immunodominance of T cell epitopes in mice is dictated by the mouse strain-dependent homogeneous MHC haplotype, a scenario different from genetically heterogeneous human populations. Thus, we expect a stronger and more diverse T cell response to N and RdRp antigens in humans following immunization with our trivalent vaccines. Likewise, although we have J o u r n a l P r e -p r o o f observed a predominantly CD8 + T cell response to our COVID-19 vaccine in murine models, a better balanced CD4 + and CD8 + T cell response is expected in humans. Indeed, while we observed a predominantly CD8 + T cell response to our HuAd-vectored TB vaccine in mice, a robust CD4 + T cell response was seen in human lungs following inhaled aerosol delivery of the same vaccine (Jeyanathan et al., 2021) . J o u r n a l P r e -p r o o f (A) Left panel, Bar graphs depicting absolute number of S1-specific IFNγ + CD8 + T cells in the BAL at 2 (red) and 4 (blue) weeks post-immunization. Right panel, Representative flow cytometric dotplots of IFNγ + CD8 + T cells in the BAL following ex vivo stimulation with overlapping peptide pools for S1. (B) Bar graphs depicting multifunctional CD8 + T cell responses in the BAL as measured by production of IFNγ, TNFα, and/or IL-2 at 4-weeks post-immunization, following ex vivo stimulation with overlapping peptide pools for S1. (C) Stacked bar graph depicting the frequency of cytotoxic CD8 + T cells in the BAL as measured by Granzyme B production at 4-weeks post-immunization, following ex vivo stimulation with DMSO (red) or overlapping peptide pools for S1 (blue). Data presented in A-I represent mean ± SEM. Statistical analysis were Mann-Whitney tests. Data is pooled from 2 independent experiments, n=3-6 mice/group. * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001.

See also Figure S2 , S4. 

Bar graphs depicting absolute number of S1, nucleocapsid, or RdRp-specific IFNγ + CD8 + T cells in the BAL at 8-weeks post-immunization, following ex vivo stimulation with overlapping peptide pools for S1, nucleocapsid, or RdRp.

(G) Sunburst plots depicting functionality (IFNγ, TNFα, and/or IL-2) of CD8 + T cells at 8weeks post-immunization, following ex vivo stimulation with either S1, nucleocapsid, or RdRp peptide pools.

Data presented in D-F represent mean ± SEM. Data is representative of 2 independent experiments, n=3-6 mice/group. See also Figure S2 , S3, S5. Data presented in D, and H represent mean ± SEM. Data is representative of 2 independent experiments, n=3-6 mice/group. Statistical analysis for panels D, and H were 1-way ANOVA with Tukey multiple comparisons test. * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001. Alveolar macrophage (AM), interstitial macrophage (IM), median fluorescence intensity (MFI). RdRp (right panels) specific IFNγ + CD8 + (top) or IFNγ + CD4 + (bottom) T cells in the BAL at 2 (red) and 4 (blue) weeks post-immunization following ex vivo stimulation with overlapping peptide pools. (B) Flow cytometric dot plots of CD44 + CD8 + T cells for CD69 and CD103 from the lung (left panels) or BAL (right panels) at 4-weeks post-immunization.

Data presented in panels B-E represent mean ± SEM. Data is representative of 1-2 independent experiments, n=3-9 mice/group. Figure S3 . Flow cytometric gating strategies, Related to Figures 1 and 3 (A) Gating strategy in this study used to distinguish antigen-specific, class switched B cells. (B) Gating strategy in this study used to distinguish bona fide pulmonary tissue-resident memory CD8 + (top) or CD4 + (bottom) T cells. (C) Gating strategy in this study used to distinguish neutrophils, alveolar macrophages (AM), and interstitial macrophages (IM) from other major pulmonary myeloid cell populations.

Examples shown are representative from BALB/c mice i.n. vaccinated with Tri:ChAd at 4-weeks post-immunization.

J o u r n a l P r e -p r o o f Figure S4 . Comparison of antigen-specific CD4 and CD8 T cells in BAL and spleen following single-dose immunization with Tri:HuAd or Tri:ChAd vaccine, Related to Figure 2 (A) Bar graphs depicting absolute number of S1 (left panels), nucleocapsid (middle panels), or RdRp (right panels) specific IFNγ + CD4 + T cells in the BAL at 2 (red) and 4 (blue) weeks post-immunization following ex vivo stimulation with overlapping peptide pools. (B) Left panel, Schema of vaccination regimen. BALB/c mice were intranasally (i.n.) vaccinated with a single dose of either Tri:HuAd or Tri:ChAd. Animals were sacrificed at 3-weeks post-immunization for immunological analysis. Bar graphs depicting frequency of IFNγ + CD4 + T cells (middle left panel), or IL4 + CD4 + T cells (middle right panel) following ex vivo stimulation with S1 overlapping peptide pools (red), or anti-CD3/CD28 (blue). Right panel, Ratio of IFNγ:IL-4 producing CD4 + T cells, based on data from middle panels. (C) Bar graphs depicting absolute number of S1 (left panels), nucleocapsid (middle panels), or RdRp (right panels) specific IFNγ + CD8 + (top) or IFNγ + CD4 + (bottom) T cells in the spleen at 2 (red) and 4 (blue) weeks post-immunization following ex vivo stimulation with overlapping peptide pools.

Data presented in panels A-C represent mean ± SEM. Data is representative of 1 independent experiment, n=3-4 mice/group. Data presented in panels E, and F represent mean ± SEM. Statistical analysis for panel F were 1-way ANOVA with Tukey multiple comparisons test. Data in panel E is representative of 1 independent experiment, n=5 mice/group. Data in panel F is pooled from 2 independent experiments, n=10 mice per group. ns, not significant. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Zhou Xing (xingz@mcmaster.ca).

All requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact or correspondence authors. All reagents including antibodies, viruses and vaccines may be made available on request after completion of a Materials Transfer Agreement.

All data supporting the findings of this study are available within the paper and are available from the Lead Contact or correspondence authors upon request. This paper does not report original code.

Age-matched 6-8-wk-old wild-type female or male BALB/c, C57BL/6J, or B6.Cg-Tg (K18- 

J o u r n a l P r e -p r o o f

The transgene cassette was constructed through a series of overlapping polymerase chain reactions (PCR) wherein transgene expression is under control of the murine CMV (mCMV) promoter and protein translation is initiated with the human tissue plasminogen (tPA) signal sequence ( Figure   1A ). The first overlapping PCR product contained the tPA signal sequence upstream of the S1 sequence of the Wuhan-Hu-1 Isolate of SARS-CoV-2 (GenBank: MN908947.3) fused to the vesicular stomatitis virus G protein transmembrane (VSVG TM) domain to facilitate trimerization and exosome targeting. This PCR product was cloned in pCY1 plasmid which contains the mCMV promoter. The second overlapping PCR product contained the porcine teschovirus-1 2A (P2A) skip sequence upstream of the full-length nucleocapsid sequence from the same SARS-CoV-2 isolate fused to a highly conserved region of nsp12 (RNA-dependent RNA polymerase (RdRp)).

The sequence of RdRp was chosen based on conserved sequence homology to bat coronaviruses and further refined to include several predicted high affinity human CD8 T cell epitopes on HLA 0101, 0201, and 0301. The second overlapping PCR product was cloned downstream of the VSVG TM domain in pCY1 to generate the complete expression cassette. The same transgene cassette was cloned in the shuttle plasmids used during co-transfection to rescue the tri-valent, replicationdefective human serotype 5 adenoviral-vectored (Tri:HuAd) and chimpanzee serotype 68 adenoviral-vectored (Tri:ChAd) COVID-19 vaccines.

Tri:HuAd was packaged and rescued in HEK293 cells through a two-plasmid co-transfection system as previously described (Wang et al., 2004) . Tri:ChAd was also constructed and rescued in HEK293 cells via direct subcloning or similarly through a two-plasmid co-transfection system.

Briefly, the transgene cassette was PCR amplified to incorporate restriction enzyme sites and cloned in a shuttle vector containing a unique FspI cut site. The shuttle vector was then linearized with FspI and used for co-transfection with an SrfI-linearized plasmid containing the E1/E3deficient ChAd68 genomic backbone. Both trivalent vaccines were further amplified in HEK293 cells and subsequently purified by cesium chloride density gradient ultracentrifugation.

Both Mono-and Bi-valent ChAd vaccines (Mono:ChAd & Bi:ChAd) were constructed, purified and characterized as described above except that they were bio-engineered to express only the S1/VSVG TM (Mono:ChAd) or N/RdRp (Bi:ChAd). J o u r n a l P r e -p r o o f

Effectiveness of the BNT162b2

Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants

Immunogenicity of Ad26.COV2.S vaccine against SARS-CoV-2 variants in humans

SARS-CoV-2 T cell immunity: Specificity, function, durability, and role in protection

A serological assay to detect SARS-CoV-2 seroconversion in humans

SARS-CoV-2 escaped natural immunity, raising questions about vaccines and therapies

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Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2

What Role Does the Route of Immunization Play in the Generation of Protective Immunity against Mucosal Pathogens?

Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice

The role of IgG Fc receptors in antibodydependent enhancement

How to redesign COVID vaccines so they protect against variants

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Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies

A SARS

Airway Macrophages Mediate Mucosal Vaccine-Induced Trained Innate Immunity against Mycobacterium tuberculosis in Early Stages of Infection

Viral targets for vaccines against COVID-19

COVID-19 herd immunity: where are we?

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity

SARS-CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-19 convalescent donors and vaccinees

Waning Immunity after the BNT162b2 Vaccine in Israel

Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia

Will SARS-CoV-2 variants of concern affect the promise of vaccines?

Combinatorial mRNA vaccination enhances protection against SARS-CoV-2 delta variant

Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence

SARS-CoV-2 variants, spike mutations and immune escape

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques

CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

A systematic review of antibody mediated immunity to coronaviruses: kinetics, correlates of protection, and association with severity

Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects. Viruses 13

Novel chimpanzee adenovirusvectored respiratory mucosal tuberculosis vaccine: overcoming local anti-human adenovirus immunity for potent TB protection

CXCR3 Signaling Is Required for Restricted Homing of Parenteral Tuberculosis Vaccine-Induced T Cells to Both the Lung Parenchyma and Airway

New Tuberculosis Vaccine Strategies: Taking Aim at Un-Natural Immunity

Immunological considerations for COVID-19 vaccine strategies

Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans

Measuring immunity to SARS-CoV-2 infection: comparing assays and animal models

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

Correlates of protection from SARS-CoV-2 infection

SARS-CoV-2 Variants and Vaccines

Intranasal vaccination with a lentiviral vector protects against SARS-CoV-2 in preclinical animal models

Exosomal vaccines containing the S protein of the SARS coronavirus induce high levels of neutralizing antibodies

Alveolar Macrophages Are the Primary Interferon-α Producer in Pulmonary Infection with RNA Viruses

Mucosal immunity and novel tuberculosis vaccine strategies: route of immunisation-determined T-cell homing to restricted lung mucosal compartments

A Mouse-Adapted SARS-CoV-2 Induces Acute Lung Injury and Mortality in Standard Laboratory Mice

Developing Covid-19 Vaccines at Pandemic Speed

Efficacy of the

Covid-19 Vaccine against the B.1.351 Variant

Elicits Spike-Independent SARS-CoV-2 Protective Immunity

Mucosal vaccines: the promise and the challenge

Thrombocytopenia and splenic platelet directed

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

and B.1.351 variants to neutralizing antibodies

Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19

Mechanisms of Mucosal and Parenteral Tuberculosis Vaccinations: Adenoviral-Based Mucosal Immunization Preferentially Elicits Sustained Accumulation of Immune Protective CD4 and CD8 T Cells within the Airway Lumen

Safety and immunogenicity of a candidate tuberculosis vaccine MVA85A delivered by aerosol in BCG-vaccinated healthy adults: a phase 1, double-blind, randomised controlled trial

Alveolar Macrophages Are Essential for Protection from Respiratory Failure J o u r n a l P r e -p r o o f and Associated Morbidity following Influenza Virus Infection

Covid-19 vaccination: evidence of waning immunity is overstated

Neutralization of SARS-CoV-2 Variants B.1.429 and B.1.351

Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant

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IgA potentiates NETosis in response to viral infection

Repeated cross-sectional sero-monitoring of SARS-CoV-2 in New York City

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Location, location, location: Tissue resident memory T cells in mice and humans

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Omicron Variant by Vaccine Sera and monoclonal antibodies

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Changes in body weight over 2-weeks post-SARS-CoV-2 infection

Viral burden (Log10TCID50) in the lung at 2-days post-SARS-CoV-2 MA10 infection

Changes in body weight of unvaccinated BALB/c, T cell depleted BALB/c, or Jh -/-mice over 2-weeks post-SARS-CoV-2 infection

Changes in body weight of i.n. Tri:ChAd vaccinated BALB/c, T cell depleted BALB/c, or Jh -/-mice for 2-weeks post-SARS-CoV-2 infection

G) Viral burden (Log10TCID50) in the lung of unvaccinated animals at 4-days post-infection. (H) Viral burden (Log10TCID50) in the lung of Tri:ChAd vaccinated animals at 4-days postinfection

Experimental Schemas

Changes in body weight of over 2-weeks post-SARS-CoV-2 infection. Black circles indicate unvaccinated mice, blue circles indicate Tri:ChAd vaccinated mice, red circles indicate Tri:ChAd vaccinated mice with continuous T cell depletion, purple circles indicate Tri:ChAd vaccinated mice with T cell depletion prior to infection

Heatmap representing cumulative acute clinical observations: ruffled fur, lethargy/depression, and erratic/laboured respiration

Gross pathological changes from the lungs of vaccinated mice at 4-(left) and 14-(right) days post-infection

Data is representative of 1-to-2 independent experiments, n=5 mice/group

Intranasal Tri:ChAd immunization protects against lethal challenge with SARS-CoV-2 variants of concern (A) Experimental schema. (B) Changes in body weight over 2-weeks post-ancestral SARS-CoV-2 infection. (C) Survival of mice post-ancestral SARS-CoV-2 SB3 infection. (D) Viral burden (Log10TCID50)

Survival of mice post-SARS-CoV-2 B.1.1.7 infection. (G) Changes in body weight over 2-weeks post-SARS-CoV-2 B.1.351 infection. (H) Survival of mice post-SARS-CoV-2 B.1.351 infection. (I) Viral burden (Log10TCID50) in the lung at 4-days post-B.1.1.7 or B.1.351 infection. (J) Viral burden (Log10TCID50)

Statistical analysis for panels D, I, and J were 1-way ANOVA with Tukey multiple comparisons test. Data in panels B, and C is pooled from 2 independent experiments, n=5-11 mice/group. Data in panels D-J is representative of 1 experiment

Comparison of protective efficacy of ChAd-vectored tri-valent vaccine with its bi-valent and mono-valent counterparts (A) Transgene cassette diagrams for bi-valent (Bi:ChAd, left panel) and mono-valent

Changes in body weight over 2-weeks post-SARS-CoV-2 infection

Viral burden (Log10TCID50) in the lung at 4-days post-SARS-CoV-2 MA10 infection

Changes in body weight over 2-weeks post-SARS-CoV-2 B.1.351 infection (open circles: 1 surviving animal)

Changes in body weight over 2-weeks post-SARS-CoV-2 B.1.351 infection

Gross pathological changes from the lungs of vaccinated mice at 4-days post-infection. Hashed circles encompass areas of visible lung damage

is pooled from 2 independent experiments, n=3-11 mice/group. Data in panels J-L is representative of 1 experiment

Data presented in B, C, and E-I represent mean ± SEM. Statistical analysis for panel C was 1-way ANOVA with Tukey multiple comparisons test. Statistical analysis for panel H was two-tailed Ttests. Data is representative of 1 independent experiment, n=3-5 mice/group

United States) were cultured at 37˚C in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1 % HEPES pH7.3, 1 mM sodium pyruvate

U/mL of penicillin-streptomycin. SARS-CoV-2 strain SB3-TYAGNC was provided by Dr

SARS-CoV-2 strain MA10 was generously provided by

NR-54000, Public Health England) and strain hCoV-19/South Africa

United States). recombinant human adenovirus (Ad) serotype 5 SARS-CoV-2 vaccine (Tri:HuAd) or 1x10 7 PFU of a recombinant chimpanzee adenovirus serotype 68 SARS-CoV-2 vaccine (Tri:ChAd). In a subset of experiments where mono-, bi-, and tri-valent ChAd-vectored vaccines were compared for protective efficacy in K18-hACE2 mouse model

) were intraperitoneally administered as a single bolus either 3-or 25-days post-vaccination. A second 100µg dose was administered 2-days following the first dose, and repeated every 7-days to maintain depletion, as per experimental requirements. Bronchoalveolar lavage, lung, and spleen mononuclear cell isolation Mice were euthanized by exsanguination

Briefly, BAL was performed by instillation with 250 µL, followed by 200 µL of PBS. This fraction was utilized for downstream soluble factor analysis. Further instillation of 3x 300 µL of PBS was performed for BAL cell retrieval. Lungs were minced into small pieces and digested with collagenase type 1

Cell numbers were quantified in Turk's Blood Dilution Fluid (RICCA Chemical

United States) were cultured at 37˚C in DMEM supplemented with 10% FBS, 1 % HEPES pH 7.3, 1% L-glutamine and 100 U/mL of penicillincarried out with Tri:HuAd (MOI 100) and Tri:ChAd (MOI 50) diluted in PBS (with Mg 2+ and Ca 2+ ). 18 hours post-infection, cells were lysed using RIPA buffer

United States) and 60 µg of each lysate was boiled at 98˚C with 1X sample buffer

The samples were run on a 4-12% SDS-PAGE gel (ThermoFisher Scientific Waltham, MA, United States) for 1.5 hours at 100 V and transferred to nitrocellulose membrane (VWR, Mississauga, ON, Canada) using wettransfer at 125 mA for 1.5 hours. The membrane was

Anti-NP (ThermoFisher Scientific Waltham, MA, United States) and 1:5000 GAPDH (MilliporeSigma, Etobicoke, ON, Canada) diluted in 5% skim milk, followed by anti-mouse and anti-rabbit IRDye secondary antibodies

Recombinant antigen production Plasmids encoding mammalian cell codon optimized sequences for the receptor binding domain (RBD) and full-length spike of SARS-CoV-2 was generously gifted from the lab of Dr

United States) according to the manufacturers' instructions and purified as previously described

United States) per 25 mL of transfected cell supernatant. The following day 10 ml polypropylene gravity flow columns

to gate out non-RBD binding B cells. The decoy tetramer was constructed through conjugating streptavidin-PE to Alexa Fluor 647 (Thermo Fisher Scientific) for 1 hour at room temperature. Excess Alexa Fluor 647 was removed through washing and centrifugation with 100 kDa Amicon spin columns

United States) were coated overnight at 4˚C with SARS-CoV-2 RBD, or full-length spike, diluted to 2 µg/mL in bicarbonatecarbonate coating buffer (pH 9.4). Plates were blocked by shaking for 1 hour at 37˚C with reagent diluent (0.5% bovine serum albumin (BSA), 0.02 % sodium azide

United States) according to the manufacturer's instructions, with volumes normalized prior to concentration. Samples were arranged such that one row contained only antigen and secondary antibodies and served as the plate blank. Following a 1 hour incubation with shaking at 37˚C, plates were washed three times with 1X Tris-Tween wash buffer

) in complete DMEM (supplemented with 10% FBS, 1% L-glutamine, 100 U/mL penicillin-streptomycin). 24 hours later, serum was inactivated by incubating at 56˚C for 30 minutes

An equal volume of SARS-CoV-2 consisting of 330 PFU per well was then added to the diluted serum. The serum-virus mixture was then incubated at 37˚C for 1 hour. The Vero E6 culture media was then replaced with 100 µL of the serum-virus mixture and was incubated at 37˚C for 72 hours. The plates were read by removing 50 µL of culture supernatant and adding 50µL of CellTiter-Glo 2.0 Reagent (Promega, Madison, WI, United States) to each well. The plates were then shaken at 282 cpm at 3 mm diameter for 2 minutes

In certain experiments, serum neutralizing antibodies were assessed utilizing a surrogate SARS-CoV-2 virus neutralization test (sVNT). sVNT assays were performed utilizing the cPass Neutralization Antibody Detection kit

Cytokine and serum chemistry Evaluation of serum and BAL cytokines were performed by Eve Technologies

Briefly, tissue isolated mononuclear cells were plated in U-bottom, 96-well plates at a concentration of 2x10 7 cells/mL in PBS. Following staining with The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit

4G2) in 0.5 % BSA-PBS for 15 min on ice and then stained with fluorochrome-labeled mAbs for 30 min on ice. Fluorochrome-labeled mAbs used for staining cells were anti-CD45-APC-Cy7 (clone 30-F11)

APC (clone HL3), anti-MHC class II (MHC II)-Alexa Fluor 700

United States), anti-CD3-V450 (clone 17A2), anti-CD45R (B220)-V450 (clone RA3-6B2)

United States)

United States), anti-Ly-6G-BV605 (clone 1A8), anti-Siglec

clone E50-2440), anti-CD4 APC-Cy7 (clone GK1.5), anti-CD8 PE-Cy7

IFNγ APC (clone XMG1.2), anti-TNFα FITC (clone MP6-XT22), anti-IL2 BV605 (clone JES6-5H4), anti-Granzyme B PE

anti-CD44 PE (clone IM7), anti-CD69 BV605 (clone H1.2F3)

Biotin (clone M290), anti-CD11a FITC (clone 2D7), anti-IgD BV711 (clone 11-26c

United States), and anti-IgG1-BV421 (clone

Stained cells were fixed and permeabilized with BD

All mAbs and reagents were purchased from BD Biosciences unless otherwise indicated. Stained cells were processed according to BD Biosciences instructions for flow cytometry and run on a BD LSR II flow cytometer. Data were analyzed using FlowJo software (version 10.1; Tree Star

Homogenates were clarified by centrifugation at 300 x g and frozen at -80˚C. Homogenates were then thawed, and serially diluted 1:10 in serum-free DMEM supplemented with 1% HEPES pH 7.3, 1 mM sodium pyruvate, 1% L-Glutamine and 100 U/mL of penicillin-streptomycin. 100 µL of viral inoculum was plated on Vero E6 cells in 96-well plates (4 x 10 4 cells per well) for 1hr at 37˚C. 5% CO2, at which point the inoculum was replaced with low-serum DMEM supplemented with 2% fetal bovine serum (FBS), 1% HEPES pH 7.3, 1 mM sodium pyruvate, 1% L-Glutamine and 100 U/mL of penicillin-streptomycin. Wells were assessed for cytopathic effect at 5-days post-infection using an EVOS M5000 microscope

Peptides were reconstituted in DMSO according to manufacturer's instructions to a final concentration of 40 µg/µL. Antigen peptide pools were generated with each pool containing 0.2 µg/µL of each peptide. Unless otherwise stated, peptide stimulations were carried for each and ****p < 0.0001) as determined using either Mann-Whitney test, two-tailed unpaired Student t test, Kruskal-Wallis test with Dunn's multiple comparisons test, or one-way ANOVA

Data are Anti-mouse CD69 BV605 (clone H1.2F3) BD BioSciences Cat# 563290 RRID:AB_2738120 Anti-mouse CD103-Biotin (clone M290) BD BioSciences Cat# 557493 RRID:AB_396730 Anti-mouse CD11a FITC (clone 2D7) BD BioSciences Cat# 553120 RRID:AB_10892820 Anti-mouse IgD BV711 (clone 11-26c.2a) BioLegend Cat# 405731 RRID:AB_2563342 Anti-IgG1-BV421 (clone RMG1-1) BioLegend Cat# 406616 RRID:AB_2562234 Goat anti-mouse IgG, human ads-BIOT SouthernBiotech Cat# 1030-08 RRID:AB_2794296 Goat anti-mouse IgG1-BIOT SouthernBiotech Cat#

Streptavidin-Alkaline phosphatase SouthernBiotech Cat#

 Figure 1 . Single-dose intranasal immunization leads to superior anti-spike protein humoral responses over intramuscular immunization (A) Transgene cassette diagram. (B) Western blot analysis of expression of S1-VSVG and N/RdRp protein from whole-cell lysates from A549 cells untransduced or transduced with Tri:HuAd or Tri:ChAd. GAPDH was used as a loading control for each condition. (C) Experimental Schema. (D) Serum anti-spike IgG reciprocal endpoint antibody titers at 2 (red) and 4 (blue) weeks postimmunization. (E) Serum anti-RBD IgG reciprocal endpoint antibody titers at 2 (red) and 4 (blue) weeks postimmunization. (F) Reciprocal endpoint titer ratios of anti-spike IgG2a:IgG1 at 2 (red) and 4 (blue) weeks postimmunization. (G) Bar graph depicting serum neutralizing antibody responses 4-weeks post-immunization, measured by percent (%) inhibition with a surrogate virus neutralization test (sVNT). Green bar (+) indicates assay positive control. Gray bar (-) indicates assay negative control. Data presented in D-H, and J-N represent mean ± SEM. Statistical analysis for panels D, E, H, and J were Kruskal-Wallis tests with Dunn's multiple comparisons test. Statistical analysis for panel G, K, and N were two-tailed T-tests. Data is from 2 pooled independent experiments, n=3-12 mice/group. ns, not significant; * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001.See also Figure S1 , S2, S3.