key: cord-0725389-90m2c2dr
authors: Routhu, Nanda Kishore; Cheedarla, Narayanaiah; Gangadhara, Sailaja; Bollimpelli, Venkata Satish; Boddapati, Arun Kumar; Shiferaw, Ayalnesh; Rahman, Sheikh Abdul; Sahoo, Anusmita; Edara, Venkata; Lai, Lilin; Floyd, Katharine; Wang, Shelly; Fischinger, Stephanie; Atyeo, Caroline; Shin, Sally A.; Gumber, Sanjeev; Kirejczyk, Shannon; Cohen, Joyce; Jean, Sherrie M.; Wood, Jennifer S.; Connor-Stroud, Fawn; Stammen, Rachelle L.; Upadhyay, Amit A.; Pellegrini, Kathryn; Montefiori, David; Shi, Pei-Yong; Menachery, Vineet D.; Alter, Galit; Vanderford, Thomas H.; Bosinger, Steven E.; Suthar, Mehul S.; Amara, Rama Rao
title: Modified Vaccinia Ankara Vector-Based Vaccine Protects Macaques from SARS-CoV-2 Infection, Immune Pathology and Dysfunction in the Lung
date: 2021-02-04
journal: Immunity
DOI: 10.1016/j.immuni.2021.02.001
sha: 6c5300b901daf10ed24bc53fb37c4b651301254c
doc_id: 725389
cord_uid: 90m2c2dr

A combination of vaccination approaches will likely be necessary to fully control the SARS-CoV-2 pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane anchored pre-fusion stabilized spike (MVA/S), but not secreted S1, induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and showed protection from SARS-CoV-2 infection and virus replication in the lung as early as day 2 following intranasal or intratracheal challenge. Single-cell RNA sequencing analysis of lung cells at day 4 post-infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities, and lowered induction of interferon stimulated genes. These results demonstrate that MVA/S vaccination induces both neutralizing antibodies and CD8+ T cells in the blood and lung and serves as a potential vaccine candidate against SARS-CoV-2.

(Control)

The novel coronavirus SARS-CoV-2 infection has emerged as a pandemic. As of January 21, 2021, the SARS-CoV-2 has infected more than 95 million people and over 2 million people have succumbed to COVID-19 disease. Thus, there is an urgent need for the development of a vaccine that can rapidly induce anti-viral immunity and prevent infection. Studies from closely related coronaviruses such as SARS-CoV and MERS-CoV demonstrate that a strong neutralizing antibody response against the spike protein can effectively prevent infection (Chan et al., 2015; Haagmans et al., 2016; Xu et al., 2019; Yong et al., 2019) . Further building on these data, a number of vaccines are currently under development for SARS-CoV-2 using a variety of platforms including mRNA, viral vectors, DNA, protein with different adjuvants (Brouwer et al., 2020; Corbett et al., 2020; Gao et al., 2020; Keech et al., 2020; Krammer, 2020; Laczko et al., 2020; Mercado et al., 2020; Poland et al., 2020; Sahin et al., 2020; Smith et al., 2020; Tostanoski et al., 2020; van Doremalen et al., 2020; Walls et al., 2020; Yu et al., 2020) . A phenomenal progress has already been made in a very short time. The mRNA (Corbett et al., 2020; Laczko et al., 2020; Sahin et al., 2020) , adenoviral vector Tostanoski et al., 2020; van Doremalen et al., 2020) and spike trimer protein (Gao et al., 2020; Keech et al., 2020) based vaccines are in the forefront and showed induction of strong neutralizing antibody response in macaques and humans. In addition, these vaccines showed protection from SARS-CoV-2 infection and replication in the lungs following intranasal and intratracheal SARS-CoV-2 challenge in macaques. Recent data from mRNA and chimp adenovirus-based vaccines in human efficacy trials also showed that these vaccines can protect from COVID-19 disease in humans and have been approved for emergency use in humans. While a remarkable progress has been made many challenges still exist for SARS-CoV-2 vaccine development. These include the lack of long-term safety and immunogenicity data in humans, poor induction of CD8 + T cell response, lack of cross-reactive protective immunity against other human beta coronaviruses with pandemic potential, failure to provide protection against SARS-CoV-2 replication in the nasopharynx and requirement of cold storage. Therefore, a combination of vaccination approaches is needed to tackle these critical challenges. Thus, there is still a great need for the development of SARS-CoV-2 vaccines using different platforms, especially that can synergize with current front runners to enhance the induction and durability of neutralizing antibody and CD8 T cell response with long term safety.

Modified vaccinia Ankara (MVA) is a highly attenuated strain of vaccinia virus. The safety, immunogenicity, and protective capacity of the replication-deficient MVA has been wellestablished and has been widely used for developing vaccines against infectious diseases and cancer in preclinical research and humans (Gilbert, 2013; Iyer and Amara, 2014) . There are several advantages to MVA-based vaccines. 1) They are safe and well-tolerated including in HIV infected individuals (Thompson et al., 2016) . 2) They induce strong antibody responses after a single vaccination and can be boosted at least 10-fold with a second dose (Amara et al., 2002; Brault et al., 2017; Domi et al., 2018; Goepfert et al., 2011; Goepfert et al., 2014) . 3) MVA vaccine-induced antibody responses in humans are durable with little contraction over a 6 month timeframe (Goepfert et al., 2014) . 4) MVA can be delivered through multiple routes and can be used to generate a mucosal antibody response. 5) MVA can accommodate large inserts (>10kb) that will allow expression of multiple antigens in a single vector. 6) MVA vectored recombinants are stable and can be produced at high titer enabling ease of vaccine manufacturing. 7) MVA vaccines can induce CD4 and CD8 T cell responses important for protection against some viral infections (Amara et al., 2002) . 8) The lyophilized MVA vaccine can be stored at 37°C for 2 weeks and 4°C for longer than a year (Zhang et al., 2007) . 9) MVAbased vaccines have been shown to protect against SARS-CoV, MERS-CoV, Zika virus and Ebola virus in animal models (Bisht et al., 2004; Brault et al., 2017; Domi et al., 2018; Haagmans et al., 2016) . In addition, MVA-based vaccines can serve as an excellent boosting agent for DNA and other viral vector-based vaccines including chimp adenovirus and Ad26 based vaccines to boost cellular and humoral immunity (Amara et al., 2001; Barouch et al., 2012; Ewer et al., 2016) .

In this study, we developed two MVA based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the soluble secreted trimeric S1 of the spike (MVA/S1). Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization in SARS-CoV-2 infected individuals (Suthar et al., 2020) . The MVA/S also incorporated two mutations that have been shown to maintain the spike protein in a prefusion confirmation (Pallesen et al., 2017; Wrapp et al., 2020) . Using a mouse model, we selected MVA/S vaccine based on its ability to induce neutralizing antibody response. Vaccination of rhesus macaques followed by SARS-CoV-2 challenge demonstrated MVA/S vaccine induces neutralizing antibodies and CD8 T cells, and protects from SARS-CoV-2 infection and replication in the lung and thus serves as a potential vaccine candidate against SARS-CoV-2 infection.

To develop the MVA recombinants we synthesized the full-length spike gene (amino acids 1-1273) with stabilizing mutations (K986P, V987P) or the S1 region with a small portion of S2 region (amino acids 14 to 780). To promote active secretion of the S1, we replaced amino acids 1-14 of the spike sequence with the signal sequence from GM-CSF (Fig. 1A) . Both sequences were optimized for MVA codon usage, corrected for poxvirus transcription termination sequences and cloned into pLW73 vector that will allow us to insert the recombinant sequences under mH5 promoter in the essential region of MVA. The recombinants were selected as described previously (Chea et al., 2019) and characterized for protein expression by flow cytometry and Western blotting. As expected, the MVA/S expressed high amounts (based on mean fluorescence intensity) of spike on the cell surface ( Fig. 1B ) and the expressed protein had a molecular mass of about 180 kDa (Fig. 1C) . Similarly, the MVA/S1 was expressed intracellularly (Fig. 1B) , and a molecular mass of about 114 kDa was also secreted into the supernatants (Fig. 1C) . The spike protein expressed by MVA/S on the surface seemed folded correctly based on strong binding to ACE2 (Fig. 1D) . The S1 protein was found to form trimers based on gel filtration profile and native-PAGE analysis (Fig. 1E) .

We immunized BALB/c mice with MVA/S or MVA/S1 at weeks 0 and 3, and measured binding antibody responses to total and different parts of spike i.e. RBD, S1, and S (S) using ELISA at 2 weeks post prime and boost (Fig. 2) . While both vaccines induced a strong binding antibody response to S, they differentially targeted binding to RBD and S1 ( Fig. 2A, Fig. S1A ). The MVA/S sera showed higher binding to RBD whereas MVA/S1 sera showed higher binding to S1. This was interesting considering that S1 protein includes the RBD region and suggests that antibody binding in sera from MVA/S1-vaccinated mice may be targeting regions outside of the RBD. We further confirmed this differential targeting of antibody using the Luminex assay (Fig.  S1B) . Analysis of immunoglobulin G (IgG) subclass and Fc-gamma receptor (FcγR) binding of RBD-specific antibody showed strong IgG2a response and binding to all three FcγRs tested with strongest binding to FcγR2 and FcγR4 in the MVA/S group (Fig. 2B) . In contrast, poor binding of RBD-specific antibody was observed in general with MVA/S1 sera. However, the S1specific antibody showed similar results in both groups (Fig. S2C ). In addition, we also observed the induction of spike specific IgG responses in the branchoalveolar lavage (BAL) (Fig. 2C) . Similar to serum, the RBD-specific IgG responses were higher in the MVA/S group compared to MVA/S1 group. These results demonstrated differential targeting of spike specific antibody with T helper 1 (Th1) profile induced by MVA/S and MVA/S1 vaccines.

We next evaluated the neutralization capacity of serum collected at 2 weeks post boost from MVA-vaccinated mice using mNeonGreen SARS-CoV-2 virus and defined 50% reduction in focus reduction neutralization titer (FRNT) (Fig. 2D) . We observed a strong neutralizing antibody response in sera from mice vaccinated with MVA/S that ranged from 20-900 with a median of 200 (Fig. 2D, Fig. S1D ). In contrast, we did not observe any detectable neutralization activity in sera from mice immunized with MVA/S1. This was despite the fact that MVA/S1 mice showed higher binding antibody response to S1 and S proteins. The neutralization titer correlated directly with the RBD binding titer and negatively correlated with the S1 binding titer (Fig. 2E) . In particular, they correlated with the RBD specific IgG2a binding titer (Fig. S1E) .

These results demonstrated that MVA/S immunogen can induce a strong neutralizing antibody response against SARS-CoV-2 and could serve as a potential vaccine for SARS-CoV-2. Importantly, they also reveal that MVA/S1 is not a good vaccine as it failed to induce a neutralizing antibody response.

To further understand the failure of MVA/S1 vaccine to induce strong RBD binding antibody and neutralizing antibody, we purified the S1 trimer protein expressed by MVA/S1 vaccine and determined its ability to bind to human ACE2 using biolayer interferometry (BLI) (Fig. S2) . We found that the affinity of S1 to ACE2 decreases by 5-fold when the protein was incubated at 25 o C for 60 min and this was not the case for RBD. These data indicated the unstable nature of the RBD in S1, as the association with ACE2 protein is decreased upon prolonged incubation at room temperature.

To determine T cell responses, we stimulated lymphocytes from spleens and lung of vaccinated mice with overlapping peptide pools specific to RBD, S1 and S2 regions of the spike protein and measured the frequency of IFNγ + CD4 and CD8 T cells using the intracellular cytokine staining (ICS) assay. Both vaccines induced comparable IFNγ + CD8 and CD4 T cell responses that primarily targeted S1 region (including RBD) both in spleen ( Fig. 3A) and lungs (Fig. 3B) . We next studied if vaccination induced formation of inducible bronchus-associated lymphoid tissue (iBALT) that have been shown to provide protection against influenza infection in the absence of peripheral lymphoid organs in mice (Moyron-Quiroz et al., 2004; Woodland and Randall, 2004)  using the immunohistochemistry at 3 weeks after the MVA boost by staining for B and T cells (Fig. 3C, 3D ). As expected, the naïve mice showed very little or no iBALT, however, the MVA/S vaccinated mice showed significant induction of iBALT indicating the generation of local lymphoid tissue. The formation of iBALT was also evident in the MVA/S1 group but to a lesser extent. The relatively lower iBALT response in MVA/S1 group could be due to overall lower spike-specific antibody response induced in MVA/S1 mice compared to MVA/S mice. While we do not know the longevity of persistence of these BALT, they are expected to help with rapid expansion of immunity in the lung following exposure to SARS-CoV-2 (Moyron-Quiroz et al., Wiley et al., 2009) .

To test the immunogenicity and protective ability of the MVA/S vaccine we immunized rhesus macaques (n=5/group) either with MVA/S vaccine or MVA/Wt vaccine delivered intramuscularly on weeks 0 and 4 with a dose of 1x10 8 pfu ( Fig. 4A) . At week 8 (4 weeks after the boost) animals were challenged intranasally and intratracheally with live SARS-CoV-2 virus. Consistent with data in mice, two MVA/S vaccinations induced a strong binding antibody against RBD (geometric mean titer of 2.4x10 4 ) and total S (geometric mean titer of 1.5x10 5 ) that persisted until 4 weeks after the boost (Fig. 4B) . The IgG subclass analysis revealed the majority of the response being IgG1 indicating Th1 dominant response (Fig. 4C) . We also detected low titer of spike-specific IgA (Fig. 4C) . The MVA/S vaccine induced antibody showed strong neutralization of live virus at 2 weeks post boost with a geometric mean titer of 177 (range 90-516) (Fig. 4D) . Similar neutralization titer was also observed in a spike (having D614G mutation) pseudotyped virus neutralization assay (Fig. 4E) . Low titer of neutralizing antibody response was detectable in 2 of the 5 animals post prime (Fig. 4D) . The binding antibody to RBD correlated directly with live virus neutralization (Fig. 4F) . In addition to the neutralizing activity, the vaccine induced sera showed strong antibody-dependent complement deposition (ADCD) activity and low antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil phagocytosis (ADNP) activities (Fig. 4G) .

The MVA/S vaccine also generated a strong spike-specific IFNγ + CD8 T cell response that was evident as early as one week post priming immunization (Fig. 4H) . The frequency of CD8 T cell response was not further boosted following the 2 nd MVA/S immunization. The vaccine-induced CD8 T cells were also positive for TNFα and IL-2 and negative for IL-17 (Fig. 4I) . The MVA/S vaccine induced very low frequencies of IFNγ + CD4 T cells (data not shown). These data demonstrated that MVA/S vaccinations induced a poly-functional CD8 T cell response capable of producing IFNγ, IL-2 and TNFα in macaques.

Following intranasal and intratracheal challenge we monitored for subgenomic viral RNA to distinguish the replicating virus from the inoculum in the lung (BAL), nasopharynx and throat on days 2, 4, 7 and 10 (day of euthanasia) ( Fig. 5) . On Day 2, all 5 controls (MVA/Wt vaccinated) tested positive for virus in BAL and throat, and 3 out of 5 tested positive in nasopharynx indicating that all animals were productively infected (Fig. 5A) . The viral RNA titer (copies/ml) was variable and ranged from 2x10 2 -7x10 6 copies/ml. These titers were maintained in the BAL until Day 4 then declined quickly with time in all animals (except in one animal in BAL) and were below our detection limit by Day 10. In contrast, MVA/S vaccinated animals rapidly controlled SARS-CoV-2 replication in the lung at Day 2 (p<0.05) and Day 4 (p<0.05) compared to controls ( Fig. 5A ) with 4 of the 5 vaccinated animals being negative in BAL. However, in the throat, all vaccinated animals tested negative at Day 2 (p<0.01) but low titer of virus replication was evident in one or two vaccinated animals on Days 4 and 7. Similarly, in nasopharynx one or two animals showed virus replication on Days 2, 4 and 7 and the virus replication was not significantly different between controls and vaccinated animals at all time points. By Day 10 all control and vaccinated animals were negative in all 3 compartments. These results demonstrated that MVA/S vaccination provides protection from SARS-CoV2 infection or replication in the lower respiratory tract.

Consistent with early virus control in the lung, vaccinated animals also showed lower lung pathology compared to control animals (Fig. 5B) . To assess lung pathology, we analyzed multiple regions of upper, middle and lower lung lobes at euthanasia. Lung pathologic analyses and scoring (considering severity and number of affected lobes) were performed by two independent pathologists in a blinded fashion. The total pathology score was lower in the vaccinated group compared to control group. Specifically, MVA/S vaccinated animals showed decreased type 2 pneumocyte hyperplasia, peribronchiolar hyperplasia, alveolar septal thickening and inflammatory cell infiltration (Fig. 5B, S3) . Overall, these data supported a beneficial role of MVA/S vaccination in reducing lung pathology. We also performed histopathologic examination of various other tissue samples including nasal turbinates, trachea, tonsils, hilar lymph nodes, spleen, heart, brain, gastrointestinal tract (stomach, jejunum, ileum, colon, and rectum) and testes. We did not observe significant histological lesions in upper respiratory tract tissues (nasal turbinates, trachea) and other examined tissues in control and vaccinated animals.

To understand the anamnestic expansion of antibody and T cells post infection, we measured binding and neutralizing antibodies in serum on Days 0, 4, 7 and 10; and T cells in blood at Day 10. Some of the control animals developed low titer of binding and virus neutralizing activity by Day 10 that was markedly lower compared to the titers in vaccinated animals (Fig. 5D, 5E) . The neutralizing antibody titer in vaccinated animals was maintained in 2 animals, and increased by 4-fold in 2 animals and by 25-fold in one animal. The control animals also showed IFNγ + CD8 and CD4 T cells in blood that were largely specific to S1 and nucleocapsid (NC) (Fig. 5F) . However, the vaccinated animals mainly showed IFNγ + CD8 T cells against S1 but not NC. The S1-specific response in vaccinated animals post infection was likely due to the persisting vaccine induced CD8 T cells. However, the lack of NC-specific CD8 T cell response suggests that these animals were not exposed to significant amount of the NC protein made by the replicating virus post infection. Thus, post infection immune responses pointed to less systemic spread of virus replication at least in some of the vaccinated animals.

To further understand the influence of infection and vaccine protection on innate and adaptive immunity in the lung early post infection, we monitored various innate cells and B cells in BAL, and GC-Tfh and GC-B cell responses in lung draining hilar LN at Day 10 post infection (Fig. 6,  6A ). Longitudinal analysis of innate cells on Days 4, 7 and 10 post infection in control (MVA/Wt) animals revealed a significant increase in the frequency of macrophages on Day 7 followed by decline on Day 10 ( Fig. 6B) . In contrast to macrophages, the frequency of total HLA-DR + DC and plasmacytoid DC (pDC) at Day 7 were higher in controls compared to MVA/S vaccinated animals and decreased over time until Day 10 ( Fig. 6C, 6D) . The frequency of BDCA-1 + DC and MDC increased by Day 7 and declined by Day 10, and the frequency of NK cells increased gradually until Day 10 ( Fig. 6D) . However, the frequency of all of these cells stayed relatively stable in vaccinated (MVA/S) animals from Days 4-10 except BDCA-1 + DC that showed a small increase on Day 10 compared to Day 4. At Day 10, the control animals showed lower frequency of HLA-DR + DC compared to vaccinated animals that also reflected in the frequency of pDC and BDCA-1 + DC. In summary, the longitudinal analysis of innate cells revealed an expansion and contraction of all subsets studied but with different kinetics in controls but not in vaccinated animals presumably due to high virus replication in controls. These data also suggested there was a potential loss of some of the DCs in controls by Day 10. It is hard to make this conclusion in the absence of data on pre-infection frequencies of innate cells in the BAL. In addition, the B cells in the controls showed lower expression of HLA-DR suggesting an impaired B cell response ( Fig. 6E ) (Titanji et al., 2010) . Furthermore, the frequency of total (Fig. 6F ) and CXCR5 + GC-Tfh (Fig. 6G) , and GC-B cells (Fig. 6H ) in the hilar LNs was higher in the control animals compared to vaccinated animals suggesting higher antigen load and proinflammatory environment in the former. Collectively, these results demonstrated distinct innate and adaptive immune profiles in the lung between controls and MVA/S vaccinated animals that point to lower antigen load and inflammation in the vaccinated animals early post infection.

J o u r n a l P r e -p r o o f SARS-CoV-2 infection of NHPs, in most cases, does not recapitulate the full spectrum of clinical symptoms of severe COVID-19 disease, with animals typically exhibiting a more moderate phenotype (Hartman et al., 2020; Munster et al., 2020) . Recently, however, we and others have observed that SARS-CoV-2 infection of rhesus macaques (Aid et al.; Hoang et al.) and African Green Monkeys (AGM) (Speranza et al.) . elicits many of the immunopathological events in the airway (elevated inflammatory cytokines and chemokines, recruitment of monocytes and neutrophils) that have been reported in the airways of patients exhibiting severe COVID-19 symptoms (Liao et al., 2020) . To assess the impact of our vaccine on these inflammatory sequelae in the airway, we performed single-cell RNA-Seq on the cellular fraction recovered from BAL at Day 4 after infection (Fig. 7) . The vast majority of cells identified in the BAL were CD68 + myeloid cells (82% in controls, 71% in vaccinated), followed by CD8 + T cells (7% in controls, 14% in vaccinated) and minor populations (< 5%) consisting of B cells, DCs, and epithelial cells (Fig. 7A, B In human and NHP studies, SARS-CoV-2 has been observed to induce a strong expression of inflammatory cytokines and chemokines in myeloid origin cells within the BAL and lungs (Hoang et al.; Liao et al.; Speranza et al.) . In the current study, control animals largely recapitulated these findings; we observed high expression of transcripts associated with IL6, TNFα and IL1β β β β production ( Fig.  7D-F) . We also noted widespread expression of CXCL8, CXCL9, CXCL10, CXCL11, CCL8 and CCL19 ( Fig. 7D-F) . In vaccinated animals, expression of IL6, TNFα, IL1b, CXCL8 and CXCL10 was reduced compared to controls (Fig. 7D-F) . Recent studies in AGMs have reported that SARS-COV-2 infection elicits a population shift within the myeloid fraction, in which interstitial macrophages/infiltrating monocytes (identified by lack of the MARCO cell marker) become predominant over MARCO + resident alveolar macrophage (Speranza et al.) . Here, we observed that vaccinated animals trended toward a higher proportion of MARCO + cells relative to unvaccinated controls (70% vs 54%), indicating a reduced recruitment of monocytes/interstitial macrophages (Fig. 7E,F ). In addition, expression of C1QB and C1QC, components of the C1q complement receptor was also higher in the control macrophages ( Fig. 7D,F) . Furthermore, the transcripts associated with activating FcγR1A (required for the effector functions of IgG1 and IgG3) was lower and the inhibitory FcγR2B was higher in the controls (Fig. 7D) . Collectively, these data demonstrate that MVA/S vaccination was able to reduce inflammatory events in the airway including reduced infiltration of activated monocytes and lowered production of inflammatory cytokines/chemokines. Additionally, our observation of attenuated expression of ISGs in the cellular fraction of the BAL of vaccinated animals, provides orthogonal validation of the ability of MVA/S vaccination to reduce airway SARS-CoV-2 virus replication.

There is an urgent global need for the development of a safe and effective COVID-19 vaccine. Both humoral and cell-mediated immune responses in systemic and mucosal compartments are crucial in preventing infection and counteracting virus replication. We focused our efforts on the development of MVA based vaccines for COVID19 based on our nearly 20 years of experience with the development of MVA based vaccines for HIV (Chea and Amara, 2017; Robinson and Amara, 2005 ) that demonstrated MVA is safe in people (Goepfert et al., 2014) including in HIVinfected individuals (Thompson et al., 2016) and induces strong humoral and cellular immunity that is long lasting (Goepfert et al., 2014; Nigam et al., 2007) . Our results showed that two doses of rMVA expressing the membrane anchored prefusion stabilized full-spike protein can induce a strong neutralizing antibody against SARS-CoV-2 and highly infectious D614G variant (Korber et al., 2020) and CD8 T cell response, and provides protection from SARS-CoV-2 replication in the lung (lower respiratory tract). In addition, vaccination blunted the virus replication very early at Day 2 in the throat. Notably, hematoxylin and eosin staining analysis in individual vaccinated (MVA/S) animals showed MVA/S vaccination retained healthy lung features like thin alveolar septal, perivascular cuffing, peribronchiolar hyperplasia and no inflammatory cells (except in one animal, few inflammatory cells were observed) in alveoli compared control (MVA/Wt) vaccinated animals. The effects of vaccination on protection in the nasopharynx was inconclusive owing to the large variation for viral load in the control group and relatively small group sizes. Our results also showed that vaccination can provide protection from inflammation, Th1 biased hyperimmune activation and immune dysfunction in the lung very early post infection.

We found that MVA expressing soluble trimeric S1 protein does not induce neutralizing antibody response despite containing the RBD domain and inducing strong S1 binding antibody. This suggests that the specificity of the antibody response induced by the S1 immunogen may be distinct from S immunogen. This was also suggested by the distinct RBD-and S1-specific humoral immune profiles in MVA/S and MVA/S1 groups where the MVA/S immunization yielded stronger RBD-specific IgG response while the MVA/S1 induced stronger S1-specifc response. This was unexpected given the fact that RBD is part of S1. It would be important to understand the mechanisms that lead to loss of neutralization when MVA/S1 is used as an immunogen. It would also be important to study if the antibody response induced by MVA/S1 vaccine possess any kind of antibody dependent enhancement of infection activity. Our preliminary data in this direction point to the instability of S1 protein to retain binding to ACE2 during prolonged incubation at room temperature. This instability of S1 protein could have contributed to induction of strong binding antibody to other regions in S1 other than RBD following immunization. Further studies are needed to understand the binding specificity of antibodies induced by these two vaccines.

Our study demonstrated that vaccination can protect from infection induced inflammation, Th1 biased hyperimmune activation and humoral immune dysfunction in the lung at single cell level at a time virus is still actively replicating very early (Day 4) after infection. Although a recent COVID19 vaccine study demonstrated that vaccination can reduce inflammation in the lung by measuring selected cytokines, we were able to show these findings at the single cell level in the lung resident macrophages using the cutting-edge technologies. The single cell RNASeq technology allowed us study several hundred genes including ISGs and immune function associated genes, and specific cell subsets that contribute to inflammation and altered expression. SARS-CoV-2 infection, despite being largely acute infection in this model, can cause significant inflammation, Th1 biased hyperimmune activation and immune dysfunction such as the loss of HLA-DR expression on B cells, lower expression of activating (FcγR1A) and higher expression of inhibitory (FcγR2B) FcγRs that can potentially lead to diminished effector functions of IgG1 and IgG3. These results are important since they demonstrate systemic vaccination can provide protection from infection induced immune abnormalities in the lung very early (Day 4) post infection. While our preliminary analyses identified important findings future analyses will dig deeper into identification of individual clusters of cell populations within myeloid cells and see how they differ between control and vaccinated animals post infection.

It will be good to compare the immunogenicity and protective ability of MVA/S vaccine with other vaccines that are based on mRNA, adenoviral (chimp Ad or Ad26) and protein-based vaccines that have either already been approved or currently being tested in human trials (Brouwer et al., J o u r n a l P r e -p r o o f Corbett et al., 2020; Gao et al., 2020; Keech et al., 2020; Krammer, 2020; Laczko et al., 2020; Mercado et al., 2020; Poland et al., 2020; Sahin et al., 2020; Smith et al., 2020; Tostanoski et al., 2020; van Doremalen et al., 2020; Walls et al., 2020; Yu et al., 2020) . However, it is difficult to do so since different studies used different neutralization assays that can provide different results. In addition, some studies reported 50% neutralization titer and others 80-90%. Similarly, for protection studies different stocks and dose of viruses, and species of macaques have been used. Due to these limitations, we refrain from making any comparisons between studies. However, it is clear that some of the protein-based vaccines with potent adjuvants (Brouwer et al., 2020; Walls et al., 2020) have induced much stronger neutralizing antibody responses compared to viral vector-based vaccines including our MVA/S vaccine. In contrast to viral vector-based vaccines, protein-based vaccines induced poor CD8 T cell response. In addition, it is important to compare the durability of these responses between different vaccines. Acknowledging the caveat of different conditions of viral challenge being used, the protection we observed in this study is quite comparable to most of the vaccines that have already been advanced into clinic.

In conclusion, these results demonstrate that MVA/S vaccination can provide protection from both intranasal and intratracheal SARS-CoV-2 challenge and support further development of this vaccine to test for immunogenicity and efficacy in humans. They also suggest that MVA/S vaccines can serve as an excellent boosting agent for mRNA, DNA and chimp adenovirus and Ad26 based COVID-19 vaccines to boost cellular and humoral immunity.

While these results are encouraging, future studies will be necessary to determine the longevity of the vaccine induced immunity and durability of protection using larger group size. This is important as this vaccine is being developed for human use. Of note, it is encouraging to see that MVA/S vaccine induced antibody response contracted only minimally (less than 2-fold) between weeks 2 and 4 post boost, the period at which maximum contraction of antibody response happens post boost (Kannanganat et al., 2016; Nigam et al., 2007) . This combined with our prior studies demonstrating the longevity of MVA vaccine induced immunity in humans (Goepfert et al., 2014) and NHPs (Nigam et al., 2007) It is highly likely that MVA/S based vaccine will provide durable protection. 

We thank Drs. Bernard Moss and Lynda Wyatt for providing the pLW73 transfer plasmid, the Yerkes Division of Pathology and Research Resources for outstanding animal care during the pandemic and Histology and Molecular Pathology Lab for help with tissue sectioning. The following reagent was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Vector pCAGGS Containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Receptor Binding Domain (RBD), NR-52309. Imaging in this research project was supported in part by the Emory University Integrated Cellular Imaging Microscopy Core. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of Health.

This work was supported in part by National Institutes of Health Grants RO1 AI148378-01S1 and Fast Grants Award #2166 to R.R.A., and NCRR/NIH base grant P51 OD011132 to YNPRC. Next generation sequencing services were provided by the Yerkes NHP Genomics Core which is supported in part by NIH P51 OD011132. Sequencing data was acquired on an Illumina NovaSeq6000 funded by NIH S10 OD026799 to S.E.B.

RRA, SG and NR are co-inventors of MVA/S vaccine technology. Emory university filed a patent on this technology. RRA serves as a SAB member for Heat Biologics Inc. Figure 1 . Construction and characterization of MVA/S and MVA/S1 recombinants. (A) Schematic representation of MVA/S and MVA/S1. MVA-SARS-CoV-2-S encodes for SARS-CoV-2 S protein with the two indicated proline mutations for prefusion stabilization. MVA/S1 contains GM-CSF signal sequence (SS) and replaces the first 14 amino acids of S protein that contains the natural SS. Recombinant inserts were cloned in the essential region in between 18R and G1L genes under mH5 promoter.

(B) Representative flow plots showing the surface expression of membrane anchored spike (MVA/S) and intracellular expression of secreted S1 (MVA/S1). (C) Western blotting analysis of expressed proteins in supernatants and lysates of MVA/S and MVA/S1 infected cells. (D) Binding of hACE2 to MVA/S expressing cells. (E) Size-exclusion chromatography (left) and blue native PAGE (right) analysis of S1 protein expressed by MVA/S1. The experiments related to B-E were repeated twice and representative data are shown. (A-E) Six-week-old female BALB/c mice (n=5 per group) were immunized via intramuscular route with MVA/S or MVA/S1 on weeks 0 and 3. The mouse immunization study was repeated twice and representative data are shown.

(A) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured in serum collected at week 2 post-prime and week 2 post-boost immunizations. Each sample was analyzed in duplicates.

(B) SARS-CoV-2 RBD-binding IgG subclass and soluble Fc receptor analysis in serum (week 3 post-boost) performed using Luminex assay. Data come from one experiment.

(C) Lung SARS-CoV-2 RBD, S1 and S -specific IgG responses measured in BAL (bronchoalveolar lavage) fluids collected three weeks after boost (at euthanasia) using ELISA. Each sample was analyzed in duplicates.

(D) Neutralization titer against live mNeonGreen SARS-CoV-2 virus was performed in serum collected at week 2 post-boost immunizations. Each sample was analyzed in duplicates and repeated in two independent times.

(E) Correlations between neutralization titer and ELISA binding titers of RBD and S1 proteins. Bars and columns show arithmetic mean values for each group ± SEM. Mann-Whitney test; * , p < 0.05; * * , p < 0.01; * * * , p < 0.001. Spearman rank test was used to perform the correlation analysis. See also Figure S1 for details. immunized animals analyzed at one-week post boost immunization after re-stimulation with indicated peptide pools. The mouse immunization study was repeated twice (n=5 per group) except for the data presented in Fig. 3C -D, and representative data are shown.

(C) Frozen lung sections from vaccinated mice were stained to analyze formation of iBALT aggregates to visualize B cell and T cell forming follicle like structure (iBALT) induced by MVA/S and MVA/S1 vaccination. The arrows in immunofluorescence images indicate iBALT structures.

(D) Total number of iBALT like structures visualized in each section per mouse was quantified and compared between the groups.

(E) Correlation between the total number of iBALT like structures and ELISA binding antibody to S protein in serum. Bars and columns show arithmetic mean values for each group ± SEM; Mann-Whitney test; * p < 0.05; * * p < 0.01; * * * p < 0.001. Spearman rank test was used to perform the correlation analysis. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured in serum. Each samples was analyzed in duplicates. (C) S protein specific IgG subclass and IgA in serum. Each samples was analyzed in duplicates. (D-E) 50% neutralization titer against mNeonGreen SARS-CoV-2 live virus at week 6 (D) and VRC7480.D614G pseudovirus at weeks 0, 4, 6 and 8 (E). Each samples was analyzed in duplicates and the assay was repeated two independent times. (F) Correlations between live virus neutralization titer and RBD-binding titer. (G) S protein specific antibody effector functions in serum.

(H) IFNγ + CD8 + T cells specific to total S protein in blood. (I) TNFα + , IL-2 + and IL-17 + CD8 + T cells specific to total S protein in blood at week 2 post boost.

Bars and columns show mean responses in each group ± SEM; Mann-Whitney test was used to compare groups: * , p < 0.05; * * , p < 0.01; * * * , p < 0.001. Spearman rank test was used to perform the correlation analysis. Dotted lines reflect limit of detection. Table S1 for details. (D-E) SARS-CoV-2 RBD-specific serum IgG responses (D) and Live SARS-CoV-2 neutralization (E) in serum following challenge. (F) IFNγ + CD8 + and CD4 + T cells in blood at Day 10 post infection after re-stimulation with peptide pool specific to indicated protein. S1, S1 region of spike; S2, S2 region of spike; NC, nucleocapsid; Sum, total response (S1+S2+NC). Bars and columns show mean responses in each group ± SEM; Mann-Whitney test: * , p < 0.05; * * , p < 0.01; * * * , p < 0.001. Dotted lines reflect limit of detection. See also Fig. S3 and Table S1 for details.

Following SARS-CoV-2 infection, BAL was collected on Days 4, 7 and 10 and various innate cells were analyzed. On Day 10 (at euthanasia), B cells were analyzed in BAL, and GC-Tfh and GC-B cells were analysed in hilar LN. N=5/group except for MVA/S group at Day 4 where data for one animal was not available. These analyses were performed only once since the macaque study was done only once. (A) Gating strategy to identify innate cells in BAL fluid. Live cells were selected using live/dead marker and CD3 + and CD20 + cells were excluded. Then, different innate cells were defined using the following combination of markers: Macrophages (HLADR + CD163 + ); PDCs (HLADR + CD163 -CD123 + BDCA1 -); BDCA1 + DC (HLADR + CD163 -CD123 -BDCA1 + ); MDCs (HLADR + CD163 -CD123 -CD11C + ) and NK cells (HLADR -NKG2A + ). LN, lymph node. Statistical differences between the groups and within the groups were analyzed using unpaired parametric t-test and paired-parametric t-test respectively. *, p<0.05, **, p<0.005, ****, p<0.0001. 

Request for further information and for resources and reagents, should be directed to and will be fulfilled by the Lead Contact, Rama Rao Amara (ramara@emory.edu).

All unique/stable reagents generated in this study are available from the Lead Contact up on reasonable request after completion of a Materials Transfer Agreement.

All data supporting the experimental findings of this study are available within the manuscript and are available from the corresponding author upon request. Supplementary figures and table are deposited in Mendeley data and are accessible using DOI: 10.17632/r7jdk7p768.1. Data tables for expression counts for single-cell RNA-Seq for BAL are deposited in NCBI's Gene Expression Omnibus and are accessible through GEO accession GSE165747.

Mice and rhesus macaques were housed at the Yerkes National Primate Research Center and animal experiments were approved by the Emory University Institutional Animal Care and Use Committee (IACUC) using protocols PROTO201700014 and PROTO202000057. All animal experiments were carried out in accordance to USDA regulations and recommendations derived from the Guide for the Care and Use of Laboratory Animals.

HEK (Human Embryonic Kidney)-293T cells, DF-1 (Chicken embryo fibroblasts), and Vero cells were obtained from ATCC. All MVA vaccines were produced in Amara's laboratory at the Emory University. SARS-CoV-2 (icSARS-CoV-2) virus was obtained from BEI resources and grown in Suthar's laboratory at the Emory University. mNeonGreen SARS-CoV-2 (2019-nCoV/USA_WA1/2020) virus was produced by Pei Yong Shi's laboratory at the University of Texas. The infectious clone SARS-CoV-2 (icSARS-CoV-2) was propagated in VeroE6 cells (ATCC) and sequenced (Xie et al., 2020) . The titer of MVA viruses was determined using DF-1 cells and SARS-CoV-2 viruses (icSARS-CoV-2 and 2019-nCoV/USA_WA1/2020) using VeroE6 cells. VeroE6 cells and DF-1 cells were cultured in complete DMEM medium consisting of 1x DMEM (Corning Cellgro), 10% Fetal bovine serum (FBS), 25 mM HEPES Buffer (Corning Cellgro), 2 mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics. Viral stocks were stored at -80ºC until further use.

Specific-pathogen-free (SPF) 6-8-week-old female BALB/c mice (00065 strain) were obtained from Jackson Laboratories (Wilmington, MA, USA) and housed in the animal facility at the Yerkes National Primate Research Center of Emory University, Atlanta, GA. Male Indian rhesus macaques (Macaca mulatta), 3-4.5 years old, were housed in pairs in standard non-human primate cages and provided with both standard primate feed (Purina monkey chow) fresh fruit, and enrichment daily, as well free access to water. Immunizations, blood draws, and other sample collections were performed under anesthesia with ketamine (5-10 mg/kg) or telazol (3-5 mg/kg) performed by trained research and veterinary staff.

The MVA recombinants expressing the full-length spike (amino acids 1-1273) carrying the prefusion-stabilized mutations (MVA/S) or only S1 portion of spike (amino acids 14-780)(MVA-S1) were generated and confirmed by standard methods. SARS-CoV-2 (MN996527.1; Wuhan strain) S ORF was codon optimized for vaccinia virus expression, synthesized using GenScript services, and cloned into pLW-73 (provided by L. Wyatt, National Institutes of Health) between the XmaI and BamHI sites under the control of the vaccinia virus modified H5 (mH5) early late promoter and adjacent to the gene encoding enhanced GFP (green fluorescent protein) (Wyatt et al., 2004) . To promote active secretion of the S1, we replaced amino acids 1-14 of the spike sequence with the signal sequence from GM-CSF (WLQGLLLLGTVACSIS). Plaques were picked for 7 rounds to obtain GFP-negative recombinants and DNA sequenced to confirm lack of any mutations. Viral stocks were purified from lysates of infected DF-1 cells using a 36% sucrose cushion and titrated using DF-1 cells by counting pfu/ml. Absence of the wildtype MVA was confirmed by PCR using recombinant specific primers, flanking the inserts.

DF-1 cells were infected with MVA/S or MVA/S1 at an MOI of 1 and stained around 36hrs postinfection. MVA/S infected cells were harvested and stained with live-dead dye and anti-SARS-CoV-2 spike antibody (GTX135356, GeneTex), followed by donkey anti-rabbit IgG coupled to PE (406421, BioLegend). Cells were then fixed with Cytofix/cytoperm (BD Pharmingen), permeabilized with permwash (BD Pharmingen), and intracellularly stained for MVA protein using mouse monoclonal anti-vaccinia virus E3L Ab (NR-4547 BEI Resources) coupled to PacBlue. MVA/S1 infected cells were stained for live dead marker, fixed, permeabilized and intracellularly stained for S1 protein using SARS-CoV-2 RBD Ab (40592-T62, SinoBiological), followed by staining with donkey anti-Rabbit IgG PE and anti -Vaccinia virus E3L-PacBlue. For detection of human ACE2 binding to surface expressed spike on MVA/S infected DF-1 cells, were incubated with biotinylated human ACE2 protein at 1:500 dilution (10108-H08H-B, Sino Biological) followed by streptavidin-PE (BD Pharmingen). Cells were then stained intracellularly for MVA as described above.

RBD-His and S1 proteins were produced in Amara laboratory by transfecting HEK293 cells using plasmids pCAGGS-RBD-His and pGA8-S1, respectively. The RBD-His plasmid was obtained from BEI resources (Cat# NR-52309). The pGA8-S1 plasmid was generated by cloning human codon-optimized S1 DNA sequence from amino acids 14-780 with GM-CSF signal sequence under the control of CMV promoter with intron A. Transfections were performed according to manufacturer's instructions (Thermo Fisher). Briefly, HEK 293F cells were seeded at a density of 2x10 6 cells/ml in Expi293 expression medium and incubated in an orbital shaking incubator at 37°C and 127 rpm with 8% CO 2 overnight. Next day, 2.5x10 6 cells/ml were transfected using ExpiFectamine TM 293 transfection reagent (ThermoFisher, cat. no. A14524) as per manufacturer's protocol. The cells were grown for 72h at 37°C,127 rpm, 8% CO 2 . The cells were harvested and collected by centrifugation at 2,000g for 10 minutes at 4°C. The supernatant was filtered using a 0.22 µm stericup filter (ThermoFisher, and loaded onto pre-equilibrated affinity column for protein purification. The SARS-CoV-2 RBD-His tag and S1 proteins were purified using Ni-NTA resin (ThermoFisher, cat.no. 88221) and Agarose bound Galanthus Nivalis Lectin (GNL) (Vector Labs, cat. no. AL-1243-5) respectively. Briefly, His-Pur Ni-NTA resin was washed with PBS by centrifugation at 2000g for 10 min. The resin was resuspended with the supernatant and incubated for 2h on a shaker at RT. Polypropylene column was loaded on the supernatant-resin mixture and washed with wash Buffer (25mM Imidazole, 6.7mM NaH 2 PO 4 .H 2 O and 300mM NaCl in PBS) four times, after which the protein was eluted in elution buffer (235mM Imidazole, 6.7mM NaH 2 PO 4 .H 2 O and 300mM NaCl in PBS). S1 protein supernatants were mixed with GNL-resin overnight on rocker at 4 o C. The supernatant-resin mix was loaded on to the column and washed 3 times with PBS and eluted using 1M methyl-α-D mannopyranoside (pH7.4). Eluted proteins were dialysed against PBS using Slide-A-lyzer Dialysis Cassette (ThermoScientific, Cat# 66030) and concentrated using either 10 kDa Amicon Centrifugal Filter Units (for RBD) or 50 kDa Amicon Centrifugal Filter Units (for S1), at 2000g at 4°C. The concentrated protein elutes were run on a Superdex 200 Increase 10/300 GL (GE Healthcare) column on an Akta TM Pure (GE Healthcare) system and collected the peak that is matching to corresponding protein. The quantity of the proteins were estimated by BCA Protein Assay Kit (Pierce) and quality by BN-PAGE (NuPAGE™, 4-12% Bis-Tris Protein Gels, ThermoScientific), SDS PAGE and Western blot.

DF-1 cells were infected either with recombinant MVA/S or MVA/S1, at MOI of 1 for 36 h. Infected cells were lysed in ice-cold RIPA buffer and supernatants were collected. Lysates were kept on ice for 10 min, centrifuged, and resolved by SDS PAGE using precast 4-15% SDS polyacrylamide gels (BioRad). Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat# 1610782 BioRad), and incubated for 1 h at room temperature with mouse monoclonal anti-SARS-CoV-2 spike antibody (Cat# GTX632604, GeneTex) for MVA/S, and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively. The membrane was washed in PBS containing Tween-20 (0.05%) and was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Southern Biotech) diluted 1:20,000. The membranes were washed, and proteins were visualized using the ECL select chemiluminescence substrate (Cat# RPN2235 GEhealthcare).

Animal Vaccination BALB/c mice of 6-8-week-old female were immunized with 10^7 plaque-forming-units (pfu) of MVA-S or MVA-S1 vaccine in the thigh muscle (dose split equally into each thigh) using 25 guage needle on weeks 0 and 3. Following 2-weeks each immunizations, the blood samples were collected by facial vein puncture in BD Microtainer® Tube for analyzing SARS-CoV-2 S (RBD, S1 and S)-specific serum antibody responses. At three weeks after the boost, animals were euthanized using CO 2 , followed by cervical dislocation. Blood, lung tissue and bronchoalveolar lavage (BAL) were collected.

10 Indian-origin adult male rhesus macaques (Macaca mulatta), 4-5 years old, were randomly allocated into two groups; one group (n=5) received MVA-empty vector (MVA-wt) and the second group (n=5) received MVA-expressing prefusion stabilized (with proline mutations) SARS-CoV-2 full-length spike protein (MVA-S). Animals received 1x10 8 pfu in 1 ml vaccines at week 0 and week 4 by the intramuscular (IM) route.

All macaques were challenged with a total of 5x10 4 pfu (2.5x10 4 pfu/ml) of SARS-CoV-2 at week 8. Virus was administered as 1 ml by intratracheal (IT) and 1ml by intranasal (IN) route (0.5 ml in each nostril). All the swab samples (nasal and throat) were collected, stored immediately in the viral transport media, and processed for viral RNA extraction the same day. The animal study was conducted at Yerkes National Primate Research Center, Emory University, and was approved by the Emory IACUC.

SARS-CoV-2 S (RBD, S1 and S) -specific IgG in serum and BAL was quantified by enzymelinked immunosorbent assay (ELISA) as described previously (Chamcha et al., 2015) . Briefly, Nunc high-binding ELISA plates were coated with 2 µg/ml of recombinant SARS-CoV-2 proteins (RBD, S1 and S) proteins in Dulbecco's phosphate-buffered saline (DPBS) and incubated overnight at 4 °C. SARS-CoV-2 RBD and S1 proteins were produced in the lab whereas, S1 and S (S1 + S2 ECD) proteins were purchased. Plates were then blocked with 5% blotting-grade milk powder and 4% whey powder in DPBS with 0.05% Tween 20 for 2h at room temperature (RT). Plates were then incubated with serially diluted serum samples (starting from 100, 3 fold, 8x) and incubated for 2h at RT followed with 6 washes. Total SARS-CoV-2 S (RBD, S1 and S)specific mouse IgG and monkey IgG antibodies were detected using HRP-conjugated antimouse (1:6000) (Southern Biotech; AL, USA) and goat anti-monkey IgG secondary antibody (1:10, 000), respectively for 1 h at RT. The plates were washed and developed using TMB (2-Component Microwell Peroxidase Substrate Kit) and the reaction was stopped using 1N phosphoric acid solution. Plates were read at 450 nm wavelength within 30 min using a plate reader (Molecular Devices; San Jose, CA, USA). ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >2-fold over background values.

Live-virus SARS-CoV-2 neutralization antibodies were assessed using a full-length mNeonGreen SARS-CoV-2 (2019-nCoV/USA_WA1/2020), generated as previously described (Xie et al., 2020) . Vaccinated mice, NHP and post-challenge sera were incubated at 56°C for 30 min and manually diluted in duplicate in serum-free Dulbecco's Modified Eagle Medium (DMEM) and incubated with 750-1000 focus-forming units (FFU) of infectious clone derived SARS-CoV-2-mNG virus (Xie et al., 2020) at 37 o C for 1 hour. The virus/serum mixture was added to VeroE6 cell (C1008, ATCC, #CRL-1586) monolayers, seeded in 96-well blackout plates, and incubated at 37 o C for 1 hour. Post incubation, the inoculum was removed and replaced with pre-warmed complete DMEM containing 0.85% methylcellulose. Plates were incubated at 37 o C for 24 hours. After 24 hours, the methylcellulose overlay was removed, cells were washed three times with phosphate-buffered saline (PBS), and fixed with 2% paraformaldehyde (PFA) in PBS for 30 minutes at room temperature. PFA is then removed and washed twice with PBS. The foci were visualized using an ELISPOT reader (CTL ImmunoSpot S6 Universal Analyzer) under a FITC channel and enumerated using Viridot (Katzelnick et al., 2018) . The neutralization titers were calculated as follows: 1 -ratio of the (mean number of foci in the presence of sera: foci at the highest dilution of respective sera sample). Each specimen is tested in two independent assays performed at different times. The focus-reduction neutralization mNeonGreen live-virus 50% titers (FRNT-mNG 50 ) were interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3. Samples that did not neutralize at the limit of detection at 50% were plotted at 10 and were used for geometric mean calculations.

SARS-CoV-2 neutralization was assessed with Spike-pseudotyped virus in 293T/ACE2 cells as a function of reductions in luciferase (Luc) reporter activity. 293T/ACE2 cells were kindly provided by Drs. Mike Farzan and Huihui Mu at Scripps. Cells were maintained in DMEM containing 10% FBS and 3 µg/ml puromycin. An expression plasmid encoding codon-optimized full-length Spike of the Wuhan-1 strain (VRC7480), was provided by Drs. Barney Graham and Kizzmekia Corbett at the Vaccine Research Center, National Institutes of Health (USA). The D614G amino acid change was introduced into VRC7480 by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies (Catalog # 210518). The mutation was confirmed by full-length Spike gene sequencing. Pseudovirions were produced in HEK 293T/17 cells (ATCC cat. no. CRL-11268) by transfection using Fugene 6 (Promega Cat#E2692) and a combination of Spike plasmid, lentiviral backbone plasmid (pCMV ∆R8.2) and firefly Luc reporter gene plasmid (pHR' CMV Luc) (Naldini et al., 1996) in a 1:17:17 ratio. Transfections were allowed to proceed for 16-20 hours at 37 o C. Medium was removed, monolayers rinsed with growth medium, and 15 ml of fresh growth medium added. Pseudovirus-containing culture medium was collected after an additional 2 days of incubation and was clarified of cells by low-speed centrifugation and 0.45 µm micron filtration and stored in aliquots at -80 o C. TCID 50 assays were performed on thawed aliquots to determine the infectious dose for neutralization assays (RLU 500-1000x background, background usually averages 50-100 RLU).For neutralization, a pre-titrated dose of virus was incubated with 8 serial 3-fold or 5fold dilutions of serum samples or mAbs in duplicate in a total volume of 150 µl for 1 hr at 37 o C in 96-well flat-bottom poly-L-lysine-coated culture plates (Corning Biocoat). Cells were suspended using TrypLE express enzyme solution (Thermo Fisher Scientific) and immediately added to all wells (10,000 cells in 100 µL of growth medium per well). One set of 8 control wells received cells + virus (virus control) and another set of 8 wells received cells only (background control). After 66-72 hrs of incubation, medium was removed by gentle aspiration and 30 µL of Promega 1X lysis buffer was added to all wells. After a 10 minute incubation at room temperature, 100 µl of Bright-Glo luciferase reagent was added to all wells. After 1-2 minutes, 110 µl of the cell lysate was transferred to a black/white plate (Perkin-Elmer). Luminescence was measured using a PerkinElmer Life Sciences, Model Victor2 luminometer. Neutralization titers are the serum dilution (ID50/ID80) or mAb concentration (IC50/IC80) at which relative luminescence units (RLU) were reduced by 50% and 80% compared to virus control wells after subtraction of background RLUs. Maximum percent inhibition (MPI) is the % neutralization at the lowest serum dilution or highest mAb concentration tested. Serum samples were heatinactivated for 30 minutes at 56 o C prior to assay.

For relative quantification of antigen-specific antibody titers, a customized multiplexed approach was applied, as previously described (Brown et al., 2012) . Therefore, magnetic microspheres with a unique fluorescent signature (Luminex) were coupled with SARS-CoV-2 antigens including spike protein (S) (provided by Eric Fischer, Dana Farber), Receptor Binding Domain (RBD), and CoV HKU1 RBD (provided by Aaron Schmidt, Ragon Institute), CoV-2 S1 and S2 (Sino Biologicals) as well as influenza as control (Immune Tech). Coupling was performed using EDC (Thermo Scientific) and Sulfo-NHS (Thermo Scientific) to covalently couple antigens to the beads. 1.2x10 3 beads per region/ antigen were added to a 384-well plate (Greiner), and incubated with diluted plasma samples (1:90 for all readouts) for 16h while shaking at 900rmp at 4°C, to facilitate immune complex formation. The next day, immune complexed microspheres were washed three times in 0.1% BSA and 0.05% Tween-20 using an automated magnetic plate washer (Tecan). Anti-mouse IgG-, IgG2a-, IgG3-, IgA-and IgM-PE coupled (Southern Biotech) detection antibodies were diluted in Luminex assay buffer to 0.65ug/ml. Beads and detection antibodies were incubated for 1h at RT while shaking at 900rpm. Following washing of stained immune complexes, a tertiary goat anti-mouse IgG-PE antibody (Southern Biotech) was added and incubated for 1h at RT on a shaker. To assess Fc-receptor binding, mouse Fcreceptor FcγR2, FcγR3, FcγR4 (Duke Protein Production facility) were biotinylated (Thermo Scientific) and conjugated to Streptavidin-PE for 10 min (Southern Biotech) before adding to immune complexes and processed as described above. Finally, beads were washed and acquired on a flow cytometer, iQue (Intellicyt) with a robot arm (PAA). Events were gated on each bead region, median fluorescence of PE for bead positive events was reported. Samples were run in duplicate for each secondary detection agents.

A Luminex assay was used to detect and quantify antigen-specific subclass, isotype and Fcreceptor (binding) factors (Brown et al., 2017) . With this assay, we measured the antibody concentration against SARS-CoV-2 RBD (kindly provided by Aaron Schmidt, Ragon Institute) and SARS-CoV-2 S (Kindly provided by Erira Ollmann Saphire, La Jolla Institute). Carboxylatemodified microspheres (Luminex) were activated using EDC and Sulfo-NHS and antigens were covalently bound to the beads via NHS-ester linkages. Antigen-coupled beads were washed and blocked. Immune complexes were formed by mixing appropriately diluted plasma (1:100 for IgG1, IgG2, IgG3, IgG4, IgA, IgM, and 1:1000 for FcγRs) to antigen-coupled beads and incubating the complexes overnight at 4˚C. Immune complexes were then washed with 0.1% BSA 0.02% Tween-20. PE-coupled secondary antibodies for each antibody isotype or subclass (Southern Biotech) was used to detect antigen-specific antibody titer. For FcRs, biotinylated FcRs were labeled with streptavidin-PE before addition to immune complexes. Fluorescence was measured with an iQue (Intellicyt) and analyzed using Forecyt software. Data is reported as median fluorescence intensity (MFI).

Antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) and antibody-dependent complement deposition (ADCD) was measured as previously described (Ackerman et al., 2011; Fischinger et al., 2019; Karsten et al., 2019) . For ADCP and ADNP, yellow-green fluorescent neutravidin beads were coupled to biotinylated SARS-CoV-2 S or RBD. For ADCD, red fluorescent neutravidin beads were coupled to biotinylated SARS-CoV-2 S or RBD. Antigen-coupled beads were then incubated with appropriately diluted plasma (ADCP 1:100, ADNP 1:50, ADCD 1:10) for 2 hours at 37˚C to form immune complexes. For ADCP, THP-1s (ATCC) were added at 1.25x10 5 cells/mL and incubated for 16 hours at 37˚C. For ADNP, leukocytes were isolated from fresh peripheral whole blood by lysing erythrocytes using ammonium-chloride potassium lysis. Leukocytes were added to immune complexes at 2.5x10 5 cells/mL and incubated for 1 hour at 37˚C. Neutrophils were detected using anti-human J o u r n a l P r e -p r o o f CD66b Pacblue (Biolegend). For ADCD, lyophilized guinea pig complement (Cedarlane) was resuspended, diluted in gelatin veronal buffer with calcium and magnesium (GVB++, Boston BioProducts) and added to immune complexes. The deposition of C3 was detected using an anti-C3 FITC antibody (Mpbio).

All functional assays were acquired with an iQue (Inellicyt) and analyzed using Forecyt software. For ADCP, event were gated on singlets and fluorescent cells. For ADNP, beadpositive neutrophils were defined as CD66b positive, fluorescent cells. For both ADCP and ADNP, a phagocytic score was defined as (percentage of bead-positive cells) x (MFI of beadpositive cells) divided by 10000. For ADCD, data was reported as median fluorescence of C3 deposition (MFI).

For mouse studies, spleens and lungs of vaccinated and control animals were removed and placed on ice in cold RPMI 1640 (1X) with 5% FBS (Company, state, USA). 1X β-Mercaptoethanol (Invitrogen, State, USA) was added to complete medium to isolate splenocytes. Whereas lungs were cut into small pieces and incubated at 37°C in RPMI (1X) medium containing Collagenase type IV and DNase I with gentle shaking for 30 minutes. After incubation, cells were isolated by forcing tissue suspensions through a 70 µM cell strainer. RBCs were removed by ACK lysis buffer and live cells counted by trypan blue exclusion. For macaques, PBMC from blood collected in sodium citrate CPT tubes were isolated using standard procedures. Post SARS-CoV-2 challenge, samples were processed and stained in BSL-3 facility.

To collect BAL fluids and processing, and single-cells isolation, up to 50 ml physiological saline was delivered through trachea to the lungs of anesthetized animals using a camera enabled fiberoptic bronchoscope. The flushed saline was re-aspirated 5 times before pulling out the bronchoscope. This collection was filtered through 70µm cell strainer and centrifuged at 2200 rpm for 5 minutes. Pelleted cells were suspended in 1ml R10 medium (RPMI(1X), 10% FBS) and stained as described in sections below.

For processing lymph-node, lymph-node biopsies were dissociated using 70µm cell strainer. The cell suspension was washed twice with R-10 media.

Functional responses of SARS-CoV-2 RBD, S1 and S2 specific CD8 + and CD4 + T cells in vaccinated animals were measured using peptide pools and intracellular cytokine staining (ICS) assay. Overlapping peptides (13 or 17 mers overlapping by 10 amino acids) were obtained from BEI resources (NR-52402 for spike and NR-52419 for nucleocapsid) and different pools (S1, S2, RBD and NC) were made. The S1 pool contained peptides mixed from 1-97, S2 pool contained peptides mixed from 98-181, RBD pool contained peptides 46-76 and NC pool contained 57 peptides. Each peptide was used at 1 µg/ml concentration in the stimulation reaction. Two million cells suspended in 200µl of RPMI 1640 medium with 10% FBS were stimulated with 1µg/ml CD28 (BD Biosciences), 1µg/ml CD49d (BD Biosciences) co-stimulatory antibodies and different peptide pools. These stimulated cells were incubated at 37°C in 5% CO2 conditioned incubator. After 2hrs of incubation, 1µl Golgi-plug and 1µl Golgi-stop/ml (both from BD Biosciences) were added and incubated for 4 more hours. After total 6 hours of incubation, cells were transferred to 4 o C overnight and were stained the next day. Cells were washed once with FACS wash (1XPBS, 2% FBS and 0.05% sodium azide) and surface stained with Live/Dead-APC-Cy7, anti-CD3, anti-CD4 and anti-CD8, each conjugated to a different fluorochrome for 30 minutes at RT. The stained cells were washed once with FACS wash and permeabilized with 200µl of cytofix/cytoperm for 30 minutes at 4 o C. Cells were washed once with perm wash and incubated with anti-cytokine antibodies for 30 minutes at 4 o C. Finally, the samples were washed once with perm wash and once with FACS wash, and fixed in 4% paraformaldehyde solution for 20 minutes before acquiring on BD LSR Fortessa flow cytometer. Data was analyzed using FlowJo software.

For visualizing iBALT structures in mouse lungs by Immunohistochemistry, the lung tissues were fixed in 4% PFA for 12h followed by PBS wash. Fixed lungs tissues were kept in 30% sucrose overnight followed by freezing in OCT solution. Frozen blocks were cryosectioned, fixed, and immunostained for iBALT structure. Sections were incubated overnight at 4°C with primary antibodies containing rat anti-mouse B220 (Cat#130-042-401) and hamster anti-mouse CD3 (Cat#550277). Next day, primary antibodies were washed with chilled PBS thrice followed by incubation with secondary antibodies containing anti-rat IgG-Alexa 488 (Cat#ab150157) and anti-hamster IgG-Alexa 546 (Cat#A-21111). Sections were incubated with secondary antibodies at room temperature for 1h followed by wash with chilled PBS thrice. Washed sections were mounted with antifade mounting media with DAPI. Imaging was performed at Olympus FV1000 confocal microscope using 20X objective. Number of iBALT structures were quantified per image section and plotted using GraphPad prism version 8.

For histopathologic examination in macaques, the animals were euthanized due to the study end point, and a complete necropsy was performed. For histopathologic examination, various tissue samples including lung, nasal turbinates, trachea, tonsils, hilar lymph nodes, spleen, heart, brain, gastrointestinal tract (stomach, jejunum, ileum, colon, and rectum), testes were fixed in 10% neutral-buffered formalin for 24h at room temperature, routinely processed, paraffin-embedded, sectioned at 4 µm, and stained with hematoxylin and eosin (H & E). The H & E slides from all tissues were examined by two board certified veterinary pathologists. For each animal, all the lung lobes were used for analysis and affected microscopic fields were scored semiquantitatively as Grade 0 (None); Grade 1 (Mild); Grade 2 (Moderate) and Grade 3 (Severe). Scoring was performed based on these criteria: number of lung lobes affected, type 2 pneumocyte hyperplasia, alveolar septal thickening, fibrosis, perivascular cuffing, peribronchiolar hyperplasia, inflammatory infiltrates, hyaline membrane formation. An average lung lobe score was calculated by combining scores from each criterion. Digital images of H&E stained slides were captured at 100 × and 200 × magnification with an Olympus BX43 microscope equipped with a digital camera (DP27, Olympus) using Cellsens® Standard 2.3 digital imaging software (Olympus).

Briefly, the cells were stained with surface antibody cocktail and incubated at RT for 30 minutes. The stained cells were given a FACS wash and permeabilized with 1ml perm buffer (Invitrogen) for 30 minutes at RT. These cells were given a perm wash (Invitrogen) and stained with an intracellular antibody cocktail for 30 minutes at RT. Finally, the cells were washed once with perm wash and a FACS wash and fixed in 4% paraformaldehyde solution for 20 minutes before acquiring on BD LSR-II flow cytometer. Samples prior to challenge were acquired without 20minute 4% paraformaldehyde fixation.

BAL innate cell surface antibody cocktail: live/dead stain-APC-cy7, anti-CD3-605, anti-CD20-605, anti-NKG2A-APC, anti-HLADR-PERCP, anti-cd11b-PE/Dazzle 594, anti-163-eflour-450, anti-CD123-PEcy7, anti-CD11c-BV655 and anti-BDCA1-BV711. BAL innate cell intracellular antibody: anti-Ki67-BV786. T-cell phenotype surface antibody cocktail: live/dead stain-APC-cy7, anti-CD3-PERCP, anti-CD4-BV655, anti-CD8-BV711, anti-PD1-BV421, anti-CXCR5-PE and anti-CXCR3-BV605. T-cell phenotype intracellular antibody: anti-Ki67-BV786. B-Cell phenotype surface antibody cocktail: live/dead stain-APC-cy7, anti-CD3-AF700 and anti-CD20-BV605, Bcell phenotype intracellular antibody: anti-BCL6-PE-CF594 and anti-Ki67-PEcy7.

SARS-CoV-2 genomic and subgenomic RNA was quantified in naso-pharyngeal (NP) swabs, throat swabs, and brocho-alveolar lavages (BAL). Swabs were placed in 1mL of Viral Transport Medium (VTM; Labscoop (VR2019-1L)). Viral RNA was extracted from NP swabs, throat swabs, and BAL on fresh specimens using the QiaAmp Viral RNA mini kit according to the manufacturer's protocol. Quantitative PCR (qPCR) was performed on genomic viral RNA using the N2 primer and probe set designed by the CDC for their diagnostic algorithm: CoV2-N2-F: 5'-TTACAAACATTGGCCGCAAA-3', CoV2-N2-R: 5'-GCGCGACATTCCGAAGAA-3', and CoV2-N2-Pr: 5'-FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ-3' (Waggoner et al., 2020) . The primer and probe sequences for the subgenomic mRNA transcript of the E gene (Wolfel et al., 2020) are SGMRNA-E-F: 5'-CGATCTCTTGTAGATCTGTTCTC-3', SGMRNA-E-R: 5'-ATATTGCAGCAGTACGCACACA-3', and SGMRNA-E-Pr: 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-3'. qPCR reactions were performed in duplicate with the Thermo-Fisher 1-Step Fast virus mastermix using the manufacturer's cycling conditions, 200nM of each primer, and 125nM of the probe. The limit of detection in this assay was about 128 copies per mL of VTM/BAL depending on the volume of extracted RNA available for each assay. To verify sample quality the CDC RNase P p30 subunit qPCR was modified to account for rhesus macaque specific polymorphisms. The primer and probe sequences are RM-RPP30-F 5'-AGACTTGGACGTGCGAGCG-3', RM-RPP30-R 5'-GAGCCGCTGTCTCCACAAGT-3', and RPP30-Pr 5'-FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3' (Waggoner et al., 2020) . A single well from each extraction was run as described above to verify RNA integrity and sample quality via detectable and consistent cycle threshold values (Ct between 25-32).

BAL samples from 5 monkeys in each group were pooled together and two technical replicate 10X captures were performed. One replicate capture failed for the control group. The libraries were run on Nova Seq 6000 lanes and the resultant bcl files were converted to count matrices using Cell Ranger v3.1 (https://support.10xgenomics.com/single-cell-geneexpression/software/downloads/latest?). Count matrices for each capture were processed using an in-house single-cell RNA-seq pipeline that uses Seurat v3.0 (Butler et al., 2018; Stuart et al., 2019) . CITE-seq-Count (https://github.com/Hoohm/CITE-seq-Count) was used along with HTODemux function in Seurat to demultiplex samples. The cells expressing nFeature _RNA < 300 and >10% mitochondrial genes, HBB, RPS or RPL genes were filtered along with doublets. One of the samples within the Vaccine group (Rcc18) was dropped due to 10-fold higher number of cells compared to other samples. Post filtration, cells from each capture were normalized using SCTransform normalization and then integrated in Seurat. After integration, Principal Component analysis was carried out. PCs 1-30 were chosen for clustering analysis, as there was very little additional variance beyond PC 30. Cell were clustered based on PC scores using the Louvain method. UMAP method (McInnes et al., 2018) was used to visualize the single cells in 2d embedding. We used Human primary cell atlas from SingleR (Aran et al., 2019) and knowledge of canonical markers to classify cells into different cell subtypes ( Figure  S4 -Canonical and Figure S5 -Top10Clusters). Differential gene expression between Control and Vaccine group was assessed by MAST (Andrew McDavid and Yajima, 2017) . Heatmaps, Dot plots, Violin plots and Feature plots were generated using seurat package in R. Additionally, we performed Gene set enrichment analysis using WebGestalt (Liao et al., 2019; Zhang et al., 2005) . 

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