COVID-19 vaccine mRNA-1273 elicits a protective immune profile in mice that is not associated with vaccine-enhanced disease upon SARS-CoV-2 challenge
Since its discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the causative agent of coronavirus disease 2019 [COVID-19]) has resulted in more than 177 million infections and more than 3.8 million deaths worldwide as of June 17, 2021. Based on a strategy of using proline substitutions to stabilize viral glycoproteins ( ), stabilization of the prefusion conformation of the neutralization-sensitive viral spike (S) glycoprotein into a form known as S-2P ( ) was key to the expedited development of many vaccines. Design and production of mRNA-lipid nanoparticle (mRNA-LNP) encoding transmembrane-anchored SARS-CoV-2 S-2P, mRNA-1273, was initiated immediately upon release of the first sequences of SARS-CoV-2. The mRNA-1273 vaccine protects from SARS-CoV-2 infection and disease in preclinical mouse ( ) and non-human primate (NHP) ( ) models, elicited neutralizing antibody and T cell responses in phase 1 clinical trials in adult and older adult populations ( ; ), and was authorized for emergency use before the end of 2020. Despite the unprecedented speed of SARS-CoV-2 vaccine development and testing, specific safety questions required close attention. A primary safety concern is the induction of vaccine-associated enhanced respiratory disease (VAERD). VAERD is a modified or more severe presentation of disease, predominantly involving the lower respiratory tract, that results from infection by a pathogen after being vaccinated for the same pathogen (for more detail, see ). Whole-inactivated viral vaccines developed in the 1960s against both respiratory syncytial virus (RSV) and measles elicited VAERD when vaccinated children were subsequently naturally infected ( ; ; ; ; ). These adverse outcomes were associated with T helper (Th)2-skewed CD4 + T cells and the induction of poor-quality antibodies with little to no neutralizing activity. Animal models, where this immune profile is elicited after vaccination, recapitulate RSV and measles VAERD with hallmarks of increased inflammation and pulmonary eosinophilia after challenge that exceeds that in unvaccinated control animals ( ). Although there has been no licensed prophylactic intervention for coronaviruses, there have been multiple findings of VAERD in preclinical studies of both Middle East respiratory syndrome (MERS)-CoV and SARS-CoV vaccines in several species, using different vaccine platforms and antigenic targets (e.g., S and nucleocapsid [N]) formulated with and without alum adjuvant ( ; ; ; ; ; ; ). Although it is unclear whether animal models can reliably predict human VAERD, it is prudent to compare immune responses elicited by candidate vaccines to the detrimental immune responses known to result in VAERD in animal models, particularly at low doses that mimic waning immunity. We assessed the immunological and safety signature of mRNA-1273 in challenge studies using the mouse-adapted SARS-CoV-2, passage 10, lethal challenge virus (MA10) ( ; ). BALB/c mice were immunized twice with whole-inactivated SARS-CoV-1 or SARS-CoV-2 virus, heat-denatured spike protein (S-2P), or mRNA-1273. Whole-inactivated virus and denatured S protein were formulated with alum to recapitulate conditions that resulted in VAERD in a prior preclinical coronavirus vaccine study ( ). These regimens consistently induced low to moderate concentrations of S-binding and neutralizing antibody and Th2-skewed S-reactive CD4 + T cells. After viral challenge, these mice were partially protected from weight loss and viral replication yet displayed enhanced pulmonary inflammation and eosinophil infiltration. In contrast, mRNA-1273 elicited potently neutralizing antibodies and a balanced or type-1-skewed response, particularly at the 1 μg dose, and mice were protected from viral replication and lung inflammation after viral challenge. Importantly, a subprotective mRNA dose of 0.1 μg was associated with reduced immunopathology after challenge compared to the control and Th2-skewing groups. These results demonstrate that mRNA-1273 elicits potent antiviral immunity and a favorable immune profile not associated with VAERD, even at subprotective doses. 

mRNA-1273 immunization elicits S-directed antibodies with high neutralizing potency compared to whole-inactivated virus and denatured S protein delivered with alum To assess the capacity of research-grade mRNA-1273 to elicit potentially adverse immune responses that could be associated with VAERD, we compared antibody and T cell responses generated from subprotective and protective doses of mRNA-1273 (0.1 and 1 μg, respectively) to those elicited by regimens previously associated with disease enhancement following infection. Due to the initial unavailability of whole-inactivated SARS-CoV-2, we elected to immunize mice with whole-inactivated SARS-CoV-1 virus or SARS-CoV-2 S protein that had been heat denatured to destroy the protein conformation. Both immunogens were delivered at doses of 0.2 or 1 μg, formulated with alum adjuvant ( A) with the goal of inducing binding antibodies with little to no neutralizing activity (for disposition of animals in the study, see ). Importantly, immunization with 0.2 μg of whole-inactivated SARS-CoV-1 in alum had previously resulted in enhanced disease following infection with mouse-adapted SARS-CoV-1 ( ). BALB/c mice were immunized twice 3 weeks apart with each vaccine or phosphate-buffered saline (PBS) as a control, and humoral and cellular responses to S were assessed 2 weeks after the boost. We first quantified binding immunoglobulin G (IgG) to S-2P protein. Binding antibody was observed in all groups except the control group, with more than a three-log range between groups ( B). Cross-reactive antibody responses elicited by whole SARS-CoV-1 inactivated with both UV irradiation and formalin (double-inactivated SARS-CoV-1 or DI CoV-1) were lower than responses elicited by the homologous, denatured SARS-CoV-2 S protein (CoV-2 DS). The 0.1 μg mRNA-1273 dose elicited lower binding antibodies than both the 0.2 and 1 μg doses of CoV-2 DS, on par with binding antibodies induced by 1 μg of heterologous DI CoV-1. The 1 μg mRNA-1273 dose elicited higher antibody responses than either dose of CoV-2 DS, consistent with potent elicitation of antibodies previously described ( ). Neutralizing activity against a SARS-CoV-2 S 614G-pseudotyped virus was measured to assess neutralization of the predominant circulating virus. Mice immunized with 1 μg mRNA-1273 had a half-maximal inhibitory concentration (IC 50 ) geometric mean titer (GMT) of 4,886. Neutralization was low or undetectable in other immunization groups, but notably, 6 of the 20 animals given 0.1 μg of mRNA-1273 had detectable neutralizing activity compared to only 3/20 and 2/20 animals immunized with 1 μg of DI CoV-1 or CoV-2 DS, respectively ( C). All but 1 of the 40 animals immunized at both doses of CoV-2 DS had binding antibody endpoint titers above 4 log 10 , and only 2 animals in those combined groups had detectable neutralizing activity. In contrast, only half (10/20) of the mice immunized with 0.1 μg mRNA-1273 had binding antibody endpoint titers above 4 log 10 , of which 6 had measurable neutralizing activity, a disparity in potency that is apparent when correlating binding to neutralizing activity ( D). Based on these results, mRNA-1273 elicited antibodies with higher average neutralizing potency than approaches aimed at Th2 skewing the S-directed response. mRNA-1273 immunization elicits S-reactive antibody with a favorable IgG2a/IgG1 ratio and a distinct CD4 + T cell profile compared to Th2-skewing regimens To gain insight into the induction of type 1 and type 2 immunity, we further assessed the IgG subclass of S-directed antibodies. In mice, although the type 1 cytokine interferon γ (IFNγ) promotes B cell secretion of IgG2a and inhibits IgG1, the type 2 cytokine interleukin-4 (IL-4) promotes IgG1 and inhibits the secretion of IgG2a antibodies ( ). As previously shown ( ), mRNA-1273 elicited high titers of both S-binding IgG2a and IgG1 ( A and 2B, respectively). mRNA-1273 given at the low dose of 0.1 μg elicited more S-binding IgG2a than seen in any group immunized with DI CoV-1 or CoV-2 DS ( A). In contrast, type-2-skewing regimens elicited IgG1 responses more similar to those elicited by 0.1 μg mRNA-1273 ( B), resulting in lower Ig2a/IgG1 ratios in mice that received either DI CoV-1 or CoV-2 DS compared to those that received 1 μg mRNA-1273 ( C). Taken together, serological assessment of S-directed antibodies confirmed the generation of a type 1 antiviral immune profile elicited by mRNA-1273 compared to vaccine approaches that elicit type-2-biased responses. We next assessed the CD4 + T cell cytokine profile of S-reactive cells in the spleen 2 weeks after the boost for all vaccine groups. We used intracellular cytokine staining (ICS) to measure the frequency of CD4 + T cells expressing three type 1 cytokines (IFNγ, tumor necrosis factor alpha [TNFα], and IL-2) and three type 2 cytokines (IL-4, IL-5, and IL-13) after in vitro restimulation with overlapping peptide pools spanning the S1 and S2 portions of the S protein ( D and 2E, respectively; gating strategy presented in ) or the SARS-CoV-2 N protein ( F). Although CD4 + Th cells were detectable in all immunization groups, the T cells of mice in the DI CoV-1 and CoV-2 DS groups exhibited a pattern of expression that included all three type 2 cytokines. As expected, only mice immunized with DI CoV-1 had responses to the N peptide pool ( F). Although IL-2 and TNFα expression exceeded background in some cases, most CD4 + T cells in the type-2-skewing immunization groups expressed one or more type 2 cytokines (cytokine co-expression profile following peptide pool restimulation shown in A). In contrast, expression of type 2 cytokines was more limited in animals immunized with mRNA-1273. IFNγ expression was only found in mice that received 1 μg of mRNA-1273, and this was the only immunization group with strong induction of S-specific CD8 + T cell responses ( B). mRNA-1273 immunization limits viral replication, morbidity, and pulmonary inflammation following mouse-adapted SARS-CoV-2 viral challenge Twenty mice from each group were challenged with 10 4 plaque-forming units (PFUs) of mouse-adapted SARS-CoV-2 MA10 (MA10) 5 weeks after the boost immunization ( A; ). The MA10 virus is capable of lethal disease in standard immunocompetent mice and recapitulates many aspects of COVID in humans ( ). Weight loss was assessed in 10 mice per group until day 7 post-infection. Control animals had the greatest weight loss, which peaked at day 4 post-infection with an average peak of 14% loss of body weight. Modest but not significant weight loss occurred through day 3 in mice immunized with DI CoV-1, but they recovered more rapidly than control mice. There was no appreciable weight loss in groups immunized with either dose of CoV-2 DS or mRNA-1273 ( A). Viral titers were measured in the nasal turbinates by plaque assay on day 2 ( B) and day 4 ( C) post-infection to assess protection in the upper airway. Low viral titers were detected in 2/5 mice in the 1 μg mRNA-1273 dose group 2 days after infection, and none had detectable virus in the nasal turbinates at day 4, as previously shown following vaccination with 1 μg of mRNA-1273 ( ). In addition to protection in the nasal turbinates, 1 μg mRNA-1273 immunization completely prevented viral infection in the lungs at both day 2 ( D) and day 4 ( E) after challenge. Mice immunized with 0.1 μg mRNA-1273 or 1 μg CoV-2 DS also had reduced viral titer in the lungs at both time points post-challenge. In all, every vaccine tested offered some protection against either weight loss or virus titer post-challenge. We examined lungs histologically on day 4 after challenge (for details and examples of scoring, see ; for scoring for each animal, see ). Assessment of inflammation in sections stained with hematoxylin and eosin (H&E) showed an inflammation and severity score in both groups of DI-CoV-immunized and the 0.2 μg CoV-2-DS-immunized mice that met or exceeded inflammation seen in control mice ( ). Mononuclear and polymorphonuclear inflammation was moderate to severe in the PBS group, severe in the 0.2 μg DI CoV-1 group, moderately severe in the 1 μg DI CoV-1 and 0.2 μg CoV-2 DS groups, mildly to moderately severe in the 1 μg CoV-2 DS and 0.1 μg mRNA-1273 groups, and mild to minimally severe in the 1 μg mRNA group (rank by severity was 0.2 μg DI CoV-1 > 1 μg DI CoV-1 = 0.2 μg CoV-2 DS > PBS > 1 μg CoV-2 DS = 0.1 μg mRNA-1273 > 1 μg mRNA-1273). Type II pneumocyte hyperplasia was evident in all groups but mild in groups immunized with mRNA-1273. Immunohistochemistry for SARS-CoV-2 antigen revealed moderately abundant and diffusely distributed SARS-CoV-2 viral antigen throughout the alveolar interstitium in the PBS group, but sparse or rare infected cells were observed in most other groups, with minimal to absent SARS-CoV-2-antigen-positive cells in 1 μg mRNA-1273-immunized mice ( ). These data are consistent with viral titer measured on day 4 ( E). Groups immunized with Th2-skewing regimens had abundant eosinophils, often in close association with and circumscribing airways and blood vessels. In contrast, eosinophils were sparsely abundant in control and mRNA-immunized mice ( ). Thus, despite limited virus-induced weight loss and partial reduction of viral replication in lungs, Th2-skewing regimes exhibited histological hallmarks of VAERD. We evaluated lung cytokines to confirm the presence of a Th2 signature and increased association with pulmonary inflammation and eosinophilia elicited by the whole inactivated and denatured spike regimens and to assess protection from inflammation in mRNA-1273-immunized mice. Cytokines were measured in clarified lung supernatants obtained on days 2, 4, and 7 post-challenge using a Bioplex 23 multiplex assay. We first measured cytokines produced by Th2 cells: IL-4; IL-5; and IL-13 ( A). Animals immunized with mRNA-1273 had lower concentrations of Th2-associated cytokines than animals in the PBS-immunized group. In contrast, animals immunized with whole-inactivated virus or denatured spike protein immunogens delivered in alum had elevated concentrations of IL-4 and IL-5 compared to the PBS group. We then evaluated the proinflammatory cytokines IL-1α, IL-1β, IL-6, and TNFα ( B). Although there was no statistical increase in proinflammatory cytokines in Th2-skewed immunization groups, with only five samples available per harvest day, there was a trend toward higher proinflammatory cytokine concentrations, particularly IL-1β and TNFα, in these groups, although concentrations of most were reduced compared to the PBS group and in animals immunized with mRNA-1273, particularly at the 1 μg dose. Finally, we assessed chemokines involved in the recruitment and activation of polymorphonuclear cells (acrophage inflammatory protein [MIP]-1α, MIP-1β, eotaxin, and CXCL1(KC); C). There was a trend toward higher concentrations of MIP-1α and MIP-1β early in Th2-skewed groups, with lower expression in mRNA-1273-immunized mice than in the control group. mRNA-1273-immunized mice similarly did not have increased concentrations of CXCL1 or eotaxin, which were higher in most groups immunized with Th2-skewing regimens, consistent with eosinophil major basic protein (EMBP) staining ( ) and with what was observed in earlier studies of DI-CoV1-enhanced disease following mouse-adapted SARS-CoV infection ( ). Overall cytokine concentrations in the lungs were consistent with the histopathological results and indicate an increase in inflammatory signatures and excess mononuclear and polymorphonuclear cell infiltration into the lungs of Th2-skewed immunization groups. Importantly, a distinct profile was observed in the 0.1 μg mRNA-1273 group despite a similar viral load. Although this dose was not sufficient to protect from viral replication, it resulted in histopathological protection and dampened inflammatory responses in the lung. Taken together, the elicitation of both protective and subprotective mRNA-1273-induced immunity resulted in reduced lung pathology without histologic evidence of VAERD. mRNA-1273 leads to protection following MA10 challenge that contrasts with enhancement observed following challenge of mice immunized with formalin-inactivated SARS-CoV-2 in alum Following the results of the first study, we performed a second study in BALB/c mice to incorporate and compare immune responses to the newly available formalin-inactivated SARS-CoV-2 immunogen (FI CoV-2; made available by the Division of Microbiology and Infectious Diseases [DMID], NIAID). The virus was inactivated in the media containing FBS, which challenged efforts to precisely quantitate viral antigen. Therefore, we selected 0.2 and 2 μg total, spanning a 10-fold dose range, and again compared responses to a control PBS group and groups immunized with 0.1 or 1 μg of mRNA-1273 ( A; see for disposition of study 2 animals). Both doses of FI CoV-2 elicited low concentrations of binding and neutralizing antibodies compared to the mRNA-1273 groups ( B and 6C, respectively). Again, although mRNA-1273 elicited both IgG2a and IgG1 subclasses of S-specific IgG, the response to 2 μg FI CoV-2 was dominated by IgG1, suggesting a Th2-biased immune response pattern ( D and 6E). Due to the low immunogenicity of the selected doses of FI CoV-2, we were unable to detect antigen-specific cytokine production by CD4 + T cells following stimulation with homologous peptide pools (S1, S2, and N) in both dose groups using ICS ( ). Responses in mRNA-1273-immunized groups largely reflected the results of the first experiment, with a dose-dependent increase in type 1 profile and CD8 + T cell induction in only the 1 μg mRNA-1273 group ( ). We elected to challenge mice for the second study with an increased dose (10-fold) of 10 5 PFUs of MA10. There was less weight loss than seen in the first study, with no group showing sustained differences from the PBS control group ( A). Consistent with the first study, mice in the 1 μg mRNA-1273 dose group demonstrated near-complete inhibition of viral replication in the nasal turbinates ( B and S5C) and no detectable virus in the lungs ( D and S5E). Mice immunized with 0.1 μg of mRNA-1273 or 2 μg of FI CoV-2 had partial protection from viral replication in the upper and lower airway at some time points ( B–S5E). On day 4, lungs from all groups had a similar degree of inflammation with mononuclear and polymorphonuclear infiltration and diffuse type II pneumocyte hyperplasia, causing a reduction in alveolar air spaces across all study groups ( F). We believe this to be due to the high viral challenge dose used in this study. This was coincident with an overall higher presence of viral antigen, which was particularly abundant in the alveolar interstitium of control animals. The highest degree of EMBP staining, and abundant eosinophils adjacent to and surrounding blood vessels, was seen in the 0.2 μg FI CoV-2 with alum group (for individual animal scoring from study 2, see ). Animals immunized with FI CoV-2 in alum experienced higher concentrations of Th2 cytokines and proinflammatory and chemotactic mediators ( ). Although the high viral inoculum appeared to blunt some differences between groups, combined data from our second challenge study confirmed the finding of higher concentrations of inflammatory cytokines in mice given whole-inactivated virus in alum and reaffirmed a distinct immune signature in mRNA-1273 groups that was associated with antiviral protection. mRNA-1273 vaccination elicits S-specific CD4 + Tfh and B cell responses We further evaluated the induction of T and B cell responses in our second study. We were particularly interested in CD4 + T follicular helper (Tfh) cells, which are critical to the generation of germinal centers (GCs) and support the clonal selection and differentiation of antigen-specific memory B cells and long-lived plasma cells ( ). We developed a flow cytometry panel to quantify total Tfh (CXCR5 + PD-1 + ) and GC B cell (GL7 + Fas + ) responses, as well as B cells capable of binding the prefusion-stabilized S protein and/or the neutralization-sensitive receptor binding domain (RBD) using tetramerized fluorochrome-conjugated capture probes ( A; for gating and representative plots, see A). 2 weeks after the boost, we found elevated total Tfh and GC B cell frequencies in the splenocytes from only the 1 μg mRNA-1273 dose group ( B and 7C, respectively). Mice that received 1 μg of mRNA-1273 also demonstrated significant induction of probe-binding, class-switched GC B cells, plasma cells (PCs) (identified as CD138 + B220 lo ), and other B cells (GL7 − Fas − ; D). Other vaccine groups had little to no detectable elicitation of S-reactive B cells. Among B cells that bound the stabilized S protein probe, approximately 20%–30% demonstrated specificity for the RBD based on co-capture of the RBD probe. This proportion is consistent with the proportion of S-directed B cells that bind RBD in SARS-CoV-2-experienced humans ( ; ). To more fully capture the repertoire of antigen-specific CD4 + T cells elicited by vaccination, we employed a more inclusive, cytokine agnostic approach called activation-induced marker (AIM) assay, which can identify activated cells even if they do not express sufficient cytokine to be measured by traditional ICS methods ( E; ). Antigen-experienced (CD44 + ) T cells were assessed for the upregulation of activation markers CD69 and CD154 (CD40L) following stimulation with S1 and S2 peptide pools in a 6-h culture in vitro . CD4 + T cells from groups immunized with 0.1 μg or 1 μg of mRNA-1273 responded in a dose-dependent manner to S-derived peptides in higher frequencies than we measured using conventional ICS ( F; for representative staining, see B). However, using this more sensitive method, we were still unable to detect T cell responses above background in groups immunized with FI CoV-2 at either dose. Further evaluation demonstrated strong induction of AIM + CD4 + Tfh cells responding to spike peptides following immunization with 1 μg of mRNA-1273 ( F), with limited reactivity detected from all other groups. Both AIM + Tfh and S-2P + GC B cells correlated well with neutralizing activity against SARS-CoV-2, suggesting a well-coordinated cellular and serological response ( G). These data further support the activation of T cell responses that promote the generation of durable immune memory following mRNA-1273 immunization. 

To assess the capacity of research-grade mRNA-1273 to elicit potentially adverse immune responses that could be associated with VAERD, we compared antibody and T cell responses generated from subprotective and protective doses of mRNA-1273 (0.1 and 1 μg, respectively) to those elicited by regimens previously associated with disease enhancement following infection. Due to the initial unavailability of whole-inactivated SARS-CoV-2, we elected to immunize mice with whole-inactivated SARS-CoV-1 virus or SARS-CoV-2 S protein that had been heat denatured to destroy the protein conformation. Both immunogens were delivered at doses of 0.2 or 1 μg, formulated with alum adjuvant ( A) with the goal of inducing binding antibodies with little to no neutralizing activity (for disposition of animals in the study, see ). Importantly, immunization with 0.2 μg of whole-inactivated SARS-CoV-1 in alum had previously resulted in enhanced disease following infection with mouse-adapted SARS-CoV-1 ( ). BALB/c mice were immunized twice 3 weeks apart with each vaccine or phosphate-buffered saline (PBS) as a control, and humoral and cellular responses to S were assessed 2 weeks after the boost. We first quantified binding immunoglobulin G (IgG) to S-2P protein. Binding antibody was observed in all groups except the control group, with more than a three-log range between groups ( B). Cross-reactive antibody responses elicited by whole SARS-CoV-1 inactivated with both UV irradiation and formalin (double-inactivated SARS-CoV-1 or DI CoV-1) were lower than responses elicited by the homologous, denatured SARS-CoV-2 S protein (CoV-2 DS). The 0.1 μg mRNA-1273 dose elicited lower binding antibodies than both the 0.2 and 1 μg doses of CoV-2 DS, on par with binding antibodies induced by 1 μg of heterologous DI CoV-1. The 1 μg mRNA-1273 dose elicited higher antibody responses than either dose of CoV-2 DS, consistent with potent elicitation of antibodies previously described ( ). Neutralizing activity against a SARS-CoV-2 S 614G-pseudotyped virus was measured to assess neutralization of the predominant circulating virus. Mice immunized with 1 μg mRNA-1273 had a half-maximal inhibitory concentration (IC 50 ) geometric mean titer (GMT) of 4,886. Neutralization was low or undetectable in other immunization groups, but notably, 6 of the 20 animals given 0.1 μg of mRNA-1273 had detectable neutralizing activity compared to only 3/20 and 2/20 animals immunized with 1 μg of DI CoV-1 or CoV-2 DS, respectively ( C). All but 1 of the 40 animals immunized at both doses of CoV-2 DS had binding antibody endpoint titers above 4 log 10 , and only 2 animals in those combined groups had detectable neutralizing activity. In contrast, only half (10/20) of the mice immunized with 0.1 μg mRNA-1273 had binding antibody endpoint titers above 4 log 10 , of which 6 had measurable neutralizing activity, a disparity in potency that is apparent when correlating binding to neutralizing activity ( D). Based on these results, mRNA-1273 elicited antibodies with higher average neutralizing potency than approaches aimed at Th2 skewing the S-directed response. 

+ T cell profile compared to Th2-skewing regimens To gain insight into the induction of type 1 and type 2 immunity, we further assessed the IgG subclass of S-directed antibodies. In mice, although the type 1 cytokine interferon γ (IFNγ) promotes B cell secretion of IgG2a and inhibits IgG1, the type 2 cytokine interleukin-4 (IL-4) promotes IgG1 and inhibits the secretion of IgG2a antibodies ( ). As previously shown ( ), mRNA-1273 elicited high titers of both S-binding IgG2a and IgG1 ( A and 2B, respectively). mRNA-1273 given at the low dose of 0.1 μg elicited more S-binding IgG2a than seen in any group immunized with DI CoV-1 or CoV-2 DS ( A). In contrast, type-2-skewing regimens elicited IgG1 responses more similar to those elicited by 0.1 μg mRNA-1273 ( B), resulting in lower Ig2a/IgG1 ratios in mice that received either DI CoV-1 or CoV-2 DS compared to those that received 1 μg mRNA-1273 ( C). Taken together, serological assessment of S-directed antibodies confirmed the generation of a type 1 antiviral immune profile elicited by mRNA-1273 compared to vaccine approaches that elicit type-2-biased responses. We next assessed the CD4 + T cell cytokine profile of S-reactive cells in the spleen 2 weeks after the boost for all vaccine groups. We used intracellular cytokine staining (ICS) to measure the frequency of CD4 + T cells expressing three type 1 cytokines (IFNγ, tumor necrosis factor alpha [TNFα], and IL-2) and three type 2 cytokines (IL-4, IL-5, and IL-13) after in vitro restimulation with overlapping peptide pools spanning the S1 and S2 portions of the S protein ( D and 2E, respectively; gating strategy presented in ) or the SARS-CoV-2 N protein ( F). Although CD4 + Th cells were detectable in all immunization groups, the T cells of mice in the DI CoV-1 and CoV-2 DS groups exhibited a pattern of expression that included all three type 2 cytokines. As expected, only mice immunized with DI CoV-1 had responses to the N peptide pool ( F). Although IL-2 and TNFα expression exceeded background in some cases, most CD4 + T cells in the type-2-skewing immunization groups expressed one or more type 2 cytokines (cytokine co-expression profile following peptide pool restimulation shown in A). In contrast, expression of type 2 cytokines was more limited in animals immunized with mRNA-1273. IFNγ expression was only found in mice that received 1 μg of mRNA-1273, and this was the only immunization group with strong induction of S-specific CD8 + T cell responses ( B). 

Twenty mice from each group were challenged with 10 4 plaque-forming units (PFUs) of mouse-adapted SARS-CoV-2 MA10 (MA10) 5 weeks after the boost immunization ( A; ). The MA10 virus is capable of lethal disease in standard immunocompetent mice and recapitulates many aspects of COVID in humans ( ). Weight loss was assessed in 10 mice per group until day 7 post-infection. Control animals had the greatest weight loss, which peaked at day 4 post-infection with an average peak of 14% loss of body weight. Modest but not significant weight loss occurred through day 3 in mice immunized with DI CoV-1, but they recovered more rapidly than control mice. There was no appreciable weight loss in groups immunized with either dose of CoV-2 DS or mRNA-1273 ( A). Viral titers were measured in the nasal turbinates by plaque assay on day 2 ( B) and day 4 ( C) post-infection to assess protection in the upper airway. Low viral titers were detected in 2/5 mice in the 1 μg mRNA-1273 dose group 2 days after infection, and none had detectable virus in the nasal turbinates at day 4, as previously shown following vaccination with 1 μg of mRNA-1273 ( ). In addition to protection in the nasal turbinates, 1 μg mRNA-1273 immunization completely prevented viral infection in the lungs at both day 2 ( D) and day 4 ( E) after challenge. Mice immunized with 0.1 μg mRNA-1273 or 1 μg CoV-2 DS also had reduced viral titer in the lungs at both time points post-challenge. In all, every vaccine tested offered some protection against either weight loss or virus titer post-challenge. We examined lungs histologically on day 4 after challenge (for details and examples of scoring, see ; for scoring for each animal, see ). Assessment of inflammation in sections stained with hematoxylin and eosin (H&E) showed an inflammation and severity score in both groups of DI-CoV-immunized and the 0.2 μg CoV-2-DS-immunized mice that met or exceeded inflammation seen in control mice ( ). Mononuclear and polymorphonuclear inflammation was moderate to severe in the PBS group, severe in the 0.2 μg DI CoV-1 group, moderately severe in the 1 μg DI CoV-1 and 0.2 μg CoV-2 DS groups, mildly to moderately severe in the 1 μg CoV-2 DS and 0.1 μg mRNA-1273 groups, and mild to minimally severe in the 1 μg mRNA group (rank by severity was 0.2 μg DI CoV-1 > 1 μg DI CoV-1 = 0.2 μg CoV-2 DS > PBS > 1 μg CoV-2 DS = 0.1 μg mRNA-1273 > 1 μg mRNA-1273). Type II pneumocyte hyperplasia was evident in all groups but mild in groups immunized with mRNA-1273. Immunohistochemistry for SARS-CoV-2 antigen revealed moderately abundant and diffusely distributed SARS-CoV-2 viral antigen throughout the alveolar interstitium in the PBS group, but sparse or rare infected cells were observed in most other groups, with minimal to absent SARS-CoV-2-antigen-positive cells in 1 μg mRNA-1273-immunized mice ( ). These data are consistent with viral titer measured on day 4 ( E). Groups immunized with Th2-skewing regimens had abundant eosinophils, often in close association with and circumscribing airways and blood vessels. In contrast, eosinophils were sparsely abundant in control and mRNA-immunized mice ( ). Thus, despite limited virus-induced weight loss and partial reduction of viral replication in lungs, Th2-skewing regimes exhibited histological hallmarks of VAERD. We evaluated lung cytokines to confirm the presence of a Th2 signature and increased association with pulmonary inflammation and eosinophilia elicited by the whole inactivated and denatured spike regimens and to assess protection from inflammation in mRNA-1273-immunized mice. Cytokines were measured in clarified lung supernatants obtained on days 2, 4, and 7 post-challenge using a Bioplex 23 multiplex assay. We first measured cytokines produced by Th2 cells: IL-4; IL-5; and IL-13 ( A). Animals immunized with mRNA-1273 had lower concentrations of Th2-associated cytokines than animals in the PBS-immunized group. In contrast, animals immunized with whole-inactivated virus or denatured spike protein immunogens delivered in alum had elevated concentrations of IL-4 and IL-5 compared to the PBS group. We then evaluated the proinflammatory cytokines IL-1α, IL-1β, IL-6, and TNFα ( B). Although there was no statistical increase in proinflammatory cytokines in Th2-skewed immunization groups, with only five samples available per harvest day, there was a trend toward higher proinflammatory cytokine concentrations, particularly IL-1β and TNFα, in these groups, although concentrations of most were reduced compared to the PBS group and in animals immunized with mRNA-1273, particularly at the 1 μg dose. Finally, we assessed chemokines involved in the recruitment and activation of polymorphonuclear cells (acrophage inflammatory protein [MIP]-1α, MIP-1β, eotaxin, and CXCL1(KC); C). There was a trend toward higher concentrations of MIP-1α and MIP-1β early in Th2-skewed groups, with lower expression in mRNA-1273-immunized mice than in the control group. mRNA-1273-immunized mice similarly did not have increased concentrations of CXCL1 or eotaxin, which were higher in most groups immunized with Th2-skewing regimens, consistent with eosinophil major basic protein (EMBP) staining ( ) and with what was observed in earlier studies of DI-CoV1-enhanced disease following mouse-adapted SARS-CoV infection ( ). Overall cytokine concentrations in the lungs were consistent with the histopathological results and indicate an increase in inflammatory signatures and excess mononuclear and polymorphonuclear cell infiltration into the lungs of Th2-skewed immunization groups. Importantly, a distinct profile was observed in the 0.1 μg mRNA-1273 group despite a similar viral load. Although this dose was not sufficient to protect from viral replication, it resulted in histopathological protection and dampened inflammatory responses in the lung. Taken together, the elicitation of both protective and subprotective mRNA-1273-induced immunity resulted in reduced lung pathology without histologic evidence of VAERD. 

Following the results of the first study, we performed a second study in BALB/c mice to incorporate and compare immune responses to the newly available formalin-inactivated SARS-CoV-2 immunogen (FI CoV-2; made available by the Division of Microbiology and Infectious Diseases [DMID], NIAID). The virus was inactivated in the media containing FBS, which challenged efforts to precisely quantitate viral antigen. Therefore, we selected 0.2 and 2 μg total, spanning a 10-fold dose range, and again compared responses to a control PBS group and groups immunized with 0.1 or 1 μg of mRNA-1273 ( A; see for disposition of study 2 animals). Both doses of FI CoV-2 elicited low concentrations of binding and neutralizing antibodies compared to the mRNA-1273 groups ( B and 6C, respectively). Again, although mRNA-1273 elicited both IgG2a and IgG1 subclasses of S-specific IgG, the response to 2 μg FI CoV-2 was dominated by IgG1, suggesting a Th2-biased immune response pattern ( D and 6E). Due to the low immunogenicity of the selected doses of FI CoV-2, we were unable to detect antigen-specific cytokine production by CD4 + T cells following stimulation with homologous peptide pools (S1, S2, and N) in both dose groups using ICS ( ). Responses in mRNA-1273-immunized groups largely reflected the results of the first experiment, with a dose-dependent increase in type 1 profile and CD8 + T cell induction in only the 1 μg mRNA-1273 group ( ). We elected to challenge mice for the second study with an increased dose (10-fold) of 10 5 PFUs of MA10. There was less weight loss than seen in the first study, with no group showing sustained differences from the PBS control group ( A). Consistent with the first study, mice in the 1 μg mRNA-1273 dose group demonstrated near-complete inhibition of viral replication in the nasal turbinates ( B and S5C) and no detectable virus in the lungs ( D and S5E). Mice immunized with 0.1 μg of mRNA-1273 or 2 μg of FI CoV-2 had partial protection from viral replication in the upper and lower airway at some time points ( B–S5E). On day 4, lungs from all groups had a similar degree of inflammation with mononuclear and polymorphonuclear infiltration and diffuse type II pneumocyte hyperplasia, causing a reduction in alveolar air spaces across all study groups ( F). We believe this to be due to the high viral challenge dose used in this study. This was coincident with an overall higher presence of viral antigen, which was particularly abundant in the alveolar interstitium of control animals. The highest degree of EMBP staining, and abundant eosinophils adjacent to and surrounding blood vessels, was seen in the 0.2 μg FI CoV-2 with alum group (for individual animal scoring from study 2, see ). Animals immunized with FI CoV-2 in alum experienced higher concentrations of Th2 cytokines and proinflammatory and chemotactic mediators ( ). Although the high viral inoculum appeared to blunt some differences between groups, combined data from our second challenge study confirmed the finding of higher concentrations of inflammatory cytokines in mice given whole-inactivated virus in alum and reaffirmed a distinct immune signature in mRNA-1273 groups that was associated with antiviral protection. 

+ Tfh and B cell responses We further evaluated the induction of T and B cell responses in our second study. We were particularly interested in CD4 + T follicular helper (Tfh) cells, which are critical to the generation of germinal centers (GCs) and support the clonal selection and differentiation of antigen-specific memory B cells and long-lived plasma cells ( ). We developed a flow cytometry panel to quantify total Tfh (CXCR5 + PD-1 + ) and GC B cell (GL7 + Fas + ) responses, as well as B cells capable of binding the prefusion-stabilized S protein and/or the neutralization-sensitive receptor binding domain (RBD) using tetramerized fluorochrome-conjugated capture probes ( A; for gating and representative plots, see A). 2 weeks after the boost, we found elevated total Tfh and GC B cell frequencies in the splenocytes from only the 1 μg mRNA-1273 dose group ( B and 7C, respectively). Mice that received 1 μg of mRNA-1273 also demonstrated significant induction of probe-binding, class-switched GC B cells, plasma cells (PCs) (identified as CD138 + B220 lo ), and other B cells (GL7 − Fas − ; D). Other vaccine groups had little to no detectable elicitation of S-reactive B cells. Among B cells that bound the stabilized S protein probe, approximately 20%–30% demonstrated specificity for the RBD based on co-capture of the RBD probe. This proportion is consistent with the proportion of S-directed B cells that bind RBD in SARS-CoV-2-experienced humans ( ; ). To more fully capture the repertoire of antigen-specific CD4 + T cells elicited by vaccination, we employed a more inclusive, cytokine agnostic approach called activation-induced marker (AIM) assay, which can identify activated cells even if they do not express sufficient cytokine to be measured by traditional ICS methods ( E; ). Antigen-experienced (CD44 + ) T cells were assessed for the upregulation of activation markers CD69 and CD154 (CD40L) following stimulation with S1 and S2 peptide pools in a 6-h culture in vitro . CD4 + T cells from groups immunized with 0.1 μg or 1 μg of mRNA-1273 responded in a dose-dependent manner to S-derived peptides in higher frequencies than we measured using conventional ICS ( F; for representative staining, see B). However, using this more sensitive method, we were still unable to detect T cell responses above background in groups immunized with FI CoV-2 at either dose. Further evaluation demonstrated strong induction of AIM + CD4 + Tfh cells responding to spike peptides following immunization with 1 μg of mRNA-1273 ( F), with limited reactivity detected from all other groups. Both AIM + Tfh and S-2P + GC B cells correlated well with neutralizing activity against SARS-CoV-2, suggesting a well-coordinated cellular and serological response ( G). These data further support the activation of T cell responses that promote the generation of durable immune memory following mRNA-1273 immunization. 

Comprehensive evaluation of safety is a critical component of every human vaccine development program. Given the history of VAERD in preclinical evaluation of emerging coronavirus vaccines, animal models should be used to evaluate the potential for vaccination against SARS-CoV-2 to enhance disease following infection. Here, we show a clear distinction between the immune profile elicited by research-grade mRNA-1273 and vaccine regimens that result in enhanced inflammation following MA10 challenge in mice. mRNA-1273, even at a relatively modest dose of 1 μg, elicits high-potency neutralizing antibodies and primes T cell immunity toward effective antiviral T cell responses. In addition to increased virus-neutralizing activity, a higher prevalence of S-directed IgG2a antibodies may increase the potential for Fc-mediated effector functions, including direct killing (antibody-dependent cellular cytotoxicity [ADCC]) and phagocytosis (antibody-dependent cellular phagocytosis [ADCP]) of target cells due to increased activating FcγR (FcγRI, FcγRIII, and FcγRIV) engagement by innate immune cells relative to IgG1 (a more functionally restricted isotype in mice; ). Importantly, immunization with as little as 0.1 μg of mRNA-1273, a dose that minimally controlled viral replication, dampened lung inflammation after challenge. Conversely, mice immunized with whole-inactivated SARS viruses or denatured S with alum induced weak neutralizing activity and Th2-biased immune responses, including evidence of increased eosinophil and neutrophil infiltration of lung that are hallmark features of disease enhancement following challenge. We provide evidence for the absence of vaccine-enhanced illness by mRNA-1273 in comparison to regimens that elicit such a response, supporting a strong safety profile for mRNA vaccination against COVID-19. Enhanced disease has long been associated with vaccines that elicit qualitative and quantitatively distinct immune responses, including Th2-biased T cells, and low or waning concentrations of antibodies, particularly if the antibodies have limited neutralization potency ( ; ). Using ICS, we determined that T cells elicited by inactivated virus and denatured protein were prone to express one or more of the prototypical Th2-associated cytokines, IL-4, IL-5, and IL-13. This was true for both S- and N-specific T cells elicited by heterologous DI CoV-1. IgG subclass determination for S-directed antibodies corroborated the ICS, with IL-4-regulated IgG1 prevailing over IFNγ-regulated IgG2a in the alum-containing immunization groups. In line with this profile, we observed no elicitation of S-directed CD8 + T cells by either of these protein-based vaccine regimens. Our data illustrate that suboptimal, pre-existing heterologous or homologous responses can upset a delicate balance and lead to cytokine deregulation and enhanced immunopathology following SARS-CoV-2 infection. Although mRNA vaccines have previously been shown to strongly induce both IgG2a and IgG1 ( ; ; ), we consistently observed a dose response in the mRNA-1273 groups, with a higher proportion of T cells expressing Th2 cytokines and a lower IgG2a/IgG1 ratio in the 0.1 μg dose group. This is not surprising, given that antigen dose is a factor that can contribute to the differentiation of type 1 versus type 2 responses ( ). Notably, 0.1 μg is a lower dose than tested for other mRNA COVID vaccines ( ; ; ) and was intentionally selected as a subprotective dose that could serve as a surrogate of limited or waning immunity and allow breakthrough infections with active viral replication in the lung. Although immunity in this group was shifted away from a strong type-1-biased toward a more balanced immune response and S-specific CD8 + T cells were undetectable, mice in this group were protected from increased proinflammatory cytokine production and eosinophilic lung inflammation following challenge. Cytokine expression by S-specific CD8 + T cells in the 1 μg mRNA-1273 group exhibited a typical IFNγ > TNFα > IL-2 hierarchy, but we noted that the hierarchy for Th1 cell cytokine expression was consistently IL-2 > TNFα > IFNγ in the 1 μg dose group, and there was a paucity of IFNγ-secreting CD4 + T cells in the 0.1 μg mRNA-1273 dose group. Other mRNA COVID-19 vaccines have more strongly elicited IFNγ in mouse models, but these studies used higher doses of mRNA ( ; ; ). The elevated production of IFNγ by T cells in the 1 μg mRNA-1273 dose group may reflect a threshold needed to achieve increased IL-12-mediated polarization toward a “type 1” immune program by activated antigen presenting cells (APCs). It is likely that higher concentrations of IFNγ reinforced both CD4 + Th1 and CD8 + effector T cell responses and, in turn, promoted antigen processing and presentation by APC through expression of transporter associated with antigen processing (TAP), the beta catalytic subunits of the immunoproteasome, and major histocompatibility complex (MHC) class I and II molecules ( ; ). During natural SARS-CoV-2 infection, IFNγ does not appear to strongly dominate the S-specific Th1 CD4 + cell profile over IL-2 and TNFα, and SARS-CoV-2 may not polarize as strongly toward the type 1 signature as influenza ( ; ; ; ). A similar pattern of cytokine expression is also observed following human vaccination with mRNA-1273 ( ) and a recombinant spike protein nanoparticle vaccine (NVX-CoV2373; ). Whether this reflects immune modulation by SARS-CoV-2 or intrinsic properties of S-specific cells remains to be determined. Importantly, approaches that focus solely on IFNγ detection, particularly IFNγ ELISpot, could severely underestimate the SARS-CoV-2 T cell response following either vaccination or infection ( ; ). We suspect that the IL-2 + IFNγ − CD4 + T cells detected 2 weeks post-boost following mRNA-1273 immunization in our study may reflect a primed but uncommitted subpopulation of memory cells with effector potential poised for differentiation into Th1 and Th2 cells depending on the cytokine microenvironment ( ). It is conceivable that the added Th cell subset flexibility of such vaccine-induced memory cells may provide the benefit of supporting type 1 responses to control viral replication and type-2-associated functions to counteract excessive Th1-cell-mediated inflammatory tissue damage that may occur following severe cases of natural infection. Evidence for additional effector functions of IL-2 + IFNγ − cells (referred to as primed precursor Th or Thpp cells) include chemokine production for immune cell recruitment to lymphoid tissues ( ; ). Given the lack of strong Th1 cell polarization at the relatively low doses of mRNA used in our studies, we posited that ICS was not capturing all S-specific T cells. Using the AIM assay, we measured 10- to 20-fold more S-specific CD4 + T cells than were detected using the ICS and demonstrated a high frequency of S-specific Tfh cells, supporting the selection of high-affinity GC B cells and the generation of potently neutralizing antibody in the 1 μg mRNA-1273 group. Strong induction of Tfh cells is one feature that makes mRNA an attractive platform for vaccination ( ). A single immunization with mRNA encoding S demonstrated a superior capacity to induce GC responses compared to adjuvanted protein ( ). This could contribute to the observed efficacy of mRNA-1273 (NIAID, NIH and Moderna) and BNT162b2 (BioNTech and Pfizer) after a single immunization in phase 3 clinical trials. In summary, our data demonstrate the absence of VAERD after MA10 challenge of mRNA-1273-vaccinated mice, even at an extremely low subprotective vaccine dose and under conditions where both whole-inactivated virus and denatured subunit protein immunization led to enhancement of lung immunopathology. mRNA-1273 elicits both B and T cell responses to the full-length S protein that contribute to effective antiviral immunity. It has demonstrated the ability to elicit potent and effective S-specific immunity in NHP ( ) and humans ( ; ; ) and demonstrated 94.1% efficacy in a phase 3 trial ( ). Continued administration of mRNA-1273 under emergency use authorization is warranted based on its high efficacy against symptomatic COVID-19 and demonstration of clinical and preclinical safety. Limitations of the study The greatest limitation of our study is the imprecise ability to predict VAERD in humans using animal models. It is particularly difficult to interpret nuanced readouts, such as weight loss, and we relied on the combination of alterations in the lung cytokine milieu and histopathology after challenge for indications of enhancement. Together, these readouts corroborate a pattern of type 2-biasing and eosinophilia post-challenge for whole-inactivated and denatured vaccine regimens and protection from inflammation in mRNA-1273-vaccinated mice. Although animal models can reproduce RSV- and measles-driven VAERD ( ; ; , ; ), it is difficult to implicate the same immunological mechanisms in humans. Despite several observations of vaccine-enhanced disease in animals for SARS-CoV-1 and MERS-CoV, the lack of human coronavirus vaccine testing has left VAERD a major open question and something that should be prospectively evaluated in ongoing clinical trials across diverse vaccine platforms while the opportunity exists. This evaluation would need clear endpoints to identify and confirm infection events; the wide and complex spectrum of COVID in the human population adds a considerable challenge for discriminating enhanced symptomology and immune pathology. To date, across all platforms, including whole-inactivated viruses, there has been no evidence of VAERD in humans, but this should be followed carefully over time as immune responses wane in the presence of emerging variant viruses of concern. Dose reduction performed in these studies is a surrogate for waning immune responses but is not the same, and long-term monitoring is recommended. We relied on using the Th2-prone BALB/c and low doses of antigen that we anticipated could elicit low-potency antibodies and are encouraged both by the measures required to drive VAERD and the absence of inflammation seen in the mRNA-1273 groups. We anticipate the use of aged mice could further drive immune pathology and have ongoing studies to address mRNA-1273-mediated protection in that setting. We are unable to parse the individual role of T cells and antibody to the VAERD seen in the whole inactivated virus and denatured S groups. T-cell-depletion studies would be needed to delineate their contribution to eosinophilic immune pathology and could help clarify the beneficial or possibly detrimental contribution of the T cell response following infection or vaccination ( ; ; ; ). The apparent lack of induction of T cells by FI CoV-2 in our second study does not rule out a potential role for primed T cells following challenge; we were unable to address the T cell response post-challenge in these studies. Although antibodies to N are more frequently implicated in VAERD ( ; ; ), enhanced immune pathology consistent with that which we observed for denatured S alone has been previously noted following S vaccination ( ). We note that antibodies to denatured S appear to be lower potency, but the sensitivity of SARS-CoV-2 neutralization assays when S-specific antibody titers are low impedes our ability to effectively compare potency and determine how relative potency relates to disease outcome. The successes of passive transfer of antibody during early infection speak to a protective role for antibody ( ; ), but selected antibodies are generally high potency. As viral mutations accumulate through adaptation and selection, the affinity, epitope specificity, and bioactivity of monoclonal countermeasures and polyclonal antibody responses to vaccination will need to be monitored. We believe that using conformationally correct, full-length S protein to elicit diverse T cell responses and potent neutralizing activity helps mitigate these risks. Combined, the high efficacy of mRNA-1273 and the preclinical data reported here provide some assurance that, even with variant strains that may be less susceptible to vaccine-induced neutralizing activity, protection against disease will be maintained. 

The greatest limitation of our study is the imprecise ability to predict VAERD in humans using animal models. It is particularly difficult to interpret nuanced readouts, such as weight loss, and we relied on the combination of alterations in the lung cytokine milieu and histopathology after challenge for indications of enhancement. Together, these readouts corroborate a pattern of type 2-biasing and eosinophilia post-challenge for whole-inactivated and denatured vaccine regimens and protection from inflammation in mRNA-1273-vaccinated mice. Although animal models can reproduce RSV- and measles-driven VAERD ( ; ; , ; ), it is difficult to implicate the same immunological mechanisms in humans. Despite several observations of vaccine-enhanced disease in animals for SARS-CoV-1 and MERS-CoV, the lack of human coronavirus vaccine testing has left VAERD a major open question and something that should be prospectively evaluated in ongoing clinical trials across diverse vaccine platforms while the opportunity exists. This evaluation would need clear endpoints to identify and confirm infection events; the wide and complex spectrum of COVID in the human population adds a considerable challenge for discriminating enhanced symptomology and immune pathology. To date, across all platforms, including whole-inactivated viruses, there has been no evidence of VAERD in humans, but this should be followed carefully over time as immune responses wane in the presence of emerging variant viruses of concern. Dose reduction performed in these studies is a surrogate for waning immune responses but is not the same, and long-term monitoring is recommended. We relied on using the Th2-prone BALB/c and low doses of antigen that we anticipated could elicit low-potency antibodies and are encouraged both by the measures required to drive VAERD and the absence of inflammation seen in the mRNA-1273 groups. We anticipate the use of aged mice could further drive immune pathology and have ongoing studies to address mRNA-1273-mediated protection in that setting. We are unable to parse the individual role of T cells and antibody to the VAERD seen in the whole inactivated virus and denatured S groups. T-cell-depletion studies would be needed to delineate their contribution to eosinophilic immune pathology and could help clarify the beneficial or possibly detrimental contribution of the T cell response following infection or vaccination ( ; ; ; ). The apparent lack of induction of T cells by FI CoV-2 in our second study does not rule out a potential role for primed T cells following challenge; we were unable to address the T cell response post-challenge in these studies. Although antibodies to N are more frequently implicated in VAERD ( ; ; ), enhanced immune pathology consistent with that which we observed for denatured S alone has been previously noted following S vaccination ( ). We note that antibodies to denatured S appear to be lower potency, but the sensitivity of SARS-CoV-2 neutralization assays when S-specific antibody titers are low impedes our ability to effectively compare potency and determine how relative potency relates to disease outcome. The successes of passive transfer of antibody during early infection speak to a protective role for antibody ( ; ), but selected antibodies are generally high potency. As viral mutations accumulate through adaptation and selection, the affinity, epitope specificity, and bioactivity of monoclonal countermeasures and polyclonal antibody responses to vaccination will need to be monitored. We believe that using conformationally correct, full-length S protein to elicit diverse T cell responses and potent neutralizing activity helps mitigate these risks. Combined, the high efficacy of mRNA-1273 and the preclinical data reported here provide some assurance that, even with variant strains that may be less susceptible to vaccine-induced neutralizing activity, protection against disease will be maintained. 

Key resources table Resource availability Lead contact Further information and requests for resources and reagents should be addressed to and will be fulfilled by the Lead Contact Tracy Ruckwardt ( truckwardt@nih.gov ). Materials availability This study did not generate unique reagents. Data and code availability Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Experimental model and subject details Mice All experiments were conducted with age-matched (6–10 weeks) female BALB/cJ mice purchased from Jackson Laboratories (strain 000651). Mice were initially maintained under specific-pathogen-free conditions on standard rodent chow and water supplied ad libitum in the animal care facility at the National Institute of Allergy and Infectious Diseases (NIAID). The protocol (19-799) was approved by the Animal Care and Use Committee of the Vaccine Research Center (VRC), NIAID, National Institutes of Health (NIH) and carried out in accordance with the recommendations and guidelines of the NIH Guide to the Care and Use of Laboratory Animals. Mice were housed in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and procedures were conducted in accordance with all relevant federal and National Institutes of Health guidelines and regulations. Upon transfer of the animals to the University of North Carolina at Chapel Hill, work was again carried out according to guidelines outlined by the AAALAC, and work performed with approved standard operating procedures and safety conditions for SARS-CoV-2 in BSL3 facilities designed to conform to the safety requirements recommended by Biosafety in Microbiological and Biomedical Laboratories (BMBL), the U.S. Department of Health and Human Services, the Centers for Disease Control and Prevention, and the NIH. The facility has been approved for use by the UNC Department of Environmental Health and Safety (EHS) and the CDC. SARS-CoV-2 MA10 virus SARS-CoV-2 MA10 was generated by serially passaging SARS-CoV-2 MA stock virus in the lungs of mice as previously described ( ; ). A clonal isolate from P10 was plaque purified to obtain SARS-CoV-2 MA10. Viral stocks were propagated on Vero E6 cells in minimal essential medium (MEM) containing 10% fetal bovine serum (HyClone) and supplemented with penicillin and kanamycin (GIBCO). Virus plaques were visualized by neutral red staining for two to three days. All viral infections were conducted under biosafety level 3 (BSL-3) conditions at negative pressure, and personnel wore Tyvek suits connected to personal powered-air purifying respirators. Cell lines The simian kidney cell line Vero E6 (ATTC # CRL1586, female) was purchased from ATCC. Vero E6 cells used in plaque assays from nasal turbinates and lungs were cultured in DMEM supplemented with 10% Fetal Clone II and 1% antibiotic–antimycotic at 37 °C and 5% CO 2 . ACE2-expressing 293T cells (female, provided by M. Farzan, Scripps Research Institute) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and 1% penicillin–streptomycin at 37 °C and 5% CO 2 . Expi293 cells (female) were used for protein expression and maintained in the manufacturer’s suggested medium. Method details Protein production Vectors encoding SARS-CoV-2 S-2P were generated as previously described ( ). Proteins were expressed by transfection of plasmids into Expi293 cells using Expifectamine transfection reagent (ThermoFisher) in suspension at 37 °C for 4–5 days. Transfected cell culture supernatants were collected, buffer exchanged into 1 × PBS, and protein was purified using Strep-Tactin resin (IBA). For SARS-CoV-2 S-2P used for mouse inoculations, tags were cleaved with addition of HRV3C protease (ThermoFisher) (1% wt/wt) overnight at 4 °C. Size-exclusion chromatography using Superose 6 Increase column (GE Healthcare) yielded final purified protein. A mammalian codon-optimized plasmid encoding foldon inserted minifibritin previously described ( ) was used to compete foldon-specific antibodies where indicated. Immunogen preparation and immunizations Products NR-3882 and NR-3883 were obtained from Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and used for lower-dose (0.2 μg) and higher-dose (1 μg) DI CoV-1 immunization, respectively, in the rear hind legs of BALB/cJ mice. These products were originally intended for active immunization for SARS-CoV-1. A seed virus was prepared, and sucrose purified prior to double inactivation with both formaldehyde and UV irradiation, and the final product was formulated with alum at a final concentration of 5 or 10 μg/mL (NR-3882 and NR-3883, respectively). Formalin-inactivated SARS-CoV-2 was produced under contract with Battelle Biomedical Research Center (BBRC) and was kindly provided by DMID. Briefly, SARS-CoV-2 (isolate USA-WA1/2020, BEI resources NR-52352) grown on Vero E6 cells was harvested on day 2, and clarified supernatant was inactivated using formalin (37% formaldehyde, Fisher Scientific) at a concentration of 0.05% at 37°C for a minimum of 48 hours. Inactivation was confirmed using plaque assays, and protein quantitated using a Bradford assay testing kit. The material was formulated with Alhydrogel® adjuvant (InvivoGen catalog vac-alu-250) at a 1:1 ratio (v/v), and doses of 0.2 or 2 μg were injected into the rear hind limbs of BALB/c mice. Heat-denatured spike protein was produced by heating SARS-CoV-S-2P protein ( ) for 10 minutes at 95°C. Denatured protein was formulated 1:1 (v/v) with Alhydrogel and doses of either 0.2 or 1 ug were injected into the rear hind limbs. Research-grade mRNA-1273, sequence-optimized mRNA encoding SARS-CoV-2 S-2P protein formulated in a lipid nanoparticle (LNP), was produced and purified at Moderna, Inc. as previously described ( ). Mice were immunized in the rear hind limb with doses of either 0.1 or 1 μg of mRNA-1273. Enzyme-linked immunosorbent assay (ELISA) Nunc Maxisorp ELISA plates (ThermoFisher) were coated with 100 ng of protein in 1X PBS at 4°C for 16 hr. After standard washes and blocks, plates were incubated with serial dilutions of sera for 1 hr at room temperature (RT). Where applicable, to block binding of foldon-specific antibodies to SARS-CoV-2 S-2P protein, sera dilutions were preincubated with 50 μg/mL of foldon protein for 1 hour at RT prior to adding to the plate. After washing, anti-mouse IgG, IgG1 or IgG2a–horseradish peroxidase conjugates (ThermoFisher) were used as secondary antibodies, and 3,3′,5,5′-tetramethylbenzidine (TMB) (KPL) was used as the substrate to detect antibody responses. Endpoint titers were calculated as the dilution that emitted an optical density exceeding four times background from secondary antibody alone and extrapolated from a 4-parameter nonlinear fit curve using GraphPad Prism software. Pseudovirus microneutralization assay To produce SARS-CoV-2 pseudoviruses, a codon-optimized CMV/R-SARS-CoV-2 S (Wuhan-1, GenBank #: MN908947.3) plasmid was constructed. A single amino acid mutation was performed to change residue 614 to a glycine (G) to better reflect the more transmissible, predominant circulating strain of SARS-CoV-2. Pseudoviruses were produced by co-transfection of plasmids encoding a luciferase reporter, lentivirus backbone, and S genes into HEK293T/17 cells (ATCC), as previously described ( ). A plasmid for expression of TMPRSS2 was also co-transfected ( ). Pseudoneutralization assays were performed as previously described ( ). Briefly, heat-inactivated (HI)-serum was mixed with pseudovirus, incubated, and then added to 293T-ACE2 cells. Three days later, cells were lysed, and luciferase activity (relative light units, RLU) was measured. Percent neutralization was calculated considering uninfected cells as 100% neutralization and cells transduced with pseudovirus as 0% neutralization. IC 50 titers were determined based on 5-parameter nonlinear curve fit using GraphPad Prism software. Intracellular cytokine staining and flow cytometry Mononuclear single cell suspensions from whole mouse spleens were generated using a gentleMACS tissue dissociator (Miltenyi Biotec) followed by 70 μm filtration and density gradient centrifugation using Fico/Lite-LM medium (Atlanta Biologicals). Cells from each mouse were resuspended in R10 media (RPMI 1640 supplemented with Pen-Strep antibiotic, 10% HI-FBS, Glutamax, and HEPES) and incubated for 6 hr at 37°C with protein transport inhibitor cocktail (eBioscience) under four conditions: no peptide (DMSO only) stimulation, stimulation with two spike peptide pools (S1 and S2 peptide pools, 85% pure, JPT), and stimulation with a nucleocapsid peptide pool (N) (PepmixTM JPT product PM-WCPV-NCAP). Peptide pools were used at a final concentration of 2 μg/ml each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: I-A/I-E (M5/114.15.2) PE, CD8α (53-6.7) BUV805, CD44 (IM7) BUV395, CD62L (MEL-14) BV605, and CD4 (RM4-5) BV480 in brilliant stain buffer (BD). After 15 min at RT, cells were washed with FC buffer, then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to manufacturer instructions. Cells were washed in perm/wash solution and stained with Fc Block (5 min at RT), followed by intracellular staining (30 min at 4°C) using a cocktail of the following antibodies purchased from BD, Biolegend, or eBioscience: CD3 (17A2) BUV737, IFNγ (XMG1.2) BV650, TNFα (MP6-XT22) BV711, IL-2 (JES6-5H4) BV421, IL-4 (11B11) Alexa Fluor 488, IL-5 (TRFK5) APC, and IL-13 (eBio13A) PE-Cy7 in 1x perm/wash diluted with brilliant stain buffer. Finally, cells were washed in perm/wash solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in . Background cytokine expression in the no peptide condition was subtracted from that measured in the S1, S2, and N peptide pools for each individual mouse. Antigen-specific CD4 + T cells, including Tfh cells were examined using an activation induced marker (AIM) assay. Splenocytes were resuspended in R10 media containing BD Fc Block and anti-CD154 (CD40L) antibody conjugated to APC (BD, clone: MR1) and incubated for 6 hr at 37°C under three conditions: no peptide (DMSO only) stimulation, and stimulation with the S1 and S2 peptide pools. Peptide pools were used at a final concentration of 2 μg/ml each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: CD3 (17A2) BUV737, CD4 (RM4-5) BV480, I-A/I-E (M5/114.15.2) PE, CD44 (IM7) BUV395, CD62L (MEL-14) PE-Cy7, CXCR5 (2G8) PE-CF594, PD-1 (J43) BV421, CD69 (H1.2F3) BV605. After 15 min at RT, cells were washed in FC stain buffer solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in A. Background cytokine expression in the no peptide condition (DMSO) was subtracted from that measured in the S1 and S2 peptide pools for each individual mouse, with representative upregulation of activation induced markers (CD69 and CD40L) shown in B. B cells and plasma cells were examined using a separate panel, which included molecular probes for identification of antigen-specific cells. Directly ex vivo , splenocytes were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend, and NIH Vaccine Research Center (VRC) generated B cell capture probes: CD3 (17A2) PE, CD19 (1D3) BUV737, B220 (RA3-6B2) BV711, CD138 (281-2) BV605, T and B cell activation marker (GL7) PE-Cy7, CD95/Fas (Jo2) BUV563, CD4 (RM4-5) BUV805, CD44 (IM7) BUV395, PD-1 (J43) APC-R700, CXCR5 (2G8) PE-CF594, IgD (11-26c.2a) APC-H7, IgM (RMM-1) BV510, S-2P probe APC, S-2P probe BB515, and RBD-SD1 probe BV421. After 15 min at RT, cells were washed in FC stain buffer solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in A. SARS-CoV-2 MA10 infection and viral titer measurements For virus challenge, mice were anesthetized (ketamine/xylazine) and infected intranasally with 10 4 (study 1) or 10 5 (study 2) PFU SARS-CoV-2 MA10 diluted in 50 μL PBS. Clinical signs of disease (weight loss) were monitored daily. Mice were euthanized by isoflurane overdose at days 2, 4, and 7 and samples for titer (caudal right lung lobe) and histopathological analyses (left lung lobe) were collected. Lung viral titers were determined using plaque assays. Briefly, right caudal lung lobes were homogenized in 1 mL PBS using glass beads and serial dilutions of the clarified lung homogenates were added to a monolayer of Vero E6 cells. After three days, plaques were visualized via staining with Neutral Red dye and counted. The left lung lobe was stored in 10% phosphate buffered formalin for seven days prior to removal from the BSL3 for processing. Cytokine and chemokine analysis BioPlex Pro mouse cytokine 23-plex assay (Bio-Rad) was utilized to analyze chemokines and cytokines in clarified lung supernatants according to manufacturer’s protocol. 50 μL of clarified lung samples were incubated with magnetic capture beads, washed, incubated with detection antibodies and streptavidin-PE. Cytokines were recorded on a MAGPIX machine (Luminex) and quantitated via comparison to a standard curve. Graphpad prism software was used for data analysis. Lung Histopathology and Immunohistochemistry Lung samples from mice were processed per a standard protocol. Briefly, the tissues were fixed in 10% neutral buffered formalin, processed with Leica ASP6025 tissue processor (Leica Microsystems), embedded in paraffin, and sectioned at 5 μm for histological analysis. Tissue sections were stained with hematoxylin and eosin (H&E) for routine histopathology. Sections were examined in a blinded manner by a board-certified veterinary pathologist using an Olympus BX51 light microscope, and photomicrographs were taken using an Olympus DP73 camera. For immunohistochemical (IHC) evaluation, formalin-fixed paraffin-embedded tissue sections (5 μm) were used to perform immunohistochemical staining using antibodies which identify SARS-CoV-2 nucleocapsid and eosinophils in tissue, respectively; for SARS-CoV-2 nucleocapsid, a rabbit polyclonal SARS-CoV-2 (GeneTex, Irvine CA, GTX135357) with dilution of 1:2000 was used. For identification of tissue eosinophils, a goat polyclonal Eosinophil Major Basic Protein (EMBP; SantaCruz, Dallas TX, sc-33938) at a dilution of 1:500 was employed. Staining was carried out on the Bond RX (Leica Biosystems) platform according to manufacturer-supplied protocols. Briefly, 5 μm-thick sections were deparaffinized and rehydrated. Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1, pH 6.0, heated to 100°C for 20 min. The specimen was then incubated with hydrogen peroxide to quench endogenous peroxidase activity prior to applying the primary antibody. Detection with DAB chromogen was completed using the Bond Polymer Refine Detection kit (Leica Biosystems CAT# DS9800). Slides were finally cleared through gradient alcohol and xylene washes prior to mounting and placing coverslips. Sections were examined by a board-certified veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera. Quantification and statistical analysis Statistical Analysis Serological responses, T and B cell readouts, and viral titer data were analyzed using Graphpad Prism using the methods outlined in each figure legend. Nonparametric methods were used when groups contained fewer than 10 mice per group. 

 

Lead contact Further information and requests for resources and reagents should be addressed to and will be fulfilled by the Lead Contact Tracy Ruckwardt ( truckwardt@nih.gov ). Materials availability This study did not generate unique reagents. Data and code availability Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. 

Further information and requests for resources and reagents should be addressed to and will be fulfilled by the Lead Contact Tracy Ruckwardt ( truckwardt@nih.gov ). 

This study did not generate unique reagents. 

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. 

Mice All experiments were conducted with age-matched (6–10 weeks) female BALB/cJ mice purchased from Jackson Laboratories (strain 000651). Mice were initially maintained under specific-pathogen-free conditions on standard rodent chow and water supplied ad libitum in the animal care facility at the National Institute of Allergy and Infectious Diseases (NIAID). The protocol (19-799) was approved by the Animal Care and Use Committee of the Vaccine Research Center (VRC), NIAID, National Institutes of Health (NIH) and carried out in accordance with the recommendations and guidelines of the NIH Guide to the Care and Use of Laboratory Animals. Mice were housed in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and procedures were conducted in accordance with all relevant federal and National Institutes of Health guidelines and regulations. Upon transfer of the animals to the University of North Carolina at Chapel Hill, work was again carried out according to guidelines outlined by the AAALAC, and work performed with approved standard operating procedures and safety conditions for SARS-CoV-2 in BSL3 facilities designed to conform to the safety requirements recommended by Biosafety in Microbiological and Biomedical Laboratories (BMBL), the U.S. Department of Health and Human Services, the Centers for Disease Control and Prevention, and the NIH. The facility has been approved for use by the UNC Department of Environmental Health and Safety (EHS) and the CDC. SARS-CoV-2 MA10 virus SARS-CoV-2 MA10 was generated by serially passaging SARS-CoV-2 MA stock virus in the lungs of mice as previously described ( ; ). A clonal isolate from P10 was plaque purified to obtain SARS-CoV-2 MA10. Viral stocks were propagated on Vero E6 cells in minimal essential medium (MEM) containing 10% fetal bovine serum (HyClone) and supplemented with penicillin and kanamycin (GIBCO). Virus plaques were visualized by neutral red staining for two to three days. All viral infections were conducted under biosafety level 3 (BSL-3) conditions at negative pressure, and personnel wore Tyvek suits connected to personal powered-air purifying respirators. Cell lines The simian kidney cell line Vero E6 (ATTC # CRL1586, female) was purchased from ATCC. Vero E6 cells used in plaque assays from nasal turbinates and lungs were cultured in DMEM supplemented with 10% Fetal Clone II and 1% antibiotic–antimycotic at 37 °C and 5% CO 2 . ACE2-expressing 293T cells (female, provided by M. Farzan, Scripps Research Institute) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and 1% penicillin–streptomycin at 37 °C and 5% CO 2 . Expi293 cells (female) were used for protein expression and maintained in the manufacturer’s suggested medium. 

All experiments were conducted with age-matched (6–10 weeks) female BALB/cJ mice purchased from Jackson Laboratories (strain 000651). Mice were initially maintained under specific-pathogen-free conditions on standard rodent chow and water supplied ad libitum in the animal care facility at the National Institute of Allergy and Infectious Diseases (NIAID). The protocol (19-799) was approved by the Animal Care and Use Committee of the Vaccine Research Center (VRC), NIAID, National Institutes of Health (NIH) and carried out in accordance with the recommendations and guidelines of the NIH Guide to the Care and Use of Laboratory Animals. Mice were housed in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and procedures were conducted in accordance with all relevant federal and National Institutes of Health guidelines and regulations. Upon transfer of the animals to the University of North Carolina at Chapel Hill, work was again carried out according to guidelines outlined by the AAALAC, and work performed with approved standard operating procedures and safety conditions for SARS-CoV-2 in BSL3 facilities designed to conform to the safety requirements recommended by Biosafety in Microbiological and Biomedical Laboratories (BMBL), the U.S. Department of Health and Human Services, the Centers for Disease Control and Prevention, and the NIH. The facility has been approved for use by the UNC Department of Environmental Health and Safety (EHS) and the CDC. 

SARS-CoV-2 MA10 was generated by serially passaging SARS-CoV-2 MA stock virus in the lungs of mice as previously described ( ; ). A clonal isolate from P10 was plaque purified to obtain SARS-CoV-2 MA10. Viral stocks were propagated on Vero E6 cells in minimal essential medium (MEM) containing 10% fetal bovine serum (HyClone) and supplemented with penicillin and kanamycin (GIBCO). Virus plaques were visualized by neutral red staining for two to three days. All viral infections were conducted under biosafety level 3 (BSL-3) conditions at negative pressure, and personnel wore Tyvek suits connected to personal powered-air purifying respirators. 

The simian kidney cell line Vero E6 (ATTC # CRL1586, female) was purchased from ATCC. Vero E6 cells used in plaque assays from nasal turbinates and lungs were cultured in DMEM supplemented with 10% Fetal Clone II and 1% antibiotic–antimycotic at 37 °C and 5% CO 2 . ACE2-expressing 293T cells (female, provided by M. Farzan, Scripps Research Institute) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and 1% penicillin–streptomycin at 37 °C and 5% CO 2 . Expi293 cells (female) were used for protein expression and maintained in the manufacturer’s suggested medium. 

Protein production Vectors encoding SARS-CoV-2 S-2P were generated as previously described ( ). Proteins were expressed by transfection of plasmids into Expi293 cells using Expifectamine transfection reagent (ThermoFisher) in suspension at 37 °C for 4–5 days. Transfected cell culture supernatants were collected, buffer exchanged into 1 × PBS, and protein was purified using Strep-Tactin resin (IBA). For SARS-CoV-2 S-2P used for mouse inoculations, tags were cleaved with addition of HRV3C protease (ThermoFisher) (1% wt/wt) overnight at 4 °C. Size-exclusion chromatography using Superose 6 Increase column (GE Healthcare) yielded final purified protein. A mammalian codon-optimized plasmid encoding foldon inserted minifibritin previously described ( ) was used to compete foldon-specific antibodies where indicated. Immunogen preparation and immunizations Products NR-3882 and NR-3883 were obtained from Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and used for lower-dose (0.2 μg) and higher-dose (1 μg) DI CoV-1 immunization, respectively, in the rear hind legs of BALB/cJ mice. These products were originally intended for active immunization for SARS-CoV-1. A seed virus was prepared, and sucrose purified prior to double inactivation with both formaldehyde and UV irradiation, and the final product was formulated with alum at a final concentration of 5 or 10 μg/mL (NR-3882 and NR-3883, respectively). Formalin-inactivated SARS-CoV-2 was produced under contract with Battelle Biomedical Research Center (BBRC) and was kindly provided by DMID. Briefly, SARS-CoV-2 (isolate USA-WA1/2020, BEI resources NR-52352) grown on Vero E6 cells was harvested on day 2, and clarified supernatant was inactivated using formalin (37% formaldehyde, Fisher Scientific) at a concentration of 0.05% at 37°C for a minimum of 48 hours. Inactivation was confirmed using plaque assays, and protein quantitated using a Bradford assay testing kit. The material was formulated with Alhydrogel® adjuvant (InvivoGen catalog vac-alu-250) at a 1:1 ratio (v/v), and doses of 0.2 or 2 μg were injected into the rear hind limbs of BALB/c mice. Heat-denatured spike protein was produced by heating SARS-CoV-S-2P protein ( ) for 10 minutes at 95°C. Denatured protein was formulated 1:1 (v/v) with Alhydrogel and doses of either 0.2 or 1 ug were injected into the rear hind limbs. Research-grade mRNA-1273, sequence-optimized mRNA encoding SARS-CoV-2 S-2P protein formulated in a lipid nanoparticle (LNP), was produced and purified at Moderna, Inc. as previously described ( ). Mice were immunized in the rear hind limb with doses of either 0.1 or 1 μg of mRNA-1273. Enzyme-linked immunosorbent assay (ELISA) Nunc Maxisorp ELISA plates (ThermoFisher) were coated with 100 ng of protein in 1X PBS at 4°C for 16 hr. After standard washes and blocks, plates were incubated with serial dilutions of sera for 1 hr at room temperature (RT). Where applicable, to block binding of foldon-specific antibodies to SARS-CoV-2 S-2P protein, sera dilutions were preincubated with 50 μg/mL of foldon protein for 1 hour at RT prior to adding to the plate. After washing, anti-mouse IgG, IgG1 or IgG2a–horseradish peroxidase conjugates (ThermoFisher) were used as secondary antibodies, and 3,3′,5,5′-tetramethylbenzidine (TMB) (KPL) was used as the substrate to detect antibody responses. Endpoint titers were calculated as the dilution that emitted an optical density exceeding four times background from secondary antibody alone and extrapolated from a 4-parameter nonlinear fit curve using GraphPad Prism software. Pseudovirus microneutralization assay To produce SARS-CoV-2 pseudoviruses, a codon-optimized CMV/R-SARS-CoV-2 S (Wuhan-1, GenBank #: MN908947.3) plasmid was constructed. A single amino acid mutation was performed to change residue 614 to a glycine (G) to better reflect the more transmissible, predominant circulating strain of SARS-CoV-2. Pseudoviruses were produced by co-transfection of plasmids encoding a luciferase reporter, lentivirus backbone, and S genes into HEK293T/17 cells (ATCC), as previously described ( ). A plasmid for expression of TMPRSS2 was also co-transfected ( ). Pseudoneutralization assays were performed as previously described ( ). Briefly, heat-inactivated (HI)-serum was mixed with pseudovirus, incubated, and then added to 293T-ACE2 cells. Three days later, cells were lysed, and luciferase activity (relative light units, RLU) was measured. Percent neutralization was calculated considering uninfected cells as 100% neutralization and cells transduced with pseudovirus as 0% neutralization. IC 50 titers were determined based on 5-parameter nonlinear curve fit using GraphPad Prism software. Intracellular cytokine staining and flow cytometry Mononuclear single cell suspensions from whole mouse spleens were generated using a gentleMACS tissue dissociator (Miltenyi Biotec) followed by 70 μm filtration and density gradient centrifugation using Fico/Lite-LM medium (Atlanta Biologicals). Cells from each mouse were resuspended in R10 media (RPMI 1640 supplemented with Pen-Strep antibiotic, 10% HI-FBS, Glutamax, and HEPES) and incubated for 6 hr at 37°C with protein transport inhibitor cocktail (eBioscience) under four conditions: no peptide (DMSO only) stimulation, stimulation with two spike peptide pools (S1 and S2 peptide pools, 85% pure, JPT), and stimulation with a nucleocapsid peptide pool (N) (PepmixTM JPT product PM-WCPV-NCAP). Peptide pools were used at a final concentration of 2 μg/ml each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: I-A/I-E (M5/114.15.2) PE, CD8α (53-6.7) BUV805, CD44 (IM7) BUV395, CD62L (MEL-14) BV605, and CD4 (RM4-5) BV480 in brilliant stain buffer (BD). After 15 min at RT, cells were washed with FC buffer, then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to manufacturer instructions. Cells were washed in perm/wash solution and stained with Fc Block (5 min at RT), followed by intracellular staining (30 min at 4°C) using a cocktail of the following antibodies purchased from BD, Biolegend, or eBioscience: CD3 (17A2) BUV737, IFNγ (XMG1.2) BV650, TNFα (MP6-XT22) BV711, IL-2 (JES6-5H4) BV421, IL-4 (11B11) Alexa Fluor 488, IL-5 (TRFK5) APC, and IL-13 (eBio13A) PE-Cy7 in 1x perm/wash diluted with brilliant stain buffer. Finally, cells were washed in perm/wash solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in . Background cytokine expression in the no peptide condition was subtracted from that measured in the S1, S2, and N peptide pools for each individual mouse. Antigen-specific CD4 + T cells, including Tfh cells were examined using an activation induced marker (AIM) assay. Splenocytes were resuspended in R10 media containing BD Fc Block and anti-CD154 (CD40L) antibody conjugated to APC (BD, clone: MR1) and incubated for 6 hr at 37°C under three conditions: no peptide (DMSO only) stimulation, and stimulation with the S1 and S2 peptide pools. Peptide pools were used at a final concentration of 2 μg/ml each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: CD3 (17A2) BUV737, CD4 (RM4-5) BV480, I-A/I-E (M5/114.15.2) PE, CD44 (IM7) BUV395, CD62L (MEL-14) PE-Cy7, CXCR5 (2G8) PE-CF594, PD-1 (J43) BV421, CD69 (H1.2F3) BV605. After 15 min at RT, cells were washed in FC stain buffer solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in A. Background cytokine expression in the no peptide condition (DMSO) was subtracted from that measured in the S1 and S2 peptide pools for each individual mouse, with representative upregulation of activation induced markers (CD69 and CD40L) shown in B. B cells and plasma cells were examined using a separate panel, which included molecular probes for identification of antigen-specific cells. Directly ex vivo , splenocytes were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend, and NIH Vaccine Research Center (VRC) generated B cell capture probes: CD3 (17A2) PE, CD19 (1D3) BUV737, B220 (RA3-6B2) BV711, CD138 (281-2) BV605, T and B cell activation marker (GL7) PE-Cy7, CD95/Fas (Jo2) BUV563, CD4 (RM4-5) BUV805, CD44 (IM7) BUV395, PD-1 (J43) APC-R700, CXCR5 (2G8) PE-CF594, IgD (11-26c.2a) APC-H7, IgM (RMM-1) BV510, S-2P probe APC, S-2P probe BB515, and RBD-SD1 probe BV421. After 15 min at RT, cells were washed in FC stain buffer solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in A. SARS-CoV-2 MA10 infection and viral titer measurements For virus challenge, mice were anesthetized (ketamine/xylazine) and infected intranasally with 10 4 (study 1) or 10 5 (study 2) PFU SARS-CoV-2 MA10 diluted in 50 μL PBS. Clinical signs of disease (weight loss) were monitored daily. Mice were euthanized by isoflurane overdose at days 2, 4, and 7 and samples for titer (caudal right lung lobe) and histopathological analyses (left lung lobe) were collected. Lung viral titers were determined using plaque assays. Briefly, right caudal lung lobes were homogenized in 1 mL PBS using glass beads and serial dilutions of the clarified lung homogenates were added to a monolayer of Vero E6 cells. After three days, plaques were visualized via staining with Neutral Red dye and counted. The left lung lobe was stored in 10% phosphate buffered formalin for seven days prior to removal from the BSL3 for processing. Cytokine and chemokine analysis BioPlex Pro mouse cytokine 23-plex assay (Bio-Rad) was utilized to analyze chemokines and cytokines in clarified lung supernatants according to manufacturer’s protocol. 50 μL of clarified lung samples were incubated with magnetic capture beads, washed, incubated with detection antibodies and streptavidin-PE. Cytokines were recorded on a MAGPIX machine (Luminex) and quantitated via comparison to a standard curve. Graphpad prism software was used for data analysis. Lung Histopathology and Immunohistochemistry Lung samples from mice were processed per a standard protocol. Briefly, the tissues were fixed in 10% neutral buffered formalin, processed with Leica ASP6025 tissue processor (Leica Microsystems), embedded in paraffin, and sectioned at 5 μm for histological analysis. Tissue sections were stained with hematoxylin and eosin (H&E) for routine histopathology. Sections were examined in a blinded manner by a board-certified veterinary pathologist using an Olympus BX51 light microscope, and photomicrographs were taken using an Olympus DP73 camera. For immunohistochemical (IHC) evaluation, formalin-fixed paraffin-embedded tissue sections (5 μm) were used to perform immunohistochemical staining using antibodies which identify SARS-CoV-2 nucleocapsid and eosinophils in tissue, respectively; for SARS-CoV-2 nucleocapsid, a rabbit polyclonal SARS-CoV-2 (GeneTex, Irvine CA, GTX135357) with dilution of 1:2000 was used. For identification of tissue eosinophils, a goat polyclonal Eosinophil Major Basic Protein (EMBP; SantaCruz, Dallas TX, sc-33938) at a dilution of 1:500 was employed. Staining was carried out on the Bond RX (Leica Biosystems) platform according to manufacturer-supplied protocols. Briefly, 5 μm-thick sections were deparaffinized and rehydrated. Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1, pH 6.0, heated to 100°C for 20 min. The specimen was then incubated with hydrogen peroxide to quench endogenous peroxidase activity prior to applying the primary antibody. Detection with DAB chromogen was completed using the Bond Polymer Refine Detection kit (Leica Biosystems CAT# DS9800). Slides were finally cleared through gradient alcohol and xylene washes prior to mounting and placing coverslips. Sections were examined by a board-certified veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera. 

Vectors encoding SARS-CoV-2 S-2P were generated as previously described ( ). Proteins were expressed by transfection of plasmids into Expi293 cells using Expifectamine transfection reagent (ThermoFisher) in suspension at 37 °C for 4–5 days. Transfected cell culture supernatants were collected, buffer exchanged into 1 × PBS, and protein was purified using Strep-Tactin resin (IBA). For SARS-CoV-2 S-2P used for mouse inoculations, tags were cleaved with addition of HRV3C protease (ThermoFisher) (1% wt/wt) overnight at 4 °C. Size-exclusion chromatography using Superose 6 Increase column (GE Healthcare) yielded final purified protein. A mammalian codon-optimized plasmid encoding foldon inserted minifibritin previously described ( ) was used to compete foldon-specific antibodies where indicated. 

Products NR-3882 and NR-3883 were obtained from Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and used for lower-dose (0.2 μg) and higher-dose (1 μg) DI CoV-1 immunization, respectively, in the rear hind legs of BALB/cJ mice. These products were originally intended for active immunization for SARS-CoV-1. A seed virus was prepared, and sucrose purified prior to double inactivation with both formaldehyde and UV irradiation, and the final product was formulated with alum at a final concentration of 5 or 10 μg/mL (NR-3882 and NR-3883, respectively). Formalin-inactivated SARS-CoV-2 was produced under contract with Battelle Biomedical Research Center (BBRC) and was kindly provided by DMID. Briefly, SARS-CoV-2 (isolate USA-WA1/2020, BEI resources NR-52352) grown on Vero E6 cells was harvested on day 2, and clarified supernatant was inactivated using formalin (37% formaldehyde, Fisher Scientific) at a concentration of 0.05% at 37°C for a minimum of 48 hours. Inactivation was confirmed using plaque assays, and protein quantitated using a Bradford assay testing kit. The material was formulated with Alhydrogel® adjuvant (InvivoGen catalog vac-alu-250) at a 1:1 ratio (v/v), and doses of 0.2 or 2 μg were injected into the rear hind limbs of BALB/c mice. Heat-denatured spike protein was produced by heating SARS-CoV-S-2P protein ( ) for 10 minutes at 95°C. Denatured protein was formulated 1:1 (v/v) with Alhydrogel and doses of either 0.2 or 1 ug were injected into the rear hind limbs. Research-grade mRNA-1273, sequence-optimized mRNA encoding SARS-CoV-2 S-2P protein formulated in a lipid nanoparticle (LNP), was produced and purified at Moderna, Inc. as previously described ( ). Mice were immunized in the rear hind limb with doses of either 0.1 or 1 μg of mRNA-1273. 

Nunc Maxisorp ELISA plates (ThermoFisher) were coated with 100 ng of protein in 1X PBS at 4°C for 16 hr. After standard washes and blocks, plates were incubated with serial dilutions of sera for 1 hr at room temperature (RT). Where applicable, to block binding of foldon-specific antibodies to SARS-CoV-2 S-2P protein, sera dilutions were preincubated with 50 μg/mL of foldon protein for 1 hour at RT prior to adding to the plate. After washing, anti-mouse IgG, IgG1 or IgG2a–horseradish peroxidase conjugates (ThermoFisher) were used as secondary antibodies, and 3,3′,5,5′-tetramethylbenzidine (TMB) (KPL) was used as the substrate to detect antibody responses. Endpoint titers were calculated as the dilution that emitted an optical density exceeding four times background from secondary antibody alone and extrapolated from a 4-parameter nonlinear fit curve using GraphPad Prism software. 

To produce SARS-CoV-2 pseudoviruses, a codon-optimized CMV/R-SARS-CoV-2 S (Wuhan-1, GenBank #: MN908947.3) plasmid was constructed. A single amino acid mutation was performed to change residue 614 to a glycine (G) to better reflect the more transmissible, predominant circulating strain of SARS-CoV-2. Pseudoviruses were produced by co-transfection of plasmids encoding a luciferase reporter, lentivirus backbone, and S genes into HEK293T/17 cells (ATCC), as previously described ( ). A plasmid for expression of TMPRSS2 was also co-transfected ( ). Pseudoneutralization assays were performed as previously described ( ). Briefly, heat-inactivated (HI)-serum was mixed with pseudovirus, incubated, and then added to 293T-ACE2 cells. Three days later, cells were lysed, and luciferase activity (relative light units, RLU) was measured. Percent neutralization was calculated considering uninfected cells as 100% neutralization and cells transduced with pseudovirus as 0% neutralization. IC 50 titers were determined based on 5-parameter nonlinear curve fit using GraphPad Prism software. 

Mononuclear single cell suspensions from whole mouse spleens were generated using a gentleMACS tissue dissociator (Miltenyi Biotec) followed by 70 μm filtration and density gradient centrifugation using Fico/Lite-LM medium (Atlanta Biologicals). Cells from each mouse were resuspended in R10 media (RPMI 1640 supplemented with Pen-Strep antibiotic, 10% HI-FBS, Glutamax, and HEPES) and incubated for 6 hr at 37°C with protein transport inhibitor cocktail (eBioscience) under four conditions: no peptide (DMSO only) stimulation, stimulation with two spike peptide pools (S1 and S2 peptide pools, 85% pure, JPT), and stimulation with a nucleocapsid peptide pool (N) (PepmixTM JPT product PM-WCPV-NCAP). Peptide pools were used at a final concentration of 2 μg/ml each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: I-A/I-E (M5/114.15.2) PE, CD8α (53-6.7) BUV805, CD44 (IM7) BUV395, CD62L (MEL-14) BV605, and CD4 (RM4-5) BV480 in brilliant stain buffer (BD). After 15 min at RT, cells were washed with FC buffer, then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to manufacturer instructions. Cells were washed in perm/wash solution and stained with Fc Block (5 min at RT), followed by intracellular staining (30 min at 4°C) using a cocktail of the following antibodies purchased from BD, Biolegend, or eBioscience: CD3 (17A2) BUV737, IFNγ (XMG1.2) BV650, TNFα (MP6-XT22) BV711, IL-2 (JES6-5H4) BV421, IL-4 (11B11) Alexa Fluor 488, IL-5 (TRFK5) APC, and IL-13 (eBio13A) PE-Cy7 in 1x perm/wash diluted with brilliant stain buffer. Finally, cells were washed in perm/wash solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in . Background cytokine expression in the no peptide condition was subtracted from that measured in the S1, S2, and N peptide pools for each individual mouse. Antigen-specific CD4 + T cells, including Tfh cells were examined using an activation induced marker (AIM) assay. Splenocytes were resuspended in R10 media containing BD Fc Block and anti-CD154 (CD40L) antibody conjugated to APC (BD, clone: MR1) and incubated for 6 hr at 37°C under three conditions: no peptide (DMSO only) stimulation, and stimulation with the S1 and S2 peptide pools. Peptide pools were used at a final concentration of 2 μg/ml each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: CD3 (17A2) BUV737, CD4 (RM4-5) BV480, I-A/I-E (M5/114.15.2) PE, CD44 (IM7) BUV395, CD62L (MEL-14) PE-Cy7, CXCR5 (2G8) PE-CF594, PD-1 (J43) BV421, CD69 (H1.2F3) BV605. After 15 min at RT, cells were washed in FC stain buffer solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in A. Background cytokine expression in the no peptide condition (DMSO) was subtracted from that measured in the S1 and S2 peptide pools for each individual mouse, with representative upregulation of activation induced markers (CD69 and CD40L) shown in B. B cells and plasma cells were examined using a separate panel, which included molecular probes for identification of antigen-specific cells. Directly ex vivo , splenocytes were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN 3 ) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend, and NIH Vaccine Research Center (VRC) generated B cell capture probes: CD3 (17A2) PE, CD19 (1D3) BUV737, B220 (RA3-6B2) BV711, CD138 (281-2) BV605, T and B cell activation marker (GL7) PE-Cy7, CD95/Fas (Jo2) BUV563, CD4 (RM4-5) BUV805, CD44 (IM7) BUV395, PD-1 (J43) APC-R700, CXCR5 (2G8) PE-CF594, IgD (11-26c.2a) APC-H7, IgM (RMM-1) BV510, S-2P probe APC, S-2P probe BB515, and RBD-SD1 probe BV421. After 15 min at RT, cells were washed in FC stain buffer solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in A. 

For virus challenge, mice were anesthetized (ketamine/xylazine) and infected intranasally with 10 4 (study 1) or 10 5 (study 2) PFU SARS-CoV-2 MA10 diluted in 50 μL PBS. Clinical signs of disease (weight loss) were monitored daily. Mice were euthanized by isoflurane overdose at days 2, 4, and 7 and samples for titer (caudal right lung lobe) and histopathological analyses (left lung lobe) were collected. Lung viral titers were determined using plaque assays. Briefly, right caudal lung lobes were homogenized in 1 mL PBS using glass beads and serial dilutions of the clarified lung homogenates were added to a monolayer of Vero E6 cells. After three days, plaques were visualized via staining with Neutral Red dye and counted. The left lung lobe was stored in 10% phosphate buffered formalin for seven days prior to removal from the BSL3 for processing. 

BioPlex Pro mouse cytokine 23-plex assay (Bio-Rad) was utilized to analyze chemokines and cytokines in clarified lung supernatants according to manufacturer’s protocol. 50 μL of clarified lung samples were incubated with magnetic capture beads, washed, incubated with detection antibodies and streptavidin-PE. Cytokines were recorded on a MAGPIX machine (Luminex) and quantitated via comparison to a standard curve. Graphpad prism software was used for data analysis. 

Lung samples from mice were processed per a standard protocol. Briefly, the tissues were fixed in 10% neutral buffered formalin, processed with Leica ASP6025 tissue processor (Leica Microsystems), embedded in paraffin, and sectioned at 5 μm for histological analysis. Tissue sections were stained with hematoxylin and eosin (H&E) for routine histopathology. Sections were examined in a blinded manner by a board-certified veterinary pathologist using an Olympus BX51 light microscope, and photomicrographs were taken using an Olympus DP73 camera. For immunohistochemical (IHC) evaluation, formalin-fixed paraffin-embedded tissue sections (5 μm) were used to perform immunohistochemical staining using antibodies which identify SARS-CoV-2 nucleocapsid and eosinophils in tissue, respectively; for SARS-CoV-2 nucleocapsid, a rabbit polyclonal SARS-CoV-2 (GeneTex, Irvine CA, GTX135357) with dilution of 1:2000 was used. For identification of tissue eosinophils, a goat polyclonal Eosinophil Major Basic Protein (EMBP; SantaCruz, Dallas TX, sc-33938) at a dilution of 1:500 was employed. Staining was carried out on the Bond RX (Leica Biosystems) platform according to manufacturer-supplied protocols. Briefly, 5 μm-thick sections were deparaffinized and rehydrated. Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1, pH 6.0, heated to 100°C for 20 min. The specimen was then incubated with hydrogen peroxide to quench endogenous peroxidase activity prior to applying the primary antibody. Detection with DAB chromogen was completed using the Bond Polymer Refine Detection kit (Leica Biosystems CAT# DS9800). Slides were finally cleared through gradient alcohol and xylene washes prior to mounting and placing coverslips. Sections were examined by a board-certified veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera. 

Statistical Analysis Serological responses, T and B cell readouts, and viral titer data were analyzed using Graphpad Prism using the methods outlined in each figure legend. Nonparametric methods were used when groups contained fewer than 10 mice per group. 

Serological responses, T and B cell readouts, and viral titer data were analyzed using Graphpad Prism using the methods outlined in each figure legend. Nonparametric methods were used when groups contained fewer than 10 mice per group. 

