key: cord-1023680-o1exzkwo
authors: DiPiazza, Anthony T.; Leist, Sarah R.; Abiona, Olubukola M.; Moliva, Juan I.; Werner, Anne; Minai, Mahnaz; Nagata, Bianca M.; Bock, Kevin W.; Phung, Emily; Schäfer, Alexandra; Dinnon, Kenneth H.; Chang, Lauren A.; Loomis, Rebecca J.; Boyoglu-Barnum, Seyhan; Alvarado, Gabriela S.; Sullivan, Nancy J.; Edwards, Darin K.; Morabito, Kaitlyn M.; Mascola, John R.; Carfi, Andrea; Corbett, Kizzmekia S.; Moore, Ian N.; Baric, Ralph S.; Graham, Barney S.; Ruckwardt, Tracy J.
title: 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
date: 2021-07-02
journal: Immunity
DOI: 10.1016/j.immuni.2021.06.018
sha: 6bb00f924c32ab820fe40382e105b4ffb205dbae
doc_id: 1023680
cord_uid: o1exzkwo

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of SARS and MERS coronavirus vaccines. We used the SARS-CoV-2 MA10 mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post-challenge, but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.

Since its discovery in late 2019, SARS-CoV-2 (the causative agent of 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 (Sanders and Moore, 2021) , stabilization of the prefusion conformation of the neutralization-sensitive viral spike (S) glycoprotein into a form known as S-2P (Pallesen et al., 2017) 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 (Corbett et al., 2020a ) and non-human primate (NHP) (Corbett et al., 2020b ) models, elicited neutralizing antibody and T cell responses in phase 1 clinical trials in adult and older adult populations J o u r n a l P r e -p r o o f Jackson et al., 2020) , 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 vaccineassociated 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 (Munoz et al., 2021) ). Whole-inactivated viral vaccines developed in the 1960's against both respiratory syncytial virus (RSV) and measles elicited VAERD when vaccinated children were subsequently naturally infected (Fulginiti et al., 1967; Kim et al., 1969; Nader et al., 1968; Polack et al., 2003; Ruckwardt et al., 2019) . 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 (Graham et al., 1993) . While there has been no licensed prophylactic intervention for coronaviruses, there have been multiple findings of VAERD in preclinical studies of both 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 Graham, 2020; Haynes et al., 2020; Lambert et al., 2020; Peeples, 2020; Smatti et al., 2018; Zellweger et al., 2020) . While 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 lethal mouse-adapted SARS-CoV-2 MA10 virus Leist et al., 2020) .

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 spike protein were formulated with alum to recapitulate conditions that resulted in J o u r n a l P r e -p r o o f 4 VAERD in a prior preclinical coronavirus vaccine study (Bolles et al., 2011) . These regimens consistently induced low to moderate concentrations of spike-binding and neutralizing antibody and Th2-skewed spike-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.

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 spike 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 (Figure 1A ) with the goal of inducing binding antibodies with little to no neutralizing activity (for disposition of animals in the study, see Table   S1 ). 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 (Bolles et al., 2011) . BALB/c mice were immunized twice three weeks apart with each vaccine or phosphate-buffered saline (PBS) as a control, and humoral and cellular responses to S were J o u r n a l P r e -p r o o f assessed two weeks after the boost. We first quantified binding 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 ( Figure 1B) . Cross-reactive antibody responses elicited by whole SARS-CoV-1 inactivated with both UV irradiation and formalin (DI CoV-1) were lower than responses elicited by the homologous, denatured SARS-CoV-2 spike 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 better antibody responses than either dose of CoV-2 DS, consistent with potent elicitation of antibodies previously described (Corbett et al., 2020a) .

Neutralizing activity against a SARS-CoV-2 spike 614G-pseudotyped virus was measured to assess neutralization of the predominant circulating virus. Mice immunized with 1 µg mRNA-1273 had an IC 50 geometric mean titer (GMT) of 4886. 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 ( Figure 1C ). 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 ( Figure 1D ). Based on these results, mRNA-1273 elicited antibodies with higher average neutralizing potency than approaches aimed at Th2skewing the spike-directed response.

To gain insight into the induction of type 1 and type 2 immunity, we further assessed the IgG subclass of spike-directed antibodies. In mice, while the type 1 cytokine IFN promotes B cell secretion of IgG2a and inhibits IgG1, the type 2 cytokine IL-4 promotes IgG1 and inhibits the J o u r n a l P r e -p r o o f secretion of IgG2a antibodies (Coffman et al., 1993) . As previously shown (Corbett et al., 2020a) , mRNA-1273 elicited high titers of both spike-binding IgG2a and IgG1 (Figure 2A and Figure 2B , respectively). mRNA-1273 given at the low dose of 0.1 µg elicited more spikebinding IgG2a than seen in any group immunized with DI CoV-1 or CoV-2 DS (Figure 2A) . In contrast, type 2-skewing regimens elicited IgG1 responses more similar to those elicited by 0.1 µg mRNA-1273 ( Figure 2B) , 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 ( Figure 2C ). Taken together, serological assessment of spike-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 spike-reactive cells in the spleen two 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, TNFα, and IL-2) and three type 2 cytokines after in vitro restimulation with overlapping peptide pools spanning the S1 and S2 portions of the spike protein ( Figure 2D and 2E, respectively, gating strategy presented in Figure S1 ) or the SARS-CoV-2 N protein ( Figure 2F ).

While 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 ( Figure 2F ). While 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 Figure   S2A ). 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 1µg of mRNA-1273, and this was the only immunization group with strong induction of spike-specific CD8 + T cell responses ( Figure S2B ). mRNA-1273 immunization limits viral replication, morbidity, and pulmonary inflammation following mouse-adapted SARS-CoV-2 viral challenge J o u r n a l P r e -p r o o f Twenty mice from each group were challenged with 10 4 PFU of mouse-adapted SARS-CoV-2 MA10 (MA10) five weeks after the boost immunization ( Figure 1A , Table S1 ). 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 ( Figure 3A ). Viral titers were measured in the nasal turbinates by plaque assay on day 2 ( Figure 3B ) and day 4 ( Figure 3C ) 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 two 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 (Corbett et al., 2020a) . In addition to protection in the nasal turbinates, 1 µg mRNA-1273 immunization completely prevented viral infection in the lungs at both day 2 ( Figure 3D ) and day 4 ( Figure   3E ) 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 Figure S3 , for scoring for each animal see Table S2 ). 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 (Figure 4) . 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 (Figure 4) . These data are consistent with viral titer measured on day 4 ( Figure 3E ). 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 mRNAimmunized mice (Figure 4) . 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-1273immunized 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 ( Figure 5A ). 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 ( Figure 5B ). While there was no statistical increase in proinflammatory cytokines in Th2skewed 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, while 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 (MIP-1α, MIP-1β, eotaxin, and CXCL1(KC), Figure 5C ). 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-J o u r n a l P r e -p r o o f skewing regimens, consistent with eosinophil major basic protein (EMBP) staining ( Figure 4) and with what was observed in earlier studies of DI CoV1-enhanced disease following mouseadapted SARS-CoV infection (Bolles et al., 2011) . 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. While 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 ( Figure 6A , see Table S3 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 ( Figure 6B and Figure 6C , respectively).

Again, while mRNA-1273 elicited both IgG2a and IgG1 subclasses of spike-specific IgG, the response to 2 µg FI CoV-2 was dominated by IgG1, suggesting a Th2-biased immune response pattern ( Figure 6D and E). 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 J o u r n a l P r e -p r o o f ( Figure S4 ). 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 ( Figure S4 ).

We elected to challenge mice for the second study with an increased dose (10-fold) of 10 5 PFU of MA10. There was less weight loss than seen in the first study, with no group showing sustained differences from the PBS control group ( Figure S5A ). 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 ( Figures S5B and S5C ) and no detectable virus in the lungs ( Figures S5D 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 timepoints ( Figure   S5B -E). 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 ( Figure S5F ). 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 Table S4 ). Animals immunized with FI CoV-2 in alum experienced higher concentrations of Th2 cytokines, and proinflammatory and chemotactic mediators ( Figure S6 ). While 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.

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 J o u r n a l P r e -p r o o f of germinal centers (GCs), and support the clonal selection and differentiation of antigen-specific memory B cells and long-lived plasma cells (Crotty, 2019) . 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 spike protein and/or the neutralization-sensitive receptor binding domain (RBD) using tetramerized fluorochrome-conjugated capture probes ( Figure 7A , for gating and representative plots, see Figure S7A ). Two 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 ( Figure 7B 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 ( Figure 7E ) (Lee et al., 2021) . 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-hour 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 spike-derived peptides in higher frequencies than we measured using conventional ICS ( Figure 7F , for representative staining see Figure S7B ). 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 ( Figure 7F) , 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 wellcoordinated cellular and serological response ( Figure 7G) . These data further support the J o u r n a l P r e -p r o o f 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 spike-directed IgG2a antibodies may increase the potential for Fc-mediated effector functions, including direct killing (ADCC) and phagocytosis (ADCP) of target cells due to increased activating FcR (FcRI, FcRIII, FcRIV) engagement by innate immune cells relative to IgG1 (a more functionally restricted isotype in mice) (Bruhns, 2012) . 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 spike 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 Graham, 2020) . Using ICS, we determined that T cells elicited by inactivated virus and J o u r n a l P r e -p r o o f 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 spike and N-specific T cells elicited by heterologous DI CoV-1. IgG subclass determination for spike-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 spike-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.

While mRNA vaccines have previously been shown to strongly induce both IgG2a and IgG1 (Corbett et al., 2020a; Laczko et al., 2020; Lederer et al., 2020) , we consistently observed a doseresponse 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 vs type 2 responses (Constant and Bottomly, 1997) . Notably, 0.1 µg is a lower dose than tested for other mRNA COVID vaccines (Laczko et al., 2020; Lederer et al., 2020; Vogel et al., 2020) , 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 spike-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 spike-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 (Laczko et al., 2020; Lederer et al., 2020; Vogel et al., 2020) . The elevated production of IFN by T cells in the 1 µg mRNA-1273 dose group may reflect a threshold needed to achieve increased IL-12-mediated polarization towards a "type 1" immune program by activated antigen J o u r n a l P r e -p r o o f presenting cells (APC). It is likely that higher concentrations of IFN 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 MHC class I and II molecules (de Araujo-Souza et al., 2015; Whitmire et al., 2005) .

During natural SARS-CoV-2 infection, IFNγ does not appear to strongly dominate the spikespecific 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 (Braun et al., 2020; Law et al., 2020; Peng et al., 2020; Sekine et al., 2020) . A similar pattern of cytokine expression is also observed following human vaccination with mRNA-1273 and a recombinant spike protein nanoparticle vaccine (NVX-CoV2373) (Keech et al., 2020) . Whether this reflects immune modulation by SARS-CoV-2 or may be intrinsic to spike-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 (Dan et al., 2016; Nakiboneka et al., 2019) . We suspect that the IL-2 + IFN -CD4 + T cells detected two 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 (Mosmann et al., 2009) . 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 + IFNcells (referred to as primed precursor Th or Thpp cells) include chemokine production for immune cell recruitment to lymphoid tissues (Mosmann et al., 2009; Yang and Mosmann, 2004) .  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 spike-specific T cells. Using the AIM assay, we measured 10 to 20-fold more spike-specific CD4 + T cells than were detected using the ICS and demonstrated a high frequency of spike-specific Tfh cells, supporting the selection of high-J o u r n a l P r e -p r o o f 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 (Pardi et al., 2018) . A single immunization with mRNA encoding spike demonstrated a superior capacity to induce germinal center responses compared to adjuvanted protein . This could contribute to the observed efficacy of mRNA-1273 (NIAID, NIH and Moderna, Inc.) 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 spike protein that contribute to effective antiviral immunity. It has demonstrated the ability to elicit potent and effective spike-specific immunity in NHP (Corbett et al., 2020b) and humans Jackson et al., 2020; Widge et al., 2020) and demonstrated 94.1% efficacy in a phase 3 trial (Baden et al., 2020) . 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.

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. While animal models can reproduce RSV-and measles-driven VAERD (De Swart et al., 2002; Johnson and Graham, 1999; Johnson et al., 2003; Johnson et al., 2004; Polack et al., 2003) , it is difficult to implicate the same 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-1273mediated 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 spike 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 (Chen and Wherry, 2020; DiPiazza et al., 2020; Jarjour et al., 2020; Lipsitch et al., 2020) . 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. While antibodies to N are more frequently implicated in VAERD (Bolles et al., 2011; Deming et al., 2006; Yasui et al., 2008) , enhanced immune pathology consistent with that which we observed for denatured spike alone has been previously noted following spike vaccination (Honda-Okubo et al., 2015) . We note that antibodies to denatured spike appear to be lower potency, but the sensitivity of SARS-CoV-2 neutralization assays when spike-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 speaks to a protective role for antibody Weinreich et al., 2020) , but selected antibodies are generally high potency. As viral J o u r n a l P r e -p r o o f 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 spike 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. shown. The limit of detection is indicated by a dotted line, and values below the limit of detection were assigned a value of half the limit of detection. See also Table S1. by a dotted line, and values below the limit of detection were assigned a value of half the limit of detection. (D-F) At week 5, splenocytes (N=10 mice per group) were isolated and stimulated in the presence of protein transport inhibitor (Thermo product 00-4980-03) and no peptides (DMSO only), or pools of overlapping peptides from the nucleocapsid (Pepmix TM JPT product PM-WCPV-NCAP) or spike (S1 and S2 peptide pools, 85% pure, JPT) proteins of SARS-CoV-2 (2 µg per mL each peptide). After 6 hours, intracellular cytokine staining (ICS) was performed to quantify CD4 + T cells producing the cytokines IFNγ, TNFα, IL-2, IL-4, IL-5, and IL-13 in response to (D) S1 peptide pool, (E) S2 peptide pool, and (F) N peptide pool restimulation.

Background cytokine expression in the no peptide condition was subtracted from that measured in the N, S1 and S2 peptide pools for each individual mouse. Error bars represent the SEM. Data were analyzed by two-way ANOVA with Dunnett's multiple comparisons tests to determine expression significantly different from the naïve group for each cytokine. Significance is indicated above for each group (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). See also Figure S1 and Figure S2 . Table S2 . See also Figure S3 and Table S2 . Probe-binding GC B cells, non-GC (GL7 -Fas -) B cells, and mature, class-switched plasma cells (CD138 + B220 lo ) were assessed. The numbers above each bar reflect the number of mice with measurable binding reactivity above 0.0% (E) Gating strategy to quantify AIM + CD4 + T cells.

(F) Frequency of AIM + CD44 + CD4 + T cells and Tfh cells following stimulation with S1 and S2 peptide pools (background subtracted) (G) Correlations of % AIM + CD4 + Tfh cells (r = 0.79) and % S-2P + GC B cells (r = 0.62) to antibody neutralization titer (IC 50 ) from mice immunized with mRNA-1273 (0.1 and 1 µg). N=10 mice per group, data were analyzed using a Kruskal-Wallis test comparing all immunization to the PBS group (*p<0.05, **p<0.01, ***p<0.001). See also Figure S7 .

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 

SARS-CoV-2 MA10 was generated by serially passaging SARS-CoV-2 MA stock virus in the lungs of mice as previously described Leist et al., 2020) . 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.

Vectors encoding SARS-CoV-2 S-2P were generated as previously described (Wrapp et al., 2020) . 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 (Corbett et al., 2020a) 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 higherdose (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 respectively) . Formalin-inactivated SARS-CoV-2 was produced under contract with Battelle Biomedical Research Center (BBRC) and was kindly provided by J o u r n a l P r e -p r o o f 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 (Wrapp et al., 2020) 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. Researchgrade 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 (Corbett et al., 2020a) . 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 hrs. 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 4parameter 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 J o u r n a l P r e -p r o o f of plasmids encoding a luciferase reporter, lentivirus backbone, and S genes into HEK293T/17 cells (ATCC), as previously described (Wang et al., 2015) . A plasmid for expression of TMPRSS2 was also co-transfected (Bottcher et al., 2006) . Pseudoneutralization assays were performed as previously described (Corbett et al., 2020a) . 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 5parameter 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. 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 Figure S1 . 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. 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 Figure 7A . 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 Figure S7B . Figure S7A .

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 L 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.

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

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 o 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 cover slips. Sections were examined by a board-certified veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera.

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.

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