response.
To address this critically important knowledge gap, we collected plasma samples from 35 49 hospitalized acutely SARS-CoV-2 infected subjects and 105 convalescent subjects 5 (Supplemental
Tables 1 and 2). Plasma was tested against the spike, N protein, ORF7a, ORF8, and NSP3, NSP9, 51 NSP10, and NSP15 of SARS-CoV-2. Notably, all subjects within the acutely infected cohort were 52 hospitalized, whereas only 9% (8/105) of subjects in the convalescent cohort had been hospitalized 53 (Supplemental Tables 1 and 2 ). 89% of acutely infected subjects and 98% of convalescent subjects 54 had detectable antibodies against one or more SARS-CoV-2 antigens (Fig. 1a) , with nearly all 55 subjects mounting a response against the spike and N protein (Fig. 1b) . We further identified that 56 convalescent subjects mounted a predominant response against the RBD of the spike protein and the 57 RNA-binding domain of the N protein (Extended data Fig. 1a and b), suggesting these domains 58 contain the immunodominant epitopes of these antigens. A larger frequency of acutely infected 59 subjects mounted antibodies against ORF7a, ORF8, and NSP antigens (Fig. 1b , Extended data Fig.   60 1c) suggesting antibodies against these antigens are either short-lived or only induced by more 61 severe infection. Moreover, the anti-N protein antibody response preceded the antibody response 62 against the spike protein and was consistently higher across all time points, peaking between the 2 nd 63 and 3 rd week after the onset of symptoms and retracting between the 3 rd and 4 th weeks after symptom 64 onset ( Fig. 1c and Extended data Fig. 1d ). Furthermore, we found a strong positive correlation 65 between the anti-N protein and anti-spike IgG titers in both the acutely infected and convalescent 66 cohorts (Extended data 1e and f), indicating subjects who generally mounted a robust antibody 67 response upon SARS-CoV-2 infection tended to mount a robust response against both antigens.
Titers against both the spike and N protein persisted even 2+ months after symptom onset (Fig. 1c) , 69 indicating the antibody response against these two antigens is stable amongst subjects with 70 symptomatic infection, a finding consistent with other reports 6,7 . We did not observe a statistical 71 difference in antibody titers against the spike and N protein by individual subjects in either the acute or 72 convalescent subject cohorts ( Fig. 1d and e) , likely due to dramatic subject-to-subject variation.
However, antibody titers against the spike and N protein were significantly higher than antibody titers 74 against ORF7a and ORF8 ( Fig. 1d and e) . Together, these data reveal the antibody response against 75 SARS-CoV-2 is largely driven against the spike and N protein and that the anti-N protein antibody 76 response precedes the response against the spike. The differences in the kinetics and magnitude of 77 the N protein response are potentially due to the differences in protein expression, as N protein 78 completely covers the entire viral genome, whereas a single virion only expresses ~26 trimers 8 .
To understand the inter-subject variability within our cohorts, we performed hierarchical 80 clustering of subjects based on antibody titers against the spike, full length and RNA-binding domain 81 of N protein, ORF7a, and ORF8 antigens. From the acutely infected cohort, we identified three 82 clusters: high, mid, and low responders (Fig. 2a and Supplemental Table 3 ). Notably, the high 83 responder cluster subjects were further from the onset of symptoms at the time of sampling and 84 ultimately were hospitalized for a longer duration than those in the mid and low responder groups ( Fig.   85 2b and c). We did not observe a statistical difference in age or sex between the three responder 86 groups (Extended data Fig. 2a and b) . Over 25% of subjects in the high responder group had a 87 severe/highest CURB-65 score (Extended data Fig. 2c ), a measure of pneumonia severity 9 , 88 suggesting subjects in the high responder group had more severe infections. We further examined 89 which features of the humoral immune response were driving subjects to segregate into these three 90 clusters. Subjects within the high and mid responder groups robustly induced antibodies against the 91 spike protein, but the high responder subjects mounted a larger response to N protein relative to the 92 mid responder subjects (Fig. 2d-f ). Additionally, subjects within the high and mid responder groups 93 were more likely to mount an antibody response against ORF8 and NSP antigens (Extended data Fig.   94 2d and e). The low responder group largely did not mount an antibody response against any of the 95 antigens tested (Fig. 2d-f and Extended Data Fig. 2d and e), although it is possible that plasma was 96 collected before the subjects mounted a significant antibody response. Our data reveal that acutely 97 infected subjects who were hospitalized for a longer duration mounted a larger antibody response 98 against N protein and were more likely to mount a response against other SARS-CoV-2 antigens.
The convalescent cohort also clustered into three distinct clusters based on the magnitude of 100 the antibody response against the spike and N protein ( Fig. 3a and Supplemental Table 4 ), similar to 101 the acutely infected cohort (Fig. 2) . To understand the relationship between infection severity and 102 antibody responses within the convalescent cohort, we scored subjects based on the severity and 103 duration of self-reported symptoms and whether subjects were hospitalized (Supplemental Table 5 ).
Notably, over 50% of subjects within the high responder group had a severe infection ( Fig. 3b and   105 Extended data Fig. 3a) , indicating infection severity is linked to increased antibody titers. Moreover, 106 subjects within the high responder group typically were older and male ( Fig. 3c and d) . Subjects within 107 each responder group had a similar duration of symptoms (Extended data Fig. 3b ), and subjects 108 within all three groups had a similar amount of time to mount a response, as determined by the 109 number of days since symptoms onset at the time of donation (Extended data Fig. 3c ). Unlike the 110 acutely infected cohort, subjects within the high responder group had higher titers against not only the 111 N protein, but also the spike and ORF8 antigens relative to subjects within the mid and low responder 112 groups ( Fig. 3e and f, Extended data Fig. 3d and e), and were trending to be more likely to 113 seroconvert against at least one of the NSP antigens tested (Extended data Fig. 3f ). Consistent with 114 these data, high and mid responder subjects had higher neutralizing titers than subjects in the low 115 responder cohorts (Fig. 3g ). In combination with the acutely infected cohort, our data reveal subjects 
(PBMCs) isolated from convalescent subjects. Notably, MBCs largely targeted the spike, whereas 121 very few MBCs targeted N protein or ORF8 (Fig. 4a) . Additionally, subjects in the serum high 122 responder group mounted a larger MBC response against the spike than subjects in the mid and low 123 responder cohorts (Fig. 4b) , with serum antibody titers against the spike positively correlating with the 124 magnitude of the anti-spike MBC response (Fig. 4c) . Despite the observed differences in anti-spike 125 MBC responses between responder groups, we did not observe any differences in the anti-N protein 126 and anti-ORF8 MBC response in the three responder cohorts (Extended data Fig. 4a and b) .
Together, these data indicate that the MBC response is largely directed against the spike protein, and is located on the interface between two subunits of the spike trimer and may impact stability of the 133 trimer 1 . As the subjects within our study were initially infected throughout March and into early April 134 (Supplemental Tables 1 and 2), they were likely infected with the D614 variant. We did not observe a 135 difference in antibody titers against the WT and D614G spike antigens within our acute cohort ( Fig.   136 5a), suggesting the D614G epitope was not a major antigenic site. Strikingly, we identified that the 137 convalescent cohort mounted a larger response against the G614 variant than the WT D614 that they 138 were likely infected with (Fig. 5b) , potentially due to the increased stability of the G614 variant 11 .
Furthermore, we observed a strong positive correlation between D614 (WT) spike titers and G614 140 titers, indicating antibodies against the WT strain likely protect against the new G614 variant (Fig. 5c ).
These data indicate that the region that encompasses the D614G mutation is not immunodominant or 142 does not affect the antigenicity of epitopes at or near this site. We also examined whether antibodies 148 5f and g). These data show that subjects who mounted a larger response against the SARS-CoV-2 149 spike protein additionally mounted a larger antibody response against conserved epitopes that cross-150 react with closely related coronaviruses.
Together, our study demonstrates that severity of SARS-CoV-2 infection is associated with an 
Subjects also mounted an antibody response against the accessory protein ORF8. ORF8 has 164 immunoregulatory properties including the ability to limit type I interferon responses 13,14 and 165 downregulate MHC-I presentation to CD8 T cells 15 . Antibodies targeting ORF8 may limit these 166 immunoregulatory properties, which could improve the host immune response and achieve better 
Our study revealed acutely infected subjects who mounted higher antibody response relative 173 to mid and low responder clusters tended to have higher pneumonia severity scores. Consistent with 174 this notion, convalescent subjects who had higher antibody titers were those subjects who had a more severe infection. A recent report identified that subjects who succumbed to COVID-19 tended to 176 mount a larger antibody response against N protein relative to the spike, whereas convalescent 177 subjects tended to focus their antibody response on the spike protein 16 . However, our study identified 178 that subjects generally had similar antibody responses against the N protein and spike, and infection 179 severity was linked to an increase in antibody responses against both the spike and N protein.
Ultimately, our findings on the relationship between infection severity and increased titers against the 181 spike is consistent with a recent surveillance study performed in Iceland 6 .
The best clinical predictors of the magnitude of the antibody responses and epitope spreading 183 within our convalescent cohort were age, sex, and hospitalization. Strikingly, the median age of the 184 high responder cluster was 10+ years greater than the mid and low responder clusters (48 years vs. For the acutely infected cohort, CURB-65 9 scores were calculated based on confusion, blood urea 274 nitrate levels, respiratory rate, blood pressure, and age of subjects. For the convalescent cohort, we 275 designed a severity scoring system (Supplemental Table 5 IgM   437  622  122  266  65  68  609  478  223  50  348  112  3  19  117  284  127  309  135  376  209  320  355  281  261  155  92  535  166  103  564  116  433  195  109  229  176  124  63  251  550  201  586  33  219  305  227  519  171  402  172  211  380  279  42  270  278  326  358  336  354  144  385  346  338  423  175  179  468  22  332  272  80  130  407  218  371  230  537  24  258  356  11  20  156  373  141  17  136  558  282  48  573  377  89  451  210  214  293  469  626  108  447  319 