key: cord-1032779-ji3dypsa
authors: Shapiro, J. R.; Park, H.-S.; Aytenfisu, T. Y.; Caputo, C.; Lee, J. S.; Johnston, T. S.; Li, H.; Hauk, P.; Jacobsen, H.; Li, Y.; Abrams, E.; Kocot, A. J.; Yang, T.; Huang, Y.; Cramer, S. M.; Betenbaugh, M. J.; Debes, A. K.; Morgan, R.; Milstone, A. M.; Karaba, A. H.; Leng, S. X.; Klein, S. L.
title: Association of frailty, age, and biological sex with SARS-CoV-2 mRNA vaccine-induced immunity in older adults
date: 2022-03-13
journal: nan
DOI: 10.1101/2022.03.11.22272269
sha: 579414cfa353ae76c3f49217d63b4492459b7273
doc_id: 1032779
cord_uid: ji3dypsa

Background: Male sex and old age are risk factors for severe COVID-19, but the intersection of sex and aging on antibody responses to SARS-CoV-2 vaccines has not been characterized. Methods: Plasma samples were collected from older adults (75-98 years) before and after three doses of SARS-CoV-2 mRNA vaccination, and from younger adults (18-74 years) post-dose two, for comparison. Antibody binding to SARS-CoV-2 antigens (spike protein [S], S-receptor binding domain [S-RBD], and nucleocapsid [N]) and functional activity against S were measured against the vaccine virus and variants of concern (VOC). Results: Vaccination induced greater antibody titers in older females than males, with both age and frailty associated with reduced antibody responses to vaccine antigens in males, but not females. ACE2 binding inhibition declined more than anti-S or anti-S-RBD IgG in the six months following the second dose (28-fold vs. 12- and 11-fold decreases in titer). The third dose restored functional antibody responses and eliminated disparities caused by sex, age, and frailty in older adults. Responses to the VOC were significantly reduced relative to the vaccine virus, with older males having lower titers to the VOC than females. Older adults had lower responses to the vaccine and VOC viruses than younger adults, with disparities being greater in males than females. Conclusion: Older and frail males may be more vulnerable to breakthrough infections due to low antibody responses before receipt of a third vaccine dose. Promoting third dose coverage in older adults, especially males, is crucial to protecting this vulnerable population.

The disproportionate burden of COVID-19 in older adults was recognized early in the 66 pandemic [1-3]. The phase III trials for the two mRNA vaccines (mRNA-1273 and BNT162b2) 67 revealed high efficacy in older adults [4, 5] , for whom immunosenescence is thought to impair 68 vaccine-induced immune responses [6] . Clinical trials, however, often fail to represent the 69 oldest and frailest subset of the population. Accordingly, wide-spread use of the vaccine in 70 long-term care facility residents revealed that old age is a risk factor for poor antibody 71 responses [7] [8] [9] . 72

Male sex also is a significant predictor of severe COVID-19 outcomes at older ages [10-73 14] . There is extensive evidence that the effects of aging on the immune system differ between 74 the sexes, including that immunosenescence occurs at a slower rate in females than males [15, 75 16 ]. The implications of biological sex are evident in the response to repeated seasonal 76 influenza vaccination in older adults, where pre-vaccination titers decrease with age in males 77 but not in females, suggesting that older females enter each influenza season with greater 78 immunity than their male counterparts [17] . In the context of SARS-CoV-2 vaccines, however, 79 studies have failed to provide sex-disaggregated data within each age group [18, 19] , and little 80 is known about how biological sex may modify the effects of age, and age-related factors like 81 frailty, on vaccine immunogenicity. Here, we investigate sex differences and sex-specific effects 82 of aging in the humoral immune response to the vaccine virus and variants of concern (VOC) 83 induced by three doses of a SARS-CoV-2 mRNA vaccine in a cohort of adults above 75 years of 84 age. We illustrate that the age-and frailty-associated declines in antibody responses occur to a 85 greater extent in males than females. 86

Longitudinal data in the older adult cohort were analyzed using mixed-effects models 131 with random intercepts on the individual to account for repeated measures, and interaction 132 terms between study timepoint (categorical) and sex (self-report), age (categorized based on 133 terciles) and frailty status. Linear regression models including interaction terms between sex 134 and age or frailty were used to investigate sex-specific effects at individual timepoints. To 135 compare the older and younger cohorts, the number of days post-dose 2 was used as a 136 continuous predictor and cubic splines were included to study non-linear relationships [25] . 137

Cubic spline knots were placed at 30-, 100-, and 160-days post-vaccination, points chosen to 138 approximately divide the data into quartiles. Mixed-effects models included an interaction term 139 between time and cohort and were repeated separately for males and females. Differences 140 between cohorts were tested at three sentinel points (14-, 90-and 180-days post dose 2). All 

Eighty-six older adults were recruited from the Baltimore area, with three participants 147 excluded from analysis due to high N titers (i.e., titer >180), suggesting prior infection 148 (Supplemental figure 1). One additional participant was excluded from analysis due to evidence 149 of severe immunosuppression, such that their responses could not be accurately captured in 150 population-level models. Characteristics of the 82 participants included in analysis are detailed 151 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint in Table 1 . The population had more females (59%) than males, and a median age of 84 years. 152

Most participants were classified as pre-frail (64%) and a greater percentage of males than 153 females were frail. All participants received two doses of a SARS-CoV-2 mRNA vaccine, with the 154 majority (70%) receiving BNT162b2. Sixty participants (73%) received a third vaccine dose at 155 least six months after the second dose. 156

Demographic information for the younger adult cohort is provided in Table 2 . Of 84 157 eligible participants from the affiliated study [21] , three were excluded due to high anti-N titers 158 (Supplemental figure 1). In the younger population included in analysis, there were more 159 females than males (60% vs 40%), most participants were between 30 and 49 years of age, and 160 a majority of samples were collected 21-43 days and 125-150 days after receipt of the second 161 vaccine dose. 162

Older females mount greater responses to vaccination than older males 164 Among older adults, IgG binding to S and S-RBD of the vaccine strain increased 165 significantly in response to the first two vaccine doses and then decreased significantly in the 6 166 months following immunization (p<0.001 for all comparisons; Figure 1A -B & D). Geometric 167 mean titers (GMT) decreased 11-and 12-fold, for S and S-RBD, respectively, from <1M_PD2 to 168 6M_PD2 (Supplemental Table 2 ). Females mounted greater IgG responses to S and S-RBD 169 relative to their baseline than males at all post-vaccination timepoints (p<0.02 for all 170 comparisons, Figure 1A -B & D). Older females also had greater titers of IgG against S and S-RBD 171 at each visit, and this difference was significant for anti-S IgG at <1M_PD1 (p=0.020) and at 172 3M_PD2 (p=0.026). After receipt of a third vaccine dose, anti-S and S-RBD titers increased 173 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint significantly in both males and females (p<0.001), leading to GMT that were 2-and 4-fold 174 greater than the post-dose 2 peak for S and S-RBD, respectively (Supplemental Table 2) . 175

The functional ability of antibodies to inhibit S from binding to ACE2 followed similar 176 kinetics as anti-S IgG in response to the primary immunization series, but then decreased more 177 rapidly in the 6-months following immunization, resulting in a 28-fold decrease in GMT from 178 <1M_PD2 to 6M_PD2 (Figure 1C & Supplemental Table 2 ). By 6M_PD2, 79% of males and 77% 179 of females had undetectable ACE2iAb. Sex differences were apparent at all timepoints and 180 were significant at 3M_PD2, with females mounting stronger responses than males (p=0.046). 181

Post dose 3, all but one participant had detectable ACE2iAb, and the geometric mean was 7-182 fold higher than the post-dose 2 peak (Supplemental Table 2 ). Despite differences in kinetics 183 over time between the binding and functional assays, the three readouts of humoral immunity 184 correlated well with each other (Supplemental figure 2). As expected, correlations became 185 weaker at the lower range of the ACE2-inhibition assay. Taken together, these data suggest that 186 older females mount stronger response to SARS-CoV-2 vaccination than males, and that a third 187 vaccine dose is necessary to boost functional antibody responses in this population. 188

The effects of age and frailty are greater in males than in females 190

We next assessed the overall and sex-specific effects of age on the humoral response to 191 vaccination. Among all older participants, age was significantly associated with reduced anti-S 192 IgG, anti-S-RBD IgG, and ACE2iAb in the six months following the primary vaccination series 193 (Figure 2A-C) . This effect was largely driven by the oldest tercile of the population (≥88 years). 194

The percent of participants with undetectable ACE2iAb by 6M_PD2 increased from 67% in the 195 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint youngest tercile (75-82 years) to 85% in the oldest tercile. In sex-disaggregated analyses 196 focusing on the 3M_PD2 timepoint (i.e., a time point when all study participants were 197 represented), age significantly impaired responses in males, but not females, leading to 198 statistically significant sex differences in the effect of age for anti-S IgG (p=0.025) and ACE2iAb 199 (p = 0.001; Figure 2D -F). The trend of greater age effects in males than females was consistent 200 at other timepoints following the primary immunization series (Supplemental figure 3A-F), and 201 by 6M_PD2, 100% of males in the oldest age group, compared to 77% of females, had 202 undetectable ACE2iAB. After receipt of a third dose, the effect of age was no longer significant 203 in the overall population or within either sex, suggesting that a third vaccine dose eliminated 204 sex and age disparities in vaccine-induced immunity ( effect of frailty was also attenuated by the third dose but remained significant for ACE2iAb 212 (p=0.005; Figure 2I ). From these data, we conclude that the effects of age and frailty in older 213 adults are largely driven by males, not females. 214 215 All rights reserved. No reuse allowed without permission.

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The breadth of vaccine-induced immunity in older adults was assessed by measuring 217 anti-S IgG to the Alpha, Delta, and Omicron variants. Antibody titers to the Alpha and Delta 218 variants were similar to each other and were both significantly reduced relative to the vaccine 219 virus (2-4-fold lower GMT, p<0.001; Figure 3A & Supplemental Table 3 ). Titers to Omicron were 220 further reduced relative to the vaccine virus (>5-fold difference in GMT) and the Alpha and 221

Delta variants (p<0.001 for all comparisons; Figure 3A & Supplemental Table 3 ). Differences 222 between anti-S IgG to the vaccine virus and the VOC were attenuated at 1M_PD3 (fold 223 difference in GMT <1.5 for Alpha and Delta and <4 for Omicron) but remained significant 224 (p<0.0001 for all comparisons). At 1M_PD3, anti-Alpha S IgG was significantly higher than anti-225 Delta S IgG (p<0.001). ACE2iAb to the Alpha and Delta variants also tended to be lower than to 226 the vaccine strain, but these differences were smaller in magnitude than for anti-S IgG and 227

were not statistically significant at all timepoints ( Figure 3B-C) . In sex-disaggregated analyses, 228 females had higher responses to the VOC than males, and this difference was significant for 229 anti-Delta S IgG (p=0.038) and ACE2iAb against the Alpha variant (p=0.034) at 3M_PD2 ( 

To investigate the crosss-reactivity of the vaccine-induced humoral response, IgG titers 234 to seasonal and epidemic β-coronaviruses were measured in the older adults. Anti-HKU1 IgG 235 titers increased in response to the primary vaccination series, but fell by 3M_PD2 (Figure4A) . 236

Titers of IgG recognizing OC43, MERS-CoV, and SARS-CoV-1 increased significantly in plasma 237 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint samples collected after SARS-CoV-2 vaccination and remained elevated above baseline levels 238 for 6 months ( Figure 4B-D) . Furthermore, titers to all four β-coronaviruses were significantly 239 elevated by the third vaccine dose (Figure 4A-D) . Notably, IgG binding to the two seasonal β-240 coronaviruses (HKU1 and OC43) was elevated at baseline, indicating widespread exposure to 241 these viruses in this population. Increased antibody titers against related β-coronaviruses 242 following SARS-CoV-2 vaccination suggests possible "back-boosting", as observed for influenza 

To further investigate the sex-specific effects of aging, antibody kinetics against vaccine, 249

Alpha, and Delta antigens were compared between the younger and older adult cohorts in the 250 six months following the primary vaccination series. In the whole population, anti-vaccine S IgG 251 was significantly lower in older than younger adults (p<0.001 at 14 days post-vaccination, 252

p=0.004 at 90 days, and p=0.026 at 180 days) ( Figure 5A ). In sex-disaggregated analyses, 253 differences between the older and younger adults were significant among males at all three 254 sentinel points (p=0.004 at 14 days post-vaccination, p=0.005 at 90 days, and p=0.019 at 180 255 days), but only significant among females at 14-days post vaccination (p=0.004) (Figure 5B-D) . 256

In addition, the magnitude of the difference between the mean of the older cohort and the 257 mean of younger cohort was consistently larger for males than for females across the three 258 sentinel points (Figure 5D ). Similar results were observed for anti-Alpha and Delta S IgG ( Figure  259 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint 5E-L). There were no significant differences in the rate of waning between older and younger 260 adults, suggesting that antibody kinetics are not age-dependent. 261 262 Discussion 263

In this longitudinal study, older adult females mounted stronger antibody responses to 264 SARS-CoV-2 mRNA vaccination than older males, and age and frailty were associated with 265 reduced responses in males but not females. While the kinetics of antibody waning in the six 266 months following immunization were not age-dependent, older adults mounted weaker initial 267 responses to vaccination, such that their antibody titers remained lower than younger adults 268 throughout the follow-up period. A sex-specific effect of age was observed both within the 269 older cohort and when comparing younger and older adults, in which age-associated reductions 270 in humoral immunity were greater among males than females. In the older adult cohort, receipt 271 of a third vaccine dose largely eliminated disparities caused by sex, age, and frailty in antibody 272 responses, with the exception of ACE2iAb, which remained lower in frail compared to non-frail 273 or pre-frail participants. The effect of age on SARS-CoV-2 vaccine responses has been studied 274 [7-9, 29-33], but the sex differential impact of age has not been reported previously. The inclusion of three measures of humoral immunity and four SARS-CoV-2 viruses 280 allowed us to capture the breadth and depth of vaccine responses in this vulnerable population. 281 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Our study had several strengths and limitations. Some of the sex-specific effects 293 observed were differences among males that were absent among females, without statistical 294 evidence of a sex difference (i.e., non-significant sex interaction terms) [45] . It is important to 295 note that our findings were generated from post-hoc analyses that were not necessarily 296 powered to investigate sex differences, and conclusions are limited by small samples sizes in 297 certain sub-groups. Given the small sample sizes, it is not surprising that statistically significant 298 sex differences were not consistently observed. Particularly for age-based analyses, however, 299 the consistency of trends between assays and timepoints, coupled with statistically significant 300 sex differences in the effect of aging at 3M_PD2, lend credibility to the conclusion that the 301 effects of age on antiviral antibody responses are driven by males. Further supporting these 302 findings are similar sex-specific effects of age observed following seasonal influenza vaccination 303 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint in both younger and older adults [17, 46] . While it is important to not over-interpret 'within-304 sex' differences as 'between-sex' differences [47] , there is considerable value in studying 305 differences within males or feamles [48, 49] . This is particularly true given the uniqueness of 306 the community-dwelling older adult cohort, which represent the 'oldest' old subset, and are 307 distinct from the population of long-term care facility residents that has been the focus of much 308 of the SARS-CoV-2 research in older adults. 309

There also were missing data in the older adult cohort, particularly at the <1M_PD1 310 timepoint. These missing data did not, however, depart from the missing at random 311 assumption, and thus multi-level models were used to account for missingness. Second, the 312 timing of sample collection was different in the older and younger cohorts. To account for this, 313

analyses that compared the two groups used days post-vaccination as a continuous variable. 314

Finally, we have not investigated the functional antibody responses to the Omicron variant, nor 315 have we included measures of cellular immunity. These analyses are on-going. 316

In conclusion, we report that both age and frailty impair antibody responses to the 317 primary series of SARS-CoV-2 vaccination in older males, and that these disparities are largely 318 eliminated by receipt of a third vaccine dose. Given that male sex is an important risk factor for 319 severe outcomes from COVID-19 [10-14], the finding that older and frail males may be 320 vulnerable to breakthrough infections due to low antibody responses, particularly before a 321 third vaccine dose is administered, is of considerable public health importance. These findings 322 emphasize that increasing third dose coverage among older males is crucial to protecting this 323 vulnerable population from SARS-CoV-2. 324 325 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. initiatives/serological-sciences-network. 396 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were measured before and at five timepoints after receipt of a SARS-CoV-2 mRNA vaccine. 522

Differences between timepoints were tested using multi-level models with study timepoint as a 523 dummy variable and random intercepts on the individual to account for repeat measures. All 524 point estimates are shown with error bars indicating the 95% confidence interval and asterisks 525 indicate significant (p<0.05) increases relative to the pre-vaccination vaccination timepoint. 526 527 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272269 doi: medRxiv preprint between cohorts are shown with 95% confidence intervals, such that confidence intervals that 545 do not span the null value of zero are statistically significant. 546 All rights reserved. No reuse allowed without permission.

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