Age and Ageing 2022; 51: 1–10 © The Author(s) 2022. Published by Oxford University Press on behalf of the British Geriatrics https://doi.org/10.1093/ageing/afac101 Society. All rights reserved. For permissions, please email: journals.permissions@oup.com This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work properly cited. For commercial re-use, please contact journals.permissions@oup.com RESEARCH PAPER Reduced humoral response 3 months following BNT162b2 vaccination in SARS-CoV-2 uninfected residents of long-term care facilities Macedonia Trigueros1 , Edwards Pradenas1 , Dolors Palacín2 , Francisco Muñoz-López1 , Carlos Ávila-Nieto1 , Benjamin Trinité1 , Josep Maria Bonet-Simó2 , Mar Isnard2 , Nemesio Moreno2 , Silvia Marfil1 , Carla Rovirosa1 , Teresa Puig1 , Eulàlia Grau1 , Anna Chamorro3 , Ana Martinez3 , Ruth Toledo3 , Marta Font3 , Jordi Ara4 , Jorge Carrillo1,5 , Lourdes Mateu3,6,7,8 , Julià Blanco1,5,7 , Bonaventura Clotet1,3,5,7 , Nuria Prat2 , Marta Massanella1,5 , on behalf of the CoronAVI@S and the KING cohort extension studies 1 IrsiCaixa-AIDS Research Institute and Germans Trias i Pujol Health Research Institute (IGTP), 08916 Badalona, Catalonia, Spain 2 Direcció d’Atenció Primària – Metropolitana Nord, 08023 Sabadell, Catalonia, Spain 3 Fight against AIDS Foundation (FLS), Germans Trias i Pujol Hospital, 08916 Badalona, Catalonia, Spain 4 Gerencia Territorial de la Metropolitana Nord, Germans Trias i Pujol Hospital, 08916 Badalona, Spain 5 Centro de Investigación Biomédica en Red de Enfermedades Infecciosas CIBERINFEC, Madrid, Spain 6 Infectious Diseases Department, Germans Trias i Pujol Hospital and Universitat Autònoma de Barcelona, 08916 Badalona, Spain 7 University of Vic–Central University of Catalonia (UVic-UCC), 08500, Vic, Catalonia, Spain 8 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, CIBERES, 28029 Madrid, Spain Address correspondence to: Marta Massanella, IrsiCaixa AIDS Research institute, Hospital Germans Trias i Pujol, Ctra de Canyet s/n, 08916 Badalona, Catalonia, Spain. Email: mmassanella@irsicaixa.es Alternative contact: Nuria Prat, Direcció d’Atenció Primària – Metropolitana Nord, Carretera de Barcelona, 473, 08203 Sabadell, Catalonia, Spain. Email: nprat@gencat.cat Abstract Background: SARS-CoV-2 vaccination is the most effective strategy to protect older residents of long-term care facilities (LTCF) against severe COVID-19, but primary vaccine responses are less effective in older adults. Here, we characterised the humoral responses of institutionalised seniors 3 months after they had received the mRNA/BNT162b2 vaccine. Methods: plasma levels of SARS-CoV-2-specific total IgG, IgM and IgA antibodies were measured before and 3 months after vaccination in older residents of LTCF. Neutralisation capacity was assessed in a pseudovirus neutralisation assay against the original WH1 and later B.1.617.2/Delta variants. A group of younger adults was used as a reference group. Results: three months after vaccination, uninfected older adults presented reduced SARS-CoV-2-specific IgG levels and a significantly lower neutralisation capacity against the WH1 and Delta variants compared with vaccinated uninfected younger individuals. In contrast, COVID-19-recovered older adults showed significantly higher SARS-CoV-2-specific IgG levels after vaccination than their younger counterparts, whereas showing similar neutralisation activity against the WH1 virus and an increased neutralisation capacity against the Delta variant. Although, similarly to younger individuals, previously infected older adults elicit potent cross-reactive immune responses, higher quantities of SARS-CoV-2-specific IgG antibodies are required to reach the same neutralisation levels. Conclusions: although hybrid immunity seems to be active in previously infected older adults 3 months after mRNA/BNT162b2 vaccination, humoral immune responses are diminished in COVID-19 uninfected but vaccinated older residents of LTCF. These results suggest that a vaccine booster dose should be prioritised for this particularly vulnerable population. 1 M. Trigueros et al. Keywords: older people, BNT162b2 vaccine, nursing homes, long-term care facilities, humoral immune response, SARS- CoV-2 Key Points • Previously infected and vaccinated older adults living in long-term care facilities (LTCF) had neutralising antibody levels comparable to those of younger individuals. • Vaccinated uninfected residents showed limited neutralisation capacity against both original and delta variants. • Hybrid immunity seems to be active in older adults and this may be relevant to the design of vaccine boosting campaigns. Introduction Material and methods Older adults have been disproportionately affected by the Study participants coronavirus disease 2019 (COVID-19) pandemic. Older A total of 98 residents of three LTCF located in the area individuals are more prone to severe infection and over of Barcelona, Spain, were included in this prospective 95% of COVID-19-related deaths have occurred in the observational study (CoronAVI@S study, Table 1). Plasma population > 60 years [1]. Among them, residents of long- samples were collected after 6 months of SARS-CoV-2 term care facilities (LTCF), who live in a congregate setting outbreaks before vaccination (September–November 2020, and thus are at increased risk of transmission and infec- N = 98) and 3 months after full vaccination schedule tion, have shown higher mortality rates than the general (April–May 2021, N = 91). Participants were recruited population of the same age [2, 3]. Therefore, SARS-CoV- irrespective of their SARS-CoV-2 infection status and SARS- 2 vaccination of LTCF residents has been a priority in CoV-2 serology was performed to subclassify participants most countries. SARS-CoV-2 vaccines are currently widely into infected and uninfected groups, depending on their available in Europe, but we are still facing several months polymerase chain reaction (PCR) and serology status of continuous new infections and new circulating variants (Supplementary Figure 1). Both study groups were vacci- of concern (VOC). In late 2021, virus outbreaks involv- nated with the BNT16b2 mRNA vaccine (Pfizer-BioNTech) ing the B.1.617.2/Delta variant were reported in nursing between January and February 2021 in accordance with the homes, despite the high vaccination rates among residents Spanish government’s COVID-19 vaccination strategy. An [4]. additional sample was collected at a median of 2.8 interquar- Currently available COVID-19 vaccines have proven to tile range (IQR) [2.4–3.1] months after vaccination. We be safe and generate effective humoral and cellular immunity performed an additional serology test against nucleoprotein [5–8], but these studies mainly involved young or middle- (NP)-protein in uninfected residents after vaccination to aged healthy adults. Relatively few studies have evaluated verify the absence of anti-NP antibodies and confirm their the immune responses generated in older adults [9–13], SARS-CoV-2-naïve status (data not shown). and most of them studied short-term (few weeks) immune For comparison purposes, we selected younger and responses after vaccination. Ageing is associated with an middle-aged adults (22–64 years) from the KING cohort immunosenescent phenotype characterised by a progres- extension study: 64 infected individuals (Table 2) for pre- sively proinflammatory state, and a diminished immune vaccine visit and 51 individuals (32 infected and 16 response to pathogens and vaccines [14]. Therefore, there uninfected, Table 3) for post-vaccine timepoint. Pre-vaccine is an urgent need to determine the quality and duration of blood samples from these groups were matched with those immune responses in the older population to adapt SARS- from the older CoronAVI@S group by time elapsed since CoV-2 vaccination calendars to their immune needs. SARS-CoV-2 infection (6 months). All matched individuals We performed a prospective study in LTCF residents to had experienced mild cases of the disease. Post-vaccine evaluate the anti-SARS-CoV-2 humoral response elicited samples were selected from among individuals in the younger before and 3 months after vaccination with the BNT162b2 group who had been vaccinated with BNT16b2 (Pfizer- mRNA COVID-19 vaccine (Pfizer-BioNTech). The study BioNTech), and blood samples were obtained a median of 3 included individuals uninfected (N = 16) and recovered IQR [2.5–3.4] months after completing the full vaccination from a previous SARS-CoV-2 infection (N = 82). Humoral schedule. responses of these older participants were also compared The CoronAVI@S study and KING cohort extension with a younger population, and a functional neutralisation were approved by the Ethics Boards of the Institut Uni- assay was performed against the original WH1 and Delta of versitari d’Investigació en Atenció Primària Jordi Gol and variants. the Hospital Universitari Germans Trias i Pujol, respectively 2 COVID-19 vaccine response in long-term care facilities residents Table 1. Descriptive data for CoronAVI@S participants Previously SARS-CoV-2 Uninfected group P-value infected group N = 82 N = 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Age, years, median [IQR] 87 [81–90] 79 [74–90] 0.26 Female, n (%) 66 (80) 8 (50) 0.021 AGM levela , n (%) 0.11 1 0 (0) 0 (0) 2 4 (5) 3 (20) 3 38 (46) 6 (40) 4 41 (49) 6 (40) Pre-vaccine visit N = 82 N = 16 PCR positive, n (%) 61 (73) 0 (0) <0.0001 Time from PCR positive to sample extraction, months, median [IQR] 6.4 [6–6.9] NA NA Detection of SARS-CoV-2-specific antibodies, n (%) 81 (98)b 0 (0) <0.0001 Medical assistance, n (%) 5 (6) NA Post-vaccine visit N = 76 N = 15 Vaccine received BNT16b2 (2 doses), n (%) 76 (100) 15 (100) 1.0 Time between vaccine doses, days, median [IQR] 21 [21–23] 21 [21–26] 0.14 Time between PCR positive and 1st vaccine dose, months, median [IQR] 9.3 [9.1–9.6] NA Time from full vaccine schedule to sample extraction, months, median [IQR] 2.4 [2.36–2.95] 2.8 [2.6–3.1] 0.05 PCR positive after vaccination, n (%) 0 (0) 1 (7) a AGM level: Stratum of Adjusted Morbidity ranging from 1 to 4 according to the number of comorbidities and their need for health care b One individual did not seroconvert despite Positive PCR. Significant p-values are indicated in bold. Table 2. Descriptive data for younger and older participants, pre-vaccine CoronAVI@S KING Cohort extension P-value Infected Infected N = 82 n = 64 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Age, years, median [IQR] 87 [81–90] 44 [38–51] <0.001 Age, years, n (%) <45 0 (0) 36 (56) 45–65 0 (0) 27 (42) 65–85 37 (45) 0 (0) >85 45 (55) 0 (0) Female, n (%) 66 (80) 47 (73) 0.33 AGM levela,b , n (%) 1 0 (0) 32 (50) <0.001 2 4 (5) 17 (27) <0.001 3 38 (46) 11 (17) <0.001 4 41 (49) 0 (0) <0.001 History of SARS-CoV-2 infection, n (%) 82 (100) 64 (100) 1 Pre-vaccine visit Time from PCR positive/symptoms to sample extraction, months, median [IQR] 6.5 [6.0–6.9] 6.6 [6.1–7.4] 0.04 Detection of SARS-CoV-2-specific antibodies, n (%) 81 (99) 52 (81) 0.0002 Medical assistance, n (%) 5 (6) 0 (0) 0.25 a AGM: Stratum of Adjusted Morbidity ranging from 1 to 4 according to the number of comorbidities and their need for health care. AGM level was not available for four individuals from the KING Cohort extension b AGM level was not available for four individuals from the KING Cohort extension. Significant p-values are indicated in bold. (20-116P and PI-20-217). All participants provided written the background signal obtained for each sample in antigen- informed consent before inclusion. free wells. Values were plotted into the standard curve, calcu- lated by plotting and fitting the log of standard dilution (in arbitrary units) versus response to a four-parameter equation Determination of anti-SARS-CoV-2 antibodies in Prism 8.4.3 (GraphPad Software) (Supplementary data). The presence of anti-SARS-CoV-2 antibodies against Spike S2 domain (S2) + Receptor Binding Domain (RBD) or NP Pseudovirus neutralisation assay in plasma samples was evaluated using an in-house sandwich- Neutralisation assay was performed using SARS-CoV- enzyme-linked immunosorbent assay (ELISA) [15]. The spe- 2.Sct19 WH1 and B.1.617.2/Delta pseudovirus [16, 17]. cific signal for each antigen was calculated after subtracting Upper and lower limits of detection were 60 and 14,580 3 M. Trigueros et al. Table 3. Descriptive data for younger and older participants, post-vaccine CoronAVI@S KING Cohort extension P-values between younger and older Infected Uninfected Infected Uninfected Infected Uninfected n = 76 n = 14 n = 32 n = 19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Age, years, median [IQR] 87 [81–90] 81 [74–90] 43 [38–50] 39 [28–48] <0.001 <0.001 Age, years, n (%) ≤45 0 (0) 0 (0) 19 (59) 12 (63) 46–65 0 (0) 0 (0) 13 (41) 7 (37) 65–85 33 (43) 9 (64) 0 (0) 0 (0) >85 43 (57) 5 (36) 0 (0) 0 (0) Female, n (%) 62 (82) 8 (57) 22 (69) 14 (74) 0.20 0.46 AGM level a , n (%) 1 0 (0) 0 (0) 19 (59) 13 (69) <0.001 <0.001 2 4 (4) 3 (21) 6 (19) 5 (26) 0.06 1.0 3 36 (48) 5 (37) 4 (13) 1 (5) <0.001 0.06 4 36 (48) 6 (42) 0 (0) 0 (0) <0.001 <0.001 Medical assistance, n (%) 4 (5) NA 2 (6) NA 1.0 NA Vaccine received, n (%) BNT16b2, 1 doses 76 (100) 14 (100) 32 (100) 19 (100) 1.0 1.0 BNT16b2, 2 doses 76 (100) 14 (100) 30 (94) 19 (100) 0.08 1.0 Time between vaccine doses, days, median [IQR] 21 [21–23] 21 [23–26] 22 [21–22] 22 [21–22] 0.34 0.23 Time between PCR positive/symptoms and complete 12.9 NA 10.6 NA <0.001 NA vaccine schedule, months, median [IQR] [12.7–13.3] [10.4–11.6] Time from complete vaccine schedule to sample 2.4 2.4 2.0 3.1 0.53 <0.001 extraction, months, median [IQR] [2.4–3.0] [2.3–2.9] [1.9–3.1] [3.0–3.4] a AGM level: Stratum of Adjusted Morbidity ranging from 1 to 4 according to the number of comorbidities and their need for health care. AGM level was not available for three individuals from the KING cohort extension. Significant p-values are indicated in bold. (reciprocal dilution). The ID50 (the reciprocal dilution groups using the AMG classification system, a measure of inhibiting 50% of the infection) was calculated by plotting the general health status [18]. The most common chronic and fitting the log of plasma dilution against response to a pathologies included hypertension (62%), arthritis (40%), four-parameter equation (Supplementary data). dementia (38%) or diabetes (33%). Individuals were tested by PCR during SARS-CoV-2 outbreaks at their respective Statistics LTCFs between March and June 2020. Continuous variables were described using medians and Serologic and PCR tests before vaccination revealed that the IQR(25th and 75th percentiles), whereas categorical 84% of the LTCF residents had experienced SARS-CoV- factors were reported as percentages. Quantitative variables 2 infection before testing. Previously infected residents were compared using the Mann–Whitney or Wilcoxon tests (n = 82) had a median age of 87 years and were 80% for unpaired and paired data, respectively; age ranges were female, whereas uninfected residents before vaccination compared using the Kruskal–Wallis (K–W) test. Percent- (n = 16) had a median age of 79 years and were 50% ages between groups were compared using the Chi-squared female (Table 1). Among infected LTCF residents, only three test. Analyses were performed with Prism 9.1.2 (GraphPad required hospitalisation due to acute infection. Software). To assess the impact of several variables (age, We took advantage of the Spanish national vaccination sex, Adjusted Morbidity Group [AMG] level and hospital- plan for LTCFs to evaluate the immune responses gener- isation) on neutralisation levels post-vaccination, univariate ated by the BNT16b2 vaccine. We confirmed that 14 out and multivariate linear models were generated using R (ver- of the 15 uninfected residents remained seronegative after sion 4.1.2), analysing infected and uninfected individuals vaccination (testing negative for antibodies against the NP- separately. The multivariate models were built using variables protein by ELISA). The infected resident after vaccination which proved to be significant in the univariate analysis, with developed a mild case and was removed from post-vaccine a P-value cut-off at ≤0.1. AMG level was excluded from analysis. the multivariate models due to its correlation with age. The quality of the models was evaluated via residual analysis. Humoral responses 3 months after vaccination The levels of SARS-CoV-2-specific IgG, IgA and IgM Results antibodies were significantly higher in individuals who were infected before vaccination compared with their uninfected Characterisation of older participants counterparts (P < 0.0001 in all cases, Figure 1A). There The 98 older participants in this study were > 65 years, was a significant increase in all isotypes of immunoglob- with 90% of them belonging to moderate- to high-risk ulins between pre- and post-vaccine samples in infected 4 COVID-19 vaccine response in long-term care facilities residents participants (Supplementary 2A), whereas uninfected indi- sequential decrease in neutralisation titres across ages (K– viduals maintained detectable levels of SARS-CoV-2-specific W P = 0.001 Figure 1D and Supplementary Figure 2G). IgGs and IgAs, but no IgMs, 3 months after vaccination Indeed, age was only negatively correlated with plasma (Supplementary Figure 2B). The levels of SARS-CoV-2- neutralisation capacity in the uninfected group (r = −0.63, specific IgGs induced after vaccination across age groups P < 0.001; Figure 1E). Overall, these results suggest that revealed higher levels in older participants (≥65 years) who uninfected older adults show lower titres of neutralising had been previously infected compared with the reference antibodies and might be at higher risk of infection than group of younger adults (Figure 1B, K-W P < 0.001, previously infected individuals of the same age. and r = 0.41, P < 0.001, Supplementary Figure 2C). To The proportion of effective neutralising antibodies among determine whether those higher levels were related to the the total SARS-CoV-2-specific IgG (ratio of plasma neu- vaccine or to the natural infection, we evaluated SARS-CoV- tralisation titre to total SARS-CoV-2 IgGs) was reduced 2-specific IgGs before vaccination across ages (Figure 2D). in infected older participants compared with their younger We determined that 6 months after infection (and prior to counterparts (Figure 1F, P < 0.001), suggesting low func- vaccination), older participants already showed higher levels tionality of antibodies in older participants. In contrast, of SARS-CoV-2-specific IgGs compared with the younger no differences were found between both uninfected and reference group (K–W, P < 0.001). vaccinated participants (Figure 1C). These results suggest The levels of circulating SARS-CoV-2-specific IgGs that mRNA vaccines induce effective neutralising antibodies in uninfected vaccinated participants tended to decrease in older uninfected individuals, but at lower levels. Levels across both age groups (K–W P = 0.06, Figure 1B) and of anti-S2 + RBD IgGs positively correlated with neutrali- were negatively correlated with age (r = −0.73, P < 0.001, sation capacity in all groups (Supplementary Figure 2H). Supplementary Figure 2C). When infected and uninfected Lastly, we performed an exploratory analysis to under- groups were compared after vaccination, the levels of SARS- stand the potential impact of different parameters (sex, age, CoV-2-specific IgGs were already significantly lower in AMG level and hospitalisation) in neutralising immune individuals between 45 and 64 years (P = 0.007), a difference response by linear regression models. In the univariate maintained in the older age group (P < 0.001 in all cases, analysis, age was significantly associated with lower neu- Figure 1B). These results highlighted some differences in tralising capacity in the uninfected group, and AMG antibody production between infected and uninfected level had a similar tendency (Supplementary Table 1A). individuals, only the uninfected younger group being able In the infected group, we observed a tendency for a to produce levels of SARS-CoV-2 antibodies similar to that positive association between neutralisation levels with age, produced by their infected counterparts. AMG level and sex, there being higher responses in males (Supplementary Table 1B). In the multivariate analysis, sex remained the only significant predictor, but we also observed Neutralisation capacity against original WH1 virus a tendency related to age and hospitalisation. SARS-CoV-2 vaccination increased the titres of antibodies with neutralising capacity in infected and uninfected participants (P < 0.0001 in both cases, Figure 1C), being Neutralisation capacity against the Delta variant significantly higher in individuals with a previous history Given the lower neutralisation responses against WH1 in of SARS-CoV-2 infection compared with the uninfected uninfected vaccinated LTCF residents, we hypothesised that counterparts (P < 0.0001). For the infected population, this particular group might experience lower neutralising there was a median fold increase of 18.7 [9.3–44.8] from responses against the VOC B.1.617.2/Delta (circulating pre- to post-vaccination. Previously infected subjects had variant in Spain since July–December 2021). Indeed, we similar levels of neutralisation independently of age (K– found a significant decrease in the plasma neutralisation W P = 0.12, Figure 1D and Supplementary Figure 2E). capacity of Delta VOC compared with WH1 in both We hypothesised that the neutralisation response after older and younger participants, regardless of their previous natural infection reached a plateau in recovered older infection status (P < 0.001 in both cases, Figure 2A participants similar to that seen in younger participants, and Supplementary Figure 3A-C). Similarly to what was therefore similar levels of neutralisation were reached in observed for WH1, we found a progressive decrease in the both groups after vaccination. To test this hypothesis, we levels of neutralisation across ages in vaccinated uninfected compared plasma neutralisation capacity before vaccination individuals (Figure 2B and Supplementary Figure 3D, K–W for older and younger individuals 6 months after SARS- P = 0.005), being barely detected in uninfected vaccinated CoV-2 infection. Surprisingly, the neutralisation titres were older individuals. Surprisingly, neutralisation capacity significantly lower in older compared with younger (45– against the Delta variant increased significantly across ages 64 years) participants 6 months after infection (K–W in individuals with prior SARS-CoV-2 infection (Figure 2B P = 0.01, Supplementary Figure 2F). These results suggest and Supplementary Figure 3E, K–W P < 0.001). Next, we a greater boost of humoral responses in infected older compared cross-neutralisation capacities by determining fold adults after vaccination compared with the younger group. change ratios between Delta and WH1 (reference spike) In contrast, uninfected vaccinated subgroups showed a variants, as a measure of the loss of neutralising capacity. 5 M. Trigueros et al. Figure 1. Comparison of humoral response and neutralising activity between uninfected and infected individuals 3 months after BNT162b2 mRNA COVID-19 vaccine. (A) Levels of SARS-CoV-2-specific immunoglobulins (IgG, IgA and IgM) against S2 + RBD proteins quantified in plasma from uninfected and infected older participants by ELISA. (B) SARS-CoV-2-specific IgG antibody levels (against S2 + RBD proteins) after vaccination across ages in infected and uninfected participants. (C) Neutralising activity against WH1 virus before and after 3 months of vaccine administration in infected and uninfected older residents of LTCF. (D) Neutralising activity against WH1 after vaccination across ages in infected and uninfected participants. (E) Correlation of neutralising activity after vaccination with age in infected and uninfected participants. Correlation coefficient and P-values were obtained from Spearman correlation. (F) Ratio of plasma neutralisation titre per total SARS-CoV-2-specific IgG antibodies in younger and older participants, sub-grouped by previous SARS-CoV-2 infection history. Median values are indicated; P-values were obtained from Mann–Whitney test for comparison between groups A, B, C, D and F), Wilcoxon test for paired tests ( C) and Kruskal–Wallis test for comparison between age ranges for infected and uninfected groups (B and D). In all panels, uninfected and infected participants at vaccination are indicated in turquoise and purple, respectively. 6 COVID-19 vaccine response in long-term care facilities residents Figure 2. Neutralising activity against B.1.617.2/Delta variant between uninfected and infected participants 3 months after BNT162b2 mRNA Covid-19 vaccine. (A) Comparison of plasma neutralising activity against WH1 and B.1.617.2/Delta variant in uninfected and infected older participants after vaccination. (B) Neutralising activity against Delta variant after vaccination across ages in infected and uninfected, younger and older participants, sub-grouped by previous SARS-CoV-2 infection history. (C) Fold change ratios between neutralisation titres against Delta variant and WH1 (reference spike) virus in infected and uninfected participants, sub-grouped by age (<65 years and ≥ 65 years). Median values are indicated. P-values were obtained from Mann– Whitney test for comparison between groups (all panels), Wilcoxon test for paired tests (A and C) and Kruskal–Wallis test for each group (B). In all panels, uninfected and infected participants at vaccination are indicated in turquoise and purple, respectively. Median fold-change of all groups was <1, highlighting Both age and hospitalisation remained significant predictors the greater difficulty in neutralising the Delta variant in the multivariate analysis. compared with WH1 (Figure 2C). Among all groups, previously infected older participants after vaccination Discussion showed significantly higher ratios compared with all other groups, demonstrating the better cross-neutralisation of the LTCF residents have been prioritised for COVID-19 vacci- Delta variant. nation in most countries due to their increased risk of mor- A linear regression model was also built for neutralisation bidity and mortality. Although the effectiveness of COVID- of the Delta variant revealing that older age was a strong 19 vaccines is very high, SARS-CoV-2 infections among predictor of a lower neutralisation capacity in the uninfected fully vaccinated individuals (i.e. breakthrough infections) are group (Supplementary Table 2A). In contrast, univariate expected, especially with the appearance of VOC with higher analysis demonstrated that higher levels of neutralising anti- transmissibility and immune resistance compared with the bodies were associated with hospitalisation, higher age and original WH1 variant. The reduced ability of older adults to AMG level in the infected group (Supplementary Table 2B). elicit effective immune responses to vaccines and pathogens 7 M. Trigueros et al. means that this population is one of the most vulnerable after vaccination, suggesting a greater resistance of this populations. variant [24] and/or a lower durability of neutralising Here we demonstrate that, 3 months after COVID- antibodies in older adults (3 weeks vs. 3 months) [9]. 19 vaccination, uninfected LTCF residents showed limited Nonetheless, the low levels of neutralising antibodies neutralisation responses compared with their younger coun- exhibited by uninfected LTCF residents against the original terparts, indicating that the uninfected older population is at WH1 variant 3 months after vaccination, and even lower higher risk. Vaccination of uninfected LTCF residents with levels against the Delta variant, suggest that this specific two doses of the BNT162b2 vaccine reduced the risk of population may require a third dose to boost their humoral infection by 90% and by >95% COVID-19-related hospital response. In fact, the administration of a third dose to admissions and mortality for up to 5 months [19]. Overall, the Israeli population over age 60 reduced the number uninfected older adults may develop a memory response and severity of COVID-19 cases [20, 25], supporting which will protect them in the event of a new infection. our recommendation. On the other hand, it has been However, the low levels of circulating neutralising antibodies demonstrated that mRNA vaccination boosts cross-variant after 3 months highlight the need for further studies to assess antibodies elicited by natural infection in the general the long-term effectiveness of SARS-CoV-2 vaccines, and population [26, 27]. Our results are in line with these suggest that this specific subgroup will particularly benefit findings and clearly reveal that previously infected older from receiving a booster vaccination [20]. adults develop the same cross-neutralisation capacity against Importantly, a potent immune responses to COVID-19 the Delta variant as the younger population. Therefore, vaccines is developed in previously infected individuals inde- previously infected older adults would not benefit from an pendently of age [10, 12, 13, 21]. Our results suggest that additional vaccine dose at this time. the combination of natural and vaccine-generated immu- Our study has some limitations. First, the number of nity (hybrid immunity [22]) elicits a larger than expected participants, especially for the uninfected group of LTCF immune response even in the older population, despite a residents, was unexpectedly low. Although 64% of the high degree of immunosenescence and increased comor- cohort tested positive by PCR, SARS-CoV-2 serologic test bidities. It has been recently shown that hybrid immunity revealed that in fact 84% of residents had already experienced improves B cells and antibodies against SARS-CoV-2 vari- COVID-19. In addition, we did not assess specific SARS- ants [23]. Older adults show a higher capacity to neutralise CoV-2 cellular responses before and after vaccination, more resistant variants (i.e. B.1.617.2/Delta), such hybrid which could also be used as a correlate of protection immunity implying a great benefit for this recovered popu- [28]. lation. In fact, hospitalisation and older age were identified Larger longitudinal studies targeting the older population as predictors of higher neutralisation levels, which could be are required to evaluate immune responses, as well as to associated with more severe disease. monitor breakthrough infections and reinfection rates in Our results demonstrate that natural infection (pre- vaccinated LTCF residents. For this purpose, determina- vaccine) generates higher virus-specific IgG antibodies in tion of the quality of humoral immune responses—using older adults compared with the younger population. In functional neutralisation assays—as well as evaluation of the contrast, in uninfected older adults, vaccination gener- features of cellular responses to SARS-CoV-2 vaccines is ates lower SARS-CoV-2-specific IgGs, similarly to other necessary to better define the immune correlates of protec- studies [9, 10, 12, 13], and these antibodies show a tion and to properly carry out tailored booster campaigns lower neutralisation capacity in the older population. On in this frail and vulnerable population. Taken together, our the other hand, previously infected older persons after results suggest that only uninfected LTCF residents, who vaccine show higher levels of total SARS-CoV-2-specific do not develop adequate immune responses, will clearly IgG compared with younger adults, with similar levels benefit from a booster vaccine dose; an adapted vaccination of neutralising antibodies. Therefore, the measurement of calendar would be necessary to respond to their immune SARS-CoV-2-specific IgGs might not be a good surrogate needs. Importantly, hybrid immunity seems to be active in marker of protection in older adults, and functional assays, older adults and is likely to be relevant to the design of future such as virus/pseudovirus-based neutralisation tests, are vaccine boosting campaigns. required. Some studies showed lower levels of neutralising titters Acknowledgement of Collaborative Authors: We wish to in uninfected older adults than in younger adults 2–3 weeks acknowledge the two collaborative group authors: after BNT162b2 vaccination [11, 12], suggesting that older 1. The CoronAVI@S collaborative group: S. Reyes Car- adults have a more limited initial immune response after rión, N. Salarich Solà, A. Vidal, R. Alvarez Viñallonga, vaccine administration. In another study, serum from older J. Tornero, E. Vilamala, C. Suarez, T. Gonzalo, L. Perez, adults 3 weeks after vaccination showed a lower albeit still D. Sans, A. Blancas Loras, A. Garcia Archer, J. Borràs, S. detectable neutralisation capacity relative to different VOC Cervelló. (Alpha, Beta or Gamma) compared with younger adults 2. The KING cohort extension collaborative group: G. [11]. Here we showed that plasma from uninfected LTCF Llados, S. España, JR Santos, C. Loste, C. López, I. Casafont, residents was unable to neutralise the Delta variant 3 months C. Estany, C. Rodriguez, J Moreno-Muñoz, A Prats. 8 COVID-19 vaccine response in long-term care facilities residents C. Herrero, A. Garcia, M. Montero, P. Tornero, N. Gon- 5. Folegatti PM, Ewer KJ, Aley PK et al. Safety and immuno- zalez Palomares, A. Grajea, L. Ortiz, C. Miranda, E. Abad, genicity of the ChAdOx1 nCoV-19 vaccine against SARS- D. Figueroa, A. Mancera, S. Gonzalez Alonso, M. Perez, L. CoV-2: a preliminary report of a phase 1/2, single-blind, Esteban, M. Ortiz, L. Valls, L. Ceron, T. Baena, C. Puig, M. randomised controlled trial. Lancet 2020; 396: 467–78. Cucurell, J. Puig. 6. Baden LR, El Sahly HM, Essink B et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med 2020; Acknowledgements: We are deeply grateful to all partici- 384: 403–16 pants and their families, to the LTCF who participated in 7. Xia S, Zhang Y, Wang Y et al. Safety and immunogenicity this study. We also thank the technical staff of Direcció of an inactivated SARS-CoV-2 vaccine, BBIBP-CorV: a ran- d’Atenció Primària de la Metropolitana Nord for sample domised, double-blind, placebo-controlled, phase 1/2 trial. collection in LTCF (S. Reyes Carrión, N. Salarich Solà, A. Lancet Infect Dis 2020; 21: 39–51. 8. Polack FP, Thomas SJ, Kitchin N et al. Safety and efficacy Vidal, R. Alvarez Viñallonga, J. Tornero, E. Vilamala, C. of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med Suarez, T. Gonzalo, L. Perez, D. Sans, A. Blancas Loras, A. 2020; 383: 2603–15. Garcia Archer, J. Borràs, S. Cervelló) and staff of IrsiCaixa 9. Van Praet JT, Vandecasteele S, De Roo A, Vynck M, De for sample processing (L. Ruiz, R. Ayen, L. Gomez, C. Vriese AS, Reynders M. Dynamics of the cellular and humoral Ramirez, M. Martinez, T Puig). We thank CERCA Program- immune response after BNT162b2 messenger ribonucleic me/Generalitat de Catalunya for institutional support and acid Coronavirus Disease 2019 (COVID-19) vaccination in the Foundation Dormeur. COVID-19-naive nursing home residents.. J Infect Dis 2021; 224: 1690–3. Declaration of Conflicts of Interest: J.B. and J.C. reports 10. Van Praet JT, Vandecasteele S, De Roo A, De Vriese AS, personal fees from Albajuna Therapeutics, outside the sub- Reynders M. Humoral and cellular immunogenicity of the mitted work. BNT162b2 messenger RNA coronavirus disease 2019 vaccine in nursing home residents. Clin Infect Dis 2021; 73: 2145–7. Declaration of Sources of Funding: Gloria Soler Founda- 11. Collier DA, Ferreira IATM, Kotagiri P et al. Age-related tion, Grifols, the Departament de Salut of the Generalitat immune response heterogeneity to SARS-CoV-2 vaccine de Catalunya (Grant DSL0016 to J.B. and Grant DSL015 BNT162b2. Nature 2021; 596: 417–22. to J.C.), the Spanish Health Institute Carlos III (Grant 12. Canaday DH, Carias L, Oyebanji OA et al. Reduced PI17/01518 and PI18/01332 to J.C.) and the crowdfunding BNT162b2 messenger RNA vaccine response in Severe initiatives ‘https://www.yomecorono.com’, BonPreu/Esclat Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)– and Correos. M.T. and C.A.N. were supported by the Early naive nursing home residents. Clin Infect Dis 2021; 73: Stage Research Staff Fellowship (FI21-B00327 and FI20- 2112–5. B00742) from the Catalan Agency for Management of Uni- 13. Tut G, Lancaster T, Krutikov M et al. Profile of humoral versity and Research Grants (AGAUR) and the European and cellular immune responses to single doses of BNT162b2 Social Fund. M.T. was then supported by a doctoral fel- or ChAdOx1 nCoV-19 vaccines in residents and staff within residential care homes (VIVALDI): an observational study. lowship from the Departament de Salut from Generalitat Lancet Heal Longev 2021; 7568: 1–10. de Catalunya (SLT017/20/000095). E.P. was supported by 14. Del Giudice G, Goronzy JJ, Grubeck-Loebenstein B et al. a doctoral grant from ANID, Chile: Grant 72,180,406. Fighting against a protean enemy: immunosenescence, vac- cines, and healthy aging. npj Aging Mech Dis 2018; 4: 1–8. 15. Massanella M, Martin-Urda A, Mateu L et al. Critical pre- References sentation of a severe acute respiratory syndrome coronavirus 2 reinfection: a case report. Open Forum Infect Dis 2021; 8: 1. Levin AT, Hanage WP, Owusu-Boaitey N, Cochran KB, 1–6. Walsh SP, Meyerowitz-Katz G. Assessing the age specificity 16. Trinité B, Tarrés-Freixas F, Rodon J et al. SARS-CoV-2 of infection fatality rates for COVID-19: systematic review, infection elicits a rapid neutralizing antibody response that meta-analysis, and public policy implications. Eur J Epidemiol correlates with disease severity. Sci Rep 2021; 11: 2608. 2020; 35: 1123–38. 17. Pradenas E, Trinité B, Urrea V et al. Clinical course impacts 2. Prieto-Alhambra D, Balló E, Coma E et al. Filling the gaps in early kinetics and long-term magnitude and amplitude of the characterization of the clinical management of COVID- SARS-CoV-2 neutralizing antibodies beyond one year after 19: 30-day hospital admission and fatality rates in a cohort of infection Edwards. medRxiv. 2021. 118 150 cases diagnosed in outpatient settings in Spain. Int J 18. Espieén ID, Vela E, Pauws S et al. Proposals for enhanced Epidemiol 2020; 49: 1930–9. health risk assessment and stratification in an integrated care 3. Meis-Pinheiro U, Lopez-Segui F, Walsh S et al. Clinical scenario. BMJ Open 2016; 6: 1–10. characteristics of COVID-19 in older adults. A retrospective 19. Cabezas C, Coma E, Mora-Fernandez N et al. Associations study in long-term nursing homes in Catalonia. PLoS One of BNT162b2 vaccination with SARS-CoV-2 infection and 2021; 16: e0255141. hospital admission and death with covid-19 in nursing homes 4. Report ET. Data collection on COVID-19 outbreaks in and healthcare workers in Catalonia: prospective cohort study. closed settings with a completed vaccination programme : BMJ 2021; 374: n1868. long-term care facilities purpose, aim and scope of this activity 20. Bar-On YM, Goldberg Y, Mandel M et al. Protection of changes in version 2 primary objectives methods inclusion BNT162b2 vaccine booster against Covid-19 in Israel. N Engl criteria for long-term care faci. 2021. J Med 2021; 385: 1393–400. 9 M. Trigueros et al. 21. Frieman M, Harris AD, Herati RS et al. SARS-CoV-2 vac- 26. Stamatatos L, Czartoski J, Wan Y-H et al. mRNA cines for all but a single dose for COVID-19 survivors. vaccination boosts cross-variant neutralizing antibodies EBioMedicine 2021; 68: 103401. elicited by SARS-CoV-2 infection. Science 2021; 372: 22. Crotty S. Hybrid immunity. Science 2021; 372: 1392–3. 1413–8. 23. Andreano E, Paciello I, Piccini G et al. Hybrid immunity 27. Reynolds CJ, Pade C, Gibbons JM et al. Prior SARS-CoV-2 improves B cells and antibodies against SARS-CoV-2 variants. infection rescues B and T cell responses to variants after first Nature 2021; 600: 530–5. vaccine dose. Science 2021; 372: 1418–23. 24. Planas D, Veyer D, Baidaliuk A et al. Reduced sensitivity of 28. San Segundo D, Comins A, Lamadrid P et al. COVID- SARS-CoV-2 variant Delta to antibody neutralization. Nature 19 mRNA based vaccine immune-response assessment 2021; 596: 276–80. for public health decision. SSRN Electron J 2021; 11: 25. Barda N, Dagan N, Cohen C et al. Effectiveness of a third dose 204–17. of the BNT162b2 mRNA COVID-19 vaccine for preventing severe outcomes in Israel: an observational study. Lancet 2021; Received 22 December 2021; editorial decision 13 March 6736: 1–8. 2022 10