[PMC free article] [PubMed] [Google Scholar] 10. 16 days). Anti\RBD total antibodies were measured by chemiluminescent immunoassay. NtAb were quantified by an Omicron S pseudotyped virus neutralization assay. SARS\CoV\2\S specific\IFN\producing CD4+ and CD8+ T cells were enumerated by whole\blood flow cytometry for intracellular cytokine staining. In total, 33/146 participants contracted breakthrough Omicron infection (symptomatic in 30/33) within 4 months after 3D. Anti\RBD antibody levels were comparable in infected and uninfected participants (21?123?vs. 24?723 BAU/ml; IL23P19 p?=?0.34). Likewise, NtAb titers (reciprocal IC50 titer, 157 vs. 95; p?=?0.32) and frequency of virus\reactive CD4+ (p?=?0.82) and CD8+ (p?=?0.91) T cells were similar across participants in both groups. anti\RBD antibody levels and NtAb titers estimated at around the time of infection were also comparable (3445?vs. 4345 BAU/ml; p?=?0.59 and 188.5 vs. 88.9; p?=?0.70, respectively). Having detectable NtAb against Omicron or SARS\CoV\2\S\reactive\IFN\producing CD4+ or CD8+ T cells after 3D was not correlated with increased protection from breakthrough infection (OR, 1.50; p?=?0.54; OR, 0.0; p?=?0.99 and OR 3.70; p?=?0.23, respectively). None of the immune parameters evaluated herein, including NtAb titers against the Omicron variant, may reliably predict at the individual level the risk of contracting COVID\19 due to the Omicron variant in nursing home residents. Keywords: anti\spike antibodies, breakthrough infection, Comirnaty? COVID\19 vaccine, neutralizing antibodies, nursing home residents, SARS\CoV\2 Omicron variant, spike\reactive T cells 1.?INTRODUCTION The SARS\CoV\2 Omicron variant has become KX2-391 dominant in many countries 1 due to its increased transmissibility compared to the Delta variant, owing at least partly to its remarkable ability to escape from SARS\CoV\2 Spike (S) neutralizing antibodies (NtAb) elicited during natural infection or after vaccination with regular or booster schedules. 2 , 3 , 4 , 5 Adaptive immunity is critically involved in preventing SARS\CoV\2 infection 6 ; nevertheless, protective thresholds remain elusive for both S\binding NtAb and T cells. 7 Herein, taking advantage of SARS\CoV\2 Omicron variant outbreaks in several nursing homes, whose congregate nature facilitates wide exposure to the virus, we investigated whether levels of antibodies targeting the receptor\binding domain (RBD) of S (anti\RBD), NtAb targeting Omicron S, and S\reactive functional T cells measured after a homologous booster dose (3D) with Comirnaty? were associated with the likelihood of subsequently contracting breakthrough infections in elderly nursing home residents. 2.?MATERIALS AND METHODS 2.1. Participants The current prospective and observational study included 146 participants (median age, 80 years; range, 66C99; 109 female; median Charlson comorbidity index of KX2-391 7, range 1C14) institutionalized in four nursing homes in the Valencian Community (Spain). Participants vaccinated with the Comirnaty? vaccine (two doses) were evaluated for their immunological response after a homologous booster dose (a median to 16 days; range, 15C18), 8 which was given to all participants irrespective of their SARS\CoV\2 infection status, and were followed for up 4 months. SARS\CoV\2 infection status at the time of 3D (na?ve vs. experienced) was defined according to historical records in the electronic Valencia Health System Integrated Databases and/or the presence or absence of anti\SARS\CoV\2 nucleocapsid (N) IgG antibodies. None of the residents included in the study had a documented immunosuppression condition or were under immunosuppressive therapy within the follow\up period. Anti\RBD and anti\N antibody detection and T\cell assays were performed at the Microbiology Service of the Hospital Clnico Universitario of Valencia. NtAb was measured at the Institute for Integrative Systems Biology, Universitat de Valencia\CSIC. Whole\genome sequencing was performed at the Foundation for the promotion of Health and Biomedical Research of the Valencian Community (FISABIO)?(Valencia, Spain). 2.2. Virological diagnosis of SARS\CoV\2 breakthrough infections Residents suspected of having developed COVID\19 were tested within 24?h after symptoms onset KX2-391 by reverse transcription polymerase chain reaction (RT\PCR) for detection of SARS\CoV\2 RNA in nasopharyngeal specimens. Asymptomatic residents were tested by RT\PCR within 48?h of diagnosis of the index case and two to three times afterward throughout the outbreak. Involvement of the SARS\CoV\2 Omicron BA.1 variant was documented by whole\genome sequencing performed at the FISABIO?(Valencia, Spain), as previously described. 8 2.3. Immunological testing Anti\RBD total antibodies and N\reactive IgGs were detected by the Roche Elecsys? Anti\SARS\CoV\2 S and the Elecsys? Anti\SARS\CoV\2 N assays (Roche Diagnostics), respectively, with values ?0.4?BAU/ml and cut\off index?1.0 considered positive results in the respective tests, according to the manufacturer. NtAb?targeting the S protein was measured using a Green fluorescent protein (GFP)\expressing vesicular stomatitis virus pseudotyped with the Omicron variant, as previously described. 8 , 9 In brief, we introduced mutations in a mammalian expression vector encoding a codon\optimized SARS\CoV\2 S sequence from the Wuhan\Hu\1 (ancestral) reference variant. First, the D614G mutation was introduced by site\directed mutagenesis. Subsequently, additional site\directed mutagenesis and/or the cloning of synthetic fragments harboring the.