key: cord-0694029-fegt2vla
authors: Bruel, T.; Pinaud, L.; Tondeur, L.; Planas, D.; Staropoli, I.; Porrot, F.; Guivel-Benhassine, F.; Attia, M.; Pelleau, S.; Woundenberg, T.; Duru, C.; Davy Koffi, A.; Castelain, S.; Fernandes-Pellerin, S.; Jolly, N.; Perrin de Facci, L.; Roux, E.; Ungeheuer, M.-N.; Van Der Werf, S.; White, M.; Schwartz, O.; Fontanet, A.
title: SARS-CoV-2 Omicron neutralization and risk of infection among elderly after a booster dose of Pfizer vaccine
date: 2022-03-30
journal: nan
DOI: 10.1101/2022.03.30.22273175
sha: c9ddef7ca3a4c24f84c0772612dc5b1ef560b2c2
doc_id: 694029
cord_uid: fegt2vla

Background: The protective immunity against Omicron following a BNT162b2 Pfizer booster dose among elderly is not well characterized. Methods: Thirty-eight residents from three nursing homes were recruited for the study. Antibodies targeting the Spike protein of SARS-CoV-2 were measured with the S-Flow assay. Neutralizing activities in sera were measured as effective dilution 50% (ED50) with the S-Fuse assay using authentic isolates of Delta and Omicron. Results: Among the 38 elderly included in the study, with median (inter-quartile range, IQR) age of 88 (81-92) years, 30 (78.9%) had been previously infected. The ED50 of neutralization were lower against Omicron than Delta, and higher among convalescent compared to naive residents. During an Omicron epidemic affecting two of the three nursing homes in December 2021-January 2022, 75% (6/8) of naive residents got infected, compared to 25% (6/24) of convalescents (P=0.03). Antibody levels to Spike and ED50 of neutralization against Omicron after the BNT162b2 booster dose were lower in those with breakthrough infection (n=12) compared to those without (n=20): median of 1256 vs 2523 BAU/mL (P=0.02) and median ED50 of 234 vs 1298 (P=0.0004), respectively. Conclusion: This study confirmed the importance of receiving at least three antigenic exposures to the SARS-CoV-2 Spike protein for achieving satisfactory neutralizing antibody levels. In this population, protection against Omicron infection was increased in individuals who had been previously infected in addition to the three vaccine doses. Thus, a fourth antigenic exposure may be useful in the elderly population to prevent infection with Omicron, a variant known for its high escape immunity properties.

The neutralization capacity of sera from vaccinated or convalescent individuals against the Omicron (B.1.1.529) (BA.1) variant of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) has been well studied among several population groups, and has been shown to be lower against Omicron compared to other variants [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . Information on the vaccine efficacy and the neutralization capacity of sera from elderly against Omicron is more limited [13, 14] , despite decreased immunogenicity, increased risk of severe forms of disease and accelerated waning of immunity in this population [15] [16] [17] . Here, we evaluated the capacity of a booster dose of BNT162b2 to elicit neutralizing antibodies against Omicron and examined levels of humoral immunity before Omicron breakthrough infections among residents living in nursing homes.

Thirty-eight residents from three nursing homes were recruited from the Covid-Oise study (NCT04644159). This community-based cohort started in November 2020 and aims to monitor the immunological response following SARS-CoV-2 infection and Coronavirus Disease 2019 (COVID- 19) vaccination.

Past infection of the residents was determined based on clinical data and detection of SARS-CoV-2 specific antibodies using two serological assays, as previously described [18, 19] . All sera samples available since inclusion of the residents in the study were evaluated with both assays. Detection of past infection relied on anti-Spike (S) and anti-Nucleocapsid (N) antibodies for pre-vaccination sera, and only on anti-N antibodies for post-vaccination sera.

. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Figure   1a ). All residents have been immunized with BNT162b2 for their primary series except four who received mRNA-1273. The two initial doses were received three weeks apart (median 21 days, IQR 21-21 days). All residents received a dose of BNT162b2 as booster eight months after the second dose (median 236 days, IQR 230-237 days).

Antibodies targeting the Spike protein of SARS-CoV-2 were measured with the S-Flow assay (N=75).

Briefly, this assay uses 293T cells stably expressing the Spike (S) protein (293T-S cells) and 293T control cells as control to detect anti-Spike antibodies by flow cytometry [20] . The sensitivity (95% confidence interval) is 99.2% (97.7-99.8) and the specificity is 100% (98.5-100) [21] . The assay is standardized with WHO international reference sera (20/136 and 20/130) and cross-validated with two commercially available ELISA (Abbott 147 and Beckmann 56) to allow calculation of BAU/mL [22] .

Neutralizing activities in sera were measured with the S-Fuse assay using authentic isolates of Delta and

Omicron. This assay uses U2OS-ACE2 GFP1-10 and GFP 11 reporter cells, also termed S-Fuse cells, that become GFP+ upon infection with SARS-CoV-2 [23, 24] . All sera were tested in limiting dilution to determine Effective Dilution 50% (ED50 or titer) values. In each well, the number of GFP+ syncytia was scored with an Opera Phenix high-content confocal microscope (PerkinElmer). ED50 were calculated with a reconstructed curve using the percentage of the neutralization at the different concentrations. Viral stock were produced on Vero E6 cells, titrated on Vero E6 or S-Fuse cells and sequenced to confirm viral lineages (GISAID accession ID: EPI_ISL_2029113 and EPI_ISL_7413964

for Delta and Omicron isolates, respectively).

We also evaluated the risk of breakthrough infection among residents from two out of the three nursing is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 30, 2022. ; https://doi.org/10.1101/2022.03.30.22273175 doi: medRxiv preprint of symptoms. RT-PCR positive test results were reanalysed with a second round of RT-PCR screening to identify SARS-CoV-2 variants of concern based on mutations E484K, E484Q and L452R, or K417N.

This study was registered with ClinicalTrials.gov (NCT04644159) and received ethical approval by the Comité de Protection des Personnes Nord Ouest IV. Informed consent was obtained from the residents, or their relatives when the residents did not have full capacity to sign legal documents. Following the second dose of vaccine, anti-Spike IgG levels were higher among convalescents compared with naive residents (Table 1 & Fig 1b) . The third dose increased the antibody titers for naive residents to levels similar to those of the convalescents after 2 doses (Table 1 & Fig 1b) . The ED50 of neutralization were lower against Omicron compared to Delta, and higher among convalescents compared to naive residents (Table 1 & Figure 1c ). For the eight naive residents, neutralization was detectable for only five (62.5%) and one (12.5%) individuals against Delta and Omicron after the second dose, respectively. The number of residents displaying neutralizing antibodies increased to eight (100%) against Delta and six (75%) against Omicron after the third dose. All convalescents except one neutralized Delta and Omicron after the second and third dose. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint In December 2021-January 2022, two out of the three nursing homes were affected by an Omicron epidemic, as reavealed by routine PCR testing (see Methods and Fig 2a) . This epidemic took place three months after administration of the third dose. Among the 32 residents (27 females and 5 males) living in these two households, 12 breakthrough infections occurred (34%). Seventy-five percent (6/8) of naive residents were infected, compared to 25% (6/24) of convalescent (P=0.03; two-sided Fisher exact test).

Anti-Spike IgG and ED50 of neutralization against Omicron post 3 rd dose were compared between those with and without breakthrough infection (corresponding to a median (IQR) of 53 (49-60) days before the breakthrough infection) (Fig 2b) . Median titers were lower in those with compared to those without is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 30, 2022. ; https://doi.org/10.1101/2022.03.30.22273175 doi: medRxiv preprint discriminative enough to separate the two groups and be used as a correlate of protection. This may have to do in part with the very high escape immunity properties of the Omicron variant.

Most previous infections occured during the first epidemic wave (February-March 2020), so that the vaccine primary series performed in early 2021 was able to boost the production of neutralizing antibodies primed one year earlier, even in this elderly population. This is comforting, and reflects the strength of the so-called hybrid immunity combining the effects of infection and vaccination [26] .

Interestingly, among these vaccinated-convalescent residents, antibody levels were slightly lower in samples collected two months after the third dose than in samples collected two months after the second dose. This may be explained by the waning of immune responses and low levels of circulating antibodies before the booster dose, so that the levels achieved after the third dose did not reach those achieved just after one infection and two doses.

Our study has limitations. First, the small sample size limits our statistical power and precludes analysis of the characteristics that may further impact vaccine efficacy, such as gender, prexisting conditions or ongoing medications. Second, we did not have access to nasopharyngeal swabs to measure antibody levels at the site of viral entry and replication. We were thus unable to link breakthrough infections to local levels of antibodies, which might represent a better correlate of protection. Further studies are needed to determine the contribution of mucosal immunity on the acquisition of SARS-CoV-2 and the severity of COVID-19. Lastly, we only tested BA.1, the initial Omicron clade, which was circulating in France at the time of the investigation and was responsible of the breaktrough infections that occurred in the nursing homes of the study. It will be worth examining the neutralization activity of the sera against other Omicron sub-lineages, such as BA.2, at the next blood sampling of the same cohort participants.

This study confirmed the importance of receiving at least three antigenic exposures to the Spike protein for achieving satisfactory neutralizing antibody levels against Omicron. Protection against Omicron was increased in those who had been previously infected in addition to the three doses, suggesting that a . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint fourth antigenic exposure may be useful in this elderly population to prevent infection with a variant known for its high escape immunity properties.

Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR Coronamito, and IDISCOVR, "TIMTAMDEN" ANR-14-CE14-0029, "CHIKV-Viro-Immuno" ANR-14-CE14-0015-01 and the Gilead HIV cure program. AF lab is funded by the INCEPTION project (PIA/ANR-16-CONV-0005) and the Labex IBEID (ANR-10-LABX-62-IBEID). The COVID-Oise cohort is funded by "Alliance Tous Unis contre le virus" Institut Pasteur, AP-HP and Fondation de France.

All data produced in the present study are available upon reasonable request to the authors . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

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