Antibody Titers and Protection against Omicron (BA.1 and BA.2) SARS-CoV-2 Infection

The emergence of the SARS-CoV-2 variants of concern has greatly influenced the immune correlates of protection, and there are little data about the antibody threshold concentrations to protect against infection with SARS-CoV-2 Omicron BA.1 or BA.2. We analyzed the antibody responses of 259 vaccinated healthcare workers, some of whom had been previously infected by SARS-CoV-2. The median follow-up was 179 days (IQR: 171–182) after blood collection. We detected 88 SARS-CoV-2 Omicron infections during the follow-up period, 55 (62.5%) with SARS-CoV-2 BA.1, and 33 (37.5%) with SARS-CoV-2 BA.2. A neutralizing antibody titer below 8 provided no protection against a BA.1 infection, a titer of 16 or 32 gave 73.2% protection, and a titer of 64 or 128 provided 78.4% protection. Conversely, the BA.2 infection rate did not vary as a function of anti-BA.2 neutralizing antibody titers. Binding antibody concentrations below 6000 BAU/mL provided no protection against Omicron BA.1 infection, 6000–20,000 BAU/mL provided 55.6% protection, and 20,000 or more provided 87.7% protection. There was no difference in BA.2 infection depending on the binding antibody concentration. Further studies are needed to investigate the relationship between antibody concentrations and infection with the Omicron BA.4/5 variants that are becoming predominant worldwide.


Introduction
Since December 2020, the coronavirus disease 2019 (COVID- 19) pandemic has been dominated by the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs). The latest to date is the Omicron (B.1.1.529) variant, further divided into distinct sublineages: BA.1, BA.2, BA.3, BA.4, and BA.5. These new variants harboring numerous spike protein mutations, particularly in the receptor binding domain (RBD), have spread rapidly worldwide because they were more transmissible or more prone to immune escape.
The SARS-CoV-2 pandemic has resulted in substantial morbidity, mortality, and social disruption. However, vaccination and a previous infection provide a degree of protection against symptomatic, severe COVID-19 [1]. The vaccines presently available to protect against such infections all use the ancestral (Wuhan-like) virus or its spike protein as immunogen.
Serological diagnosis is becoming increasingly important in attempts to understand the extent to which COVID-19 is spreading in the community and to identify individuals who are immunized and potentially "protected" against re-infection. The most widely accepted marker of protection against a SARS-CoV-2 infection is the concentration of neutralizing antibody [2,3] that prevent viral infection mostly by blocking the early step of infection, viral entry, especially in interfering with virions binding to their receptor. There is also a correlation between the concentrations of neutralizing antibody and those of binding antibodies measured by immunoassays that use ancestral strain antigens [4][5][6]. The concentration of anti-spike IgG produced by vaccination or natural infection is associated with protection against infections of SARS-CoV-2 strains that were present before the Omicron variant emerged in November 2021 [7][8][9]. Goldblatt et al. used a random effects meta-analytic approach to calculate protective thresholds in WHO units for ancestral strain SARS-CoV-2 and Alpha (B.1.1.7) of 154 (95% CI 42, 559) and 171 (95% CI 57, 519) anti-S binding antibody units (BAU/mL), respectively [10]. It was consistent with the threshold of 141 BAU/mL conferring more than 89% protection, according to another study [7]. A slightly higher protective threshold of 264 BAU/mL was also proposed [8]. Gilbert et al. reported geometric mean neutralization titers ∼247 IU/mL [9], compared to 1057 IU/mL in Khoury et al. [2], suggesting that different protection thresholds could be obtained depending on the neutralization assay used.
The emergence of these SARS-CoV-2 variants of concern (VOC) with increased transmissibility and the capacity to escape natural and vaccine immunity [11][12][13][14] has greatly influenced the immune correlates of protection. In late 2020, the Delta variant (B.1.617) was detected in India and spread rapidly worldwide, displacing other variants. Notable mutations in the B.1.617.2 variant included L452R, T478K, and E484Q in the S RBD. The combination of mutations in the Delta variant seems to impart the virus a selective advantage compared to the original virus and other variants, as evidenced by high transmissibility and infectivity, and immune evasion [11]. In late 2021, the B.1.1.529 variant emerged in Southern Africa and contains several mutations present in other variants, such as N501Y (Alpha), E484A~E484K (Beta and Gamma), and T478K (Delta), although in total it has more than 50 mutations with more than 30 identified in the S gene alone [15]. These mutations are associated with enhanced infectivity and transmissibility, and Omicron has also been demonstrated to escape neutralization by monoclonal antibodies, convalescent serum, and post-vaccine antibody [12]. Overall, with the exception of the Alpha VOC, the emerging VOCs have been associated with reductions in neutralizing activity of antibodies derived from previously infected or individuals who have undergone primary vaccination [16][17][18][19]. Evidence based on in vitro neutralization assays suggests that, for immune responses to Omicron in individuals who have already been exposed to ancestral SARS-CoV-2 antigens (whether through infection or vaccination), an Omicron correlate of protection may be higher than for ancestral SARS-CoV-2 or other VOCs, due to the reduced effectiveness of antibodies directed against the spike protein. To that point, Pfizer-BioNTech has reported a 25-fold reduction in neutralization titres against Omicron compared to ancestral SARS-CoV-2 in individuals vaccinated with two doses of BNT162b2 [20]. Studies from South Africa and Germany report a reduction in neutralization up to 41-fold [17], despite two or three doses of BNT162b2 or mRNA-1273 and previous infection. We therefore have determined the antibody threshold concentrations (binding and neutralizing antibodies) needed to protect against infection with SARS-CoV-2 Omicron BA.1 or BA.2.

SARS-CoV-2 Detection, Variant Screening, and Genome Sequencing
Nucleic acids were extracted from nasopharyngeal swab samples on an MGI SP-960 instrument using the MGIEasy Nucleic Acid Extraction kit and amplified with the Thermofisher TaqPath RT-PCR assay (ThermoFisher, Waltham, MA, USA) on QuantStudio 5 Real-Time PCR systems. Positive results were then classified, according to the TaqPath S gene profile, as: S gene target failure (SGTF), S gene target late detection (SGTL), or non-SGTF/SGTL [21]. The SGTL profile was defined as a difference of at least 4 Ct between the N and S genes. The Omicron BA.1 variant was identified by its SGTF/SGTL TaqPath profiles (69-70 S gene deletion). Positive specimens with non-SGTF/SGTL profiles (only samples with N Ct ≤ 30) were then tested using the IDTM SARS-CoV-2/VOC Revolution Pentaplex assay (ID solutions, Montpellier, France). This multiplex RT-PCR assay targets the K417N, L452R, and E484K mutations. The Omicron BA.2 variant was identified by the presence of the K417N mutation. Our VOC screening strategy was validated by sequencing a large number of positive nasopharyngeal samples using the Pacific Biosciences (Pacbio) SMRT System [22].

Neutralizing and Binding Antibodies against SARS-CoV-2 Spike Protein
Neutralizing antibody (NAb) titers were measured by end-point dilution using Vero cells (ATCC, CCL-81™) [14] and clinical strains of SARS-CoV-2 Omicron BA.1 (EPI_ISL_10316329) and BA.2 (EPI_ISL_13540703). Anti-spike Immunoglobulin G (anti-S) concentrations were measured with an electrochemiluminescent assay, which is a binding antibody assay based on the RBD of the Spike protein (IgG II Quant, Alinity, Abbott, Sligo, Ireland) [4]. Raw data in AU/mL were converted in BAU/mL by using the conversion factor (0.142) recommended by the manufacturer.

Statistical Analysis
Correlations were identified using a Spearman test and groups were compared with the Chi 2 or Fisher's exact tests. Statistical significance was set at: p-value < 0.05.
The anonymized data were analyzed using Stata version 14 (StataCorp LP, College Station, TX, USA) and MATLAB 2018b.
Only 1/75 (1.3%) of the HCWs who were not infected before vaccination and given a booster dose of vaccine had an anti-BA.1 NAb titer exceeding 64, or a BAb concentration above 6000 BAU/mL. None of the 16 vaccinated (2 doses, no booster) HCWs had a neutralizing antibody titer above 64 or a Bab concentration above 6000 BAU/mL.
Only 1/75 (1.3%) of the HCWs who were not infected before vaccination and given a booster dose of vaccine had an anti-BA.1 NAb titer exceeding 64, or a BAb concentration above 6000 BAU/mL. None of the 16 vaccinated (2 doses, no booster) HCWs had a neutralizing antibody titer above 64 or a Bab concentration above 6000 BAU/mL.

Discussion
Several pre-oOmicron clinical [2,5,[7][8][9] and animal [23] studies found that the neutralizing and binding antibody concentrations were good biomarkers of protection against a SARS-CoV-2 infection. We now find a similar relationship between the concentrations of anti-BA.1 binding and neutralizing antibodies and protection against a BA.1 SARS-CoV-2 infection.
We also find that there is no clear antibody concentration above which Omicron infection does not occur, in contrast to infections with the alpha variant or other earlier strains (D614G) [7]. Although we found an inverse link between the risk of a SARS-CoV-2 BA.1 infection and the anti-BA.1 NAb and Bab concentration, there seems to be no such link for BA.2. This could be due to differences in the time between antibody assay and SARS-CoV-2 BA.1 and BA. protection against infection with a D614G strain, and a BAb concentration of 1700 BAU/mL or more provided 100% protection. Our present results indicate that a concentration of 20,000 BAU/mL is needed for an equivalent degree of protection, approximately 142-times the 141 BAU/mL threshold concentration. This is probably due to differences in the antigenicity of the spike proteins in Omicron BA.1 and the ancestral SARS-CoV-2 strain used in current vaccines and immunoassays [24]. Similarly, 8.5 times more binding antibody is required to neutralize the Delta variant than to neutralize the Alpha variant [14]. Our results partially agree with those of a recent Danish study [25] that demonstated that the inverse relationship between the IgG level and the risk of contagion observed for the Delta variant demonstrated the protective effect of vaccines, but this association was not observed for the Omicron variant. This suggests that the quantitative level of anti-spike IgG has a limited impact on the risk of breakthrough infection with Omicron. Note, however, that the study did not distinguish between infections caused by the BA.1 strain or the BA.2 strain of Omicron. Another study on nursing home residents demonstrated correlates of protection between the level of (binding and neutralizing) antibodies and Omicron BA.2 infection [26]. A BA.2 NAb title below 8 provided 19.2% protection against Omicron BA.2, titles of 16 or 32 gave 85.3% protection and an NAb title of 64 or more provided 95.6% protection. A BAb concentration below 1000 BAU/mL provided only 25% protection against Omicron BA.2; 1000-6000 BAU/mL provided 77.9% protection; while >6000 provided 97.1% protection. It should be noted, however, that this population, which is particularly at risk, continued to benefit from restrictive sanitary measures, unlike the general population, and that in this study it was very little exposed to Omicron BA.1.
Although current vaccines reduce the risk of a symptomatic SARS-CoV-2 infection, hospitalization and death, their effectiveness against the Omicron variant [11,17,27,28] is altered due to lower neutralizing antibody titers. We found that a greater percentage of previously infected, vaccinated HCWs had high NAb and BAb titers than did uninfected vaccinated HCW. This is consistent with studies indicating that vaccination of previously infected individuals gives greater protection than the vaccination of naive individuals [29].
The limitations of this study concern both vaccination and antibody measurement. First, antibodies were not measured at the time of SARS-CoV-2 breakthrough. Waning antibody titers are associated with loss of protection [30] and the post-vaccination total antibody titer decreases faster in vaccinated people with no previous SARS-CoV-2 infection than in previously-infected, vaccinated individuals [30]. Second, all the HCWs were not given a booster vaccination at the time of analysis, particularly those HCWs who were infected before being vaccinated. The majority were given a complete (2-dose) schedule without booster. Third, we did not discriminate between infection and symptomatic disease. The antibody concentrations providing robust protection from severe infection require further investigation in other populations. Last, the contribution of the T-cell system to protection was not assessed.

Conclusions
This is, to our knowledge, the first study of protective antibody thresholds against infection with Omicron BA.1 and BA.2. We provide further evidence in a longitudinal cohort that robust antibody levels against the ancestral strain fail to establish sufficient protection against antigenically distant variants. Further, we demonstrate that a full booster vaccination schedule without infection may not adequately confer protection against breakthrough infection with the Omicron variant [25]. In order to achieve more robust immune protection and limit SARS-CoV-2 transmission, the next generation of SARS-CoV-2 vaccine preparations will include Omicron subvariant's spike determinants in addition to ancestral strain antigens [31].
While our results provide markers of protection against BA.1 infection, further studies are needed to investigate the relationship between antibody concentrations and infection with the Omicron BA.4/5 variants that are becoming predominant worldwide.  Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of CHU de Toulouse (COVID BioToul, ID-RCB 2020-A01292-37, ClinicalTrials.gov Identifier: NCT04385108).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.