Serum neutralization of SARS‐CoV‐2 Omicron BA.2, BA.2.75, BA.2.76, BA.5, BF.7, BQ.1.1 and XBB.1.5 in individuals receiving Evusheld

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Omicron variant is undergoing continuous evolution and convergent mutation. These new subvariants are raising concerns that they may evade neutralizing monoclonal antibodies (mAbs). We investigated the serum neutralization efficacy of Evusheld (cilgavimab and tixagevimab) against SARS‐CoV‐2 Omicron BA.2, BA.2.75, BA.2.76, BA.5, BF.7, BQ.1.1, and XBB.1.5. A total of 90 serum samples from healthy individuals were collected in Shanghai. Anti‐RBD antibodies were measured and symptoms of infection with COVID‐19 were compared among those individuals. The neutralizing activity of serum against Omicron variants was analyzed by pseudovirus neutralization assays in 22 samples. Evusheld retained neutralizing activity against BA.2, BA.2.75, and BA.5, albeit with somewhat reduced titers. However, the neutralizing activity of Evusheld against BA.2.76, BF.7, BQ.1.1, and XBB.1.5 significantly decreased, with XBB.1.5 showing the greatest escape activity among the subvariants. We also observed that Evusheld recipients displayed elevated antibody levels in their serum, which efficiently neutralized the original variant, and exhibited different characteristics of infection than those who did not receive Evusheld. The mAb has partial neutralization activity against Omicron sublineages. However, the increasing doses of mAb and a larger size of population should be further investigated.

SARS-CoV-2 spike protein have been isolated from convalescent individuals and are effective in preventing or treating COVID-19. [3][4][5] Therefore, exploring the efficacy of therapeutic mAbs can meet the above needs.
Previous studies have shown that Omicron BA.4 and BA.5 escape most neutralizing mAbs and can be only targeted by cilgavimab and bebtelovimab with high potency. [6][7][8] The combination of cilgavimab and tixagevimab (Evusheld) and casirivimab and imdevimab (Ronapreve) both displayed a drop in potency against BA.4 and BA.5 compared with Delta, which was less decreased for Evusheld than Ronapreve. 9 But the neutralizing activity against the recent Omicron BF.7, BQ.1.1, and XBB.1.5 after receiving Evusheld is still unclear.
Evusheld/AZD7442 is a combination of two fully human longacting monoclonal antibodies, tixagevimab (AZD8895) and cilgavimab (AZD1061), that bind nonoverlapping epitopes of the SARS-CoV-2 spike protein RBD and sterically block RBD binding to angiotensinconverting enzyme 2 (ACE2). 10 Evusheld has been approved in many countries or regions worldwide for pre-exposure prophylaxis (PrEP) of SARS-CoV-2 in adults and adolescents aged ≥12 years and weighing ≥40 kg. 11 A phase III clinical trial showed that administration of Evusheld reduced the relative risk of symptomatic infection by 82.8% within 6 months. 12 In addition, serum from individuals receiving Evusheld as PrEP had detectable serum-neutralizing activity against BA.5 for up to 6 months. 9 However, mutations in the RBD portion of the S subunit occur frequently in SARS-CoV-2 sublineages, and these mutations can increase the binding affinity of the RBD to ACE2, resulting in higher infectivity. 13 As of December 2022, the most predominant Omicron sublineages in China are BA.5.2 and BF.7, while BQ.1.1 has become the predominant strain, and XBB.1.5 is outcompeting BQ.1.1 in many other countries. 14,15 Given the emergence of new subvariants and the clinical application of Evusheld, we investigated the neutralizing activity against the current prevalent strains worldwide in Evusheld recipients, measured the in vivo anti-RBD antibody titers and analyzed the correlation between Evusheld administration and clinical symptoms of breakthrough COVID-19. This study aims to optimize the utilization of Evusheld and improve our understanding of its efficacy against SARS-CoV-2 subvariants.

| Sample collection
A total of 90 serum samples from healthy individuals were collected in our hospital from January 9, 2023 to February 3, 2023. Among them, 29 samples were from participants receiving Evusheld as PrEP, and 61 samples were from those who did not receive Evusheld. The demographic characteristics, main infection symptoms, doses, and time of vaccination or Evusheld administration were obtained from the participants who answered the questionnaire, and their test results were compared. All participants provided written informed consent before any study procedures were performed. This study was approved by the Ruijin Hospital Ethics Committee in Shanghai.

| Construction and production of variant pseudoviruses
The Omicron sublineage spike genes (plasmids containing target genes were obtained by Yiqiao Shenzhou) were mammalian codonoptimized and inserted into the pCAGGS vector. HEK293T cells were grown to 90% density before transfection with the indicated spike gene using Lipofectamine 3000 (Invitrogen). After 24 h of culture at 37°C with 5% CO 2 , the supernatants were discarded, and the cells were washed three times with DMEM. The cells were then infected with VSV-G pseudotyped ΔG-luciferase (G*ΔGluciferase, Kerafast) at a multiplicity of infection of 10 for 2 h. Next, the cells were washed three times with DMEM, and the culture media were replaced with 3% FBS DMEM containing anti-VSV-G rat serum. The cells were cultured for 24 h, and then the cell supernatant containing pseudotyped virus was harvested and filtered through a 0.45 μm filter after being centrifuged at 1250 rpm for 10 min. The pseudoviruses were aliquoted and stored at −80°C. Titers of the pseudoviruses were measured before the pseudoviruses were used.

| Pseudovirus neutralization assays
Serum was serially diluted and incubated with the pseudoviruses in 96-well plates for 1.5 h at 37°C. Freshly trypsinized BHK-ACE2 cells were then added to each well and cultured for 20-28 h in 5% CO 2 incubators at 37°C. One hundred microliters of supernatant were discarded from each well, leaving~100 μL of liquid in each well, and 100 μL of luciferase substrate (Beyotime, RG056S) was added before incubation of the cells in the dark for 2 min. The samples were mixed by pipettor action, and 150 μL was transferred to a corresponding 96-well chemiluminescence detection plate (Beyotime, FCP968). Chemiluminescence signals were collected by a luminescence meter (Promega).

| Finecare TM 2019-nCoV RBD antibody test
The 2019-nCoV RBD Antibody Titer Assay Kits were purchased from Guangzhou Wondfo Biotech Co. The antibody test is based on fluorescence immunoassay technology, specifically the sandwich immunodetection method. Detection buffer was added to the specimen, and the sample was mixed well. When the specimen was added into the sample well of the Test Cartridge, the fluorescencelabeled detector 2019-nCoV RBD protein bound to anti-RBD antibodies in blood specimens and formed immune complexes. As the complexes migrated on the nitrocellulose membrane by capillary action, the anti-2019-nCoV RBD antibodies were captured by another RBD protein that had been immobilized on the test strip.
In brief, 20 µL of plasma was added to the provided buffer tube and mixed for 45 s. Then, 75 µL was added to the test cartridge, incubated for 15 min at room temperature (RT), and then inserted in the test cartridge holder of Finecare™ FIA Meters holder for measurement on quick mode. The default results unit of this test is displayed as relative fluorescence unit (RFU, AU/mL) or binding antibody units per milliliter (BAU/mL). Readings ≥1 AU/mL or ≥20 BAU/mL indicate positive results.

| Statistical analysis
The statistical analyses for the pseudovirus neutralization assessments were performed using GraphPad Prism for the calculation of The statistical tests were performed in a two-sided manner, and a p value <0.05 was considered to indicate statistical significance.

| Characteristics of blood donors
A total of 90 blood samples were collected from healthy donors, of which 29 had received Evusheld as PrEP, and 61 had not. We classified the donors into two groups: the Evusheld (n = 29; 300 mg) and non-Evusheld groups (n = 61; individuals not receiving Evusheld). A complete description of the donors' characteristics is provided in Table 1. The mean age of the Evusheld group was higher than that of the non-Evusheld group (41.31 ± 6.54 vs. 32.43 ± 6.37 years; p < 0.001), with a significant difference in sex composition between the two groups (p < 0.001). Only three people had asthma or chronic liver disease complications.

| Serum neutralization of Omicron sublineages
We constructed a panel of vesicular stomatitis virus (VSV)   These results may be related to the structure of the spike protein of SARS-CoV-2, which is composed of the S1 and S2 subunits. The RBD on the S1 subunit helps the virus recognize ACE2 on the surface of host cells. 17 Some researchers confirmed that an anti-RBD antibody test and neutralization tests were well correlated and could effectively identify convalescent COVID-19 individuals. 18 Quantification of anti-RBD antibodies can reflect neutralizing activity against SARS-CoV-2 strains. In our study, we found that individuals receiving Evusheld had significantly higher levels of anti-RBD antibodies than those in the group without Evusheld. Moreover, the antibody titers in individuals without Omicron infection from the Evusheld group were also higher than those of individuals without Evusheld but infected with SARS-CoV-2. Evusheld recipients had a relatively strong neutralizing capacity against the original Wuhan SARS-CoV-2 strain compared to those who did not receive Evusheld. Some researchers have found that several mAbs have similar efficacy against the original variant and Omicron variant. 6,19 Interestingly, the anti-RBD antibody titers in females were significantly lower than those in males. The trend that the anti-RBD titers were higher in males compared to females was also observed among convalescent individuals. 20 However, some studies indicated that female participants had significantly higher anti-RBD antibody concentrations

AUTHOR CONTRIBUTIONS
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

ACKNOWLEDGMENTS
This study was supported by the innovative research team of highlevel local universities in Shanghai. We thank all the participants involved in this study.