Evaluation of Neutralizing Capacity of Tixagevimab plus Cilgavimab (AZD7442) against Different SARS-CoV-2 Variants: A Case Report Study with Comparison to a Vaccinated Population

AZD7442 (150 mg of tixagevimab plus 150 mg of cilgavimab) has been approved for the preexposure prophylaxis of COVID-19 and for the treatment of adults and adolescents with COVID-19 who do not require supplemental oxygen and who are at increased risk of severe COVID-19. Thus, the aim of the present study is to evaluate the neutralizing capacity of tixagevimab and cilgavimab across different SARS-CoV-2 variants in two patients who received AZD7442 for immunoprophylaxis. A cohort of subjects (n = 45) who had received the BNT162b2 mRNA COVID-19 vaccine has been included to compare these two preventive strategies. Neutralizing antibody (NAb) titers against several variants were assessed against the wild-type, alpha, beta, gamma, delta, omicron BA.5, and XBB.1.5 variants. Binding antibodies have also been measured. NAbs T1/2 for AZD7442 was 8.1 days (95% CI: 5.1–19.5 days) and was 11.8 days (95% CI: 7.9–23.7 days) for the primo-vaccination cohort. The time to reach neutralization negativity was 108.3 days (95% CI: 66.9–130.7) for AZD7442 compared to 95.4 days (95% CI: 31.0–119.7 days) for the primo-vaccination cohort. The time to reach NAbs' negativity differs between variants with the maximum value obtained for alpha (i.e., 101.1 days (95% CI: 30.0–135.4 days)) and the minimum obtained for beta (i.e., 61.2 days (95% CI: 37.8–77.1 days)). Our results reinforce the need of reviewing the use of AZD7442 in relation to variants of concern and potentially adapting its administration schedule. AZD7442 could be indicated for short-term prophylaxis in frail patients who may be acutely exposed to SARS-CoV-2.


Introduction
Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reduced the burden of coronavirus disease 2019 (COVID-19) [1].However, some patients, including immunocompromised persons such as recent transplant patients or patients sufering from lymphoid hemopathy, remain at risk for severe COVID-19 despite having been vaccinated with regular booster doses [2,3].Tere are several alternatives for these individuals such as the use of convalescent COVID-19 patient plasma (CCP) or the use of monoclonal antibodies (mAbs) against SARS-CoV-2.CCPs collected from recovered COVID-19 patients are supposed to be rich in neutralizing antibodies (NAbs) [4,5].CCP will almost always be the frst antibody therapy available to treat an outbreak with a new SARS-CoV-2 variant [6].Tey are injected just after the development of the symptoms and aim at treating the disease [4].CCPs need to be analyzed to determine their neutralizing capacity before use, although the determination of their neutralizing capacity is not always related to the diferent variants of interest (VOIs).Focosi et al. reported that CCPs with NAb titer >160 are efective [7].However, there are several limitations to the use of CCPs, such as injection timing, injection volume, which can be very large (up to 2,400 mL), and the phenomenon of antibody-dependent enhancement (ADE) [8].Terefore, there is a need to have access to therapies which are at least as efcient as CCPs to protect individuals but without the need to continuously feed biobanks with plasma from donors, which is clearly also dependent on patient's recruitment.Monoclonal antibodies, which protect against disease irrespective of immune system status and provide rapid protection, are potential options for COVID-19 immunoprophylaxis [9,10].
AZD7442, an association of 150 mg of tixagevimab and 150 mg of cilgavimab (Evusheld ® , AstraZeneca, Södertälje, Sweden), has been approved for the preexposure prophylaxis of COVID-19 and for the treatment of adults and adolescents with COVID-19 who do not require supplemental oxygen and who are at increased risk of progressing to severe COVID-19 [11].Tese mAbs are modifed at their Fc regions with the aim to increase their half-life time (T  [14][15][16].Tus, the efcacy of tixagevimab and cilgavimab against some circulating SARS-CoV-2 variants with decreased in vitro susceptibility is still uncertain.Due to this observed decrease in neutralization activity against the omicron subvariants such as BA.4,BA.5, XBB 1.5, and recently XBB 1.16, the duration of the protective efect of AZD7442 for these subvariants is currently unknown.
As of today, only the omicron variant XBB 1.5 is considered as VOIs [17].Te emergence of these variants raised concerns regarding the duration of vaccine efcacy as assessed by comparing the residual neutralizing capacity of the immune response generated with vaccines encoding for the ancestral spike protein [18][19][20][21][22][23][24][25].Tis observation has led the marketing authorization holders to adapt their vaccines to these VOIs.Such strategy could also apply to mAbs if there is evidence that their neutralizing capacity is signifcantly reduced with the current VOIs.Tus, the aim of the present study is to evaluate the neutralizing capacity of tixagevimab and cilgavimab across diferent SARS-CoV-2 variants.

Study Design and Population
Two patients received AZD7442 for COVID-19 immunoprophylaxis.One subject (female, 28 years) was seropositive to SARS-CoV-2 before the administration of AZD7442, i.e., antinucleocapsid antibody level over the positive threshold, and the other subject (female, 32 years) was seronegative, i.e., anti-nucleocapsid antibody level below the positive threshold [26].Blood samples were obtained at diferent times after injection of AZD7442, i.e., 0, 7, 14, 28, 56, and 90 days following injection.No breakthrough infection occurred during this period in these AZD7442 patients.
To compare the immunity acquired following AZD7442 administration to another prophylactic strategy, a cohort from the CRO-VAX study was used.Te CRO-VAX-HCP study is a Belgian multicenter, prospective, and interventional study that was designed to assess the humoral response in a population of healthcare workers (HCWs) from 18 to 65 years of age having received two doses of the BNT162b2 mRNA COVID-19 vaccine (Comirnaty, Pfzer-BioNTech) [27].Te study was approved by a central ethical committee (CHU UCL Namur, Yvoir, Belgium; approval number: 2020-006149-21).A total of 231 participants were initially enrolled.Participants provided written informed consent to take part in the study [28].Only subjects without breakthrough infection were included.Only samples (n � 45) collected after 0, 7, 14, 28, 56, and 90 days after vaccination were included in this study to match the sampling scheme of AZD7442 participants [18,29].

Neutralizing Antibodies. Neutralizing antibodies were analyzed using a pseudovirus neutralization test (pVNT).
Pseudoviruses were from E-enzyme (Gaithersburg, MD, USA).SARS-CoV-2 pseudoviral particles are replicationdefcient Maloney murine leukemia virus (MLV or MuLV) pseudotyped with the SARS-CoV-2 spike protein carrying a genotype depending on the variant used.Tey also contain the open reading frame (ORF) for frefy luciferase as a reporter.Briefy, HEK293T hACE2 cells (catalog n °: hkb-hace2, InvivoGen, CA, USA) were seeded at a density of 8,500 cells/well in a white 384-well cell culture plate.Te sera used were heat-inactivated in a water bath at 54 °C for 30 min and then serially diluted in a culture medium, Dulbecco's modifed Eagle's medium (DMEM, catalog n °: L0102-500, VWR, PA, USA), supplemented with 10% of fetal bovine serum (FBS, catalog n °: S181B-100, VWR, PA, USA).Tereafter, samples are mixed in a 1 : 4 ratio with pseudovirus and incubated for 2 h at 37 °C and 5% CO 2 .Tis mixture was added to the cell culture plates and incubated for 48 h at 37 °C and 5% CO 2 .Te reading is done on the Spectramax 3 iD (Molecular Devices, LLC, CA, USA) after emptying the plate and flling with frefy luciferase reagent to measure the activity of luciferase which is proportional to the cells infected by the pseudovirus.In this study, this technique was used to assess the neutralizing capacity of AZD7442 against diferent variants: the wild-type SARS-CoV-2 spike protein (D614G), the alpha (UK B.1.1.7),beta (South Africa B.1.351),gamma (Brazil P.1), delta (Indian B.1.617.2), and omicron subvariants BA.5 and XBB.1.5.Te antibody titer was defned as the dilution of serum at which 50% of the infectivity potency is inhibited (IC 50 ) using a nonlinear sigmoid model.A sample is considered negative if its dilution titer is below the 20-fold dilution.Tis technique has already been described in detail elsewhere [27].

Binding Antibodies.
Total binding antibodies against the receptor-binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike protein were measured by the Elecsys anti-SARS-CoV-2 S assay which measured total antibodies (application code n °:10230, Roche Diagnostics, Machelen, Belgium) with a positivity cutof of 0.8 BAU/mL.Te analyzer performs an automatic 100-fold dilution for the signal above 250 BAU/mL to extend the measurement range up to 25,000 BAU/mL.In addition, total antibodies against the nucleocapsid (Roche Diagnostics) were measured using the Elecsys anti-SARS-CoV-2 assay.Results above 0.165 cutof index were considered positive as previously determined [26].

Statistical Analysis.
Te mean and 95% confdence intervals (95% CI) were used to describe the data.Diferences in antibody titers between groups were analyzed using a oneway ANOVA with the Geisser-Greenhouse correction.Correlations were computed using a nonparametric Spearman correlation test.Kinetic models were computed using non-log-transform data and using the following equation: In this equation, "a" stands for the maximal antibody response, "b" for the baseline response, "c" for the antibody production rate, and "d" for the antibody elimination rate.Te half-life time (T 1/2 ) was obtained from this model, which permitted the calculation of the elimination rate of the antibodies.Te area under the curve (AUC) from day 0 to day 90 has been computed.Te time required to reach the negativity threshold of the pVNT tests, i.e., a dilution titer below 1/20, was also computed.Te signifcance level was set at a p value < 0.05.Analyses were performed using GraphPad Prism 9.0.1 (GraphPad Software, San Diego, CA, USA) and JMP Pro 16.0.0(JMP ® , version 16.0.0SAS Institute Inc., Cary, NC, USA, 1989-2023).

AZD7442 Neutralizing Capacities among Diferent SARS-
CoV-2 Variants.Maximum titers were all reached 14 days after administration of AZD7442, regardless of the variant.Te maximum mean titers difered depending on the variant.For the WT SARS-CoV-2, the maximum mean titer reached the upper limit of the test, i.e., 5,120, while for the alpha variant and the beta variant, maximum mean titers were 3,010 and 253.6, respectively.Regarding the gamma and delta variants, the maximum mean titers were 993.8 and 864.8.Finally, for the omicron BA.5 and XBB.1.5subvariants, the maximum mean titer was 701.9 and 335.0, respectively (Table 1 and Figure 2).

AZD7442 Total Binding Antibody.
Te total binding antibody titer reached the maximum measurable signal (25,000 BAU/mL) after 7 and 14 days after AZD7442 Case Reports in Infectious Diseases administration for seronegative and seropositive subjects, respectively.However, there was no signifcant diference in total antibody titers between the two cases.Te correlation between anti-RBD total binding antibodies and NAb titer is shown in Figure 3.

Discussion
According to our results, AZD7442 provided a higher maximal neutralizing capacity response than the level of NAbs obtained following primary vaccination.Te T max was also shorter after the injection of mAbs compared to the vaccination (14 vs. 28 days).Tis rapid and sharp elevation of NAb titers agrees with the data of Levin et al. that reported NAb positivity at 8 days postinjection with the WT strain with a NAb titer of 493.1 (95% CI: 469.3-518.1).However, they reported an increase up to day 28 with a titer of 677.3

4
Case Reports in Infectious Diseases   Case Reports in Infectious Diseases 30 days after administration of AZD7442 [31].Another major diference between those who received conventional vaccination and those who received mAbs was the elimination kinetics of the NAbs.According to our results, the T 1/2 of the neutralizing capacity induced by AZD7442 was 8.1 days and 11.8 days for the NAbs generated by vaccination, i.e., T1/2 approximately 1.5 times lower.AstraZeneca reported a T 1/2 of 80 days for tixagevimab and 84 days for cilgavimab in the summary of product characteristics (SmPCs) [11].Tis may be related to the diference between the measurement of the circulating concentration of AZD7442 and the measurement of the neutralizing capacity induced by the injection of this product, the latter being the technique used in our study.Tese diferences between primary vaccination and administration of AZD7442 raise questions about the prescription of these mAbs.According to the SmPCs, the combination of tixagevimab plus cilgavimab is indicated for both the prophylaxis and treatment of SARS-CoV-2 infections [11].However, the time to negative reported for the diferent variants shows a loss of early protection ranging from 101.1 days (days (95% CI: 30.0-135.4 days) for the alpha variant to 65.9 days (95% CI: 29.3-92.1 days) for the XBB 1.5 variant, which raises questions about the product's indication for long-term prophylaxis.Tese results contrast with those reported in the PROVENT study (conducted before the emergence of omicron and its subvariant), which reported protection for up to 180 days postadministration [13].Tese worries about the use of AZD7442 for prophylaxis are shared by the expert panel of the National Institutes of Health responsible for guidelines relating to COVID-19 treatment.In the latest update (March 6, 2023), the expert panel decided on the use of the combination of tixagevimab plus cilgavimab for preexposure prophylaxis of COVID-19 [32].Te current recommended administration schedule for AZD7442 consists of a single 150 mg dose of tixagevimab followed immediately by a single 150 mg dose of cilgavimab.Tis administration schedule applies to both prophylactic and therapeutic uses.As mentioned by the company, there are no data on the safety and efcacy of repeated doses, but our results suggest that in order to maintain protection against the latest VOIs, several close doses are necessary with a regimen of 1 dose every 2 months [11].Vaccination and injection of monoclonal antibodies are two diferent strategies, on the one hand, 150 mg of tixagevimab and 150 mg of cilgavimab could represent more antibodies than the body produces, but on the other hand, the response produced postvaccination is more sustained, demonstrating a continuous production process.Tis hypothesis is supported by the signifcant diference between AUCs calculated with a higher AUC for vaccine response (66,752 pVNT titer dilution −1 × day (95% CI: 27,363-164,389) compared to 78,857 pVNT titer dilution −1 × day (95% IC: 42,697-118,382)).Among the diferent correlations computed between NAbs and binding antibodies, there are two of them that are not signifcant, for the alpha variant and delta variant (p value � 0.07).Moreover, there are discrepancies between the two tests for the results obtained at day 90.Indeed, the total antibodies always provide a positive result, while the NAbs are negative (Figure 3).Tis result description suggests that binding antibodies alone cannot be considered a marker of protection against reinfection.Tis discrepancy between NAbs and total antibodies has already been reported in the vaccinology literature [18,33,34].
At the beginning of the pandemic, CCPs were presented as a solution to the lack of efective therapies.Te comparison between CCPs and mAbs is mandatory because CCPs represent an alternative to mAbs administration whereas vaccination is not.Our results show a decrease in the neutralizing capacity of the mAbs tested as a function of the variants analyzed, with the lowest peak in NAbs obtained for the beta variant and the lowest time to negative for the most recent VOI, XBB.1.5.Tis trend towards a gradual decrease of neutralizing capacity would require the mAbs to be adapted to the circulating VOIs, but this would mean creating new mAbs where the CCPs are, by defnition, naturally adapted to the VOIs currently circulating in the population.Franchini et al. observed that CCPs are adapted to the VOIs circulating at the time of plasma collection [35].However, this also requires frequent collection of CCPs in order to have adequate stocks [36].

Conclusions
We show a diference in the neutralizing capacity of AZD7442 recipients compared to a vaccinated cohort.Tis diference can be explained by the mechanism of action of the two approaches.Indeed, mAbs are directly related to their T 1/2 , whereas vaccination allows the immune system to generate its own antibodies.Whether with vaccination or mAbs, a modifcation of the neutralizing capacity according to the VOIs was observed.Tis observation strengthens the need to assess the efectiveness of these antibodies in patients who have received AZD7442 over time.Our results reinforce the need of reviewing the use of AZD7442 in relation to VOIs and potentially adapting its administration schedule.Based on these and depending on the variant, it could be of interest to consider a bimonthly administration of AZD7442, especially considering the XBB1.5 variant.AZD7442 could be indicated for short-term prophylaxis in frail patients who may be acutely exposed to SARS-CoV-2.Nevertheless, further investigations are necessary to confrm these results, and continuous surveillance of VOIs is needed.

Figure 1 :
Figure 1: Kinetics models of neutralizing antibodies' response in (a) AZD7442 cases and (b) primo-vaccination BNT162b2 cohort.Means and standard deviation are shown whenever possible for diferent time points.Te dotted line represents the positivity test of the pVNT technique, i.e., an NAb titer of 20.

Figure 2 :Figure 3 :
Figure 2: Evolution of the neutralizing antibody (NAb) titers over time following administration of AZD7442 according to the variant of interest.Te dotted line represents the positivity threshold of neutralizing antibodies: 1/20.

Table 1 :
Mean neutralizing antibody titers after AZD7442 administration were obtained for each time point with the diferent SARS-CoV-2 variants tested.
[30] CI: 647.1-709.0)[13].Our results indicate an earlier peak of NAbs at 14 days as well as an earlier decay starting before 28 days.Tese results are in accordance with those of Loo et al. who reported a similar T max at 14 days[30].Bruel et al. reported positivity in 5 out of 8 patients at three days after injection and in 7 out of 8 patients between 3 and