key: cord-0977210-yv9ygtwe
authors: Kumar, Deepali; Hu, Queenie; Samson, Reuben; Ferreira, Victor H.; Hall, Victoria G.; Ierullo, Matthew; Majchrzak‐Kita, Beata; Hardy, William; Gingras, Anne‐Claude; Humar, Atul
title: Neutralization against Omicron variant in transplant recipients after three doses of mRNA vaccine
date: 2022-03-21
journal: Am J Transplant
DOI: 10.1111/ajt.17020
sha: 511904234b07857aebc102d87ec9bfe5cdb33af3
doc_id: 977210
cord_uid: yv9ygtwe

The SARS‐CoV‐2 virus Omicron variant has now supplanted wild‐type virus as the dominant circulating strain globally. Three doses of mRNA COVID‐19 vaccine are recommended for transplant recipients as their primary vaccine series. However, the immunogenicity of mRNA vaccines as they specifically relate to the Omicron variant are not well studied. We analyzed Omicron‐specific neutralization in transplant recipients after three‐doses of mRNA‐1273 vaccine. Neutralization was determined using a SARS‐CoV‐2 spike pseudotyped lentivirus assay with constructs for Omicron and Delta variants. A total of 60 transplant patients (kidney, kidney‐pancreas, lung, heart, liver) were analyzed 1 month and 3 months after completion of three doses of mRNA‐1273. At 1 month, 11/60 (18.3%) patients had detectable neutralizing antibody responses to Omicron (log(10)ID50 of 2.38 [range 1.34–3.57]). At 3 months, 8/51 (15.7%) were positive (median log(10)ID50 [1.68; range 1.12–3.61; approximate fivefold reduction over time]). The proportion of positive patients was lower for Omicron versus wild‐type, and Omicron vs. Delta (p < .001). No demographic variables were found to be significantly associated with Omicron response. Many patients with a positive anti‐RBD response still had undetectable Omicron‐specific neutralizing antibody. In conclusion, three doses of mRNA vaccine results in poor neutralizing responses against the Omicron variant in transplant patients.

AJT KUMAR et Al.

immunogenicity and COVID-19 infections occurring in immunocompromised hosts may be a critical factor for variant evolution. 4 Therefore, it is important to understand vaccine immunogenicity against circulating variants over time in these patients. We analyzed serum from organ transplant recipients one month and three months after three doses of mRNA-1273 vaccine for anti-RBD titers and neutralization activity against Omicron SARS-CoV-2 variant compared with Wild-type and Delta.

Patients were identified and enrolled from previous trials of mRNA-1273 vaccine. 1 Additional informed consent was obtained for longterm follow-up, blood collection and testing of serum samples against circulating variants. mRNA-1273 was administered at a 0-,1-, and 3-month dosing schedule with blood collected at months 4 and 6. Sera were analyzed for anti-RBD and neutralization against wildtype (WT), Delta and Omicron SARS-CoV-2 pseudoviruses.

Neutralization assays were performed using a previously validated SARS-CoV-2 spike pseudotyped lentivirus assay with constructs for Omicron and Delta variants. Spike cDNAs encoding full-length wildtype SARS-CoV-2 bearing the D614G mutation, or the full-length Delta variant, were obtained from Twist Bioscience. The spike cDNA encoding the full-length Omicron variant was obtained from Thermo Fisher Scientific. For expression in mammalian cells, the aforementioned cDNAs were cloned downstream of the CMV promoter/enhancer in the HDM expression plasmid (kindly provided by Dr. Jesse Bloom). These spike expression plasmids are freely available through CoVaRR-Net (https://nbcc.lunen feld.ca/resou rces/). The pseudovirus neutralization assay was performed as described previously, 5,6 with a minor modification of the starting dilution for Omicron to 1:40 and Delta to 1:45. Briefly, pseudotyped lentivirus particles were generated from co-transfection of the viral packaging (psPAX2, Addgene), the ZsGreen and luciferase reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W, kindly provided by Jesse Bloom) and the spike protein were first prepared and serially diluted 3-fold (2.5-fold for Omicron testing) over 7 dilutions, followed by incubation with diluted virus at a 1:1 ratio for 1 h at 37°C. The virus and serum mixture were then added to HEK293T-ACE2/TMPRSS2 cells and incubated for 48 h prior to lysis using the BrightGlo Luciferase Assay System (Promega, Madison, WI). Luminescence signals were detected using a PerkinElmer Envision instrument. The 50% neutralization titers (ID50s) were calculated in GraphPad Prism 9 (GraphPad Software, San Diego, CA) using a nonlinear regression (log[inhibitor] versus normalized response -variable slope) algorithm. A positive neutralization assay can be defined as any dilution that results in 50% viral neutralization as calculated based on the above generated curve (log 10 ID50 > 0). Both the HEK293TN and HEK293T-ACE2/TMPRSS2 cells were maintained at 85% confluency for no more than 25 passages.

Anti-receptor binding domain (RBD) testing was performed using the Roche Elecsys anti-SARS-COV-2 S enzyme immunoassay as per manufacturer's instructions. The detection threshold for this assay is 0.4 U/ml although positive detection is defined as ≥0.8 U/ml. Absence of previous diagnosis of COVID-19 was confirmed by testing for antinucleocapsid protein antibody using the 1-month post-third dose sample (Abbott Laboratories) as per manufacturer's instructions. A cut-off of <1.4 was considered negative as per manufacturer's instructions. (n = 15). Median time from transplant was 3.57 years (IQR 1.99-6.75). Tacrolimus, prednisone, and mycophenolate were the most common immunosuppression agents. Baseline demographic data are shown in Table 1 . No patient had previous COVID-19 as confirmed by negative anti-nucleocapsid antibody.

Neutralization at 1 month and 3 months post-third-dose vaccination is shown in Figure 1A for WT, Delta, and Omicron. One month postthird dose, 11/60 (18.3%) patients had detectable neutralizing antibody responses to Omicron with a median log 10 ID50 of 2.38 (range 

We assessed demographic factors and immunosuppression in relation to those who developing Omicron specific neutralization response vs. those who did not. The data are shown in Table 1 We also show that many patients had detectable anti-RBD titers at 1 month (70%) and 3 months after third vaccine dose. However, despite having detectable RBD antibodies, the vast majority of patients did not have neutralizing capacity against the Omicron variant. Although RBD titers were usually higher in those that were able to neutralize Omicron, the significant overlap in values between groups especially at titers >1000 U/ml suggests that standard antibody measurements require cautious interpretation.

Limitations of our study are that no clear protective correlate exists for current immunogenicity assays (including neutralization) although estimates suggest that log 10 ID5 values between 1.0 to Moderna had no role in funding the study or in the design, conduct, analysis or any other aspect of the study. We are grateful to

Ms. Ilona Bahinskaya, and Ms. Natalia Pinzon for their contributions to the study.

The authors of this manuscript have conflicts of interest to dis- 

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

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