Serum Neutralization of Omicron BA.5, BA.2 and BA.1 in Triple Vaccinated Kidney Transplant Recipients

Rune M. Pedersen, Line L. Bang, Ditte S. Tornby, Anna C. Nilsson, Christian Nielsen, Lone W. Madsen, Isik S. Johansen, Thomas V. Sydenham, Thøger G. Jensen, Ulrik S. Justesen, the COVAC-TX study group, Lars Vitved, Yaseelan Palarasah, Claus Bistrup, Thomas E. Andersen Department of Clinical Microbiology, Odense University Hospital and Research Unit for Clinical Microbiology, University of Southern Denmark, Odense, Denmark; Department of Clinical Immunology, Odense University Hospital and Research Unit for Clinical Immunology, University of Southern Denmark, Odense, Denmark; Department of Infectious Diseases, Odense University Hospital and Research Unit for Infectious Diseases, University of Southern Denmark, Odense, Denmark; Department of Cancer and Inflammation, University of Southern Denmark, Odense, Denmark; and Department of Nephrology, Odense University Hospital and the Nephrology Research Unit, University of Southern Denmark, Odense, Denmark


INTRODUCTION
K idney transplant recipients (KTRs) develop a lowerthan-normal antibody (Ab) response against the COVID-19 mRNA vaccines and are consequently more vulnerable to breakthrough infections. 1,2 Although booster vaccinations given to these patients increase serum-levels of spike Abs, 3 SARS-CoV-2 variants have emerged with increasing vaccine-evading properties, the most recent being the Omicron variant of concern. The original Omicron lineage, BA.1 (also known as B.1.1.529), is neutralized less efficiently by booster vaccinated KTRs than the ancestral and the Delta variant, as demonstrated by authentic virus and pseudovirus neutralization assays conducted with blood from these patients. [4][5][6] During spring 2022, Omicron BA.1 was rapidly replaced by the Omicron BA.2 subvariant in Europe and the United States and a descendant from Omicron BA.2, the even more transmissible Omicron BA.5 subvariant, which has now replaced other subvariants worldwide and to date (October 2022) continues to dominate the COVID-19 pandemic with more than 85% prevalence in most countries. 7 BA.5 deviates from BA.2 by 4 additional mutations, including 2 in the receptor binding domain of the spike protein, which makes this subvariant even more immune-evasive in otherwise healthy individuals. 8 Although the neutralization status in KTRs postvaccination against the original Omicron BA.1 was earlier estimated, 4-6 the neutralization capacity in KTRs and solid organ transplanted recipients in general, against the currently dominating descendants of Omicron BA.1, remains to be elucidated.
Here, we report the spike Ab levels and serum neutralization capacity against isolates of Omicron BA.1, BA.2, and BA.5 in KTRs who have received 3 doses of the BNT162b2 Pfizer-BioNTech mRNA vaccines (n ¼ 44); and compare these values to the BA.5 neutralization capacity in healthy controls at the same level of vaccination (n ¼ 20). In addition, we report the SARS-CoV-2 reactive T-cell response in a subset of the KTRs (n ¼ 25).

RESULTS
The KTRs in this cross-sectional study is part of a KTR cohort that has been described previously, together with the plaque reduction neutralization test (PRNT). 1 The PRNT 90 , which indicates the serum titer that reduces the plaque forming ability of the virus by >90%, was measured. A titer of 10 was applied as the cut-off, indicating the lowest titer that yields actual neutralization in our assay, because a PRNT 90 titer of 8.8 was recently shown to indicate the threshold of real-world protection against the Omicron variant of concern in humans. S1 Three clinical Omicron strains, BA.1, BA.2, and BA.5, and 1 Delta strain, were used for the PRNT 90 (Genome sequences: GenBank accession no. ON055874 for BA.1, ON055857 for BA.2, OP225643 for BA.5, and ON055856 for the Delta strain). All experimental work with SARS-CoV-2 was conducted in approved biosafety level 3 facilities. The serum samples were also analyzed for spike Ab levels using the LIAISON SARS-CoV-2 TrimericS IgG assay (DiaSorin, Saluggia, Italy). Finally, we performed flow cytometry on freshly collected blood to quantify SARS-CoV-2 inducible T cells in 25 KTRs and 3 healthy controls (for methods, statistics, and patient flow chart, see Supplementary Cohort Data, Supplementary Methods, Supplementary Statistics, and Supplementary  Figures S1 and S2).
The PRNT 90 was performed on serum collected at a median of 38 days (interquartile range [IQR] 33À49) after the third BNT162b2 vaccination, from 44 KTRs (19 females, 25 males) with a median age of 60 years (IQR 51-67). As a reference for BA.5 neutralization, PRNT 90 was also performed on serum collected at a median of 42 days (IQR 41-42) after the third BNT162b2 vaccination, from 20 healthy control subjects (12 females, 8 males) with a median age of 57 years (IQR 50À60). The characteristics of the KTR cohort are shown in Table 1. We found that 19 of 44 (43%), 15 of 44 (34%), 18 of 44 (41%) and 17 of 44 (39%) of the KTRs displayed above threshold neutralization of the Delta, Omicron BA.1, Omicron BA.2, and Omicron BA.5 strains, respectively ( Figure 1a). The PRNT 90 titer range of the KTRs toward the different strains were as follows: Delta <10 to 160, Omicron BA.1 <10 to 80, Omicron BA.2 <10 to 80, and Omicron BA.5 <10 to 40; with titer levels toward Omicron BA.5 being significantly lower for the KTRs than for the healthy controls (P < 0.0001, Mann-Whitney U test) ( Figure 1a). The median Ab levels in sera from the KTRs as measured on the Liaison platform was 193 Binding Antibody Units (BAU)/ml (IQR 8À1743), whereas the median Ab level of the controls was 4115 BAU/ml (IQR 2943À5800). This difference was statistically significant (P < 0.0001, t-tests). Ab levels correlated with the PRNT 90 titer of Delta (r ¼ 0.88, P < 0.0001, Spearman's correlation [SC]), Omicron BA.1 (r ¼ 0.82, P < 0.0001, SC), Omicron BA.2 (r ¼ 0.86, P < 0.0001, SC), and Omicron BA.5 (r ¼ 0.86, P < 0.0001, SC) among the KTRs as well as with Omicron BA.5 among the controls (r ¼ 0.74, P ¼ 0.0002, SC) (Figure 1b; Supplementary Figure S3). Finally, cellular immunity was evaluated in a subset of KTRs (n ¼ 25) and in 3 healthy controls using T cell flow cytometry. This analysis showed that 21 of 25 KTRs (84%) had detectable levels of cytotoxic CD8þ T cells directed against the SARS-CoV-2 spike protein, and 24 of 25 KTRs (96%) had spike protein-reactive  CD4þ T helper cells. The KTRs had similar levels of T helper cells compared to both of the vaccinated controls, and similar levels of cytotoxic T cells compared to the 2-times vaccinated, infection-naïve control. The infection convalescent, 2-times vaccinated control had considerably higher levels of cytotoxic T cells than all others, indicating the significant boost in these cells upon infection (Figure 1c).

DISCUSSION
Our results show that despite the splitting of the original Omicron variant of concern into increasingly In conclusion, our results indicate that the KTRs' neutralization capacity against the currently dominating Omicron BA.5 approximates their ability to neutralize earlier Omicron subvariants. Moreover, a considerable proportion of the KTRs show a relatively robust neutralization response against all tested Omicron subvariants after the first BNT162b2 booster. In addition, we observed a tendency toward a division of KTRs into low responders and high responders, with the first group, despite a general increase in Ab levels, still being unable to neutralize recent SARS-CoV-2 variants. Additional boosters including the now available bivalent mRNA vaccines may increase Ab levels and affinities even in this group, thus providing an important fundamental level of protection against currently circulating SARS-CoV-2 strains.

DISCLOSURE
All the authors declared no competing interests.

Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.  Figure S1. Flow cytometry gating strategy. Figure S2. Patient flow chart showing the inclusion of kidney transplant recipients for plaque reduction neutralization test and T cell analysis. NC, nucleocapsid. Figure S3. Neutralization of authentic SARS-CoV-2 Delta, Omicron BA.1 and Omicron BA.2 strains in relation to spike antibody levels.