key: cord-0303669-cguufxdg
authors: Jergovic, M.; Coplen, C. P.; Uhrlaub, J. L.; Beitel, S. C.; Burgess, J. L.; Lutrick, K.; Ellingson, K. D.; Watanabe, M.; Nikolich-Zugich, J.
title: Resilient T cell responses to B.1.1.529 (Omicron) SARS-CoV-2 variant
date: 2022-01-16
journal: nan
DOI: 10.1101/2022.01.16.22269361
sha: 94ff046cb1ce76093fbe9788dd38ac956fe83980
doc_id: 303669
cord_uid: cguufxdg

Emergence of the SARS-CoV-2 variant-of-concern (VOC) B.1.1.529 (Omicron) in late 2021 has raised alarm among scientific and health care communities due to a surprisingly large number of mutations in its spike protein. Public health surveillance indicates that the Omicron variant is significantly more contagious than the previously dominant VOC, B.1.617.2 (Delta). Several early reports demonstrated that Omicron exhibits a higher degree (~10-30-fold) of escape from antibody neutralization compared to earlier lineage variants. Therefore, it is critical to determine how well the second line of adaptive immunity, T cell memory, performs against Omicron in people following COVID-19 infection and/or vaccination. To that purpose, we analyzed a cohort (n=345 subjects) of two- or three- dose messenger RNA (mRNA) vaccine recipients and COVID-19 post infection subjects (including those receiving 2 doses of mRNA vaccine after infection), recruited to the CDC-sponsored AZ HEROES research study, alongside 32 pre-pandemic control samples. We report that T cell responses against Omicron spike peptides were largely preserved in all cohorts with established immune memory. IFN-gamma producing T cell responses remained equivalent to the response against the ancestral strain (WA1/2020), with some (<20%) loss in IL-2 single- or IL-2+IFN-gamma+ poly-functional responses. Three-dose vaccinated participants had similar responses to Omicron relative to convalescent or convalescent plus two-dose vaccinated groups and exhibited responses significantly higher than those receiving two mRNA vaccine doses. These results provide further evidence that a three-dose vaccine regimen benefits the induction of optimal functional T cell immune memory.

The B.1.1.529 (Omicron) SARS-CoV-2 variant likely originated in Botswana and was first reported to the World Health Organization (WHO) by the South African government on November 24 th , 2021 1 . Several days later, the WHO and the United States designated Omicron as a variant of concern (VOC). Omicron has accumulated a surprisingly large number of mutations compared to the original Wuhan strain; a total of 60 out of which 50 were nonsynonymus 2 . 32 of those mutations reside in the Spike (S) protein, which is the sole target of the majority of the currently approved SARS-CoV-2 vaccines and immunoglobulin therapies. The large number of mutations raised concerns about the potential for increased transmissibility, escape from vaccine protection, and potential pathogenicity. Although peer-reviewed epidemiological and clinical studies remain scarce, considerable evidence points to increased transmissibility of Omicron as compared to previous VOCs, including the highly transmissible B.1.617.2 (Delta) variant. A cohort analysis of household transmission with >72 thousand index cases in England showed that two-fold more Omicron index cases gave rise to a secondary household case in comparison to Delta 3 . Omicron is now the dominant SARS-CoV-2 variant in many parts of the world 4,5 , with a recent study from the University of Hong Kong reporting that the Omicron variant multiplies about 70 times faster in human respiratory tract tissues as compared to Delta 6 . Modeling studies further showed that mutations in the receptor binding domain (RBD) result in stronger binding of Omicron S protein to human ACE2 receptor 7 . Early studies demonstrated a substantial reduction in antibody neutralizing capacity against Omicron, and in vitro findings using authentic SARS-CoV-2 viruses indicated that compared to Delta the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced 8 .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01. 16.22269361 doi: medRxiv preprint This was independently confirmed by the findings of Rössler et al. which demonstrated that antibody neutralization capacity against Omicron was maintained best in sera from individuals who experienced infection and were subsequently vaccinated 9 .

Of interest, the spread of Omicron in South Africa has been followed by a decrease in hospitalizations 10 and death rates 11 . Latest reports from UK also showed lower hospitalization rates and mortality over the period when Omicron became the dominant VOC 3 . Thus, the current consensus is forming that disease severity may be reduced with the Omicron variant as compared to Delta. Given the large degree of escape from antibody response, T-cell mediated immunity could be essential to prevent Omicron-induced severe COVID-19. Indeed, in studies with the original, Wuhan strain, depletion of T cells in convalescent macaques resulted in impaired immunity against rechallenge with SARS-CoV-2, suggesting a significant role for T cells in the context of subprotective antibody titres 12 . Human clinical studies also demonstrated a link between SARS-CoV-2 T cell responses and reduced disease severity 13, 14 . Thus, it is paramount to determine how well T cell immunity against Omicron is preserved in vaccinees and COVID-19 convalescents. We and others have shown that T cell immunity against Delta, Gamma and other variants was largely preserved in mRNA vaccine recipients 15, 16 . However, earlier variants contained drastically lower numbers of mutations in the spike protein.

To assess cross-reactive T cell immune memory to the Omicron variant we have analyzed a large cohort of participants recruited into the CDC-sponsored AZ HEROES research study 17 . Participants in this cohort included subjects sampled post SARS-CoV-2 infection; fully vaccinated subjects (2 doses of mRNA vaccine); subjects post . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 16, 2022. that three doses of mRNA should be considered as an optimal, full vaccination regimen to induce robust T cell immunity.

This study was approved by the University of Arizona IRB (protocol #2102460536 and #1410545697) and the Oregon Health and Science University IRB (protocol #00003007, pre-pandemic controls) and was conducted in accordance with all federal state and local regulations and guidelines. The cohort included 215 recipients of two doses of mRNA SARS-CoV-2 vaccines, 25 recipients of three doses of mRNA . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 1 ), stained with the live dead fixable blue dye (Thermofisher) and then fixed and permeabilized using the FoxP3 Fix/Perm kit (eBioscience, San Diego, CA). Samples were acquired using a Cytek Aurora cytometer (Cytek, Fremont, CA) and analyzed by FlowJo software (Tree Star, Ashland, OR). Dead cells and doublets were excluded prior to analysis.

Graph Pad Prism v9 was used for statistical analysis. Upon inspection of data distribution by Shapiro-Wilks normality test, differences between paired samples treated with different peptide pools were calculated by two-tailed Wilcoxon rank test.

Differences between the subject groups were calculated either by Mann Whitney U-test, Kruskal Wallis test with Dunn's post hoc correction and. For all statistical differences *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 1A ). Spike peptide pool induced a 5-fold increase in IFN-γ SFU in immunized post pandemic participants while no effect was evident in the pre-pandemic samples (Suppl. Figure 1A) . Pre-pandemic samples overall showed little crossreactive responses to SARS-CoV-2 peptide pools. This limited cross reactivity was slightly higher against the WA1/2020 peptide pool compared to Omicron, as evidenced by a higher number of IFN-γ SFU (Suppl. Figure 1B) . When comparing samples from all immunized participants, the number of IFN-γ SFU between Omicron and WA1/2020 peptide stimulated wells was equal (Figure 1B, left panel) .

However, the number of IL-2 SFU was reduced ( Figure 1B, middle panel) from an . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01.16.22269361 doi: medRxiv preprint average of 167 with WA1/2020 to 139 with the Omicron peptide pool, representing a 16.8% reduction. There was no difference in GrB SFU between the peptide pools ( Figure 1B, right panel) . Next, we analyzed the number of polyfunctional (double or triple cytokine positive) T cells. We detected a decrease in IFN-γ IL-2 double positive cells, but not IFN-γ GrB double positive cells (Figure 1C) , while triple positive (IFN-γ, IL-2, GrB) were also decreased with Omicron peptide pool stimulation. Based on this large sample (N=345 exposed/immunized participants) we can conclude that the decrease in T cell immunity to mutated Omicron spike peptide is minimal and that functional responses are largely preserved.

We next investigated the differences in T cell responses between the groups of study subjects. When stimulated with the WA1/2020 peptide pool, recipients of two doses of mRNA vaccine had significantly lower numbers of IFN-γ SFU (Figure 2A , left panel) than all other groups. A similar trend was measured with the Omicron peptide pool, albeit without reaching statistical significance (Figure 2A, right panel) . Similarly, number of IL-2 SFU was decreased in the 2X VAX group stimulated with WA1/2020 peptides ( Figure 2B) . Double positive (IFN-γ+IL-2+) and triple positive (IFN-γ+IL-2+GrB+) SFU were also lower in the 2X VAX group (Figure 2C) . Interestingly, the magnitude of polyfunctional triple positive cells to Omicron was the highest in the 3X VAX group (Figure 2D, right panel) . Jointly, these results imply that two doses of mRNA vaccine induced sub-maximal T cell immunity, and that at least three doses are required to achieve the same level of antigen specific T cells found in post-infection subjects. To compare the relative preservation of T cell responses to each variant between subjects vaccinated with courses of vaccines produced by different manufacturers (Pfizer and . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01.16.22269361 doi: medRxiv preprint Moderna), we pooled the results from the 2X and 3X vaccinated subjects and divided them into groups based on vaccine manufacturer. We found that while there was no statistical difference in IFN-γ T cell responses between ancestral and Omicron pools within vaccine manufacturer, there was a higher number of IL-2+ SFU detected in vaccines who received Moderna's vaccine (Supp. Fig. 2A ). We measured a higher number of IL-2+ SFU in samples from Moderna vaccinated subjects in response to either WA1/2020 or Omicron as compared to their Pfizer vaccinated counterparts (Supp. Fig. 2B ). The subjects grouped by vaccine manufacturer did not differ in the number of GrB SFU (Supp. Fig. 2C ). Next, we investigated whether T cell responses were dependent on the age of the subject or time elapsed from vaccination. The 2X VAX subjects were the largest group (N=215), and therefore the most appropriate cohort for this specific analysis. Additionally, analysis of all groups with immune memory demonstrated that IL-2 production was the cytokine most impacted by the mutations in Omicron Spike peptides as compared to WA1/2020 (Fig. 1) so we analyzed the impact of age or time from vaccination by IL-2+ SFU. We have detected a minimal, but measurable, negative correlation of IL-2 responses with age (r=-0.1575, p=0.0224) while there was no statistically significant association of IL-2 and time from vaccination ( Figure 3A, B) . There was no impact of age for double positive (IFN-γ+IL-2+) SFU, whereas triple positive (IFN-γ+IL-2+GrB+) SFU were negatively correlated to age of the subject (r=-0.2386, p<0.0001) but not to time-from-vaccination ( Figure 3C) . Overall, changes in function of T cell responses with the age of the participant was minimal.

Additionally, given that the 3X vaccinated group was on average 8 years older than 2X VAX (58 vs 50 years average age, respectively, Table 1 ) and had higher T cell . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022. Figure 3A , representative FCM in Figure 4A ). As previously reported 19 , we have detected more CD4 helper T cells than cytotoxic CD8 T cells upregulating CD137 (4-IBB) and OX-40 in response to spike peptide pools (Figure 4B) . This was further reinforced by higher correlation of the FLUORISpot response with flow cytometric CD4+CD137+OX-40+ cells than CD8+CD137+OX-40+ for both IFN-γ ( Figure 4C ) and IL-2 ( Figure 4D) . We did detect a slight decrease in the number of ag-specific CD4 and CD8 T cells in response to Omicron peptides vs. WA1/2020. However, the difference was not statistically significant ( Figure 4B) . We also analyzed the naïve and memory phenotype of circulating T cells for possible correlation to ag-specific T cell responses. We did not find any association of the naïve circulating T cell phenotype (CCR7+CD45RA+CD28+CD95-) or the advanced differentiation/senescence-related marker CD57 with antigen specific response (Supplemental Figure 3B and C.) . Given that CD4+ helper T cells represent the bulk of the Spike peptide response we have . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01.16.22269361 doi: medRxiv preprint additionally investigated the expression of Th polarizing cytokines IL-17a and IL-4 in the 2X VAX group. We found that both IL-17a ( Figure 4E ) and IL-4 ( Figure 4F ) were reduced in response to Omicron peptides, which could be interpreted as a positive finding, given that Th17 and Th2 responses would be expected to be counterproductive against viral infection.

Overall, our findings highlight resilience of T cell responses generated in response to a pre-Omicron infection and/or to Wuhan-derived spike protein-based vaccines in the face of the B1.1.529 Omicron variant, particularly as measured by the IFN-γ production, which was by far the largest component of the antiviral T cell response. It is tempting to speculate that this finding may in part explain resistance against severe forms of disease and death in breakthrough Omicron cases. We did measure decreased IL-2 and IL-2 plus other cytokines (polyfunctional) responses, however these were generally reduced by less than 20-30%, and the extent of reduction depended on the exposure/vaccination, being less reduced, if at all, in three-doses vaccine recipients. Stratified analysis of subjects immunized by vaccines or prior infection clearly revealed that 2X VAX participants exhibited inferior responses to both Omicron and the ancestral strain relative to infected, infected and vaccinated, and in particular, 3X VAX participants, that trended, or were significantly higher, by most measures of immunity. Therefore, our results stress the need for a three-dose vaccine regimen to achieve robust T cell immunity.

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The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01.16.22269361 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. D. Figure 1 .

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The copyright holder for this preprint this version posted January 16, 2022. ; . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 16, 2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01.16.22269361 doi: medRxiv preprint C.

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The copyright holder for this preprint this version posted January 16, 2022. ; https://doi.org/10.1101/2022.01.16.22269361 doi: medRxiv preprint

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We would like to thank the personnel of UArizona Health Sciences Biobank for expert processing and storage of human blood samples. We would like to thank our CDC colleagues Drs Natalie J. Thornburg, Mark G. Thompson and Julie Mayo Lamberte for logistical help, input into study design and comments on the manuscript.

Dr Nikolich is receiving research support, as well as honoraria as co-Chair of the Scientific Advisory Board for Young Blood Institute, Inc.,(YBI), a nonprofit company with the goal to explore the efficacy of use of human plasma and other blood products to delay adverse effects of aging and/or mitigate chronic diseases. YBI had no impact on the design, execution or conclusions of the present study or on the contents of this manuscript.