key: cord-0300211-hevwu4ux
authors: Hadjadj, J.; Planas, D.; Ouedrani, A.; Buffier, S.; Delage, L.; Nguyen, Y.; Bruel, T.; Stolzenberg, M.-C.; STAROPOLI, I.; Ermak, N.; Macraigne, L.; Morbieu, C.; Henriquez, S.; Veyer, D.; Pere, H.; Casadevall, M.; Mouthon, L.; Rieux-Laucat, F.; Chatenoud, L.; Schwartz, O.; Terrier, B.
title: Immunogenicity of BNT162b2 vaccine Against the Alpha and Delta Variants in Immunocompromised Patients
date: 2021-08-09
journal: nan
DOI: 10.1101/2021.08.08.21261766
sha: 56cf22c3ffd487b1881c356426097851cb49dac2
doc_id: 300211
cord_uid: hevwu4ux

Background. The emergence of strains of SARS-CoV-2 exhibiting increase viral fitness and immune escape potential, such as the Delta variant (B.1.617.2), raises concerns in immunocompromised patients. To what extent Delta evades vaccine-induced immunity in immunocompromised individuals with systemic inflammatory diseases remains unclear. Methods. We conducted a prospective study in patients with systemic inflammatory diseases (cases) and controls receiving two doses of BNT162b2. Primary end points were anti-spike antibodies levels and cross-neutralization of Alpha and Delta variants after BNT162b2 vaccine. Secondary end points were T-cell responses, breakthrough infections and safety. Results. Sixty-four cases and 21 controls not previously infected with SARS-CoV-2 were analyzed. Kinetics of anti-spike IgG and IgA after BNT162b2 vaccine showed lower and delayed induction in cases, more pronounced with rituximab. Administration of two doses of BNT162b2 generated a neutralizing response against Alpha and Delta in 100% of controls, while sera from only one of rituximab-treated patients neutralized Alpha (5%) and none Delta. Other therapeutic regimens induced a partial neutralizing activity against Alpha, even lower against Delta. All controls and cases except those treated with methotrexate mounted a SARS-CoV-2 specific T-cell response. Methotrexate abrogated T-cell responses after one dose and dramatically impaired T-cell responses after 2 doses of BNT162b2. Conclusions. Rituximab and methotrexate differentially impact the immunogenicity of BNT162b2, by impairing B-cell and T-cell responses, respectively. Delta fully escapes the humoral response of individuals treated with rituximab. These findings support efforts to improve BNT162b2 immunogenicity in immunocompromised individuals (Funded by the Fonds IMMUNOV; ClinicalTrials.gov number, NCT04870411).

The course of Coronavirus-induced disease 2019 (Covid-19) is less favorable in patients with systemic inflammatory diseases. Older age, male gender, cardiovascular disease and obesity are risk factors of severe forms and Covid-19-related death in this immunocompromised population [1] [2] [3] [4] , as it is in the general population 5, 6 . Disease-specific factors including disease activity and treatments, especially glucocorticoids, mycophenolate mofetil and rituximab, are additional risks factors [1] [2] [3] . The efficacy of vaccine has been recently mitigated by variants of SARS-CoV-2 exhibiting increase viral fitness and immune escape potential. Among them, the Delta variant (B.1.617. 2) was first identified in India in October 2020 and rapidly became the predominant strain across the globe 10 . While in vitro data indicate reduced sensitivity of Delta variant to antibody neutralization 11 , only modest differences in vaccine effectiveness are noted with Delta as compared to Alpha 12 . In patients with systemic inflammatory diseases, the use of rituximab and methotrexate, commonly used to induce and maintain remission, decreases seroprotection rate following vaccination against influenza, pneumococcus and ancestral and Alpha variants of SARS-CoV-2 [13] [14] [15] [16] . Yet, how the different immunosuppressive or immunomodulatory drugs tune humoral and cellular responses, and how the Delta variant impacts vaccine effectiveness in this population remains unclear.

In this study, we measured seroconversion, cross-neutralization of Alpha and Delta variants and T-cell responses induced by BNT162b2 in immunocompromised patients with systemic inflammatory diseases according to the treatments received. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021.

Extended B cell and T cell immunoprofiling were performed on whole blood as described in the Supplementary Appendix and Supplementary Figures 1-2 .

The S-Flow assay was used to detect antibodies bound to 293T cells stably expressing the spike protein (S) at their surface using flow cytometry. This assay is highly sensitive and allows quantification of antibodies through a standardized MFI (referred as Binding Unit ; BU), which is calculated using an anti-spike monoclonal antibody as reference. The method is described in the Supplementary Appendix.

The Alpha (B.1.1.7) variant originated from an individual returning from the United Kingdom.

The Delta (B.1.617.2) variant originated from a hospitalized patient returning from India. The variant strains were isolated from nasal swabs using Vero E6 cells and amplified by two passages. Additional information is described in the Supplementary Appendix.

The S-Fuse neutralization assay was used to assess the neutralizing activity of sera against emerging variants. The method is described in the Supplementary Appendix.

SARS-CoV-2-specific IFNγ-producing T cells were identifed by using commercially available pools derived from a peptide scan through SARS-CoV-2 N-terminal (pool S1) and C-terminal (pool S2) fragments of spike glycoprotein (JPT Peptide Technologies GmbH, BioNTech AG, Berlin, Germany). Results are expressed as Spot Forming Unit (SFU)/10 6 CD3+ T cells after subtracting background values from wells with non-stimulated cells. The method is described in the Supplementary Appendix. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.08.21261766 doi: medRxiv preprint

No statistical methods were used to predetermine sample size. The experiments were performed in blind regarding to the allocation groups. Flow cytometry data were analyzed with FlowJo v.10 software (TriStar). Calculations were performed using Excel 365 (Microsoft). Figures were drawn using GraphPad Prism 9. Statistical analyses were conducted using GraphPad Prism 9. Statistical significance between different groups was calculated using the tests indicated in each figure legend. Detailed statistical analysis is described in the Supplementary Appendix.

Between January and April 2021, 77 cases and 28 controls were included in the study. Twenty participants (13 cases and 7 controls) with positive SARS-CoV-2 serological tests at baseline were excluded from the main analysis (Figure 1) . Finally, 64 cases and 21 controls were analyzed. Baseline characteristics of patients are shown in Table 1 Compared to controls, cases showed lower total lymphocytes count. As expected, in the "rituximab" treatment group, circulating B cells were not detected (except in one patient) and levels of IgG, IgA and IgM were lower.

First, we analyzed the kinetics of induction of anti-spike IgG and IgA in patients' sera after the first and second dose of BNT162b2 vaccine. We observed a delayed response in cases compared to controls. Anti-spike IgG and IgA inductions were detectable mainly after the All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.08.21261766 doi: medRxiv preprint second dose in cases, whereas it was noted from the first dose in controls (Supplementary Figure 5) . On samples collected after the 2 doses, at 3 months, all treatment groups except the "other" group showed significantly lower anti-spike IgG levels than controls (Figure 2A) . The "rituximab" group showed the lowest response. Then, we categorized individuals who seroconvert in IgG or IgA at M3 as "responders". All controls and cases from the "other" treatment group seroconverted in IgG ( Figure 2B) . "Rituximab" showed again the lowest response, with only 50% of individuals who seroconverted at M3 (Figure 2B ). "Methotrexate" and "cDMARDs" treatment groups showed intermediate levels of anti-spike IgG levels at 3 months, with 93% and 68% of individuals who seroconverted, respectively (Figure 2A-B) . A large inter-individuals variability was observed in these two groups. The use of azathioprine or mycophenolate mofetil did not discriminate between responders and non-responders in the cDMARDs group. Anti-spike IgA levels after two doses of BNT162b2 were lower than for IgG in all cases and controls (Figure 2A-B) . Anti-spike IgA levels after the first and second doses of BNT162b2 vaccine were significantly lower in patients treated with rituximab and methotrexate compared to controls and cases from the "cDMARDS" and "other" treatment groups ( 

We next examined whether BNT162b2 vaccine-elicited antibodies at month 3 neutralized the Alpha and Delta variants in cases and controls ( Figure 2C-D) . Median ED50 for Alpha in controls and in cases from the "rituximab", "methotrexate", "cDMARDs" and "other" treatment All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ; groups were 1942, <7.5, 199, 65 and 2173, respectively; and 539, <7.5, 31, <7.5 and 270 for Delta ( Figure 2C) . Delta was 4-fold less sensitive to neutralization than Alpha in the controls, confirming previous observation 11 . Among cases, titers were reduced by 6-fold between Delta and Alpha in the "methotrexate" group, 9-fold in the "cDMARDs" group, 8-fold in the "other" group. The lack of neutralization in the "rituximab" group impaired the calculation of a fold decrease.

Then, we arbitrarily classified individuals as neutralizers according to the detection of neutralizing antibodies at a serum dilution of 1:30 and non-neutralizers. Administration of two doses of BNT162b2 generated a neutralizing response against the Alpha and Delta variants in 100% of controls. Only one individual in the "rituximab" group neutralized Alpha (5%) and none neutralized Delta ( Figure 2C-D) . Sera of 87% of patients in the "methotrexate" group neutralized Alpha, dropping to 57% against Delta ( Figure 2D ). Sera from patients in the "cDMARDs" group neutralized Alpha and Delta in 53% and 42%, respectively. Nine (14%) cases neutralized Alpha but not Delta, including 5 patients treated with methotrexate, 2 with cDMARDs, 1 with rituximab and 1 with anti-TNF- therapy. Correlation between Alpha and Delta neutralization titers, and between IgG production and ED50 of Alpha variant was strong in all participants except for those receiving rituximab and cDMARDs (Supplementary Figure   7) .

The lack of neutralization of Delta was associated with active disease (P<0.001), the use of rituximab (P<0.001), glucocorticoids (P=0.007) and low IgM (P=0.047) and IgG2 (P=0.05) levels (Supplementary Table 3 ). In multivariate analysis, ED50 of Delta remained negatively associated with rituximab (P<0.001), methotrexate (P<0.001) and cDMARDs (P<0.001) (Supplementary Table 4 ).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ;

Overall, B-cell response to BNT162b2 vaccine was impaired in immunocompromised patients at different levels depending on the treatments received. The effect was further amplified when evaluating the efficacy of sera to neutralize the Delta variant.

We then quantified anti-spike IgG and IgA and neutralization activity 3 months after vaccination in the 7 controls and 13 cases who had been previously infected with SARS-CoV-2 and excluded from the main analysis (Supplementary Figure 8) . In convalescent controls, vaccination boosted levels of anti-spike IgG and IgA as well as neutralizing antibody titres against both variants, as compared to the uninfected vaccinated control group. In previously infected cases under immunosuppressive or immunomodulatory drugs, a low response remained after vaccination.

We next investigated whether controls and cases mounted a SARS-CoV-2 specific T-cell response following the first and second doses of BNT162b2 vaccine (Figure 3 and Supplementary Figure 9 ). All controls (excepte one) and cases except those from the "methotrexate" treatment group had similar levels of specific T-cells in response to S1 pool ( Figure 3A) . Methotrexate completely abrogated T-cell responses after one dose and dramatically impaired T-cell responses after 2 doses of BNT162b2 compared to controls and cases from other treatment groups (Figure 3A and 3B) . Similar results, but less pronounced, were observed for S2 peptide pool ( Figure 3A and Supplementary Figure 9) . Importantly, despite the absence of neutralizing activity in response to BNT162b2, patients receiving rituximab showed increased levels of specific T-cell responses that reached after a delay the same levels as controls (Figure 3A and 3B) . The relationship between humoral and cellular All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.08.21261766 doi: medRxiv preprint immune responses against SARS-CoV-2 is shown in Figure 4 , highlighting the impact of the different treatment groups on both humoral and cellular responses. The lack of T-cell response was associated with the use of methotrexate (P=0.045) and glucocorticoids (P=0.012). In multivariate analysis, no variable correlated with SARS-CoV-2-specific IFNγ-producing T cells. Also, no significant differences in the proportion of circulating CD4+ memory T cells and Th1 T cells after the first and second dose of BNT162b2 were found in all groups (Supplementary Figure 6) .

Overall, T-cell responses to S1 and S2 peptide pools were similar in cases compared to the controls except methotrexate treated patients showing significantly decreased T-cell responses. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.08.21261766 doi: medRxiv preprint

As the Delta variant spreads across the globe, aggregating data on the effectiveness of Covid-19 vaccines in specific immunocompromised populations is a critical issue. Data from solid organ transplant recipients, patients with malignant hemopathy or with chronic inflammatory arthritis suggested that risk factors for reduced SARS-CoV-2 vaccine immunogenicity included older age and treatments with glucocorticoids, rituximab, mycophenolate mofetil and abatacept 15, [17] [18] [19] . However, levels of anti-spike antibodies were mainly measured and few studies used neutralization assay or assessed T-cell response.

Additional studies specifically reported that B cell depletion by rituximab blocked humoral but not T cell response to vaccination, using anti-RBD IgG measurement and IFNγ ELISPOT Tcell response. SARS-CoV-2 antibody response was reported in 0 to 39% of the vaccinated Bcell-depleted patients, whereas T cell responses were noted in 58 to 100% 20,21 . This early assessment showed that humoral immunity to one or two doses of BNT162b2 was also impaired by methotrexate treatment 22-24 . However, conflicting results were found for cellular responses showing either preserved 22 or impaired T-cell activation 24 . Most of these studies assessed very early timepoints that may not allow an appropriate assessment of immune response after complete vaccination.

Sera from convalescent and vaccinated individuals neutralize less efficiently the Delta variant than the Alpha 11 . However, this was studied in the general population and assessing the sensitivity of the Delta variant to antibody neutralization in immunocomprised populations is thus necessary.

In this study, we focused on patients with systemic inflammatory diseases that were receiving rituximab, methotrexate and/or other cDMARDs, and provided important data regarding sensitivity to Delta variant according to the treatments used. We analyzed patients after the first and the second doses of the BNT162b2 vaccine. We report a delayed and lower induction of All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.08.21261766 doi: medRxiv preprint anti-spike IgG and IgA compared to controls, much more pronounced with rituximab. While two doses of BNT162b2 generated a neutralizing response against Alpha and Delta variants in 100% of controls, 95% of sera from patients treated with rituximab did not neutralize these two variants. In contrast, SARS-CoV-2 specific T-cell response were similarly measured in controls and cases with the exception of methotrexate-treated patients. This differential impairement of immunogenicity after BNT162b2 vaccine according to the treatments received, mainly for rituximab and methotrexate, is critical to identify patients in which optimization of vaccine strategies should be evaluated.

To counteract this impaired immunogenicity, the administration of a third dose of mRNA-based vaccine has been proposed. Recent data in solid-organ transplant recipients showed that a third dose of BNT162b2 vaccine increased the prevalence of seroconversion and antibody titers, without serious adverse events [25] [26] [27] . A third dose also increased specific cellular response even in patients who remained seronegative 27 .

Our study has several limitations. The findings are observational and based on small numbers and should be interpreted with caution. Differences in treatment groups where highly associated with the type of underlying inflammatory disease, and there may be differences among the populations. However, except for more frequent renal involvement at diagnosis in the "rituximab" group and younger age in the "cDMARDs" group, patients' characteristics were comparable between treatment groups.

Overall, we found that rituximab and methotrexate differentially impact the immunogenicicty of BNT162b2 vaccine, by imparing B-cell and T-cell responses, respectively. The Delta variant fully escapes the sub-optimal humoral response of individuals treated with rituximab. Our findings support efforts to improve effectiveness of mRNA vaccines in this immunocompromised population. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

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