key: cord-0715190-wqeiyi8w
authors: Espi, Maxime; Charmetant, Xavier; Barba, Thomas; Koppe, Laetitia; Pelletier, Caroline; Kalbacher, Emilie; Chalencon, Elodie; Mathias, Virginie; Ovize, Anne; Cart-Tanneur, Emmanuelle; Bouz, Christine; Pellegrina, Laurence; Morelon, Emmanuel; Fouque, Denis; Juillard, Laurent; Thaunat, Olivier
title: The ROMANOV study found impaired humoral and cellular immune responses to SARSCov-2 mRNA vaccine in virus unexposed patients receiving maintenance hemodialysis.
date: 2021-07-18
journal: Kidney Int
DOI: 10.1016/j.kint.2021.07.005
sha: 3b3e2e23c15a470d285d4a97de57bd75ae1871d4
doc_id: 715190
cord_uid: wqeiyi8w

Patients on maintenance hemodialysis (MHD), which are at high risk of infection by SARS-CoV-2 virus and death due to COVID-19, have been prioritized for vaccination. However, because they were excluded from pivotal studies and have weakened immune responses, it is not known whether these patients are protected after the “standard” two doses of mRNA vaccines. To answer this, anti-spike receptor binding domain (RBD) IgG and interferon gamma-producing CD4(+) and CD8(+) specific-T cells were measured in the circulation 10-14 days after the second injection of BNT162b2 vaccine in 106 patients receiving MHD (14 with history of COVID-19) and compared to 30 healthy volunteers (four with history of COVID-19). After vaccination, most (72/80, 90%) patients receiving MHD naïve for the virus generated at least one type of immune effector, but their response was weaker and less complete than that of healthy volunteers. In multivariate analysis, hemodialysis and immunosuppressive therapy were significantly associated with absence of both anti-RBD IgGs and anti-spike CD8(+) T cells. In contrast, previous history of COVID-19 in patients receiving MHD correlated with the generation of both types of immune effectors anti-RBD IgG and anti-spike CD8(+) T cells at levels similar to healthy volunteers. Patients receiving MHD naïve for SARS-Cov-2 generate mitigated immune responses after two doses of mRNA vaccine. Thus, the good response to vaccine of patients receiving MHD with a history of COVID-19 suggest that these patients may benefit from a third vaccine injection.

center in shared vehicles, increase the risk for disease transmission 3 . As a result, the reported incidence of COVID-19 in hemodialysis centers was high, particularly during the peaks of the pandemic 1,4,5 . Furthermore, because of their comorbid profile and chronic kidney disease-induced immunosuppression [6] [7] [8] , the risk of death due to COVID-19 was consistently and dramatically higher in MHD patients infected with SARS-CoV-2 than in the general population 9-11 . Due to their higher risk for both infection by SARS-CoV-2 and death due to COVID-19, MHD patients were prioritized for vaccination in France 12 . Pivotal studies using lipid nanoparticle-encapsulated mRNA-based vaccines that encodes the full-length spike protein of SARS-CoV-2, indeed shown excellent efficacy (95%) at preventing COVID-19 illness in the general population after two doses of the vaccine administered intramuscularly three weeks apart 13, 14 . However, whether these good results are generalizable to individuals living with kidney disease, in particular those on MHD, is not certain because the latter were not enrolled in these studies 15 . Furthermore, several lines of evidence suggest that in MHD patients, immune response (in particular after vaccination) may be blunted 7, 8 . Hospital and compared these results to that of a cohort of 30 healthy volunteers.

J o u r n a l P r e -p r o o f

According to the recommendations of the French health authority 12 , vaccination with mRNA BNT162b2 COVID-19 vaccine was offered to all patients on MHD in the two centers of Lyon University Hospital (France) that did not have any of the following contra-indications: i) diagnosis of COVID-19 within the last 3 months, organ transplantation within the last 3 months, Rituximab injection within the last 3 months, ongoing flare of vasculitis, acute sepsis, major surgery within the last 2 weeks.

All adult patients who received a standard (prime + boost 3 to 5 weeks apart depending on availability of the dose) vaccination with BNT162b2 vaccine and gave consent for the use of their blood, collected at the time of a routine biological evaluation, for analysis of the post-vaccinal immune response were enrolled in ROMANOV study.

In the absence of validated correlates of vaccine-induced protection against SARS-Cov-2, i.e. measurable parameter indicating that a person is protected against becoming infected and/or developing COVID-19 disease 14 , we reasoned that HD patients would have the same excellent level of protection than the general population 14 if they were able to generate similar amount of specific humoral (antibodies) and cellular (helper and cytotoxic T lymphocytes) effectors. We therefore compared the Briefly, 10µL of serum were incubated in the appropriate buffer with magnetic microbeads covered with S-RBD recombinant antigen, in order to form immune complexes. After precipitation in a magnetic field and washing, ABEI (N-(4-Aminobutyl)-N-ethylisoluminol)-stained anti-human IgG antibodies were added to the samples. After a second magnetic separation and washing, the appropriate reagents were added to initiate a chemiluminescence reaction. When necessary, sera were diluted sequentially up to 1:1000.

Spike specific CD4+ and CD8+ T cells response were quantified in the circulation of the HV and HD patients using the QuantiFERON® SARS-CoV2 test (Qiagen, Netherlands), a commercially available Interferon Gamma Releasing Assay (IGRA), according to the manufacturer's instructions.

Briefly, after collection, 1mL blood was distributed in each tube of the assay : (i) uncoated tube : negative control/background noise, (ii) tube coated with mitogen : positive control, (iii) tube coated with HLA-II restricted 13-mers peptides derived from the entire SARS-CoV2 Spike glycoprotein used to stimulate CD4+ T cells and (iv) tube coated with HLA-II and HLA-I 8-and 13-mers derived from the entire SARS-CoV2 spike glycoprotein used to stimulate both CD4+ and CD8+ T cells. After 20 hours of culture at 37°C, tubes were centrifugated 15 minutes at 2500g, and stored at 4°C before INFᵧ quantification by ELISA.

The CD4+ T cell assay value was the difference between tube (iii) and the negative control. The CD8+ T cell assay value was the value obtained for tube (iv), with subtraction of the CD4 tube (iii) and the negative control (i).

All the analyses were carried out using R software version 4.0.4 (R Foundation for Statistical Computing, Vienna, Austria, 2021, https://www.R-project.org) and or GraphPad Prism v8.0 (San Diego, California USA). Categorical variables were expressed as percentages and compared with the chi-squared test. Continuous variables were expressed as mean ± SD and compared using one-way ANOVA and multiple t-tests post-hoc analyses or as median ± IQR and compared using Mann Whitney test for variables with non-normal distribution.

Logistic regression models were used in both univariate and multivariate analyses.

Non colinear explanatory variables associated with outcomes (ie. optimal humoral and cellular responses) in univariate analysis (p<0.1) were included in multivariate models.

The Firth's bias-correction method was used in cases of complete separation 17 .

Stepwise regression analyses with bidirectional elimination were then performed, using Aikake Information Criterion to select the most fitting final multivariate models.

Venn diagrams were computed using R with the "ggplot2" and "ggVennDiagram" packages.

Among the 150 MHD patients dialyzing at Lyon University Hospital, 38 refused the vaccine or had contra-indications to the injection. Of the 112 who were vaccinated, 1 declined participating to the study and 5 were lost during the follow-up (Figure 1) . The general characteristics of the 106 MHD patients available for analysis, including 14 with a previous history of COVID-19 dating more than >3 months (black circles), are summarized in Table 1 . Mean age was 65 years, most of them were male (65%) and had a high burden of comorbid conditions (including cardiovascular disease in 45%, and diabetes in 44%). In addition, 22% had a previous history of kidney transplantation and 12% were on immunosuppressive drugs (crossed circles).

These 106 MHD patients were compared to a cohort of 30 unmatched healthy volunteers (HV), 4 of which had a past history of COVID-19 dating of more than 3 months (black triangles, Figure 1) . The general characteristics of HV are presented in Table 1 .

According to the manufacturer, the threshold of detection of the assay for anti-RBD IgG is 1 A.U (Figure 2A 

The generation of IgG against a target protein requires a cognate interaction between antigen-specific B cells and antigen-specific CD4+ T cells 18, 19 .

In line with their strong anti-RBD IgG response, all HV (30/30, 100%) and MHD patients with previous history of COVID (14/14, 100%) had detectable spike-specific CD4+T cells in their circulation ( Figure 3A) . This percentage was 70% (48/69) for Responders but dropped to 18% (2/11) for Non-Responders among naïve MHD patients without immunosuppressive therapy ( Figure 3A ). As expected, MHD patients on immunosuppressive therapy had almost never detectable spike-specific CD4+T cells ( Figure 3A) . A correlation was therefore established between the presence of spikespecific CD4+T cells and the titer of anti-RBD IgG (Figure 3B) .

Complementing the role of antibodies, virus-specific CD8+ T cells are involved in the elimination of infected cells (virus "factories"). Like the humoral response, CD8+ T cell response of MHD patients appeared more heterogeneous than that of HV ( Figure 4A) .

Spike-specific CD8+ T cells could be detected in the large majority of HV (21/30, 70%) and MHD patients with previous history of COVID (12/14, 86%; Figure 4B ). This percentage was 43% (30/69) for Responders but only 18% (2/11) for Non-Responders among naïve MHD patients without immunosuppressive therapy ( Figure 4B) . Again, MHD patients on immunosuppressive therapy had almost never (1/12, 8%) detectable spike-specific CD8+T cells ( Figure 4B) .

with the presence of spike-specific CD8+ T cells in the circulation after vaccination Table 3 ). Among naïve MHD patients without immunosuppressive therapy, there were no difference in clinical and biological characteristics between patients that had or had not generated specific CD8+ T cells (Suppl Table 4 ).

Color-coded Venn diagrams were used to analyze the logical relation between the individual components of the immune response (IgG, CD4+ and CD8+T cells) induced by SARS-Cov-2 mRNA vaccine. Because immunosuppressive therapy has been shown above to strongly impair the response to the vaccine, these patients were analyzed separately (Figure 5A) Furthermore, it has also been suggested that uremic milieu of end-stage renal disease may be associated with antibody dysfunction 20 . It is therefore tempting to speculate that one could improve immune function (and therefore response to vaccine) of MHD patients by optimizing uremic toxins elimination. A theory in line with the data reported by Kovacic et al, demonstrating that higher Kt/V values were associated with better antibody response to HBV vaccine 21 .

The parameter that predicted the best an optimal response to vaccination of MHD patients was a previous history of COVID-19. Indeed, while previous history of COVID-19 did not significantly impact the generation of any of the 3 types of immune effectors in vaccinated HV, this parameter had a massive impact in MHD patients. In contrast with MHD patients naïve for the virus, those with a previous history of COVID-19 had a response to vaccine, which was indistinguishable from that of HV. This result may indicate that increasing the exposure to viral antigens could circumvent the immune dysfunction of HD patients. It is therefore tempting to speculate that naïve MHD patients with suboptimal immune response after 2 doses of vaccine might benefit from J o u r n a l P r e -p r o o f Table 1 Clinical description of healthy volunteers and MHD patients. N (%) or mean ± SD.

Abbreviations are: MHD, maintenance hemodialysis; HV, healthy volunteers; BMI, body mass index; SOT : solid organ transplantation ; min, minutes; CRP : C reactive protein; Kt/V was used for the quantification of dialysis adequacy by the formula : 

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Supplementary Table 1 -Univariate analysis relating characteristics responders VS non-responders in the whole cohort (106 MHD patients + 30 HV)

Supplementary Table 2 -Univariate analysis relating comparisons between High-Responders and Low + No-Responders in naïve MHD patients without IS drugs

Supplementary Table 3 -Univariate analysis relating characteristics of CD8

responders VS CD8+ non-responders in the whole cohort (106 MHD patients + 30 HV)

Supplementary Table 4 -Univariate analysis relating comparisons between CD8-Responders and CD8-Non-Responders in naïve MHD patients without IS drugs

The authors are indebted to the members of the GRoupe de REcherche Clinique 

Abbreviations are: Non-R, non-responders ; Low-R, low responders, High-R, high responders (low and high responders are defined by the median titer value of responders MHD patients without immunosuppressive therapy and naïve for the virus). BMI, body mass index; SOT, solid organ transplantation; Kt/V was used for the quantification of dialysis adequacy by the formula: dialysis clearance of urea (K) multiplied by t (dialysis time) divided by the volume of distribution of urea (V); CRP, c reactive protein.

Non-R N=11