Abstract
Patients with kidney failure are at increased risk for SARS-CoV-2 infection making effective vaccinations a critical need. It is not known how well mRNA vaccines induce B and plasma cell responses in dialysis patients (DP) or kidney transplant recipients (KTR) compared to healthy controls (HC). We studied humoral and B cell responses of 35 HC, 44 DP and 40 KTR. Markedly impaired anti-BNT162b2 responses were identified among KTR and DP compared to HC. In DP, the response was delayed (3-4 weeks after boost) and reduced with anti-S1 IgG and IgA positivity in 70.5% and 68.2%, respectively. In contrast, KTR did not develop IgG responses except one patient who had a prior unrecognized infection and developed anti-S1 IgG. The majority of antigen-specific B cells (RBD+) were identified in the plasmablast or post-switch memory B cell compartments in HC, whereas RBD+ B cells were enriched among pre-switch and naïve B cells from DP and KTR. The frequency and absolute number of antigen-specific circulating plasmablasts in the cohort correlated with the Ig response, a characteristic not reported for other vaccinations. In conclusion, these data indicated that immunosuppression resulted in impaired protective immunity after mRNA vaccination, including Ig induction with corresponding generation of plasmablasts and memory B cells. Thus, there is an urgent need to improve vaccination protocols in patients after kidney transplantation or on chronic dialysis.
INTRODUCTION
COVID-19 leads to a high morbidity and mortality especially among patients with kidney failure (1). Dialysis patients (DP) and kidney transplant recipients (KTR) are at increased risk of developing COVID-19 and experiencing a severe infection, due to exposure risk in the health care system, their co-morbidities, and their impaired immune function from kidney failure or immunosuppressive medications. For this vulnerable population, vaccination is of the utmost importance.
The mRNA SARS-CoV-2 vaccine BNT162b2 (BioNTech/Pfizer) has demonstrated efficacy in healthy individuals in a clinical study (2) and under real-world conditions (3). Recent data described a lower serological response to an mRNA vaccine in dialysis patients (4) and kidney transplant recipients (5), suggesting an overall diminished vaccine response. Whereas numerous studies have addressed the consequences of conventional vaccines on B and plasma cells (6–8) and corresponding Ig levels, nothing is known yet about the B lineage consequences in response to an mRNA vaccine among healthy controls and immunocompromised patients. The ongoing uremic state in kidney failure patients leads to an immune dysfunction on various levels of innate and adaptive immunity. Restoring kidney function by kidney transplantation does not fully restore cellular and adaptive immunity while immunosuppressive drugs impair protective immunity further. Thus, kidney failure patients (with or without kidney transplant) show an increased susceptibility for infection and viral-associated cancers (9–11).
Previous studies in kidney failure patients (with or without kidney transplant) report markedly diminished response to vaccinations. This has led to an adaption of vaccination protocols with either higher initial vaccine doses or more frequent booster doses (12, 13). If such adaptations of the protocol are required for the COVID-19 mRNA vaccines or if alternate adjuvanted vaccines are necessary is not yet known.
The induction of B cell memory by mRNA vaccines and the relation to humoral immune response is largely under-investigated, especially studies of immunocompromised cohorts. Upon natural acute SARS-CoV-2 infection, immunological memory (antibodies and memory B cells) is shown to last for at least 8 months (14–16). In patients with chronic kidney disease, such data are largely lacking, although prolonged time of viral shedding with impaired virus clearance is reported and likely related to impaired cell-mediated immunity (17).
In this study, we compared the characteristics of the humoral and antigen-specific B cell immune response against the mRNA vaccine BNT162b2 between healthy controls and patients with kidney failure treated by maintenance hemodialysis or kidney transplantation. We found a diminished humoral response to BNT162b2, and a lack of proper B lineage memory formation including RBD-specific plasmablasts and post-switch memory B cells.
RESULTS
Cohorts and patient characteristics
For this study, we recruited 35 healthy controls (HC), 41 patients on maintenance hemodialysis, 4 peritoneal dialysis patients and 40 KTR. Hemodialysis and peritoneal dialysis (PD) patients did not significantly differ in age and vaccine response and were therefore grouped together. After written informed consent, serum and peripheral blood mononuclear cells (PBMCs) were collected before vaccination (baseline) and 7 ± 2 days after boost vaccination (second dose), respectively. Serological follow-up was available in DP and KTR patients 3-4 weeks after boost. Due to local vaccination guidelines, HC, who were mainly health care workers, were significantly younger than DP (p< 0.01). DP were significantly older than KTR. As known for patients with kidney failure (18), a majority of DP and KTR were male. The median time on dialysis was 5.5 years (IQR, 2.0, 9.0). Among KTR only one patient was transplanted less than one year ago and median time after transplantation was 5.0 years (IQR. 2.0, 10.0). KTR were on a uniform immunosuppressive regimen with mycophenolate mofetil (MMF) in 39/40, steroid in 37/40 and calcineurin inhibitor (CNI) in 37/40 patients. Demographics are summarized in Table 1. To identify previously SARS-CoV-2 infected individuals we measured anti-nucleocapsid protein (NCP) antibodies 7 ± 2 days after boost, which is not a component of BNT162b2. Therefore, positivity of NCP originated from natural infection. One HC, one DP and one KTR were identified anti-NCP positive (Figure S1).
Substantially impaired serological response upon mRNA vaccination with BNT162b2 in DP and even more pronounced in KTR patients
Antibody response to BNT162b2 was assessed in all individuals 7 ± 2 days after boost using the Euroimmun ELISA for the detection of IgG and IgA against the S1 domain of the SARS-CoV-2 spike. All HC seroconverted, were positive for both anti-S1 IgG and anti-S1 IgA (Fig. 1 A, B), and showed SARS-CoV-2 neutralization (Fig. 1C). Anti-S1 IgA and anti-S1 IgG titers were markedly diminished 7 ± 2 days after boost in DP patients compared to HC (Fig. 1A, B). In the S1 IgG assay, 31/44 (70.5%) of the DP were positive and 30/44 (68.2%) developed anti-S1 IgA antibodies.
Humoral immune response was delayed in DP and markedly reduced in KTR. (A-C) Humoral immune response against SARS-CoV-2 was assessed by Euroimmune ELISA for (A) spike protein S1 IgG, (B) spike protein S1 IgA and (C) virus neutralization by a blocking ELISA in HC (n=34), DP (n=44) and KTR (n=40) 7 ± 2 days after 2nd vaccination with BNT162b2 in the total cohort. (D-F) Humoral immune response with each cohort divided according to age (>60 n=71 (HC n=13, DP n=35, KTR n=23) and <60 years n=47 (HC n=21, DP n=9, KTR n=17)) into two subgroups and the corresponding results are shown for (D) spike protein S1 IgG, (E) spike protein S1 IgA and (F) virus neutralization by a blocking ELISA. (G-I) Follow-up sera were collected from 37 DP and 26 KTR patients, respectively 3-4 weeks after 2nd vaccination and investigated for (G) spike protein S1 IgG, (H) spike protein S1 IgA a and (I) virus neutralization by a blocking ELISA . (A-I) Threshold of upper limit of normal is indicated as dotted lines. (A-F) Kruskal-Wallis with Dunn´s post-test. Previously infected individuals are indicated in red. (G-I) Two-way ANOVA with Šidák´s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Humoral immune response was delayed in DP and markedly reduced in KTR. (A-C) Humoral immune response against SARS-CoV-2 was assessed by Euroimmune ELISA for (A) spike protein S1 IgG, (B) spike protein S1 IgA and (C) virus neutralization by a blocking ELISA in HC (n=34), DP (n=44) and KTR (n=40) 7 ± 2 days after 2nd vaccination with BNT162b2 in the total cohort. (D-F) Humoral immune response with each cohort divided according to age (>60 n=71 (HC n=13, DP n=35, KTR n=23) and <60 years n=47 (HC n=21, DP n=9, KTR n=17)) into two subgroups and the corresponding results are shown for (D) spike protein S1 IgG, (E) spike protein S1 IgA and (F) virus neutralization by a blocking ELISA. (G-I) Follow-up sera were collected from 37 DP and 26 KTR patients, respectively 3-4 weeks after 2nd vaccination and investigated for (G) spike protein S1 IgG, (H) spike protein S1 IgA a and (I) virus neutralization by a blocking ELISA . (A-I) Threshold of upper limit of normal is indicated as dotted lines. (A-F) Kruskal-Wallis with Dunn´s post-test. Previously infected individuals are indicated in red. (G-I) Two-way ANOVA with Šidák´s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Of particular interest, anti-S1 IgG and anti-S1 IgA responses were substantially diminished in KTR compared to HC and DP, respectively. Only one out of 40 patients (2.5%) was positive for IgG (apparently after prior unrecognized infection) and 4 patients for IgA (10%). Virus neutralization was observed in 30/44 (68.2%) DP patients (Fig. 1C), while 0/40 KTR had inhibiting antiviral antibodies (Fig. 1C). Interestingly, the patient’s serum with IgG and prior infection did not achieve neutralizing effects. Previously infected individuals are indicated in red in (Fig. 1A-C). Their levels of antibody and neutralization was in the range of other individuals of the respective group.
To further address the effect of age in our cohort, we divided the group into individuals 60 years of age. HC >60 years showed a lower anti-S1 IgG and IgA than HC 60 exhibited a lower neutralization capacity compared to DP<60 years (Fig. 1D-F). DP and KTR 60 years of age showed an overall diminished anti-S1 IgG and IgA as well as neutralization capacity compared to HC60 years of age, respectively (Fig. 1D-F). Anti-S1 IgG and IgA correlated with age in HC while this correlation was weak in DP and KTR (Figure S2).
HC showed no significant further increase of humoral response later than 28 days post initial vaccination with BNT162b2 (19). A delayed immune response might have explained the initial limited serologic response in immunocompromised individuals (DP and KTR) with mRNA vaccines. Therefore, we collected additional follow-up samples from KTR and DP 3-4 weeks after boost. Interestingly, anti-S1 IgG increased significantly in DP (Fig. 1G), while anti-S1 IgA and surrogate neutralization remained stable (Fig. 1H) during the additional observation. In contrast, KTR patients did not develop additional anti-S1 IgG, anti-S1 IgA and neutralizing antibodies until the second follow-up investigation 3-4 weeks after the boost (Fig. 1G-I). In summary, KTR showed a significantly reduced serological response including lack of further increases up to 3-4 weeks after BNT162b2 boost.
DP and KTR showed reduced B cells numbers but similar distribution among memory subsets
B cell lymphopenia is described for DP (20) and KTR (21) and might affect proper humoral immune responses. To initially address the frequency, distribution, and phenotype of peripheral blood B cells in DP, KTR compared to HC, we analyzed the distribution of B cell subsets at baseline (pre-vaccination) and 7 ± 2 days after boost (Fig. 2A). Of interest, the frequency of CD19+ B cells was significantly diminished only in KTR compared to DP at the assessment 7 ± 2 days after boost and compared to HC at baseline, while no differences were otherwise observed (Fig. 2B). However, substantial reductions in absolute B cell counts were identified between KTR patients and HC at baseline as well as HC versus the DP and KTR cohorts, 7 ± 2 days after boost, respectively (Fig. 2C).
B cells were reduced in DP and KTR but show a similar distribution after 2nd BNT162b2 vaccination. (A) Representative pseudocolor plots of CD19+ B cell gating into plasmablasts and mature B cells, and representative pseudocolor plots of IgD/CD27 based classification. (B) Frequency of CD19+ B cells (gates shown in (A)) in HC, DP and KTR before vaccination (n=46: HD n=11, DP n=21 and KTR n=14) and 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40) with BNT162b2. (C) Corresponding absolute numbers (per μl blood) measured by BD Trucount . Frequencies of plasmablasts and mature B cells according to CD27/IgD (gates shown in (A)) at (D) baseline (n=46: HD n=11, DP n=21 and KTR n=14) and (E) 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40). (F) Representative pseudocolor plot of IgA and IgG expression in B cells from HC. (G) Distribution of surface immunoglobulin isotype expression among HC, DP and KTR 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40). Two-way ANOVA with Šidák´s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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B cells were reduced in DP and KTR but show a similar distribution after 2nd BNT162b2 vaccination. (A) Representative pseudocolor plots of CD19+ B cell gating into plasmablasts and mature B cells, and representative pseudocolor plots of IgD/CD27 based classification. (B) Frequency of CD19+ B cells (gates shown in (A)) in HC, DP and KTR before vaccination (n=46: HD n=11, DP n=21 and KTR n=14) and 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40) with BNT162b2. (C) Corresponding absolute numbers (per μl blood) measured by BD Trucount . Frequencies of plasmablasts and mature B cells according to CD27/IgD (gates shown in (A)) at (D) baseline (n=46: HD n=11, DP n=21 and KTR n=14) and (E) 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40). (F) Representative pseudocolor plot of IgA and IgG expression in B cells from HC. (G) Distribution of surface immunoglobulin isotype expression among HC, DP and KTR 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40). Two-way ANOVA with Šidák´s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
The frequency of plasmablasts among total CD19+ B cells did not differ between groups (Fig. 2D, E) at baseline and after boost. DP and KTR patients carried lower frequencies of pre-switch B cells, while KTR had an increased frequency of naïve B cells before vaccination but not after vaccination. Post-switch memory B cells were higher in DP before but not after vaccination. Double negative (DN, CD27-IgD-) B cells did not differ significantly among groups (Fig. 2D,E). Interestingly, immunoglobulin isotype distribution among B cell subsets was not different among study groups (Fig. 2F,G). In summary, KTR and DP showed a characteristic reduction of absolute B cells with certain differences in the pre-memory (naive and pre-switch) but no differences within B memory compartments.
Impaired induction of anti-BNT162b2 B cell and plasmablast responses in KTR and HD patients
In order to better understand the underlying B and plasma cell differentiation upon vaccine challenge, we developed a flow cytometric method to identify and quantify RBD-specific B cells in human peripheral blood. B cells (CD3−CD14−CD19+) able to bind simultaneously RBD-AF488 and RBD-AF647 were validated as antigen-specific (Fig. 3A). The specificity of RBD binding was further confirmed by blocking with unlabeled RBD prior to staining (Fig. 3A). We identified an RBD-specific clone (CDRH3: ARDYGGNANYFHY, CDRL3:QQYDNLPIT) in 3 different vaccines (HC) with highly identical amino acid sequence as reported before upon mRNA vaccinations (22). Subsequently RBD+ B cells were further analyzed according to their distribution among subsets and isotypes (gated as shown for general B cells in Fig. 2A,F.
RBD-specific B cells were present in DP and KTR patients after BNT162b2 vaccination but populate different B cell subsets. (A) Representative dot plot of double positive cells RBD-specific B cells before and after blocking with unlabeled RBD are shown. (B) Frequencies and (C) absolute numbers of RBD+ cells among total CD19+ B cells measured before (n=59: HD n=10, DP n=23 and KTR n=26) and 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40). (D) Frequencies of plasmablasts, naïve, pre-switch, post-switch and double negative B cells (bar) and immunoglobulin isotype distribution among subsets (cakes) (HD n=10, DP n=23 and KTR n=26). (E) Immunoglobulin isotype expression among total RBD+ cells in HC, DP and KTR and 7 ± 2 days after 2nd vaccination (HD n=35, DP n=44 and KTR n=40). (F) Two-dimensional t-SNE of all RBD+ cells in HC (n=21), DP (n=23) and KTR (n=34). Color code indicates expression of CD27 (upper panel), CD38 (middle panel) and IgG (lower panel). Previously infected individuals are marked red (E). (B-D) Two-way ANOVA with Šidák´s post-test. (E) Kruskal-Wallis with Dunn´s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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RBD-specific B cells were present in DP and KTR patients after BNT162b2 vaccination but populate different B cell subsets. (A) Representative dot plot of double positive cells RBD-specific B cells before and after blocking with unlabeled RBD are shown. (B) Frequencies and (C) absolute numbers of RBD+ cells among total CD19+ B cells measured before (n=59: HD n=10, DP n=23 and KTR n=26) and 7 ± 2 days after 2nd vaccination (n=119: HD n=35, DP n=44 and KTR n=40). (D) Frequencies of plasmablasts, naïve, pre-switch, post-switch and double negative B cells (bar) and immunoglobulin isotype distribution among subsets (cakes) (HD n=10, DP n=23 and KTR n=26). (E) Immunoglobulin isotype expression among total RBD+ cells in HC, DP and KTR and 7 ± 2 days after 2nd vaccination (HD n=35, DP n=44 and KTR n=40). (F) Two-dimensional t-SNE of all RBD+ cells in HC (n=21), DP (n=23) and KTR (n=34). Color code indicates expression of CD27 (upper panel), CD38 (middle panel) and IgG (lower panel). Previously infected individuals are marked red (E). (B-D) Two-way ANOVA with Šidák´s post-test. (E) Kruskal-Wallis with Dunn´s post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Overall, an increased frequency of RBD-specific B cells among CD19+ B cells was found 7 ± 2 days after boost compared to baseline for HC, DP and KTR (Fig. 3B). The absolute number of antigen-specific B cells was significantly increased in HC at 7 ± 2 days after boost only in contrast to DP and KTR patients (Fig. 3C).
Subsequent analyses addressed the distribution of the RBD-specific B cells among B cell subsets (gating as seen in Fig. 2A). Most notably, a large number of RBD+ B cells were found in the plasmablast compartment in HC, which was significantly lower in DP and KTR (Fig. 3D and Figure S3). The very limited antigen-specific B cells in KTR resided preferentially within the naïve and pre-switch compartment compared to HC (Fig. 3D and Figure S3). In contrast, antigen-specific B cells from HC were detected mainly within post-switch and double negative B cells belonging largely to the memory compartment (Fig. 3D). Consistent with impaired (not completely executed) B memory induction, the frequency of IgM RBD+ B cells (defined as IgG-IgA-) was more frequently detected in KTR and DP patients compared to HC, in whom antigen-specific IgG+ B cells dominated. The frequency of IgA+ RBD+ B cells was comparable across groups (Fig. 3E).
Two-dimension t-SNE plots clustering all RBD+ B cells according to expression patterns, analyzed with a color axis for CD27, CD38 and IgG, illustrated the notable differences between groups including the substantially reduced plasmablasts (CD27++, CD38++) and IgG expressing RBD+ B cells in the KTR cohort (Fig. 3F). In summary, KTR patients were not only characterized by a reduced overall number of antigen specific B cells, but also exhibited signatures of abnormal B cell memory formation.
Unique correlation of anti-BNT162b2 serological and B cell responses
Our earlier vaccination studies against tetanus, diphtheria and KLH (Keyhole Limpet Hemocyanin) do not reveal a typical relation between plasmablast/ B cell responses and the serologic Ig outcome (6–8) in contrast to such relation for polysaccharides, such as meningococcal and pneumococcal vaccine (23, 24). Therefore, we wondered how the anti-BNT162b2 humoral immune and B cell specific responses against an mRNA vaccine are interrelated. A correlation matrix including all groups and patients was carried out. As previously described (25, 26), the neutralization capacity strongly correlates with anti-S1 IgG as well as anti-S1 IgA (Fig. 4A). The frequency of total RBD+ cells did not correlate with anti-S1 IgG, IgA and neutralization capacity, respectively. The frequency and total number of RBD+ plasmablasts correlated with all parameters of humoral response (anti-S1 IgG, anti-S1 IgA and the neutralization capacity). Age and the total number of RBD+ B cells correlated in HC, while it did not in DP and KTR (Figure S2). Subsequent analyses addressed how non-responders with a negative neutralization test (30%). Responders and non-responders were significantly different in the frequency and number of RBD+ plasmablasts and RBD+ pre-switch memory B cells as well as in the frequency of RBD+ naïve B cells (Fig. 4B). This data suggested clear interdependence of the distinct memory B and plasmablast compartments being a characteristic of this mRNA vaccine.
Correlation of anti-BNT162b2 serological and B cell responses. (A) Spearman´s correlation matrix showing the correlation of frequency of RBD+ cells in each B cell subset in the cohort. Corresponding correlations are represented by red (negative) or blue (positive) circles; size and intensity of color refer to the strength of correlation (HD n=35, DP n=44 and KTR n=40). Only correlations with p ≤ 0.05 are indicated. (B) Frequency (upper panel) and absolute numbers (lower panel) of RBD+ plasmablasts (PB), naïve B cells and post-switch B cells in non-responders (surrogate virus neutralization capacity ((NC) NC30%, n=63). Each point represents a donor. Unpaired two-sided Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Correlation of anti-BNT162b2 serological and B cell responses. (A) Spearman´s correlation matrix showing the correlation of frequency of RBD+ cells in each B cell subset in the cohort. Corresponding correlations are represented by red (negative) or blue (positive) circles; size and intensity of color refer to the strength of correlation (HD n=35, DP n=44 and KTR n=40). Only correlations with p ≤ 0.05 are indicated. (B) Frequency (upper panel) and absolute numbers (lower panel) of RBD+ plasmablasts (PB), naïve B cells and post-switch B cells in non-responders (surrogate virus neutralization capacity ((NC) NC30%, n=63). Each point represents a donor. Unpaired two-sided Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.