Immune response to the mRNA COVID-19 vaccine in hemodialysis patients: cohort study

Background: End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines. Methods: A transcriptomic analysis of the immune response to the Covid-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells (PBMCs) was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and seven days after the second dose (V2D7) using anti-Spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified six months after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response. Results: Transcriptomic analyses demonstrated differing time courses of immune responses, with predominant T cell activity in controls one week after the first vaccination dose, compared to predominant myeloid cell activity in HD at this time point. HD demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p < 0.05). Anti-spike IgG remained elevated above baseline at six months in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development. Conclusion: Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance hemodialysis subjects (HD) comparable to healthy controls (HC) and identify transcriptomic and clinical predictors of anti-Spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of end stage renal disease (ESRD). Funding: F30HD102093, F30HL151182, T32HL144909, R01HL138628 This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003

differential counts obtained during standard of care monthly blood draws for the three months 145 preceding vaccination. Within our analyses, ferritin was coded as either low risk (200ng/ml -1200 146 ng/ml) or high risk (<200 ng/ml or >1200 ng/ml), since ferritin levels 200ng/ml -1200 ng/ml have been 147 shown to be associated with lowest all-cause mortality in HD patients 20 . Baseline clinical lab values were 148 calculated as the median of three lab values across the three months prior to vaccination. Demographic 149 and clinical data was collected from a medical questionnaire at time of consent for control subjects, and 150 included medical history, medications, and self-reported prior SARS-CoV-2 positive test results. 151

RNA extraction and RNA Sequencing (RNAseq) 152
RNA sequencing was performed on PBMCs at all V1 and V2 time points for all subjects for whom RNA 153 libraries were successfully built at > 5 time points. PBMCs stored in RNAlater were thawed and diluted 154 1:1 with 1X phosphate buffered saline. The mixture was then pelleted and RNA was extracted using the 155 PureLink RNA Mini kit (Invitrogen). DNase treatment to remove genomic DNA contamination was 156 performed using either the PureLink DNase kit or DNA-free DNA Removal Kit (Invitrogen). Purified RNA 157 in sterile water was stored at -80°C. Each RNA sample was quantified using the Qubit RNA High 158 Sensitivity kit (Invitrogen) and Bioanalyzer RNA Pico kit (Agilent) with RIN>=8. 159 For library construction, 50ng of RNA from each sample was aliquoted in 96 well plates. Libraries were 160 generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with the optional 161 NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). Each individual sample 162 library was barcoded during PCR amplification using unique dual indexed i5 and i7 primers from the 163 NEBNext Multiplex Oligos for Illumina kit. Each sample library was quantified using the Qubit DNA High 164 Sensitivity kit and Bioanalyzer DNA High Sensitivity kit. Samples were then pooled and sequenced using 165 the MiSeq Nano V2 kit (Illumina) to check read proportions between samples. Samples with lower-than-166 expected percentage of reads detected were supplemented with an additional spike-in of sample library 167 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 to the main pool. The supplemented pooled library was sequenced again using the MiSeq Nano V2 kit to 168 verify adequate adjustment. The finalized library was sequenced using a NovaSeq S2 flow cell configured 169 for 75bp paired end output. 170

Differential Gene Expression Analysis 171
Raw demultiplexed reads were filtered using fastp to remove adapters and short reads 21 . Trimmed 172 reads were then quantified using the Salmon pipeline with an hg38 reference transcriptome index 22 . 173 Quantified data was imported into R using the tximeta package 23 to convert Salmon quantification and 174 index data to a count matrix. Transcript names were extracted and matched using Entrez IDs with the 175 AnnotationHub package 23 . This finalized count matrix was then imported into a DESeqDataset object 176 and normalized using the variance stabilizing transformation in DESeq2. 177 The DESeq2 R package was used to identify genes that were differentially expressed at each time point 178 after vaccination for each subject group. Specifically, we implemented a design incorporating group-179 specific condition effects with individual subjects nested within groups. We performed the classical 180 Deseq2 workflow of estimation of size factors, estimation of dispersion, and negative binomial GLM 181 fitting for βi and Wald statistics, increasing the maximum number of iterations for estimation of the 182 negative binomial distribution to 500. We then generated contrasts to obtain differentially expressed 183 genes for controls at V1D1 and V1D7 (compared to V1D0), and at V2D1 and V2D7 (compared to V2D0). 184 Differentially expressed genes for HD were similarly obtained at V1D2 and V1D7 (compared to V1D0), 185 and at V2D2 and V2D7 (compared to V2D0). We also directly compared gene expression between 186 controls and HD at V1D7 and at V2D7. The significance threshold to determine differential expression 187 was FDR-adjusted (p < 0.05). 188

Anti-Spike (trimer) IgG Titer Quantification 189
The Human SARS-CoV-2 Spike (Trimer) IgG ELISA Kit from Invitrogen was used to quantitate IgG to the 190 . CC-BY 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 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 SARS-CoV-2 spike protein in serum samples at V1D0, V2D7, and M6 time points. All samples were 191 initially diluted 1:100 (in addition to the 1:10 assay buffer dilution on the 96-well plate) and assayed in 192 duplicate, with two-fold serial dilution of the 150,000 units/mL standard control in duplicate for relative 193 quantification. Absorbance at 450 nm was quantified using a Spark® multimode microplate reader. 194 Samples that produced signals greater than the upper limit of the standard curve were diluted 1:2000 195 and assayed again. IgG concentration was calculated by fitting four-parameter logistic curves to the 196 standard controls and taking the average concentrations of duplicates. 197

Antibody Neutralization Assays 198
Neutralization assays were performed on serum samples from V1D0 and V2D7 using SARS-CoV-2 199 pseudotyped virus (pseudovirus). To produce pseudoviruses, an expression plasmid bearing codon-200 optimized SARS-CoV-2 full length S plasmid was co-transfected into HEK293T cells using the SARS-CoV-2 201 Spike-pseudotyped lentiviral particle Kit (BEI # R-52948). The cell supernatants were collected 72h after 202 transfection, divided into aliquots and cryopreserved at −80 °C. 203 To titrate the pseudovirus, 5x10 3 293T-ACE2 cells were seeded per well in a 96-well plate in DMEM 204 containing 10% FBS and 1% penicillin streptomycin. Twenty-four hours later, the pseudovirus was 205 diluted 1:10, followed by five-fold serial dilutions for a total of nine dilutions, with each dilution 206 performed in six replicate wells. After incubation at 37 °C and 5% (vol/vol) CO2 for 72h, the luciferase 207 substrate was added to the 96-well plate for chemiluminescence detection. The 50% tissue culture 208 infectious dose (TCID50) of the pseudovirus was calculated according to the Reed-Muench method in 209 the titration macro template 24 . 210 Neutralization activity against SARS-2-CoV was measured in a single-round-of-infection assay with 211 pseudoviruses as previously described 25 . 5x10 3 293T-ACE2 cells were seeded per well in a 96-well plate. 212 Twenty-four hours later, serial dilutions of the serum samples were performed, incubated for one hour 213 . CC-BY 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 input dilution of serum was 1:20, thus 20 is the lower limit of quantification. 222

BTM module enrichment analysis 223
Gene set enrichment analysis was performed for each contrast generated in the DESeq2 analysis above 224 using blood transcription module (BTMs) gene sets 28 . BTMs with FDR-adjusted p < 0.05 were considered 225 significantly enriched. Enriched BTMs were further characterized using the distribution of Wald statistics 226 of membership genes from DESeq2. To summarize BTM analyses, BTMs were categorized into different 227 families: B cells, cell cycle, dendritic cell/antigen presentation, type I interferon (IFN type I), myeloid 228 activity/inflammation/ T/NK cells, and "others" 29 . The percentage of BTMs in each BTM family with 229 significant enrichment at each time point was then quantified over time. 230

Statistical analysis of antibody response 231
To determine the effect of vaccination on anti-spike IgG titers at V2D7 and M6, Kruskal-Wallis tests were 232 performed separately for HD subjects and controls. For each group, anti-spike IgG titer levels were 233 compared to assess for the significant effect of time (V1D0, V2D7, M6), and Wilcoxon rank sum tests 234 were performed with FDR correction to assess significant differences between each pair of time points 235 (V2D7 vs. V1D0, M6 vs. V1D0, M6 vs. V2D7). To determine the effect of vaccination on antibody 236 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 neutralization activity (ID50) at V2D7, Wilcoxon rank sum tests were performed for each group to 237 compare V2D7 vs. V1D0. 238 Linear models were constructed to establish the effect of prior SARS-CoV-2 infection and subject group 239 on anti-spike IgG titer development at V2D7 and M6 and neutralization activity at V2D7. Specifically, log-240 transformed V2D7 anti-spike IgG titers or V2D7 neutralization activity (ID50) were modeled as the 241 dependent variable, with subject group (HD or controls), log-transformed V1D0 anti-spike IgG titers or 242 V1D0 neutralization activity (ID50), gender, age, race, and ethnicity as independent predictors. To 243 determine predictors of anti-spike IgG at six months, a linear model was constructed with the log-244 transformed M6 anti-spike IgG titers as the dependent variable, and V2D7 anti-spike IgG titers, SARS-245 CoV-2 history, gender, age, race, and ethnicity as independent predictors. 246

Identification of BTM and clinical predictors of Ab response in HD 247
BTM predictors of antibody response in HD were identified by first calculating a representative 248 expression level of each BTM per sample, which we will refer to as the eigengene. Specifically, the first 249 principal component of each BTM was calculated using DESeq2-derived variance-stabilized gene counts 250 from each module's member genes across the HD V1 time points, and then across the HD V2 time 251 points. Signs (positive or negative) were assigned to the eigengenes such that samples with higher 252 expression of member genes in a BTM would be given a positive sign, while those with lower overall 253 gene expression would be given a negative sign. This was accomplished for each BTM by (1)  . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. Finally, clinical laboratory values responding to vaccination that predicted antibody titer response in the 270 HD subjects were identified. Linear models were separately constructed using log-fold change (LFC) from 271 baseline measurements of ferritin (continuous instead of binarized low-and high-risk), transferrin 272 saturation, and WBC count to predict log-transformed anti-spike IgG titers at V2D7 and M6. 273 274

Demographic and Clinical Characterization 276
Demographic and clinical data of the 20 maintenance hemodialysis (HD) and controls (HC) are 277 summarized in Table 1. The racial distribution differed between cohorts with more Black/African 278 American subjects in the HD cohort. The cohorts were otherwise demographically similar. The subjects 279 within the HD cohort had significantly more comorbidities, most notable of which include type 2 280 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. There were eight HD subjects who previously tested positive for SARS-CoV-2, with positive test dates 294 ranging from 7 months to four weeks preceding vaccination. Five control subjects self-reported a prior 295 positive SARS-CoV-2 test, with positive test dates ranging from 8 months to four weeks preceding 296 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. WBCs (k/ul) 3.9 -12 6.0 (2.1) 2) * indicates value outside of normal range 301 302 All subjects received two BTN162b2 vaccination doses with the second dose (V2) administered three 303 weeks after the first (V1). Anti-spike IgG binding and neutralizing assay data were obtained for all 304 subjects prior to V1 (V1D0) and seven days after V2 (V2D7). RNA sequencing data was obtained for all 305 control subjects prior to each vaccination dose (D0), and at one day (D1) and seven days (D7) after each 306 dose, corresponding to six time points: V1D0, V1D1, V1D7, V2D0, V2D1, V2D7. One control subject is 307 missing V2D0 data, and one is missing V2D1 data. RNA sequencing data was obtained for 12 HD subjects 308 prior to each vaccination dose, and at two days (D2) and seven days after each dose, corresponding to 309 six time points: V1D0, V1D2, V1D7, V2D0, V2D2, V2D7. Two HD subjects are missing V2D2 data. 310 Sequencing data was not obtained for subjects with fewer than five time points of successfully 311 constructed RNA libraries, due to time points without sample collection or failure to extract high quality 312 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. To characterize the molecular basis of immune responses to vaccination in HC and HD, we performed 319 differential gene expression analyses of the PBMC RNA sequencing data. There are substantially more 320 differentially expressed genes (DEGs) in response to V2 compared to V1, and at D1 and D2 post-321 vaccination compared to D7 (Figure 1). For HC, the largest number of DEGs is found at V2D1, indicating 322 the most transcriptional activity immediately after the 2 nd vaccine dose, followed by V2D7, V1D1, and 323 V1D7. HD follows a similar pattern, with the largest number of DEGs found at V2D2, followed by V2D7, 324 V1D2, and V1D7. Notably, HD subjects with no SARS-CoV-2 history (n = 6) have substantially lower 325 numbers of DEGs than HD subjects with positive SARS-CoV-2 history (n = 6) at each time point, and 326 particularly at V2 time points. 327 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ; https://doi.org/10.1101/2023.01.19.23284792 doi: medRxiv preprint In contrast, HD demonstrate early (V1D2) enrichment of 12 BTMs after the first vaccination dose, most 361 significantly involving upregulation of innate antiviral responses (Figure 2, TableS5). The immune 362 response transitions to V1D7 enrichment of 17 BTMs, with substantial upregulation of myeloid modules 363 (Figure 2, TableS6). The V1D7 positive enrichment of monocyte/myeloid modules in HD contrast the 364 negative enrichment of these modules in HC (Figure 2, Figure S1). Following the second vaccination 365 dose, HD demonstrate early (V2D2) enrichment of 27 BTMs most significantly involving upregulation of 366 dendritic cell activity and proinflammatory cytokines and chemokines (Figure3, TableS7). The immune 367 response progresses to V2D7 enrichment of one BTM: PLK signaling events (Figure 2). 368 369 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ; https://doi.org/10.1101/2023.01.19.23284792 doi: medRxiv preprint negative SARS-CoV-2, which show primary enrichment of myeloid BTMs (Figure 2). The remainder of 381 these enrichments are shown in Figure S2. 382 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. activity that dissipate by V1D7 in both HC and HD (Figure 3). However, HC show early positive and 392 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 negative enrichments of myeloid/inflammatory family activity that dissipate by V1D7, while HD show 393 many early positive enrichments of myeloid/inflammatory family activity that persist and increase at 394 V1D7. Following V2, HC show early predominance of dendritic cell (DC)/antigen presenting cell (APC), 395 IFN Type I, and myeloid/inflammatory family activity transitioning to B cell and cell cycle activity at 396 V2D7, while HD show predominant early IFN type I family activity transitioning to just one detectable 397 cell cycle module enrichment. 398 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 399 . CC-BY 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.

Antibody Binding and Neutralization Assay Response 408
We next aimed to assess immune protection conferred by the vaccine through quantification of anti-409 spike IgG antibodies and functional assessment of neutralizing antibodies. All subjects demonstrated an 410 increase in anti-spike IgG at V2D7, with titers for all subjects except one still elevated above baseline at 411 six months. The exception was one HD subject with prior SARS-CoV-2 infection who demonstrated the 412 highest baseline titers of all subjects prior to vaccination. Both HC and HD subjects demonstrated a 413 statistically significant increase in anti-spike IgG and neutralization activity (ID50) from V1D0 to V2D7 (p 414 < 0.001), followed by an expected decrease at M6 from V2D7 levels (p < 0.001) (Figure 4). Despite this 415 decrease, M6 titers were still increased compared to baseline (p< 0.001). 416 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  Higher anti-spike IgG at V2D7 was significantly predicted by higher pre-vaccination anti-spike IgG, 425 control group assignment, and younger age (p <0.01, p<0.05, p<0.05, respectively), while gender, race, 426 and ethnicity were not. Higher anti-spike IgG at M6 was significantly predicted by higher V2D7 anti-spike 427 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ; https://doi.org/10. 1101/2023 IgG (p < 0.001), with no additional predictive value conferred by SARS-CoV-2 history, subject group, age, 428 gender, race, or ethnicity. Higher neutralization activity (ID50) at V2D7 was significantly predicted by 429 higher pre-vaccination ID50, with no additional predictive value conferred by subject group, age, gender, 430 race, and ethnicity. 431 432

Transcriptomic and clinical predictors of antibody binding response in HD 433
Linear models to predict anti-Spike IgG at V2D7 and at M6 in HD using enriched BTMs, controlling for 434 SARS-CoV-2 history, identified BTM predictors at all time points except for V1D2. Of the 18 enriched 435 BTMs at V1D7, increased expression (from V1D0) of "LI.M156.1 plasma cells, immunoglobulins" was 436 predictive of higher anti-spike IgG at V2D7 (p < 0.05, FDR-corrected), controlling for SARS-CoV-2 history. 437 Of the 30 enriched BTMs at V2D2, increased expression of 18 BTMs was predictive of higher anti-Spike 438 IgG at V2D7 (p < 0.05, FDR-corrected). These include innate immune, antigen presentation, and T cell 439 pathways ( Figure 5). Increased expression of "LI.M4.2 PLK1 signaling events" at V2D7 compared to 440 V2D0, which was the only enriched module at this time point for HD subjects with no SARS-CoV-2 441 history, was predictive of higher anti-spike IgG at both V2D7 and M6 (p < 0.05). 442 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ; https://doi.org/10. 1101/2023 449 Linear models to predict anti-Spike IgG at V2D7 and at M6 In HD using clinical predictors yielded 450 significant baseline and post-vaccination predictors. Baseline ferritin levels in the intermediate range 451 (200 -2000 ng/ml) were associated with higher anti-spike IgG at V2D7 and M6 (p < 0.01, p < 0.05), 452 controlling for history of SARS-CoV-2. URR, WBC counts, transferrin saturation, and hemoglobin were 453 not significant predictors of antibody development. Figure 6A shows anti-spike IgG at V2D7 as a function 454 of baseline ferritin levels, identifying the intermediate range of ferritin which has previously been 455 associated with lowest all-cause mortality 20 456 The LFC of WBCs from baseline after the first vaccination dose was significantly predictive of antibody 457 titer levels at both V2D7 (p <0.01) and M6 (p < 0.05), controlling for SARS-CoV-2 history and number of 458 days after vaccination that labs were collected ( Figure 6B). The predictive value of LFC of WBCs is 459 predominantly driven by increased lymphocyte counts; LFC of absolute lymphocyte counts was 460 predictive of V2D7 (p < 0.01) and M6 (trend-level, p = 0.056) antibody titers, controlling for initial 461 antibody titers and date of clinical labs. 462 463 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  showed prolonged upregulation of myeloid activity at V1D7, while controls showed downregulation of 481 these modules at V1D7 (Figure 2, Figure S1). Overall, these observations indicate delayed resolution of 482 innate myeloid responses in the HD cohort. 483 Following the second vaccination dose, both groups demonstrated early enrichment of innate immune 484 modules, with HC alone transitioning to a plasma cell response by V2D7 (Figure 3). Direct group 485 comparisons at V2D7 did not show differences of plasma cell response, but metabolic activity was 486 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 decreased in HD compared to controls ( Figure S1). Further, HD demonstrated increased V2D7 487 expression compared to controls of pro-apoptotic Bcl-2 family member BH3, a mediator of lymphocyte 488 apoptosis. A prior study showed accelerated in vitro apoptosis of lymphocytes in uremia, with a 489 particularly pronounced effect on B cells, mediated by dysregulation of Bcl-2. These results suggest a 490 state of heightened cellular stress in HD after vaccination leading to increased apoptotic signaling. 491 Despite differing transcriptomic time courses in the two group, our results demonstrate significant 492 elevation of anti-spike IgG titers after two doses of BNT162b2 mRNA COVID-19 vaccination in both HD 493 and controls. HD demonstrated only a slight decrease of IgG levels at V2D7 when controlling for SARS-494 CoV-2 history (p < 0.05) and no statistically significant difference at six months. Prior studies comparing 495 short-term antibody response to BNT162b2 mRNA COVID-19 vaccination in HD versus controls find 496 antibody response rates of 84-96% in HD after two vaccination doses, but with lower mean IgG levels 497 compared to controls [16][17][18][19][32][33][34] . Notably, the HD population studied here is younger and more racially 498 and ethnically diverse. The average age of HD cohorts in prior studies was predominantly in the 60s, 499 compared to an average age of 54 in our study. Jahn et al. found in a subset analysis that HD patients 500 under 60 years of age responded equally to healthy controls, suggesting an interaction between 501 increasing age and less effective antibody response in HD patients 17 . 502 HD subjects with documented SARS-CoV-2 infection prior to vaccination had wider variance of antibody 503 titers at all time points in this study, with two subjects demonstrating V1D0 antibody titer levels similar 504 to that of uninfected subjects. These two subjects consistently had the lowest titer levels at V2D7 and 505 M6 within the group of previously infected subjects and amongst the lowest titers across all subjects. 506 One subject is the oldest enrolled patient, and both are diagnosed with hyperlipidemia. 507 Given previously and presently demonstrated the wider variance of protective immune responses in HD 508 and altered interactions with risk factors including age, it is valuable to identify predictors of the 509 . CC-BY 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.

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The copyright holder for this preprint this version posted January 19, 2023. ; https://doi.org/10. 1101/2023 strength of immune response to vaccination in this population. We identified both transcriptomic and 510 clinical predictors of anti-spike IgG development at both V2D7 and six months after the second 511 vaccination dose (M6). Increased gene expression of blood transcription modules (BTMs) including 512 monocyte activity, dendritic cell and antigen presentation activity, IFN type I activity, and T cell 513 activation two days after the second vaccination dose (V2D2) in HD were predictive of V2D7 anti-spike 514 IgG. Additionally, increased expression of PLK1 signaling events, indicating increased cell cycle activity, at 515 V2D7 was predictive of V2D7 and M6 anti-spike IgG. Clinically, serum ferritin values in the intermediate 516 range at baseline predicted stronger anti-spike IgG development. A prior study of 58,058 maintenance 517 HD subjects found serum ferritin levels between 200 and 1200 ng/ml to be associated with lower all-518 cause mortality, due to ferritin <200 ng/ml representing low iron status, and >1200 ng/ml representing a 519 hyper-inflammatory state due to ferritin's status as an acute phase reactant 20 . Iron deficiency has been 520 linked to impaired immune response and vaccine efficacy in other infections, while inflammation 521 induces macrophage release of the heavy chain component of ferritin, FTH, which has been reported to 522 inhibit lymphocyte proliferation and function 34,35 . Additionally, increased LFC in WBC count 1-3 weeks 523 after vaccination was predictive of higher antibody titers. 524 Our study is limited by different early time points between controls and HD (Day 1 vs Day 2 after each 525 vaccination dose) and by sample size, particularly when subdividing SARS-CoV-2 history. The smaller 526 sample size additionally limits our ability to characterize differential immune pathways in clinical subsets 527 of the dialysis population, such as those with low, medium, and high baseline ferritin levels. Future 528 studies are needed for more comprehensive characterization of the immune pathway recruitment in 529 response to the Covid-19 vaccinations in this population. The Covid-19 mRNA vaccines are proving more 530 efficacious than other vaccines in the ESRD population; for example, while more than 90% of patients 531 without chronic kidney disease develop protective antibodies against hepatitis B after vaccination, only 532 50-60% of patients with ESRD seroconvert 9 . One explanation is that, in mRNA vaccines, the mRNA both 533 . CC-BY 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 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 encodes the viral antigen and acts as an adjuvant due to its innate immunostimulatory properties; the 534 mRNA is recognized by endosomal and cytosolic innate sensors upon cell entry, resulting in cellular 535 activation and production of type I interferons and other inflammatory mediators 36   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 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023