key: cord-0726204-umg0ms2i
authors: Lederer, K.; Parvathaneni, K.; Painter, M. M.; Bettini, E.; Agarwal, D.; Lundgreen, K. A.; Weirick, M.; Goel, R. R.; Xu, X.; Drapeau, E. M.; Gouma, S.; Greenplate, A. R.; Le Coz, C.; Romberg, N.; Jones, L.; Rosen, M.; Besharatian, B.; Kaminiski, M.; Weiskopf, D.; Sette, A.; Hensley, S. E.; Bates, P.; Wherry, E. J.; Naji, A.; Bhoj, V.; Locci, M.
title: Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals
date: 2021-09-21
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.09.16.21263686
sha: f34a0fd8a8f125647f5d35aa015eea714de8202d
doc_id: 726204
cord_uid: umg0ms2i

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. In this study, through a fine-needle-aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant (KTX) recipients. We found that, unlike healthy subjects, KTX recipients presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cells, SARS-CoV-2 receptor-binding-domain-specific memory B cells and neutralizing antibodies. KTX recipients also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals, and suggest a GC-origin for certain humoral and memory B cell responses following mRNA vaccination.

after primary immunization, which were further enhanced by the booster vaccination ( Figure 1C ). 148 The increase in GC B cell frequencies was measurable when the responses were evaluated 149 following a longitudinal (11 individuals) or an orthogonal (15 individuals) approach (Figures 1D 150 and S1B). No correlation between age and GC B cell frequencies was observed ( Figure S1C ). 151 Next, by using a combination of fluorescently-labelled SARS-CoV-2 Full S and RBD tetrameric 152 probes, we identified SARS-CoV-2-specific GC B cells as GC B cells binding the Full S but not 153 the RBD probes (Full S + RBD -) or simultaneously binding the Full S and RBD probes (Full S + 154 RBD + ), while failing to bind an irrelevant tetrameric probe (influenza hemagglutinin, HA from 155 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.16.21263686 doi: medRxiv preprint 7 A/Puerto Rico/8/34) ( Figure 1E and Figure S1A ). The specificity of the probes is indicated by the 156 lack of Full S and RBD-specific GC B cells in pre-pandemic tonsil samples ( Figure 1E) . Overall, 157 a boost in both Full S RBDand RBD + GC B cell frequencies following the second vaccine dose 158 was observed, especially when a longitudinal evaluation was performed ( Figure 1F and S1D). 159 Similar to total GC B cells, SARS-CoV-2-specific GC B cells did not correlate with age ( Figure   160 S1E-F). Next, to determine if mRNA vaccine-induced GC responses were detectable in non- 161 draining lymph nodes, we collected contralateral axillary lymph nodes, which do not directly drain preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

) and Th17 (CCR6 + )-polarized cells (Morita et al., 2011) . CCR4 was also used in this analysis to 187 help refine the delineation of Th2 (CCR6 -CCR4 + ) and Th17 (CCR6 + CCR4 + )-biased cells ( Figures 3A and S1A ). Next, SARS-CoV-2 Full S-specific memory B cells were stratified into 213 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. RBDand RBD + cells ( Figure 3B ), as previously described for GC B cells ( Figure 1E ). A paired 214 (longitudinal) analysis of matched FNA samples highlighted the generation of Full S and RBD-215 specific memory B cells after the first vaccine dose, which was significantly higher after the 216 administration of a second mRNA vaccine dose ( Figure 3C ). An orthogonal analysis approach 217 confirmed similar trends ( Figure S3A ). SARS-CoV-2-specific memory B cells were also 218 detectable at low frequencies in healthy vaccinee peripheral blood samples after two 219 immunizations ( Figure S3B ), and only S-specific RBD -, but not RBD-specific memory B cell in 220 peripheral blood correlated with the respective SARS-CoV-2-specific memory B cell populations 221 in FNAs ( Figure S3C ). A population of plasmablasts was also measurable by flow cytometry as 222 IgD -IgM -CD38 hi CD20 lo/cells ( Figure 3D ). This population was more abundant after the booster 223 immunization (Figures 3D-E) and was also detectable at variable levels in peripheral blood 224 samples of vaccinated healthy donors ( Figure S3D ). However, no correlation was found between 225 the plasmablast populations detected in FNA and blood samples ( Figure S3E ). In sum, the data 226 obtained in our study indicate that two doses of SARS-CoV-2 mRNA vaccines can elicit SARS- 232 We then sought to determine the capacity of immunocompromised individuals to form GC 233 responses to SARS-CoV-2 mRNA vaccines. To this end, 13 individuals who underwent kidney 234 transplant were enrolled. Due to withdrawal of consent (n=1), failure to undergo collection 235 procedures (n=1), and lack of sufficient cells for analysis (n=1), 10 patients who were a median 236 12.6 months post-transplant (range -0.3-63 months) were included in further analysis (Table 1) .

Although the draining lymph nodes of KTX recipients were not significantly smaller than HDs 238 (data not shown), the FNA cell recovery yield was scarcer in KTX recipients and, due to limited were visualized by a dimensionality-reduction approach ( Figure 4A ). The most striking 242 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 4B-C) . Importantly, the generation of SARS-CoV-2-specific GC B cells in 248 response to vaccination was completely abrogated even in the few KTX recipients who could 249 mount low but detectable GC B cell responses ( Figure 4D ). 250 We next questioned whether the dramatic reduction in SARS-CoV-2-specific GC B cells was preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CoV-2 mRNA in HDs, whereas KTX plasmablast frequencies were, for the most part, reduced in 274 both locations and time points analyzed ( Figure 4F and S4E ). In line with these data, only ~40-275 50% of the KTX recipients in our FNA cohort produced Full S-and RBD-specific IgG within the 276 lower range of HDs after two immunizations, whereas the remaining ~60-50% of the patients had 277 SARS-CoV-2 binding Abs below the limit of detection ( Figure 4G -H). To shed light on the quality 278 of the Ab responses driven by SARS-CoV-2 vaccination in KTX patients after two vaccine doses, 279 we measured SARS-CoV-2 nAbs by pseudotyped lentivirus-based in vitro assays. In these assays, 280 the large majority of HD samples collected after two vaccine doses could efficiently neutralize a 281 pseudovirus containing the D614G mutation ( Figure 4I ) and, less efficiently, a pseudovirus 282 containing the mutation of the SARS-CoV-2 beta strain ( Figure 4J ). By contrast, KTX patients 

CoV-2-specific T cell responses. 296 Next, we aimed at determining whether KTX recipients are capable of generating T cell responses 297 to the vaccines, which could counterbalance the impaired B cell responses observed in these 298 patients ( Figure 4) . As GC B cell responses were heavily impaired in KTX recipients, we predicted 299 reduced Tfh cell frequencies in the KTX group. As anticipated, a viSNE analysis of antigen-300 experienced CXCR5 + CD4 T cells in FNA samples revealed a deep reduction of a cell population 301 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.16.21263686 doi: medRxiv preprint expressing the Tfh cell signature markers PD-1, BCL6 and ICOS in the KTX group ( Figure 5A ).

Concordantly, a significant reduction of Tfh cells in KTX patients in comparison to HDs also 303 emerged by a direct flow cytometry analysis ( Figures 5B-C) . 304 We then asked whether KTX recipients are, more broadly, incapable of mounting efficient antigen-305 specific T cell responses to the SARS-CoV-2 mRNA vaccines. Since a direct evaluation of SARS- Figure 6E ) similar to what was observed in draining lymph node bona fide Tfh cells ( Figure 2F ). 331 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In the few KTX recipients that had detectable AIM + cells after two vaccinations, we did not preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Results in KTX are discussed at length below). Although to a lower degree, we also found that 381 bona fide Tfh cells in draining lymph nodes correlated with nAb production, while activated Tfh 382 cells in blood were a less reliable predictor of nAb generation. Additionally, in our study GC 383 formation appeared to be tightly connected with the capacity to produce RBD-specific memory B 384 cells. These findings, which we would have been unable to observe by studying blood alone, 385 provide valuable insights on the otherwise poorly understood processes by which nAbs and 386 memory B cells are formed in humans after immunization with SARS-CoV-2 mRNA vaccines. 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 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 perpetuity. and Sarah Benchimol for administrative assistance, Susan Rostami for assistance with sample 510 All rights reserved. No reuse allowed without permission. 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 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. Analysis was performed on samples from ipsilateral lymph nodes of HDs at V2 and V3. 585 All rights reserved. No reuse allowed without permission. 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 perpetuity. In (C and E), n = 11; red data points = V2 and blue data points = V3. Statistical analysis: In (C and 615 E), a paired Mann-Whitney U test with continuity correction was performed. * P £ 0.05, ** P £ 

CD38 + CD27 lo/int BCL6 + ) in ipsilateral lymph node samples from HDs and KTX recipients at V3. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In (A), for HD and KTX n=3 at V3. In (C-F), for HD: n = 13 for at V2 and V3; For KTX: n = 3 at 638 V2 and n = 7 at V3. In (G-J), for HD: n = 12 at V1 and V2 and n = 13 at V3; For KTX: n = 7 at 639 V1, n = 2 at V2, and n = 8 at V3. In (C-J), a circle is used to represent HDs, a triangle is used to 640 represent KTX recipients, and a square is used to indicate a KTX recipient with a prior SARS- 663 All rights reserved. No reuse allowed without permission. 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 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 perpetuity. The AIM assay was performed as previously described (Painter et al., 2021) . Briefly, after thawing 769 and counting, cells were resuspended in fresh R10 to a final density of 10x10 6 cells/mL, and 2x10 6 770 cells in 200µL were plated in duplicate wells in 96-well round-bottom plates. After resting 771 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.16.21263686 doi: medRxiv preprint overnight, CD40 blocking antibody was added to both duplicate wells for 15 minutes prior to 772 stimulation. One of the duplicate wells was then stimulated for 24 hours with costimulation (anti- Table 4 . 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 perpetuity.

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.16.21263686 doi: medRxiv preprint pseudotypes was harvested 28-30 hours after infection and clarified by centrifugation twice at 830 6000g then aliquoted and stored at -80 °C until used for antibody neutralization analysis.

Antibody neutralization assay using VSVΔG-RFP SARS-CoV-2: All sera were heat-inactivated for 832 30 minutes at 55 ⁰C prior to use in neutralization assay. Vero E6 cells stably expressing TMPRSS2 p-values are corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure 858 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. values of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). 860 All rights reserved. No reuse allowed without permission. 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 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. In (A and B), n=13 for V2 and V3. In (C -E), n =13 for V2 and n = 11 for V3. In ( preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In (A), for HD: n = 13 for V2 and V3; for KTX: n = 5 for V2 and n = 6 for V3. In (B and E), for 940 HD: n =13 for V2 and n = 11 for V3; for KTX: n = 2 for V2 and n = 8 for V3. In (C and D), for 941 HD: n = 13 for V2 and V3; for KTX: n = 3 for V2 and n = 7 for V3. In (F-H), for HD: n = 12 for 942 V2 and V3; for KTX: n = 2 for V2 and n = 7 for V3. In (A-H) , a circle is used to represent HDs, a 943 triangle is used to represent KTX recipients, and a square is used to indicate a KTX recipient with In (B-H), for HD: n = 11 for V1and V3, n = 9 for V2; for KTX: n = 5 for V1, n = 6 for V2, and n 965 = 7 for V3. In (B and E-F), a circle is used to represent HDs and a triangle is used to represent 966 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In (B-H), the Wald-Wolfowitz runs test was used to perform an exact comparison between the two 968 data distributions of interest. * P £ 0.05, ** P £ 0.01, *** P £ 0.001, **** P £ 0.0001.

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Acosta-Rodriguez, E.V., Rivino, L., Geginat, J., Jarrossay, D., Gattorno, M., Lanzavecchia, A., Medicine 5, 176ra32-176ra32. 999 All rights reserved. No reuse allowed without permission. 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 perpetuity. 

All rights reserved. No reuse allowed without permission. 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 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.

The copyright holder for this this version posted September 21, 2021. 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 perpetuity.

The copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.16.21263686 doi: medRxiv preprint

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