key: cord-0331000-4ck9glnl authors: Bruminhent, J.; Sethaudom, C.; Chaumdee, P.; Boongird, S.; Kiertiburanakul, S.; Malathum, K.; Nongnuch, A.; Phuphuakrat, A.; Jirasiritham, S.; Janphram, C.; Thotsiri, S.; Upama, S.; Assanatham, M. title: SARS-CoV-2-specific Humoral and Cell-mediated Immune Responses after Immunization with Inactivated COVID-19 Vaccine in Kidney Transplant Recipients (CVIM 1 Study) date: 2021-08-04 journal: nan DOI: 10.1101/2021.08.02.21261095 sha: 8fdff2764a82816e631e71601484b59045a6fc58 doc_id: 331000 cord_uid: 4ck9glnl Immunogenicity following inactivated SARS-CoV-2 vaccination among solid organ transplant recipients has not been assessed. Seventy-five patients (37 kidney transplant [KT] recipients and 38 non-transplant controls) received two doses, at 4-week intervals, of an inactivated whole-virus SARS-CoV-2 vaccine. SARS-CoV-2-specific humoral (HMI) and cell-mediated immunity (CMI) were measured before, 4 weeks post-first dose, and 2 weeks post-second dose. The median age of KT recipients was 50 years (IQR, 42 to 54) and 89% were receiving calcineurin inhibitors/mycophenolate/corticosteroid regimens. The median time since transplant was 4.5 years (IQR, 2 to 9.5). Among 35 KT patients, anti-RBD IgG titer after vaccination was not significantly different to baseline, but was significantly lower than in controls (7.8 [95%CI 0.2 to 15.5] vs 2,691 [95%CI 1,581 to 3,802], p<0.001) as well as the percentage of surrogate virus neutralizing antibody inhibition (2 [95% CI -1 to 6] vs 71 [95%CI 61 to 81], p<0.001). However, the mean of SARS-CoV-2 mixed peptides-specific T-cell responses measured by enzyme-linked immunospot assays was significantly increased compared with baseline (66 [95%CI 36 to 99] vs. 34 [95%CI 19 to 50] T-cells/10^6 PBMCs, p=0.02) and comparable to that in controls. Our findings revealed weak HMI and marginal CMI responses in fully vaccinated KT recipients receiving inactivated SARS-CoV-2 vaccine. (Thai Clinical Trials Registry, TCTR20210226002). CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint Herein, we decided to conduct an immunogenicity study among kidney transplant (KT) 113 recipients following a full course of inactivated SARS-CoV-2 vaccine. Both SARS-CoV-2 specific 114 humoral (HMI) and cell-mediated immune (CMI) responses were investigated along with the 115 safety profile. 116 117 Between April 2021 and July 2021, we performed a prospective cohort study of adult KT 120 recipients who received a two-dose, 4-week interval vaccination with an inactivated whole-virus 121 SARS-CoV-2 vaccine, CoronaVac ® (Sinovac Biotech Ltd., China), which contains 3 μg of inactivated 122 whole-virus SARS-CoV-2 in 0.5 mL, given intramuscularly into the deltoid muscle. We included KT 123 recipients from three hospitals; Ramathibodi Hospital, Praram 9 Hospital, and Samitivej 124 Sukhumvit Hospital, all located in Bangkok, Thailand. 125 HMI and CMI were measured before, 4 weeks after the first dose, and 2 weeks after the 126 second dose, using a SARS-CoV-2 immunoglobulin G (IgG) assay that tests for antibodies against 127 the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, SARS-CoV-2 surrogate virus 128 neutralization test (sVNT), and an enzyme-linked immunospot (ELISpot) assay for interferon-γ 129 (IFN-γ), respectively (Supplementary Figure 1) . 130 Participants were eligible if they were KT recipients aged 18 to 59 years old, at least 1 131 month post-transplant, and stable in their allograft function and immunosuppressive regimen. In 132 addition, patients with suspected respiratory tract infection in the preceding 3 days, concurrent 133 active infection, recent diagnosis of allograft rejection requiring intense immunosuppressants 134 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint (methylprednisolone pulse therapy with 500 mg IV daily for 3 days, antithymocyte globulin 135 therapy within 3 months, rituximab therapy within 6 months, or prednisolone more than 15 136 mg/day), receiving other vaccination within 4 weeks, previous history of COVID-19, or prior 137 administration of COVID-19 vaccine were excluded. All included patients were screened for active 138 respiratory tract infection, recent COVID-19 exposure, and comorbidities by history. 139 Nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 reverse transcription polymerase 140 chain reaction (RT-PCR) were not performed before vaccination. Thirty-eight non-transplant 141 adult patients who did not receive immunosuppressants were included and referenced as a 142 control. They also received the same type and interval of COVID-19 vaccination and were 143 assessed for immunity as described above. 144 SARS-CoV-2 anti-RBD IgG antibodies were measured using the Abbott SARS-CoV-2 IgG II 146 Quantification assay (Abbott Diagnostics, U.S.A). Plasma samples were run on the Abbott Alinity 147 instrument following the manufacturer's instructions. The assay is a chemiluminescent 148 microparticle immunoassay for the quantitive detection of IgG in human serum against the RBD 149 of the SARS-CoV-2 spike protein. The quantitative results of anti-RBD IgG were reported in 150 The function of the anti-SARS-CoV-2 spike protein S1 RBD antibody was determined by 152 using a SARS-CoV-2 NeutraLISA surrogate neutralization test assay (Euroimmun, Germany). The 153 neutralizing antibodies in plasma were inhibit binding between RBD and angiotensin-converting 154 enzyme 2 (ACE2) receptor. The percentage of neutralizing antibody inhibition was reported. 155 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint According to the manufacturer's protocol, heparinized whole blood samples from 157 participants were collected, and peripheral blood mononuclear cells (PBMCs) were isolated using 158 the EasySep™ Direct Human PBMC Isolation Kit (Stemcell Technologies, Vancouver, Canada). 159 Isolated cells were counted, and the cell suspension was normalized at a final concentration of 160 2.5×10 6 cells/mL in AIM V media (Gibco, Waltham, MA) followed by manually plating of cells into 161 strip plates (2.5×10 6 PBMCs/well) for stimulation with peptide pool or cell stimulation cocktail. 162 ELISpot assays assessed IFN-γ production by activated PBMCs using a human IFN-γ ELISpot control. The final concentration was 2 µg/mL of each peptide. After incubation for 40 hours at 171 37°C and 5% CO2, cells were removed, and IFN-γ production was determined using biotinylated 172 anti-human IFN-γ mAb 7-B6-1 (1 μg/mL in AIMV; Mabtech) for 2 hours at room temperature, 173 followed by incubation with streptavidin-alkaline phosphatase (1:1,000 in AIM V), and finally 174 treatment with 100 μL ready-to-use BCIP ® /NBTLiquid substrate (Gibco). After each step, plates 175 were washed five times with distilled water. Emerged spots were counted using an ImmunoSpot 176 analyzer (Cellular Technology Limited, Shaker Heights, OH), and spot quality was checked using 177 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint ImmunoSpot Software v5.0.9.15. Results were reported as mean and 95% confidence interval 178 (CI) of IFN-γ-producing spot forming units (SFUs) per 10 6 PBMCs for each peptide pool. 179 All patients underwent vital sign measurement and physical examination before is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint CoV−2-specific IFN-γ-producing SFUs/10 6 PBMCs were expressed as the mean with 95% CI, 200 presented as a dot plot generated with GraphPad Prism 6.0 (GraphPad Software, Inc, San Diego, 201 CA). A prospective study was conducted between April and July 2021. Table 1 . Among 214 35 eligible participants, the median age was 50 years (IQR, 42-54), and 60% were male. All (100%) 215 had received a deceased allograft and the majority (97%) had undergone first KT. The median 216 time since transplant was 4.5 years (IQR, 2-9.5). The maintenance immunosuppression regimen 217 included tacrolimus (57%), cyclosporine (29%), corticosteroids (97%), mycophenolic acid (91%) 218 sirolimus (3%), and everolimus (3%). The controls' median age was 40 years (IQR, 35-44) and 82% 219 were female. There were no immunosuppressive conditions in this group. 220 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. At 4 weeks after a single dose of the vaccine, mean (95% CI) S1 and SNMO-specific T-cell 233 responses were not significantly different compared with before vaccination in all participants 234 34 [95% CI specific T-cells/10 6 PBMCs, p=0.02). However, S1 and S2N-specific T-cell 239 responses were not significantly different compared with baseline (p=NS; Table 3) . 240 Two representative KT recipients without and with SARS-CoV-2-specific IFN-γ-producing 241 T-cell responses to the S1 protein, S2N protein, and the SMNO protein detected by IFN-γ ELISpot 242 assay at 2 weeks post-second dose of inactivate COVID-19 vaccine; Figure 4 . 243 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. fever (17%), pain at the injection site (14%), sleepiness (9%), muscle aches (6%), increased 248 appetite (3%) and others (6%). Two (6%) unsolicited AEs were reported, including asthmatic 249 attack (n=1) and subconjunctival hemorrhage (n=1). Both were evaluated and deemed not 250 inadequate immune response in a small recent study. 15 Our study also confirmed a weak HMI 274 response and a optimal CMI production. Although no threshold has been established for 275 protective immunity, antibody levels and the amount of neutralizing property of those were well 276 below those observed in the vaccinated immunocompetent group. We also observed that the 277 mean RBD-specific IgG antibody titer and neutralizing antibody inhibition in our participants was 278 lower than that of immunocompetent patients vaccinated with CoronaVac â in phase 1 and 2 279 studies. 16 However, neutralizing antibody measured by plaque reduction test is believed to be a 280 valid test to assess protective immunity, although an appropriate cut-off value to determine 281 those with sufficient neutralizing titer has not yet been established and requires a post-marketing 282 study to prove its effectiveness. 17 283 We are among the first to report the induction of CMI, focusing on quantification of T-284 cells stimulated with SARS-CoV-2 peptides in a post-transplant immunocompromised population. 285 Although SARS-CoV-2-reactive T-cell responses to an isolated S1 domain of the spike protein 286 alone or combined with nucleocapsid protein were not significantly increased after complete 287 vaccination, we observed increased responses to mixed peptides (SNMO) after the second dose. 288 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint The development of responses to SARS-CoV-2 mixed peptides could be due to a natural 289 characteristic of the whole virus we selected. S1-specific T-cell responses would be more 290 significant among those receiving mRNA-based or viral vector vaccines which targeted the S1 291 domain of the spike protein. Moderate generation of IFN-γ-producing T-cell responses among 292 those receiving 3-6 μg inactivated virus-containing vaccines were 3.4 and 1.2 SFU/10 6 PBMCs, 293 respectively (the former produced more) in a relatively new cohort, which was lower compared 294 with our results even in those with intact immunity. 16 Our study also revealed a relatively 295 comparable CMI after immunization to the control group and supported that 3 μg inactivated 296 virus-containing vaccine to robust CMI should be adequate. The wash step in the ELISpot assay 297 could subside an immunosuppressive effect to the T cells and create an intact response. CMI 298 response was also detectable after mRNA-based vaccination in SOT recipients in a recent study. 18 299 We believe an intact CMI induced by memory T-cells is essential and could be activated during 300 natural infection, thus decreasing the severity of the disease. 301 Several factors could diminish immune responses after vaccination in SOT recipients, not 302 least the fact that renal allografts must be maintained by immunosuppressive agents. 303 Mycophenolate mofetil treatment of greater than 1 g per day, as in our recipients, has been 304 reported in the literature as a critical factor to blunt an immune response. The virus contained in 305 the CoronaVac â vaccine should be more than 3 mg to produce adequate immunogenicity in 306 patients receiving immunosuppressants. These data are compatible with immunogenicity 307 generated following a standard dose of inactivated influenza vaccine in KT recipients, which 308 revealed lower antibody titers than non-transplant immunocompromised populations such as 309 patients living with HIV or end-stage renal disease. 19 Therefore, influenza vaccine formulations 310 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint with a higher dose of hemagglutinin is encouraged for those in need, such as elderly individuals 311 or SOT recipients, to generate a stronger immune response compared with the standard dose. 20-312 22 A recent study evaluating immunogenicity after triple doses of an mRNA COVID-19 vaccine in 313 SOT recipients indicated that this approach could achieve an optimal response and be 314 promising. 14 There is the possibility that additional vaccine doses would be needed, or switching 315 to another vaccine platform could be intriguing. Heterogenous vaccine studies have been more 316 focused on investigation of the immunocompetent population while our specific post-transplant 317 population is often excluded from the study. Although immunosuppressive agents could blunt 318 our patients' immunity, we observed more HMI effects than CMI. The responses of CMI in KT 319 recipients were not statistically significant compared to the controls could be explained by a wash 320 step of ELISpot assay, which attempts to decrease the effect of T cell immunosuppressants on 321 their responses. 322 Safety is another issue of concern among SOT recipients. Adverse reactions during the 323 early period were reported to be mild, confirmed by a large cohort prospective study of mRNA 324 vaccine provided to SOT recipients. The most common AE reported in a phase 1/2 study of the 325 inactivated whole virus vaccine was injection-site pain, reported by approximately one in five 326 participants; this was higher than the rate reported in our study of 14%. 16 Our study confirmed 327 that only minimal and mild adverse reactions were observed following vaccination in these 328 unexplored populations. However, immediate and short-term AEs are tolerable. Long-term 329 adverse events and allograft profiles such as allograft rejection require further follow-up. 330 Limitations of this study include the small sample size. Future large-scale studies are 331 needed to confirm our findings and further explore predictors of inadequate immune responses 332 . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint in these specific populations. The strength of this study is it represents one of the first studies to 333 investigate immunogenicity and safety in SOT recipients vaccinated with an inactivated SARS-334 CoV-2 vaccine. Although poor anti-RBD antibody and surrogate neutralization antibody 335 responses were observed compared with immunocompetent individuals, the assumption of 336 inadequate humoral responses cannot yet be completely elucidated, as further studies using 337 standardized plaque reduction neutralization tests are necessary to define a better cut-off 338 antibody titer that correlates well with neutralization. However, we instead attempted to assess 339 CMI, which is believed to boost a prolonged protective memory response in our susceptible 340 patients. However, the most important thing is adherence to strict basic infection prevention 341 measures remains crucial after immunization. 342 So far, research focused on the effectiveness of COVID-19 vaccines in SOT recipients has 343 not been fully explored. Our study could not report the effectiveness of this vaccine in preventing 344 natural infection because of the short follow-up period after vaccination. Furthermore, vaccine 345 effectiveness varies depending on the study population, the dynamics of local virus transmission, 346 the dominance of variants of concern, and healthcare resources. Thus, post-marketing 347 investigations will be required to determine the efficacy of vaccination in SOT recipients. Allograft 348 safety profiles and long-term data on safety also need to be followed up. Nevertheless, we 349 believe our findings could provide preliminary data on SARS-CoV-2 immune responses following 350 whole virus SARS-CoV-2 vaccination and be beneficial in designing an appropriate strategy for 351 vaccination in SOT recipients. 352 Our study revealed that KT recipients develop weak antibody responses and their 353 neutralizing effect to the spike protein, but with marginal SARS-CoV-2-specific T-cell responses 354 . CC-BY-NC-ND 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. CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint . CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint transplant; PBMC, peripheral blood mononuclear cell; SARS-CoV-2, severe acute respiratory 436 syndrome coronavirus 2; SFU, spot forming unit, S1, S1 domain of spike protein; S2N, spike and 437 . CC-BY-NC-ND 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. CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. Clinical 457 characteristics and risk factors for coronavirus disease 2019 (COVID-19) among patients under 458 investigation in Thailand COVID-19 in Immunocompromised Hosts: What We Know So Far. Clin 460 Infect Dis Outcomes of COVID-19 in Solid Organ 462 Transplant Recipients: A Propensity-matched Analysis of a Large Research Network International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity A family cluster of 465 diagnosed coronavirus disease 2019 (COVID-19) kidney transplant recipient in Thailand A case of successful treatment of 468 severe COVID-19 pneumonia with favipiravir and tocilizumab in post-kidney transplant 469 recipient Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Safety and Efficacy of the BNT162b2 mRNA Covid-473 Messenger RNA Vaccines in Recipients of Solid Organ Transplants Antibody Response to 2-Dose SARS-CoV-2 mRNA Vaccine Series in Solid Organ Transplant Recipients Immunogenicity of a Single Dose Messenger RNA Vaccine in Solid Organ Transplant Recipients What do we know about China's covid-19 vaccines? International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity COVID-19 in Solid Organ Transplant Recipients: a Review of the 486 Three Doses of an mRNA 488 Covid-19 Vaccine in Solid-Organ Transplant Recipients Letter to Editor: COVID-19 in Kidney Transplant 491 Recipients Vaccinated with Oxford-AstraZeneca COVID-19 vaccine (Covishield): A Single SARS-CoV-2 vaccine in healthy adults aged 18-59 years: a randomised, double-blind, placebo-496 controlled, phase 1/2 clinical trial Neutralizing antibody levels are highly predictive 498 of immune protection from symptomatic SARS-CoV-2 infection Cellular and humoral response after mRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients Comparison of the immunogenicity of a monovalent influenza A/H1N1 2009 vaccine between 504 healthy individuals, patients with chronic renal failure, and immunocompromised populations CC-BY-NC-ND 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. CC-BY-NC-ND 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. The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.02.21261095 doi: medRxiv preprint