key: cord-0810606-tmzcvbxn
authors: Bruminhent, Jackrapong; Setthaudom, Chavachol; Chaumdee, Pongsathon; Boongird, Sarinya; Kiertiburanakul, Sasisopin; Malathum, Kumthorn; Nongnuch, Arkom; Phuphuakrat, Angsana; Jirasiritham, Sopon; Janphram, Chitimaporn; Thotsiri, Sansanee; Upama, Supparat; Assanatham, Montira
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: 2021-12-06
journal: Am J Transplant
DOI: 10.1111/ajt.16867
sha: 49dc962def41e8012d8a68d13918fd83cb58902f
doc_id: 810606
cord_uid: tmzcvbxn

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 healthy 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 (IQR) age of KT recipients was 50 (42–54) years and 89% were receiving calcineurin inhibitors/mycophenolate/corticosteroid regimens. The median (IQR) time since transplant was 4.5 (2–9.5) years. Among 35 KT patients, the median (IQR) of anti‐RBD IgG level measured by CLIA after vaccination was not different from baseline, but was significantly lower than in controls (2.4 [1.1–3.7] vs. 1742.0 [747.7–3783.0] AU/ml, p < .01) as well as percentages of neutralizing antibody inhibition measured by surrogate viral neutralization test (0 [0–0] vs. 71.2 [56.8–92.2]%, p < .01). However, the median (IQR) of SARS‐CoV‐2 mixed peptides‐specific T cell responses measured by ELISpot was significantly increased compared with baseline (30 [4–120] vs. 12 [0–56] T cells/10(6) PBMCs, p = .02) and not different from the controls. Our findings revealed weak HMI but comparable CMI responses in fully vaccinated KT recipients receiving inactivated SARS‐CoV‐2 vaccination compared to immunocompetent individuals (Thai Clinical Trials Registry, TCTR20210226002).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged pathogen causing coronavirus disease 2019 , which spread worldwide. The clinical manifestations vary from asymptomatic to mild upper or severe lower respiratory tract disease. 1 Solid organ transplant (SOT) recipients are among those who are potentially compromised for this particular infection, resulting in significant morbidity and substantial mortality in this demographic. [2] [3] [4] [5] Vaccination against SARS-CoV-2 is recommended to ameliorate this potentially serious infection and its unfavorable consequences. Various COVID-19 vaccines have been developed across a range of platforms and have been deployed among immunocompetent individuals. However, immunogenicity and safety data following COVID-19 vaccination among SOT recipients receiving immunosuppressants remain limited.

A messenger RNA (mRNA)-based COVID-19 vaccine has been shown to produce immune responses and adequate efficacy to prevent natural infection in immunocompetent recipients. 6, 7 However, recent studies focusing on immunogenicity following a two-dose, 4-week interval mRNA-based COVID-19 vaccination strategy revealed suboptimal immune responses among immunocompromised patients. Only 17% and 54% of participants generated robust immune responses after single and double doses, respectively, of the mRNA-based COVID-19

vaccine. [8] [9] [10] An inactivated SARS-CoV-2 vaccine has been shown to be primarily adequate to prevent death (86% efficacy), with reportedly lower effect against clinical infection (65.9%). 11 However, a study focusing on immunogenicity and safety following vaccination with an inactivated whole-virus SARS-CoV-2 vaccine among SOT recipients has not been assessed. Furthermore, safety concerns for these immunocompromised patients have not been investigated.

Herein, we decided to conduct an immunogenicity study among kidney transplant (KT) recipients following a full course of inactivated SARS-CoV-2 vaccine. Both SARS-CoV-2-specific humoral (HMI) and cell-mediated immune (CMI) responses were investigated along with the safety profile.

Between April 2021 and July 2021, we performed a prospective cohort study of adult KT recipients who received a two-dose, 4-week interval vaccination with an inactivated whole-virus SARS-CoV-2 vaccine, CoronaVac ® (Sinovac Biotech Ltd.), which contains 3 μg of inactivated whole-virus SARS-CoV-2 in 0.5 ml, given intramuscularly into the deltoid muscle.

HMI and CMI were measured before, 4 weeks after the first dose, and 2 weeks after the second dose, using a SARS-CoV-2 immunoglobulin G (IgG) assay that tests for antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, SARS-CoV-2 surrogate virus neutralization test (sVNT), and an enzyme-linked immunospot (ELISpot) assay for interferonγ (IFNγ), respectively ( Figure S1 ).

Participants were eligible if they were KT recipients aged 18-59 years old, at least 1 month post-transplant, and stable in their allograft function and immunosuppressive regimen. KT recipients who attended the outpatient care during the study period were evaluated by their transplant nephrologist and recruited for vaccination.

In addition, patients with suspected respiratory tract infection in the preceding 3 days, concurrent active infection, recent diagnosis of allograft rejection requiring intense immunosuppressants (methylprednisolone pulse therapy with 500 mg IV daily for 3 days, antithymocyte globulin therapy within 3 months, rituximab therapy within 6 months, or prednisolone more than 15 mg/day), receiving other vaccination within 4 weeks, previous history of COVID -19, was defined as tacrolimus ≤5 ng/ml or cyclosporine ≤150 ng/ml. 12 A low therapeutic dose of mycophenolic acid was defined as mycophenolate mofetil (MMF) ≤1 g/day or mycophenolate sodium (MPS) ≤720 mg/day. 13 Healthy controls aged 18-59 years old who did not receive immunosuppressants were voluntarily recruited and referenced as a control. They also received the same type and interval of COVID-19 vaccination and were assessed for immunity as described above. 

According to the manufacturer's protocol, heparinized whole blood samples from participants were collected, and peripheral blood mononuclear cells (PBMCs) were isolated using the EasySep™ Direct 

All patients underwent vital signs measurement and physical examination before vaccination and were then monitored for immediate adverse events (AEs) up to 30 minutes after each vaccination, including local and systemic adverse reactions ( Figure S2 ). In addition, solicited AEs were monitored by a phone call on days 3 and 7 after each vaccination ( Figure S3 ), and the patients were encouraged to report unsolicited AEs recorded in their diary ( Figure S4 ). We then determined the causal association between vaccination and AEs.

Participants were also encouraged to contact us to report any possible infections, especially those who developed respiratory symptoms. Those in need of medical attention were asked to visit our facility for further evaluation of adverse reactions or investigation of COVID-19 diagnosis. Nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 RT-PCR were performed if needed to confirm the diagnosis, and treatment was provided according to a standard of care.

Categorical variables were presented as absolute, and frequencies and continuous variables were expressed as median with interquartile range (IQR). Accordingly, the chi-square test, Fisher exact test, and Mann-Whitney U test were used to assess differences between categorical and continuous variables as appropriate.

In addition, the distribution of anti-RBD IgG level, the percentage of neutralization inhibition, and SARS-CoV-2-specific IFNγproducing SFUs/10 6 PBMCs were presented as a dot plot with a bar representing median with IQR. These were generated by GraphPad

A prospective study was conducted between April and July 2021.

A total of 75 adult patients were vaccinated, including 37 KT recipients and 38 healthy controls. Among the former, two were excluded owing to denial participation and prior COVID-19 diagnosis ( Figure   S1 ). Clinical characteristics of KT recipients are shown in Table 1 .

Among 35 eligible participants, the median (IQR) age was 50 years (42-54), and 60% were male. All (100%) had received a deceased allograft and the majority (97%) had undergone first KT. The median (IQR) time since transplant was 4.5 (2-9.5) years. The maintenance immunosuppression regimen included tacrolimus (68%), cyclosporine (29%), corticosteroids (97%), mycophenolic acid (97%), sirolimus (3%), and everolimus (3%).

The controls' median (IQR) age was 39 (34-42) years, which was significantly younger compared to the KT group (p < .01). Of those, 82% were female and 47% were health care workers.

An anti-RBD antibody level in KT recipients from before to after the first and second doses in KT recipients compared to the controls is present in Figure 1 and Table 2 Table 2 ). Similarly, seroconversion significantly occurred less in KT recipients compared to healthy controls (9% vs. 100%, p < .01).

Seroconverted KT recipients were more likely to receive low therapeutic doses of mycophenolic acid (100% vs. 33%, p < .01) and cyclosporine-based regimen (75% vs. 26%, p = .08) as a maintenance immunosuppression compared to non-seroconverted individuals.

There was no significant difference in the proportion of participants with and without seroconversion in terms of onset after transplant and C 0 level of CNI (Table S1 ). 

A change in SARS-CoV-2-specific CMI in KT recipients compared to the controls is described in Figure 3 and Table 3 

The prevalence of SARS-CoV-2 RBD-specific IgG antibody level before, 4 weeks post-first dose, and 2 weeks post-second dose in healthy controls and KT recipients. Bar represents median with IQR. Dash horizontal line indicated SARS-CoV-2 RBDspecific IgG antibody level of 50 AU/ml (seroconversion). *p value < .05 AJT BRUMINHENT ET al.

We herein present a pilot study investigating immunogenicity, fo- SOT recipients are considered to have comorbidities and are at greater risk of severe respiratory tract disease. 17 Among several COVID-19 vaccines available, the total anti-SARS-Cov-2 antibodies seroconversion rate after a two-dose regimen of SARS-CoV-2 mRNA vaccine in SOT recipients was 40%, and a third dose was required to boost a more significant response to 68%. 18 KT recipients receiving adenovirus-vectored vaccine could still be vulnerable to infection, reflecting a possible inadequate immune response in a small recent study. 19 Our study also confirmed a weak HMI response, although no threshold has been established for protective immunity. In our cohort, 9% of KT recipients seroconverted while 100% of healthy controls seroconverted. Anti-RBD antibody levels were well below those observed in immunocompetent patients vaccinated with

CoronaVac ® in phase 1 and 2 studies. 20 However, a direct comparison may not be possible due to the lack of standardization among assays and patients' demographics variations.

We observed increasing SARS-CoV-2-reactive T cell responses to an isolated S1 domain of the spike protein in KT recipients parallel and comparable with healthy controls. Surprisingly, SARS-CoV-2-reactive T cell responses to spike protein combined with nucleocapsid protein significantly decreased 1 month after the first dose and later increased 2 weeks after complete vaccination. Furthermore, we also observed significantly increased responses to mixed peptides (SNMO) after the second dose in KT recipients and the controls which could be due to a natural characteristic of the whole virus we selected. Although S1-specific T cell responses would expect to be more significant among those receiv- Moderate generation of IFNγ-producing T cell responses among those receiving 3-6 μg inactivated virus-containing vaccines was 3.4 and 1.2 SFU/10 6 PBMCs, respectively (the former produced more), in a relatively new cohort, which was lower compared with our results even in those with intact immunity. 20 Our study also revealed a relatively comparable CMI after immunization to the control group and supported that 3 μg inactivated virus-containing vaccine to robust CMI should be adequate.

Although immunosuppressive agents could blunt our patients' immunity, we observed more HMI effects than CMI. The responses of CMI in KT recipients were not statistically significant compared to the controls could be explained by a wash step of ELISpot assay, which attempts to decrease the effect of T cell immunosuppressants on their responses. This assumption was probably supported by comparable numbers of IFNγ-producing T cells after stimulation with anti-CD3 antibodies (p = NS) in both groups. CMI response was also detectable after mRNA-based vaccination in SOT recipients in a recent study. 22 We believe an intact CMI induced by memory T cells is essential and could be activated during natural infection, thus decreasing the severity of the disease.

The prevalence of neutralizing antibody inhibition measured by surrogate virus neutralization test (sVNT) at 2 weeks post-second dose in healthy controls and kidney transplant recipients. Bar represents median with IQR. Dash horizontal line indicated the percentage of neutralizing antibody inhibition of 35% (positive test). *p value < .05 F I G U R E 3 SARS-CoV-2-specific IFNγ-producing T cell responses reactive to the S1 protein (A), S2N protein (B), and the SMNO protein (C) detected by IFNγ ELISpot assay before vaccination, 4 weeks post-first dose, and 2 weeks post-second dose in KT recipients. Bar represents median with IQR. *p value < .05. IFNγ, interferonγ; PBMC, peripheral blood mononuclear cell; S, spike glycoprotein; S1, S1 domain of spike protein; S2N, spike and nucleoproteins; SFU, spot forming unit; SNMO, peptide pool of spike protein, nucleoprotein, membrane protein, and open reading frame proteins

Several factors could diminish immune responses after vaccination in SOT recipients; not least, immunosuppressive agents must maintain renal allografts, especially antimetabolites. 9 More specifically, mycophenolate mofetil treatment greater than 1 g per day and mycophenolate sodium greater than 720 mg per day have been reported in the literature as a critical factor to blunt an immune response along with our result. 13 Furthermore, we observed a slightly better trend of immune responses in those receiving a cyclosporinebased immunosuppressive regimen. However, a low plasma C 0 concentration of CNIs was not correlated.

The virus contained in the CoronaVac ® vaccine should be more than 3 μg to produce adequate immunogenicity in patients receiv- in SOT recipients indicated that this approach could achieve an optimal response and be promising. 18 There is the possibility that additional vaccine doses would be needed, or switching to another vaccine platform could be intriguing. Heterologous vaccine studies have been more focused on investigation of the immunocompetent population while our specific posttransplant population is often excluded from the study.

Safety is another issue of concern among SOT recipients.

Adverse reactions during the early period were reported to be mild, confirmed by a large cohort prospective study of mRNA vaccine provided to SOT recipients. The most common AE reported in a phase 1/2 study of the inactivated whole virus vaccine was injection site pain, reported by approximately one in five participants; this was higher than the rate reported in our study of 14%. 20 Our study confirmed that only minimal and mild adverse reactions were observed following vaccination in these unexplored populations. However, immediate and short-term AEs are tolerable. Long-term adverse events and allograft profiles such as allograft rejection require further follow-up.

Limitations of this study include the small sample size, and the controls were not age matched with KT recipients, although they were all adults within the same age group (<60 years old). A previous study revealed that age older than 80 could impact the ability to neutralize the virus. 27 Future large-scale, with age-and sex-matched control, studies are needed to confirm our findings and further explore independent predictors of inadequate immune responses in this specific population. In addition, neutralizing antibody in our study is measured by a sVNT and rather be described as an ACE2

receptor competing for antibody test. Therefore, neutralizing antibody measured by plaque reduction test is believed to be a valid test to assess protective immunity, although an appropriate cut-off value TA B L E 3 SARS-CoV-2-specific T cell responses assessed by the IFN-γ ELISpot assay in KT recipients and healthy controls vaccinated with inactivated SARS-CoV-2 vaccine to determine those with sufficient neutralizing titer has not yet been established and requires a postmarketing study to prove its effectiveness. 28 The strength of this study is it represents one of the first studies to investigate immunogenicity and safety in SOT recipients vaccinated with an inactivated SARS-CoV-2 vaccine. Although poor anti-RBD antibody and surrogate neutralization antibody responses were observed in the KT recipients compared with immunocompetent individuals, the assumption of inadequate humoral responses cannot yet be completely elucidated, as further studies using standardized plaque reduction neutralization tests are necessary to define a better cut-off antibody level that correlates well with neutralization. However, we instead attempted to assess CMI, which is believed to boost a prolonged protective memory response in our susceptible patients. However, the most important thing is adherence to strict basic infection prevention measures, which remains crucial after immunization.

So far, research focused on the effectiveness of COVID-19 vaccines in SOT recipients has not been fully explored. Our study could not report the effectiveness of this vaccine in preventing natural infection because of the short follow-up period after vaccination.

Furthermore, vaccine effectiveness varies depending on the study population, the dynamics of local virus transmission, the dominance of variants of concern, and health care resources. Thus, postmarketing investigations will be required to determine the efficacy of vaccination in SOT recipients. Allograft safety profiles and long-term data on safety also need to be followed up. Nevertheless, we believe our findings could provide preliminary data on SARS-CoV-2 immune responses following whole virus SARS-CoV-2 vaccination and be beneficial in designing an appropriate strategy for vaccination in SOT recipients.

Our study revealed that KT recipients develop weak antibody responses and their neutralizing effect to the spike protein, but with potentially optimal SARS-CoV-2-specific T cell responses after completing a two-dose course of inactivated SARS-CoV-2 vaccine with acceptable adverse reactions and favorable short-term outcomes.

Therefore, future directives are encouraged to study the role of the third dose with the same platform or another heterologous vaccine in these vulnerable populations to prevent this potentially devastating infection. 

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Data available on request from authors. 

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Additional supporting information may be found in the online version of the article at the publisher's website. How to cite this article

SARS-CoV-2-specific humoral and cell-mediated immune responses after immunization with inactivated COVID-19 vaccine in kidney transplant recipients (CVIM 1 study)