Immune response after COVID-19 vaccination among patients with chronic kidney disease and kidney transplant

Graphical abstract


Introduction
Patients with chronic kidney disease (CKD), including kidney transplant (KT) recipients, and those on dialysis represent a special subgroup of patients requiring protection during the severe coronavirus disease 2019 (COVID-19) pandemic [1,2]. Patients with CKD usually have a compromised immune response [3,4], require higher dosages of vaccine and more frequent dosing because the vaccine response is short lived and achieves a lower response, especially among patients undergoing dialysis [5,6].
Related reports of vaccination among patients with CKD mainly considered mRNA vaccines [8,7]. Recent reports describing seroconversion rates among patients undergoing dialysis receiving two doses of the BNT 162b2 vaccine (Pfizer BioNtech) were lower than those of controls [9,10]. One study reported a weak antibody response of patients with HD to the viral vector COVID-19 vaccine [11]. In Thailand, the main vaccines available are Coronavac (Sinovac Life Science, Beijing, China), BBIBP-Cor V vaccine (Sinopharm) and ChadOx1 nCoV-19 (Oxford-Astra Zeneca). Zhang et al. conducted a pilot, prospective study to survey the safety and humoral response to inactivated SARS-CoV-2 vaccine among 45 patients with CKD receiving a 2-dose immunization of inactivated (Sinovac and Sinopharm). They showed that the majority (84 %) of patients with CKD acquired detectable neutralizing antibody lower than those of controls [12]. Bruminhent

Materials and methods
This prospective cohort study included four different patient groups: patients with CKD, those on hemodialysis (HD) and contin-uous ambulatory peritoneal dialysis (CAPD), recipients of KT, and a control group without kidney failure from the Faculty of Medicine, Vajira Hospital, Navamindradhiraj University. Participants were enrolled between July and December 2021. The inclusion criteria were CKD stages 3-5 (eGFR 60 mL/min/1.73 m 3 ), patients with stage 5 CKD undergoing HD, CAPD and KT > 3 months. The healthy control group consisted of volunteer healthcare workers that had eGFR ! 60 mL/min/1.73 m 3 . Participants in every group were 18-90 years old. Every participant received the same vaccine type in both first and second doses. The exclusion criteria included allergy to the components of the vaccines, inability to receive the vaccine according to their schedule, fever or concomitant serious illnesses and side effects from the first dose of vaccination. Patients  Table 1 Baseline characteristics of patients.

Trial procedure
The enrolled patients received the COVID-19 vaccine according to the vaccination protocol approved in Thailand, that is, two doses of ChAdox-1 nCOV-19 vaccine, 12-week interval, Coronavac, 3week interval or BBIBP-Cor V, 4-week interval. All participants provided a blood sample for antibody and cellular immunity measurement at the following time periods: T0 (before the first injection), T1(before the second injection) and T2 (12 weeks after the second injection). Immunogenicity analysis was performed at one and three months post-infection.

Determination of antibodies against SARS-CoV-2
All SARS-CoV-2 antibody assays were performed and analyzed using the EUROIMMUN Analyzer I-2P Ò (Euroimmun Medizinische Labordiagnostika, Lubeck, Germany) at the Central Laboratory and Blood Bank, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University. Controls and calibrators were used in the test kit for each run. The ratios of diluted serum, optical density and cut-off values in this study were used according to the manufacturer's instructions.

Quantitative determination of anti-SARS-CoV-2 S1 (IgG)
The anti-SARS-CoV-2 S1/ (RBD) IgG QuantiVac ELISA IgG (Euroimmun, Lübeck, Germany) Kit was used for quantitative determination of human antibodies against immunoglobulin class IgG against the S1 domain of the SARS-CoV-2 spike protein of in serum samples (see Appendix).   To detect the presence of NA against the S1 receptor-binding domain (RBD) of SARS-CoV-2 to ACE2 receptors in the plasma samples, the ELISA-based surrogate virus neutralization test was used (SARS-CoV-2 NeutraLISA (Euroimmun, Lübeck, Germany) (see Appendix).

Assessment of the T cell response by quantitative determination of interferon-c release by SARS-CoV-2-specific T cells
Cellular immunogenicity was measured by calculating the secretion of interferon gamma (IFN-c) using peripheral blood mononuclear cells upon SARS-Co-V2 glycoprotein stimulation and subsequent determination of released IFN-c by ELISA (Euroimmun, Lübeck, Germany) (see Appendix).

Participants
Demographic information, including age, sex and body mass index, was obtained at the first enrolment. The vaccine type, date of vaccination, use of immunosuppressed agents, number and types of comorbidities and history of transplantation were recorded. Primary outcomes included humoral and cellular responses after COVID-19 vaccination at T0, T1 and T2, as measured by SARS-CoV2 spike S1-specific IgG antibody levels and the viral neutralization test by surrogate virus neutralization test. The percentages of responders in different cohorts (CKD, HD, CAPD and KT) were compared with the controls, within and between cohorts to define the seropositivity rate (individuals who developed detectable anti-SARS-Co-V antibodies). The secondary outcomes were rates of AEs after vaccination and the incidence of COVID-19 breakthrough infection after vaccination, including illness severity.

Statistical analysis
The sample size calculation is provided in detail in the Appendix. Values are presented as median (interquartile range) for con-  tinuous variables. Antibody levels were compared between timepoints and analyzed using the paired sample t-test or Wilcoxon matched-pairs signed-ranks test. Categorical variables were reported as frequencies and percentages. Proportions were compared using Fisher's exact test (or the Kruskal-Wallis test as appro-priate). Correlation between two continuous parameters was calculated using Spearman's correlation. Logistic regression models were used in both univariate and multivariate analyses, and statistical significance was set at p < 0.05. Statistical analysis was

Monitoring of adverse events
AE assessments, including vaccine and drug side effects after the first and second vaccine doses, were monitored.

Baseline characteristics
Between June 2021 and December 2021, 212 patients with CKD at various stages and controls were vaccinated with COVID-19 vaccines, CoronaVac, BBIBP-Cor V, or ChAdOx1 nCoV-19 vaccine (AZD 1222). Totally, 31 patients (15.20 %) had underlying heart problems and none of the patients had either lung or liver diseases. Fourteen patients were lost to follow-up. Eleven patients died during the study period (COVID-19, eight;underlying diseases, two; sepsis,one:underlying disease). Finally, 212 patients (104 men, 49.06 %) with a mean age 54.8 ± 16.07 years were enrolled in the study (Fig. 1). The vaccination distribution was as follows: 190 patients (89.62 %) received ChAdOx1 nCoV-19, 20 (9.43 %) Corona-Vac and two (0.94 %) BBIBP-Cor V. One hundred and thirty-four (63.20 %) patients were undergoing HD, four (1.88 %) were undergoing CAPD and seven (3.30 %) were KT recipients, twelve (5.66 %) were nondialysis patients with CKD, and 55 (25.94 %) were the controls. The median duration of HD was 3.04 years (IQR 1.42-5.29 years). Almost all patients and the control group received the ChAdOx1 nCoV-19 vaccine being the main vaccine scheme adopted in our country at the time of the study; the baseline characteristics of the population are detailed in Table 1.
The KT recipient group revealed an average age of 50.86 ± 11. 11 years; 42.86 % were women; and median time since transplantation was 9.83 years (IQR 5.08-20.5) The maintenance immunosuppressant regimens included calcineurin inhibitors (87 %), corticosteroids (45.4 %), antimetabolites (82.4 %) and mTor inhibitors (10.4 %). The antimetabolite treatments used included mycophenolate mofetil (85.2 %), mycophenolic acid (11.5 %) and azathioprine (3.3 %). The mean age in the HD group was 57.34 ± 1 4.84 years, 45.52 % were women.Subjects in the control group were aged 46.69 ± 17.65 years and 60 % were women. Only four patients were treated with peritoneal dialysis, with a mean age of 58.00 ± 11.66 years. None of the patients had a prior or current diagnosis of COVID-19 and all tested negative for the anti-SARS-CoV-2 NCP IgG.
Diabetes is the most common cause of end-stage renal disease (ESRD  Fig. 2).

Anti-SARS-CoV-2 antibody response
Patients on HD and nondialysis patients with CKD exhibited nonsignificant different antibody responses compared with those in the control group. In the CKD group, the median antibody titer was 3. 20 (Fig. 3).The antibody levels in the CAPD group at T2 were significantly lower than those in the control and HD groups (p = 0.01; CAPD vs control, p = 0.016 CAPD vs HD). A positive antibody level was detected in only one KT recipient at T2.
Vaccine response was evaluated for 151 patients after the second dose in vaccine types. The response rate was 70.59 % in the control group of the ChAdOx1 nCoV-19 vaccine. The CKD and dialysis group had similar response rates of 60 and 59.62 % respectively. The KT group revealed a weak response of 33.33 % (Fig. 4). The CAPD group also showed a poor immunological response, with none being seropositive at T2. The NA and IFNc seropositive rates followed a similar pattern to anti-SARS-CoV-2 antibodies with the lowest response rate in the KT and CAPD groups, and the level of immunity and response rate in the inactivated vaccine groups were satisfactory in CKD, HD, and KT groups compared with controls ( Table 2). NA showed a good correlation with levels of anti-spike IgG antibodies at T1 and T2 (r = 0.876 at T1, r = 0.819 at T2, p <0.001) (Fig. 5) (Tables 2 and 4).
Of these, 13 (92.85 %) received only one dose of a vaccine, with a median interval of 52 [IQR 44-61] days after the first vaccination. One patient developed COVID-19 after completing the second dose on day 64. Infection in two controls was resolved uneventfully. Overall, 85.17 % of cases were in the HD group. Factors associated with SARS-CoV2 infection were male sex and blood group (p = 0.005 and p = 0.039, respectively) (Supplementary Table 1). Only one patient received an inactivated vaccine. Among the patients diagnosed with COVID-19 during follow-up, the median anti-spike IgG, NA and IFNc levels significantly increased at 1one and three months after diagnosis, and natural immunity was robust and significantly higher than vaccine-induced immunity for as long as three months (Table 6).

Vaccine type and immune response
Of the 20 patients receiving inactivated vaccines, seven, one and 14 were in the HD, CKD and control groups, respectively. Antibodies were detected at a positive level (>35 BAU/mL) at T1 and increased progressively to a median of 217.  at T2 among patients with HD. NA levels were detected at low titers at T2 in both CKD and HD groups. All patients in the control group responded to the inactivated vaccine with an antibody titer above

Adverse events
Among vaccine recipients, mild-to-moderate pain at the injection site was the most commonly reported local reaction, which Table 6 Comparison of immunogenicity between non-COVID-19 and COVID-19 patients.  resolved within 1-2 days. Fever was the second most common symptom. The local reactions did not increase after the second dose. Fever occurred more frequently in the control group (p = 0.025) and no serious AEs were recorded (Table 7).

Discussion
Patients with CKD, especially ESRD undergoing dialysis, are at a very high risk of death following COVID-19 [14,15]. Evidence suggests that patients with CKD may have a less robust antibody response after vaccination than healthy controls [16][17][18][19][20]. Our study consisted of a diverse group of patients with CKD receiving different therapies. Our major finding was that patients with CKD, including those on maintenance HD, developed a substantial humoral response following the two vaccine doses (inactivated and ChAdOx1 nCoV-19 vaccines). Humoral seroconversion responses were maintained for as long as 12 weeks after completing the second dose, and the responses were equivalent to those of healthy individuals. However, patients with KT developed fewer humoral immune responses than those in the other groups. Immunosuppression may induce a weak anti-SARS-CoV-2 antibody response.
The immune response from inactivated whole-virus SARS-CoV-2 vaccine among patients with HD was demonstrated to be satisfactory. However, fewer patients achieved humoral immune responses compared with healthy individuals [21]. In our study, >50 % of all patients except recipients of KT experienced seroconversion after receiving the second dose of inactivated vaccines. Related studies have reported variable responses to COVID-19 vaccines among patients with CKD, with most studies reporting on mRNA vaccines [22][23][24][25][26]. However, the durability of this immune response and the extent to which it translates to protective immunity remain unclear. A systematic review of 18 studies found that the antibody response to full vaccination with two doses of COVID-19 mRNA vaccines among patients undergoing HD, CAPD and KT was lower than that in the healthy population [27]. In phase 3 trials, BNT162b2, mRNA-1273 and ChAdOx1 nCoV-19 prevented COVID-19 in 95, 94.1 and 70.4 % of participants [28][29][30], respectively, suggesting that the mRNA vaccines might induce protective immunity more reliably than ChAdOx1 nCoV-19. In addition, both mRNA vaccines and viral-vector vaccines induce balanced humoral and T cell immunity [31].
Our study measured cellular immunity to better explore the immunogenicity of these specific populations using IFNc levels. We found a significant correlation between IFNc, SARS-CoV-2specific antibodies and NA.Cytotoxic CD8+ T cells help accelerate the clearance of many respiratory viruses [32] and are essential in reducing the risk of SARS-CoV-2. Here, we demonstrated a good T cell response among patients with CKD and those on HD, which occurred as early as after the first dose of the ChAdOx1 nCoV-19 vaccine. The level of cellular immunity in this study correlated well with anti-SARS-CoV-2 antibody and NA, as in related studies [33]. Cellular immunity was then extrapolated to a good humoral immune response.
The antibody responses and NA levels in both vaccine groups did not significantly different except in the control group at T1. After the second dose, the level of immunity was similar (Supplementary Tables 3-7). The sample size in the inactivated vaccine group was small, and the protocol for the inactivated vaccine was only 3-4 weeks apart. This implied that antibody levels in the inactivated group declined more rapidly than in the other groups and this vaccine should not be recommended among patients with CKD and those on HD. The inappropriately high level of anti-spike IgG in the control group at T1 after inactivated vaccine might have been caused by natural infection. Since antinucleocapsid was performed only before participants recruitment.
The new cases of COVID-19 were detected after the first doses of vaccination; more than one half of these patients were in the HD group (85.71 %). Our study suggested a more rapid vaccination response among patients with CKD and on dialysis. The results also implied that patients on HD should not be considered for a delayed second vaccination dose. To prevent new cases of COVID-19, the second dose should be scheduled early as four to eight weeks after the first dose. Most people with symptomatic SARS-CoV-2 undergo seroconversion to produce a detectable, specific antibody response in the acute phase (Table 6). However, they should be re-vaccinated because the specific IgM rises in the acute phase and the IgG peaks appear later but decline after three to four months [34,35].
Most studies have not reported an association between antibody response and other factors, such as age or sex. Our findings showed significant associations for blood group O, which constitutes a novel finding. Related studies have revealed that blood group O is associated with less viral infection and illness severity [36][37][38].Blood group A was found to be associated with an increased risk of infection and mortality but a decreased risk of intubation and death [39]. The molecular mechanisms by which ABO polymorphism impacts risks of SARS-CoV2 infection might involve ABO antibodies inhibiting the interaction between angiotensin converting enzyme-2 receptor and the virus, related to the presence of the anti-A antibody [40] or anti-A isohemaglutinin titers [41]. Further studies need to confirm these findings. This study showed the effectiveness of the ChAdOx1 nCoV-19 vaccine over inactivated vaccines among patients with CKD. We found no difference in serious AEs between the two vaccine formulations, except for fever and numbness, which resolved in a few days.
Our study exhibits several strengths, including a comprehensive overview of the immunogenicity of both humoral and cellular responses to COVID-19 vaccines in a broad sample of patients with CKD. The sizes of the HD and CKD cohorts were sufficiently large for the control group to allow us to identify differences. The results presented here have a long follow-up to three months after vaccination in contrast to only <four weeks in other studies. This may have implications for treatment and policy, because the ChAdOx1 nCoV-19 vaccine remains one of the main COVID-19 vaccine used in many countries. Nevertheless, our findings were limited by the small sample size and unequal distribution of the CKD population The number of patients other than those undergoing HD was insufficient to draw a meaningful conclusion concerning the other subgroups. The loss follow-up rate was also high in the control group.

Conclusion
Immunity among patients on HD and those with CKD after completing two vaccinations with candidate vaccines was strong, although the responses among recipients of KT and patients on CAPD were below acceptable levels, reinforcing the idea that this population should be vaccinated as soon as possible and receive a booster dose with the same or a different vaccine platform, such as an mRNA vaccine. A timely second dose of the COVID-19 vaccine seems necessary to ensure protection of patients with kidney disease from SARS-CoV-2. Blood group O and vaccine type were associated with good immune response.

Ethics approval
This study protocol was reviewed and approved by the Vajira Institutional Review Board, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, approval number 94/2564.

Consent to participate
Written informed consent was obtained from all participants, and the experiment was performed in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice.

Data availability
The data supporting the findings of this study are available from the corresponding author. Data supporting the findings of this study are openly available in ''figshare" at http://doi.org/10.6084/m9.figshare. 19552222.

Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thananda Trakarnvanich reports financial support was provided by Navamindradhiraj University. Thananda Trakarnvanich reports a relationship with Navamindradhiraj University that includes: funding grants and non-financial support. None to be declared.