DOI: https://doi.org/10.21203/rs.3.rs-2316160/v1
Introduction: We evaluated neutralizing antibody and anti-spike antibody (anti-S) response against omicron variant in solid organ (SOT) or hematopoietic stem cell (HSTC) receivers after third dose of BNT162b2 (BNT) or CoronaVac (CV) following two doses of CV.
Methods: In total, 95 participants who underwent SOT (n=62; 44 liver, 18 kidney) or HSCT (n=27; 5 allogeneic, 22 autologous) were included from five centers in Turkey. The median time between third doses and serum sampling was 154 days. The vaccine-induced antibody responses of both neutralizing antibodies and Anti-Spike antibodies were assessed by plaque neutralizing assay and immunoassay.
Results: Neutralizing antibody and anti-spike IgG levels were significantly higher in transplant patients receiving BNT compared to those receiving CV (GMT:26.76 vs 10.89; p=0.03 and 2116 Au/ml vs 172.1 Au/ml; p<0.001). Solid organ transplantation recipients, particularly liver transplant recipients, showed lower antibody levels than HSCT recipients. Thus, among HSCT recipients, the GMT after BNT was 91.29 and it was 15.81 in the SOT group (p<0.001). In SOT, antibody levels after BNT in kidney transplantation recipients was significantly higher than that in liver transplantation recipients (GMT:48.32 vs 11.72) (p<0.001). Besides, the neutralizing antibody levels after CV were very low (GMT: 10.81) in kidney transplantation recipients and below the detection limit (<10) in liver transplant recipients. There was a weak correlation between the neutralizing and anti-Spike antibody responses (r=0.36).
Conclusion:
This study highlights the superiority of BNT responses against omicron as a third dose among transplant recipients after two doses of CV. Lack of neutralizing antibody against omicron after CV in liver transplant recipients should be taken into consideration particularly in countries where inactivated vaccines are available in addition to mRNA vaccines.
Since the beginning of the pandemic, the novel coronavirus (SARS-CoV-2) has infected nearly 635 million individuals and caused almost 6.5 million deaths worldwide 1. Hematopoietic stem cell transplantation (HSCT) and solid organ transplantation (SOT) patients are at an increased risk for COVID-19 because of immunosuppressive medication and other comorbidities. Furthermore, their response to vaccination, which is the most effective way to control the pandemic, is substantially lower than that of immunocompetent people, occurring at rates comparable to those of unvaccinated individuals in some cases 2. Despite the successful maintenance of vaccination processes, even past SARS-CoV-2 infection and post-vaccination immunity were not adequate to prevent infections with emerging variants, which highlights the significance of booster doses, particularly in high-risk individuals 3,4. It also took time to establish an immunization schedule for immunosuppressed patients, which is still a matter of debate regarding the needs and unique properties of various immunosuppressed states, such as HSCT and SOT.
SOT and HSCT patients who had received two vaccine doses of inactivated vaccine, the only vaccine available in our country at the beginning of the pandemic, were offered a third dose optionally with either inactivated vaccine or mRNA vaccine. There are limited studies comparing the responses of mRNA vaccine and inactivated vaccine as third dose in transplant recipients, and no study comparing SOT and HSCT patients.
The aim of this study was to compare immunological responses to mRNA and inactivated vaccines by measuring neutralizing antibodies and anti-spike IgG in SOT and HSCT patients after the third dose, following two doses of inactivated vaccine administered 28 days apart.
The overall study design is shown in Fig. 1. Seventy (73.7%) of the 95 participants received BNT162b2 (BNT) as booster dose (38 liver transplantation, 21 allogeneic or autologous HSCT, 11 kidney transplantation) and 25 (26.3%) of the participants received CV as third dose (14 liver transplantation, 5 allogeneic and autologous HSCT, 6 from kidney transplantation). The median time between third doses and serum sampling was 154 days (range 15 to 381 days).
The demographic characteristics of the participants were presented in Table 1.
| Total (n = 95) | BNT162b2 group (n = 70) | CoronaVac group (n = 25) | |
|---|---|---|---|
| Median age (IQR*) | 56 (42–63) | 56.5 (43–65) | 52 (39.5–60) |
| Female gender (n/ %) | 28 (29.5) | 24 (34.3) | 4 (16.0) |
| Type of transplantation (n/ %) SOT** Liver Kidney HSCT*** Autologous Allogeneic | 69 (72.6) 52 (54.7) 17 (17.9) 26 (27.4) 21 (22.1) 5 (5.3) | 49 (70.0) 38 (54.3) 11 (15.7) 21 (30.0) 16 (22.9) 5 (7.1) | 20 (80.0) 14 (56.0) 6 (24.0) 5 (20.0) 5 (20.0) 0 (0) |
| Median time after transplantation (IQR) | 4 (2–6) | 4 (2–6) | 4 (3–6) |
| Time after the booster dose (n/ %) < 6 months ≥ 6 months | 82 (86.3) 13 (13.7) | 60 ( 85.7) 10 (14.3) | 22 (88.0) 3 (12.0) |
| Antimetabolite usage for SOT (n/ %) | 28 (40.6) | 23 (32.9) | 5 (20.0) |
| *Inter qurtile range; IQR | |||
| **Solid organ transplantation; SOT | |||
| ***Hematopoietic stem cell transplantation; HSCT | |||
The neutralizing antibody levels against the omicron variant in the transplantation group were significantly higher in BNT receivers than in CV receivers, with GMT levels of 26.76 for the BNT and 10.89, the CV (p = 0.03). Likewise, the third dose of BNT showed significantly higher AntiSpike IgG antibody than CV with a GMT of 2116 Au/ml vs 172.1 Au/ml, respectively (p < 0.001). In the healthy control group, BNT induced significantly higher neutralizing (GMT:50.40 vs 10.48, p = 0.06) and Anti-Spike IgG antibodies (GMT:29328 AU/ml vs. 3204 AU/ml, p < 0.001)) than CV (Fig. 2).
In the Pearson correlation test for neutralizing antibody and anti-spike antibody levels in the transplantation group, the correlation coefficient was found to be 0.36.
In HSCT receivers, both vaccines elicited higher neutralizing antibodies levels than SOT receivers. Thus, in the HSCT group, the GMT was 91.29 after BNT and it was 15.81 in the SOT group (p < 0.001). Likewise, the GMTs after CV were 34.82, and < 10 in the HSTC and SOT groups, respectively.
In the HSTC group, BNT induced significantly higher levels of neutralizing antibodies than CV (GMT:91.29 vs 34.82) (p = 0.03). However, the difference in the Anti-Spike IgG Ab levels after BNT and CV was not significant (GMT: 3259 vs 720 AU/ml, p = 0.28) (Fig. 3.A and Fig. 3.B). Among the SOT receivers, the antibody response after third dose of CV was below the detection limit (GMT:<10). Furthermore, Anti-Spike IgG levels was found to be very low after CV (GMT:120.3 AU/ml)
In the analysis of neutralizing antibody levels based on the duration after the 3rd dose of vaccines, no significant decline was detected in either vaccine in either group. In the HSCT group with BNT, the GMT was reduced to 80 from 94 after 6 months. In the SOT group with BNT, the GMTs were 15.80 and 20 before and after 6 months (p = 0.7271). The Anti-Spike IG levels were at the same trend with those of the neutralizing antibodies.
Lastly, we evaluated the neutralizing antibody and Anti Spike IgG levels based on transplantation types (liver, kidney, allogeneic, and autologous HSCT) (Fig. 4. A and Fig. 4.B).
In the auto-SCT group, BNT induced significantly higher neutralizing antibody response (GMT:108.3) than CV (34.82) (p = 0.04), increase in the Anti-Spike IgG levels was not significant (GMT: 1879 Au/ml vs 720.8 Au/ml, respectively, p = 0.55).
The neutralizing antibody response to BNT in kidney transplantation recipients was significantly higher than that in liver transplantation recipients (GMT:48.32 vs 11.72) (p < 0.001). The CV elicited a weak neutralizing antibody production in kidney transplantation recipients (GMT: 10.81) and the antibody levels were below the detection limit (< 10) in liver transplant recipients. Likewise, anti- Spike IgG levels in liver recipients were found to be very low (GMT: 97.04 AU/ml) after CV.
In the comparison of vaccine efficacy in kidney transplantation recipients, BNT induced significantly higher neutralizing and Anti-Spike IgG antibody (GMT:48.32 and GMT:1962 AU/ml) than CV (GMT:10.81 and GMT:198.6 AU/ml) (p = 0.01 and p = 0.03).
Immunosuppressive regimens in SOT patients revealed no significant differences in neutralizing activity and anti-spike IgG levels between the antimetabolite-using and non-using groups (p = 0.061 and 0.682, respectively). Among the HSCT recipients, only three patients received immunosuppressive therapy.
In this multicenter study, including SOT and HSCT recipients who received two doses of CV administered 28 days apart, the GMT of neutralizing antibody against omicron was found to be 2.45-fold higher and the GMT of anti-spike IgG antibody was found to be 18-fold higher after the third dose of BNT than that of CV. Compared with CV, BNT had considerably better responses in both groups of transplant patients. Solid organ transplantation patients, particularly liver transplant recipients, showed lower antibody levels than HSCT recipients when antibody responses were examined according to the transplantation type. A lower humoral response was not found to be related to the immunosuppressive regimen, contrary to current publications 5–8. For BNT recipients of HSCT, all individuals had positive neutralizing antibody titers regardless of the time elapsed after vaccination. In the autologous HSCT group, the immune response was similar to that in the healthy controls. Despite the fact that BNT positivity remained consistent in SOT recipients before and after 6 months, no positivity of neutralizing antibodies was seen in any period with CV. While the neutralizing antibodies were negative, the anti-spike IgG antibodies were positive.
In phase 2/3 research and field tests conducted at the beginning of the outbreak in healthy adults, inactivated vaccines were found to be effective in preventing symptomatic or severe illness 9–11. However, as research on immunosuppressive patients grew and novel variants appeared, several studies have demonstrated that mRNA vaccines are more protective against illness and more effective for hospitalization and death in both immunosuppressive patients and the general population 2,12,13. After recognizing that antibody responses to SARS CoV 2 decrease over time, additional doses are needed in both healthy and immunosuppressed individuals. Crucially, in immunocompromised patient groups, such as SOT or HSCT, antibody responses have been shown to decline substantially sooner than in healthy controls 14. Based on this finding, the CDC recommended additional doses and defined the primary scheme for this patient group as three doses, beginning in October 2021 15. In addition, vaccine effectiveness was found to be better with three doses than two doses of SARS-COV-2 vaccines according to hospitalization, intensive care requirement, and mortality16.
Recent studies indicated that heterologous vaccination (BNT after two doses of CV) provides neutralizing activity higher than three doses of CV in healthy adults 17. These findings are similar to the responses developed after two doses of BNT and protective levels for new emerging variants, such as Omicron, which can escape from vaccine or infection-induced immunity 18. However, there are limited studies in transplant recipients that show differences in immunological responses in countries where various vaccine types are available. Our study was different in that the testing for neutralizing antibodies was against the current variant omicron. Dib et al. reported a better humoral response after three doses of mRNA vaccines than after heterologous regimens in SOT recipients 19. However, in countries in which only inactivated vaccines were available at the beginning of the pandemic, patients had access to the mRNA vaccine only as the third dose, and studies indicated better immunogenicity after mRNA boosters. Pestana et al. reported higher seroprevalence, seroconversion, and IgG antibody values with a booster with BNT after two doses of CV as a primary regimen in kidney transplant recipients 5. In our study, additional BNT was found to be more effective than CV after two doses of CV were administered 28 days apart regarding the type of transplantation and time passed after vaccination, similar to recent studies 18. This finding is particularly notable in countries where there is availability of a variety of vaccinations with varying efficacy in preventing illness onset, hospitalization, and mortality.
According to current studies, immune responses among HSCT recipients after COVID-19 vaccination are better than SOT recipients and immune responses, and the duration of protection in autologous transplant recipients is higher than that in allogeneic transplantation, in line with our study 6,16,20−22. These results are important because current vaccination schemes are prepared without considering the reasons for immunosuppression and differences in vaccine type.
Previous studies have shown a strong correlation between neutralizing activity and anti spike IgG level 10,23. However, our results show a weak correlation (correlation coefficient:0.36). The reason for this difference is thought to be that our study's neutralizing activity against the Omicron variant was different from that of earlier research on neutralizing antibody activities, which used the Wuhan or Delta variants.
The strengths of our study are the comparison of immune responses following different COVID-19 vaccines in various transplant types, the evaluation of spike antibody and neutralizing antibody activities against mRNA and inactivated vaccines, and the evaluation of neutralizing antibody responses against the current variant omicron.
The limitations of our study are the small number of participants and absence of real-life data. At the time of the study, there were no established recommendations for COVID-19 vaccination for the immunosuppressed population; therefore, we could not define additional doses as boosters or the last dose of the primary scheme.
This study highlights the superiority of BNT responses as the third dose when compared with CV responses among SOT and HSCT recipients after two doses of CV. Emerging variants are of pivotal importance for the protection level of vaccines in real life; therefore, the effects of the variants should be taken into consideration in neutralization studies. These findings are more significant in countries such as Turkey, where inactivated vaccines are available, in addition to mRNA vaccines. Further studies are needed to establish vaccination schedules for immunosuppressed groups in various countries.
In this multicenter observational study, 95 participants who underwent SOT or HSCT with no history of COVID-19 were recruited. (n = 44 liver transplantation, n = 5 allogeneic HSCT, n = 22 autologous HSCT, and n = 18 kidney transplantation). For patients with SOT, all living donors were blood relatives or spouses. Patients were selected from four centers in Turkey: Başkent Ankara Hospital, Ankara City Hospital, Ankara University School of Medicine, and Adana City Hospital. After collection, serum samples were transferred to the Koç University-İşbank Center for Infectious Diseases (KUISCID) for laboratory tests and stored at -80°C until use.
All participants included in the study received two doses of the inactivated vaccine, CoronaVac (CV), as their primary vaccination. Nineteen healthy control samples from volunteer Koç University Hospital healthcare workers (n = 10 for BNT third-dose receivers, n = 9 for CV third-dose receivers) were included in the study.
The study was approved by the Başkent University Institutional Review Board (KA/22/84). All experiments were performed in accordance with Declaration of Helsinki. Informed consent was obtained from all the participants.
Before testing, the serum samples were inactivated at 56oC °C for 30 min. The SARS-CoV-2 Omicron variant (hcov-19/Turkey/koc_23122021_VK107/2021 (GISAID) Omicron BA.1.1), which was previously isolated from the SARS-CoV-2 RdRp PCR-positive nasopharyngeal specimen of a patient admitted to Koç University Hospital, was used for plaque neutralization assays. Plaque assays were conducted under BSL-3 conditions. Vero E6 cells were cultured with DMEM High-Glucose (Sigma-Aldrich®, cat.no: D6429) supplemented with 10% fetal bovine serum (FBS), (HyClone™, cat.no:SV30160.03HI), 1% Penicillin-Streptomycin (HyClone™, cat.no:SV30010), and Amphotericin B (HyClone™, cat.no:SV30078.01). Serial serum dilutions of 300 µL were incubated with 300 µl SARS-CoV-2 at a multiplicity of infection (MOI) 0.01 for 1 h at 37°C, 5% CO2, and then 600 µl mixture was inoculated onto the Vero E6 cells at 100% confluency. After 1 h of incubation at 37°C, 5% CO2, the serum-virus mixture was discarded. The cell monolayers were coated with 2% methylcellulose (Sigma, M0512, cat.no: 9004–67–5) and 5%FBS-DMEM mixture (1:1). Five days after infection, the methylcellulose/DMEM mixture was discarded. Plates were washed and cells were fixed with 4% PFA (Electron Microscopy Sciences, cat.no: 15710-S) followed by Gram's crystal violet solution staining (Merck millipore, cat.no: 109218). Plaques were counted with the naked eye, and the Celigo Image cytometer (Nexcelom, Celigo Image Cytometer 200-BFFL-5C) and plaque reduction titers (PRNT50) were calculated. The viral control was studied in duplicate for each assay.
Anti-SARS-CoV-2 Spike (S) IgG was measured using the Abbott™ Alinity™ ci-series Integrated Clinical Chemistry and Immunoassay System (cat. no.04S1750) according to the manufacturer’s instructions.
Statistical analysis of unpaired samples was performed using an unpaired nonparametric Mann–Whitney U test to compare two dependent groups. For the correlation analysis between neutralizing antibody and Anti-Spike IgG levels, Pearson’s correlation test was used. GraphPad Prism 8.0.2 Software was used for analysis and visualization of the obtained data.
Conflict of interests
The authors declare that there is no conflict of interest.
Data availability statement
The corresponding authors (Füsun Can and Özlem Kurt Azap), upon request, will provide information supporting the conclusions of the study. Owing to patient privacy, the data are not publicly accessible.
Funding information
The study was supported by Koc University Isbank Center for Infectious Diseases (KUISCID)
Author contributions
Erol, Ç. and Kuloğlu, Z.E. contributed to data interpretation, statistic analysis and writing.
Kayaaslan,B., Altınsoy, A., Çınar, G., Hasanoğlu, İ., Oruç, E., Korkmaz, G., Turan Gökçe, D., Kırımker O.E., Coşkun Yenigün, E., Ölçücüoğlu, E., Ayvazoğlu Soy, E., Çetinkünar, S. contributed to data interpretation.
Kuloğlu, Z.E., Esken, G., Barlas, T., İncir, S. and Can, F. contributed to laboratory analysis.
Kurt Azap, Ö., Can, F., Azap, A. and Haberal, M. contributed to proof‐reading and supervision.