Key words : Antibody titer, BNT162B2 mRNA COVID-19 vaccine, Immunosuppressive medicine, Kidney transplant, SARS-CoV-2

Introduction

We previously reported that the seropositivity rate in patients vaccinated with the BNT162b2 mRNA COVID-19 vaccine after transplant was clearly lower than the seropositivity rate in healthy patients.1 Our report demonstrated 3 main points. (1) After vaccination in transplant recipients, the seropositivity rate was 41%, which was significantly lower than the seropositivity rate (100%) in healthy patients. (2) In the viral fragment analysis, healthy patients showed a stable seropositivity rate for each viral fragment without interindividual variation, whereas in the transplant recipients the seropositivity rate for each fragment was not maintained and showed interindividual variation in the seropositivity rate for each fragment. (3) Variations in antibody production were dependent on the patients’ various immuno-suppression regimens. At our facility, even in the era of the COVID-19 pandemic, we have been perfor-ming kidney transplants while maintaining preventative measures against coronaviruses to the extent that we are abled. At the time of this writing, the SARS-CoV-2 vaccination program has been progressing well in Japan, and the third doses of vaccination are underway.

The objective of this study was to analyze antibody production after vaccination in pretransplant and posttransplant patients. The study group consisted of 19 patients with chronic renal failure who were vaccinated before transplant, 111 recipients vaccinated after transplant, and 10 healthy patients (as a control group) for whom the same measurement was performed. As in our previous study,¹ we analyzed the time course of antibody production against each viral fragment and differences in the type of antibodies produced against 5 protein antigens of the virus.

Materials and Methods

Study
The study group consisted of 111 patients who were 2-dose vaccinated after transplant and 19 patients with renal failure who were 2-dose vaccinated in preparation for eventual transplant. In all patients, immunosuppressive therapy was started with tacrolimus, mycophenolate mofetil, steroids, and basiliximab. Patients vaccinated before transplant underwent living donor kidney transplant, on average, about 1 month after the second dose of vaccine. For the control group of healthy patients, serum samples obtained from 10 health care workers (5 male, 5 female) of our hospital were used. Blood sampling was performed 1 month and 3 months after vaccination, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody measurement was performed. Kidney transplants were performed from living donors who were fourth-degree relatives.

This study was designed as a retrospective study and was approved by the Ethics Committee (Figure 1). Informed consent was obtained from each patient, and the study was conducted in accordance with the Helsinki Declaration.

Methods
To assess the status of immunoglobulin G (IgG) antibody production in transplant recipients, the antibodies to 5 proteins constituting the SARS-CoV-2 virus, ie, extracellular domain (ECD), spike proteins (S1, S2), receptor-binding domain (RBD), and nucleocapsid, were measured simultaneously in the patients’ sera collected 1 month and 3 months after the second vaccination, before or after kidney transplant, using LABScreen COVID Plus (One Lambda) and LABScan3D. These data were analyzed with regard to rate of antibody seropositivity and percentage of responding viral fragments (Figure 1).

SARS-CoV-2 antibody measurement
Each serum sample was processed by enzyme-linked immunosorbent assay in a 96-well tray. The serum (2 μL) was combined with 1× phosphate-buffered saline (17 μL), and 0.02 M EDTA (1 μL), followed by addition of LABScreen COVID Plus beads (5 μL). The mixture was incubated for 30 minutes in a dark place at a temperature of 20 to 25 °C with gentle agitation. The 1× washing buffer (150 μL) was added to each well of the plate, followed by centrifugation at 1300 g for 5 minutes. The washing buffer was removed from all of the plate wells by flicking the plate, after which 1× washing buffer (200 μL) was added to each well, followed by 5-minute centrifugation at 1300 g (washing done twice). Then, 100× phycoerythrin-conjugated anti-human IgG (100 μL) was added to each well, followed by 30-minute incubation in a dark place at 20 to 25 °C with gentle agitation. After centrifugation for a further 5 minutes at 1300 g, the supernatant was removed from the wells by the standard method, ie, flicking the plate (washing done twice). Then, 1× phosphate-buffered saline (80 μL) was added to each well, followed by measurement with a LABScan or LABScan3D system. The data in the form of trimmed mean values were analyzed with HLA Fusion software (version 4.5).

Statistical analyses
The data are shown as mean ± SD or median values. We used t test to analyze continuous variables that possibly assumed a normal distribution. P < .05 (2-tailed) was considered statistically significant.

Results

Patients
Details of the patients are shown in Figure 2. The study consisted of 111 recipients vaccinated twice after transplant and 19 patients vaccinated twice before transplant. There were no significant differences between the 2 groups in terms of sex and age. The proportion of patients who underwent blood type-incompatible transplant was higher in the group vaccinated after transplant. Therefore, the proportion of rituximab was also significantly higher in the group vaccinated after transplant. The proportion of patients who received double filtration plasmapheresis at the time of transplant was similar between the 2 groups. In addition, there were no significant differences in underlying diseases and serum creatinine levels before and after transplant, as shown in Figure 2.

Differences in seropositivity rates after vaccination and viral fragments responded to vaccination by the timing of vaccination
As shown in Figure 3, 46 (42%) of the 111 patients who were vaccinated after living donor kidney transplant tested positive for antibody. On the other hand, 19 (100%) of the 19 patients vaccinated twice before transplant tested positive for antibody. Also, 10 of the 10 healthy patients in the control group showed antibody production. As shown in Figure 3, antibody analysis by viral fragment showed various patterns of seropositivity in recipients vaccinated after transplant who tested positive for antibody. This shows that antibodies, once produced against various fragments in transplant recipients, become negative against several fragments.

On the other hand, there was a trend in patients vaccinated before transplant that the fragment antibodies produced after vaccination remained positive to almost all the fragments. The pattern of seropositivity was similar to those observed in healthy patients, as shown in Figure 1. In healthy patients, antibodies were produced against all nonnucleoside fragments (ECD, S1, RBD, and S2).

Distribution of positivity for each viral fragment at 1 month after vaccination
Figure 4 shows the distribution of seropositivity for each viral fragment at 1 month after vaccination. In healthy patients, antibodies were produced against nonnucleoside fragments. The mean fluorescence intensities (MFI) of all the antibodies produced were high, exceeding 20 000. On the other hand, in recipients vaccinated after transplant, even in those with antibody production, the MFI levels of the antibodies against viral fragments other than ECD were significantly lower versus healthy patients. In addition, in patients vaccinated before transplant, the pattern of positivity distribution was intermediate between those observed in healthy patients and recipients vaccinated after transplant. More specifically, MFI levels of antibodies against S1 and RBD were around 20 000 to 25 000, which were almost equal to the levels observed in healthy patients, whereas the MFI of antibodies against S2 was low (7800). In addition, the mean of cumulative MFI of antibodies against all the fragments was 94 780 in patients vaccinated before transplant and 59 073 in recipients vaccinated after transplant (Figure 5). The cumulative MFI of all antibodies produced was significantly higher in patients vaccinated before transplant versus recipients vaccinated after transplant.

Time course of antibody mean fluorescence intensity at 1 month and 3 months after vaccination
Figure 6 shows the time course of antibody MFI at 1 month and 3 months after vaccination. We analyzed antibodies against each viral fragment. At 1 month after the second vaccination, the MFI of antibodies against ECD was similar (about 27 000) in patients vaccinated before transplant and recipients vaccinated after transplant. However, the MFI levels of antibodies against S1, S2, and RBD were significantly higher in patients vaccinated before transplant versus recipients vaccinated after transplant (for S1, MFI was 19 720 for pretransplant vaccination vs 11 421 for posttransplant vaccination; for S2, 7876 vs 6825; and for RBD, 23 713 vs 3906, respectively). After 3 months from vaccination, in patients vaccinated before transplant, the high MFI values of antibodies against S1, S2, and RBD decreased markedly to the levels in recipients vaccinated after transplant (S1, pretransplant MFI was 10 670 vs 3 months post-transplant MFI of 10 274; S2, 3130 vs 5450; and RBD, 16 505 vs 13 981, respectively). In recipients vaccinated after transplant, antibody MFI did not decrease during the period from 1 month to 3 months after vaccination. However, in patients vaccinated before transplant, antibody MFI decreased from 19 720 at 1 month postvaccination to 10 670 at 3 months postvaccination for S1; from 7876 to 3130 for S2; and from 23 713 to 16 505 for RBD, respectively. During the period from 1 month to 3 months after vaccination, all patients vaccinated before transplant underwent kidney transplant and started oral immunosup-pressive therapy. Regarding the production of antibodies against nucleosides, no seropositivity was observed either before and after transplant or at any time point.

Discussion

At our facility, even in the era of COVID-19 the pandemic, we have performed kidney transplants while maintaining highly rigorous preventative measures against COVID-19 infection and transmission. In our previous study,1 we reported 3 main points. (1) In transplant recipients on oral immunosuppressants, the seropositivity rate after the second vaccination is significantly lower versus healthy patients. (2) The seropositivity rate varies depending on the type of immunosuppressants in use. (3) The seropositivity rates vary depending on viral fragments that constitute the coronavirus. At present, in 2022, vaccination is progressing well in Japan, and people are currently receiving the third dose of vaccine. To date, many reports have been produced about mortality from coronavirus infection in transplant recipients and patients with end-stage renal failure, as well as about seropositivity rate after coronavirus vaccination.

Azzi and colleagues² reported the mortality from coronavirus infection in transplant recipients was as high as 30%. The study analyzed mortality for each organ transplanted, such as heart, lung, and liver. However, no significant difference was found in the mortality of patients, particularly when analyzed according to the organ type. The mortality from coronavirus infection was around 10% to 30% in recipients of any organ. Based on this results, it can be said that patient survival outcomes depend on the oral immunosuppressants in use and other comorbidities of the patient, rather than the transplanted organ. In addition, Azzi and colleagues reported the mortality from coronavirus infection was 20% to 30% in patients with end-stage renal failure who were vaccinated before transplant, but it was review of recent publications and provided no detailed information on patient demographic characteristics, such as vaccination. However, it is now common knowledge that transplant recipients and patients with renal failure have a higher mortality from coronavirus infection than healthy individuals.

Rincon-Arevalo and colleagues³ scientifically proved the low seropositivity rate after vaccination in such transplant recipients and patients with end-stage renal failure, by analyzing immature B cells and plasma cells found in transplant recipients. They proposed the importance of more strict and frequent vaccination protocols for transplant recipients. Boyarsky and colleagues⁴ published the first report on the need for at least 2 doses of vaccine for transplant recipients, especially in transplant recipients on oral treatment with metabolic antagonist, for whom the seropositivity rate was low (17%) after a single dose of vaccine but significantly increased after the second dose. Furthermore, Basic-Julic and Ivo⁵ reported that antibody production did not occur, because 2 doses of vaccine were insufficient to maintain the antigenicity in kidney recipients, but 3 or more doses of vaccine were capable of enhancing the antigenicity and therefore improved the seropositivity rate. Kamar and colleagues⁶ also reported that the third dose of vaccine enhanced the antigenicity of the vaccine, leading to an increase in the antibody production rate after vaccination.

On the other hand, Bertrand and colleagues⁷ have reported that, for the anti-coronavirus antibodies produced after the third dose of vaccine, the production of these antibodies is maintained until 1 month after vaccination, but a rapid decline in antibody titers starts after 6 months. In addition, in our previous report,¹ regarding the antibody production as a postvaccination response in kidney transplant recipients, we showed that it is extremely difficult before vaccination to predict and discriminate responders (likely to produce antibodies) and nonresponders (less likely to produce antibodies), because the analysis involves various complicated background factors, such as the doses and types of their immunosuppressants in use.⁸

In this study, we analyzed the seropositivity rate in patients vaccinated with the new coronavirus vaccine before and after kidney transplant. The study was conducted with reagents (LABScreen COVID Plus assay) capable of simultaneous identification and quantification of responses to the 5 proteins (ECD, S1, S2, RBD, and nucleocapsid) that constitute SARS-CoV-2. This product contains antigens for SARS-CoV-2 spikes, receptor-binding domains, nucleocapsid, spike S1, seasonal coronaviruses, Middle East respiratory syndrome, and SARS. Therefore, it has been reported that a major feature of the LABScreen COVID Plus assay is the low likelihood of cross-reactivity. According to a previous report, this assay has good specificity (98.6%) and sensitivity (100%).⁹

The patients that we investigated in this study had received kidney transplants at our department. There were 111 transplant recipients who received 2 doses of BNT162b2 mRNA COVID-19 vaccine after transplant and 19 who completed the 2-dose vaccination series before transplant. As a control group, 10 healthy patients were evaluated. The mean number of days from transplant to vaccination was 3257 days in recipients vaccinated after transplant. The patients vaccinated before transplant underwent transplant, on average, about 1 month (day 42) after the second dose of vaccine (Figure 2). Forty-six (42%) of the 111 recipients vaccinated after transplant tested positive for COVID-19 antibodies. On the other hand, 19 (100%) of the 19 patients vaccinated twice before transplant had seropositivity by 1 month after vaccination (Figure 3). In addition, all the 10 healthy patients had seropositivity. The antibody titers were significantly lower in patients vaccinated before transplant and recipients vaccinated after transplant versus healthy patients (data not shown). This finding is consistent with the findings by Danthu and colleagues, who reported that in the posttransplant immunosuppressive state about half of the antibody production after vaccination is reduced, whereas with the metabolic abnormalities in patients with uremia of chronic renal failure before transplant, antibody production occurs but is reduced.¹⁰

Table 1 shows cases of recipients vaccinated after transplant who showed no antibody production at 1 month after vaccination but showed seropositivity at 3 months after vaccination. To match demographic characteristics, 6 patients who were receiving oral cyclosporine treatment were excluded from the 111 patients. Of the remaining 105 patients, 43 patients (43/105, 41%) had antibody production at 1 month after vaccination, and 15 patients (15/105, 15%) produced antibodies for the first time at 3 months after vaccination. Thus, these results support the findings of Arevalo and colleagues,³ who reported that oral immunosuppressants in transplant recipients inhibit the maturation of B cells and prevent the differentiation of the B cells into mature plasma cells capable of antibody production.

Figure 7 shows the analysis of MFI levels of antibodies against each viral fragment in 43 patients who were producing antibodies at 1 month after vaccination and 15 patients who were producing antibodies for the first time at 3 months after vaccination. The comparison of the MFI measu-rements of antibodies produced against each fragment showed that the MFI levels in the 15 patients in whom antibodies were detected for the first time at 3 months were significantly lower versus the 43 patients with antibody production at 1 month. As shown in Table 1, 42 (89%) of 47 patients who tested negative for antibody production were on rituximab treatment to suppress B cells before and after transplant.

The analysis of each viral fragment showed that the production of antibodies against each fragment was stable in patients vaccinated before transplant and in healthy patients. Antibodies were produced against almost all spike proteins, but not nucleosides. On the other hand, in recipients vaccinated after transplant, various patterns of seropositivity were observed. More specifically, there was no antibody production against some of the fragments in recipients vaccinated after transplant, and therefore seropositivity was not sustainable.

Figure 3 shows the antibody titer against each fragment, expressed as MFI by flow cytometry analysis, on the vertical axis. In healthy patients, consistent antibody production (MFI ≥20 000) was observed against all spike antigens, but not nucleosides. On the other hand, in recipients vaccinated after transplant, the antibody titer was high only for ECD and was low (around 10 000) for the spike proteins. In pretransplant patients with renal failure, the seropositivity rate was 100% at 1 month after vaccination, but their antibody titers were intermediate between the antibody titers in healthy patients and transplant recipients.¹⁰ The cumulative MFI levels of antibodies against the various fragments in Figure 4 also show that the seropositivity rate for each fragment was consistent in each of the patients vaccinated before transplant, whereas the proportion of each fragment to all the fragments was not consistent in recipients vaccinated after transplant. In addition, the cumulative MFI of antibodies for all the fragments in patients vaccinated before transplant was 94 780, which is nearly twice as high as the MFI (59 073) in recipients vaccinated after transplant.

The time course of MFI of coronavirus antibodies produced after vaccination is shown in Figure 5. In the 19 patients vaccinated before transplant, they underwent living donor kidney transplant, on average, 42 days after the second dose of vaccine. That is, oral immunosuppressive therapy was started, followed by kidney transplant, between 1 month and 3 months after the second vaccination. Figure 5 shows the analysis of antibodies by viral fragment, expressed as MFI. The analysis revealed that there are temporal differences in the production of antibodies among the different fragment epitopes. Immunosuppressants had a very minor effect, if any, on the ECD, and the MFI of its antibodies was unchanged between 1 month and 3 months after the second dose of vaccine. On the other hand, for the spike proteins (S1, RBD, and S2), the antibody MFI showed a rapid decline after the start of immuno-suppressant therapy after transplant and was reduced to the same level observed in recipients vaccinated after transplant. In patients vaccinated twice after transplant, no such rapid change was observed by 1 month and 3 months after vaccination. The antibody MFI gradually decreased during the period from 3 to 6 months after vaccination (data not shown).

Many studies have shown the relationship between antibodies produced after vaccination and immunosuppressants, not only for transplant but also for rheumatic diseases.¹¹

This study revealed that a 2-dose vaccination regimen with BNT162b2 mRNA COVID-19 vaccine enabled antibody production at a considerably high likelihood in patients before transplant, whereas the proportion of recipients vaccinated after transplant who had antibody production was about half of the pretransplant levels (42%). Fifteen percent of these patients showed a delayed production of antibodies, with no production at 1 month but with production for the first time at 3 months. As reported in our previous study,¹ in the viral fragment analyses, the proportion of antibodies produced against each fragment versus all the fragments was consistent in patients vaccinated before transplant, whereas in recipients vaccinated after transplant, there was a great interindividual variation in the type of unresponsive fragment antibodies. Therefore, the proportion of antibodies produced against each fragment versus all the fragments was not maintained. Also, the cumulative MFI of produced antibodies was about twice higher in patients vaccinated before transplant versus recipients vaccinated after transplant. The most noteworthy is that the coronavirus antibodies produced after the second dose of vaccine, therefore became seropo-sitive once, rapidly decreased after the start of perioperative immunosuppressive therapy.

In Japan, we currently live our daily lives after the sixth wave of the pandemic, preparing for the seventh wave. It is also the fact that the vaccination with BNT162b2 mRNA COVID-19 vaccine has reduced the burden of self-restraint in our social life and drastically reduced the number of severe and fatal cases of the viral infection. However, it appears that the level of coronavirus antibodies produced by this vaccine seems to decrease with the lifespan of IgG. Thus, the antibody-based immunity is rather fragile. Moreover, our findings suggest that immuno-suppressants, which are drugs used to induce an immunosuppressive state in transplant recipients and patients with autoimmune diseases, may drastically reduce the level of antibodies produced by the vaccination. Furthermore, the seropositivity rate in a posttransplant immunosup-pressive state was less than 50%, whereas it was 100% in patients vaccinated twice before transplant. Even if immuno-suppression for the subsequent transplant causes antibody reduction, vaccination is considered essential for standby patients scheduled to undergo transplant in the near future. Further vaccination may be necessary in the future to safely survive in the world where we have to coexist with coronavirus.

Conclusions

All patients with renal failure who were vaccinated before transplant showed a high seropositivity rate, similar to that in healthy patients. The seropositivity rate for each of the viral fragment antibodies in patients vaccinated before transplant was maintained, as seen in healthy patients. However, in patients vaccinated before transplant who tested positive for antibody production at 1 month after vaccination, the antibody MFI at 3 months after vaccination (posttransplant) was remarkedly lower versus at 1 month after vaccination. Immunosup-pressive medicines administered at the time of transplant should link to remarkable reduction of antibody elicited by vaccines. The timing of booster vaccination should be a consideration, although further study is needed.

References:

1. Ishida H, Furusawa M, Unagami K, Omoto K, Iizuka J, Takagi T. Antibody response to SARS-CoV-2 mRNA vaccine among kidney transplant recipients: a retrospective cohort study at a single transplant institute in Japan. Exp Clin Transplant. 2022;20(5):463-471. doi:10.6002/ect.2022.0020
   CrossRef - PubMed
   
2. Azzi Y, Bartash R, Scalea J, Loarte-Campos P, Akalin E. COVID-19 and solid organ transplantation: a review article. Transplantation. 2021;105(1):37-55. doi:10.1097/TP.0000000000003523
   CrossRef - PubMed
   
3. Rincon-Arevalo H, Choi M, Stefanski AL, et al. Impaired humoral immunity to SARS-CoV-2 BNT162b2 vaccine in kidney transplant recipients and dialysis patients. Sci Immunol. 2021;6(60):eabj1031, . doi:10.1126/sciimmunol.abj1031
   CrossRef - PubMed
   
4. Boyarsky BJ, Werbel WA, Avery RK, et al. Antibody response to 2-dose SARS-CoV-2 mRNA vaccine series in solid organ transplant recipients. JAMA. 2021;325(21):2204-2206. doi:10.1001/jama.2021.7489
   CrossRef - PubMed
   
5. Basic-Jukic N, Ivo J. SARS-CoV-2 infection after two doses of mRNA vaccine in renal transplant recipients. Transpl Infect Dis. 2021;23(4):e13628. doi:10.1111/tid.13628
   CrossRef - PubMed
   
6. Kamar N, Abravanel F, Marion O, Couat C, Izopet J, Del Bello A. Three doses of an mRNA Covid-19 vaccine in solid-organ transplant recipients. N Engl J Med. 2021;385(7):661-662. doi:10.1056/NEJMc2108861
   CrossRef - PubMed
   
7. Bertrand D, Lemee V, Laurent C, et al. Waning antibody response and cellular immunity 6 months after third dose SARS-Cov-2 mRNA BNT162b2 vaccine in kidney transplant recipients. Am J Transplant. 2022;22(5):1498-1500. doi:10.1111/ajt.16954
   CrossRef - PubMed
   
8. Charmetant X, Espi M, Barba T, et al. Predictive factors of a viral neutralizing humoral response after a third dose of COVID-19 mRNA vaccine. Am J Transplant. 2022;22(5):1442-1450. doi:10.1111/ajt.16990
   CrossRef - PubMed
   
9. Bray RA, Lee JH, Brescia P, et al. Development and validation of a multiplex, bead-based assay to detect antibodies directed against SARS-CoV-2 proteins. Transplantation. 2021;105(1):79-89. doi:10.1097/TP.0000000000003524
   CrossRef - PubMed
   
10. Danthu C, Hantz S, Dahlem A, et al. Humoral response after SARS-CoV-2 mRNA vaccination in a cohort of hemodialysis patients and kidney transplant recipients. J Am Soc Nephrol. 2021;32(9):2153-2158. doi:10.1681/ASN.2021040490
    CrossRef - PubMed
    
11. Friedman MA, Curtis JR, Winthrop KL. Impact of disease-modifying antirheumatic drugs on vaccine immunogenicity in patients with inflammatory rheumatic and musculoskeletal diseases. Ann Rheum Dis. 2021;80(10):1255-1265. doi:10.1136/annrheumdis-2021-221244
    CrossRef - PubMed