Blood unconjugated bilirubin and tacrolimus are negative predictors of specific cellular immunity in kidney transplant recipients after SAR-CoV-2 inactivated vaccination.

The immunogenicity of SARS-CoV-2 vaccines is poor in kidney transplant recipients (KTRs). The factors related to poor immunogenicity to vaccination in KTRs are not well defined. Here, observational study demonstrated no severe adverse effects were observed in KTRs and healthy participants (HPs) after first or second dose of SARS-CoV-2 inactivated vaccine. Different from HPs with excellent immunity against SARS-CoV-2, IgG antibodies against S1 subunit of spike protein, receptor-binding domain, and nucleocapsid protein were not effectively induced in a majority of KTRs after the second dose of inactivated vaccine. Specific T cell immunity response was detectable in 40% KTRs after the second dose of inactivated vaccine. KTRs who developed specific T cell immunity were more likely to be female, and have lower levels of total bilirubin, unconjugated bilirubin, and blood tacrolimus concentrations. Multivariate logistic regression analysis found that blood unconjugated bilirubin and tacrolimus concentration were significantly negatively associated with SARS-CoV-2 specific T cell immunity response in KTRs. Altogether, these data suggest compared to humoral immunity, SARS-CoV-2 specific T cell immunity response are more likely to be induced in KTRs after administration of inactivated vaccine. Reduction of unconjugated bilirubin and tacrolimus concentration might benefit specific cellular immunity response in KTRs following vaccination.

Tacrolimus toxicity due to enzyme inhibition from ritonavir.

Tacrolimus is commonly used for immunosuppression in patients following solid organ transplantation. For transplant patients with COVID-19 infection, early treatment is indicated due to the risk of progression to severe disease. However, the first line agent, nirmatrelvir/ritonavir, has multiple drug-drug interactions. We report a case of tacrolimus toxicity in a patient with a history of renal transplant due to enzyme inhibition related to nirmatrelvir/ritonavir. An 85-year-old woman with a history of multiple comorbidities presented to the emergency department (ED) with weakness, increasing confusion, poor oral intake, and inability to walk. She had been recently diagnosed with COVID-19 infection and was prescribed nirmatrelvir/ritonavir due to her underlying comorbidities and immune suppression. In the ED, she was dehydrated and had an acute kidney injury (creatinine 2.1 mg/dL, up from a baseline of 0.8 mg/dL). The tacrolimus concentration on initial labs was 143 ng/mL (5-20 ng/mL) and it continued to rise despite being held, to a peak of 189 ng/mL on hospital day 3. The patient was treated with phenytoin for enzyme induction and the tacrolimus concentration began to fall. She was discharged to a rehabilitation facility after a 17 day hospitalization. ED physicians must be cognizant of drug-drug interactions when prescribing nirmatrelvir/ritonavir and evaluating patients recently treated with the drug to identify toxicity due to these interactions.

Analysis of Antibody Responses After COVID-19 Vaccination in Liver Transplant Recipients: A Single-Center Study.

BACKGROUND: Liver transplant (LT) recipients were considered a vulnerable population during the coronavirus disease 2019 (COVID-19) pandemic. The clinical efficacy of the COVID-19 vaccine is unknown in immunocompromised patients. The purpose of this study was to provide evidence of antibody responses after COVID-19 vaccination in LT recipients. METHODS: This study enrolled 46 patients who underwent LT at Samsung Medical Center (Seoul, Korea) before implementation of the one-dose vaccine in Korea. Those who completed the two-dose COVID-19 vaccine between August 2021 and September 2021 were included and followed through December 2021. Semiquantitative anti-spike serologic testing was performed using the Roche Elecsys anti-SARS-CoV-2 S enzyme immunoassay (Roche Diagnostics, Rotkereuz, Switzerland) with a positive cutoff of at least 0.8 U/mL. RESULTS: Among all 46 participants, 40 (87%) demonstrated an antibody response after the second dose of a COVID-19 vaccine, while six (13%) had no antibody response after the second dose. Upon univariate analysis, patients with higher antibody titer had longer years since LT (2.3 ± 2.8 vs. 9.4 ± 5.0, P < 0.001). A lower median tacrolimus (TAC) level before vaccination and after the second dose of COVID-19 vaccine indicated a significantly higher antibody response (2.3 [1.6-3.2] vs. 7.0 [3.7-7.8], P = 0.006, 2.5 [1.6-3.3] vs. 5.7 [4.2-7.2], P = 0.003). Period between 2nd vaccination and serologic testing was significantly higher in the antibody-response group compared to the no-antibody-response group (30.2 ± 24.0 vs. 65.9 ± 35.0, P = 0.012). A multivariate analysis of antibody responses revealed TAC level before vaccination as a statistically significant factor. CONCLUSION: A higher TAC level before vaccination resulted in less effective vaccination in LT patients. Booster vaccinations are required, especially for patients in the early stage after LT who have compromised immune function.

Nirmatrelvir/ritonavir treatment in SARS-CoV-2 positive kidney transplant recipients - a case series with four patients.

BACKGROUND: Despite vaccination coronavirus disease 2019 (COVID-19)-associated mortality caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains high in kidney transplant recipients. Nirmatrelvir is a protease inhibitor with activity against SARS-CoV-2. Nirmatrelvir reduces the risk for mortality and hospitalization, which is approved for treating adults at risk for severe disease. Nirmatrelvir is metabolized by the cytochrome P-450 (CYP) 3A4 isozyme CYP3A4 and is therefore co-administered with the irreversible CYP3A4 inhibitor ritonavir, which results in a drug interaction with tacrolimus. A limited number of patients with nirmatrelvir/ritonavir and tacrolimus therapy after kidney transplantation have been reported to date. It has been reported that tacrolimus was paused during the five-day nirmatrelvir/ritonavir therapy and subtherapeutic tacrolimus levels were observed after finishing nirmatrelvir/ritonavir in two patients. Therefore, optimization of tacrolimus dosing is urgently needed in transplant recipients receiving nirmatrelvir/ritonavir treatment. CASE PRESENTATION: Here, we present our first-hand experience with four patients receiving tacrolimus therapy following kidney transplantation and nirmatrelvir/ritonavir therapy due to COVID-19. Tacrolimus was paused during nirmatrelvir/ritonavir therapy in all patients, which resulted in stable therapeutic tacrolimus levels. Tacrolimus was continued directly after finishing nirmatrelvir/ritonavir to avoid subtherapeutic levels in the first patient treated. This patient received his usual tacrolimus maintenance dose, which resulted in toxic levels. Based on this observation, tacrolimus therapy was continued 24 h after finishing nirmatrelvir/ritonavir treatment at a reduced dose in the subsequent patients. In these patients, therapeutic to supratherapeutic tacrolimus levels were observed despite the therapeutic break and dose reduction. DISCUSSION AND CONCLUSIONS: Based on altered CYP3A4 metabolism, tacrolimus levels have to be closely monitored after treatment with nirmatrelvir/ritonavir. Our study suggests that tacrolimus treatment should be paused during nirmatrelvir/ritonavir medication and be continued 24 h after completing nirmatrelvir/ritonavir therapy at a reduced dose and under close monitoring. Based on the limited number of patients in this study, results must be interpreted with caution.

Nirmatrelvir/ritonavir Use With Tacrolimus in Lung Transplant Recipients: A Single-center Case Series.

BACKGROUND: Limited data and guidelines exist for using nirmatrelvir/ritonavir in solid organ transplant recipients stabilized on tacrolimus for the treatment of mild-to-moderate coronavirus disease. Concern exists regarding the impact of utilizing a 5-d course of nirmatrelvir/ritonavir with calcineurin inhibitors because of significant drug-drug interactions between ritonavir, a potent cytochrome P450 3A inhibitor, and other cytochrome P450 3A substrates, such as tacrolimus. METHODS: We report the successful use of nirmatrelvir/ritonavir in 12 outpatient lung transplant recipients with confirmed severe acute respiratory syndrome coronavirus 2 infection stabilized on tacrolimus immunosuppression. All patients stopped tacrolimus and started nirmatrelvir/ritonavir 10 to 14 h after the last dose of tacrolimus. Tacrolimus was withheld and then reinitiated at a modified dose 48 h following the completion of nirmatrelvir/ritonavir therapy. Tacrolimus trough levels were checked during nirmatrelvir/ritonavir therapy and tacrolimus reinitiation. RESULTS: Ten (10/12) patients were able to resume their original tacrolimus dose within 4 d of completing nirmatrelvir/ritonavir therapy and maintain therapeutic levels of tacrolimus. No patients experienced tacrolimus toxicity or acute rejection during the 30-d postcompletion of nirmatrelvir/ritonavir therapy. CONCLUSIONS: In this cohort of lung transplant recipients on tacrolimus, we demonstrated that nirmatrelvir/ritonavir can be safely used with close monitoring of tacrolimus levels and appropriate dose adjustments of tacrolimus. Further confirmatory studies are needed to determine the appropriate use of therapeutic drug monitoring and tacrolimus dose following completion of nirmatrelvir/ritonavir in the solid organ transplant population.

A multicentre, patient- and assessor-blinded, non-inferiority, randomised and controlled phase II trial to compare standard and torque teno virus-guided immunosuppression in kidney transplant recipients in the first year after transplantation: TTVguideIT.

BACKGROUND: Immunosuppression after kidney transplantation is mainly guided via plasma tacrolimus trough level, which cannot sufficiently predict allograft rejection and infection. The plasma load of the non-pathogenic and highly prevalent torque teno virus (TTV) is associated with the immunosuppression of its host. Non-interventional studies suggest the use of TTV load to predict allograft rejection and infection. The primary objective of the current trial is to demonstrate the safety, tolerability and preliminary efficacy of TTV-guided immunosuppression. METHODS: For this purpose, a randomised, controlled, interventional, two-arm, non-inferiority, patient- and assessor-blinded, investigator-driven phase II trial was designed. A total of 260 stable, low-immunological-risk adult recipients of a kidney graft with tacrolimus-based immunosuppression and TTV infection after month 3 post-transplantation will be recruited in 13 academic centres in six European countries. Subjects will be randomised in a 1:1 ratio (allocation concealment) to receive tacrolimus either guided by TTV load or according to the local centre standard for 9 months. The primary composite endpoint includes the occurrence of infections, biopsy-proven allograft rejection, graft loss, or death. The main secondary endpoints include estimated glomerular filtration rate, graft rejection detected by protocol biopsy at month 12 post-transplantation (including molecular microscopy), development of de novo donor-specific antibodies, health-related quality of life, and drug adherence. In parallel, a comprehensive biobank will be established including plasma, serum, urine and whole blood. The date of the first enrolment was August 2022 and the planned end is April 2025. DISCUSSION: The assessment of individual kidney transplant recipient immune function might enable clinicians to personalise immunosuppression, thereby reducing infection and rejection. Moreover, the trial might act as a proof of principle for TTV-guided immunosuppression and thus pave the way for broader clinical applications, including as guidance for immune modulators or disease-modifying agents. TRIAL REGISTRATION: EU CT-Number: 2022-500024-30-00.

Adjustment of Immunosuppressants to Facilitate Anti-COVID-19 Antibody Production after mRNA Vaccination in Liver Transplant Recipients.

Liver transplant recipients are immunocompromised and have low immunogenicity to produce antibodies in anti-COVID-19 vaccination. Whether immunosuppressant adjustment could facilitate anti-COVID-19 antibody production in anti-COVID-19 mRNA vaccination is undetermined. Our patients were informed to temporarily suspend mycophenolate mofetil (MMF) or everolimus (EVR) for 2 weeks during both the 1st and 2nd doses of Moderna mRNA-1273 vaccine. A total of 183 recipients receiving two doses of Moderna mRNA-1273 vaccine were enrolled and grouped into tacrolimus monotherapy (MT, n = 41), and dual therapy with non-adjustment (NA, n = 23), single suspension (SS, n = 19) and double suspension (DS, n = 100) of MMF/EVR in two-dose mRNA vaccination. A total of 155 (84.7%) patients had a humoral response to vaccines in this study. The humoral response rates were 60.9%, 89.5%, 91.0% and 80.5% in NA, SS, DS, and MT group patients, respectively (p = 0.003). Multivariate analysis showed that favorable factors for humoral response were temporary suspension of MMF/EVR and monotherapy, and unfavorable factors were deceased donor liver transplantation, WBC count < 4000/uL, lymphocyte < 20% and tacrolimus trough level ≥ 6.8 ng/mL. In conclusion, temporary two-week suspension of anti-proliferation immunosuppressants could create a window to facilitate antibody production during anti-COVID-19 mRNA vaccination. This concept may be applied to other vaccinations in liver transplant recipients.

"Saving lives with nirmatrelvir/ritonavir one transplant patient at a time".

BACKGROUND: Solid organ transplant (SOT) recipients are at risk of complications from COVID-19. Nirmatrelvir/ritonavir (Paxlovid) can reduce mortality from COVID-19 but is contraindicated in patients receiving calcineurin inhibitors (CI), which depend on cytochrome p4503A (CY3PA). In this study, we aim to show the feasibility of nirmatrelvir/ritonavir administration to SOT recipients receiving CI with coordination of medication management and limited tacrolimus trough monitoring. METHODS: We reviewed adult SOT recipients treated with nirmatrelvir/ritonavir from 4/14 to 11/1/2022 and assessed for changes in tacrolimus trough and serum creatinine after therapy. RESULTS: Of 47 patients identified, 28 were receiving tacrolimus and had follow-up laboratory testing. Patients had a mean age of 55 years, 17 (61%) received a kidney transplant and 23 (82%) received three or more doses of SARS-CoV-2 mRNA vaccine. Patients had mild-moderate COVID-19 and started nirmatrelvir/ritonavir within 5 days of symptom onset. Median baseline tacrolimus trough concentration was 5.6 ng/mL (Interquartile range 5.1-6.7), while median follow-up tacrolimus trough concentration was 7.8 ng/mL (Interquartile range 5.7-11.5, p = 0.0017). Median baseline and follow-up serum creatinine levels were 1.21 mg/dL (Interquartile range 1.02-1.39) and 1.21 mg/dL (interquartile range 1.02-1.44, p = 0.3162), respectively. One kidney recipient had a follow up creatinine level >1.5 times baseline. No patients were hospitalized or died from COVID-19 in the follow up period. CONCLUSION: While administration of nirmatrelvir/ritonavir resulted in a significant increase in tacrolimus concentration, this did not result in significant nephrotoxicity. Early oral antiviral treatment in SOT recipients is feasible with medication management, even with limited tacrolimus trough monitoring.

Improved immunogenicity following the third dose of BNT162b2 mRNA vaccine in heart transplant recipients.

OBJECTIVES: The immunogenicity of two-dose severe acute respiratory syndrome coronavirus 2 vaccine is lower among heart transplant (HTx) recipients, compared with the general population. Our aim was to assess the immunogenicity of a third-dose vaccine in HTx recipients. METHODS: This is a prospective cohort study of HTx recipients who received a third dose of the BNT162b2 vaccine. Immunogenicity was assessed by serum levels of anti-spike immunoglobulin G (S-IgG), taken at baseline and 14-28 days after the third dose. Titres above 50 U/ml were interpreted positive. RESULTS: We Included 42 HTx recipients at a median age of 65 years [interquartile range (IQR) 58-70]. At baseline, the median of 27 days (IQR 13-42) before the third dose and the median titre of the whole group was 18 U/ml (IQR 4-130). Only 14 patients (33%) were S-IgG seropositive. After the third dose, the proportion of seropositive patients increased significantly to 57% (P = 0.05) and the median titre increased significantly to 633 U/ml (IQR 7-6104, P < 0.0001). Younger age at HTx (OR per 1-year decrease 1.07, P = 0.05), low tacrolimus serum level (OR per 1-unit decrease 2.28, P = 0.02), mammalian target of rapamycin use (OR 13.3, P = 0.003), lack of oral steroids use (OR 4.17, P = 0.04) and lack of calcineurin inhibitor use (71% of responders vs 100% non-responders received calcineurin inhibitors, P = 0.01) were predictors of seropositive result after the third dose. However, no significant association was detected following adjustment for baseline S-IgG titre. CONCLUSIONS: Third-dose booster of BNT162b2 vaccine significantly increased immunogenicity among HTx recipients who previously received a two-dose vaccine.

Nirmatrelvir increases blood tacrolimus concentration in COVID-19 patients as determined by UHPLC-MS/MS method.

OBJECTIVE: Transplant recipients have a higher risk of SARS-CoV-2 infection owing to the use of immunosuppressive drugs like tacrolimus (FK506). FK506 and nirmatrelvir (NMV) (an anti-SARS-CoV-2 drug) are metabolized by cytochrome P450 3A4 and may have potential drug-drug interactions. It is important to determine the effect of NMV on FK506 concentrations. PATIENTS AND METHODS: Following protein precipitation from blood, FK506 and its internal standard (FK506-13C,2d4) were detected by ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). Total 22 blood samples (valley concentrations) from two coronavirus disease 2019 (COVID-19) patients were collected and analyzed for FK506 concentrations. RESULTS: Blood levels of FK506 (0.5-100 ng/mL) showed good linearity. The UHPLC-MS/MS method was validated with intra- and inter-batch accuracies of 104.55-107.85%, and 99.52-108.01%, respectively, and precisions of < 15%. Mean blood FK506 concentration was 12.01 ng/mL (range, 3.15-33.1 ng/mL). Five-day co-administration with NMV increased the FK506 concentrations from 3.15 ng/mL to 33.1 ng/mL, returning to 3.36 ng/mL after a 9-day-washout. CONCLUSIONS: We developed a simple quantification method for therapeutic drug monitoring of FK506 in patients with COVID-19 using UHPLC-MS/MS with protein precipitation. We found that NMV increased FK506 blood concentration 10-fold. Therefore, it is necessary to re-consider co-administration of FK506 with NMV.

Tacrolimus Drug-Drug Interaction with Nirmatrelvir/Ritonavir (Paxlovid™) Managed with Phenytoin.

INTRODUCTION: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19) pandemic has had a significant impact on communities and health systems. The Federal Drug Administration (FDA) authorized Pfizer's nirmatrelvir/ritonavir (Paxlovid™) through an EUA for the treatment of mild to moderate cases of COVID-19 at high risk for progression to severe disease. Patients with a history of transplant who test positive for COVID-19 are considered high risk because of their immunosuppression and are therefore candidates for nirmatrelvir/ritonavir. CASE REPORT: This is a case of a 67-year-old female with a past medical history of orthotopic heart transplant who received tacrolimus as part of her immunosuppressive regimen. She originally presented with complaints of dyspnea and cough for several days in the setting of COVID-19. The patient was started on nirmatrelvir/ritonavir due to her high risk for progression to severe disease. Four days after starting nirmatrelvir/ritonavir, she presented to the ED for slowed speech, fatigue, weakness, and loss of appetite. Upon admission she was found to have a supratherapeutic tacrolimus level of 176.4 ng/mL and an acute kidney injury. In this case, phenytoin was used as a CYP3A4 inducer to quickly decrease the tacrolimus level to within therapeutic range. CONCLUSION: This case highlights the strong and important drug-drug interaction between tacrolimus and nirmatrelvir/ritonavir leading to toxic levels of tacrolimus. It also demonstrates the utility and effectiveness of phenytoin as a "rescue" medication for tacrolimus toxicity.

Favipiravir therapy for Covid-19 infection and tacrolimus toxicity in a kidney transplant patient on chronic eculizumab therapy.

Although the latest data show that complement activation has an essential role in the pathogenesis and severity of Covid-19, the data on the prognosis of patients using complement inhibitors during Covid-19 infection are scarce. There is no specific treatment for Covid-19 yet. The introduction of novel agents such as favipiravir may affect metabolism of immunosuppressive drugs. We report the clinical course of Covid-19 in a kidney transplant patient with atypical haemolytic uraemic syndrome on chronic eculizumab therapy. The patient had mild Covid-19 but had severe tacrolimus toxicity, which may be associated with favipiravir and eculizumab. The mild course of Covid-19 in our patient is encouraging for eculizumab use; on the other hand, unusually high levels of tacrolimus that we observed underlines the importance of frequent drug level monitoring in transplanted patients who are receiving new drugs.

SARS-CoV-2 vaccine alleviates disease burden and severity in liver transplant recipients even with low antibody titers.

BACKGROUND AND AIMS: We retrospectively assessed the clinical Pfizer's mRNA SARS-CoV-2 BNT162b2 vaccination outcomes and the serologic impact on liver transplant (LT) recipients. PATIENTS AND METHODS: One hundred and sixty-seven LT cases followed between March 1, 2020 and September 25, 2021, and were stratified into two groups: (1) 37 LT recipients after SARS-CoV-2 infection before vaccine era and (2) 130 LT recipients vaccinated with 2 doses without earlier SARS-CoV-2 exposure. Serum SARS-CoV-2 spike immunoglobulins (anti-S) were assessed 7 days following vaccination (Liaison assay). RESULTS: In addition to the 37 nonvaccinated cases (22.2% of total group) who experienced SARS-CoV-2 infection (34 symptomatic and 3 asymptomatic), another 8 vaccinated symptomatic recipients (4.8%) were infected (5 from the third and three from the fourth waves). Three of the 45 infected cases died (6.7%) before the vaccine program. Vaccinated group: of the 130 LT vaccinated recipients, 8 (6.2%) got infected postvaccination (added to the infected group) and were defined as clinical vaccine failure; 38 (29.2%) were serological vaccine failure (total failure 35.4%), and 64.6% cases were serological vaccine responders (anti-S≥19 AU/mL). Longer post-LT interval and lower consumption of immunosuppressants (steroids, FK506, and mycophenolate mofetil) correlated with favorable SARS-CoV-2 vaccine response. Mammalian target of rapamycin inhibitors improved vaccine outcomes associated with lower FK506 dosages and serum levels. Patients with anti-S levels <100 AU/mL risked losing serologic response or being infected with SARS-CoV-2. A booster dose achieved an effective serologic response in a third of failures and most responders, securing better and possibly longer protection. CONCLUSION: Pfizer's BNT162b2 vaccine seems to lessen SARS-CoV-2 morbidity and mortality of LT recipients even with weak serological immunogenicity. Switching mycophenolate mofetil to mammalian target of rapamycin inhibitors might be effective before boosters in vaccine failure cases. A booster vaccine should be considered for nonresponders and low-responders after the second dose.

Drug-drug interactions of ritonavir-boosted SARS-CoV-2 protease inhibitors in solid organ transplant recipients: experience from the initial use of lopinavir-ritonavir.

OBJECTIVES: To review the drug-drug interactions between tacrolimus and lopinavir/ritonavir in 23 patients who received solid organ transplant during the first wave of COVID-19 and to determine the efficacy as well as safety of prednisone monotherapy. METHODS: Observational study performed between March and June 2020 in solid organ transplant recipients admitted with an established diagnosis of SARS-CoV-2 infection who received lopinavir/ritonavir (≥2 doses). Once lopinavir/ritonavir therapy was initiated, calcineurin inhibitor treatment was temporarily switched to prednisone monotherapy (15-20 mg/d) to avoid drug-drug interactions and toxicity. After lopinavir/ritonavir treatment completion, immunosuppressive treatment was restarted with reduced doses of prednisone-tacrolimus (target minimum blood concentration -C0- approximately 5 ng/mL). Patients were observed for 3 months to confirm the absence of rejection. RESULTS: The median time from discontinuation of tacrolimus to initiation of lopinavir/ritonavir was 14 hours (interquartile range [IQR], 12-15) and from discontinuation of lopinavir/ritonavir to resumption of tacrolimus 58 hours (IQR, 47-81). The duration of lopinavir/ritonavir treatment was 7 days (IQR, 5-7). Nine of the 21 (42.8%) patients on tacrolimus treatment had C0 above the cutoff point after lopinavir/ritonavir initiation, despite having been substituted with prednisone before lopinavir/ritonavir initiation. Three patients had very high concentrations (>40 ng/mL) and developed toxicity. No episodes of acute rejection were diagnosed. DISCUSSION: We did not observe toxicity in patients for whom tacrolimus was discontinued 24 hours before starting lopinavir/ritonavir and reintroduced at half dose 48 to 72 hours after lopinavir/ritonavir discontinuation. Prednisone monotherapy during lopinavir/ritonavir therapy was safe with no episodes of acute rejection. Experience with lopinavir/ritonavir may be applicable to the use of nirmatrelvir/ritonavir, but larger multicentre studies are needed to confirm these findings.

Translation suppression underlies the restrained COVID-19 mRNA vaccine response in the high-risk immunocompromised group.

Background: Immunocompromised (IC) patients show diminished immune response to COVID-19 mRNA vaccines (Co-mV). To date, there is no 'empirical' evidence to link the perturbation of translation, a rate-limiting step for mRNA vaccine efficiency (VE), to the dampened response of Co-mV. Materials and methods: Impact of immunosuppressants (ISs), tacrolimus (T), mycophenolate (M), rapamycin/sirolimus (S), and their combinations on Pfizer Co-mV translation were determined by the Spike (Sp) protein expression following Co-mV transfection in HEK293 cells. In vivo impact of ISs on SARS-CoV-2 spike specific antigen (SpAg) and associated antibody levels (IgGSp) in serum were assessed in Balb/c mice after two doses (2D) of the Pfizer vaccine. Spike Ag and IgGSp levels were assessed in 259 IC patients and 50 healthy controls (HC) who received 2D of Pfizer or Moderna Co-mV as well as in 67 immunosuppressed solid organ transplant (SOT) patients and 843 non-transplanted (NT) subjects following three doses (3D) of Co-mV. Higher Co-mV concentrations and transient drug holidays were evaluated. Results: We observed significantly lower IgGSP response in IC patients (p<0.0001) compared to their matched controls in 2D and 3D Co-mV groups. IC patients on M or S showed a profound dampening of IgGSP response relative to those that were not on these drugs. M and S, when used individually or in combination, significantly attenuated the Co-mV-induced Sp expression, whereas T did not exert significant influence. Sirolimus combo pretreatment in vivo significantly attenuated the Co-mV induced IgMSp and IgGSp production, which correlated with a decreasing trend in the early levels (after day 1) of Co-mV induced Sp immunogen levels. Neither higher Co-mV concentrations (6µg) nor withholding S for 1-day could overcome the inhibition of Sp protein levels. Interestingly, 3-days S holiday or using T alone rescued Sp levels in vitro. Conclusions: This is the first study to demonstrate that ISs, sirolimus and mycophenolate inhibited Co-mV-induced Sp protein synthesis via translation repression. Selective use of tacrolimus or drug holiday of sirolimus can be a potential means to rescue translation-dependent Sp protein production. These findings lay a strong foundation for guiding future studies aimed at improving Co-mV responses in high-risk IC patients.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.