Outcomes of COVID-19 in Solid Organ Transplant Recipients in Canada 2020-2021: A Prospective, Multicenter Cohort Study

Purpose: To understand the outcomes and changes in disease severity of COVID-19 in Solid Organ Transplant (SOT) recipients over time in the context of therapeutic advances. Method(s): We performed a multicenter, prospective cohort study of all SOT recipients diagnosed with COVID-19, across 9 transplant programs in Canada, from March 2020-November 2021. Baseline characteristics, demographics, treatment and disease severity outcomes were collected. The primary outcome was need for supplemental oxygen. Factors associated with the primary outcome and changes in outcomes over time were analyzed. Pandemic time periods were divided into four time frames coinciding with 4 waves in North America. Result(s): We enrolled 509 SOT recipients with confirmed COVID-19 during the study period. The risk factors associated with oxygen requirement are outlined in Table 1. Severe disease and mortality were greatest in lung transplant recipients compared to other organ types (15/48 (31.3%) lung deaths vs 63/461(13.7%) nonlung organs, (p=0.001). There was no influence of 2-dose vaccination and 3 patients were infected after 3-dose vaccine. Disease with alpha or delta variant was not associated with increased oxygen requirement. In a subgroup analysis of participants requiring oxygen (n=190), remdesivir was associated with less death (p=0.035). Over the pandemic period (Figure 1), there were no significant changes in the proportion of patients requiring oxygen, ICU admission, ventilatory support or death. (Table Presented) Conclusion(s): COVID-19 is especially severe in lung transplant recipients and immunosuppression plays a significant role. The outcomes associated with COVID-19 in SOT have not appreciably changed over time despite the emergence of novel variants and changes in therapeutic regimens.

SARS-CoV-2 Infection is Associated with Greater Neutralizing Antibody Response Compared to Three Doses of Mrna-1273 Vaccine in Solid Organ Transplant Recipients

Purpose: Prior studies suggest that two doses of mRNA vaccine in SOTR may result in lower antibody and T-cell responses relative to levels seen following natural SARS-CoV-2 infection. In this study, we evaluated whether three doses of mRNA-1273 vaccine result in immune responses more comparable to, or greater than, natural infection. Method(s): Serum was collected 4-6 weeks from symptom onset in n=74 SOTR recovered from SARS-CoV-2 infection, and in n=60 SOTR receiving a third dose of mRNA-1273. Disease severity in the infection cohort ranged from mild to severe, but no deaths were reported. Vaccinated SOTR all had negative anti-nucleoprotein antibody results to confirm absence of infection. SARS-CoV-2 serology was assessed using an anti-spike (S) receptor binding domain (RBD) immunoassay (Roche). Neutralizing antibodies (nAb) were assessed using a commercial surrogate virus neutralization test (SVNT) targeting wildtype (WT), alpha, beta and delta strains (GenScript). A subset of participants underwent spike-specific T-cell testing (infection n=50, three doses n=34). PBMCs were stimulated overnight with overlapping peptides and frequencies of S-specific polyfunctional CD4+ and CD8+ T-cells (expressing IFN-gamma and IL-2) were measured by intracellular cytokine staining. Mann Whitney U, and Chi-square tests were used for statistical comparisons;significance was defined at p<0.05. Result(s): Anti-S RBD antibodies in SOTR recovered from infection were similar to levels in those receiving three doses of mRNA-1273 (median U/mL [IQR]: 73.5 [14.9-240.1] vs. 313.8 [313.8-2191.0];p=0.17). Relative to SOTR recovered from infection, the proportion of SOTR positive for nAb after three doses of vaccine was significantly lower. This was true for WT (93.2% vs. 60.0%, p<0.0001) and all variants tested - alpha: 90.5% vs. 56.7%, p<0.0001;beta: 67.6% vs. 50%, p=0.039;and delta: 85.1% vs. 55%, p=0.0001. Spike-specific polyfunctional CD4+ T-cell frequencies were similar between infection and three doses of vaccine (median cell frequency [IQR]: 241.7 [50-539.7] vs. 432.4 [50-1226];p>0.05). Spike-specific polyfunctional CD8+ T-cells were uncommonly detected following infection or vaccination. Vaccinated participants were significantly older than infected SOTR (p<0.001), and some differences in type of transplant were found between groups. However, sex and type of immunosuppressive medications were similar between infected and vaccinated SOTR cohorts (p>0.05). Conclusion(s): Three doses of mRNA vaccine may be required to optimize binding antibody, and to a lesser extent, CD4+ T-cell immunity, to levels similar to natural infection. However, nAb responses to wild-type virus and variants of concern were highest in SOTR recovered from infection when compared to vaccinated patients. These data provide further evidence of impaired SARS-CoV-2 vaccine responses in SOTR.

Longitudinal antibody response and viral loads in COVID-infected organ transplant recipients

Purpose: The full spectrum of COVID-19 disease and the impact of disease severity on antibody response and viral shedding dynamics in transplant patients is unclear. The aims of this study were to determine the outcomes COVID-19 in SOT recipients, and correlate disease severity with antibody response and viral dynamics following SARS-CoV-2 infection. Methods: We performed a single-centre, prospective, observational study of adult SOT patients infected with COVID-19 and followed patients for 4 weeks. Severe disease was defined as either hospitalization attributable to COVID-19 or death. SARS-CoV-2 serology using available sera was assessed by a commercial antinucleoprotein (NP) assay (Abbott). Viral loads on serial nasopharyngeal swabs were assessed using real time RT-qPCR (Norgen Biotek). Results: Between March and November 2020, 55 SOT recipients had PCRconfirmed SARS-CoV-2 infection. 78.2% were male with a median age 55 years (IQR 43-65), median time post-transplantation of 6 years (IQR 1.6-11.5). Transplant types were kidney (53.7%), liver (20.4%), lung (13.0%), kidney-pancreas (9.3%) and heart (3.7%). The majority of patients (65.5%) had >=2 comorbidities other than transplantation. Hospitalization occurred in 55.6% and 33.6% required supplemental oxygen. Other outcomes were ICU admission (16.7%), mechanical ventilation (13.0%), ECMO (1.9%), and all-cause mortality (5.6%). All deaths were lung transplant recipients. On univariate analysis, factors significantly associated with severe disease were >=2 comorbidities (p=0.034), and African-American race (p=0.015). Immunosuppression was reduced in 66.7% of cases, most commonly the antiproliferative agent. A subgroup of patients (n=26) underwent SARS-CoV-2 antibody testing and 23/26 (88%) had antibodies by day 14 post-symptom onset. The three negative patients had mild disease. A subgroup of patients (n=23) had serial nasopharyngeal swabs for viral load. The median duration of positivity was 15 days (IQR 10-24) (Fig 1,2). The median peak VL measured was 4,669 copies/ mL (IQR 274 to 103,038 copies/mL). Peak viral load and duration of shedding were not significantly different between hospitalized and non-hospitalized groups (p=0.59 and p=0.52 respectively). Conclusions: SOT patients experience a spectrum of COVID-19 although mortality was low in our cohort likely due to greater capture of mild cases in the outpatient population. Virus is shed for long durations despite most transplant recipients generating SARS-CoV-2 directed antibody responses.

A mechanistic evaluation to guide the optimal immunosuppression adjustment strategy in transplant patients with COVID-19

Purpose: The optimal management of immunosuppression in transplant patients infected with COVID-19 is unknown. Reduction in calcineurin inhibitors or antimetabolite doses is often performed in order to enhance immune responses, but there are minimal data to guide decisions. We performed an in vitro study to determine the effect of individual immunosuppressive agents and doses on SARSCoV- 2 specific T-cell cytokine expression. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from nine non-immunosuppressed patients diagnosed with COVID-19 at least 14 days prior. Cells were pre-incubated for four hours with clinically relevant, differing concentrations of immunosuppressive drugs (tacrolimus (TAC), mycophenolate (MPA), sirolimus, prednisone). PBMCs were then stimulated with SARS-CoV-2 spike and nucleoprotein peptide pools for 24 hours with appropriate positive and negative controls. Supernatants collected were analyzed by a 14-plex high sensitivity T-cell cytokine array (Eve Technologies). Results: PBMCs stimulation with SARS-CoV-2 peptides resulted in broad cytokine responses. In the presence of TAC at medium (6ng/ml) and high concentrations (24ng/ml) (both are commonly achieved trough/peak clinical concentrations respectively), there were significantly reduced levels of IL-2 (p=0.0078 for both), and at high concentrations, lower amounts of IFN-γ (p= 0.0391) in response to peptide stimulation. TAC (24ng/ml) also resulted in a skewing of the response from a TH1 to a TH2 phenotype as indicated by lower IFN-γ: IL-13 ratio (p=0.0273), and IFN-γ: IL-4 ratio (p=0.0234). In contrast, differing clinically relevant concentrations of MPA and prednisone did not appear to influence the cytokine response postpeptide stimulation. Interestingly, Sirolimus at medium (4ng/ml) and high (16ng/ ml) concentrations was found to be significantly associated with pro-inflammatory cytokine release in response to SARS-CoV-2 peptides;TNF-α (p=0.0078 for both), IL-6 (p=0.0234 for both) and IL-1β (p=0.0156 and p=0.0078, respectively). When expressed as a pro- vs. anti-inflammatory ratio (with IL-10 as the anti-inflammatory cytokine), this finding remained consistent. Conclusions: These in-vitro results could help guide clinical decisions. Greater concentrations of TAC led to an inhibition of Th1 responses, whereas sirolimus led to an unexpected proinflammatory response, and MPA was neutral. This suggests that for transplant patients with COVID-19, a reduction of TAC may be of greater benefit than MPA. For those on sirolimus, reduction should be considered if evidence of an inflammatory phase.

T-cell responses in solid organ transplant recipients with SARSCOV- 2 infection

Purpose: Preliminary data suggest solid organ transplant (SOT) recipients may be at high-risk for developing severe COVID-19. This may be due in part to alterations in T-cell physiology owing to use of immunosuppressive agents to prevent rejection. In this study we evaluated convalescent T-cell responses against SARS-CoV-2 in SOT recipients who had COVID-19. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of 20 SOT recipients and 15 non-transplant controls (NTCs), all of whom had COVID >=14 days prior (convalescent samples). A total of 106 PBMCs were stimulated for 16 hours with megapools of overlapping 15mer peptides corresponding to the spike (S), nucleoprotein (NP) or membrane (Mb) protein of SARS-CoV-2 (each peptide at 5 μg/mL). After incubation, flow cytometry was performed for intracellular cytokines (IFN-γ, TNF-α, IL-2) and cell-surface T-cell exhaustion markers (CTLA4, PD-1, TIM-3). Total and SARS-CoV-2 antigen-specific CD4+ and CD8+ T-cells were identified. Polyfunctional T-cells were defined as those expressing ≥ 2 of the cytokines investigated. Results: The median age among SOT recipients was 54 years (range 24-86). The majority (15/20) were male, and kidney (12/20) transplant recipients. The majority (60.0%) of SOT recipients were hospitalized with COVID-19;three (15.0%) required ICU admission and mechanical ventilation. SOT recipients had significantly lower total CD4+ T-cells (51.6% vs. 62.2%, p=0.002) but significantly higher proportions of total CD8+ T-cells relative to NTCs (41.9% vs. 31.2% of live CD3+ cells, p=0.0016). SOT recipients also had significantly higher proportions of PD-1 on total CD4 (15.2% vs. 3.7%, p<0.0001) and CD8 T-cells (9.4% vs. 4.1% of live CD3+ cells, p=0.014). The majority of SOT recipients and NTCs generated S, NP and Mb specific CD4 and CD8 T-cells. More specifically, compared to NTCs, SOT recipients had increased proportions of IFN-γ, or IL-2 producing CD4+ T-cells, as well as polyfunctional CD4+ T-cells reactive to S peptides. SOT recipients also had increased proportions of IFN-γ, IL-2 or TNF-α producing CD4+ T-cells, and CD4+ polyfunctional T-cells reactive to NP peptides. NTCs were also characterized by lower proportions of IL-2 producing CD8+ T-cells reactive to S peptides. Hospitalization of SOT recipients (severe illness) was associated with higher proportions of total PD-1+ CD4 T-cells (22.2% vs 13.3% of CD4 T-cells, p=0.02) and low frequencies of CD8+ polyfunctional T-cells reactive to NP peptides (5.80 vs. 49.9 per 106 polyfunctional CD8 T-cells, p=0.014). Conclusions: Despite immune suppression, SOT recipients mount SARS-CoV-2 reactive T-cells at magnitudes often exceeding non-transplant controls. However, perturbations in global T-cell proportions, and increased expression of T-cell exhaustion markers, such as PD-1, may compromise the SARS-CoV-2-specific immune response.

Prospective observational study and serosurvey of SARS-CoV-2 infection in asymptomatic healthcare workers at a Canadian tertiary care center

Health care workers (HCWs) are at higher risk for SARS-CoV-2 infection and may play a role in transmitting the infection to vulnerable patients and members of the community. This is particularly worrisome in the context of asymptomatic infection. We performed a cross-sectional study looking at asymptomatic SARS-CoV-2 infection in HCWs. We screened asymptomatic HCWs for SARS-CoV-2 via PCR. Complementary viral genome sequencing was performed on positive swab specimens. A seroprevalence analysis was also performed using multiple assays. Asymptomatic health care worker cohorts had a combined swab positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) relative to a comparative cohort of symptomatic HCWs, where 54/1597 (3.4%) tested positive for SARS-CoV-2 (ratio of symptomatic to asymptomatic 6.8:1). SARS-CoV-2 seroprevalence among 996 asymptomatic HCWs with no prior known exposure to SARS-CoV-2 was 1.4-3.4%, depending on assay. A novel in-house Coronavirus protein microarray showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Our study demonstrates the utility of routine screening of asymptomatic HCWs, which may help to identify a significant proportion of infections.