Longitudinal immune profiling of a SARS-CoV-2 reinfection in a solid organ transplant recipient

The underlying immunologic deficiencies enabling SARS-CoV-2 reinfections are currently unknown. Here we describe a renal-transplant recipient who developed recurrent, symptomatic SARS-CoV-2 infection 7 months after primary infection. To elucidate the immunological mechanisms responsible for reinfection, we performed longitudinal profiling of cellular and humoral responses during both primary and recurrent SARS-CoV-2 infection. We found that the patient responded to the primary infection with transient, poor-quality adaptive immune responses that was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we identified the development of neutralizing antibodies and humoral memory responses prior to SARS-CoV-2 reinfection. However, these neutralizing antibodies failed to confer protection against reinfection, suggesting that additional factors are required for efficient prevention of SARS-CoV-2 reinfection. Further, we found no evidence supporting viral evasion of primary adaptive immune responses, suggesting that susceptibility to reinfection may be determined by host factors rather than pathogen adaptation.


Introduction
The dynamics and duration of adaptive immune responses to SARS-CoV-2 infection have been described in association with disease severity and the rate of viral clearance, yet the correlates of adaptive immunity responsible for preventing reinfection remain incompletely characterized. In studies of SARS-CoV-2 infection in animal models (mice 1,2 , hamsters 3,4 , and rhesus macaques [5][6][7][8] ), both vaccine-induced and natural infection-induced immunity are su cient for protection from SARS-CoV-2 rechallenge. Recent Phase III vaccine clinical trials 9 , as well as epidemiologic studies of natural infection 10 , have also demonstrated robust development of protective immunity in humans. Taken together, these data unambiguously demonstrate that adaptive immunity confers protection against SARS-CoV-2 infection in the majority of cases. However, rare case reports of SARS-CoV-2 reinfection by antigenically similar variants have also been documented as soon as 48 days from primary symptom onset [11][12][13][14][15][16][17][18] (Extended   Data Table 1). Whether these reinfections are the direct result of de cient adaptive immune responses to the primary infection, or are the result of waning adaptive immunity, is currently unknown.
Notably, cases of persistent SARS-CoV-2 infection among patients with underlying genetic defects (such as X-linked agammaglobulinemia 19 ) or acquired defects (B cell depletion therapy 20 ) in humoral immunity have been reported. Immunocompromised COVID-19 patients with prolonged infection also achieved viral clearance when treated with multiple doses of convalescent plasma 21 , demonstrating the su ciency of humoral responses in clearing SARS-CoV-2 infection. The role of cellular immunity in protection from SARS-CoV-2 infections is also a subject of intense investigation. Studies demonstrate that most COVID-19 patients develop SARS-CoV-2 speci c CD4 + and CD8 + T cells 22 and reports of durable T-cell memory responses to related SARS-CoV-1 infection lasting up to 17 years after initial infection 23 . While neutralizing antibodies are a correlate of protection, non-human primate models have demonstrated reduced virological control in the upper respiratory tract in CD8 + depleted convalescent animals upon reinfection 24 , suggesting that both arms of the adaptive immune response may be required for optimal clearance and protection against SARS-CoV-2 infection.
Due to the rarity and complexity involved in investigation of human SARS-CoV-2 reinfections, complete immune pro les exploring the magnitude and extent of these adaptive immune responses in paired primary infection and reinfection are lacking. Identifying the de cient features of initial adaptive immune responses that enables subsequent SARS-CoV-2 reinfection will help to further de ne the correlates of immune protection in humans.

Results
Clinical presentation of immunocompromised solid organ transplant recipient with SARS-CoV-2 reinfection.
In March 2020, a 66-year-old man residing in a transitional group living facility with a medical history notable for bipolar disorder and end-stage renal disease due to lithium toxicity, for which he had undergone living-donor renal transplantation two years prior, was hospitalized with fevers, fatigue, and dry cough (Fig. 1). Induction immunosuppression for renal transplantation had consisted of antithymocyte globulin, while maintenance immunosuppression initially included tacrolimus (a calcineurin inhibitor that inhibits T-cell cytokine production), mycophenolate mofetil (MMF, a B and T lymphocyte anti-proliferative agent), and low-dose prednisone. By the time of hospitalization, belatacept (a T lymphocyte costimulation blocker) had been substituted for tacrolimus due to the development of calcineurin-induced neurotoxicity, and prednisone had been discontinued due to perceived exacerbation of psychiatric illness. Persistent neutropenia complicated the post-transplantation course, requiring substitution of prophylactic inhaled pentamidine for trimethoprim-sulfamethoxazole and frequent infusions of lgrastim. Upon hospitalization, SARS-CoV-2 infection was diagnosed via reversetranscriptase polymerase chain reaction (RT-PCR) performed on a nasopharyngeal swab (NP) specimen.
He was subsequently enrolled in the Yale Implementing Medical and Public Health Action Against Coronavirus CT (IMPACT) study, a biospecimen repository housing clinical and demographic data as well as respiratory, blood, and other tissue samples from patients with con rmed COVID-19 at Yale New Haven Hospital. He developed symptomatic moderate COVID-19 for which he received hydroxychloroquine and atazanavir for 5 days and a single dose of tocilizumab at 8 milligrams/kilogram (mg/kg). MMF was paused and a reduced dose of belatacept was administered in the setting of acute infection. The oxygen requirement peaked at 4 liters per minute by nasal cannula; by 13 days from symptom onset (DFSO), the patient was transitioned to room air. Though the patient was asymptomatic thereafter, nasopharyngeal (NP) swabs and saliva (SL) from the patient remained positive for SARS-CoV-2 by PCR throughout the hospital stay (Extended Data Table S2). The patient was discharged from the hospital on 27 DFSO to the transitional group residential facility after a 14-day period without hypoxia, reemergence of symptoms, or other clinical signs of infection. MMF was restarted on discharge.
Approximately 10 weeks after discharge, a kidney allograft biopsy was performed because of increasing serum creatinine and was notable for evidence of acute T-cell-mediated rejection (TMCR) and antibodymediated rejection (AMR) of the transplanted organ (Fig. 1). He was readmitted and treated with 400 mg of antithymocyte globulin and 125 mg of methylprednisolone. Belatacept was continued, low-dose prednisone was restarted, and the MMF dose was increased. Notably, a nasopharyngeal swab collected at the time was negative for SARS-CoV-2 using a non-quantitative transcription-mediated ampli cation (TMA) test. The patient remained asymptomatic and was discharged back to the transitional living facility. He received rituximab 1 week after discharge to address AMR.
Approximately 15 weeks after this hospitalization, the patient underwent repeat renal allograft biopsy for evaluation of polyomavirus-associated nephropathy that demonstrated evidence of mild remnant AMR ( Fig. 1). Ongoing neutropenia necessitated additional infusions of lgrastim. At 220 DFSO, a NP swab collected from the patient was again negative for SARS-CoV-2 using TMA.
Approximately 4 months after the diagnosis of rejection and 7 months from his primary COVID-19 diagnosis, the patient was readmitted to the hospital with fatigue and nonproductive cough (Fig. 1). Repeat SARS-CoV-2 PCR of NP samples returned positive at 236 DFSO / 5 days from reinfection symptom onset (DFSO*) with cycle thresholds to targets N1 and N2 of 27.34 and 27.15, respectively. The patient did not develop fevers or hypoxia, had no evidence of pneumonia on chest imaging, and did not require COVID-19-speci c therapy. SARS-CoV-2 IgG was reactive at 5 DFSO*. Isolation precautions were reinstituted for the 10-day duration of hospitalization and were maintained after his return to the group living facility.
Genome sequencing reveals two distinct lineages of SARS-CoV-2 during primary infection and reinfection Following symptom onset during the primary infection in March 2020, both nasopharyngeal and saliva specimens tested positive by PCR, and nasopharyngeal specimens were whole genome sequenced for phylogenetic analysis 25 . Additional nasopharyngeal and saliva specimens were collected and sequenced during the reinfection episode in November 2020 (Extended Data Table 2). To rule out the possibility of persistent SARS-CoV-2 infection, which has been previously reported 26-29 , we compared the virus genomes sequenced from specimens collected 7 DFSO in the primary infection (NP swab), and 5 DFSO* during the reinfection (NP swab and saliva). Phylogenetic analysis revealed that viruses from the primary  S2). Importantly this mutation is not located within the SARS-CoV-2 spike receptor binding domain, which is the primary target of neutralizing antibodies (Extended Data Fig. S2), nor has it been reported among SARS-CoV-2 variants of concern (VOC) B.1.1.7, B.1.351, or P.1 that display variable evasion of humoral immune responses 31 .
Our phylogenetic analysis also demonstrates that the distinct viral lineages identi ed from the patient's primary infection and subsequent reinfection diverged from their common ancestor around March 2020 (Fig. S1), suggesting intra-host evolution in the setting of persistent infection to be an unlikely explanation for this case and providing unambiguous evidence of reinfection. To rule out the remote possibility of the presence of multiple SARS-CoV-2 lineages during reinfection, we also sequenced virus genomes from both saliva and nasopharyngeal swabs collected during the reinfection (Fig. 1c) and found them to be identical. Lastly, we analyzed the geographic distributions of circulating SARS-CoV-2 lineages and discovered that the sub-lineage of viruses identi ed in the reinfection likely rst circulated in the Southern US in June of 2020 before being reintroduced to the Northeast US. This patient's primary residence is located within the Northeast, and he reported no travel since discharge from the primary SARS-CoV-2 infection in March of 2020 ( Fig. S1), con rming that his SARS-CoV-2 reinfection was likely the result of a broad geographic reintroduction and unlikely to represent an instance of persistent SARS-CoV-2 infection.
Given our ndings of the distinct genetic lineages of each SARS-CoV-2 isolate, the lack of multiple strains of SARS-CoV-2 during reinfection, and the congruent geographic patterns of the patient's clinical narrative, we established that our case represents a genetically con rmed SARS-CoV-2 reinfection and next sought to identify the speci c immune correlates that conferred this susceptibility.
Immunologic pro ling reveals naive lymphocyte depletion and poor humoral immunity During the patient's primary SARS-CoV-2 infection, we performed longitudinal whole blood sampling which was separated into peripheral blood mononuclear cells (PBMC) and serum fractions at 7, 15, and 23 DFSO. PBMCs were analyzed by multidimensional ow cytometry and serum was analyzed with multiplex ELISA to measure 71 cytokines ( In comparison to disease severity and DFSO matched patients from our larger IMPACT cohort, we found that the patient differed signi cantly in both immune cell subtype composition as well as cytokine expression during his primary infection. Notably, during the primary SARS-CoV-2 infection, the patient maintained very high levels of circulating T-cells and did not suffer from a T-cell lymphopenia as is characteristic of symptomatic COVID-19 patients 32 (Fig. 3a). Not only was general lymphopenia absent, there also was no speci c loss of CD8 + T cells, as can be seen in more severe cases of COVID-19 33,34 (Fig. 3b). Importantly the patient also demonstrated a relatively higher, rather than characteristically depressed, CD8 + /CD4 + ratio primarily as a result of his diminished CD4 + populations. (Fig. 3b-c,e; Extended Data Fig S3b). With regards to functionality, the patient's CD8 + and CD4 + T-cells exhibited broad increases activation markers (CD38 + , HLA-DR + ), exhaustion/terminal differentiation markers (PD1 + , TIM-3 + ), and effector T regulatory cell markers (PD1 + , TIM-3 + , CD25 + , CD127 − , HLA-DR + ) (Fig. 3b-d). In comparison to the larger IMPACT cohort, this patient's immunological pro le was uncharacteristic of either moderate or severe SARS-CoV-2 infection, and instead resembled an immunophenotype consistent with chronic antigen exposure. Importantly, we found that the patient also had very low numbers of circulating naïve CD4 + and CD8 + T cells at the time of primary SARS-CoV-2 infection, which are required for the generation of potent de novo antiviral response.
To assess whether alterations in immune cell composition contributed to reinfection, we again performed multidimensional ow cytometry on PBMCs isolated from the patient at 5 DFSO* (Fig. 3a-c, orange). In comparison to results from the primary infection (Fig. 3a-c, green), we found a general loss of circulating lymphocytes, while myeloid cell subsets remained at similar levels as seen during his primary infection (Extended Data Fig. 3S). We suspect that this broad depletion of lymphocytes was due to intervening treatment with antithymocyte globulin and rituximab during an episode of graft rejection 3 months prior to his reinfection (Fig. 1). It is also possible that the SARS-CoV-2 reinfection exacerbated this global depletion, although decreases in circulating B cell populations have not been widely reported in COVID-19 patients. Among the patient's remaining T cell populations, and in the context of recent anti-rejection treatment, the patient again presented with largely depleted pools of naïve CD4 + and CD8 + T-cells and with continued activation and exhaustion among effector CD4 + and CD8 + T cell populations. In contrast to the primary infection, we found an almost complete depletion of CD19 + B cells, likely as a result of intervening rituximab treatment (Fig. 3a). Consistent with our ndings during the primary infection, the patient again presented with an immunophenotype suggestive of chronic antigen engagement, but with globally reduced lymphocytes likely by the treatment for TMCR and AMR.
To investigate the full extent of immunological dysfunction present in the patient during the primary SARS-CoV-2 infection, we next explored whether altered cytokine signaling could have contributed to the patient's poor initial adaptive immune response. Accordingly, we performed multiplex cytokine analysis from the patient's serum and found that the patient had globally elevated cytokines (Extended Data Fig.  S4) including IL-10, IFN , IFNλ, IL-1 , TNF , TRAIL, and IL-27 at all sampled points during primary infection. Other markers of T-cell functionality, including secreted cytokine IFNγ and T cell activating cytokines IL-18 and IL-12, remained elevated through the patient's course of infection even after improvement in COVID-19 symptoms (Fig. 4a). In contrast to this patient, a disease-severity matched COVID-19 cohort showed either no elevation, or conversely, a reduction in levels of these cytokines over their course of infection. Additionally, the patient's IL-15 and IL-7 levels, required for maintenance of naïve T-cell pools, were also persistently elevated (Fig. 4b). These data suggest that persistent utilization of Tcell populations -likely a result of continual immunological response to the patient's allograft -rather than poor production of cytokines may be responsible for low numbers of naïve T cells at the time of primary SARS-CoV-2 infection.
Given the patient's loss of B cells prior to reinfection following the administration of rituximab (Fig. 1,   Fig. 3), we initially hypothesized that SARS-CoV-2 reinfection may have also been the result of loss of humoral immunity. Accordingly, we rst assessed anti-SARS-CoV-2 IgG and IgM levels by ELISA during primary infection and found that the patient produced typical levels of SARS-CoV-2 speci c antibodies (S1 and RBD) compared with other hospitalized COVID-19 patients (Fig. 5a). Increasing S1 IgG and IgM levels positively correlated with rising RT-qPCR CT values speci c for SARS-CoV-2 genomes (i.e. decreasing viral load), suggesting their role in resolution of the primary SARS-CoV-2 infection. During the patient's reinfection, and in the setting of few circulating B-cells, we found an accelerated S1 IgG response that was again positively correlated with RT-qPCR CT values, suggestive of a memory response upon pathogen rechallenge (Fig. 5b-c). Moreover, there was a complete absence of S1 speci c IgM during reinfection, consistent with a memory response to SARS-CoV-2 infection (Fig. 5b). These results suggest that antiviral antibodies were not lost during rituximab treatment as initially hypothesized, and furthermore that the source of S1-speci c IgG during the reinfection was likely due to long-lived plasma cells (which are not depleted by rituximab 35 ) generated during initial SARS-CoV-2 infection rather than a de novo response to the reinfection.
To assess the neutralizing capacity of anti-SARS-CoV-2 antibodies present during both the primary and recurrent SARS-CoV-2 infections, we performed longitudinal PRNT 50 assays and calculated the corresponding serum dilution IC 50 values for each time point (Fig. 5d-e). While the patient developed neutralizing antibodies by 15 DFSO, they were transient in nature and signi cantly declined in potency by 23 DFSO. This atypical neutralizing antibody response is not consistent with other large-scale studies that show persistence of neutralization capacity following SARS-CoV-2 infection (t 1/2 =90 days; 95% CI: 70-125 days). Furthermore, the neutralization capacity was notably reduced even in comparison to other hospitalized COVID-19 patients of matched disease severity (Extended Data Fig. S5a-b). Longitudinal analysis of serum samples was not performed during the intervening period between primary infection and reinfection; however, early hospital clinical laboratory serologic assays showed persistence of anti-SARS-CoV-2 IgG at 5 DFSO*. We were able to assess neutralizing antibodies during the reinfection, and found that neutralizing antibodies were present at 8 DFSO* and increased slightly by 12 DFSO*. Similar to the primary infection they were of poor neutralizing capacity relative to other COVID-19 patients ( Fig   S5a; Extended Data Fig. S6). Given that the patient was depleted of naive circulating B cells, had no IgM response, and had detectable circulating antibodies as early as 5 DFSO*, we hypothesized that these neutralizing antibodies observed during the reinfection re ected antibodies generated from the primary infection, rather than a new humoral response to the reinfection. To examine whether these neutralizing antibodies targeted the same regions with in the SARS-CoV-2 spike protein, we performed linear epitope mapping of this patient's antibody binding using Serum Epitope Repertoire Analysis (SERA) -a random bacterial display peptide library -coupled with a recently described bioinformatic method that enriches for antigen-speci c antibody binding signals relative to healthy (uninfected) controls (Protein-based Immunome Wide Association Study, "PIWAS") 36 . Using this approach, we found two characteristic PIWAS peaks -signifying locations of peak patient antibody binding -at identical locations in both the primary infection and reinfection (Fig. 5f, black arrows). These peaks of antibody binding were centered on amino acid 141 in the N-terminal domain of S1 and on amino acid 1112 in the S2 domain of Spike. The high degree of concordance in peak locations between primary infection and reinfection suggests the same antibody-secreting population responded to both infections. Importantly, this peak is distinct from the Spike amino acid mutation at 1078 that was found only in the reinfection isolate (Extended Data Fig. S2), suggesting that viral evasion of the antibody response generated during the primary infection was unlikely to be responsible for reinfection.
In summary, we found that the patient developed an antigen-speci c, neutralizing antibody response during his primary SARS-CoV-2 infection; that this neutralizing antibody response likely developed into a long-lived plasma cell population; and that it was insu cient to provide protection against reinfections with a novel lineage of SARS-CoV-2 that bore no evidence of viral immune evasion.

Discussion
We have described a case of symptomatic SARS-CoV-2 reinfection in a solid organ transplant recipient and pro led the unique immunological dysfunctions present during both initial SARS-CoV-2 infection and reinfection. Through extensive clinical investigation and phylogenetic analysis of virus sequences, we con rmed that the patient was reinfected with a genetically distinct lineage of SARS-CoV-2, which was neither the result of persistent infection nor the result of infection by an antigenically distinct SARS-CoV-2 variant. Accordingly, we investigated the potential mechanistic causes of this patient's multiple SARS-CoV-2 infections by performing longitudinal immunologic pro ling during both initial SARS-CoV-2 infection and reinfection.
Multiple recent longitudinal studies have shown that the majority of COVID-19 patients, even those with mild or asymptomatic infection, develop long-lasting SARS-CoV-2 speci c cellular and humoral adaptive immunity for as long as 8 months 37 . In contrast, a series of case reports and this manuscript report that SARS-CoV-2 reinfections can occur between 48 and 236 days from initial infection (Extended Data Table 1). The discrepancy between frequent, durable protective immune responses generated during most SARS-CoV-2 infections and rare cases of reported reinfection by antigenically similar variants is currently unexplained. While the protective capacity of humoral responses against SARS-CoV-2 infection is apparent, large variability in magnitude of responses between patients has been shown in multiple longitudinal studies. The underlying immunologic correlates of this variability, and ultimately what is required to develop strong humoral responses, have not been fully elucidated.
In this case study, we found that a failure of humoral immunity may have led to this patient's SARS-CoV-2 reinfection. We investigated both the dynamics of antibody production as well as the general quality of antibodies produced by the patient. Our initial analysis of humoral responses indicated that the patient mounted a typical IgM and IgG response to SARS-CoV-2 primary infection, as assessed by longitudinal ELISA measurements. While total anti-SARS-CoV-2 antibody production was not particularly hampered in this patient, the neutralizing antibody response was clearly defective.
To address the underlying cellular defects that may have led to a poor neutralizing antibody response, we performed ow cytometry analysis of PBMC populations during primary infection and reinfection. We found signi cant differences in the patient's T cell composition and phenotype relative to other patients with COVID-19. While T cell lymphopenia is common in even mild cases of COVID-19 and a characteristic response to many other viral infections, this patient did not develop T cell lymphopenia, and but instead presented with a profound and relatively speci c reduction in naive T cell pools, most signi cantly in their CD4 compartment. Reduced naive T cell pools are a characteristic feature of aging and may contribute to the impaired immune responses observed in elderly individuals 38 . Depletions in naive T cell populations, and the corresponding de cits in adaptive immune responses, have also been reported in in ammatory states like chronic hepatitis C infection 39 and chronic granulomatous disease 40 ; however, this phenomenon has been less well documented in solid organ transplantation. Additionally, it has been consistently observed that solid organ transplant recipients develop poor adaptive response to new antigens either during immunization or new infections -including SARS-CoV-2 mRNA vaccination [41][42][43] . It is unlikely that the patient's immunosuppression prior to primary infection led to naive T cell speci c lymphopenia as MMF, a purine biosynthesis inhibitor, would be expected to inhibit both T and B cell proliferation non-speci cally, and this patient, having received Belatacept, a CTLA-4 F c fusion protein and co-stimulatory inhibitor, would also be expected to inhibit T cell activation and differentiation, which would more likely lead to increased, rather than decreased, naive T cell pools. Additionally, cytokine pro ling revealed high levels of IL-7 and IL-15, both of which promote naive T cell pool expansion 44,45 . While it is clear that the patient had su cient cytokines to replenish naive T cell pools, naive T cell populations were not replenished, possibly due to either over-utilization (via repeated antigen engagement), insu cient thymic reserve, or some combination of both. Interestingly, immunophenotyping also revealed high levels of activation, terminal differentiation, and exhaustion in both CD4 + and CD8 + pools, possibly as a result of chronic antigen exposure from the transplanted organ 46 . We suspect that the lack of naive T cell pools may have contributed to a de cient humoral immune response during initial SARS-CoV-2 infection. Whether similar impaired cellular dynamics may lead to impaired humoral immunity to SARS-CoV-2 in other long-term organ graft recipients, or other populations with aspects of repeated antigen exposure such as chronic infection and cancer, warrants further investigation.
While the patient's overall anti-SARS-CoV-2 response was not speci cally impaired by low naïve CD4 T cell pools, we suspect that the patient's poor neutralization response may have been and that the insu cient T cell support resulted in either extrafollicular or dysfunctional germinal center B cell responses 34,47−49 . In line with these ndings, the patient demonstrated a transient, relatively poor-quality neutralizing antibody response during initial infection consistent with a short-lived extrafollicular response or de cient germinal center dynamics.
SARS-CoV-2 speci c IgG antibodies were detected as early as 5 DFSO* during reinfection, suggesting a memory response given the accelerated humoral kinetics relative to rst infection. Additionally, the absence of circulating B cell and antiviral IgM during the reinfection indicates that it was likely only SARS-CoV-2 speci c plasma cells established after the primary infection that provided neutralizing antibodies during reinfection. To better characterize these neutralizing antibodies, we performed linear epitope pro ling of SARS-CoV-2 speci c antibodies from the primary infection and reinfection against the spike protein. This revealed binding peaks at identical amino acid locations, suggesting that a humoral memory response was indeed generated during the patient's primary infection and was also present during reinfection. Importantly, the spike protein amino acid change we identi ed in the virus strain causing the SARS-CoV-2 reinfection did not correspond to the location of antibody binding generated during the primary infection, suggesting viral evasion of primary humoral responses to be an unlikely explanation for reinfection in this case (Fig. 5f, Extended Data Fig. S2). These two binding sites within the spike protein corresponded to amino acid 141 and 1112, which did not correspond to areas of high antigenicity or neutralization with in our larger IMPACT cohort (Extended Data Fig. S6), We hypothesize that the patient's underlying immune de ciencies (low naïve CD4 pools) led to poor neutralizing antibody quality (IC 50 titers approximately 1:10 to 1:30), which were insu cient to protect against SARS-CoV-2 reinfection in vivo.
By means of a case study of a solid organ transplant recipient with COVID-19, we demonstrate that the mere presence of neutralizing antibodies during primary infection was insu cient to confer protection against reinfection. Further investigation into additional immunological correlates of protection, including the roles of cellular immunity and tissue-resident immune cell populations, are warranted.

Limitations Of The Study
As with all case studies, a limitation on the generalizability of our ndings to wider patient populations is present. Also, while the lack of immunological responsiveness to vaccination or acute infection in immunosuppressed and solid organ transplant populations is well documented, there may be additional mechanisms contributing to these defects beyond those discussed in this manuscript -particularly with regards to SARS-CoV-2 infection. Our analysis of the immunophenotype of the patient was limited to surveys of circulating immune dynamics; however, numerous studies have also described perturbations in immunity at tissue sites not easily amenable to direct interrogation. We also did not directly analyze antigen speci c T-cell responses during either infection, which may reveal additional dysfunction not discussed within this manuscript. Lastly, we also did not fully address every potential avenue of viral immune evasion to immune responses and accordingly suggest that a greater understanding of the virusintrinsic and host-intrinsic features determining susceptibility to SARS-CoV-2 reinfections is required.
Future studies should investigate not only the circulating and systemic adaptive immune responses during SARS-CoV-2 reinfections, but also the possibility that local defects in immune responsiveness among barrier tissue sites may also enable recurrent SARS-CoV-2 infection.  data is shown in red (IgG) and blue (IgM) (right y-axis, red). Solid lines correspond to S1, dashed lines