*3.3. Immunological Response after COVID-19 in LT Recipients*

Regarding the immunological response after SARS-CoV-2 infection in LT patients, only four studies were considered, including a total of 91 LT recipients. However, all of these studies examined different types of tests/assays directed toward different targets, and data could not always be extrapolated to LT recipients alone, as shown in Table 3, making a pooled analysis not feasible.


**Table 3.** Summary of the included studies concerning immunological response of LT recipients after COVID-19.

Abbreviations: SOT: solid organ transplantation; LT: liver transplant.

Caballero-Marcos et al. [48] showed a decline over time of IgG-antinucleocapsid, with lower incidence at 3 months (77.4% vs. 100%, *p* < 0.001) and at 6 months (63.4% vs. 90.1%, *p* < 0.001) when compared with a matched cohort of immunocompetent subjects. A more comprehensive analysis performed in 28 SOT patients (of which five were LT patients) of the immunological response, which also considered T-cell responses, showed that the overall response was not impaired in the SOT patients. However, when the humoral response was considered alone, there was some delay in mounting a response compared to the immunocompetent control group [49].

#### *3.4. COVID-19 Vaccine Immunogenicity in LT Recipients*

Regarding response to COVID-19 vaccines, we considered five studies. Overall, the studies included 269 LT recipients (Table 4). However, analogously to those regarding the immunological response after COVID-19, the included studies considered different types of tests/assays, and data could not always be extrapolated to LT recipients alone, making comparisons difficult. Two studies evaluated side effects after receiving one dose of mRNA-1273 or BNT162b2 vaccine or two doses of BNT162b2 vaccine. Both studies showed local and systemic side effects in proportions comparable to those in pivotal studies for RNA vaccines.


**Table 4.** Summary of the included study concerning vaccination against SARS-CoV-2 response in LT

Concerning immunogenicity, four studies, although using different assays, evaluated the humoral response recording seroconversion rates between 29 and 50% [52,53]. Two studies emphasized that the use of antimetabolites as immunosuppression was a risk factor for reduced serum conversion rates [53,54]. Only one study also analyzed T-cell response, which was described as reduced [55], and only one of the included studies considered only LT recipients [53].

#### **4. Discussion**

At the beginning of the COVID-19 pandemic, SOT recipients, including LT recipients, were considered a vulnerable population, raising the question as to whether they would be at particular risk for severe disease and graft injury given their immunocompromised state and high prevalence of metabolic comorbidities. The aim of our study was to systematically pool all the available literature on this topic. We found that middle-aged men with metabolic comorbidities were the main target for the infection. Even though respiratory problems represented the main clinical feature in LT patients, a high percentage of gastrointestinal symptoms were also reported. Approximately 70% of LT patients with SARS-CoV-2 infection were hospitalized. Modification/reduction of IS was common, particularly for MMF, although complete withdrawal of all IS was rarely observed. With respect to outcome, a case fatality rate ranging between 12 and 22% was described in the major reports accessible for this analysis. Interestingly, when compared to the control population, outcomes were not worse in the LT recipient group.

There seems to be a difference in the immune response to the SARS-CoV-2 infection and the immune response as acquired by vaccination. In the first case, the data conflict, but if we consider the response mediating the neutralizing activity (anti-spike protein IgG and T-cell mediated), it seems that there is a similar response, albeit probably slightly delayed, in LT recipients compared to immunocompetent subjects. On the other hand, vaccine-induced immunogenicity seems to be defective.

Although a considerable number of patients were included in the present study, the quality of the manuscripts analyzed makes it difficult to consolidate associations and predictive factors regarding COVID-19 and the LT population.

The epidemiological distribution of the disease is superimposable to that of the general population [2,57], with COVID-19 being mainly prevalent in middle-aged males.

Recently, new findings have highlighted how obesity [58], diabetes type II [59], and arterial hypertension [60] are associated with a more severe course of COVID-19 and hence a poorer outcome. Despite the high prevalence of these metabolic conditions in LT recipients [61,62] and more specifically in the present cohort, this did not seem to negatively affect the prognosis of the current study population.

Interestingly, the presence of gastrointestinal complaints (28%) was considerably higher among LT recipients. A recent review on gastrointestinal symptoms in COVID-19 [63] showed a high heterogeneity in incidence (ranging between 3 and 79%), with other large cohorts of patients reporting rates of gastrointestinal symptoms between 5 and 15% [60,64]. It is widely accepted that SARS-CoV-2 enters host mucosal cells via the cell receptor angiotensin-converting enzyme-2 (ACE-2) and the transmembrane serine protease 2 (TMPRSS2), which are also highly expressed in the absorptive enterocytes from the ileum and colon [65]. Once the virus enters the enterocytes, it can start replication and its cytopathic effect [66]. The gut microbiome can be significantly altered by SARS-CoV-2 through several mechanisms (e.g., proinflammatory cytokines, perturbation in the gut–lung axis, medications, changing ratio of pathogenic organisms) leading to clinical manifestations such as diarrhea and vomiting [67]. LT patients are also known to have an extremely vulnerable gut microbiomes [68,69], and immunosuppressive fluctuation in trough level can interfere with gut flora stability [70]. Furthermore, it was observed that patients with digestive symptoms, probably not recognized from the outset as symptoms associated with COVID-19, had a significantly longer time from onset to admission than patients without digestive symptoms (9.0 days vs. 7.3 days, *p* = 0.013) [71]. Interestingly, a recent study aiming to analyze the gut inflammatory response in immunocompetent subjects infected with SARS-CoV-2 highlighted the absence of a proinflammatory response in the gastrointestinal tract despite detection of SARS-CoV-2. Additionally, this study showed reduced mortality in patients with COVID-19 presenting with GI symptoms. Therefore, the authors speculated on a potential role of the gastrointestinal tract in attenuating SARS-CoV-2-associated inflammation [72].

In the current cohort, the mortality rate and case-fatality rate did not seem to exceed those expected in the general population. Indeed, the hypothesis that LT is a possible associated factor for a pejorative outcome could not be confirmed. However, of note was the high rate of hospitalization, with 64.8% of LT recipients being admitted to a ward and 22.9% of such patients requiring intensive care. This may of course reflect a certain selection bias, with more LT recipients being hospitalized per se. Additionally, one must keep in mind that during the first wave, there were many logistical difficulties, which probably led to an underdiagnosis of asymptomatic or paucisymptomatic cases [73].

With regard to the management of immunosuppression, not all the information can be extrapolated, as most studies have had descriptive designs. The different nature of the immunosuppressive regimens adopted, often multiple, and the modification of these regimens during the course of infection make it difficult to provide any clear guidelines to this respect. However, it seems that a complete discontinuation of IS therapy was very rare and limited to extremely severe cases. Unfortunately, complete cessation of IS was not associated with improved prognosis [17] and should therefore only be considered as a last resort in selected cases.

In the Spanish cohort, patients receiving MMF were more prevalent in the severe COVID-19 group. Baseline immunosuppression containing MMF was identified as an independent predictor of mortality, whereas the withdrawal of IS was not. However, data on modifications in immunosuppressive therapy during the infection were not extensively available in this study [17] and reduction or discontinuation of MMF was recommended by the guidelines shared by experts at the beginning of the pandemic [74]. Therefore, whether the impact on outcome is attributable to the MMF itself or to its reduction/discontinuation remains objects of further investigation. In a preclinical setting, MMF showed promising results against Middle East respiratory syndrome (MERS); however, in vivo studies suggested that its use is likely to cause more harm than benefit against coronavirus (CoV) infection [75]. Bearing in mind that MMF acts on activated lymphocytes with a cytostatic effect [76] and that SARS-CoV-2 has a cytotoxic effect on the same target [77], these synergistic effects may represent an additional risk factor and worsen the prognosis of LT patients taking MMF.

In Belli et al. [14], TAC was found to protect against worse outcomes in COVID-19 LT recipients. In vitro experiments have shown that TAC, and CNIs in general, are capable of inhibiting human CoV growth, mainly by acting on the cyclophilin pathway [78]. Additionally, by modulating T-cell activation, CNIs may act on reducing the deleterious effect of the COVID-19 late inflammatory phase [79].

Concerning the immunological response to SARS-CoV-2 infection, contrasting results were seen. However, most of the studies considered analyzed only the humoral response and in particular used an assay, the antinucleocapsid test, more suitable to evaluate preexisting exposure to the virus than to assess protective efficacy. Indeed, these antibodies have low or no neutralizing activity [80]. On the other hand, the analysis of the T-cell-mediated response in LT recipients showed similar results compared to that for immunocompetent subjects. Further studies are therefore necessary that take into account the complexity of the immune response in vivo and of the interplay among native, humoral, and T-cell immunity.

In contrast, all studies evaluating vaccine immunity have demonstrated reduced immunogenicity of LT recipients, and SOT recipients in general. It is possible, as pointed out by Mazzola et al. [52], that this reduced response is more evident in other SOT recipients, such as those for kidney or heart SOTs, than in LT recipients. In this line, the serum

conversion data of the only cohort that included only LT recipients were actually higher than the average of the other studies, which included mixed cohorts. A deterrent role could be played again by more sustained immunosuppression (with dual or triple regimens) and by the use of antimetabolites [53]. Reduced immunogenicity in LT recipients has already been demonstrated with other respiratory virus (e.g., influenza) vaccines [81], suggesting that more than the standard dose may be needed to achieve protective immunogenicity [82].

Several limitations affected the current study, mainly because of the high heterogeneity and quality of the majority of the studies considered, leading in several cases to incomplete information. Therefore, more than a few research questions remain open and will need future investigation (Figure 2).

**Figure 2.** Graphical representation of the key unresolved issues for SARS-CoV-2 infection in LT recipients.

#### **5. Conclusions**

Despite the heterogeneity of the reports included in this study, we are able to show that the middle-aged man with metabolic comorbidities represents the main target of COVID-19 among LT recipients, as is the case in nontransplanted patients. Gastrointestinal symptoms are very common in LT recipients with COVID-19, and particular attention should be paid to these complaints as a surrogate marker for COVID-19, even in the absence of fever or respiratory problems. The outcome of the LT population is nevertheless similar to that of the general population. This is, in part, also likely due to a more cautious management of immunosuppressive therapy, paying particular attention to the use of MMF and TAC while discouraging the complete discontinuation of all immunosuppression. LT recipients should be vaccinated, and great attention to protective measures should be maintained in these individuals even after a regular course of vaccination.

**Author Contributions:** C.B. contributed to this paper with conception, literature review, statistical analysis, writing the manuscript, critical revision, and editing. S.G.G. participated in drafting and literature review. V.B. and J.-F.D. contributed with critical revision and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** Chiara Becchetti received financial support from the Stiftung für Leberkrankheiten.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
