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Is there a role for immune-enhancing therapies for acutely ill patients with coronavirus disease 2019?

Purpose of review Although the so-called cytokine storm has been early described and related to a dramatic evolution in severe COVID-19 patients, it soon became clear that those patients display clinical and biological evidence of an immunosuppressive state characterized, among other, by a profound lymphopenia. The negative role of this immune suppression on the outcome raises the question on immune therapies that might improve patient's condition. Recent findings Important positive effects of active immune therapies, such as IL-7 or thymosin-α are already described and warrant confirmation in larger prospective trials. For other therapies, such as interferons, firm conclusions for critically ill COVID-19 patients are lacking as those patients were often excluded from the published trials. Treatment with immunoglobulins or convalescent plasma is a passive strategy to provide specific immunity. Unfortunately, results from large RCTs do not support their use presently. Summary In this article, we provide a review on active and passive immune boosting strategies that might help treating the most severe COVID-19 patients. We mainly focus on active strategies that include IL-7, thymosin-α, interferons, and vitamin D. Although some positive effects are described, they certainly warrant confirmation in large randomized controlled trials.


INTRODUCTION
The initial description of the coronavirus disease-2019 (COVID-19) pathophysiology put forward a prominent role of the so-called cytokine storm, a condition previously described in other pathological states after some treatments, such as chimeric antigen receptor T (CAR-T) cells infusion. However, although median value of IL-6 in patients with COVID-19 with Acute Respiratory Distress Syndrome (ARDS) is reportedly high, it does not reach those described in non-COVID-19 ARDS patients or peak IL-6 level found in patients who develop Cytokine Release Syndrome after CAR-T cells infusion [1].
Another paradigm of the disease soon emerged: a concomitant immunodeficiency involving among other a (profound) decreased lymphocyte count and an impaired type-I interferon response [2,3]. Lymphopenia occurs in up to 68-80% of patients [3,4], is correlated to severity [4], and involves all subsets including CD4þ and CD8þ cytotoxic T cells, natural killer (NK) cells, memory and regulatory T cells along with B cells [3,5]. Furthermore, blood mononuclear cells obtained from COVID-19 patients produce lower levels of cytokines upon stimulation than those from septic or nonseptic critically ill patients [5], consistent with a marked impairment of immune effector cell function.
Hence, any therapy that might improve immune function and the COVID-19 related immune suppression warrant attention. They may be classified into two major modes of action: therapies that directly improve immune function (IL7, thymosin alpha, etc.) and those therapies providing immune support (immunoglobulins, convalescent plasma, etc.) Interleukin-7 (IL-7), a common g-chain pleiotropic cytokine, displays various properties including the prevention of lymphocyte apoptosis, the induction of CD4þ and CD8þ T-cell proliferation and the improvement of lymphocyte function. It has, therefore, been proposed in various pathological states including patients with cancer, hematopoietic stem cell transplantation, AIDS, mycobacterial infection, multiple sclerosis, inflammatory bowel disease [6]. IL-7 has been tested in various models of infection and sepsis and in a recent prospective, multicenter, randomized, double-blind, placebo-controlled phase IIb trial in patients with septic shock and severe lymphopenia [7]. With a dose of 10 mg/kg CYT107 (a glycosylated recombinant human IL-7) twice a week for a total of 4 weeks, treated patients displayed an increased absolute lymphocyte count persisting after the end of treatment. An initial transient decrease followed by a significant increase in CD4þ and CD8þ T cells, and, a decreased IL-7 receptor a (CD127) expression on CD4þ and CD8þ T cells were described. Treatment related adverse effect included reversible 'at the site of injection' skin reaction consisting of a raised red rash characterized by a CD3-positive lymphocytic infiltration in skin biopsies [7].
Recently, Laterre et al. [8 & ] reported on a case series of 12 patients with COVID-19 and severe lymphopenia (defined as two consecutive absolute lymphocyte counts of less than 700/ml) treated with an initial safety dose of 3 mg/kg, followed by a dose of 10 mg/kg by intramuscular injection twice a week for 2 weeks. Treated patients were matched to 13 patients presenting similar severity of illness and comorbidities. As described by François et al., an initial decrease followed by an increase in total lymphocyte count was observed with significant differences starting from day 15 after the first injection and reaching levels more than two-fold greater than the control group. IL-7 was well tolerated without any significant change in clinical (temperature, blood pressure, or PaO 2 : FiO 2 ratio) or biological variables (tumor necrosis factor a, IL-1b, IL-12p70, or IL-6 concentrations). Secondary infections were less frequent but, as in the previous study [7], this study was not powered to detect such difference.
Monneret et al. [9] provided compassionate IL-7 to a 74 years old COVID patient who presented severe immunosuppression (assessed by HLA-DR and persisting lymphopenia) and recurrence of nosocomial infections, including Aspergillus fumigatus-related ventilator-associated pneumonia. After an inaugural injection of 3 mg/kg, the patient received 10 mg/kg injections twice a week for 4 weeks. Improvement in total lymphocyte count (including CD4þ cells and NK cells) and mHLA-DR expression toward reference ranges was rapidly observed. Likewise, the IFN score started to decrease while circulating IFN-g returned to normal range, without any increase in cytokine levels, such as IL-6, IFN-g, IL-10, or TNF-a.
Taken together, those data suggest IL7 not only improves lymphocyte count but also restores lymphocyte function. Further studies are urgently warranted to confirm these potential benefits. Interestingly, some raise the hypothesis that the positive outcome of dexamethasone might, partially at least, be explained by its capacity to enhance levels of the IL-7 receptor a [10].

Thymosin-alpha
Thymosin alpha1 (Ta1), originally isolated from the thymus, is a peptide of 28 amino acids, sharing similarities with IL-7 [11]. It is used worldwide as an immunomodulatory agent in a wide range of clinical indications, such as for the treatment of chronic hepatitis B and C, and, as a vaccine enhancer. Pharmacological studies showed that Ta1 stimulates endogenous IFN-g secretion and enhances T cells and the whole immune system by stimulating innate and adaptive immune responses [11].
Previous meta-analysis [12] and review of the literature [13] confirmed reduced mortality and modulation of immunity with increased level of HLA-DR, and improvement of lymphocyte subsets

KEY POINTS
A 'cytokine storm' with markers of acute inflammation was thought to be the major pathophysiologic event in COVID-19 patients. It soon became obvious that immunodeficiency characterized among other by a profound lymphopenia was also a hallmark of severe COVID-19.
Treatment of this CRIS (for COVID-19-related immune suppression) could include either direct or indirect immune boosters.
Reports on the effects of IL-7, thymosin-a, vitamin D, and interferons look promising for some of them but we clearly need larger randomized controlled trials to draw firm conclusions.
Reports on passive immune booster (such as convalescent plasma) are currently disappointing but several studies are still under way.
The efficacy of Ta1 was assessed ex vivo in blood samples drawn from COVID-19 patients and incubated for 8 h with 50 mg/ml Ta1 (SciClone Pharmaceuticals) [14]. The authors demonstrate Ta1 affects genes associated with immune response, inflammation, and response to infection pathways. Moreover, Ta1 decreases some Cytokine-Related Gene Transcriptional Expression found to have a higher transcriptional expression in COVID-19 patients including IL-6, IL-1b, and TNFa. At the opposite, some other genes (such as IL-10) were upregulated. Finally, the authors show Ta1 inhibits lymphocyte activation specifically in a CD8þ T cell [14].
In a retrospective study of 76 patients classified as severe or critical COVID-19 patients from two hospitals in Wuhan, China, 36 patients received subcutaneous injections of 1.6 mg Ta1 once a day for at least 7 consecutive days while 40 patients received usual cares [15 & ]. Decreased mortality and lower need for invasive mechanical ventilation are observed in the treated patients while an effective restored T-cell numbers is also described only for those patients presenting with lymphopenia (counts of CD8þ T cells or CD4þ T cells lower than 400 or 650/mL, respectively).
Finally, results from a prospective randomized trial performed in Rhodes Island Hospital are awaited (NCT04487444). This study aims at recruiting 80 participants presenting with COVID-19 infection and lymphopenia. The primary objective is to demonstrate an improved time to recovery and the secondary objective will assess the improvement in severity of infection. Treatment protocol is similar to the Chinese study, that is, Ta1 (1.6 mg) administered subcutaneously (s.c.) daily for 1 week.
Interestingly, some authors hypothesized that Ta1, thanks to its potential to stimulate IFN-g secretion, might also be of benefit in patients developing pulmonary aspergillosis after Sars-Cov-2 infection [16]. This, of course, warrants further studies.

Interferons
Interferons (IFN), produced by leucocytes, T lymphocytes, and fibroblasts, act as a 'first alarm bell' effector of the host immune response during viral infection. There are three main types of IFNs: types I (including alpha and beta), II (gamma), and III (lambda), type I being the largest IFN class. Through activation of various IFN-stimulated genes (ISG), type I IFN display several roles and functions, such as direct antiviral action, inhibition of cellular proliferation, immunomodulation, and desensitization after activation of immune response [17].
Interestingly, Hadjadj et al. demonstrated in COVID-19 patients a severity-related impaired IFN type I response associated with a persistent blood viral load, an exacerbated inflammatory response and a lower IFN activity in serum from severe or critical patients as compared with mild-to-moderate patients [18]. The persistence of these effects over time was confirmed by others in critically ill COVID-19 patients with ARDS [19]. These observed dysregulated IFN responses suggest the effective immunomodulatory strategies used by coronaviruses [20] and certainly raise the hypothesis of a potential therapeutic effect of IFN.
While type-I IFN is being evaluated in a large number of trials, either alone or in combination, study design, studied population, and outcomes are very variables.
In a first randomized trial, IFN b-1a, administered subcutaneously at a dose of 12 million IU/ml three times weekly for two consecutive weeks, was superior in terms of 28-day mortality [21]. This conclusion is limited by the small sample size (92), confounding factors (more steroids and intravenous immunoglobulins in patients receiving IFN) and the absence of benefit in other outcomes, such as hospital and ICU length of stay or duration of mechanical ventilation. Interestingly, early administration looked more favorable in terms of reduced mortality.
In the COVIFERON trial, Alavi Darazam et al. [22 & ] randomized 60 patients to receive on top of standard of cares (oral Lopinavir/Ritonavir and a single dose of 400 mg hydroxychloroquine on the first day), either IFNb1a (subcutaneous injections of 12 000 IU on days 1, 3, 6), IFNb1b (subcutaneous injections of 8 000 000 IU on days 1, 3, 6) or placebo. 75 percent of the patients were in ICU and 35% were on mechanical ventilation. The primary endpoint, defined as the time from enrollment to discharge from hospital or a decline of two steps on a seven-step ordinal scale, was reached 2 days earlier for both treated groups (5 versus 7), reaching statistical significance when both treated groups were analyzed together and for IFNb1a when each group were analyzed separately. However, despite mortality rate in the control group was more than two-fold higher than that of the IFNb1a group, mortality difference did not reach statistical significance.
In another double-blind, placebo-controlled, phase 2 pilot trial at nine sites in the UK, 101 patients were randomized to assess the efficacy of a 6 MIU inhaled interferon beta-1a formulation (SNG001) in patients admitted to hospital [23]. Although the primary outcome (defined as change in clinical condition on the WHO Ordinal Scale for Clinical Improvement) reached statistical significance, it is unclear if those results may be translated to critical care patients as only 67% of the patients received oxygen and only 2% required oxygenation through HFNC. The odds of improvement were more than two-fold greater in the SNG001 group on day 15 or 16 and more than three-fold greater on day 28 but treated patients did not present a decreased likelihood of intubation or time to intubation or death.
An open-label, randomized clinical trial also assessed the efficacy of 250 mg subcutaneous IFN b-1b administration every other day for two consecutive weeks [24]. Despite effective in shortening the time to clinical improvement and decreasing admission in ICU and need for invasive mechanical ventilation, the translation of those results to critical COVID-19 patients warrant further evaluation as only three patients required HFNC or NIV at the time of randomization.
Results from other ongoing trials (such as NCT04449380 [25]) will be available soon. However, the most severe patients [such as those requiring mechanical ventilation (MV)] are often excluded from those studies [25].
Interferon lambdas or type III IFN display similar antiviral effect than IFN alfa or beta but use a distinct receptor complex, and, usually result in fewer systemic side-effects. The potential effects of a pegylated form of IFNl was assessed in two studies of outpatients and resulted in conflicting data [26,27]. This form of IFN is, to our knowledge, not evaluated in the most severe patients.

Vitamin D
For years, it is known that vitamin D is linked to the innate immune system leading to induction of the defensin b2 and cathelicidin antimicrobial peptides, which can block virus entry into cells as well as suppress viral replication [28,29]. It also increases the phagocytic ability of immune cells and reinforces the physical barrier function of epithelial cells. Vitamin D promotes autophagy, one of the mechanisms by which cells deal with viruses. Finally, vitamin D also modulates the adaptative immune response but research on this led to conflicting results, depending, among others, on the type of disease. Because of these positive effects and the results on the role of vitamin D in respiratory (viral) infections, several experts argued for the evaluation of the use of vitamin D in COVID-19 patients [29,30].
Moreover, lower circulating levels of vitamin D and metabolites is common in critical care and are associated with worse outcomes in critically ill patients, an observation also confirmed in some retrospective studies of COVID-19 patients, showing an inverse relationship between a low level of vitamin D and the incidence of COVID-19 [29,31,32].
More than 90 studies from all over the world are registered on trials.gov. and available results on the potential of vitamin D as adjunctive treatment in COVID-19 were recently reviewed [29][30][31]. It is uneasy to draw any firm conclusions from the published studies as there was considerable variation in number and type of patients, dosing regimen, duration of treatment, and outcome measures reported. Furthermore, although ICU admission may be an outcome in some studies, ICU patients are most of the time excluded, which makes any generalization of limited interest for ICU patients.

Immune checkpoint inhibitors
Immune checkpoint molecules are negative regulatory receptors expressed on immune cells, acting as a brake for the immune system. However, these molecules may induce T-cell dysfunction in a variety of diseases, such as cancer and infection. Immune checkpoints inhibitors (ICIs) include anti-PD-1, anti-PD ligand-1 (PD-L1), anti-TIM-3, and anti-CTLA-4 antibodies. They activate gd T cells and mucosal-associated invariant T (MAIT) cells and restore individual cellular-mediated immune-competence.
Interestingly, higher PD-1 expression on T cells characterizes severely affected COVID-19 patients when compared with healthy patients [33]. Furthermore, when patients deteriorated, an enhanced PD-1 and Tim-3 expression on T cells were observed.
Despite ICIs overturned the management of several malignancies and was proposed to treat septic patients [34], a very limited number of studies is recorded on trials.gov. to address the efficacy of ICIs in COVID-19 patients. Among them, a clinical study will evaluate the role of nivolumab in 120 obese patients by assessing the proportion of patients able to be weaned from oxygen at D15 after randomization (NCT04413838). Study completion is expected in June 2021.

PASSIVE IMMUNE BOOSTERS
The treatments described in the following paragraph may not be considered as a booster of patient's own immunity, but rather a way to provide a passive immune protection against a broad range of pathogens [intravenous immunoglobulins (IvIg)] or against a more specific pathogen [hyperimmune globulins (HIGs) and convalescent plasma]. Those treatments may also display some immune

Intravenous immunoglobulins
Immunoglobulins obtained from healthy donors act through various mechanisms as an immunomodulatory therapy [35]. Case series [36,37] and retrospective studies [38,39] display variable results and prevent from any firm conclusions as there were confounding factors (concomitant treatment, timing of IvIg administration, or dosing) and the number of critical patients looks very limited. Two recent randomized trials, performed in Iran, also display conflicting results. In the first study on 59 patients (30 patients receiving IvIg), mortality rate was significantly lowered (20 versus 48.3%) and IVIg was an independent determinant of mortality in the multivariate regression analysis [40]. In the other trial evaluating IvIg in 52 patients versus 32 patients receiving standard of care, none of the primary outcomes (need for invasive mechanical ventilation and oxygenation, need for admission to ICU, and mortality rate) were positive [41]. At best, a relationship between early timing of treatment and decreased ICU and hospital length of stay is described in survivors.

Hyperimmune globulins
Hyperimmune globulins (HIG) derived from a large pool of individuals with high antibody titers to specific pathogens has been used successfully in the treatment of infections, such as cytomegalovirus and H1N1 influenza [35]. A 'simple' modified caprylic acid method allows for HIG production from pooled convalescent plasma of COVID-19-recovered individuals, leading to a highly purified immunoglobulin G product with more concentrated neutralizing antibody activity [42,43].
One RCT assessing HIG in 50 patients (NCT04521309) is completed but unpublished so far [44], whereas another one with a larger number of patients will be completed in July 2021. ]NCT04546581 -The Inpatient Treatment with Anti-Coronavirus Immunoglobulin (ITAC) study].

Therapeutic plasma exchange
Therapeutic plasma exchange (TPE) has been proposed to treat the so-called cytokine storm described in the early stages of the Sars-Cov-2 infection, reduce viral burden, clear antifibrinolytic mediators and fibrin degradation products, decrease the levels of injurious free radicals, and viscous components [45]. Case reports [46], case series [47], retrospective studies [48,49], and matched controlled studies [50] provide contrasting results on a potential mortality benefit probably related to the heterogeneous studied populations. Whenever reported, a decreased cytokine level is described and importantly some authors also demonstrate an increased lymphocyte count after TPE [51]. The specific role of TPE and the mechanism of action to explain this improvement, as well as its clinical consequences remain a matter of research. The role of TPE on the improvement of other markers of immunosuppression beyond lymphocyte count certainly warrants further research. Interestingly, Guo et al. [52] report on similar results for lymphocytes, by using an artificial blood liver purification system, which consists of modules for plasma replacement, plasma adsorption, and blood/ plasma filtration.
Some authors advocate for TPE with convalescent plasma from recovered individuals as the replacement solution [53,54] or with transfusion of convalescent plasma after the TPE procedure [55]. This is hypothesized to improve the benefit of each technique performed alone. The sequential therapy is reported in a case series of 14 patients on mechanical ventilation with apparent good outcomes when compared with the existing literature [55]. The limited number of patients and the absence of control group limit firm conclusions.
Finally, TPE was also used to treat other conditions associated with COVID-19, such as neurological or gastro-intestinal involvement [45].

Convalescent plasma
Plasma from convalescent patients is a form of therapy used as early as 1918 during the 'flu epidemics' and in recent years for SARS, Middle-East respiratory syndrome (MERS), H1N1, and Ebola pandemics [53,56]. In a first report on five critically ill on mechanical ventilation because of severe COVID-19, Shen et al. [57] described improved clinical condition and a resolution of ARDS in four out of five patients within 12 days. Since that time, the United States Food and Drug Administration (US FDA) approved the use of convalescent plasma therapy for COVID-19, more than 150 studies from all over the world were recorded on https://clinicaltrials.gov/, and several meta-analysis and systematic reviews have been conducted. Despite the abundant literature, firm conclusions are still a matter of debate. Various designs, setting, titer of antibody [58], timing of treatment [59], and other factors may explain those discordant results. Large recent metaanalysis, including more than 20 000 patients, were unable to demonstrate any significant positive effect on mortality or other secondary outcomes (length of stay in hospital, need for MV) [60,61].
Although some studies in the particular setting of ICU are still ongoing (NCT04558476) [62], the group of ICU patients randomized in larger trials did not benefit from convalescent plasma [63][64][65][66]. In the recently published RECOVERY trial, 617 patients were receiving invasive mechanical ventilation at randomization [66]. Twenty-eight percent were successfully weaned from invasive ventilation in the convalescent plasma group versus 34% in the usual care groups, which confirms that patients undergoing invasive ventilation at time of randomization are unlikely to benefit from convalescent plasma. Two other large convalescent plasma trials were stopped for futility and final publications are awaited: CONCOR-1 (NCT04348656) and REMAP-CAP (NCT02735707).

Monoclonal antibodies
Studies on monoclonal antibodies (mAb) directed against pro-inflammatory molecules (not only IL-6 but also IL-1ra, IL-8, IL-1b, IL-17A, TNFa, etc.) or their receptor are still underway or were recently terminated. In this particular setting, tocilizumab, a recombinant humanized mAb directed against IL-6 receptor inhibiting its signal transduction pathway, certainly is the most studied drug [67]. Although some clinical signs such as fever reportedly improve, results on mortality are somewhat conflicting, some studies demonstrating improved outcome [68], others showing no benefit at all [69]. mAb may also be directed against viral components (for instance, the S protein on the surface of the virus particle). Other mechanisms of action include binding to ACE2 protein (and block the combination of the virus and its receptor) or acting as an ACE2 analog that competitively binds to the viral S protein [70]. Among those mAb, LY-CoV555, an effective antispike neutralizing mAb, received FDA approval in end 2020.

CONCLUSION
Immune therapies in COVID-19 seem of particular interest to treat this condition and its associated well confirmed immune suppression. Among the immune boosting therapies, some positive effects are described for IL-7, thymosin-a, interferons, and vitamin D. Confirmation in large randomized controlled trials are certainly warranted.
The other strategy involve a passive improvement of the immune function through the administration of IvIg or convalescent plasma. Unfortunately, results from large randomized controlled trial (RCT) in this setting were contrasting, and could currently not serve as a recommendation for treating critically ill. The debate remains opened as results from many trials will be available soon.