key: cord-0865377-y8ry0cbg
authors: Sokolowska, M; Lukasik, Z; Agache, I; Akdis, CA; Akdis, D; Akdis, M; Barcik, W; Brough, H; Eiwegger, T; Eliaszewicz, A; Eyerich, S; Feleszko, W; Gomez Casado, C; Hoffmann‐Sommergruber, K; Janda, J; Jiménez‐Saiz, R; Jutel, M; Knol, E; Kortekaas Krohn, I; Kothari, A; Makowska, J; Moniuszko, M; Morita, H; O’Mahony, L; Nadeau, K; Ozdemir, C; Pali‐Schöll, I; Palomares, O; Papaleo, F; Prunicki, M; Schmidt‐Weber, CB; Sediva, A; Schwarze, J; Shamji, MH; Tramper‐Stranders, G; van, W; de Veen,; Untersmayr, E
title: Immunology of COVID‐19: mechanisms, clinical outcome, diagnostics and perspectives – a report of the European Academy of Allergy and Clinical Immunology (EAACI)
date: 2020-06-25
journal: Allergy
DOI: 10.1111/all.14462
sha: 3bf35229ccfe7afe922247c6de28039723f65699
doc_id: 865377
cord_uid: y8ry0cbg

With the worldwide spread of the novel Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) resulting in declaration of a pandemic by the World Health Organization (WHO) on March 11, 2020, the SARS‐CoV‐2‐induced Coronavirus disease‐19 (COVID‐19) has become one of the main challenges of our times. The high infection rate and the severe disease course led to major safety and social restriction measures worldwide. There is an urgent need of unbiased expert knowledge guiding the development of efficient treatment and prevention strategies. This report summarizes current immunological data on mechanisms associated with the SARS‐CoV‐2 infection and COVID‐19 development and progression to the most severe forms. We characterize the differences between adequate innate and adaptive immune response in mild disease and the deep immune dysfunction in the severe multi‐organ disease. The similarities of the human immune response to SARS‐CoV‐2 and the SARS‐CoV and MERS‐CoV are underlined. We also summarize known and potential SARS‐CoV‐2 receptors on epithelial barriers, immune cells, endothelium and clinically involved organs such as lung, gut, kidney, cardiovascular and neuronal system. Finally, we discuss the known and potential mechanisms underlying the involvement of comorbidities, gender and age in development of COVID‐19. Consequently, we highlight the knowledge gaps and urgent research requirements to provide a quick roadmap for ongoing and needed COVID‐19 studies.

Infections with the novel coronavirus SARS-CoV-2 resulting in COVID-19 development represent the major medical and scientific challenges of our time. Knowledge on SARS-CoV-2 infection pathways and mechanisms associated with immune defense or immunopathology is growing exponentially, as it is indispensable to design the proper diagnostic and therapeutic strategies. However, there are several knowledge gaps and urgent unmet research needs in our understating of the current pandemics ( Table 1) .

A group of experts in basic and clinical immunology has joined forces under the umbrella of the European Academy of Allergy and Clinical Immunology (EAACI) to provide a consensus report on the basic molecular and immune mechanisms associated with susceptibility, clinical presentations and severity of COVID-19.

On the basis of sequence homology, all human coronaviruses have animal origins: SARS-CoV, SARS-CoV-2, MERS-CoV, HCov-NL63 and HCoV-229E are considered to have originated from bats, 1 whereas HCoV-OC43 and HKU1 likely originated from rodents ( Fig. 1) . 2 SARS-CoV-2 has a significant structural similarity to SARS-CoV and MERS-CoV and other human and animal coronaviruses. 3, 4 It has been quickly determined that SARS-CoV-2, similarly to SARS-CoV, utilises the membrane bound form of angiotensin converting enzyme 2 (ACE2) to enter human cells via its spike protein (S). 5 After SARS-CoV-2 has bound ACE2, ACE2 will be internalized and its membrane expression decreased. Whereas ACE2 is an important regulator of bradykinin, its reduced expression in the lung environment results in local vascular leakage leading to angioedema in the affected lung tissue. 6 The host serine protease TMPRSS2 cleaves spike protein into S1 and S2 fragments, which enables fusion with the cellular membrane, entrance to the cell and start of the replication process. 7 In addition to TMPRSS2, other proteins such as furin or human endosomal cysteine proteases are potentially capable of cleaving S, such as cathepsin L (CTSL) and cathepsin B (CTSB). 8, 9 ACE2 is highly expressed in the lungs, small intestine, kidney and heart, but it is not expressed on innate and adaptive immune cells. [10] [11] [12] [13] As recently shown, SARS-CoV-2 can also use CD147 (also called basigin (BSG) or extracellular matrix metalloproteinase inducer (EMMPRIN), to enter T-cell lines, as well as cells of epithelial origin, but it is not yet clear if then virus can efficiently replicate or it leads to the cell death 14, 15 CD147 is utilized as a receptor by other viruses including SARS-CoV and HIV-1, as well as by malaria to enter erythrocytes. [16] [17] [18] It is, however, not yet clear if SARS-CoV-2 can replicate inside immune cells or just infects them and causes cell death. CD147 is a transmembrane immunoglobulin-like receptor, which also exists in a secreted form. 13, 19 At the cellular membrane, it is activated by several extracellular ligands such

This article is protected by copyright. All rights reserved as cyclophilins A and B (PPIA and PPIB), S100A9 or platelet glycoprotein VI (GP6). 20-23 Its extracellular glycosylation sites bind to complex proteoglycans such as syndecan-1. 24 CD147 often creates membrane complexes with CD44, one of the receptors for hyaluronan, an extracellular matrix component. 25 Coronaviruses incorporate host cyclophilins during their cellular replication cycle, which further enables them to bind to CD147. 26,27 CD147 is expressed in human airway and kidney epithelium, as well as in innate cells (granulocytes, macrophages, dendritic cells (DC), innate lymphoid cells (ILCs) and lymphocytes. 10 Other receptors potentially utilized by SARS-CoV-2 are CD26 (encoded by DPP4; a receptor for MERS-CoV), an important T cell and also epithelial cell receptor, amino peptidase N (ANPEP; a receptor for human and porcine coronaviruses), 13 ,28 ENPEP and a glutamyl aminopeptidase 29 , as well as DC-SIGN 30 (Fig.2) 

In the upper and lower airways ACE2 and TMPRSS2 are highly co-expressed, [10] [11] [12] [13] but there is no expression of SLC6A19, which potentially blocks the access of TMPRSS2 to ACE2 and subsequently reduces active infection. 31, 32 In the nasal and the pharyngeal epithelium, in goblet and ciliated cell, ACE2 is expressed at high levels and co-expressed with TMPRSS2 representing the sites of initial viral replication and a main source of infectious particles. [10] [11] [12] [13] 31 The lower airways, bronchial epithelium and type II pneumocytes (AT2 cells) highly express ACE2 and TMPRSS2, which may provide virus entrance to the lung and lead to COVID-19 pneumonia. Moreover, CD147, CD26, ANPEP and ENPEP are also expressed in the airway epithelium, as well as in many innate and adaptive immune cells, 10, 13 both in bronchoalveolar lavage (BAL) and peripheral blood (Fig. 3a) .

Once the virus enters the host cell, it releases its RNA into the cytoplasm and uses the host translation machinery to translate its polyproteins pp1a and pp1b, also known as replicases and viral essential proteases 3CLpro and PLpro. These proteases cleave polyprotein complex into several non-structural proteins (Nsp), which together with the viral RNA-dependent RNA polymerase form the replication complex, where the negative strand and mRNA for structural proteins (S, nucleocapsid (N), envelope (E), and membrane (M)) and accessory proteins for the virus are created. 2, 33, 34 After protein translation, they traffic through the ER to the Golgi apparatus, where the mature virions are assembled in budding vesicles and are exocytosed from the cell. Inside infected cells, there are several innate immune mechanisms responsible for recognizing the virus at different stages of its replication and leading to the production

This article is protected by copyright. All rights reserved interferons type I (IFN and ), type III, and proinflammatory cytokines. 35 Genes encoding these interferons form the type-1 (E1) epithelial response profile. Also ACE2 is a typical E1 gene. 36 This response also includes mechanisms such as the expression of helicases or cytidine deaminases targeting viral RNAs (Fig. 3a) .

Viruses use various strategies to evade those mechanisms. 37 Epithelial cells produce type I and type III IFNs upon viral infection. Type I IFN act through receptors expressed in a vast number of cells. In contrast, type III IFNs seem to exert their effect mostly on epithelial cells, are less inflammatory and are activated faster than type I IFN. 39, 40 IFNs are one of the most potent antiviral components of the innate immune response. They work on various levels i.e. blocking viral attachment, entry, trafficking, protein production and genome amplification and also viral assembly and egress. 39 Moreover, IFNs also activate other innate and adaptive immune responses. However, in case of COVID-19 these responses seem to be diminished 41 or dysregulated. 42 SARS-CoV and MERS-CoV inhibit IFN signaling on various levels. 43 The nsp 16 mediated 2'O methylation of viral mRNA cap structure prevents coronaviruses recognition by MDA5. 44 The sequestration of viral dsRNA within double membrane vesicles (DMVs) also protects coronaviruses from detection through cytosolic PRRs 45 . Moreover, coronaviruses produce many non-structural proteins which inhibit induction of IFNs (inhibition of IRF3 and IRF 7) and/or interferon signaling (inhibition of STAT 1 signaling). 43 A reduced antiviral response via IFN pathway inhibition, together with an ongoing pro-inflammatory response, presumably heightened by increased viral load, may lead to excessive inflammation 42 and worsening of the disease. In an animal model of SARS-CoV, a delayed type I interferon response resulted in accumulation of inflammatory monocytes/macrophages, leading to elevated lung cytokine/chemokine levels, vascular leakage and impaired virus-specific T cell responses. 42 A recent study in humans showed that SARS-CoV-2 infection induces weak IFN responses from infected pneumocytes, even weaker than in SARS-CoV infection. 41 Interestingly, ACE2 has been recently shown to be an interferon stimulated gene. 12

This article is protected by copyright. All rights reserved ACE2 is also known to protect mice against acute lung injury. Therefore, it needs to be determined whether upregulation of ACE2 after the initial antiviral response is used by SARS-CoV-2 to enhance infection, but also if the delay of IFN responses potentially leads to impairment of ACE2-related protection against lung injury.

Based on the current knowledge of SARS-CoV-2 receptors' expression on the epithelial barrier sites, the gastrointestinal tract requires special attention. Human ACE2 is homogeneously distributed on the brush border of enterocytes across the small intestine and in the lung epithelium. 46, 47 In the oral mucosa, the basal layer of non-keratinized, squamous epithelial cells was reported to be ACE2-positive, while stomach epithelial cells and colon enterocytes remained negative. 46 TMPRSS2 and TMPRSS4 mediate infection of small intestinal epithelial cells. 48 These enzymes might additionally be an interesting target for therapeutic intervention, since a clinically approved protease inhibitor is available. 7 Less is known regarding the gastrointestinal distribution of CD147. 14 Enteric CD147 seems to play a role in carcinogenesis and inflammation, 49 which might shed a new light on patients' group at risk for severe SARS-CoV-2 infections and needs further attention. Of note, CD26 expression was reported to be high in ileum and jejunum, low in duodenal samples and not detectable in colon epithelial cells (Fig. 3b) . 50 Gastrointestinal symptoms like vomiting and diarrhea in COVID-19 are gaining attention. 51 In the previous SARS outbreak and in MERS patients, gastrointestinal complaints were found in approximately 30% of patients. In SARS-CoV-2 infections, diarrhea and abdominal pain occur in 20-50% of COVID-19 patients and might even precede onset of respiratory symptoms. 52,53 SARS-CoV active replication was detected in small intestinal enterocytes 54 and enteroids derived from human ileum and colon in case of SARS- This is highly relevant as viral excretion was detected in fecal samples and anal swabs of COVID-19 patients. 55 While first evidence is available that human colonic fluids might rapidly inactivate SARS-CoV-2 in vitro, 47 MERS-CoV was found to resist gastrointestinal fluids simulating conditions with elevated pH levels after food ingestion, while the virus rapidly lost infectivity when exposed to an acidic gastric fluid simulating fasted state. 56 These reports might explain, at least partially, the fact that even though SARS-CoV-2 RNA was detected in stool samples from patients, the isolates were not infective. 57 Thus, it remains unclear whether the fecal-oral-route might propagate disease transmission especially in reduced hygienic conditions. 58

This article is protected by copyright. All rights reserved Further understanding of the relationship between disease and the digestive tract is essential to prevent transmission and disease progression as well as to design efficient treatment of COVID-19.

Recent reports indicate that in COVID-19 the skin might also be affected. An Italian and a French study reported that 20.4% to 50% of COVID-19 cases, respectively developed nonpruritic, erythematous rashes, urticaria or varicella-like lesions affecting the trunk and sometimes the limbs. 59,60 In general, the rashes occurred 3 days after development of COVID-19 symptoms and the median duration was 8 days. 61 In addition, acrolated ischemic, self-healing lesions at toes and fingers have been observed mainly in children and young adults shortly before COVID-19 symptom appearance. 62 To put otherwise healthy kids in quarantine upon detection of these lesions might help to prevent infection from spreading. Apparently, cutaneous manifestations of COVID-19 are similar to skin rashes observed in other common viral infections. There is no evidence that they are related to the severity of the disease or an indication that the virus can replicate in the skin.

Unfortunately, the few studies available so far did not detect SARS-CoV-2 presence in skin lesions, which questions if the skin manifestations are indeed infectious or para-infectious driven. Given that ACE2 and TMPRSS2, the receptors for SARS-CoV-2 entry into human cells, are absent or weakly expressed in the skin, 63 para-infectious events seem to be more likely. Furthermore, the possibility of adverse drug reactions as causative for skin manifestations in COVID-19 is being strongly considered in certain cases.

Some of the protective measures taken during the SARS-CoV-2 pandemic (use of gloves, masks or goggles) can affect the skin. Masks and goggles often induce pressure injury due to not properly fitted material, and a study in Chinese health care workers indicated that 71% suffer from skin barrier damage such as dryness, scales, papules or erythema. 64 Causative is the inevitable hand hygiene procedure -66.1% stated to wash their hands more than 10 times per day and only 22.1% used appropriate skin care products afterwards. Moreover, long-term usage of gloves over 6 hours per day is common in health care workers leading to overhydration and dysbiosis with damage to the stratum corneum and subsequent skin infection or sensitization. 65 Thus, proper education regarding the use of skin care products after the hand hygiene procedure is essential to protect the skin barrier and prevent further skin complications. 

Neutrophils are one of the predominant lung infiltrating leukocytes in severe SARS-CoV-2 infection, and neutrophilia predicts poor clinical outcome. 84 Post-mortem analysis of lung samples from COVID-19 Functional exhaustion of NK cells and CD8 + T cells was described in relation to severe SARS-CoV-2 infection (Fig 4) . Exhausted NK and CD8 + T cells expressed CD94/NK group 2 member A (NKG2A), which functions as an inhibitory receptor, and showed diminished production of CD107a, IFN-γ, IL-2, granzyme B, and TNF-α. 101 During infection IFN-γ induce expression of the non-classical human leukocyte antigen E (HLA-E). 102 HLA-E is the ligand of NKG2A, which is expressed on epithelial cells. NKG2A blockade with

This article is protected by copyright. All rights reserved monoclonal antibodies (Monalizumab) prevents the binding of HLA-E, which may be a target for COVID-19 therapy.

The complement system is engaged in both coagulation and inflammatory pathways. 

Stimulation of innate immune cells with specific microbial antigens induces long lasting epigenetic and metabolic re-programming leading to enhanced responses upon a second challenge by the same or unrelated microbial insults, a process coined as "trained innate immunity". 106 

This article is protected by copyright. All rights reserved increased production of pro-inflammatory cytokines, increased protection against infections and reduced mortality. 109 Increased expression of PRRs in monocytes isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals 1 year after BCG vaccination has marked the importance of trained immunity. 108 Although further studies are required, lower number of cases and deaths per population during COVID-19 pandemic seem to be reported in countries with BCG vaccination programs than those that did not have or ceased it, which could be attributed to potential BCG vaccination-induced trained immunity effects. 110 

responses (Fig 4) 113 . T cells are instrumental in developing immunological memory in the form of virus specific CD8 + and CD4 + T cells as shown in case of SARS-CoV. [114] [115] [116] In fact, SARS-CoV specific CD8 + T cells have been detected in humans up to 11 years post-infection, which is longer than the specific antibodies 116 . SARS-CoV-2-specific CD8 + and CD4 + T cells were also recently identified in ~70% and 100% of COVID-19 convalescent patients, respectively. CD4 + T cells responded to spike (S) protein, which correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. Importantly, SARS-CoV-2 reactive CD4 + T cells were also detected in ~40-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating 'common cold' coronaviruses and SARS-CoV-2 117 , which was confirmed by others. 118 Profound lymphopenia, with the subsequent shifts in the T cell subsets composition, is often reported in SARS-CoV-2 infection, similarly to SARS-CoV and some other viruses. 96,100,119 Total numbers of CD4 + T cells and CD8 + T cells are below normal levels in most COVID-19 patients, with the lowest numbers in the severe cases.

Moreover, the number of Treg cells is also decreased, 100 whereas a recent case report of non-severe COVID-19 showed a progressive increase in the proportion of CD4 + CXCR5 + ICOS + PD-1 + circulating follicular helper T (T FH ) cells. 98 Delayed development of adaptive responses, together with prolonged virus clearance has been reported in cases of severe SARS-CoV-2 infection (Fig 4) . 120 Unfortunately, the mechanisms involved in the lymphocytopenia are still not known in SARS-CoV and SARS-CoV-2 patients. T

This article is protected by copyright. All rights reserved cells can be infected through highly expressed CD147 14, 15 or potentially through CD26, as ACE2 expression on lymphocytes is very low 10 , except in certain tissue-derived T cells. 121 It is yet unclear whether such infection is the reason of the death of infected T cells. Secondly, as in the case of SARS-CoV, an alteration in the antigen presenting cells (APCs) function and subsequent impairment of T cell priming might lead to an inefficient/delayed formation of virus-specific T cells [122] [123] [124] . Finally, also a high cytokine response from the infected cells might induce apoptosis of T cells 125 . The causes of lymphopenia need to be extensively studied, as it correlates with the higher risk of severe disease and increased length of hospitalization 126, 127 . In addition to decrease in numbers, there are also other defects reported in the function of T cell subsets in SARS-CoV-2 infection. In severe pneumonia in COVID-19 patients, it has also been shown that highly cytotoxic, activated CD8+ T cells and Th17 cells, can also participate in the CRS, together with macrophages and epithelial cells 119 

Human SARS-CoV-2 infection activates mechanisms of B and T cell immunity that result in the generation of neutralizing antibodies. 130 Initially, B cells appear to recognize SARS-CoV-2 through the nucleocapsid protein, which induces their activation and subsequent interaction with cognate CD4 + T cells. The antibody response is mounted 4-8 days after the onset COVID-19 symptoms and dominated by IgM. 131

This initial IgM-response is followed by IgA and then IgG production (10-18 days).

The development of mucosal IgA likely prevents SARS-CoV-2 re-infection while circulatory IgA may contribute to systemic SARS-CoV-2 neutralization and to dampen inflammation during active infection (Fig   4) . 132 The extent and quality of the IgG response to neutralize SARS-CoV-2 is critical. Based on previous 

It has yet to be determined whether SARS-CoV-2 shows significantly more pronounced lung tropism than other respiratory viruses. COVID-19-associated viral pneumonia is relatively frequently complicated by ARDS. Lung CT images of COVID-19 pneumonia patients revealed mostly diffuse patchy ground glass opacities under the pleura with partial consolidation which, in clinically improving individuals, can be further absorbed and followed by formation of fibrotic tissue. 144 Postmortem analysis of COVID-19 patients revealed extensive alveolar damage complicated by the formation of hyaline membranes, diffuse remodeling of alveolar wall and accumulation of immune cells (mostly macrophages) infiltrating air spaces. 119, 145 Macrophages accumulating in lungs secrete type I and type III IFNs that enhance local antiviral defenses in surrounding epithelial cells. Lung-associated macrophages contribute to development of CRS by producing IL-6 and IL-1β, cytokines promoting further recruitment of cytotoxic T cells and neutrophils. In consequence, activated neutrophils produce reactive oxygen species and leukotrienes that directly contribute to acute lung injury. 146 Even successful eradication of the virus does Accepted Article not prevent from continuous lung damage, development of frequently progressive and irreversible fibrotic consequences. 147 At this relatively early phase of the pandemic, the exact fraction of COVID-19

patients burdened with persistent fibrotic interstitial lung disease cannot be precisely determined.

Nevertheless, available and novel anti-fibrotic therapies should also be considered as candidate strategies to manage post-COVID-19 long-term lung fibrosis. 148

Cardiac injury is a prominent feature in COVID-19 developed by a considerable proportion of patients, and is associated with an increased mortality. 149 The pathogenesis of COVID-19 in the cardiovascular system likely results from a combination of several mechanisms such as direct viral toxicity, systemic CRSmediated and stress-related injury. These mechanisms promote cardiomyocyte and endothelial apoptosis, endothelial shedding, plaque destabilization and increase wall shear stress, leading to myocarditis, endotheliitis, ischemia, cardiac arrhythmias and hypercoagulability. ACE2 is highly expressed on cardiomyocytes and endothelial cells, possibly facilitating direct viral damage. However, it is unknown whether vascular derangements in COVID-19 patients are due to endothelial cell involvement by the virus.

A recent study found that endothelial cells can be infected by SARS-Cov-2, as postmortem analysis of kidney by electron microscopy revealed viral inclusion structures within endothelial cells. 150 The postmortem histology from patients with multiorgan failure in COVID -19 showed endothelitis in the lung, heart, kidney, liver and small intestine, with an accumulation of inflammatory cells associated with endothelium 150 . These findings suggest that the endotheliitis may be a combination of direct consequence of the viral involvement (i.e. presence of intracellular viral bodies) and the host inflammatory response (Fig. 5) .

In cardiomyocytes SARS-CoV-2 appears to downregulate ACE2 and diminish its cardioprotective role, promoting left ventricular failure and hypertrophy, as well as pro-thrombotic and pro-oxidant pathways 151 . Few cases documented myocarditis with diffuse T-lymphocytic inflammatory infiltrates with interstitial oedema and without fibrosis, suggesting an acute inflammatory process. A recent study presenting data from autopsy series also demonstrated SARS-CoV-2 viral load in heart tissue [152] [153] [154] . Since SARS-CoV-2 may predispose patients to coagulopathies with clinical manifestations ranging from arterial and venous embolisms to disseminated intravascular coagulation, with very poor prognosis, early prophylactic anticoagulation in hospitalized patients is recommended 155 . Taken together, direct viral involvement,

imbalanced host immune response and systemic inflammation are proposed as important mechanisms of myocardial/endothelial injury.

Abnormal coagulation parameters such as mild thrombocytopenia, prolonged prothrombin time, disseminated intravascular coagulation [156] [157] [158] and elevated D-dimers are seen in 36% to 43% of COVID-19

patients. 157, 159 In a meta-analysis of 4 published studies, higher D-dimers were found in patients with more severe COVID-19. 159 Also, thrombocytopenia was reported to be associated with more severe COVID-19 and increased risk of death (Fig. 5) . 158, 160 In a trial of severe COVID patients (n=99), anticoagulant therapy (e.g. low molecular weight heparin) was associated with better prognosis. 161 Activation of endothelium, platelets, and leukocytes leads to enhanced local and systemic production of thrombin, which in turn leads to deposition of fibrin, microangiopathy, and eventual organ damage. Both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) initiate these processes. 162 In 16 patients with severe COVID-19 a correlation between IL-6 and fibrinogen levels was found, supporting a link between hyperinflammation and increased venous thromboembolism (VTE) risk. 163 Although thrombocytopenia has been implicated in patients infected with SARS-CoV-2, the association between platelets and the disease mortality is not clear. In COVID-19 patients from Wuhan, China, platelet count increase was an independent risk factor reversely associated with in-hospital mortality, as an increase of 50x109/L platelets was associated with a 40% decrease in mortality 158 . Another study of 548 patients from China found that while platelet levels were decreased when hospitalization for COVID-19, platelet levels increased in survivors over time, but maintained lower levels or dropped significantly over time in non-survivors. 164 Thus, baseline platelet levels and changes over time appear to be associated with subsequent mortality and monitoring platelet levels is important in predicting prognosis of patients with SARS CoV-2 infection.

Together with the choroid plexus, the blood-brain-barrier protects the brain from invading microorganisms. 165 Nevertheless, several pathogens, including viruses, are still able to traverse the

This article is protected by copyright. All rights reserved barriers, 166 especially in cases of systemic inflammation 167 causing potential alterations of the central nervous system (CNS). In particular, coronaviruses might exhibit neurotropic properties, 168 and SARS-CoV was detected in human brain 169, 170 . Of interest, ACE2 is expressed in the human brain 46 . Based on the knowledge from animals studies, SARS-CoV can enter the brain via the olfactory nerve leading to a rapid, transneuronal spread to connected areas of the brain. 171 

Recent evidence indicates that kidney injury occurring during SARS-CoV-2 infection can result not only from CRS and on-going sepsis but also from direct virus-induced impairment. 179, 180 In fact, ACE2 is highly expressed on renal tubular cells. 181 Clinical observations of COVID-19-related kidney damage have been confirmed by an elegant experiment demonstrating that SARS-CoV-2 can directly infect human kidney organoids. 182 Moreover, this infection led to further efficient shedding of progeny viruses capable of infecting Vero E6 cells. This finding suggests that kidneys are an active player in the process of viral spread rather than only a site of virus-induced tissue damage. The process of kidney infection by SARS-CoV-2 was significantly, but not completely inhibited by human recombinant soluble ACE2, which

indicates that there might be other than ACE2 receptors accounting for the entry of SARS-CoV-2 to kidney cells. 182 The putative candidate can be CD147 being highly expressed on proximal tubular epithelium.

CD147 together with one of its ligands, cyclophilin, plays a crucial role in renal inflammation and renal fibrosis. 183 Moreover, cyclophilins efficiently control the process of coronavirus replication. 184 Thus, therapeutic strategies could aim at breaking the CD147-cyclophillins.

This article is protected by copyright. All rights reserved 185, 186 In the nation-wide report from China including 1590 patients with COVID-19

one comorbidity was present in 25.1% and two or more comorbidities in 130 8.2% patients (Fig 6) . 187 Several mechanisms directly linked to the underlying pathological condition can contribute to the unfavorable clinical outcome. A recent study suggested that hypertension and diabetes resulted in COPD and ongoing smoking contribute to COVID-19 severity 194 . COPD and active smokers had significantly increased expression of ACE2 and its gene expression inversely related to the lung function, suggesting a dose-dependent response. 195 Multimorbidity is also associated with elevated levels of plasminogen. Plasmin, and other proteases, may cleave a newly inserted furin site in the S protein of SARS-CoV-2, extracellularly, which increases its infectivity and virulence. Hyperfibrinolysis associated with plasmin leads to elevated D-dimer in severe patients. Thus, the plasminogen system may prove a promising therapeutic target in Accepted Article

This article is protected by copyright. All rights reserved

The increased vulnerability of males compared to females to severe COVID-19 has been reported during the pandemic. A direct endocrine link is involved as androgen receptor activity is required for the transcription of TMPRSS2 gene. 197, 198 Male vulnerability may be further enhanced by X-linked inheritance of genetic polymorphisms as both the androgen receptor and the ACE2 genes loci are on chromosome X.

Old age was also associated with an increased risk of infection and worse outcome (Fig. 6) . Frailty is characterized by multisystem dysregulation leading to reduced physiologic reserve. Although not formally assessed in the COVID-19 trials, frailty may be linked to infectious disease through common pathways that reduce immunity. 199, 200 Frailty has also been shown to be associated with poor post-vaccination immune response. 199 The aged immune system is characterized by a low-grade chronic systemic inflammatory state marked by elevated inflammatory markers such as IL-6 and C-reactive protein and an increased susceptibility to infection. 201 The expression of ACE2 and TMPRSS2 genes in the type II alveolar cells of elderly and young patients is comparable. Therefore, it does not seem to be responsible for the worse outcomes observed in COVID-19 affected elderly, but the expression of other receptors is agedependent 10 .

Drug hypersensitivity (11.4%) and urticaria (1.4%) were self-reported by patients with COVID-19. 88 In contrast, respiratory allergies and asthma were not reported as risk factors for SARS-CoV-2 infection. 84,88,202-205 However, a report from the CDC of U.S. hospitalizations described contradicting findings in adults with asthma. Among hospitalized patients with COVID-19, 27.3% of 18-49 year old adults had asthma, 13.2% of 50-64 years, and 12.9% of those of 65 years or older. 206 Currently, patients with allergic rhinitis and patients treated with allergen-specific immunotherapy are advised to continue their therapies. [207] [208] [209] Another study elucidated the impact of comorbid respiratory allergy or asthma on COVID-19 susceptibility and disease severity. 210 Children with asthma and moderate to severe allergic sensitization showed reduced ACE2 gene expression compared to children with non-atopic asthma. An additional trial including 23 patients with asthma confirmed reduced ACE2 expression in lower airway epithelial cells post-allergen

This article is protected by copyright. All rights reserved challenge. Finally, in vitro experiments using nasal and bronchial airway epithelium showed that IL-13 reduced the ACE2 expression. 210 However, adult patients with asthma seem to have higher expression of TMPRSS2 and CD44, which forms a functional complex with CD147 in bronchial epithelium 10 .

The development of serious complications and even fatal outcome in SARS-CoV-2 infection is strongly linked to the patients' immune response resulting in CRS. 211 There is an urgent need for biomarkers that predict patients developing severe complications. 212 To date there is limited information on the biomarkers associated with, or even predicting severe complications in COVID-19. However, there is much similarity on the biomarkers that have been described before for MERS-CoV and SARS-CoV, also βcoronaviruses, but also with sepsis. Many markers have been demonstrated to be increased In SARS-CoV-2 infected individuals. These markers are related to innate as well as adaptive immunity, endothelial cell activation, thrombocyte activation and leukocyte infiltration. 204 The list of markers related to severe disease, ICU and even lethality is more limited. In ICU-admitted COVID-19 patients, significant increases of D-dimer, ferritin, LDH, IL-6, high sensitivity cardiac troponin, IL-2, IL-7, G-CSF, MCP-1, MIP-1α and TNF-α were reported. 204 An even more restricted group of markers (IL-10, MCP-3, IL-1ra) were increased in severe and lethal cases. 213 Differences in the biomarkers described are most probably due to the different sampling time during disease and the large heterogeneity between the patients. 204 Most likely, single biomarkers will not be predictive. On the other hand, a combination of markers (a biosignature) will help in patient stratification and may even guide patients-tailored therapy.

6. Urgent research needs for mechanistic, diagnostic approaches, therapeutic and preventive insight

Children experience milder COVID-19 as compared to adults, and a larger proportion of children remains asymptomatic (Fig. 6) . 206 

This article is protected by copyright. All rights reserved indicating that Th1/Th2 balance may significantly influence course of SARS-CoV-2 infection. 210 Therefore, type 1 IFNs driving anti-viral immunity may paradoxically promote SARS-CoV-2 expansion by upregulating ACE2 expression. Inflammatory responses differ throughout life, for example pre-existing chronic inflammation is common in elderly while absent in children. In addition, children have less potent PAMP activation, suboptimal, and Th2-skewed cytokine production, all resulting a hypo-inflammatory immune response. 216 This confers decreased protection against infections, but seem beneficial in preventing a CRS in SARS-CoV-2 infection. Hence, preferential Th2-skewed cytokine production observed in children is presumably protective (Fig. 6) 

PCR tests are useful for detecting SARS-CoV-2 RNA in an upper respiratory (preferably a nasopharyngeal)

specimens. In addition, a number of diagnostic procedures to assess immunity built against SARS-CoV-2 are still being developed, validated and optimized.

Antibody testing is evolving, and the market is flooded with test kits (both ELISA and rapid tests in the form of lateral flow immunoassays). However, only a small number of these kits are certified, and the results need to be interpreted with caution. Preliminary data indicate that COVID-19 presents with a classical antibody response consisting of early induction of IgM, followed by IgA and IgG antibodies (Fig.   7) . 131 IgG seems to appear early in the course of clinical presentation probably due to the relatively long incubation period. However, there is not yet enough evidence with regard to the development of longterm protective immunity. Antibody testing is so far more valuable in mapping the situation in individual populations, as planned by the WHO in the Solidarity II project. 220 Test kits for the assessment of SARS-CoV-2-specific T cell responses for diagnostic use are currently not available.

This article is protected by copyright. All rights reserved Table 2) . Eculizumab targets complement protein C5

preventing activation of complement terminal complex, which was used off-label in patients with SARS-CoV-2 infection and severe pneumonia or ARDS and is now evaluated in an ongoing trail (SOLID-C19). 246 Additionally, clinical trials with type I and III interferons in COVID-19 are currently conducted. 247, 248 Targeting T cell exhaustion to reverse the dysfunctional state and restore immune responses can be achieved by anti-PD-1 and LAG-3 therapies, 249,250 revealing novel therapeutic opportunities for persisting infections. In conclusion, prospective, randomized, placebo-controlled trials are needed to elucidate the clinical potential of immunomodulatory or passive immunization therapies.

Mucosal anti-viral immunity can be regulated by the microbiota via multiple mechanisms. The immune response to microbes is a form of host defence and entails a variety of intimate interactions with important symbiotic physiological effects on the host. 251 Specific bacterial components and specific metabolites can promote immune maturation and polarization, which ensures appropriate defence against occasional pathogens, while strongly promoting immune tolerance networks that dampen aberrant inflammatory responses 252, 253 . The composition of the gut and lung microbiome is strongly associated with the induction of polarized immune responses within the human lung. 190, 253 Bacterialderived metabolites such as short chain fatty acids (SCFAs) promote anti-viral responses in the lungs, while also reducing inflammation. 254, 255 Composition and metabolic activity of the gut microbiome has been associated with blood proteomic biomarkers predictive of severe COVID-19. 256 The integration of

This article is protected by copyright. All rights reserved microbial diagnostics with traditional immunological biomarkers will improve patient´s stratification and prognosis. In addition, the combination of microbial-derived therapeutics with immune modifying drugs, such as biologicals, will enhance response to treatment and better protect from damaging inflammatory processes.

Apart from the well-known measures of social distancing, washing hands and disinfection, which have proven to limit the SARS-CoV-2 spread, several prevention strategies can be considered from an immunological point of view. The WHO Strategic and Technical Advisory Group for Infectious Hazards (STAG-IH) regularly reviews and updates its risk assessment of COVID-19 to make recommendations. 257 For the future, it is essential to define the actual prevalence of COVID-19 in the population. Confirmation of infection at present consists of PCR for acute infection and serological tests to identify antibodies. 258 However, this may not be sufficient. The implementation of immune tests detecting neutralizing antibodies is key to define protection against SARS-CoV-2 (Fig 7) . This can only be achieved by implementing massive testing. Moreover, multiple vaccines are under development with the aim of preventing infection, reducing disease severity and viral shedding. A complete and continually updated list is available from the WHO. 259, 260 Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years.

Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. The COVID-19 pandemic is an extreme reminder of the role which animal reservoirs play in public health. Also, it reinforces the urgent need for globally operationalizing a One Health approach focusing on a broad surveillance for SARS-CoV-2 among different animals, and the possibility of reverse zoonosis. 261 Moreover, the current pandemic highlights the essential need for a broad understanding of immunological mechanisms underlying infectious diseases to design suitable therapeutic and preventive strategies. 

This article is protected by copyright. All rights reserved Tables   Table 1 . Summary of knowledge gaps and research needs pertaining to SARS-CoV-2 and COVID-19 (as of May 20, 2020).

Origin 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved characterized by a systemic cytokine release syndrome (CRS), increased levels of LDH and CRP, hypoalbuminemia, deepening decrease in lymphocyte counts and immune exhaustion of T cells. 

Bats are natural reservoirs of SARS-like coronaviruses

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A Genomic Perspective on the Origin and Emergence of SARS-CoV-2

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Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Kallikrein-kinin blockade in patients with COVID-19 to prevent acute respiratory distress syndrome

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Protease inhibitors targeting coronavirus and filovirus entry

Cell entry mechanisms of SARS-CoV-2

Distribution of ACE2, CD147, cyclophilins, CD26 and other SARS-CoV-2 associated molecules in various human tissues and immune cells in health and disease

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues

Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses

SARS-CoV-2 invades host cells via a novel route: CD147-spike protein

SARS-CoV-2 infects T lymphocytes through its spike protein-mediated membrane fusion

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Presence of SARS-CoV-2 reactive T cells in COVID-19 patients and healthy donors. medRxiv

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

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Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients

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Suspected myocardial injury in patients with COVID-19: Evidence from front-line clinical observation in Wuhan, China

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COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-up

Coagulation disorders in coronavirus infected patients: COVID-19, SARS-CoV-1, MERS-CoV and lessons from the past

Laboratory abnormalities in patients with COVID-2019 infection

Association between platelet parameters and mortality in coronavirus disease 2019: Retrospective cohort study

D-dimer is Associated with Severity of Coronavirus Disease 2019: A Pooled Analysis

Procalcitonin in patients with severe coronavirus disease 2019 (COVID-19): A meta-analysis

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study

Renal Involvement and Early Prognosis in Patients with COVID-19 Pneumonia

Kidney International and the COVID-19 infection

Focus on Receptors for Coronaviruses with Special Reference to Angiotensin-converting Enzyme 2 as a Potential Drug Target -A Perspective

Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2

The roles of CD147 and/or cyclophilin A in kidney diseases

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Does comorbidity increase the risk of patients with COVID-19: evidence from meta-analysis

Eleven faces of coronavirus disease 2019

Comorbidity and its impact on 1590 patients with Covid-19 in China: A Nationwide Analysis

Hypertension and Diabetes Delay the Viral Clearance in COVID-19 Patients. medRxiv

Comorbid diabetes results in immune dysregulation and enhanced disease severity following MERS-CoV infection

Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients

Binding of SARS coronavirus to its receptor damages islets and causes acute diabetes

Network-based analysis of fatal comorbidities of COVID-19 and potential therapeutics

Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications

Susceptibility Analysis of COVID-19 in Smokers Based on ACE2

ACE-2 Expression in the Small Airway Epithelia of Smokers and COPD Patients: Implications for COVID-19

Elevated Plasmin(ogen) as a Common Risk Factor for COVID-19 Susceptibility

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is likely to be androgen mediated

Increased androgen receptor gene copy number is associated with TMPRSS2-ERG rearrangement in prostatic small cell carcinoma

Comorbidities in the Elderly and Their Possible Influence on Vaccine Response

Frailty is associated with elevated CRP trajectories and higher numbers of neutrophils and monocytes

Inflammaging: a new immune-metabolic viewpoint for age-related diseases

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical Characteristics of Coronavirus Disease 2019 in China

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention

Disease 2019 in Children -United States

Intranasal corticosteroids in allergic rhinitis in COVID-19 infected patients: An ARIA-EAACI statement

Handling of allergen immunotherapy in the COVID-19 pandemic: An ARIA-EAACI statement

Asthma and COVID-19: is asthma a risk factor for severe outcomes? Allergy

Association of Respiratory Allergy, Asthma and Expression of the SARS-CoV-2 Receptor, ACE2

COVID-19: consider cytokine storm syndromes and immunosuppression

Advanced forecasting of SARS-CoV-2 related deaths in Italy

Exuberant elevation of IP-10, MCP-3, and IL-1ra during SARS-CoV-2 infection is associated with disease severity and fatal outcomes

High-Resolution Computed Tomography Manifestations of 5 Pediatric Patients With 2019 Novel Coronavirus

An analysis of SARS-CoV-2 viral load by patient age

Is the developmentally immature immune response in paediatric sepsis a recapitulation of immune tolerance?

Hyperinflammatory shock in children during COVID-19 pandemic

Multisystem inflammatory syndrome in children and adolescents temporally related to COVID-19

WHO. Report of the WHO-China Joint Mission on Coronavirus Disease

These are answers we need. WHO plans global study to discover true extent of coronavirus infections. Science Magazine

A single center observational study of the clinical characteristics and short-term outcome of 20 kidney transplant patients admitted for SARS-CoV2 pneumonia

Tocilizumab, an anti-IL6 receptor antibody, to treat Covid-19-related respiratory failure: a case report

Covid-19 pneumonia in a kidney transplant recipient successfully treated with Tocilizumab and Hydroxychloroquine

Rapid and Severe Covid-19 Pneumonia with Severe Acute Chest Syndrome in a Sickle Cell Patient Successfully Treated with Tocilizumab

Favorable changes of CT findings in a patient with COVID-19 pneumonia after treatment with tocilizumab. Diagn Interv Imaging

Targeting the inflammatory cascade with anakinra in moderate to severe COVID-19 pneumonia: case series

Interleukin-1 blockade with high-dose anakinra in patients with COVID-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study

Favorable Anakinra Responses in Severe Covid-19 Patients with Secondary Hemophagocytic Lymphohistiocytosis

Baricitinib therapy in COVID-19: A pilot study on safety and clinical impact

Treatment with convalescent plasma for COVID-19 patients in Wuhan

Eculizumab treatment in patients with COVID-19: preliminary results from real life ASL Napoli 2 Nord experience

COVID-19: lambda interferon against viral load and hyperinflammation

Type 1 interferons as a potential treatment against COVID-19

Restoring function in exhausted CD8 T cells during chronic viral infection

Clinical blockade of PD1 and LAG3--potential mechanisms of action

Recent developments and highlights in mechanisms of allergic diseases: Microbiome

The influence of the microbiome on respiratory health

The Role of Lung and Gut Microbiota in the Pathology of

High levels of butyrate and propionate in early life are associated with protection against atopy

Dietary Fiber Confers Protection against Flu by Shaping Ly6c(-) Patrolling Monocyte Hematopoiesis and CD8(+) T Cell Metabolism

Gut microbiota may underlie the predisposition of healthy individuals to COVID-19. medRxiv

Strategic and Technical Advisory Group for Infectious Hazards (STAG-IH)

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WHO. DRAFT landscape of COVID-19candidate vaccines -20

From SARS to COVID-19: A previously unknown SARS-related coronavirus (SARS-CoV-2) of pandemic potential infecting humans -Call for a One Health approach

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