{'fraction_non_alphanumeric': 0.03750753595728189, 'fraction_numerical': 0.02514856601498579, 'mean_word_length': 5.002326182476092, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The immunology and immunotherapy for COVID-19

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and significantly impacts the world economy and daily life. Symptoms of COVID-19 range from asymptomatic to fever, dyspnoea, acute respiratory distress and multiple organ failure. Critical cases often occur in the elderly and patients with pre-existing conditions. By binding to the angiotensin-converting enzyme 2 receptor, SARS-CoV-2 can enter and replicate in the host cell, exerting a cytotoxic effect and causing local and systemic inflammation. Currently, there is no specific treatment for COVID-19, and immunotherapy has consistently attracted attention because of its essential role in boosting host immunity to the virus and reducing overwhelming inflammation. In this review, we summarise the immunopathogenic features of COVID-19 and highlight recent advances in immunotherapy to illuminate ideas for the development of new potential therapies.


Introduction
At the end of 2019, a total of 41 cases of pneumonia of unknown aetiology were first reported and then spread rapidly throughout the world (Ref. 1). Further studies identified this novel zoonotic virus as an enveloped, positive-sense single-stranded RNA coronavirus belonging to the subgenus Sarbecovirus of the genus Betacoronavirus (Ref. 2). It is named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the disease caused by SARS-CoV-2 was termed corona virus disease 2019 (COVID-19) by the World Health Organization (WHO). On 31 January 2020, the WHO announced that COVID-19 is listed as Public Health Emergency of International Concern. By the end of July 2021, SARS-CoV-2 has spread rapidly and affected more than 200 countries, resulting in more than 230 million identified cases and 4.7 million confirmed deaths (Ref. 3). SARS-CoV-2 is the seventh coronavirus known to infect humans, and the third coronavirus emerged as a public health issue over the past two decades. SARS-CoV-1, MERS-CoV and SARS-CoV-2 can cause serious illness, whereas HKU1, NL63, OC43 and 229E are associated with mild symptoms (Ref. 4). In a previous study of 44 672 patients with COVID-19 in China, 81% of infected patients had a broad spectrum of clinical manifestations ranging from asymptomatic to cough, fever, coagulation dysfunction and metabolic acidosis. A total of 14% had severe manifestations, and 5% had critical manifestations, such as hyper-inflammation, multiple organ dysfunction syndromes and acute respiratory distress syndrome (ARDS) (Ref. 5). Most mortalities happened in elderly patients or patients with multiple comorbidities, including cardiovascular diseases, respiratory diseases, diabetes mellitus, hypertension and immune-compromised patients, such as cancer (Ref. 6).
The pathogenesis of COVID-19 is thought to be determined by two main courses. In the early phase of infection, SARS-CoV-2 identifies host cell angiotensin-converting enzyme 2 (ACE2) receptors and invades the host cell to complete the replication cycle. The cellular damage caused during the replication cycle can lead to respiratory disease, a decrease in lymphocytes and local/systemic inflammation. As the infection progresses, immune pathologies such as cytokine storm and lymphopoenia occur (Ref. 7).
There is currently no specific treatment available. Treatment strategies for COVID-19 infection are early detections, the quarantine of new cases and supportive therapies for the confirmed individual (Ref. 8). Immunotherapy plays an important role in inhibiting viral infection or modifying the overactivated immune response against SARS-CoV-2. In this review, we summarise the immunopathogenic features of COVID-19 and highlight current advances in immunotherapies to combat COVID-19, hoping to enlighten ideas of developing new potential therapies in the future.

Viral entry and replication
Epidemiological and virological studies suggest that SAR-CoV-2 is transmitted to others mainly by symptomatic and asymptomatic individuals through close contact via respiratory droplets or direct contact with infected individuals, sometimes through contaminated objects and surfaces (Ref. 8). The spike (S) protein of SARS-CoV-2 determines the tropism of receptors in host cells and plays a vital role in the invasion process. Figure 1 summarises the recognition and replication cycle of SARS-CoV-2. The S protein is composed of two subunits: S1 and S2. S1 is responsible for the binding of the viral receptorbinding domain (RBD) to the host cell ACE2, and S2 ensures the fusion of the virus with the host cell membrane (Ref. 9). SARS-CoV-2 employs the cellular transmembrane serine protease 2 (TMPRSS2) for S protein priming (Ref. 10). Most recently, neuropilin 1 has been identified as an important cofactor for entry, particularly in cells with low-level ACE2 expression (Ref. 11). Studies also show that the interaction of the S protein with the CD26 and CD209L could be a possible way of viral entry, but the mechanism is still unclear (Refs 12,13). During the fusion process, binding of the virus to ACE2 causes stabilisation of the RBD, which triggers conformational changes in the S-complex, leading to the release of the S1 subunit and activation of the fusogenic activity of S2 (Ref. 14). A study demonstrated that strains isolated from COVID-19 patients could use the host cell protease TMPRSS2 and cathepsins B/L to prime the S protein in vitro (Ref. 15). Structural changes in the S protein may prevent the immune system from recognising the virus. For example, the current delta variant has three mutations at the S1 subunits, which increases the affinity of RBD to bind to ACE2 and enhances its ability to escape our immune system (Ref. 16).
ACE2 is an enzyme that belongs to the renin-angiotensin system and is located on the cell surface of type II alveolar epithelial cells in the lungs and other tissues (Ref. 17). ACE2 plays a critical role in controlling vasoactive effects in the body and is highly co-expressed with genes related to TMPRSS in the upper airways ( Refs 18,19). The affinity of SARS-CoV-2 to ACE2 is 10-20 times higher than that of SARS-CoV, possibly because of the RBD of SARS-CoV-2 having a more compact conformation (Ref. 20). Several residue changes in the RBD of SARS-CoV-2 stabilise two virus-binding hotspots at the RBD-ACE2 interface and increase ACE2 binding affinity (Ref. 21).
Once the virus enters the host cell cytoplasm, translation begins using viral RNA as a template to generate virus-specific mRNAs. After the translation of virus-specific mRNAs is completed, the production of structural and nonstructural viral proteins is incorporated into the endoplasmic reticulum or Golgi membrane (Ref. 22). Finally, the vesicles containing the virus particles fuse with the plasma membrane to release the virus (Ref. 23). Another possible mechanism for SARS-CoV-2 entry is that the virus binds to its corresponding antibody to form an antigen-antibody complex and then enters the target cell through the Fc receptor (Ref. 24).

Innate and adaptive immunity against SARS-COV-2
Activated natural killer (NK) cells exert a major histocompatibility complex-independent immune response against SARS-CoV-2 and may limit viral pathogenesis in the early stages of infection (Ref. 25). The respiratory tract is the first front line of the immune system, which contains many immune effector cells such as epithelial cells, type 1 macrophages (M1) and dendritic cells (DCs). After infection, epithelial cells produce immune cytokines and express adhesive molecules, recruiting immune cells to the lung tissue to combat the virus. After virus recognition, activation of interferon regulatory factor 3 (IRF-3) in M1 leads to the production of various immune cytokines such as type I interferon (IFN-I) and phagocytosis (Ref. 26). IFN-I plays an essential role in the immune response against SARS-CoV. One of the mechanisms of anti-viral immunity by IFN-I is activating IFN-induced transmembrane family proteins, which inhibit virus entry into the host cell. DCs can serve as the antigen-presenting cell (APC)-mediated immune response to SARS-CoV-2 and produce IFN-I and interleukin 6 (IL-6) to inhibit viral infection. A study shows that excessive production of IFN-I by DCs leads to severe inflammation and ARDS in severe patients (Ref. 27). The APCs, such as monocytes and DCs, distinguish the viral antigen on infected cells, introduce the antigen to the helper T (Th) cells, and produce cytokines that direct the anti-viral immune response of the T cells. Serum levels of Th1-associated cytokines, including IFN-γ, tumour necrosis factor (TNF)-α and IL-2, are reported to be elevated in COVID-19 patients (Ref. 28). These cytokines activate cytotoxic T cells (CTLs) to attack the virus-infected cells and destroy them by producing perforin and granzymes. In addition, Th2 cells present the viral antigen to B lymphocytes, which subsequently produce neutralising antibodies against the S protein of the virus. The neutralising antibodies inhibit the replication of the virus in the body and generate humoral immunity, which is one of the most important concepts for developing vaccines against SARS-CoV-2 (Ref. 23). Figure 2 briefly summarises the immunopathology of COVID-19.
Humoral immunity plays a vital role in the induction of adaptive immunity to coronaviruses. Activation of B cells and plasma cells leads to the production of neutralising antibodies that prevent further infection with the virus. Appropriate anti-viral antibodies can prevent patients from reinfecting the virus. Some cases of reinfection because of insufficient serum antibody levels have been reported, questioning the humoral immunity of SARS-CoV-2 (Ref. 32). Further research is needed to understand the exact mechanism of the immune response to SARS-CoV-2.

Cytokine storm
As illustrated in Figure 3, cytokine storm is a state of uncontrolled systemic hyper inflammation caused by an excess of cytokines, leading to multiorgan failure and even death. Rather than direct cytopathic effects of the COVID-19, cytokine storm activates inflammatory immune cells to attack alveolar cells, causing lung tissue damage (Ref. 33). The inflamed lung tissue also stimulates fibroblast cells to form fibrotic tissue in the lungs. In addition, the leakage of fluid into the alveoli and the accumulation of the inflammatory exudate lead to the formation of hyaloid membranes and further ARDS (Ref. 34). Damaged lung cells and M1 produce chemotactic factors that attract other immune cells and trigger an uncontrolled inflammatory response in the lung. Then the inflammatory cells begin an uncontrolled production of pro-inflammatory cytokines and chemokines contribute to the cytokine storm, including IL-1, IL-6 (Ref. 35), IL-8, IL-10, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), CXCL-1, -3, -10 and CCL-2, -3 (Refs 36-38). Increased IL-6 and IL-8 lead to severe inflammation in critical patients (Ref. 26).
Currently, one article introduces the concept of dividing the cytokine storm into two phases: the first phase is a temporary state of immunodeficiency, and the second phase is an overactive state of the immune system to compensate for the failure of target clearance, resulting in a cytokine storm (Ref. 39). Another report described the effects of human coronavirus on cytokines, noting . These studies underscore that the cytokine storm results from the failure of the initial response to the type I and III IFNs of SARS-CoV-2, leading to the delayed response of the immune system the inability to clear the virus. According to the pathogenesis of cytokine storm, appropriate treatments such as IL-1, IL-6 and TNF-α inhibitors can reduce lung tissue damage and lead to a better outcome in COVID-19 patients (Ref. 42). Several strategies, such as injection of immunomodulatory drugs and mesenchymal stem cells (MSCs), have been used to prevent lung injury and multiple organ failure in severe patients with cytokine storm. Targeting inflammatory cytokines could also reduce the severity of cytokine storm. Abnormalities in granulocytes and monocytes have also been observed in COVID-19 patients (Fig. 2). The number of neutrophils is significantly higher in severe patients than in non-severe patients (Ref. 52). At the same time, a lower percentage of eosinophils, basophils and monocytes was observed in the serve patients (Ref. 53). The mechanisms are currently unclear, and further research is needed.

Immunotherapy of COVID-19
Currently, there is no proven specific treatment modality for COVID-19. Initial treatments are mainly supportive, including oxygen therapy, ventilator support in patients with respiratory failure, antibiotics to prevent secondary bacterial infections, antiinflammatories and fluid management (Ref. 54). Since the COVID-19 outbreak, scientists have begun to evaluate the impact of currently available anti-viral drugs on the virus. Because the Immunotherapy has achieved remarkable results in the treatment of many diseases such as cancer and viral infections. The use of   Yixin Liu et al.
immune-enhancing agents to boost and strengthen the immune system could have a positive impact on the treatment of COVID-19. Figure 4 summarises the immunotherapy of COVID-19, which will be discussed step by step below. The clinical trials of immunotherapy for COVID-19 are also summarised (Table 1).    Llama-derived single-domain antibodies, also known as nanobodies, are generally more heat stable, accessible and cheaper to produce and can be better used for protein engineering than conventional antibodies. Combining nanobodies with different epitopes or complementary epitopes increases resistance to mutant escape (Ref. 80).

IFN therapy
Intravenous immunoglobulin (IVIG) is widely used as an alternative therapy to treat immunodeficiency and reduce inflammatory responses (Ref. 81). The use of high-dose IVIG in hospitalised COVID-19 patients may improve their clinical progress by regulating their immune status. For example, IVIG may reduce the excessive inflammation observed in individuals infected with SARS-CoV-2 by reducing the levels of cytokines, chemokines and some complement factors (C5a) involved in immune activation and cell migration (Ref. 82). A 5% anti-COVID-19 IVIG solution contains 90% immunoglobulins (IgG, IgM and IgA), and the total anti-SARS CoV-2 antibody titre is three times higher than pooled convalescent plasma (Ref. 83).

Vaccine therapy
The first COVID-19 vaccine urgently approved by the U.S. FDA was BNT162b2, an mRNA encoding S protein, which showed 95% efficacy (Ref. 84). The second vaccine, mRNA-1273, had almost the same efficacy as BNT162b2, and it remains to be investigated whether BNT162b2 can elicit a long-term anti-SARS-CoV-2 immune response. The third vaccine, CVnCoV/CV2CoV, has been shown to prevent disease and death caused by the VOCB. Protein vaccines require adjuvants to enhance the immune response. Novavax's vaccine uses a genetically modified full-length S protein (NVX-CoV2373) that has higher structural stability. When administered to volunteers, the NVX-CoV2373 vaccine has a protective efficacy of 89.7% against SARS-CoV-2 infection and shows effective protection against the B.1.1.7 variant (Ref. 89). Studies have shown that the neutralising antibody response elicited by the Ad5-nCoV vaccine against COVID-19 with two doses of the atomised adenovirus type 5 vector is similar to that elicited by one dose of the intramuscular injection, and nebulisation at 28 days after the first intramuscular injection enhanced the vaccination and induced a strong IgG and neutralising antibody response (Ref. 90). Table 2 lists the COVID-19 vaccines within WHO Emergency Use Listing (EUL) and prequalification (PQ) evaluation process.
The humoral and cellular immune responses elicited by the different vaccines differed considerably. In particular, inactivated vaccines showed relatively low levels of neutralising antibodies and T-cell responses. Compared with three consecutive doses of inactivated vaccines to enhance the immune response, the use of recombinant subunits, adenoviral vectors or mRNA vaccines after two doses of inactivated vaccines can further boost neutralising antibodies and specific Th1 cell responses (Ref. 91). Modified vaccinia Ankara (MVA) vectors expressing membrane-anchored prefusion-stabilised spike (MVA/S) but not secreted S1 induce strong neutralising antibody responses against SARS-CoV-2 in mice. MVA/S vaccination triggered potent neutralising antibodies and CD8+ T-cell responses in macaques and protected them from SARS-CoV-2 infection, demonstrating that MVA/S is a potential vaccine candidate (Ref. 92).  TNF-α is an inflammatory cytokine that triggers leucocyte recruitment to the site of infection in response to SARS-CoV-2 (Ref. 99). IFN-γ is a vital biomarker promoting the pathogenesis of ARDS and acute lung injury. The synergistic effect of TNF-α and IFN-γ leads to inflammatory cell death, tissue damage and mortality in SARS-CoV-2 infection (Ref. 100). Therefore, inhibition of TNF-α and IFN-γ is a promising immunotherapy method to inhibit ARDS progression in severe COVID-19 patients. For example, XPro1595, a soluble TNF-α-neutralising protein that inhibits the interaction between soluble TNF-α and its receptor, is currently evaluated in a clinical trial to treat COVID-19 (NCT04370236). Infliximab, an approved mouse/human chimeric monoclonal antibody, is undergoing clinical trials to evaluate its therapeutic effect on COVID-19 (NCT04922827, NCT04425538 and NCT04593940). GM-CSF is an immunoregulatory cytokine that plays a central role in initiating and maintaining inflammatory diseases. Several clinical trials have considered the use of otilimab (NCT04376684), lenzilumab (NCT04351152), gimsilumab (NCT04351243), mavrilimumab (NCT04492514), TJ003234 (NCT04341116) and sargramostim (NCT04707664) to block GM-CSF as potential treatments for COVID-19.

Management of cytokine storm
The complement system could cause some pathophysiological aspects of COVID-19 infections, such as thrombotic microangiopathy

Ligands on virus-infected cells induce NK cells to activate and release IFN-γ and TNF-α, thereby lysing virus-infected cells.
Ongoing clinical trials NCT04280224 and NCT04324996 in China will investigate the role of NK cells in severe pneumonia or lymphopoenia triggered by COVID-19. Recent studies have shown that chimeric antigen receptor (CAR)-NK cell therapy exhibits effective antitumor activity in haematologic cancers with minimal side effects (Ref. 110). CAR-NK cells use the scFv of S309, a neutralising antibody that targets the highly conserved region of the SARS-CoV-2 S protein can kill target cells expressing SARS-CoV-2 S protein (Ref. 111).
SARS-CoV-2-specific memory CD8+ T cells were detectable in SARS-CoV-2 convalescent individuals (Ref. 112). Furthermore, the peptide-specific memory CD8+ T cells of SARS-CoV-2 could proliferate when the antigen was re-attacked, suggesting that SARS-CoV-2-specific CD8+ T cells are not exhausted but functional (Ref. 113). Overexpression of inhibitory receptors CTLA4 and PD-1/PD-L1 is a typical characteristic of the exhaustion of CD8+ T cells, leading to decreased effector activity and proliferation ability. The total number of NK and CD8+ T cells was significantly decreased, and the function of NK and CD8+ T cells was exhausted in patients with SARS-CoV-2 infection (Ref. 114). Functional blockade of CTLA4 and PD-1/PD-L1 may unleash exhausted NK and CD8+ T cells, enhancing antiviral immunity against SARS-CoV-2 ( Refs 115,116).
DC is a crucial bridge between innate and adaptive immunity in anti-viral infection. COVID-19 DCs are shown to promote the secretion of cytokines and chemokines, regulate the inflammatory response and induce the differentiation of CD4+ and CD8+ T cells against pathogens (Ref. 117). In addition, plasma cell-like DCs inhibit viral replication by rapidly producing IFN-I (Ref. 118). It may be helpful to inhibit the functions of DC to control the excessive inflammatory process in severe COVID-19 patients.

Other immunotherapies
Fulminant activation of coagulation and consumption of clotting factors occurs in severe cases of COVID-19, leading to disseminated intravascular coagulation and patient death (Ref. 119). On the other hand, coagulation factors can increase inflammation by acting on specific cell receptors. Considering these two aspects, inhibition of blood clotting may decrease the mortality of COVID-19 patients by reducing the risk of small intravascular thrombosis and severe inflammation. In a retrospective study, COVID-19 patients taking anticoagulants at moderate doses (7 mg/kg per 12 h) during hospitalisation were associated with a significantly lower cumulative incidence of in-hospital death compared with patients not receiving anticoagulants (Ref. 120). Lowdose radiotherapy (LDRT, <100 cGy) has been used to treat a variety of chronic inflammatory diseases. LDRT is reported to have an anti-inflammatory effect by downregulating proinflammatory macrophages and upregulating anti-inflammatory macrophages and NK T cells. Thus, LDRT has the potential to counter the immune reaction incited by 122). The latest research studies suggest that LDRT is feasible in COVID-19 patients and has shown promising results in COVID-19 pneumonia ( Refs 123,124). IL-15 is expressed by myeloid cells to support T-cell responses, activate NK cells and modulate inflammation, and is a key immunomodulatory cytokine with antiviral properties (Ref. 125). Induction of IL-15 can elicit a robust immune response against SARS-CoV-2 (Ref. 126). IL-15 and IL-21 combination therapy for COVID-19 has also been performed (Ref. 127).

Conclusion
In this review, we have reported several aspects of the immunopathogenesis of COVID-19 and analysed the mechanism of the host immune response against this disease. Among immunotherapeutic approaches for blocking viral attachment or entry, monoclonal antibodies are preferred because of their specificity, purity, low risk of contamination with bloodborne pathogens. A monoclonal antibody cocktail or combination of different monoclonal antibodies recognising different epitopes on the viral surface may increase the efficacy of virus neutralisation.
COVID-19 is an infectious disease that causes an imbalance in the immune system and an inflammatory cytokine storm. Immunosuppressants and anti-inflammatory drugs are potential agents for the treatment of cytokine storm. For example, immunomodulators with IL-6 blockers have the therapeutic potential to specifically inhibit the status of hyperinflammation. Controlling the excessive inflammatory response is crucial to prevent the progression of ARDS in severe cases. On the other hand, enhancing host immunity is crucial to ensure that the host is sufficiently able to fight viral infections. These two aspects are essential concerns of immunotherapy against COVID-19. However, further studies are needed to evaluate the efficacy of immunotherapy in patients infected with COVID-19.