Immune Response
Following the viral entry, the host immune system recognizes the whole virus or its epitopes, initiating both the innate and adaptive immune response. SARS-CoV-2 infects through the naso-oral route, infecting cells expressing ACE2 receptor first in the upper respiratory tract and then to the lower respiratory tract. Macrophages, neutrophils, and NK cells are activated to kill infected cells. However, the infiltration of macrophages, neutrophils, and other adaptive immune cells leads to increased pro-inflammatory cytokines. The stimulation of Th1/Th17 cells also leads to aggravated inflammatory responses, which results in “cytokine storms” that lead to immunopathologies like pulmonary edema and pneumonia. Cytotoxic T cells recruited to the site of infection induce apoptosis of virus-infected cells. B cells/plasma cells also recognize viral proteins and are activated to produce antibodies specific to SARS-CoV-2 [17]. Figure 5 shows an overview of immune response against SARS-CoV-2.
Fig. 5. Plausible host immune responses during COVID-19 infection.
Innate Immune Response
After the virus enters the cell, the infected cell detects virus replication through pattern recognition receptors (PRRs), that can bind to both pathogens inside and outside of the cell. For SARS-CoV-2 viral infection, viral replication is recognized by intracellular PRRs via aberrant RNA structures produced during virus replication. The interaction between virus-specific RNA structures and PPRs results in oligomerization of receptors and activating downstream transcription factors, such as interferon regulator factors (IRFs) and nuclear factor kB (NF-kB). The activation of IRFs and NF-kB initiates two antiviral programs. The first is cellular antiviral defenses, which are mediated by type I and III interferons (IFN-I and IFN-III) and subsequent upregulation of IFN-stimulated genes (ISGs). The second antiviral response involves the recruitment of leukocytes, which are coordinated by chemokines [18].
Toll-like Receptors (TLR)
The first line of defense against infections is innate immunity. Particularly, the virus is recognized by Toll-like receptors (TLR). TLRs recognize pathogen-associated molecular patterns (PAMPs) and activate downstream immune responses. TLR3, TLR4, TLR7, and TLR8 interact with SARS-CoV-2 and activates Myd-88 (myeloid differentiation primary response 88) and TRIF (TIR-domain-containing adapter-inducing interferon-β), which stimulate the production of inflammatory cytokines and chemokines.
Studies on SARS-CoV and MERS showed TLR3 mediates the development of immune responses against coronaviruses. High expression of TLR3 is found in dendritic cells, placenta, and pancreas, and it recognizes viral RNA at the endosome. Then its activation through the TRIF pathway results in the activation of transcription factors, such as IRF3 and NF-kB, associated with increased production of type I interferons (IFN alpha and beta), inflammatory cytokines (IL-6, TNF), and IFN-gamma [18,20,24].
TLR4 usually recognizes LSP in gram-negative bacteria similar to TLR3 and then activates transcription factors such as NF-kB and IRF3, initiating an inflammatory cascade of TLR3. TLR4 is also found to be triggered by oxidized phospholipids (OxPLs) induced by SARS-CoV2 infection. Similar to TLR3, TLR4 then activates MyD88 and TRIF, which leads to the production of type I interferon and inflammatory cytokines such as IL6 and TNF. IL6 may contribute to lung damage.
However, there are some distinctions between TLR3 and TLR4 pathways (shown in Figure 6). TRIF is associated with TLR 3 and determines the activation of IFR3 and NK-kB, while Myd-88 interacts with TLR4 and several proteins involved in IL1 function, such as IRAK1–2 and interleukin 1 (IL1) [16].
TLR7 and TLR8 also recognize viral RNA at the endosome and through MyD88 and TRIF and activate IRF3 and IRF7. TLR7 is expressed at high levels in human plasmacytoid dendritic cells and B cells. TLR7 binds ssRNA and activates the MyD88 pathway, which subsequently activates mitogen-activated protein kinase (MAPK) cascade, NF-kB, and other pathways. This activation increases the expression of TNF-alfa, IL1beta, IL-6, IL12, and IFN-alfa. TLR8 is expressed in myeloid cells and low levels in human plasmacytoid dendritic cells. TLR8 binds with RNA degeneration product and leads to the activation of MyD88 with a downstream pathway similar to TLR7 (Figure 6) [16].
Fig. 6. Role of toll-like receptor in response to coronavirus infection.
Cytokines and Interferon
Innate immunity is initiated by PAMPs, characteristics of pathogens to which PRRs bind. The binding triggers subsequent signaling pathways that activate transcription factors including NF-κB, IRF3, and AP-1, which promote type I interferon (IFN-I) production. These cytokines induce the expression of interferon-stimulated genes (ISGs) [36]. Interferon-stimulated genes play a significant role in innate antiviral defense, by limiting viral entry and restricting viral replication. Interferon-stimulated gene expression is driven predominantly by IFNAR-mediated activation of the Jak/STAT pathway, resulting in the binding of STAT1 homodimers and STAT1/2 heterodimers to the promoter region of ISGs [23]. Interferons (IFN) are essential in the inhibition of viral replication, through different effector proteins. There are three types of interferons, Type I (interferon α β), Type II (Interferon γ), and Type III (Interferon λ). All three types of interferons demonstrate protection from coronavirus infection. The production of type I IFN is enhanced by viral RNA via two cytosolic proteins: RIG-1 (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated protein 5). The recognition of viral RNA by these cytosolic receptors leads to the activation of IRF3 and determines the initiation of the transcription of type I IFN [22].
Figure 7 illustrates an overview of the potential innate immune responses against severe SARS-CoV-2 infection. There are increased levels of macrophages and neutrophils in the lung. Inflammatory macrophages secrete IL-1b, activating T cells. Activated DCs present viral antigens to naive T cells. NK cells, inflammatory macrophages, and activated neutrophils could kill infected type II alveolar epithelial cells. Additionally, the MAC may result in the lysis of infected cells. Complement proteins and chemokines attract additional immune cells to the site of infection [20].
Neutrophils
The complete blood count of patients infected with SARS-CoV-2 showed an increase in neutrophils with lymphopenia. The pathogenetic role of neutrophils in coronaviruses infections is unclear, a significant neutrophil infiltration is reported in autopsies of COVID-19 patients [16,22]. However, studies on the pathogenic role of neutrophils from other coronavirus infections showed that activated neutrophils help clear the virus by killing infected cells (Figure 7) [16,20]. The role of neutrophils seems dichotomous. On the one hand, neutrophils recruit inflammation cells at the early stage of infection and kill infected cells. On the other, they contribute to tissue damage when their concentration is adequately controlled.
Macrophages
Macrophages reduce early viral replication by initiating an IFN-I response and an inflammatory response to recruit immune cells [25]. However, excess inflammation, a cytokine storm, contributes to the mortality associated with COVID-19. Significant dysregulation of monocytes and macrophages is a feature of severe COVID-19 [20].
The specific role of macrophages can be inferred from immune responses observed in other coronavirus infections because genome analysis shows that there is a high similarity between the SARS-CoV-2 genome and the SARS-CoV. SARS-CoV has an accessory protein, Open Read Frame 8 (ORF-8), that activates intracellular stress mechanisms, lysosomal damage, and autophagy. At a macrophage level, the intracellular aggregation caused by ORF-8 interact with Cryopyrin, a structural protein of the inflammasome that activates Inflammasome. The Inflammasome is responsible for the activation of inflammatory responses. Inflammasome recruits pro-caspase-1 that cleaves pro-inflammatory cytokines, such as pro-IL-1 β and pro-IL18, thus activating them into IL-1 β and IL18. This leads to a programmed cell death called pyroptosis, in which intracellular pathogen is recognized and infected cells undergo programmed cell death. In addition, IL18 stimulates the production of IFN gamma, which facilitates the development of TH1. IL-1 released by macrophages through the inflammasome also contributes to the cytokine storm [16].
Natural Killer (NK) cells
Reduced NK cell count in peripheral blood is frequently observed in severe COVID-19 patients. Killer-immunoglobulin-like receptors (KIRs) expressed on the membrane of NK cells as well as CD16 plays an important role in the cytotoxic functions of NK cells. The decreasing number of NK cells in peripheral blood in SARS-CoV and SARS-CoV-2 patients suggest migration of circulating NK cells into the peripheral tissues. Additionally, a lower NK cell count correlates with a higher IL-6 plasma concentration in SARS-CoV-2 infection because of impaired perforin produced by healthy NK cells. Furthermore, cytokines such as IL-12 secreted by macrophages and dendritic cells promote NK cell proliferation, cytotoxicity, survival, and IFN-γ production, which suggests that early IFN-γ production and NK cell stimulation could limit SARS-CoV-2 infection [23].
Fig. 7. The Innate Immune Response to Severe SARS-CoV-2 Infection of the Lung. Solid lines represent interactions that have been reported. Dashed lines represent interactions that have not been reported and warrant future studies. DC, dendritic cell; NK cell, natural killer cell; MAC, complement membrane attack complex.
Adaptive Immune Response
Both T and B cell responses against SARS-CoV-2 are detected in the blood around 1 week after the first appearance of COVID-19 symptoms.
T cell immunity
Dendritic cells from the lungs present antigens to activate naïve CD8+T cells, resulting in the differentiation and proliferation of cytotoxic T cells. CD8+ T lymphocytes employ cell-associated mediators such as FasL, perforin, or granzyme B to initiate apoptosis of infected cells that present abnormal MHC. CD8+ T cells are cytotoxic and directly attack and kill virus-infected cells, while CD4+ T cells are crucial to the production of virus‐specific antibodies by activating T‐dependent B cells. CD4+ T cells are also responsible for cytokine production to recruit additional immune cells [19,26]. Because of CD8+ T cells' cytotoxic nature, they play a vital role in clearing CoVs in infected cells and inducing immune injury. An autopsy of a patient with COVID-19 infection revealed that mononuclear cells (monocytes and T cells) accumulated in the lungs, and a low level of hyperactive T cells were found in the peripheral blood. Lymphopenia and reduced peripheral T cell levels suggest that T cells are attracted to the infected site to control the viral infection. Increased T cell exhaustion and reduced functional diversity are an indication of severe infections. After the virus is cleared, most effector T cell undergoes apoptosis and specific memory T cells are generated by patients recovered from SARS-CoV infection. CD4+ memory T cells, once being re-stimulated, can activate B cells and other immune cells by cytokines, and cytotoxic memory T cells kill the infected cells during the re-infection [18,19].
CD4+ T cells express IFNγ, TNF, and IL-2, indicating that patients with SARS-CoV infection exhibit TH1 response. T helper cells help in the regulation of B cell proliferation, differentiation, and class switching. T follicular helper cells also play crucial roles in the somatic hypermutation of B cells in the germinal centers [19]. CD4+ T cells contribute to the production of antibodies, induce the activity of cytotoxic T cells, and function as memory cells. Although the pro-inflammatory feature can contribute to immunopathogenesis, studies have shown that CD4+ T cells control SARS, as a reduction in the number of CD4+ T cells resulted in slower clearance of the virus from the host and severer lung inflammation [19].
B cell immunity
Humoral response against SARS-CoV-2 is similar to that of other coronaviruses, involving the IgG and IgM production. B cell responses occur concomitantly with Tfh cell responses, from around 1 week after the onset of symptoms. For SARS-CoV infection, B cells elicit an initial response against N protein, while antibodies against S protein are found within 4–8 days after symptom onset [18,19].
Neutralizing antibodies start to develop after week 2. Antibodies can be effective against SARS-CoV-2 since serum samples from recovered patients were applied to treat COVID-19 patients and good clinical results were shown [19].
Although the specific titre and specificity of the antibody repertoire remain unconfirmed, neutralization is hypothesized to be an important action for antibodies. In SARS-CoV, the primary target of neutralizing antibodies is the RBD, consisting of 193 amino acid region (amino acids 318–510) in the S protein. Although a few identified monoclonal antibodies to SARS-CoV also neutralize SARS-CoV-2, the majority of antibodies do not. This could be due to significant differences in the RBDs of SARS-CoV-2 and SARS-CoV due to the unconserved amino acid sequences. However, neutralizing epitopes overlap between SARS-CoV and SARS-CoV-2, since mouse antiserum against SARS-CoV can cross-neutralize SARS-CoV-2 pseudovirus [19].