COVID-19 antibodies preferentially target a different part of the virus in mild cases of COVID-19 than they do in severe cases, and wane significantly within several months of infection, according to a new study by researchers at Stanford Medicine.
The findings identify new links between the course of the disease and a patient’s immune response. They also raise concerns about whether people can be re-infected, whether antibody tests to detect prior infection may underestimate the breadth of the pandemic and whether vaccinations may need to be repeated at regular intervals to maintain a protective immune response.
“This is one of the most comprehensive studies to date of the antibody immune response to SARS-CoV-2 in people across the entire spectrum of disease severity, from asymptomatic to fatal,” said Scott Boyd, MD, Ph.D., associate professor of pathology.
“We assessed multiple time points and sample types, and also analyzed levels of viral RNA in patient nasopharyngeal swabs and blood samples. It’s one of the first big-picture looks at this illness.”
The study found that people with severe COVID-19 have low proportions of antibodies targeting the spike protein used by the virus to enter human cells compared with the number of antibodies targeting proteins of the virus’s inner shell.
Boyd is a senior author of the study, which was published Dec. 7 in Science Immunology. Other senior authors are Benjamin Pinsky, MD, Ph.D., associate professor of pathology, and Peter Kim, Ph.D., the Virginia and D. K. Ludwig Professor of Biochemistry. The lead authors are research scientist Katharina Röltgen, Ph.D.; postdoctoral scholars Abigail Powell, Ph.D., and Oliver Wirz, Ph.D.; and clinical instructor Bryan Stevens, MD.
Virus binds to ACE2 receptor
The researchers studied 254 people with asymptomatic, mild or severe COVID-19 who were identified either through routine testing or occupational health screening at Stanford Health Care or who came to a Stanford Health Care clinic with symptoms of COVID-19.
Of the people with symptoms, 25 were treated as outpatients, 42 were hospitalized outside the intensive care unit and 37 were treated in the intensive care unit. Twenty-five people in the study died of the disease.
SARS-CoV-2 binds to human cells via a structure on its surface called the spike protein. This protein binds to a receptor on human cells called ACE2. The binding allows the virus to enter and infect the cell. Once inside, the virus sheds its outer coat to reveal an inner shell encasing its genetic material. Soon, the virus co-opts the cell’s protein-making machinery to churn out more viral particles, which are then released to infect other cells.
Antibodies that recognize and bind to the spike protein block its ability to bind to ACE2, preventing the virus from infecting the cells, whereas antibodies that recognize other viral components are unlikely to prevent viral spread. Current vaccine candidates use portions of the spike protein to stimulate an immune response.
Boyd and his colleagues analyzed the levels of three types of antibodies—IgG, IgM and IgA—and the proportions that targeted the viral spike protein or the virus’s inner shell as the disease progressed and patients either recovered or grew sicker.
They also measured the levels of viral genetic material in nasopharyngeal samples and blood from the patients. Finally, they assessed the effectiveness of the antibodies in preventing the spike protein from binding to ACE2 in a laboratory dish.
“Although previous studies have assessed the overall antibody response to infection, we compared the viral proteins targeted by these antibodies,” Boyd said. “We found that the severity of the illness correlates with the ratio of antibodies recognizing domains of the spike protein compared with other nonprotective viral targets. Those people with mild illness tended to have a higher proportion of anti-spike antibodies, and those who died from their disease had more antibodies that recognized other parts of the virus.”
Substantial variability in immune response
The researchers caution, however, that although the study identified trends among a group of patients, there is still substantial variability in the immune response mounted by individual patients, particularly those with severe disease.
“Antibody responses are not likely to be the sole determinant of someone’s outcome,” Boyd said. “Among people with severe disease, some die and some recover. Some of these patients mount a vigorous immune response, and others have a more moderate response. So, there are a lot of other things going on.
There are also other branches of the immune system involved. It’s important to note that our results identify correlations but don’t prove causation.”
As in other studies, the researchers found that people with asymptomatic and mild illness had lower levels of antibodies overall than did those with severe disease. After recovery, the levels of IgM and IgA decreased steadily to low or undetectable levels in most patients over a period of about one to four months after symptom onset or estimated infection date, and IgG levels dropped significantly.
“This is quite consistent with what has been seen with other coronaviruses that regularly circulate in our communities to cause the common cold,” Boyd said. “It’s not uncommon for someone to get re-infected within a year or sometimes sooner.
It remains to be seen whether the immune response to SARS-CoV-2 vaccination is stronger, or persists longer, than that caused by natural infection. It’s quite possible it could be better. But there are a lot of questions that still need to be answered.”
Boyd is a co-chair of the National Cancer Institute’s SeroNet Serological Sciences Network, one of the nation’s largest coordinated research efforts to study the immune response to COVID-19.
He is the principal investigator of Center of Excellence in SeroNet at Stanford, which is tackling critical questions about the mechanisms and duration of immunity to SARS-CoV-2.
“For example, if someone has already been infected, should they get the vaccine? If so, how should they be prioritized?” Boyd said.
“How can we adapt seroprevalence studies in vaccinated populations?
How will immunity from vaccination differ from that caused by natural infection?
And how long might a vaccine be protective? These are all very interesting, important questions.”
Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome packaged by nucleocapsid phosphoproteins . Coronaviruses are one of the largest RNA viruses (26−32 kb); the SARS-CoV-2 genome varies from 29.8–29.9 kb and shares 79.6 and 50 % homology with SARS-CoV and MERS, respectively . The SARS-CoV genome comprises 14 open reading frames (ORFs) encoding 27 proteins .
The first ORF (ORF1a and ORF1b) represents nearly 70 % of the virus genome and encompassed 15 nonstructural proteins (nsps) (Fig. 1 A) . The remaining ORFs in the 3’-terminal region encoded for four structural proteins, namely membrane (M), envelope (E), spike (S) and nucleocapsid (N) proteins (Fig. 1B) and eight accessories proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and ORF14) (Fig. 1A) .
The S protein is composed of two subunits; S1 holds the receptor-binding domain (RBD), which binds to a receptor on the host cell’s surface, while S2 is needed for the fusion of the virus with the cellular membrane host . In the inner side of the envelope, the nucleocapsid (N) proteins are bound to the positive-sense single-stranded RNA genome . SARS-CoV-2 is characterized by a low mutation rate (6.7 mutations per sample) when comparing to a reference genome from Wuhan (NC_045512.2) [14,15].
The phylogenic analysis of the genomic RNA sequences shared by the public database of the Global Initiative on Sharing All Influenza Data (GISAID) (https://www.gisaid.org/) grouped SARS-CoV-2 into several distinctive viral clades characterized by specific mutations. Each clade clusters related sequences that refer to a common ancestor. For example, the substitution of aspartic acid by glycine at position 614 (D614 G) in the spike protein is the most frequently sequenced in a clade observed predominantly in Europe .
However, no experimental evidence demonstrated a biological difference acquired by the mutations, suggesting that a single strain of the SARS-CoV-2 virus is currently present.
Life cycle of the virus
Viruses are obligate intracellular parasites and the SARS-CoV-2 life cycle debuts when the transmembrane S structural glycoprotein binds to its receptor, angiotensin-converting enzyme 2 (ACE2), on the host cell . ACE2 was also identified as a functional receptor for SARS-CoV and human coronavirus NL63 [17,18].
The RBD of the S1 subunit binds to the peptide domain of ACE2; this interaction triggers the priming of S1 at the S1/S2 junction by cellular proteases such as furin or endosomal proteases and exposes a secondary site (S2’) cleaved by the transmembrane serine protease 2 (TMPRSS2), which reveals the fusion peptide of the S2 subunit [12,19,20].
The cleavage modifies the conformation of the cleaved protein S irreversibly and allows the S2 subunit to insert into the host membrane and guide the fusion of the viral and cellular membranes . Previous studies have reported the essential role of TMPRSS2 in the cellular entry of SARS-CoV-2, as well as SARS-CoV and MERS [, , , ].
Hoffmann et al. demonstrated that camostat mesylate, an inhibitor of TMPRSS2, partially blocked the cellular entry of SARS-CoV-2 in vitro . In addition to ACE2, CD147 was mentioned as a potential transmembrane receptor to mediate the cellular entry of SARS-CoV-2 .
CD147, also known as basigin or extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein, a member of the immunoglobulin superfamily, expressed on the surface of red blood cells, lymphocytes, dendritic cells (DCs), monocytes, macrophages, and many tissues [26,27]. CD147 is a known target for malaria treatment, as anti-CD147 antibodies prevent the red blood cell invasion by the protozoan Plasmodium falciparum , which has also been shown to facilitate the cellular entry of various viruses, including HIV-1  and measles virus . Wang et al. demonstrated the direct interaction, and colocalization of protein S and CD147 and the blocking of CD147 by humanized anti-CD147 antibody (Meplazumab) decreased SARS-CoV-2 replication in vitro .
The SARS-CoV-2 entry into the cells, similarly as for SARS-CoV, is achieved by receptor-mediated endocytosis . The viral RNA is released in the cytoplasm, uncoated, and the translations of the ORF1a and 1b are initiated. ORFs 1a and 1b code for the polyproteins pp1a (nsp1-11) and pp1ab (nsp1-16), respectively, depending on a ribosomal (-1)-frameshift demonstrated in coronavirus 31 years ago .
The polyproteins processing is coordinated spatially and temporally by viral proteases such as Mpro (nsp5) and PLpro (nsp3) into nsps involved in the replication/transcription complex [33,34]. The nsps rearranged portion of the membrane derived from the rough endoplasmic reticulum into vesicles where the viral assembly is organized . Viruses are then released from the host cell by exocytosis via secretory vesicles .
The pathophysiology of SARS-CoV-2 is similar to that observed with SARS-CoV and mainly affects the respiratory system, but other organs may be involved [, , , ]. The presenting symptoms are a fever with dry cough and dyspnea  accompanied by headaches, dizziness, muscle pain, joint pain, and fatigue with gastrointestinal symptoms, including vomiting and diarrhea . A multisystem inflammatory syndrome related to SARS-CoV-2 has been reported in older children and associated with severe abdominal pain, cardiac dysfunction, and shock, bearing similarities with the Kawasaki disease .
Like SARS-CoV and MERS, respiratory droplets are the primary source of transmission of SARS-CoV-2 from human to human. The similarities of SARS-CoV-2 with SARS-CoV and MERS also suggest the possibility of fecal-oral transmission that remains unproven . The median incubation period of SARS-CoV-2 varies from 4 to 6 days before developing symptoms [37,, , ].
Several studies reported a high viral load before or on symptom onset similarly to influenza, suggesting a high transmission potential even with mildly symptomatic cases; this appears to differ from the observation made with SARS-CoV showing a delayed peak of the viral load of several days following the symptoms’ onset [, , , ].
Patients infected with SARS-CoV-2 may have minimal symptoms or experience acute respiratory distress syndrome (ARDS) with uncontrolled inflammation that may lead to multiple organ failures. Most patients with mild symptoms showed ground-glass opacity on chest computed tomography (CT) on admission . Similar to SARS-CoV and MERS, the severity of the symptoms is strongly associated with the patients’ age that may be explained by a weaker or dysregulated immune system [37,38,50].
SARS-CoV-2 is a cytopathic virus that kills the cells and damages the tissue host as part of its replication cycle . ACE2 was described as the primary receptor promoting the cellular entry of SARS-CoV-2 . ACE2 is highly expressed on the alveolar epithelial type II cells’ surface, critical for the lungs’ gas exchange function preventing the alveoli from collapsing . However, ACE2 is also present on the surface of type I pneumocytes, endothelial cells, and in extrapulmonary tissues, including heart, intestine, blood vessels, kidneys, and urinary bladder [53,54].
The alveolar epithelial type II cells were also described as a potential viral reservoir that may disseminate the virus to other organs and individuals [54,55]. The epithelial cell death following viral infection has been associated with apoptosis and a pro-inflammatory program cell death named pyroptosis, which triggers the release of pro-inflammatory cytokines, for example, interleukin-1β (IL-1β) and IL-18 (Fig. 2 A-B) [56,57].
Previous studies have reported the increased level of pro-inflammatory cytokines in patients infected with SARS-CoV-2, including IL-1β, IL-6, IL-8, TNF-α, IP10, MCP-1, and RANTES [38,58] (Fig. 2C). Studies on SARS-CoV and MERS also reported the elevation of pro-inflammatory cytokines in patients’ serum and their association with pulmonary inflammation and extensive lung damage (for review, see ).
The secretion of cytokines and chemokines recruit monocytes and macrophages, which release cytokines to prime T-cell adaptive immune response. In most patients, the immune cells’ recruitment will clear the virus, and inflammation will recede. However, in a few patients with a weak immune system and/or other unknown preexisting conditions, an uncontrolled inflammatory response linked to a cytokine storm, characterized by the increased plasma level of IL-2, IL-7, IL-10, GCSF, IP-10, MCP-1, MIP-1α, and TNF-α, has been associated with the severity of the pulmonary inflammation, and likely the leading cause of death (Fig. 2D) .
As described above, epithelial cells, macrophages of the alveoli, and DCs underneath the epithelium are the main components for the innate immunity to the virus . Antigen presentation by DCs and macrophages will initiate the T-cell responses. SARS-CoV-2 enters these cells either by phagocytosis of apoptotic cells infected by virus-like epithelial cells  or via pattern recognition receptors (PRRs) . The SARS-CoV-2 may also use the ACE2, expressed in macrophages and DCs [62,63].
The SARS-CoV-2 virus may bind to the dendritic cell intercellular adhesion molecules 3-grabbing non-integrin (DC-SIGN), and L-SIGN [, , ]. These APCs then present viral antigens to T-cells. CD4+ and CD8+ T-cell in the draining lymph nodes. CD4+ T-helper cells induce and help B-cells to produce the virus-specific antibody. The cytotoxic CD8+ T-cells can kill virally infected cells.
It has been observed in patients with severe diseases a storm of pro-inflammatory cytokines, mainly IL-6, TNF-α, G-CSF in addition to the anti-inflammatory cytokine IL-10 and the chemokines MCP-1, MIP-1α, and IL-8, but the more severe disease was noted with higher levels of IL-6 [38,, , ].
In the patients never exposed to SARS-CoV-2, the T-cells’ cross-reactivity was detected in 20 % of the cases, probably due to previous exposure to common cold coronaviruses . Specific T-cells against the proteins S, M, and N were detected in the peripheral blood mononuclear cells from convalescent SARS-CoV-2 patients .
Furthermore, CD4+ T-cells showed strong reactivity against M and S proteins, mostly in mild SARS-CoV-2 patients . In severe SARS-CoV-2 cases, CD4+ with memory phenotype and CD8+ T-cells with effector phenotype were detected in the blood . Further studies will be required to understand the T-cells’ involvement in the progression of severe SARS-CoV-2 symptoms.
Furthermore, thrombosis and pulmonary embolism have been observed in severe SARS-CoV-2 diseases . Platelets were hyperactivated, and aggregation occurred at thrombin suboptimal concentrations . This may be due to the massive inflammation enhancing cytokines and increasing the liver’s production of clotting factors.
Furthermore, the cytokine TNF-α highly produced in SARS-CoV-2 has been implicated in promoting overexpression of tissue factor (TF) in platelets and macrophages . TF acts as a receptor and cofactor for coagulation factors VII and VIIa [76,77]. Thus, the cytokine-induced TF synthesis and release can significantly impact the coagulation responses . Another mechanism noted in the pathogenesis of COVID-19 is the interaction with hemoglobin via CD147 and CD26 expressed on erythrocytes .
The immune response and associated molecular events to SARS-CoV-2 infection remain poorly understood; it is critical to identify these elements to direct an efficient therapeutic approach. A recent study reported that individuals previously infected with COVID-19, which triggered an antibody, was sufficient to protect most individuals from reinfection in the six months following infection; the long term protection remains to be determined .
According to the World Health Organization (WHO), there are no specific medicines or vaccines for SARS-CoV-2. In the early days of the pandemic, many governments worldwide have to some degree implemented measures recommended by WHO to limit the spread of the virus such as self-isolation, social distancing, hand washing, closure of schools and universities, and wearing a mask in public places . These measures proved to be efficient, when followed, and saved countless lives .
Several medications are currently evaluated in clinical trials; 3947 clinical trials are presently listed on clinicaltrials.gov, including 490 phase-III and 104 phase IV clinical trials as of November 19, 2020. The management provided to the patients is mainly supportive and critical for at least 5% of patients developing severe symptoms and requiring hospitalization .
Available drugs have been repurposed for the treatment of SARS-CoV2, including lopinavir-ritonavir, remdesivir, favipiravir, arbidol, chloroquine and analogs hydroxychloroquine, and azithromycin and tested in clinical trials [, , , , ]. However, results were not very promising for any of them to be a definite therapy yet. Fig. 3 schematized some of the various therapeutic strategies presently assessed in various clinical trials.
Inhibitors of virus entry
Arbidol, an anti-influenza virus drug, was also efficient in inhibiting SARS-CoV-2 infection in vitro . Arbidol prevents the interaction between ACE2 and the viral protein S by blocking its trimerization of the viral protein S . In a small non-randomized controlled study, arbidol showed a tendency to improve recovery and decrease mortality . A comparison of the safety and efficacy of arbidol or lopinavir/ritonavir to the standard of care in a phase IV interventional clinical trial (NCT04252885) showed little benefit of both treatment regimen for improving the clinical outcome of patients hospitalized with mild/moderate COVID-19 over the standard of care . However, further power randomized placebo-controlled studies are required to validate these observations.
Recombinant soluble form of human ACE2
A similar initiative trying to block the interaction between the viral protein S and ACE2 is currently evaluated in a phase I clinical trial using a recombinant soluble form of human ACE2 (NCT00886353) based on the previous clinical trial demonstrating its safety . In a case report, a 45-year-old woman infected by SARS-CoV-2 initially received hydroxychloroquine (400 mg twice daily) and anticoagulant nadroparin (4 mg once daily); as her condition worsened, she was intubated. On the 9th day after the onset of symptoms, she was given human recombinant soluble ACE2 (APN01; 0·4 mg/kg, i.v., twice daily) .
After 7 days, the treatment was well-tolerated, and her condition improved gradually; she was extubated on day 21. The recombinant soluble ACE2 decreased the plasma concentration of angiotensin II while promoting the increase of its metabolites’ angiotensin 1–7 and angiotensin 1–5, and cleaving angiotensin I into angiotensin 1–9 . Angiotensin II is elevated in lung injury and is associated with worsening conditions such as hypertension, diabetes, inflammation, and affecting the physiology of the heart, lungs, kidneys, vasculature, and liver . The treatment was also associated with a decrease of pro-inflammatory cytokines (IL-6 and IL-8) and other inflammation markers (TNF-α, ferritin) .
Chloroquine and analog hydroxychloroquine
Chloroquine and analogs hydroxychloroquine act as viral entry inhibitors; by increasing endosomal pH required for virus/cell fusion and interfering with the cellular receptors’ glycosylation for SARS-CoV-2 (ACE-2) . In contrast to the early reports of poorly controlled studies, chloroquine and analogs hydroxychloroquine failed to demonstrate any benefit for treating this disease (for review, see ).
The interim report of WHO solidarity trial involving 11,266 hospitalized COVID-19 patients involving 405 hospitals in 30 countries assessed the efficacy of four repurposed antiviral drugs, including remdesivir (intravenous, day 0: 200 mg, day 1 to 9: 100 mg), hydroxychloroquine (oral, 800 mg twice over 6 h, then 400 mg twice daily for 10 days), lopinavir/ritonavir (oral, 400 mg lopinavir +1 g ritonavir twice daily for 14 days), and interferon-β1a (subcutaneous, 3 doses 44 μg over 6 days or intravenous., 10 μg once daily over 6 days) (WHO registration: ISRCTN83971151, NCT04315948).
Overall, most patients hospitalized were aged between 50–69 years (45 %) and had underlying conditions including diabetes (25 %), heart disease (21 %), chronic lung disease (6%), asthma (5%), and chronic liver disease (1%) . The report concluded that remdesivir, hydroxychloroquine, lopinavir/ritonavir, and interferon-β1a had little to no effect on COVID-19 patients as measured by the overall death, placement under ventilation, or duration of hospitalization .
Inhibitor of proteolysis
Lopinavir-ritonavir, a combination medication used for HIV/AIDS treatment, is a protease inhibitor that inhibits viral protein synthesis, potentially targeting the SARS-CoV-2 viral protease Mpro essential for the replication. Lopinavir-ritonavir also demonstrated efficacy in vitro, inhibiting SARS-CoV-2 replication . The combination failed to procure any benefit beyond standard care in clinical trials [98,99].
Inhibitors of the viral RNA-dependent RNA polymerase
Remdesivir (adenosine analog prodrug) and Favipiravir (guanine analog prodrug) act as inhibitors of the viral RNA-dependent RNA polymerase (RdRp), demonstrated potent antiviral activity in vitro [94,100,101].
Favipiravir was approved for the treatment of SARS-CoV-2 in Russia and India and currently assessed in many countries. The drug is given orally and improves the patients’ conditions in the clinic compared to the standard of care but no significant difference in viral clearance, the requirement for oxygen support, and side effect profile .
In an open-label clinical trial (ChiCTR2000029600), COVID-19 patients were treated with favipiravir (1600 mg twice daily on Day 1 and 600 mg twice daily on days 2–14, orally) or lopinavir-ritonavir (400 mg/100 mg twice daily orally). All patients received interferon (IFN)-α by aerosol inhalation (5 × 106 international unit twice daily . The favipiravir group had an early viral clearance (median 4 days) compared to patients treated with lopinavir-ritonavir (median 11 days); the chest CT findings were improved in the favipiravir group at day 14 (91.4 % vs. 62,2 %) .
The patients experienced a lower incidence of side effects in the favipiravir group (11.4 % vs. 55.6 %) . Another clinical trial (ChiCTR2000030254) compared favipiravir treatment (1600 mg twice a day on the first day followed by 600 mg twice a day for 10 days) to arbidol (200 mg three times per day for 10 days) . Clinically confirmed patients and not the virally confirmed ones were allocated randomly to each group; only 46.5 and 38.3 % of patients were nucleic acid positive at enrollment in the favipiravir and arbidol group, respectively .
The clinical recovery rate and auxiliary oxygen therapy or noninvasive mechanical ventilation did not significantly differ between the two groups; favipiravir tended to relieve adverse effects more rapidly . Favipiravir is currently evaluated in 17 phase-III clinical trials. The data obtained for favipiravir in clinical trials are encouraging, but the completion of large, randomized double-blinded, placebo-controlled clinical trials will provide unbiased data.
Remdesivir results in clinical trials were conflicting. Remdesivir failed to demonstrate any statistically significant clinical benefits for severe SARS-CoV-2 patients in a randomized, double-blind, placebo-controlled, multicentre trial in Wuhan, China, but underpowered (clinical trial: NCT04257656) . In a double-blind, randomized, placebo-controlled trial sponsored by the US National Institute of Allergy and Infectious Diseases (NIAID) (NCT04280705), patients received 200 mg on day 1, followed by 100 mg daily for up to 9 days given intravenously. Remdesivir was shown to improve the recovery time from 15 to 10 days for COVID-19 patients (rate ratio for recovery, 1.29 [95 % CI, 1.12–1.49]) .
Patients had preexisting conditions such as type 2 diabetes (30.6 %), hypertension (50.7 %), or obesity (45.6 %) . However, no significant differences in survival were observed; mortality was decreased by 3.8 % in the remdesivir versus placebo groups (hazard ratio, 0.73; 95 % CI, 0.52–1.03) . The Food and Drug Administration (FDA) approved remdesivir on October 22nd, 2020. More recently, following the WHO SOLIDARITY trial results, the WHO suggested against the use of Remdesivir for hospitalized patients with Covid-19 regardless of the severity .
More recently, avifavir, introduced as the first favipiravir-based drug, was approved for the treatment of SARS-COV-2 in Russia. An interim report of a phase II/III clinical trial of 60 hospitalized patients with mild COVID-19 symptoms randomized equally in three groups receiving avifavir 1600 mg twice on day 1 followed by 600 mg twice on days 2–14, or avifavir 1800 mg twice on day 1 followed by 800 mg twice on days 2–14, or standard of care as a control group . Both avifavir dosages had a similar effect on viral clearance on day 5, which was achieved in 25/40 patients (62.5 %) compared to 6/20 patients treated by the standard of care . Despite the small sample size, avifavir was granted fast-track marketing authorization by the Russian Ministry of Health .
6.4. Alternative strategies
6.4.1. Targeting host proteins and immune response modulators
Alternative strategies targeting host proteins and immune response modulators are currently developed. Camostat mesylate and nafamostat mesylate target the TMPRSS2, an essential serine protease critical for the cellular entry of SARS-CoV-2 into the lung cells [12,109]. Both compounds are approved in Japan to treat pancreatitis and are currently assessed in several clinical trials for the treatment of SARS-CoV-2. Immunomodulators as Nitazoxanide were also used for interference with host-regulated pathways involved in viral replication, amplifying cytoplasmic RNA sensing, and type I IFN pathways .
6.4.2. Targeting inflammation and coagulation
In severe cases of SARS-CoV-2, anti-inflammatory and anti-Coagulative agents have been proposed and implemented because of the profound inflammatory response and the thrombotic phenomena described in the lungs of patients with severe SARS-COV-2, including administration of low-molecular-weight heparin as a standard measure in hospitalized patients with SARS-CoV-2 .
IL-6 is an inflammatory cytokine targeted in neutralizing therapies in several inflammatory diseases . Increased serum concentrations of IL-6 were noted during severe infections with Coronavirus SARS-CoV-2, suggesting possible implications for anti-IL-6 therapy in SARS-CoV-2 disease . Tocilizumab, an inhibitor of IL-6, might reduce the risk of invasive mechanical ventilation or death in patients with severe SARS-CoV-2 . Tocilizumab is currently registered in 71 clinical trials for the treatment of SARS-CoV-2, a randomized double-blinded phase III clinical trial (NCT04320615) compared tocilizumab (8 mg/kg, i.v.) to the standard of care in severe COVID-19 patients . The outcome of the treatment was measured at day 28; also, tocilizumab did not improve the clinical status or mortality when compared to the standard of care; it decreased the stay in the intensive care unit by 5.8 days (9.8 days) and shorter the discharge time by 8 days (20 days) . Additional clinical trials will be necessary to determine the advantage of tocilizumab on the duration of the hospitalization.
6.4.3. Immunomodulation by glucocorticoids
Glucocorticoids are also assessed in several clinical trials; their immunosuppressive effect requires careful consideration. It may influence the viral clearance, while their anti-inflammatory effect may limit the tissue damage induced by the cytokine storm and favor their use in severe COVID-19 cases . A randomized, controlled clinical trial in the United Kingdom using dexamethasone in severely ill patients reduced deaths by about one-third of patients on ventilators [116,117]. The randomized evaluation of the COVID-19 therapy (RECOVERY) trial (NCT04381936) demonstrated that a dose of dexamethasone (6 mg) once daily for up to 10 days reduced the mortality of patients under respiratory assistance but may be harmful to patients who do not require oxygen support . The WHO has recently recommended the use of corticosteroids to treat hospitalized patients with severe and critical Covid-19 . Additional glucocorticoids were evaluated in clinical trials, including methylprednisolone (NCT04343729, 2020-001934-37, and IRCT20200404046947N1), hydrocortisone (NCT02735707, NCT02517489). Methylprednisolone Both clinical trials assessing hydrocortisone were stopped earlier for failing to meet the prespecified criteria for statistical superiority [119,120].
6.4.4. Passive immunotherapy and neutralizing antibodies
Passive immunotherapy using plasma from recovering patients was considered a promising option in the treatment of SARS-CoV-2 infections. The WHO and the FDA have permitted plasma therapy for severe conditions upon failure of trial medications. Several clinical trials are underway to test the effectiveness of hyperimmune plasma . A recent study demonstrated that antibodies in the serum of patients infected with SARS-CoV effectively neutralized SARS-CoV-2 in vitro . The administration of convalescent plasma to 5,000 COVID-19 hospitalized patients demonstrated to be safe as the occurrence of serious adverse events was low and likely to be beneficial when given at an early stage of the development of COVID-19 .
LY-CoV555 (bamlanivimab, LY3819253), a neutralizing IgG1 monoclonal antibody that binds with high affinity to the SARS-CoV-2 spike protein, was isolated from some of the first patients in North America . In a phase II clinical trial (NCT04427501) sponsored by Eli Lilly and Company, patients received a single dose of either LY-CoV5555 at 700, 2,800, or 7,000 mg or placebo. Only the single intravenous infusion of LY-CoV555 (2,800 mg) appeared to accelerate the viral load’s natural decline .
The LY-CoV555 treated patients had fewer hospitalizations than the placebo group, 1.6 vs. 6.3 %, respectively, and 4.2 vs. 14.6 % when considering high-risk patients, respectively . Eli Lilly has been granted emergency use on November 10, 2020, by the FDA, to treat mild to moderate Covid-19 adults and pediatric patients older than 12 years of age, weighing 40 kg, and at risk of developing severe Covid-19 and /or hospitalization.
AZD7442 is a combination of two long-acting antibodies derived from convalescent patients after SARS-CoV-2 infection. The antibodies (COV2-2196 and COV2-2130), isolated by the Vanderbilt University Medical Center, recognized non-overlapping sites of the viral spike protein and were shown to neutralize the SARS-CoV-2 infection synergistically in two murine models .
These antibodies were licensed by AstraZeneca in June 2020 and modified to extend their half-life for 6–12 months and reduce the Fc receptor binding. AZD7442 entered phase I clinical trial (NCT04507256) in August 2020. AstraZeneca started two phase III clinical trials in November 2020, Storm chaser (NCT04625972, 1125 participants) and Provent (NCT04625725, 5,000 participants). The Provent trial will evaluate the safety and efficacy for the prevention of SARS-CoV-2 infection for up to 12 months following a single injection, while the Storm Chaser trial will evaluate post-exposure prophylaxis and pre-emptive treatment following a single injection. Recently, the US government secured 100,000 doses of AZD7442.
REGN-COV2, manufactured by Regeneron Pharmaceuticals, is a cocktail of two neutralizing synthetic antibodies, casirivimab (REGN10933) and imdevimab (REGN10987), targeting non-overlapping epitopes of the viral spike protein. On November 21st, 2020, REGN-COV2 received an emergency use authorization by the FDA to treat mild to moderate COVID-19 adults and pediatric patients older than 12 years of age, weighing 40 kg, and at high risk of developing severe COVID-19.
This emergency use authorization includes subjects who are 65 years or older or who have chronic medical conditions. In a preclinical study, REGN-COV2 reduced the viral load in upper and lower airways when given as a prophylactic or as a treatment in rhesus macaques . In an animal model of severe COVID-19, REGN-COV2 decreased the viral lung titers and evidence of pneumonia .
Regeneron Pharmaceuticals announced preliminary data from 275 patients in a phase I/II/III trial (NCT04425629, NCT04426695, and NCT04452318), showing that REGN-COV2 reduced viral load, alleviate symptoms in non-hospitalized patients infected with COVID-19, and decreased patient medical visits (https://investor.regeneron.com/press-releases). For patients at high risk for disease progression, hospitalizations, and emergency room visits occurred in 3 % of the REGN-COV2 patients compared to 9 % of the placebo-treated patients (https://www.fda.gov/news-events/fda-newsroom/press-announcements).
Additional neutralizing antibodies are currently evaluated in multiple phase I clinical trials, including VIR-7831, 47D11, CT-P59, ALVR109, STI-1499, IVIG, and COVID-HIG (for review see .
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706592/
More information: Katharina Röltgen et al. Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome, Science Immunology (2020). DOI: 10.1126/sciimmunol.abe0240