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CB6 can effectively reduce viral load and alleviated infection-related lung damage in rhesus macaques a, Nine rhesus macaques (6 males and 3 females) were divided into pre-exposure (prophylactic), post-exposure (treatment) and control groups with three macaques (2 males and 1 female) in each group. Before infection, the macaques in the pre-exposure group were infused with 50 mg kg⁻¹ body weight CB6(LALA) intravenously. One day later, all macaques were inoculated with 1 × 10⁵ TCID50 SARS-CoV-2 via intratracheal intubation. The post-exposure group were also infused with 50 mg kg⁻¹ CB6(LALA) on days 1 and 3 after exposure, and the three macaques in the control group were given PBS as a control. Viral RNA loads in throat swabs, determined by RT–qPCR, were monitored for 7 days. Data are average values from 3 macaques for the first 5 days, from 2 macaques at 6 dpi, and from 1 macaque at 7 dpi. To evaluate the viral loads for each macaque at the indicated time point, RT–qPCR were performed with two replicates. b–d, Histopathology and immunohistochemical examination of lung tissues from pre-exposure, post-exposure and control macaques. One macaque from each group was euthanized and necropsied at 5 dpi. Samples for histological examination were stored in formalin for 7 days, embedded in paraffin, sectioned and stained before examination by light microscopy. Haematoxylin and eosin (H&E) sections exhibited the interstitial pneumonia and inflammatory-factor infiltration in tissues. Masson’s trichrome showed lung tissue fibrosis. Scale bar, 200 μm. Source data
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An outbreak of the coronavirus disease 2019 (COVID-19)1–3, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4 spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. In this study, we report the isolation of 2 specific human monoclonal antibodies (MAbs) from a convalescent COVID-...
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Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-C...
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... Among them, Paxlovid has shown clinical benefit of reducing the risk of progression to severe COVID-19 or death [11][12][13] . Neutralizing antibodies have been used clinically against respiratory syncytial virus and Ebolavirus disease 14,15 , and also quickly been developed against SARS-CoV-2 infection to exert potent neutralizing activity in preclinical models and clinic [15][16][17][18][19] . Angiotensin-converting enzyme 2 (ACE2) is a key entry receptor for SARS-CoV-2 1 . ...
Neutralizing antibodies exert a potent inhibitory effect on viral entry; however, they are less effective in therapeutic models than in prophylactic models, presumably because of their limited efficacy in eliminating virus-producing cells via Fc-mediated cytotoxicity. Herein, we present a SARS-CoV-2 spike-targeting bispecific T-cell engager (S-BiTE) strategy for controlling SARS-CoV-2 infection. This approach blocks the entry of free virus into permissive cells by competing with membrane receptors and eliminates virus-infected cells via powerful T cell-mediated cytotoxicity. S-BiTE is effective against both the original and Delta variant of SARS-CoV2 with similar efficacy, suggesting its potential application against immune-escaping variants. In addition, in humanized mouse model with live SARS-COV-2 infection, S-BiTE treated mice showed significantly less viral load than neutralization only treated group. The S-BiTE strategy may have broad applications in combating other coronavirus infections.
... Through single-cell sorting of S glycoprotein-specific memory B cells from COVID-19 convalescent individuals, 453 NAbs were identified, the most potent of which could neutralize the virus at very low doses, exhibiting significant prophylactic and therapeutic efficacy in a hamster COVID-19 model [13]. Furthermore, two potent NAbs were isolated from a convalescent patient; the most potent of these demonstrated therapeutic potential through inhibition of infection in rhesus monkeys in both prophylactic and therapeutic settings [14]. Table O R I G I N A L U N E D I T E D M A N U S C R I P T 1 summarizes the representative NAbs for the wild-type SARS-CoV-2 identified and characterized in these studies. ...
The ongoing COVID-19 pandemic caused by SARS-CoV-2 has raised global concern for public health and the economy. The development of therapeutics and vaccines to combat this virus are continuously progressing. Multi-omics approaches, including genomics, transcriptomics, proteomics, metabolomics, epigenomics, and metallomics, have helped understand the structural and molecular features of the virus, thereby assisting in the design of potential therapeutics and accelerating vaccine development for COVID-19. Here, we provide an up-to-date overview of the latest applications of multi-omics technologies in strategies addressing COVID-19, in order to provide suggestions towards the development of highly effective knowledge-based therapeutics and vaccines.
... For most viruses, there are a wide variety of cell lines that can be used to assess the potency of polyclonal and monoclonal antibodies with antiviral activity. For example, for SARS-CoV-2 alone, cell lines that have been used to assess antibody neutralization activity include Vero/Vero E6 (African green monkey kidney), 293/293T (human embryonic kidney), HeLa (human cervical carcinoma), Huh7 (human hepatocellular carcinoma), Calu-3 (human lung carcinoma), HT1080 (human fibrosarcoma), U2OS (human osteosarcoma), and HOS (human osteosarcoma) cells, which are often engineered to stably express human angiotensin-converting enzyme 2 (ACE2), which is the SARS-CoV-2 receptor, and/or transmembrane serine protease 2 (TMPRSS2), which is a protease involved in SARS-CoV-2 entry [65,67,70,[153][154][155][156][157][158]. ...
Viral diseases represent a major public health concerns and ever-present risks for developing into future pandemics. Antiviral antibody therapeutics, either alone or in combination with other therapies, emerged as valuable preventative and treatment options, including during global emergencies. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well-suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.
... 16L and VI are codes for A1016L and A1016V/ A1020I mutations. immunodominant antibody target in natural infection [10] and highly potent NAbs directed to the RBD can block infection by binding to the ACE2 receptor-binding motif (RBM) and directly blocking ACE2 binding, via steric blockade of ACE2 binding, by locking RBDs in the down orientation to preclude ACE2 binding, or by triggering premature refolding of S1-S2 into the post-fusion state with shedding of S1 [11][12][13][14][15][16][17][18]. The N-terminal domain (NTD) of S1 has also been identified as a supersite of vulnerability and comprises multiple antigenic sites [12,15,[19][20][21]. ...
The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.
... Currently, most therapeutic neutralizing antibodies (nAbs) and promising vaccine candidates are designed to target the RBD or use RBD as the sole antigen. [21][22][23][24][25] However, RBD is poorly conserved, with the newly emerged Omicron subvariants harboring multiple mutations in RBD that are capable of immune evasion from the majority of existing neutralizing antibodies (nAbs). Consequently, vaccine efficacy has markedly reduced, thus, posing new challenges for the prevention and treatment of SARS-CoV-2. ...
... In contrast, the CB6 mAb targeting RBD does not, possibly because 76E1 binds to S after ACE2 binding, whereas CB6 competes with ACE2 for the RBD epitope. 24,53 More recently, a FP-targeted nAb, fp.006, isolated from COVID-19 convalescents, binds to a core epitope (S 813 RRSFIEDLLFNK 825 ) containing the S2ʹ site and the helical FP segment. 54 Structural Fig. 2 Sequence features of targetable elements (fusion peptide, stem helix, and heptad repeats 1 and 2) in coronavirus S2 subunit. ...
The ongoing global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), has caused devastating impacts on the public health and the global economy. Rapid viral antigenic evolution has led to the continual generation of new variants. Of special note is the recently expanding Omicron subvariants that are capable of immune evasion from most of the existing neutralizing antibodies (nAbs). This has posed new challenges for the prevention and treatment of COVID-19. Therefore, exploring broad-spectrum antiviral agents to combat the emerging variants is imperative. In sharp contrast to the massive accumulation of mutations within the SARS-CoV-2 receptor-binding domain (RBD), the S2 fusion subunit has remained highly conserved among variants. Hence, S2-based therapeutics may provide effective cross-protection against new SARS-CoV-2 variants. Here, we summarize the most recently developed broad-spectrum fusion inhibitors (e.g., nAbs, peptides, proteins, and small-molecule compounds) and candidate vaccines targeting the conserved elements in SARS-CoV-2 S2 subunit. The main focus includes all the targetable S2 elements, namely, the fusion peptide, stem helix, and heptad repeats 1 and 2 (HR1-HR2) bundle. Moreover, we provide a detailed summary of the characteristics and action-mechanisms for each class of cross-reactive fusion inhibitors, which should guide and promote future design of S2-based inhibitors and vaccines against new coronaviruses.
... The emerging COVID-19 global outbreak has had widespread effects, and its causative agent, SARS-CoV-2, continues to evolve. Neutralizing mAbs that target the RBD of the SARS-CoV-2 spike protein are reported as the most promising antibody-based therapeutics against SARS-CoV-2 infection [1][2][3]. However, the numerous emerging genetically distinct SARS-CoV-2 VOCs have become the major concern of the COVID-19 pandemic, particularly the more transmissible and more immune evasive VOCs with substantial mutations, such as the Delta variant and the Omicron variant, which undermine the efficacy of existing vaccines and therapeutic neutralizing antibodies. ...
... As previously described [2], 200 ng of human ACE2 protein per well was coated on ELISA plates at 4 °C overnight followed by washing 3 times with PBST. The ELISA plates were blocked with 100 μL of blocking buffer per well at 37 °C for 1 h as above, meanwhile, fivefold serial dilutions of parental mAbs or a cocktail or Bi-Nab were incubated with 4 ng/mL SARS-CoV-2 RBD-mouse Fc protein for 1 h at 25 °C. ...
Potent neutralizing antibodies (nAbs) against SARS-CoV-2 are a promising therapeutic against the ongoing COVID-19 pandemic. However, the continuous emergence of neutralizing antibody escape variants makes it challenging for antibody therapeutics based on monospecific nAbs. Here, we generated an IgG-like bispecific antibody (bsAb), Bi-Nab, based on a pair of human neutralizing antibodies targeting multiple and invariant sites of the spike receptor binding domain (RBD): 35B5 and 32C7. We demonstrated that Bi-Nab exhibited higher binding affinity to the Delta spike protein than its parental antibodies and presented an extended inhibition breadth of preventing RBD binding to angiotensin-converting enzyme 2 (ACE2), the cellular receptor of SARS-CoV-2. In addition, pseudovirus neutralization results showed that Bi-Nab improved the neutralization potency and breadth with a lower half maximum inhibitory concentration (IC 50 ) against wild-type SARS-CoV-2, variants being monitored (VBMs) and variants of concern (VOCs). Notably, the IgG-like Bi-Nab enhanced the neutralizing activity against Omicron variants with potent capabilities for transmission and immune evasion in comparison with its parental monoclonal antibody (mAb) 32C7 and a cocktail (with the lowest IC 50 values of 31.6 ng/mL against the Omicron BA.1 and 399.2 ng/mL against the Omicron BA.2), showing evidence of synergistic neutralization potency of Bi-Nab against the Omicron variants. Thus, Bi-Nab represents a feasible and effective strategy against SARS-CoV-2 variants of concern.
... In some cases, the rationale may be relatively strong when the principal mechanisms of drug action are similar between the original and the new applications. For example, repurposing RNA polymerase nucleos(t)ide drugs such as remdesivir and molnupiravir to inhibit viral RNA synthesis would be expected to have a relatively high probability of success, but it remains a trial-and-error endeavour to identify nucleos(t)ide analogues that escape the SARS-CoV-2 proofreading mechanism 252 . By contrast, repurposing DNA polymerase inhibitors such as tenofovir to inhibit the RNA synthesis of SARS-CoV-2 is doomed to failure because of their different mechanisms of action. ...
The coronavirus disease 2019 (COVID-19) pandemic has stimulated tremendous efforts to develop therapeutic strategies that target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or human proteins to control viral infection, encompassing hundreds of potential drugs and thousands of patients in clinical trials. So far, a few small-molecule antiviral drugs (nirmatrelvir–ritonavir, remdesivir and molnupiravir) and 11 monoclonal antibodies have been marketed for the treatment of COVID-19, mostly requiring administration within 10 days of symptom onset. In addition, hospitalized patients with severe or critical COVID-19 may benefit from treatment with previously approved immunomodulatory drugs, including glucocorticoids such as dexamethasone, cytokine antagonists such as tocilizumab and Janus kinase inhibitors such as baricitinib. Here, we summarize progress with COVID-19 drug discovery, based on accumulated findings since the pandemic began and a comprehensive list of clinical and preclinical inhibitors with anti-coronavirus activities. We also discuss the lessons learned from COVID-19 and other infectious diseases with regard to drug repurposing strategies, pan-coronavirus drug targets, in vitro assays and animal models, and platform trial design for the development of therapeutics to tackle COVID-19, long COVID and pathogenic coronaviruses in future outbreaks.
... For most viruses, there are a wide variety of cell lines that can be used to assess the potency of polyclonal and monoclonal antibodies with antiviral activity. For example, for SARS-CoV-2 alone, cell lines that have been used to assess antibody neutralization activity include Vero/Vero E6 (African green monkey kidney), 293/293T (human embryonic kidney), HeLa (human cervical carcinoma), Huh7 (human hepatocellular carcinoma), Calu-3 (human lung carcinoma), HT1080 (human fibrosarcoma), U2OS (human osteosarcoma), and HOS (human osteosarcoma) cells, often engineered to stably express human angiotensin-converting enzyme 2 (ACE2, the SARS-CoV-2 receptor) and/or transmembrane serine protease 2 (TMPRSS2, a protease involved in SARS-CoV-2 entry) 50,52,55,[145][146][147][148][149][150] . ...
Viral diseases represent a major public health concern and an ever-present risk for developing into a future pandemic. Antiviral antibody therapeutics, either alone or in combination with other therapies, have emerged as valuable preventative and treatment options, including during a global emergency. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.
... Since the early COVID-19 pandemic, a large number of potent neutralizing antibodies against SARS-CoV-2 have been reported 27,[35][36][37][39][40][41][42] . However, the continuous emergence of SARS-CoV-2 variants, especially the Omicron sublineages, with high levels of mutations has led to serious concerns about immune evasion, vaccine failure, and the lack of effective neutralizing antibodies for clinical treatment 2,3,6,8,9,11,33,43 . ...
Due to the continuous evolution of SARS-CoV-2, the Omicron variant has emerged and exhibits severe immune evasion. The high number of mutations at key antigenic sites on the spike protein has made a large number of existing antibodies and vaccines ineffective against this variant. Therefore, it is urgent to develop efficient broad-spectrum neutralizing therapeutic drugs. Here we characterize a rabbit monoclonal antibody (RmAb) 1H1 with broad-spectrum neutralizing potency against Omicron sublineages including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3 and BA.4/5. Cryo-electron microscopy (cryo-EM) structure determination of the BA.1 spike-1H1 Fab complexes shows that 1H1 targets a highly conserved region of RBD and avoids most of the circulating Omicron mutations, explaining its broad-spectrum neutralization potency. Our findings indicate 1H1 as a promising RmAb model for designing broad-spectrum neutralizing antibodies and shed light on the development of therapeutic agents as well as effective vaccines against newly emerging variants in the future.
... It contains the receptor-binding domain (RBD) responsible for human ACE2 (hACE2) receptor binding and mediating virus entry [12,13]. Neutralizing antibodies (NAbs) specific for RBD in the S1 region of the S protein play critical roles in COVID-19 protection [14,15]. Thus far, SARS-CoV-2 S protein-encoding mRNA vaccines have shown high protection efficacy in human populations [2]. ...
... The generation of NAbs is known to be crucial for protecting people from virus infection. NAb levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection in humans [14,15]. To gain insight on pAD10160-generated NAbs in vivo, antisera from pAD1016-immunized C57BL/6 mice and rabbits were tested for their ability to block the mimic infection of ACE2-transgenic HEK293T cells by pseudoviruses displaying the S protein of the SARS-CoV-2 wildtype virus or variants. ...
Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants currently pose a global challenge to the control of the COVID-19 pandemic. We previously reported a pVAX1-based DNA vaccine candidate, pAD1002, that encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 wildtype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1 RBD-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-γ+ cellular responses in BALB/c and C57BL/6 mice. More importantly, pAD1016 vaccination in mice, rabbits and pigs generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These preliminary data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad-spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy of further translational study as a COVID-19 vaccine candidate.
