RESUMEN
Bats carry genetically diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Some of them utilize human angiotensin-converting enzyme 2 (hACE2) as a receptor and cannot efficiently replicate in wild-type mice. Our previous study demonstrated that the bat SARSr-CoV rRsSHC014S induces respiratory infection and lung damage in hACE2 transgenic mice but not wild-type mice. In this study, we generated a mouse-adapted strain of rRsSHC014S, which we named SMA1901, by serial passaging of wild-type virus in BALB/c mice. SMA1901 showed increased infectivity in mouse lungs and induced interstitial lung pneumonia in both young and aged mice after intranasal inoculation. Genome sequencing revealed mutations in not only the spike protein but the whole genome, which may be responsible for the enhanced pathogenicity of SMA1901 in wild-type BALB/c mice. SMA1901 induced age-related mortality similar to that observed in SARS and COVID-19. Drug testing using antibodies and antiviral molecules indicated that this mouse-adapted virus strain can be used to test prophylactic and therapeutic drug candidates against SARSr-CoVs. IMPORTANCE The genetic diversity of SARSr-CoVs in wildlife and their potential risk of cross-species infection highlights the importance of developing a powerful animal model to evaluate the antibodies and antiviral drugs. We acquired the mouse-adapted strain of a bat-origin coronavirus named SMA1901 by natural serial passaging of rRsSHC014S in BALB/c mice. The SMA1901 infection caused interstitial pneumonia and inflammatory immune responses in both young and aged BALB/c mice after intranasal inoculation. Our model exhibited age-related mortality similar to SARS and COVID-19. Therefore, our model will be of high value for investigating the pathogenesis of bat SARSr-CoVs and could serve as a prospective test platform for prophylactic and therapeutic candidates.
Asunto(s)
Quirópteros , Ratones , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Ratones/virología , Quirópteros/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/clasificación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Ratones Endogámicos BALB C , COVID-19/mortalidad , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/mortalidad , Pase Seriado , Antivirales/farmacología , Antivirales/uso terapéutico , Anticuerpos Antivirales/farmacología , Anticuerpos Antivirales/uso terapéutico , Zoonosis Virales/tratamiento farmacológico , Zoonosis Virales/transmisión , Zoonosis Virales/virología , Enfermedades Pulmonares Intersticiales/tratamiento farmacológico , Enfermedades Pulmonares Intersticiales/virología , Envejecimiento , Evaluación Preclínica de MedicamentosRESUMEN
SARS, or severe acute respiratory syndrome, is caused by a novel coronavirus (COVID-19). This situation has compelled many pharmaceutical R&D companies and public health research sectors to focus their efforts on developing effective therapeutics. SARS-nCoV-2 was chosen as a protein spike to targeted monoclonal antibodies and therapeutics for prevention and treatment. Deep mutational scanning created a monoclonal antibody to characterize the effects of mutations in a variable antibody fragment based on its expression levels, specificity, stability, and affinity for specific antigenic conserved epitopes to the Spike-S-Receptor Binding Domain (RBD). Improved contacts between Fv light and heavy chains and the targeted antigens of RBD could result in a highly potent neutralizing antibody (NAbs) response as well as cross-protection against other SARS-nCoV-2 strains. It undergoes multipoint core mutations that combine enhancing mutations, resulting in increased binding affinity and significantly increased stability between RBD and antibody. In addition, we improved. Structures of variable fragment (Fv) complexed with the RBD of Spike protein were subjected to our established in-silico antibody-engineering platform to obtain enhanced binding affinity to SARS-nCoV-2 and develop ability profiling. We found that the size and three-dimensional shape of epitopes significantly impacted the activity of antibodies produced against the RBD of Spike protein. Overall, because of the conformational changes between RBD and hACE2, it prevents viral entry. As a result of this in-silico study, the designed antibody can be used as a promising therapeutic strategy to treat COVID-19.
Asunto(s)
COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Epítopos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/metabolismo , Internalización del Virus , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Antivirales/farmacología , Anticuerpos Antivirales/metabolismo , SARS-CoV-2/metabolismo , Unión ProteicaRESUMEN
The recently circulating SARS-CoV-2 Omicron BA.5 is rampaging the world with elevated transmissibility compared to the original SARS-CoV-2 strain. Immune escape of BA.5 was observed after treatment with many monoclonal antibodies, calling for broad-spectrum, immune-escape-evading therapeutics. In retrospect, we previously reported Kansetin as an ACE2 mimetic and a protein antagonist against SARS-CoV-2, which proved potent neutralization bioactivity on the Reference, Alpha, Beta, Delta, and Omicron strains of SARS-CoV-2. Since BA.5 is expected to rely on the interaction of the Spike complex with human ACE2 for cell entry, we reasonably assumed the lasting efficacy of the ACE2-mimicking Kansetin for neutralizing the new SARS-CoV-2 variant. The investigation was accordingly performed on in vitro Kansetin-Spike binding affinity by SPR and cell infection inhibition ability with pseudovirus and live virus assays. As a result, Kansetin showed dissociation constant KD and half inhibition concentration IC50 at the nanomolar to picomolar level, featuring a competent inhibition effect against the BA.5 sublineage. Conclusively, Kansetin is expected to be a promising therapeutic option against BA.5 and future SARS-CoV-2 sublineages.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Humanos , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Inhibidores Enzimáticos/farmacologíaRESUMEN
Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.
Asunto(s)
COVID-19 , Inmunoglobulina A , SARS-CoV-2 , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Antivirales/farmacología , Epítopos/química , Humanos , Inmunoglobulina A/farmacología , Inmunoglobulina G , Ratones , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del CoronavirusRESUMEN
The major challenge to controlling the COVID pandemic is the rapid mutation rate of the SARS-CoV-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-CoV-2 variants. Here, we report a synthetic nanobody (named C5G2) obtained by phage display and subsequent antibody engineering. C5G2 has a single-digit nanomolar binding affinity to the RBD domain and inhibits its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assays indicated that monovalent C5G2 could protect the cells from infection with SARS-CoV-2 wild-type virus and most of the viruses of concern, i.e., Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron BA.1 among all the variants, with an IC50 of 4.9 ng/mL. The cryo-EM structure of C5G2 in complex with the spike trimer showed that C5G2 binds to RBD mainly through its CDR3 at a conserved region that does not overlap with the ACE2 binding surface. Additionally, C5G2 binds simultaneously to the neighboring NTD domain of the spike trimer through the same CDR3 loop, which may further increase its potency against viral infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may serve as an effective drug for prophylaxis and therapy against Omicron as well as future variants.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , SARS-CoV-2 , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , COVID-19 , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del CoronavirusRESUMEN
The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/farmacología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/farmacología , Anticuerpos Antivirales/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/genética , COVID-19/inmunología , COVID-19/virología , Cricetinae , Citidina/análogos & derivados , Combinación de Medicamentos , Hidroxilaminas , Indazoles , Lactamas , Leucina , Ratones , Nitrilos , Prolina , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Triazinas , TriazolesRESUMEN
SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 µg mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.
Asunto(s)
SARS-CoV-2 , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Unión Proteica , Ingeniería de Proteínas , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacología , Proteínas Recombinantes de Fusión/farmacología , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/genética , Modelos Moleculares , Pruebas de Neutralización , Unión Proteica/efectos de los fármacos , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genética , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaAsunto(s)
Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Receptores Virales/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Animales , COVID-19/inmunología , COVID-19/virología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/virología , Ratones , Unión Proteica/efectos de los fármacos , Receptores Virales/genética , Receptores Virales/inmunología , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Many antibody-based antivirals, including broadly neutralizing antibodies (bnAbs) against various influenza virus strains, suffer from limited potency. A booster of the antiviral activity of an antibody is expected to facilitate development of antiviral therapeutics. In this study, a nanodisc (ND), a discoidal lipid bilayer encircled by membrane scaffold proteins, is engineered to provide virucidal properties to antibodies, thereby augmenting their antiviral activity. NDs carrying the Fc-binding peptide sequence form an antibody-ND complex (ANC), which can co-endocytose into cells infected with influenza virus. ANC efficiently inhibits endosome escape of viral RNA by dual complimentary mode of action. While the antibody moiety in an ANC inhibits hemagglutinin-mediated membrane fusion, its ND moiety destroys the viral envelope using free hemagglutinins that are not captured by antibodies. Providing virus-infected host cells with the ability to self-eliminate by the synergistic effect of ANC components dramatically amplifies the antiviral efficacy of a bnAb against influenza virus. When the efficacy of ANC is assessed in mouse models, administration of ANCs dramatically reduces morbidity and mortality compared to bnAb alone. This study is the first to demonstrate the novel nanoparticle ANC and its role in combating viral infections, suggesting that ANC is a versatile platform applicable to various viruses.
Asunto(s)
Anticuerpos Antivirales , Envoltura Viral , Animales , Anticuerpos Antivirales/farmacología , Antivirales/farmacología , Anticuerpos ampliamente neutralizantes , Hemaglutininas , RatonesRESUMEN
Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.
Asunto(s)
Anticuerpos Antivirales/farmacología , Antivirales/farmacología , Anticuerpos Anti-VIH/farmacología , Receptores Fc/efectos de los fármacos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Epítopos/efectos de los fármacos , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina G/inmunología , Ratones Endogámicos C57BL , Receptores de IgG/efectos de los fármacos , Receptores de IgG/inmunologíaAsunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos , Expresión Génica , Humanos , Evasión Inmune , Modelos Moleculares , Mutación , Pruebas de Neutralización , Unión Proteica/efectos de los fármacos , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Virales/genética , Receptores Virales/inmunología , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Omicron was designated by the WHO as a VOC on 26 November 2021, only 4 days after its sequence was first submitted. However, the impact of Omicron on current antibodies and vaccines remains unknown and evaluations are still a few weeks away. We analysed the mutations in the Omicron variant against epitopes. In our database, 132 epitopes of the 120 antibodies are classified into five groups, namely NTD, RBD-1, RBD-2, RBD-3, and RBD-4. The Omicron mutations impact all epitopes in NTD, RBD-1, RBD-2, and RBD-3, with no antibody epitopes spared by these mutations. Only four out of 120 antibodies may confer full resistance to mutations in the Omicron spike, since all antibodies in these three groups contain one or more epitopes that are affected by these mutations. Of all antibodies under EUA, the neutralisation potential of Etesevimab, Bamlanivimab, Casirivimab, Imdevima, Cilgavimab, Tixagevimab, Sotrovimab, and Regdanvimab might be dampened to varying degrees. Our analysis suggests the impact of Omicron on current therapeutic antibodies by the Omicron spike mutations may also apply to current COVID-19 vaccines.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Antivirales/farmacología , Simulación por Computador , Mutación/inmunología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Neutralizantes/farmacología , Bases de Datos Factuales , Epítopos/inmunología , Humanos , Inmunoglobulina G/farmacología , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/farmacología , SARS-CoV-2/inmunología , Proteínas Virales/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Anticuerpos Monoclonales/clasificación , Anticuerpos Antivirales/inmunología , COVID-19/terapia , COVID-19/virología , Células HEK293 , Humanos , Proteínas Recombinantes/inmunología , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Orthomyxoviridae/efectos de los fármacos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/aislamiento & purificación , Células CHO , Cricetinae , Cricetulus , Reacciones Cruzadas , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Ratones , Orthomyxoviridae/clasificación , Orthomyxoviridae/inmunologíaRESUMEN
SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, including with a P681R mutation, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Antivirales/inmunología , Furina/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Anticuerpos Antivirales/farmacología , Humanos , Mutación , Nariz/enzimología , Proproteína Convertasas/metabolismo , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoAsunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Tratamiento Farmacológico de COVID-19 , Epítopos/química , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Anticuerpos Antivirales/farmacología , Sitios de Unión , COVID-19/inmunología , COVID-19/virología , Línea Celular Tumoral , Chlorocebus aethiops , Microscopía por Crioelectrón , Epítopos/inmunología , Epítopos/metabolismo , Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptores Virales/química , Receptores Virales/inmunología , Receptores Virales/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
The severe acute respiratory syndrome (SARS-CoV-2), a newly emerging of coronavirus, continues to infect humans in the absence of a viable treatment. Neutralizing antibodies that disrupt the interaction of RBD and ACE2 has been under the spotlight as a way of developing the COVID-19 treatment. Some animals, such as llamas, manufacture heavy-chain antibodies that have a single variable domain (VHH) instead of two variable domains (VH/VL) as opposed to typical antibodies. Nanobodies are antigen-specific, single-domain, changeable segments of camelid heavy chain-only antibodies that are recombinantly produced. These types of antibodies exhibit a wide range of strong physical and chemical properties, like high solubility, and stability. The VHH's high-affinity attachment to the receptor-binding domain (RBD) allowed the neutralization of SARS-CoV-2. To tackle COVID-19, some nanobodies are being developed against SARS-CoV-2, some of which have been recently included in clinical trials. Nanobody therapy may be useful in managing the COVID-19 pandemic as a potent and low-cost treatment. This paper describes the application of nanobodies as a new class of recombinant antibodies in COVID-19 treatment.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Anticuerpos de Dominio Único , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , COVID-19/inmunología , COVID-19/terapia , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Human noroviruses (NoVs) are the most common cause of acute gastroenteritis worldwide. One major obstacle in developing NoV vaccines is the lack of robust cell culture for efficacy evaluation. In this study, we successfully developed a NoV virus-like particle (VLP) entry assay based on split NanoLuc luciferase (LgBiT and HiBiT) complementation. HiBiT-tagged NoV GII.4 VLP (VLP-HiBiT) can be efficiently produced in Pichia pastoris and retain binding activity towards NoV receptor histo-blood group antigens (HBGAs). A 293T-FUT2-LgBiT cell line was established and was shown to stably express cell surface HBGAs and intracellular LgBiT. GII.4 VLP-HiBiT can bind and enter into the 293-FUT2-LgBiT cells, producing strong luminescence signals in live cells. Anti-GII.4 sera can inhibit VLP-HiBiT entry into the 293-FUT2-LgBiT cells in a dose-dependent manner, and neutralizing titers well correlate with their blocking titers measured by HBGAs-binding blockade assay. Moreover, such a surrogate infection/neutralization assay can be applied to other NoV genotypes such as GI.1 and GII.17. Together, the VLP-HiBiT entry assay can mimic both NoV attachment and internalization in live cells and thus facilitate reliable and comprehensive evaluation of NoV vaccine and antibodies.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Anticuerpos Antivirales/farmacología , Luciferasas/genética , Norovirus/genética , Norovirus/inmunología , Internalización del Virus , Anticuerpos Antivirales/inmunología , Infecciones por Caliciviridae/virología , Prueba de Complementación Genética/métodos , Prueba de Complementación Genética/normas , Genotipo , Células HEK293 , Humanos , Luciferasas/metabolismo , Mediciones Luminiscentes , Saccharomycetales/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/inmunología , Acoplamiento ViralRESUMEN
Passive antibody therapy and vectored antibody gene delivery (VAGD) in particular offer an innovative approach to combat persistent viral diseases. Here, we exploit a small animal model to investigate synergies of VAGD with the host's endogenous immune defense for treating chronic viral infection. An adeno-associated virus (AAV) vector delivering the lymphocytic choriomeningitis virus (LCMV)-neutralizing antibody KL25 (AAV-KL25) establishes protective antibody titers for >200 days. When therapeutically administered to chronically infected immunocompetent wild-type mice, AAV-KL25 affords sustained viral load control. In contrast, viral mutational escape thwarts therapeutic AAV-KL25 effects when mice are unable to mount LCMV-specific antibody responses or lack CD8+ T cells. VAGD augments antiviral germinal center B cell and antibody-secreting cell responses and reduces inhibitory receptor expression on antiviral CD8+ T cells. These results indicate that VAGD fortifies host immune defense and synergizes with B cell and CD8 T cell responses to restore immune control of chronic viral infection.