Alpaca-derived Nanobody Of SARS-CoV-2

The Coronavirus Disease 2019(COVID-19)pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has posed serious threat to human health.Since late 2020,SARS-CoV-2 variants have emerged,from the initial variants of concerns known as Alpha,Beta,Gamma,Delta to the current Omicron variant in dominant,driving new epidemic waves.In March 2023,the latest SARS-CoV-2 Omicron variant sub lineages CH.1.1,CH.1.1.1,EA.1,XBB.2.3,were reported by the chinese center for disease control and prevention.The emergence of SARS-CoV-2 variants,particularly those with immune escape capacity,severely compromised the protective efficacy of the existing COVID-19 vaccines and antibodies.Therefore,the development of a broad-spectrum neutralizing antibody against SARS-CoV-2 is of great interest.In the current study,we constructed a phage display nanobody library and screened nanobodies with cross neutralizing activity and high affinity from the library.Nanobody monomers were further fused to the human IgG Fc domain to obtain long-acting nanobodies.The in vivo protective efficacy of candidate nanobodies was evaluated using two well-established infection models,the SARS-CoV-2 mouse-adapted model and a golden hamster infection model.In addition,antibodies recognizing different antigenic epitopes were identified by random peptide repertoire,and were further selected for the construction of bispecific antibody.The current study provided novel neutralizing antibody candidates with broad-spectrum neutralizing activity for the prevention and control of COVID-19.1.Construction of an anti-SARS-CoV-2 nanobody phage library and screening of nanobodiesSARS-CoV-2 S-trimer protein and S DNA vaccine were utilized as immunogens.Alpacas were immunized at an interval of 3 weeks for a total of 6 immunizations.Simultaneously,neutralizing antibody titers in immunized alpaca were monitored.Blood was taken and PBMCs were isolated after the 6th boost,and total RNA was extracted,reverse transcribed to form cDNA.After two rounds of PCR amplification,the heavy chain antibody variable region gene,also named VHH,was amplified with a size of 450 bp.The recombinant plasmids were constructed by ligation of VHHs with the phagemid vector pComb3XSS after digestion and electro transfer to TG1 competent cells.After addition of helper phage,a phage library of anti-SARS-CoV-2 nanobodies was successfully prepared after PEG/NaCl precipitation,and the library capacity was identified to reach 8 × 1013 pfu/mL and the positive rate was 96%.Using purified SARS-CoV-2 receptor binding domain(RBD)protein as the coating antigen,192 monoclonal colonies were randomly selected after three rounds of screening,which were used for phage ELISA identification and pseudovirus neutralization assay.The positive rate of colonies was 86.46%,20 of the supernatants of the single phage exhibited an inhibition rate of above 97%.Three candidate nanobodies with high binding and neutralization capacity were further selected by sequence analysis.2.Expression,identification,and evaluation of anti-SARS-CoV-2 nanobodiesIn this study,three SARS-CoV-2 candidate nanobody monomers were expressed as fusions with the Fc domain of human IgG,respectively termed as aVHH-11-Fc,aVHH13-Fc,and aVHH-14-Fc.As identified by SDS-PAGE,the purified nanobodies were all about 37 KD in size.Identified by indirect ELISA and competitive ELISA,the results showed that all three nanobodies could specifically bind to RBD protein and competitively inhibit the ACE2 receptor binding to RBD.According to the pseudovirus neutralization assay,aVHH-11-Fc,aVHH-13-Fc,and aVHH-14-Fc neutralized SARS-CoV-2 WTD614G,Alpha,Beta,Gamma,Delta and Omicron sublines BA.1,BA.2,BA.4 and BA.5 pseudovirus to varying degrees.In the SARS-CoV-2 mouse-adapted lethal infection model,intranasal administration of aVHH-11-Fc,aVHH-13-Fc,and aVHH-14-Fc at 0.5 h,1 h,and 2 h post-challenge effectively protected mice from lethal challenge with the virus and significantly reduced the viral loads in the upper and lower respiratory tracts,of which the protective efficacy of aVHH-13-Fc was better than that of the other two nanobodies.In golden hamsters,intranasal administration of aVHH-13-Fc at 0.5 h,1 h,2 h post challenge effectively protected golden hamsters from challenge of SARS-CoV-2 WT,Delta,Omicron BA.1 and BA.2,significantly reduced viral load in the lungs and alleviated pathological lung damage.Molecular docking simulation structure analysis revealed that aVHH-13-Fc binded to different amino acid residues in the RBD of different variants of SARS-CoV-2 to block RBD-ACE2 interactions,thus exerting broad-spectrum neutralization.3.Construction,expression,and preliminary evaluation of an anti-SARS-CoV2 bispecific antibodyIn this study,Ph.D.-12 phage display peptide library kit was used to identify antigenic epitopes of aVHH-13-Fc and aVHH-14-Fc.The results showed that aVHH-13-Fc and aVHH-14-Fc respectively recognized different antigenic epitopes.Then three different configurations of bispecific antibodies named 13-14-Fc,13-Fc-14,13-14-His were designed.The results of indirect ELISA showed that the binding activity of 13-14-Fc was slightly stronger than that of 13-Fc-14 and significantly stronger than that of 13-14-His.According to the VSV-SARS-CoV-2 surrogate virus neutralization assay,13-14-Fc showed better neutralizing ability than 13-Fc-14 and 13-14-His against SARS-CoV-2 WT,Alpha,Beta,Delta,Gamma,and Omicron BA.4 surrogate virus.In conclusion,we obtained alpaca-derived SARS-CoV-2 nanobody monomers and bispecific antibodies with cross-neutralizing ability against the SARS-CoV-2 variants.Among them,aVHH-13-Fc was proved to be a broad-spectrum candidate.In a mouseadapted infection model,aVHH-13-Fc protected mice from lethal challenge with a mouse adapted strain of SARS-CoV-2.In golden hamster infection model,aVHH-13-Fc provided protection against challenge of multiple SARS-CoV-2 variants.In conclusion,this study provided alpaca-derived nanobodies with broad-spectrum neutralizing activity,which exhibiting potent protective efficacy in animal models that can reflect different human COVID-19 clinical signs and severity.These results may provide references for the selection of potential therapeutic antibodies for COVID-19 patients in clinical,and simultaneously provide a basis for the development of a broad-spectrum anti-SARS-CoV2 antibody.