title: Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants.
abstract: BACKGROUND
  Experimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties.
  
  
  RESULTS
  Human mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318-510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one.
  
  
  CONCLUSIONS
  The combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.
authors.affiliation: Crucell Holland B.V., Leiden, Netherlands. j.termeulen@crucell.com
authors.forename: Jan
authors.initials: J
authors.lastname: ter Meulen
authors.forename: Edward N
authors.initials: EN
authors.lastname: van den Brink
authors.forename: Leo L M
authors.initials: LL
authors.lastname: Poon
authors.forename: Wilfred E
authors.initials: WE
authors.lastname: Marissen
authors.forename: Cynthia S W
authors.initials: CS
authors.lastname: Leung
authors.forename: Freek
authors.initials: F
authors.lastname: Cox
authors.forename: Chung Y
authors.initials: CY
authors.lastname: Cheung
authors.forename: Arjen Q
authors.initials: AQ
authors.lastname: Bakker
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Bogaards
authors.forename: Els
authors.initials: E
authors.lastname: van Deventer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Preiser
authors.forename: Hans Wilhelm
authors.initials: HW
authors.lastname: Doerr
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Chow
authors.forename: John
authors.initials: J
authors.lastname: de Kruif
authors.forename: Joseph S M
authors.initials: JS
authors.lastname: Peiris
authors.forename: Jaap
authors.initials: J
authors.lastname: Goudsmit
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000940:Antigenic Variation; D000956:Antigens, Viral / Q000276:immunology*; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured / Q000821:virology; D002522:Chlorocebus aethiops; D004196:Disease Outbreaks; D004306:Dose-Response Relationship, Immunologic; D004357:Drug Synergism; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D007116:Immunization, Passive; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D008264:Macrophages / Q000821:virology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D053827:Nandiniidae / Q000821:virology; D009500:Neutralization Tests; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000276:immunology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology*; D045169:Severe Acute Respiratory Syndrome / Q000188:drug therapy / Q000453:epidemiology / Q000517:prevention & control* / Q000628:therapy / Q000821:virology; D064370:Spike Glycoprotein, Coronavirus; D020349:Surface Plasmon Resonance; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D014779:Virus Replication
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D007106:Immune Sera; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV