Wed that the HR1 and HR2 binding HmAbs are more effective in inhibiting the entry of the RBD surrogate clinical isolates. Those HmAbs did not inhibit the entry of VSV-G pseudotyped virus (data not shown).AZ876 combinations of SARS-CoV HmAbs Targeted to Different Regions of the S Glycoprotein More Efficiently Inhibit the Entry of RBD Surrogate Clinical IsolatesNext, we tested combinations of 4D4 (binds to S1, N-terminal of RBD), 1F8 (binds to HR1) and 5E9 (binds to HR2) HmAbs to see if they can more effectively inhibit viral entry. The combinations of 4D4/1F8, 4D4/5E9 and 1F8/5E9 HmAbs were more effective in blocking Urbani pseudovirus entry compared to the individual antibodies (p value ,0.05). The same pattern of inhibition was seen with 
the Sin845-S, GZ-C-S and GZ0402-S pseudoviruses (p values = 0.005?.04). However, these HmAb combinations exhibited similar levels of GD01 pseudovirus blocking as seen with the 1F8 or 5E9 HmAbs when used individually. Maximum inhibition of 90?5 (p values = 0.003?.04) was noted 
when a combination of 4D4/1F8/5E9 HmAbs was used (Fig. 4). These results indicated that a cocktail of HmAbs targeting different conserved regions of the S protein is likely to be more effective in neutralizing different SARS-CoV clinical isolates than individual antibodies with specificity to those regions.S Proteins Containing RBD Sequences of Sin845, GD01, GZ0402 and GZ-C Isolates do not Affect Pseudovirus EntryWe prepared pseudoviruses expressing S proteins containing RBD sequences of Sin845, 1662274 GD01, GZ0402 and GZ-C isolates to serve as “RBD surrogates” for those clinical isolates. The S protein and the HIV p24 Ag incorporation into the viral particles were confirmed by western blot (Fig. S3A). In HIV/DE, as expected, no surface glycoprotein was detected. Pseudoviruses expressing the mutant S proteins entered 293 cells, stably expressing ACE2, with equal efficiency when compared to the HIV/S positive control (Fig. S3B).SARS-CoV Neutralization by Human AntibodiesFigure 1. Reactivity of the18 Neutralizing HmAbs with SARS 23727046 CoV 12-510-S1 proteins. Medisorp ELISA plates were coated with 100 ng/well of Urbani and RBD mutant 12-510S1-IgG proteins and 2.5 mg/ml of each HmAb was used as the primary antibody. Anti-human IgG2 HRP mouse monoclonal antibody was used as secondary antibody. OD was measured at 450 nm. Error bars represent SD of a representative experiment performed in triplicates. (A) Urbani versus Sin845 mutant. (B) Urbani versus GD01 mutant. (C) Urbani versus GZ0402 mutant. (D) Urbani versus GZ-C mutant. doi:10.1371/journal.pone.0050366.gDiscussionTherapies that are directed towards RNA viruses, including SARS-CoV, must consider the quasispecies nature of the viral population, the ability of the virus to Eledoisin mutate and recombine in response to host selection pressure [32]. Such changes likely allowed the SARS-CoV to jump from the intermediate hosts to humans and resulted in the 2002?003 outbreak [33]. Therefore, therapies against SARS-CoV, including passive immunotherapy with HmAbs, must be able to neutralize awide range of clinical isolates and prevent or minimize generation of escape mutants. In this study, we found that the anti-S1 HmAbs were unable to bind to the recombinant mutant 12-510 S1 fragments (i.e. Sin845, GD01 and GZ0402) except for the 4D4 antibody, which showed only a decreased binding. All anti-S1 HmAbs showed enhanced binding to the GZ-C-S1 fragment. The 4D4 HmAb binds to an epitope that resides N-terminal to RBD and.Wed that the HR1 and HR2 binding HmAbs are more effective in inhibiting the entry of the RBD surrogate clinical isolates. Those HmAbs did not inhibit the entry of VSV-G pseudotyped virus (data not shown).Combinations of SARS-CoV HmAbs Targeted to Different Regions of the S Glycoprotein More Efficiently Inhibit the Entry of RBD Surrogate Clinical IsolatesNext, we tested combinations of 4D4 (binds to S1, N-terminal of RBD), 1F8 (binds to HR1) and 5E9 (binds to HR2) HmAbs to see if they can more effectively inhibit viral entry. The combinations of 4D4/1F8, 4D4/5E9 and 1F8/5E9 HmAbs were more effective in blocking Urbani pseudovirus entry compared to the individual antibodies (p value ,0.05). The same pattern of inhibition was seen with the Sin845-S, GZ-C-S and GZ0402-S pseudoviruses (p values = 0.005?.04). However, these HmAb combinations exhibited similar levels of GD01 pseudovirus blocking as seen with the 1F8 or 5E9 HmAbs when used individually. Maximum inhibition of 90?5 (p values = 0.003?.04) was noted when a combination of 4D4/1F8/5E9 HmAbs was used (Fig. 4). These results indicated that a cocktail of HmAbs targeting different conserved regions of the S protein is likely to be more effective in neutralizing different SARS-CoV clinical isolates than individual antibodies with specificity to those regions.S Proteins Containing RBD Sequences of Sin845, GD01, GZ0402 and GZ-C Isolates do not Affect Pseudovirus EntryWe prepared pseudoviruses expressing S proteins containing RBD sequences of Sin845, 1662274 GD01, GZ0402 and GZ-C isolates to serve as “RBD surrogates” for those clinical isolates. The S protein and the HIV p24 Ag incorporation into the viral particles were confirmed by western blot (Fig. S3A). In HIV/DE, as expected, no surface glycoprotein was detected. Pseudoviruses expressing the mutant S proteins entered 293 cells, stably expressing ACE2, with equal efficiency when compared to the HIV/S positive control (Fig. S3B).SARS-CoV Neutralization by Human AntibodiesFigure 1. Reactivity of the18 Neutralizing HmAbs with SARS 23727046 CoV 12-510-S1 proteins. Medisorp ELISA plates were coated with 100 ng/well of Urbani and RBD mutant 12-510S1-IgG proteins and 2.5 mg/ml of each HmAb was used as the primary antibody. Anti-human IgG2 HRP mouse monoclonal antibody was used as secondary antibody. OD was measured at 450 nm. Error bars represent SD of a representative experiment performed in triplicates. (A) Urbani versus Sin845 mutant. (B) Urbani versus GD01 mutant. (C) Urbani versus GZ0402 mutant. (D) Urbani versus GZ-C mutant. doi:10.1371/journal.pone.0050366.gDiscussionTherapies that are directed towards RNA viruses, including SARS-CoV, must consider the quasispecies nature of the viral population, the ability of the virus to mutate and recombine in response to host selection pressure [32]. Such changes likely allowed the SARS-CoV to jump from the intermediate hosts to humans and resulted in the 2002?003 outbreak [33]. Therefore, therapies against SARS-CoV, including passive immunotherapy with HmAbs, must be able to neutralize awide range of clinical isolates and prevent or minimize generation of escape mutants. In this study, we found that the anti-S1 HmAbs were unable to bind to the recombinant mutant 12-510 S1 fragments (i.e. Sin845, GD01 and GZ0402) except for the 4D4 antibody, which showed only a decreased binding. All anti-S1 HmAbs showed enhanced binding to the GZ-C-S1 fragment. The 4D4 HmAb binds to an epitope that resides N-terminal to RBD and.
