IesFigure 1. Reactivity of the18 Neutralizing HmAbs with SARS 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 Hesperidin site 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 neutralizes the SARS-CoV by inhibiting a post-binding step in the viral entry [11,19]. This HmAb continued to react albeitSARS-CoV Neutralization by Human AntibodiesFigure 2. Reactivity of Urbani SARS-CoV-S protein antibodies with Urbani S1 protein and mutant S1 proteins. (A) Different dilutions of a rabbit anti-Urbani SARS-CoV-S protein immune serum were tested in an ELISA against Urbani as well as mutant S1-IgG proteins. Anti-rabbit donkey polyclonal HRP antibody was used as the secondary antibody. (B) Competitive ELISA assay: Different protein concentrations of Urbani or GZ-C proteins were pre-incubated with 5A7 antibody then the protein/Ab mixtures were tested for binding to the other protein by ELISA. OD was measured at 450 nm. doi:10.1371/journal.pone.0050366.gto a lesser extent with surrogate clinical isolates. Moreover, when used in combination with other HmAbs, such as HmAb 3C7, it showed a synergistic effect [11]. Accordingly, our earlier as well as current results highlight the importance of the HmAb 4D4 in neutralizing SARS-CoV mutants and its ability to compliment other HmAbs. The Identification of S2 domain specific neutralizing HmAbs is consistent with a previous study which showed B-cell responses against the S2 domain in patients who recovered from SARS-CoV infection [34], and other studies which showed that a fragmentconsisting of amino acids 1055 to 1192 can induce neutralizing antibodies [29,30]. Therefore, our finding of Avasimibe site thirteen neutralizing HmAbs that bind to HR2 domain is consistent with the previous reports on mouse HR2 specific 11967625 monoclonal antibodies. However those Abs were neither of human origin nor were tested for their ability to neutralize different clinical isolates [27,35]. Our finding of nine HR1 binding neutralizing HmAbs is novel as there are no reported HR1 specific neutralizing antibodies to date. We believe that the HmAbs targeted to epitopes within the S1 domain failed to bind and neutralize because of the mutationsSARS-CoV.IesFigure 1. Reactivity of the18 Neutralizing HmAbs with SARS 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 neutralizes the SARS-CoV by inhibiting a post-binding step in the viral entry [11,19]. This HmAb continued to react albeitSARS-CoV Neutralization by Human AntibodiesFigure 2. Reactivity of Urbani SARS-CoV-S protein antibodies with Urbani S1 protein and mutant S1 proteins. (A) Different dilutions of a rabbit anti-Urbani SARS-CoV-S protein immune serum were tested in an ELISA against Urbani as well as mutant S1-IgG proteins. Anti-rabbit donkey polyclonal HRP antibody was used as the secondary antibody. (B) Competitive ELISA assay: Different protein concentrations of Urbani or GZ-C proteins were pre-incubated with 5A7 antibody then the protein/Ab mixtures were tested for binding to the other protein by ELISA. OD was measured at 450 nm. doi:10.1371/journal.pone.0050366.gto a lesser extent with surrogate clinical isolates. Moreover, when used in combination with other HmAbs, such as HmAb 3C7, it showed a synergistic effect [11]. Accordingly, our earlier as well as current results highlight the importance of the HmAb 4D4 in neutralizing SARS-CoV mutants and its ability to compliment other HmAbs. The Identification of S2 domain specific neutralizing HmAbs is consistent with a previous study which showed B-cell responses against the S2 domain in patients who recovered from SARS-CoV infection [34], and other studies which showed that a fragmentconsisting of amino acids 1055 to 1192 can induce neutralizing antibodies [29,30]. Therefore, our finding of thirteen neutralizing HmAbs that bind to HR2 domain is consistent with the previous reports on mouse HR2 specific 11967625 monoclonal antibodies. However those Abs were neither of human origin nor were tested for their ability to neutralize different clinical isolates [27,35]. Our finding of nine HR1 binding neutralizing HmAbs is novel as there are no reported HR1 specific neutralizing antibodies to date. We believe that the HmAbs targeted to epitopes within the S1 domain failed to bind and neutralize because of the mutationsSARS-CoV.
