Therapies 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 
. Such changes likely allowed the SARS-CoV to jump from the intermediate hosts to humans and resulted in the 2002–2003 outbreak 
. Therefore, therapies against SARS-CoV, including passive immunotherapy with HmAbs, must be able to neutralize a wide 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 
. This HmAb continued to react albeit
to a lesser extent with surrogate clinical isolates. Moreover, when used in combination with other HmAbs, such as HmAb 3C7, it showed a synergistic effect 
. 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 
, and other studies which showed that a fragment consisting of amino acids 1055 to 1192 can induce neutralizing antibodies 
. Therefore, our finding of thirteen neutralizing HmAbs that bind to HR2 domain is consistent with the previous reports on mouse HR2 specific monoclonal antibodies. However those Abs were neither of human origin nor were tested for their ability to neutralize different clinical isolates 
. 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 mutations which most likely disrupted the conformation of the protein and resulted in the loss of expression of specific epitopes. In contrast, S2 domain reactive HmAbs were able to neutralize different RBD surrogate isolates even better than the 4D4 HmAb. Interestingly, analyses of the amino acid sequences of the S protein of 94 SARS-CoV clinical isolates revealed no mutations that are localized to HR1, and only K1163E mutation in the HR2 of six isolates (i.e. SZ3, GZ0402, HSZ-Cb, SZ16, A022, and GZ02), and Q1183R and Q1183K mutations in the HR2 of BJ182-12 and GZ-C isolates respectively. Other isolates were found to be free of any mutations in either HR1 or HR2 domains.
Most of the previously reported HmAbs recognize epitopes within the RBD in which mutations that allow viruses to escape neutralization without loss of infectivity are often found 
. This is further substantiated in the current study by the loss of neutralization by different RBD binding antibodies due to a single mutation in the S protein. However, HR1 and HR2 regions contain highly conserved neutralization epitopes in which mutations are likely lethal due to their critical role in the membrane fusion required for virus entry. Consequently, as shown by our results, the HR1 and HR2 specific antibodies can neutralize a broad spectrum of SARS-CoV variants with very limited potential, if any, for the emergence of escape mutants, especially when they are used in combination.
Based on results obtained using a combination of mAbs against HBV and RSV, and our previous demonstration of highly efficient neutralization of SARS-CoV using combinations of HmAbs 
, we reasoned that a combination of HmAbs targeting different regions of the S protein would likely confer better protection against different isolates. Combining the S1 binding 4D4 HmAb with either 1F8 (HR1) or 5E9 (HR2) resulted in increased virus neutralization of the mutants Sin845, GZ-C and GZ0402 compared to the individual HmAbs. Failure of the 4D4/1F8, 4D4/5E9 and 1F8/5E9 combinations to increase GD01 pseudovirus inhibition, when compared to the inhibition seen either with 5E9 or 1F8 alone, was likely due to enhanced binding and neutralization of this virus by these HmAbs when used individually. However, a combination comprising of HmAbs 4D4, 1F8 and 5E9 showed further significant increase in virus neutralization compared to each of the individual HmAbs or any of the pairs. These results suggested that the use of a cocktail consisting of HmAbs that can bind to different conserved regions of the S protein may be more desirable for therapeutic use against SARS-CoV infection. This speculation is supported by earlier studies that have shown that it is far more difficult to select for viral variants in the presence of either a polyclonal neutralizing antibody or a cocktail of monoclonal neutralizing antibodies 
Although others have demonstrated the utility of cocktails consisting of mAbs with similar mode of action 
, the present study shows the utility of a cocktail of HmAbs with different specificities and likely with different mechanisms of action for neutralizing a broad spectrum of SARS-CoV clinical isolates. SARS-CoV S protein is a class I fusion protein that contains HR1 and HR2 regions 
, which are highly conserved. Presence of similar structures in many other class I viral fusion proteins 
, point to a common fusion mechanism used by different viruses. Therefore, monoclonal antibodies against such conserved regions might constitute the most effective passive therapy. Our findings are not only relevant to designing a highly effective passive therapy for SARS-CoV but have implications for the development of passive therapy for other viral infections including influenza and HIV