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.

Methods and Findings

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.

Editors' Summary

Background.

Late in 2002, severe acute respiratory syndrome (SARS) emerged in the Guangdong province of China. In February 2003, an infected doctor from the province carried this new viral threat to human health to Hong Kong. Here, people staying in the same hotel caught the disease and took it to other countries. SARS was on the move, hitching lifts with international travellers. Because the virus responsible for SARS—SARS-CoV—spread by close person-to-person contact and killed 10% of the people it infected, health experts feared a world-wide epidemic. This was avoided by the World Health Organization issuing a global alert and warning against unnecessary travel to affected areas and by public-health officials isolating patients and their close contacts. By July 2003, the first SARS epidemic was over. 8,098 people had been infected; 774 people had died. Since then, sporadic cases of SARS have been contained locally.

Why Was This Study Done?

The first epidemic of SARS was caused by an animal virus that became adapted to spread between people. There is no reason this process won't be repeated. If it is, stringent quarantine measures could again prevent a global epidemic, but at considerable economic cost. What is needed is a way to prevent SARS developing in healthy people who have been exposed to SARS-CoV and to treat sick people so that they are less infectious and can fight the virus. In this study, researchers have been investigating “passive immunization” as a way to limit SARS epidemics. In passive immunization, short-term protection against illness is achieved by injecting antibodies—proteins that recognize specific molecules (called antigens) on foreign organisms such as bacteria and viruses and prevent those organisms from causing disease. Antibodies for passive immunization can be isolated from blood taken from people who have had SARS, or they can be manufactured as so-called “human monoclonal antibodies” in a laboratory. One of these human monoclonal antibodies—CR3014—had been previously made and shown to prevent lung damage in ferrets infected with SARS-CoV and to stop the infected animals from infecting others. But for effective disease prevention in people, a single monoclonal antibody might not be enough. There are strains of SARS-CoV that CR3014 does not recognize and therefore cannot act against. Also, the virus can alter the antigen recognized by CR3014 when it is grown at a low antibody concentration, producing so-called escape variants; if this happens CR3014 can no longer prevent these escape variants from killing human cells.

What Did the Researchers Do and Find?

The researchers tested how well a combination of two monoclonal antibodies controlled SARS-CoV killing of human cells. First, they showed that CR3014 escape variants all had the same small change in a part of the virus surface that interacts with human cells. CR3014 blocked this interaction in the parent SARS-CoV strain but not in the escape variants. They then made a new monoclonal antibody—CR3022—that prevented both the parent SARS-CoV stain and the CR3014 escape viruses from killing human cells. The two antibodies bound to neighboring parts of the virus surface, and both of them could bind at the same time. CR3022 also bound to surfaces of SARS-CoV strains to which CR3014 does not bind. And when they tried, the researchers could not generate any viral escape variants to which CR3022 was unable to bind. Finally, the effect of the two antibodies together on inhibition of SARS-CoV killing of human cells was more than the sum of their individual effects.

What Do These Findings Mean?

A combination of two (or more) human monoclonal antibodies that recognize different parts of the SARS-CoV surface that interacts with human cells might be a good way to immunize people passively against SARS-CoV. It might minimize the possibility of escape variants arising, broaden the range of virus strains against which protection is provided, and reduce the amount of antibody needed for effective protection. Before the approach is tried in people, it will have to be tested in animals—results from experiments done on human cells in dishes are not always replicated in whole animals or people. If the approach passes further tests, the hope is that passive immunization of people with SARS and their close contacts might reduce disease severity in infected people and reduce viral spread as effectively as dramatic quarantine measures

Additional Information.

Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030237.

• Medline Plus pages on SARS

• US Centers for Disease Control and Prevention information on SARS

• US National Institute of Allergy and Infectious Diseases factsheet about research on SARS

• Wikipedia page on SARS and monoclonal antibodies (note: Wikipedia is a free online encyclopedia that anyone can edit)

Abstract

Two human monoclonal antibodies that bind to different parts of the viral glycoprotein spike show synergistic effects in virus neutralization and suppress the emergence of resistant virus in vitro.