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.

Figure 1. Neutralization of Wild-Type SARS-CoV and a CR3014-Neutralization Escape Variant (E6) with mAbs CR3014 and CR3022 Individually and in Combination

Neutralization of 100 TCID ₅₀ of each virus was performed in octuplicate. Data are also shown in Table 1.

Figure 2. Binding of mAbs CR3014 and CR3022 to Recombinant Wild-Type and P462L-Substituted S318–510 Fragments Analyzed by ELISA

Bars represent the means ± standard deviation.

Figure 3. Competition ELISA on Immobilized S318–510

Binding of biotinylated CR3014 (A) and CR3022 (B) was analyzed in the presence of competitor CR3014 (open circles), CR3022 (closed circles), and control mAb (open squares). Binding is expressed as percentage of binding without competitor.

Figure 4. Binding of Human mAbs to S318–510 Fragments in ELISA

Wild-type and variant fragments were synthesized according to published sequences of SARS-CoV strains. Wild-type S318–510 from strain Frankfurt 1, variant fragments from strains GZ02 (K344R), Sin3408 (S353F), Shanghai LY (R426G and N437D), GZ-C (Y436H), Sino1–11 (Y442S), BJ302 cl. 2 (N479S), SZ3 (K344R, F360S, N479K, and T487S), GD03T0013 (K344R, F360S, L472P, D480G, and T487S), and GD01 (K344R and F501Y). Bars represent the means ± standard deviation.

Figure 5. Infection of Differentiated Human Macrophages with SARS-CoV (HKU-39849) in the Presence of CR3014 or a Control mAb

One representative experiment of three independent experiments is shown. Total RNA was extracted at 6 and 24 hours postinfection, and the copy number of positive (P) and negative (N) strand RNA of SARS-CoV ORF1b was determined by real-time quantitative RT-PCR and normalized for the levels of β-actin mRNA. The bars represent mean of duplicate viral load titrations. In the control experiment, * indicates an aberrant result.