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Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants.

ter Meulen J, van den Brink EN, Poon LL, Marissen WE, Leung CS, Cox F, Cheung CY, Bakker AQ, Bogaards JA, van Deventer E, Preiser W, Doerr HW, Chow VT, de Kruif J, Peiris JS, Goudsmit J - PLoS Med. (2006)

Bottom Line: 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.Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization.The combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection.

View Article: PubMed Central - PubMed

Affiliation: Crucell Holland B.V., Leiden, Netherlands. j.termeulen@crucell.com

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.

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Infection of Differentiated Human Macrophages with SARS-CoV (HKU-39849) in the Presence of CR3014 or a Control mAbOne 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-CoVORF1b was determined by real-time quantitative RT-PCR and normalized for the levels ofβ-actin mRNA. The bars represent mean of duplicate viral load titrations. In the control experiment, * indicates an aberrant result.
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pmed-0030237-g005: Infection of Differentiated Human Macrophages with SARS-CoV (HKU-39849) in the Presence of CR3014 or a Control mAbOne 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-CoVORF1b was determined by real-time quantitative RT-PCR and normalized for the levels ofβ-actin mRNA. The bars represent mean of duplicate viral load titrations. In the control experiment, * indicates an aberrant result.

Mentions: SARS-CoV did not replicate to measurable titers after day 2 on macrophages in the absence or presence of serial dilutions of mAb CR3014, a control mAb, or serum from a convalescent patient (unpublished data).Figure 5 shows the detection of SARS-CoV positive (P) or negative (N) strand RNA (replication intermediate) by RT-PCR in the same assay. Positive-strand RNA is detectable at 6 and 24 h postinfection, indicating uptake of virus by the macrophage. In the presence of 2.8 × 10−5 to 2.8 × 10−3 μg/ml of CR3014 a slight increase in virus uptake into macrophages is suggested by the increased copy number of positive-strand RNA (Figure 5). In contrast, negative-strand RNA, which indicates viral replication, is detectable at much lower levels that are constant over time, with no difference between the virus incubated with CR3014 or control antibody. Taken together, these results show that with or without monoclonal or polyclonal antibody, macrophages take up SARS-CoV, but this uptake does not lead to the productive virus replication and release of infectious virus.


Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants.

ter Meulen J, van den Brink EN, Poon LL, Marissen WE, Leung CS, Cox F, Cheung CY, Bakker AQ, Bogaards JA, van Deventer E, Preiser W, Doerr HW, Chow VT, de Kruif J, Peiris JS, Goudsmit J - PLoS Med. (2006)

Infection of Differentiated Human Macrophages with SARS-CoV (HKU-39849) in the Presence of CR3014 or a Control mAbOne 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-CoVORF1b was determined by real-time quantitative RT-PCR and normalized for the levels ofβ-actin mRNA. The bars represent mean of duplicate viral load titrations. In the control experiment, * indicates an aberrant result.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1483912&req=5

pmed-0030237-g005: Infection of Differentiated Human Macrophages with SARS-CoV (HKU-39849) in the Presence of CR3014 or a Control mAbOne 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-CoVORF1b was determined by real-time quantitative RT-PCR and normalized for the levels ofβ-actin mRNA. The bars represent mean of duplicate viral load titrations. In the control experiment, * indicates an aberrant result.
Mentions: SARS-CoV did not replicate to measurable titers after day 2 on macrophages in the absence or presence of serial dilutions of mAb CR3014, a control mAb, or serum from a convalescent patient (unpublished data).Figure 5 shows the detection of SARS-CoV positive (P) or negative (N) strand RNA (replication intermediate) by RT-PCR in the same assay. Positive-strand RNA is detectable at 6 and 24 h postinfection, indicating uptake of virus by the macrophage. In the presence of 2.8 × 10−5 to 2.8 × 10−3 μg/ml of CR3014 a slight increase in virus uptake into macrophages is suggested by the increased copy number of positive-strand RNA (Figure 5). In contrast, negative-strand RNA, which indicates viral replication, is detectable at much lower levels that are constant over time, with no difference between the virus incubated with CR3014 or control antibody. Taken together, these results show that with or without monoclonal or polyclonal antibody, macrophages take up SARS-CoV, but this uptake does not lead to the productive virus replication and release of infectious virus.

Bottom Line: 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.Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization.The combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection.

View Article: PubMed Central - PubMed

Affiliation: Crucell Holland B.V., Leiden, Netherlands. j.termeulen@crucell.com

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.

Show MeSH
Related in: MedlinePlus