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Identifying Monoclonal Antibodies that Potently Inhibit MERS-CoV

Docked complexes of MERS-CoV RBD with mAbs (A) m336, (B) m337, and (C) m338. (D) Superposition of the docked complexes of RBD-m336,7,8 and the crystal structure of the RBD-DPP4 complex.
Docked complexes of MERS-CoV RBD with mAbs (A) m336, (B) m337, and (C) m338. (D) Superposition of the docked complexes of RBD-m336,7,8 and the crystal structure of the RBD-DPP4 complex.

The Middle East respiratory syndrome coronavirus (MERS-CoV), first isolated in September 2012, infects cells lining the human airway, causing severe flu-like symptoms that, in some cases, lead to death. As of July 2, 2014, 824 confirmed cases of MERS-CoV infection, including at least 286 related deaths, have been reported to the World Health Organization. While there are currently no effective therapies against the virus, monoclonal antibodies (MAbs) may be a promising candidate. Having previously developed MAbs against other viruses, including the related severe acute respiratory syndrome coronavirus or SARS-CoV, Dimiter Dimitrov, Ph.D., of CCR’s Laboratory of Experimental Immunology (LEI), and his colleagues decided to pan a library of antigen binding fragments (Fab) for activity against MERS-CoV.

Using B cells from the blood of 40 healthy donors, Tianlei Ying, Ph.D., from Dimitrov’s group constructed a very large library of about 1011 Fab and expressed them on the surface of bacteriophage, a technique known as phage display. They panned the library against the receptor binding domain (RBD) of the MERS-CoV spike protein (S), which recognizes DPP4 on the surface of cells and is important for virus entry, to enrich for high affinity binders. After four rounds of panning, the investigators identified 12 Fab with significant affinities for the RBD. The three with the highest affinity, m336, m337, and m338, all derived from the VH gene 1-69, which has been the source of other antiviral antibodies, were selected for further study.

The scientists generated full-size IgG1 MAbs from m336, m337, and m338, and all three MAbs continued to display high affinity to the RBD. To investigate the MAbs’ abilities to neutralize MERS-CoV infection, the researchers in collaboration with Shibo Jiang, Ph.D., from Fudan University, first used a pseudovirus system. In this system virus-like particles containing a reporter gene and the MERS-CoV S protein on the surface are mixed with DPP4-expressing cells, with or without MAbs. After washing, the cells are analyzed for reporter gene expression, indicating infection of the cells. The three MAbs exhibited potent neutralizing activity, with m336 inhibiting over 90 percent of pseudovirus infection at a concentration of 0.039 μg/mL. The investigators in collaboration with KY Yuen, M.D., from the University of Hong Kong, then tested the MAbs against live MERS-CoV and found that all three could inhibit infection, again with m336 being the most potent. These results are significant since these are the first fully human MAbs to neutralize pseudovirus and live virus with such high efficacy.

The researchers next wanted to better define the MERS-CoV epitopes recognized by the MAbs. First, they looked for MAb binding to fragments of the S protein. As expected, the MAbs bound only the S fragments containing the RBD, specifically residues 377 to 588. The scientists next created a series of alanine mutants within the RBD and analyzed MAb binding to each. The three MAbs had overlapping as well as distinct binding sites within the RBD. For example, residues 510 and 553 were important for binding all three MAbs. Residues 536 and 539, on the other hand, were uniquely bound by m336, suggesting they may be crucial for m336’s potent interaction and could be important for vaccine development.

Finally, the investigators examined the neutralization mechanism of the MAbs, hypothesizing that the antibodies neutralize MERS-CoV infection by competing with DPP4 binding. The researchers found that the MAbs competed with each other for binding the MERS-CoV RBD as well as with a soluble version of DPP4, supporting the idea that the MAbs have overlapping epitopes and neutralize MERS-CoV by competing with receptor binding. The scientists then used their mutagenesis data and the MERS-CoV RBD crystal structure to generate three-dimensional molecular models of the MAbs and the MAbs interacting with the RBD. They identified disulfide bond-forming cysteine residues in m336 that might be important for stabilizing its conformation and a number of tyrosine residues in m337 and m338 that could provide structural support and antigen recognition. When they superimposed the structure of DPP4 on the MAb-RBD structures, the investigators confirmed the overlap in the MAb and receptor binding sites and identified interactions that may be important for vaccine and drug design.

Together, these studies identified and characterized novel, human MAbs with exceptionally high neutralizing activity for MERS-CoV. They suggest that the RBD, particularly the regions recognized by these MAbs, has great vaccine development potential. Likewise, the antibody library methodology the researchers used may be a rapid and effective way to respond to emerging coronaviruses, which are likely to pose serious human health threats.

Reference
Ying T, Du L, Ju TW, Prabakaran P, Lau CCY, Lu L, Liu Q, Wang L, Feng Y, Wang Y, Zheng BJ, Yuen KY, Jiang S, Dimitrov DS. Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies. J Virology. April 30, 2014. PubMed Link