Abstract
Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123–421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5–80.4 °C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17–1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.
Highlights
Since its first reported isolation from a patient in Saudi Arabia in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) has posed a global threat to public health
We described the use of these monoclonal antibodies (mAbs) for the development of a novel enzyme-linked immunosorbent assay (ELISA) format called ACCEL ELISATM, which we anticipate can be used as a rapid diagnostic test with a high sensitivity for point-of-care testing (POCT) of MERS-CoV infection
A mouse anti-229E coronavirus nucleoprotein OC-43 antibody (MERCK, Darmstadt, Germany), a mouse anti-coronavirus antibody, human coronaviruses (hCoV) OC-43 (LifeSpan BioScience, Seattle, WA, USA), and a rabbit polyclonal anti-hCoV-HKU1 spike protein antibody (Sino Biological) were incubated with MRC5 cells infected with the hCoV 229E or OC-43 strain, and with LLC-MK2 cells infected with the hCoV NL63 strain, with both types of cells used as positive controls
Summary
Since its first reported isolation from a patient in Saudi Arabia in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) has posed a global threat to public health. Neutralizing monoclonal antibodies (mAbs) are rapidly emerging as an alternative approach complementing vaccines against viral infections [14]. Due to its critical role in the interaction with host cell receptors, tremendous efforts have been focused on the discovery of neutralizing mAbs against the RBD of the MERS-CoV S1 subunit [15,16,17,18]. The first MERS-CoV S2 subunit (MERS-S2P)-targeting antibody, called G4, was generated using mouse immunization and was demonstrated to recognize a variable loop in the S2 connector domain and to neutralize infection by the virus [13,21]. We panned a synthetic human Fab phage display library against MERS-S2P and obtained monoclonal antibodies (mAbs) that detected the viral S2 subunit. We described the use of these mAbs for the development of a novel ELISA format called ACCEL ELISATM, which we anticipate can be used as a rapid diagnostic test with a high sensitivity for point-of-care testing (POCT) of MERS-CoV infection
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