Monoclonal Antibodies Targeting the HR2 Domain and the Region Immediately Upstream of the HR2 of the S Protein Neutralize In Vitro Infection of Severe Acute Respiratory Syndrome Coronavirus

Abstract

We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. The virus-cell membrane fusion event is an essential step in the entry process of all enveloped animal viruses, including important human pathogens such as influenza virus, human immunodeficiency virus (HIV) (8, 23), and the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) (9). Following the binding to their receptors on the cell surface, virus-encoded membrane fusion proteins mediate the fusion process. In many but not all cases, the viral fusion proteins are proteolytically processed by host proteases into 2 subunits that remain closely associated with each other: a surface subunit with a receptor-binding site and a transmembrane subunit with a fusion peptide consisting of two or more heptad repeat domains. Upon interaction of the fusion protein with a cellular receptor, the buried fusion peptide is exposed and inserted into the membrane of the target cell. A series of conformational changes trigger virus-cell fusion activity (9) and lead to the unloading of the viral genome into cells. Additionally, many viral fusion proteins also induce cell-cell fusion, i.e., the formation of multinucleated syncytia, facilitating the rapid spread of virus infection.TThe spike (S) protein of coronaviruses is responsible for receptor binding and membrane fusion. It shares similarity with class I virus fusion proteins (2, 3). Typically, it is a type I integral membrane protein, which is N-glycosylated and trimerized in the endoplasmic reticulum. The N-terminal S1 protein contains the receptor-binding site (10,18,22,34). The C-terminal S2 protein is a fusion subunit and anchors on the viral envelope through a transmembrane domain. The S2 protein ectodomain contains two 4,3 hydrophobic heptad repeats (HR1 and HR2) and a putative, internal fusion peptide (3, 23). For the SARS-CoV S protein, the HR2 is located adjacent to the transmembrane domain, whereas the HR1 is 140 to 170 residues upstream of the HR2.Crystallographic, biophysical, and biochemical analysis of the fusion core of SARS-CoV S protein (2,12,19,27,30,35) and other class I fusion proteins (8, 25) supports a model of membrane fusion probably adopted by these enveloped viruses. After the attachment of the receptor-binding subunit to the receptor, the HR1 and HR2 domains in the membrane fusion subunit interact with each other and form a six-helix bundle, a complex consisting of a homotrimeric HR1 coiled coil surrounded by three HR2 helices. The spacer domain (or link, or interhelical domain) between HR1 and HR2 forms a loop and reverses the direction of the polypeptide chain so that the HR2 helices pack against the HR1 coiled coil in an antiparallel manner. This conformational change results in a close apposition of the fusion peptide, already exposed and inserted into th...

Template-based coiled-coil antigens elicit neutralizing antibodies to the SARS-coronavirus

“…Thus the HRN coiled-coil regions analyzed did not show the potential for eliciting neutralizing antibodies. These results are in good agreement with recent studies by Duan et al (2005), Keng et al (2005), Lai et al (2005), and Lip et al (2006) which have mapped the location of neutralizing antibodies generated from mice immunized with truncated sections of the S2 domain. These studies show no neutralizing antibodies corresponding to the HRN region.…”

A monoclonal antibody binds to threonine 49 in the non-structural 1 protein of influenza A virus and interferes with its ability to modulate viral replication

“…Variable heavy (V H ) and variable light (V L ) genes were obtained from RNA extracted from the 2H6 or 7G12 hybridoma as described previously (Dang et al, 2013). 7G12 is a monoclonal antibody targeting the spike protein of severe acute respiratory syndrome (SARS) coronavirus (Lip et al, 2006). Subsequently, the 2H6-scFv and 7G12-scFv were constructed by using overlap PCR to link them via a GGGSGGGSGGGS linker.…”

“…2B). The 7G12 antibody, which binds to the spike protein of SARS coronavirus (Lip et al, 2006), was used as a negative control for this experiment. In contrast to the results for 2H6-Fab, 7G12-Fab and NS1(RBD) eluted out of the column separately when the mixture was run on a Superdex 200 column (see Supplementary Fig.…”

“…4A, the viral growth in the 2H6-scFv transfected A549 cells was reduced by approximately 0.5 log PFU/mL at 24 and 48 hpi when compared to cells transfected with vector only. To determine if viral inhibition by 2H6-scFv is specific, the scFv of an irrelevant antibody 7G12, which binds to the spike protein of SARS coronavirus (Lip et al, 2006), was also used in this experiment. The viral growth in the 7G12-scFv transfected A549 cells did not show reduction when compared to cells transfected with vector only.…”

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV cross-react with the newly-emerged SARS-CoV-2

“…Purification of antibody 7 1 The hybridoma for mAb 1A9 was previously generated [19]. All mAbs were purified 7 2 from cell culture supernatant using a HiTrap protein G HP affinity column (GE 7 3 Healthcare) and stored at -80 °C.…”