We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S(e), were not cleaved to any significant degree. They ran at about 180-200kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17-537 and 272-537 but not 17-276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450-454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors.

epitope ͉ paratope ͉ vaccine ͉ therapeutic

The severe acute respiratory syndrome coronavirus (SARSCoV, or SCV), which caused a world-wide epidemic in 2002 and 2003, binds to a receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) of its envelope (spike, S) glycoprotein. The RBD is very immunogenic; it is a major SCV neutralization determinant and can elicit potent neutralizing antibodies capable of out-competing ACE2. However, the structural basis of RBD immunogenicity, RBD-mediated neutralization, and the role of RBD in entry steps following its binding to ACE2 have not been elucidated. By mimicking immune responses with the use of RBD as an antigen to screen a large human antibody library derived from healthy volunteers, we identified a novel potent cross-reactive SCV-neutralizing monoclonal antibody, m396, which competes with ACE2 for binding to RBD, and determined the crystal structure of the RBD-antibody complex at 2.3-Å resolution. The antibody-bound RBD structure is completely defined, revealing two previously unresolved segments (residues 376 -381 and 503-512) and a new disulfide bond (between residues 378 and 511). Interestingly, the overall structure of the m396-bound RBD is not significantly different from that of the ACE2-bound RBD. The antibody epitope is dominated by a 10-residue-long protruding ␤6 -␤7 loop with two putative ACE2-binding hotspot residues (Ile-489 and Tyr-491). These results provide a structural rationale for the function of a major determinant of SCV immunogenicity and neutralization, the development of SCV therapeutics based on the antibody paratope and epitope, and a retrovaccinology approach for the design of anti-SCV vaccines. The available structural information indicates that the SCV entry may not be mediated by ACE2-induced conformational changes in the RBD but may involve other conformational changes or/and yet to be identified coreceptors.The severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV) 4 infected more than 8000 humans with a fatality rate of ϳ10% (1-4). Although there have been no recent outbreaks, the need to develop potent therapeutics and vaccines against a re-emerging SCV or a related virus remains of high priority. The amazingly rapid pace of SARS research for the last few years has resulted in a wealth of information for the virus, especially about its interactions with the host leading to disease and immune responses, which could also be helpful for the development of strategies to cope with other viral pathogens including influenza and HIV.Entry of viruses into animal cells is initiated by binding to cell-surface-associated receptors and can be prevented by neutralizing antibodies (nAbs) targeting the virus receptor-binding site (5, 6). In the case of SCV entry, the spike (S) glycoprotein (7, 8) binds to a receptor, angiotensin-converting enzyme 2 (ACE2) (9), through the receptor-binding site of its receptor-binding domain (RBD) (7, 10, 11). The RBD is an attractive target for neutralizing antibodies that could prevent SCV entry by blocking the a...

Several human monoclonal antibodies (hmAbs) including b12, 2G12 and 2F5 exhibit relatively potent and broad HIV-1 neutralizing activity. However, their elicitation in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. We have hypothesized that HIV-1 has evolved a strategy to reduce or eliminate the immunogenicity of the highly conserved epitopes of such antibodies by using “holes” (absence or very weak binding to these epitopes of germline antibodies that is not sufficient to initiate and/or maintain an efficient immune response) in the human germline B cell receptor (BCR) repertoire. To begin to test this hypothesis we have designed germline-like antibodies corresponding most closely to b12, 2G12 and 2F5 as well as to X5, m44 and m46 which are cross-reactive but with relatively weak neutralizing activity as natively occurring antibodies due to size and/or other effects. The germline-like X5, m44 and m46 bound with relatively high affinity to all tested Envs. In contrast, germline-like b12, 2G12 and 2F5 lacked measurable binding to Envs in an ELISA assay although the corresponding mature antibodies did. These results provide initial evidence that Env structures containing conserved vulnerable epitopes may not initiate humoral responses by binding to germline antibodies. Even if such responses are initiated by very weak binding undetectable in our assay it is likely that they will be outcompeted by responses to structures containing the epitopes of X5, m44, m46, and other antibodies that bind germline BCRs with much higher affinity/avidity. This hypothesis, if further supported by data, could contribute to our understanding of how HIV-1 evades immune responses and offer new concepts for design of effective vaccine immunogens.

Abstract-Angiotensin-converting enzyme 2 (ACE2) is considered an important therapeutic target for controlling cardiovascular diseases and severe acute respiratory syndrome (SARS) outbreaks. Recently solved high-resolution crystal structures of the apo-bound and inhibitor-bound forms of ACE2 have provided the basis for a novel molecular docking approach in an attempt to identify ACE2 inhibitors and compounds that block SARS coronavirus spike protein-mediated cell fusion. In this study, Ϸ140 000 small molecules were screened by in silico molecular docking. In this structure-activity relation study, the molecules with the highest predicted binding scores were identified and assayed for ACE2 enzymatic inhibitory activity and for their ability to inhibit SARS coronavirus spike protein-mediated cell fusion. This approach identified N-(2-aminoethyl)-1 aziridine-ethanamine as a novel ACE2 inhibitor that also is effective in blocking the SARS coronavirus spike protein-mediated cell fusion. Thus, the molecular docking approach resulting in the inhibitory capacity of N-(2-aminoethyl)-1 aziridine-ethanamine provides an attractive small molecule lead compound on which the development of more effective therapeutic agents could be developed to modulate hypertension and for controlling SARS infections. Key Words: angiotensin-converting enzyme Ⅲ cardiovascular diseases Ⅲ hypertension A ngiotensin-converting enzyme 2 (ACE2) is a newly discovered membrane-bound aminopeptidase. 1-3 This enzyme has been proven to be critical in impacting cardiovascular and immune systems by 2 distinct physiologically important mechanisms. ACE2 catalyzes the production of vasodilatory peptides, including angiotensin 1 to 7, 4,5 and thus is responsible in counterbalancing the potent 6 -9 vasoconstrictor effects of angiotensin II. 6 -9 This counterbalancing property of ACE2 is proposed to be important for the development of novel pharmacotherapy against hypertension and related cardiovascular diseases. 10 -14 In addition to its critical role in regulation of hypertension, ACE2 has been demonstrated to be a functional receptor for the coronavirus that causes severe acute respiratory syndrome (SARS). The coronavirus, SARSCoV, the primary cause of SARS, gains entry into pulmonary endothelial cells by membrane fusion on binding to this ectoenzyme. This interaction is mediated by the SARS-CoV spike protein. [15][16][17][18][19] This conclusion is further supported by recent observations that pulmonary endothelial cells express high levels of ACE2. 20 These data provide the impetus to develop ACE2 modulators with anticipation that in vivo activation of ACE2 would lead to protection and successful treatment for hypertension and other cardiovascular diseases. In contrast, ACE2 inhibitors would be expected to block ACE2/SARS-CoV spike protein interactions and inhibit SARS-CoV infection. In addition, ACE2 inhibitors could be potentially important in the mechanisms of hypertension. Despite this urgency, only a few studies have been attempted to develop potentia...