The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins which are cleaved at specific sites by a cysteine 3C-like protease (3CL pro) in a post-translational processing step that is critical for coronavirus replication. The 3CL pro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CL pro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CL pro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19. (~450 kDa) and pp1ab (~750 kDa) that contain overlapping sequences and include a 3C-like cysteine protease (3CL pro). The function of this internally encoded 3CL pro is integral to the processing of these proteins and critical for viral replication. 7 The SARS CoV-1 3CL pro shares a high degree of structural homology and similar substrate specificity with the coronavirus 3C-like cysteine proteases of hCoV-229E and TGEV 8 , but is most similar to the SARS CoV-2 3CL pro. Specifically, the SARS CoV-1 and SARS CoV-2 share 96% identity between their respective 3CL pro sequences and 100% identity in the active site. 8 There are numerous reports of reversible cysteine protease inhibitors which include aldehydes 9-12 , thio-or oxymethylketones 13 , cyclic ketones 14 , amidomethylketones 15 , nitriles 16,17 or various 12-283,039 ± 22,586 13 Me 220 ± 0.5 14 cyc-propyl 182 ± 6 15 tert-butyl 230 ± 5 16 Ph 86 ± 3 17 4-OMe-Ph 79 ± 3 18 4-Me-Ph 87 ± 2 19 4-CN-Ph 53 ± 1 20 4-F-Ph 82 ± 3 21 4-Cl-Ph 97 ± 3 22 2,6-(Cl)2-Ph 62,993 ± 2,501 23 2,6-(F)2-Ph 12,776 ± 594 24 2-OH-4-Cl-Ph 11,525 ± 40 25 2-F, 4-CN-Ph 13,321 ± 2,309 26 2,6-(Me)2-Ph 74 ± 4 27 2,6-(MeO)2-Ph 205 ± 2 28 2-CN-Ph 17 ± 2 a See Experimental Section for details on assay methods, values were calculated from at least eight data points with at least two independent determinations.

The poor correlation between cellular immunity to respiratory virus infections and the numbers of memory CD8+ T cells in the secondary lymphoid organs suggests that there may be additional reservoirs of T cell memory to this class of infection. Here we identify a substantial population of Ag-specific T cells in the lung that persist for several months after recovery from an influenza or Sendai virus infection. These cells are present in high numbers in both the airways and lung parenchyma and can be distinguished from memory cell populations in the spleen and peripheral lymph nodes in terms of the relative frequencies among CD8+ T cells, activation status, and kinetics of persistence. In addition, these cells are functional in terms of their ability to proliferate, express cytolytic activity, and secrete cytokines, although they do not express constitutive cytolytic activity. Adoptive transfer experiments demonstrated that the long-term establishment of activated T cells in the lung did not require infection in the lung by a pathogen carrying the inducing Ag. The kinetics of persistence of Ag-specific CD8+ T cells in the lung suggests that they play a key role in protective cellular immunity to respiratory virus infections.

Although CD4+ T cells have been shown to mediate protective cellular immunity against respiratory virus infections, the underlying mechanisms are poorly understood. For example, although phenotypically distinct populations of memory CD4+ T cells have been identified in different secondary lymphoid tissues, it is not known which subpopulations mediate protective cellular immunity. In this report, we demonstrate that virus-specific CD4+ T cells persist in the lung tissues and airways for several months after Sendai virus infection of C57BL/6 mice. A large proportion of these cells possess a highly activated phenotype (CD44hi, CD62Llo, CD43hi, and CD25hi) and express immediate effector function as indicated by the production of interferon γ after a 5-h restimulation in vitro. Furthermore, intratracheal adoptive transfer of lung memory cells into β2m-deficient mice demonstrated that lung-resident virus-specific CD4+ T cells mediated a substantial degree of protection against secondary virus infection. Taken together, these data demonstrate that activated memory CD4+ T cells persisting at mucosal sites play a critical role in mediating protective cellular immunity.

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by b-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection. (Marra et al., 2003;Rota et al., 2003) and by experimental infection of macaques to fulfil Koch's postulates . 0008-1579 G 2006 SGM Printed in Great BritainCurrently, there is no effective treatment for SARS. Prevention through contact-reduction or transmission-blocking measures has been the only means available to modify the devastating impact of this illness. Prevention through vaccination would be an attractive alternative that is less reliant on individual case detection to be effective. No vaccines are currently licensed for any of the human CoVs, but effective vaccines have been produced for some animal CoVs, such as certain strains of Infectious bronchitis virus (poultry), Bovine coronavirus and Canine coronavirus (Cavanagh, 2003;Enjuanes et al., 1995;Pratelli et al., 2003;Saif, 2004;Takamura et al., 2002). Individuals convalescing from SARS develop high titres of neutralizing antibodies (Tan et al., 2004) and the appearance of antibodies coincides with the onset of resolution of SARS pneumonia Woo et al., 2004). Thus, there is some optimism that an effective vaccine against SARS-CoV may also be possible.Coronavirus spike (S) proteins have long been known to be a major determinant in coronavirus pathogenesis, given that this viral protein interacts with cellular receptors as well as con...

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