Following intranasal administration, the severe acute respiratory syndrome (SARS) coronavirus replicated to high titers in the respiratory tracts of BALB/c mice. Peak replication was seen in the absence of disease on day 1 or 2, depending on the dose administered, and the virus was cleared within a week. Viral antigen and nucleic acid were detected in bronchiolar epithelial cells during peak viral replication. Mice developed a neutralizing antibody response and were protected from reinfection 28 days following primary infection. Passive transfer of immune serum to naïve mice prevented virus replication in the lower respiratory tract following intranasal challenge. Thus, antibodies, acting alone, can prevent replication of the SARS coronavirus in the lung, a promising observation for the development of vaccines, immunotherapy, and immunoprophylaxis regimens.

SummaryInfluenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS The etiologic agent of severe acute respiratory syndrome (SARS) has been shown to be a new human coronavirus (SARS-CoV) (18,21,23,25). The significant morbidity and mortality, and potential for reemergence, make SARS-CoV a continued worldwide public health threat. Genome sequences of SARS-CoV isolates have provided important information regarding the organization of the genome and its relationship to other coronaviruses (21, 25). The significant conservation of deduced amino acid sequence and predicted protein domains in the replicase polyprotein across multiple coronaviruses indicates that these proteins likely play important, conserved roles in replication (29, 32).Coronavirus replication in cells is initiated by translation of the two overlapping open reading frames (ORF1a and ORF1b) of the 5Ј replicase gene to yield two polyproteins, pp1a and pp1ab (see Fig. 1) (25, 32). Based on studies of other coronaviruses, co-and posttranslational proteolytic processing of the nascent SARS-CoV replicase polyproteins is predicted to be mediated by two virus-encoded proteinases, a 3C-like proteinase (3CLpro) and a papain-like proteinase (PLP) (12,29,32). The proteolytic precursors and mature replicase proteins likely mediate the processes of replication complex formation, subgenomic mRNA transcription, and genome replication. Comparison of known polyprotein cleavage sites of other coronaviruses with the deduced amino acid (39) sequence of the SARS-CoV replicase polyprotein has resulted in prediction of 16 mature replicase nonstructural proteins (nsps) (32). However, detection of these mature products in SARSCoV-infected cells has not been reported.The number of mature proteins produced from coronavirus replicase polyproteins suggests a level of complexity and possible virus-encoded functions greater than that of any other known positive-strand RNA virus family. Enzymatic activities have been demonstrated for coronavirus proteinases and the RNA helicase and have been predicted for an RNA-dependent RNA polymerase (Pol) and several RNA processing enzymes (19, 27, 39) (see Fig. 1). Recent bioinformatics analyses of the SARS-CoV replicase gene also have predicted functions for mature replicase proteins in RNA processing (29). The development of reverse genetic systems for SARS-CoV and other coronaviruses should make it possible to experimentally determine the role of replicase proteins in replication and pathogenesis (9,15,31,(36)(37)(38).In addition to their functions in RNA synthesis, new roles for rep...

BackgroundRecent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic.Methods and FindingsLive, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 106 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses.ConclusionsThe promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.