Kathryn V. Holmes

Peptide Nanoparticles as Novel Immunogens: Design and Analysis of a Prototypic Severe Acute Respiratory Syndrome Vaccine

Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel coronavirus that cost nearly 800 lives. While there have been no recent outbreaks of the disease, the threat remains as SARS coronavirus (SARS‐CoV) like strains still exist in animal reservoirs. Therefore, the development of a vaccine against SARS is in grave need. Here, we have designed and produced a prototypic SARS vaccine: a self‐assembling polypeptide nanoparticle that repetitively displays a SARS B‐cell epitope from the C‐terminal heptad repeat of the virus’ spike protein. Biophysical analyses with circular dichroism, transmission electron microscopy and dynamic light scattering confirmed the computational design showing α‐helcial nanoparticles with sizes of about 25 nm. Immunization experiments with no adjuvants were performed with BALB/c mice. An investigation of the binding properties of the elicited antibodies showed that they were highly conformation specific for the coiled‐coil epitope because they specifically recognized the native trimeric conformation of C‐terminal heptad repeat region. Consequently, the antisera exhibited neutralization activity in an in vitro infection inhibition assay. We conclude that these peptide nanoparticles represent a promising platform for vaccine design, in particular for diseases that are characterized by neutralizing epitopes with coiled‐coil conformation such as SARS‐CoV or other enveloped viruses.

Global Distribution of Novel Rhinovirus Genotype

Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years.

Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein–protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

Multiplex MassTag-PCR for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade

Abstract Background Respiratory infections are the most common infectious diseases in humans worldwide and are a leading cause of death in children less than 5 years of age. Objectives Identify candidate pathogens in pediatric patients with unexplained respiratory disease. Study design Forty-four nasopharyngeal washes collected during the 2004–2005 winter season from pediatric patients with respiratory illnesses that tested negative for 7 common respiratory pathogens by culture and direct immunofluorescence assays were analyzed by MassTag-PCR. To distinguish human enteroviruses (HEV) and rhinoviruses (HRV), samples positive for picornaviruses were further characterized by sequence analysis. Results Candidate pathogens were detected by MassTag PCR in 27 of the 44 (61%) specimens that previously were rated negative. Sixteen of these 27 specimens (59%) contained picornaviruses; of these 9 (57%) contained RNA of a recently discovered clade of rhinoviruses. Bocaviruses were detected in three patients by RT-PCR. Conclusions Our study confirms that multiplex MassTag-PCR enhances the detection of pathogens in clinical specimens, and shows that previously unrecognized rhinoviruses, that potentially form a species HRV-C, may cause a significant amount of pediatric respiratory disease.

Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation

Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH4Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.

Detection of four human coronaviruses in respiratory infections in children: a one-year study in Colorado.

Lower respiratory tract infections are the leading cause of death in children worldwide. Studies on the epidemiology and clinical associations of the four human non‐SARS human coronaviruses (HCoVs) using sensitive polymerase chain reaction (PCR) assays are needed to evaluate the clinical significance of HCoV infections worldwide. Pediatric respiratory specimens (1,683) submitted to a diagnostic virology laboratory over a 1‐year period (December 2004–November 2005) that were negative for seven respiratory viruses by conventional methods were tested for RNA of four HCoVs using sensitive RT‐PCR assays. Coronavirus RNAs were detected in 84 (5.0%) specimens: HCoV‐NL63 in 37 specimens, HCoV‐OC43 in 34, HCoV‐229E in 11, and HCoV‐HKU1 in 2. The majority of HCoV infections occurred during winter months, and over 62% were in previously healthy children. Twenty‐six (41%) coronavirus positive patients had evidence of a lower respiratory tract infection (LRTI), 17 (26%) presented with vomiting and/or diarrhea, and 5 (8%) presented with meningoencephalitis or seizures. Respiratory specimens from one immunocompromised patient were persistently positive for HCoV‐229E RNA for 3 months. HCoV‐NL63‐positive patients were nearly twice as likely to be hospitalized (P = 0.02) and to have a LRTI (P = 0.04) than HCoV‐OC43‐positive patients. HCoVs are associated with a small, but significant number (at least 2.4% of total samples submitted), of both upper and lower respiratory tract illnesses in children in Colorado. Our data raise the possibility that HCoV may play a role in gastrointestinal and CNS disease. Additional studies are needed to investigate the potential roles of HCoVs in these diseases. J. Med. Virol. 81:1597–1604, 2009.

CD13 is a novel mediator of monocytic/endothelial cell adhesion

During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction‐dependent manner involving monocytic cytoskeletal rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13‐dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept is strengthened by the fact that activated monocytic cells adhere to immobilized recombinant CD13. Furthermore, treatment with anti‐CD13 antibodies in a murine model of peritonitis results in a decrease in leukocyte infiltration into the peritoneum, suggesting a potential role for CD13 in leukocyte trafficking in vivo. Therefore, this work supports a new direction for CD13 biology, where these cell surface molecules act as true molecular interfaces that induce and participate in critical inflammatory cell interactions.

Alphacoronaviruses in New World Bats: Prevalence, Persistence, Phylogeny, and Potential for Interaction with Humans

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

Crystal Structure of Bovine Coronavirus Spike Protein Lectin Domain

Background: Coronavirus spike protein N-terminal domains (NTDs) bind sugar or protein receptors. Results: We determined crystal structure of bovine coronavirus NTD and located its sugar-binding site using mutagenesis. Conclusion: Bovine coronavirus NTD shares structural folds and sugar-binding sites with human galectins and has subtle yet functionally important differences from protein-binding NTD of mouse coronavirus. Significance: This study explores origin and evolution of coronavirus NTDs. The spike protein N-terminal domains (NTDs) of bovine coronavirus (BCoV) and mouse hepatitis coronavirus (MHV) recognize sugar and protein receptors, respectively, despite their significant sequence homology. We recently determined the crystal structure of MHV NTD complexed with its protein receptor murine carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which surprisingly revealed a human galectin (galactose-binding lectin) fold in MHV NTD. Here, we have determined at 1.55 Å resolution the crystal structure of BCoV NTD, which also has the human galectin fold. Using mutagenesis, we have located the sugar-binding site in BCoV NTD, which overlaps with the galactose-binding site in human galectins. Using a glycan array screen, we have identified 5-N-acetyl-9-O-acetylneuraminic acid as the preferred sugar substrate for BCoV NTD. Subtle structural differences between BCoV and MHV NTDs, primarily involving different conformations of receptor-binding loops, explain why BCoV NTD does not bind CEACAM1 and why MHV NTD does not bind sugar. These results suggest a successful viral evolution strategy in which coronaviruses stole a galectin from hosts, incorporated it into their spike protein, and evolved it into viral receptor-binding domains with altered sugar specificity in contemporary BCoV or novel protein specificity in contemporary MHV.

The Spike Glycoprotein of Murine Coronavirus MHV-JHM Mediates Receptor-Independent Infection and Spread in the Central Nervous Systems of Ceacam1a−/− Mice

ABSTRACT The MHV-JHM strain of the murine coronavirus mouse hepatitis virus is much more neurovirulent than the MHV-A59 strain, although both strains use murine CEACAM1a (mCEACAM1a) as the receptor to infect murine cells. We previously showed that Ceacam1a−/− mice are completely resistant to MHV-A59 infection (E. Hemmila et al., J. Virol. 78:10156-10165, 2004). In vitro, MHV-JHM, but not MHV-A59, can spread from infected murine cells to cells that lack mCEACAM1a, a phenomenon called receptor-independent spread. To determine whether MHV-JHM could infect and spread in the brain independent of mCEACAM1a, we inoculated Ceacam1a−/− mice. Although Ceacam1a−/− mice were completely resistant to i.c. inoculation with 106 PFU of recombinant wild-type MHV-A59 (RA59) virus, these mice were killed by recombinant MHV-JHM (RJHM) and a chimeric virus containing the spike of MHV-JHM in the MHV-A59 genome (SJHM/RA59). Immunohistochemistry showed that RJHM and SJHM/RA59 infected all neural cell types and induced severe microgliosis in both Ceacam1a−/− and wild-type mice. For RJHM, the 50% lethal dose (LD50) is <101.3 in wild-type mice and 103.1 in Ceacam1a−/− mice. For SJHM/RA59, the LD50 is <101.3 in wild-type mice and 103.6 in Ceacam1a−/− mice. This study shows that infection and spread of MHV-JHM in the brain are dependent upon the viral spike glycoprotein. RJHM can initiate infection in the brains of Ceacam1a−/− mice, but expression of mCEACAM1a increases susceptibility to infection. The spread of infection in the brain is mCEACAM1a independent. Thus, the ability of the MHV-JHM spike to mediate mCEACAM1a-independent spread in the brain is likely an important factor in the severe neurovirulence of MHV-JHM in wild-type mice.