Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line.

While employing deep sequencing and de novo assembly to characterize the mRNA transcript profile of a cell line derived from the microbat Myotis velifer incautus, we serendipitously identified mRNAs encoding proteins with a high level of identity to herpesviruses. A majority were closely related to proteins of equine herpesvirus 2 (EHV-2), a horse gammaherpesvirus. We demonstrated by electron microscopy the presence of herpesvirus-like particles in the microbat cells. Passage of supernatants from microbat cells to Vero cells resulted in syncytium formation, and expression of viral genes and amplification of viral DNA were demonstrated by quantitative PCR. Susceptibility of human cell lines to productive infection was also demonstrated. Next-generation sequencing and de novo assembly of the viral genome from supernatants from Vero cells yielded a single contig of approximately 130 kb with at least 77 open reading frames (ORFs), predicted microRNAs (miRNAs), and a gammaherpesvirus genomic organization. Phylogenic analysis of the envelope glycoprotein (gB) and DNA polymerase (POLD1) revealed similarity to multiple gammaherpesviruses, including those from as-yet-uncultured viruses of the Rhadinovirus genus that were obtained by deep sequencing of bat tissues. Moreover, the assembled genome revealed ORFs that share little or no homology to known ORFs in EHV-2 but are similar to accessory proteins of other gammaherpesviruses. Some also have striking homology to predicted Myotis bat proteins. Cumulatively, this study provides the first isolation and characterization of a replication-competent bat gammaherpesvirus. IMPORTANCE Bats are of significant interest as reservoirs for zoonotic viral pathogens; however, tools to dissect bat-virus interactions are limited in availability. This study serendipitously identified, in an established bat cell line, a fully replication-competent gammaherpesvirus; determined the complete genome sequence of the virus; and generated a viral transcript map. This virus can replicate in select human and nonhuman primate cell lines. However, analyses of viral sequences support a bat origin for this virus; we therefore refer to the virus as bat gammaherpesvirus 8 (BGHV8). The viral genome contains unique open reading frames that likely encode modulators of bat innate and adaptive immune signaling pathways and expresses viral miRNAs. The virus and its gene products should provide a unique tool to dissect both bat and gammaherpesvirus biology.

A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

1976 and 2009 H1N1 influenza virus vaccines boost anti-hemagglutinin stalk antibodies in humans.

BACKGROUND: Infection with pandemic H1N1 influenza A viruses (IAVs) containing hemagglutinin (HA) proteins with globular heads that differ substantially from seasonal strains results in a boost in broadly cross-reactive antibodies that bind to the HA stalk. Boosting these antibodies has become an attractive strategy for creating a universal IAV vaccine. Therefore, it was essential to determine whether vaccines containing H1N1 viruses whose head domains differ substantially compared to seasonal strains could also achieve this boost. METHODS: Prospective samples of subjects who had received the A/New Jersey/1976 (NJ/76) vaccine and healthy, age-matched controls were assessed for the presence of anti-HA stalk antibodies before and after receiving the A/California/04/2009 (Cal/09) vaccine between October 2009 and January 2010. RESULTS: Individuals who received either the NJ/76 vaccine or the Cal/09 vaccine experienced a robust boost in HA stalk-reactive, neutralizing antibodies similar to what has been observed in individuals infected with Cal/09. CONCLUSIONS: These results demonstrate that vaccines containing viruses whose HA head domains that differ substantially from seasonal strains are capable of boosting titers of HA stalk antibodies. Furthermore, anti-HA stalk antibodies elicited by vaccination appear to be long-lived and therefore could be targeted for the generation of a universal IAV vaccine.

Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2). The 1F1 heavy chain also reaches into the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

Human monoclonal antibodies to pandemic 1957 H2N2 and pandemic 1968 H3N2 influenza viruses.

Investigation of the human antibody response to the 1957 pandemic H2N2 influenza A virus has been largely limited to serologic studies. We generated five influenza virus hemagglutinin (HA)-reactive human monoclonal antibodies (MAbs) by hybridoma technology from the peripheral blood of healthy donors who were born between 1950 and 1968. Two MAbs reacted with the pandemic H2N2 virus, two recognized the pandemic H3N2 virus, and remarkably, one reacted with both the pandemic H2N2 and H3N2 viruses. Each of these five naturally occurring MAbs displayed hemagglutination inhibition activity, suggesting specificity for the globular head domain of influenza virus HA. When incubated with virus, MAbs 8F8, 8M2, and 2G1 each elicited H2N2 escape mutations immediately adjacent to the receptor-binding domain on the HA globular head in embryonated chicken eggs. All H2N2-specific MAbs were able to inhibit a 2006 swine H2N3 influenza virus. MAbs 8M2 and 2G1 shared the V(H)1-69 germ line gene, but these antibodies were otherwise not genetically related. Each antibody was able to protect mice in a lethal H2N2 virus challenge. Thus, even 43 years after circulation of H2N2 viruses, these subjects possessed peripheral blood B cells encoding potent inhibiting antibodies specific for a conserved region on the globular head of the pandemic H2 HA.

Epitope-specific human influenza antibody repertoires diversify by B cell intraclonal sequence divergence and interclonal convergence.

We generated from a single blood sample five independent human mAbs that recognized the Sa antigenic site on the head of influenza hemagglutinin and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the V(H)3-7 and J(H)6 gene segments, but at least four independent clones were identified by junctional analysis. High-throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the Ab genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the Ag combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus Ags, such as influenza hemagglutinin. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human Abs.

A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin.

The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. We isolated human monoclonal antibody 5J8, which neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Fine mapping of the interaction unexpectedly revealed a novel epitope between the receptor-binding pocket and the Ca2 antigenic site on HA. This antibody exposes a new mechanism underlying broad immunity against H1N1 influenza viruses and identifies a conserved epitope that might be incorporated into engineered H1 virus vaccines.

Protection of mice against lethal challenge with 2009 H1N1 influenza A virus by 1918-like and classical swine H1N1 based vaccines.

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918-1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.

Naturally occurring human monoclonal antibodies neutralize both 1918 and 2009 pandemic influenza A (H1N1) viruses.

The 2009 pandemic influenza A (H1N1) virus exhibits hemagglutinin protein sequence homology with the 1918 pandemic influenza virus. We found that human monoclonal antibodies recognized the Sa antigenic site on the head domains of both 1918 and 2009 hemagglutinins, a site that is hypervariable due to immune selection. These antibodies exhibited high potency against the 2009 virus in vitro, and one exerted a marked therapeutic effect in vivo.

Neutralizing anti-influenza virus monoclonal antibodies: therapeutics and tools for discovery.

The human antibody response to influenza virus infection plays a protective role against re-infection, yet little molecular detail is available regarding how human antibodies, when characterized at the monoclonal level, neutralize this important human pathogen. Recent studies, using a diverse array of strategies, have isolated and characterized human anti-virus neutralizing antibodies and shed light not only on the specificity and origin of these antibodies but on their potential for therapeutic use against influenza virus infection.

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors.

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.