H epatitis C virus (HCV) has emerged as the major etiological agent of liver disease. Approximately 170 million individuals are infected worldwide, and the majority are at risk for developing serious progressive liver disease, with HCV being the leading indication for liver transplantation. The HCV single-stranded RNA genome encodes a single polyprotein, which is cleaved by viral and cellular proteases to produce the structural proteins; core E1 and E2 and nonstructural proteins; p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The only approved treatment for HCV infection is interferon-␣ in combination with ribavirin, which is toxic and only effective in 50% of individuals with genotype I infections. Clearly, there is a need for more effective therapies and for the development of prophylactic and/or therapeutic vaccines.Cellular and humoral responses are generated during acute infection, but they are insufficient to achieve viral clearance in the majority of individuals, with approximately 60%-80% of new infections becoming persistent. 1,2 Neutralizing antibody (nAb) responses often provide the first-line adaptive defense against infection by limiting virus spread. However, little is known about the impact of the humoral immune response on HCV pathobiology. Serum antibodies (Abs) from chronically HCVinfected individuals demonstrate broadly reactive neutralizing properties in vitro and yet fail to control viral infection in vivo. [3][4][5] The reasons for their lack of effect are poorly understood. HCV may escape neutralization by

Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver. Hepatitis C virus (HCV) establishes chronic infection in 3%of the world's population, resulting in a progressive liver disease that is one of the leading indications for liver transplantation. HCV has evolved several immune evasion strategies in order to persist within the infected host (15,20,40), including genetic escape from humoral immune responses (25,46). However, functional constraints may restrict antigenic change in some regions of the virally encoded E1E2 envelope glycoproteins, such as the CD81 receptor binding site (9,11,33). The observation that glycoprotein-specific antibodies from chronically infected subjects neutralize the infectivity of laboratory prototype HCV strains yet demonstrate a limited ability to control HCV replication in vivo (40) suggest that additional means of evading antibody responses may exist.How virus particles disseminate within an immune-competent host has been a relatively neglected area of study; however, it is becoming increasingly clear that viruses employ multiple strategies to infect new target cells. Diffusion through the pericellular environment or the vascular circulation introduces a rate-limiting step in virus entry and exposes particles to the humoral immune system. Consequently, a number of viruses have evolved direct cell-to-cell modes of transmission that maximize particle delivery, often in a neutralizing antibody (nAb)-resistant manner (reviewed in reference 30).We (44) and others (48) previously reported that HCV strain JFH-1 could be transmitted via cell-free and cell-to-cell routes in vitro. We extend these observations and show that disruption of HCV particle assembly or physical separation of target and producer cells ablates transmission, demonstrating that intact virions are transferred via cell-cell conta...

Many aspects of the assembly of hepatitis C virus (HCV) remain incompletely understood. To characterize the role of NS2 in the production of infectious virus, we determined NS2 interaction partners among other HCV proteins during productive infection. Pulldown assays showed that NS2 forms complexes with both structural and nonstructural proteins, including E1, E2, p7, NS3, and NS5A. Confocal microscopy also demonstrated that NS2 colocalizes with E1, E2, and NS5A in dot-like structures near lipid droplets. However, NS5A did not coprecipitate with E2 and interacted only weakly with NS3 in pulldown assays. Also, there was no demonstrable interaction between p7 and E2 or NS3 in such assays. Therefore, NS2 is uniquely capable of interacting with both structural and nonstructural proteins. Among mutations in p7, NS2, and NS3 that prevent production of infectious virus, only p7 mutations significantly reduced NS2-mediated protein interactions. These p7 mutations altered the intracellular distribution of NS2 and E2 and appeared to modulate the membrane topology of the C-terminal domain of NS2. These results suggest that NS2 acts to coordinate virus assembly by mediating interactions between envelope proteins and NS3 and NS5A within replication complexes adjacent to lipid droplets, where virus particle assembly is thought to occur. p7 may play an accessory role by regulating NS2 membrane topology, which is important for NS2-mediated protein interactions and therefore NS2 function.

The UspA1 and Hag proteins have previously been shown to be involved in the ability of the Moraxella catarrhalis wild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library of M. catarrhalis DNA fragments, which was introduced into a nonadherent Escherichia coli strain. Recombinant E. coli bacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. The unencapsulated, gram-negative bacterium Moraxella catarrhalis is one of the leading causes of otitis media in young children and of lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). In developed countries, more than 80% of children under the age of 3 years will be diagnosed at least once with otitis media, and M. catarrhalis is responsible for 15 to 25% of all of these cases (6, 7, 10, 12, 26-29, 41, 51, 60). Lower respiratory tract infections (exacerbations) have been shown to contribute to the progression of COPD, and of the approximately 20 million cases of exacerbations documented each year in the United States, up to 35% result from M. catarrhalis infections (3,41,43,54,55).A vaccine that provides protection against M. catarrhalis is highly desirable due to this substantial morbidity as well as the growing concern over antibiotic resistance observed in clinical isolates (30). Toward this end, current research is focused on the identification of potential antigens with emphasis on the outer membrane proteins (OMPs) (26,34,35). While M. catarrhalis OMPs with a wide variety of functions have been identified, adhesins are particularly attractive vaccine candidates because they are surface-exposed antigens. Furthermore, adhesins generally play a crucial role in colonization and pathogenesis. For many bacteria, adherence to epithelial surfaces contributes to the evasion of host clearance mechanisms (4,25,59). Previous studies with M. catarrhalis have identified UspA1 (32, 36), UspA2 (2, 8, 36), Hag (14,15,17,19,24,45,47; M. M. Holm and E. R. Lafontaine, unpublished data), lipooligosaccharide (24), OMPCD (49), and UspA2H (32) as potential adhesins. Of these, only UspA1 and UspA2H have been directly shown to mediate adherence to human cells (32,36). However, while UspA1 is expressed by virtually all strains tested to date, only ϳ20% of clinical isolates contain an uspA2H gene (32, 37).The present study describes the identification of the novel M. catarrhalis OMP McaP. While this protein was identified based on its ability to function as an adhesin, we report that it also displays the enzymatic activity of an esterase and a phospholipase B (PLB). MATERIALS AND METHODSStrains, plasmids, tissue culture (TC) cell lines, and growth conditions. The bacterial strains and plasmids described in this study are listed in Table 1. M. catarrhalis was routinely cultured at 37°C on Todd-Hewitt medium (Difco). When cultured on agar plates, M. catarrhalis was incubated in an at...

The gram-negative bacterium Moraxella catarrhalis is a significant health problem, causing approximately 20% of all episodes of bacterial otitis media in children (23) and up to 10% of instances of lower respiratory tract infections in elderly patients suffering from chronic obstructive pulmonary disease (COPD) (45). Furthermore, diseases such as sinusitis (8) and conjunctivitis (7) can be added to the growing list of ailments caused by the organism. The development of a vaccine to reduce the risks of M. catarrhalis infections is therefore desirable and would have a substantial impact on the overall health status of the young and elderly.Several surface antigens expressed by M. catarrhalis have been studied for their vaccinogenic potential. Proteins such as OMPE (6,46,47), OMPCD (28,44,[48][49][50], and OMPG1a and OMPG1b (1-3) are promising candidates because they are highly conserved among strains, expressed by most isolates tested to date, and contain surface epitopes. Furthermore, immunization with these outer membrane (OM) proteins elicits the production of antibodies that bind to the surface of intact bacteria, and COPD patients recovering from M. catarrhalis infections produce antibodies against OMPCD, OMPE, and OMPG1a/OMPG1b (1-3, 6, 28, 44, 46-50). The adhesins UspA1 (15,35,39,41,43) and Hag/MID (10,27,39,(41)(42)(43)61), the serum resistance factor UspA2 (5,15,39,41,43,61), and the iron acquisition proteins CopB (39,41,43,59,61), TbpA (52), TbpB (14, 43, 52, 67), LbpA (18), and LbpB (18, 67) also exhibit most of the aforementioned vaccinogenic qualities, with the exception that these proteins are more variable at the amino acid level among isolates of various origins. Nevertheless, these types of molecules play key roles in pathogenesis by most bacterial pathogens (e.g., adherence, serum resistance, and iron acquisition) and targeting them in a vaccine may have the added benefit of interfering with the ability of M. catarrhalis to establish itself in the respiratory tract of individuals that are at risk of infection by the bacterium. This hypothesis is supported by the recent demonstration that UspA1, Hag, and UspA2 are the major targets of new immunoglobulin A antibodies in the sputum of COPD patients with M. catarrhalis infections who have successfully cleared the bacterium (43). This protective immune response, however, appears to be strain specific, as COPD patients often get reinfected by different strains of M. catarrhalis (45).These observations suggest that an effective vaccine for M. catarrhalis will need to include a mixture of antigens expressed by this unencapsulated bacterium. There is clearly a need to identify the regions of vaccine candidates having the best vaccinogenic properties, as well as to identify new and highly

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10-to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

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