Distinct roles of IL-23 and IL-17 in the development of psoriasis-like lesions in a mouse model.

Psoriasis is an inflammatory disease with dynamic interactions between the immune system and the skin. The IL-23/Th17 axis plays an important role in the pathogenesis of psoriasis, although the exact contributions of IL-23 and IL-17 in vivo remain unclear. K5.Stat3C transgenic mice constitutively express activated Stat3 within keratinocytes, and these animals develop skin lesions with histological and cytokine profiles similar to those of human plaque psoriasis. In this study, we characterized the effects of anti-mouse IL-17A, anti-mouse IL-12/23p40, and anti-mouse IL-23p19 Abs on the development of psoriasis-like lesions in K5.Stat3C transgenic mice. Treatment with anti-IL-12/23p40 or anti-IL-23p19 Abs greatly inhibited 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia in the ears of K5.Stat3C mice, whereas the inhibitory effect of an anti-IL-17A Ab was relatively less prominent. Treatment with anti-IL-12/23p40 or anti-IL-23p19 Abs markedly lowered transcript levels of Th17 cytokines (e.g., IL-17 and IL-22), ß-defensins, and S100A family members in skin lesions. However, anti-IL-17A Ab treatment did not affect mRNA levels of Th17 cytokines. Crossing IL-17A-deficient mice with K5.Stat3C mice resulted in partial attenuation of 12-O-tetradecanoylphorbol-13-acetate-induced lesions, which were further attenuated by anti-IL-12/23p40 Ab treatment. FACS analysis of skin-draining lymph node cells from mice that were intradermally injected with IL-23 revealed an increase in both IL-22-producing T cells and NK-22 cells. Taken together, this system provides a useful mouse model for psoriasis and demonstrates distinct roles for IL-23 and IL-17.

Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes.

Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab' fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.

Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches.

Chimeric 101F (ch101F) is a mouse-human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422-438 spanning antigenic site IV, V, VI was analysed. Residues 423-436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus.

To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies.

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function.

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32 degrees C, 37 degrees C, or 39.5 degrees C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.

The cytoplasmic domain of the F protein of Human respiratory syncytial virus is not required for cell fusion.

The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.

Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein.

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.

Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication. NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b. Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein. Differences in thermal stability may partially account for the inability to efficiently oligomerize. Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration. To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function.

Resistance profile of a hepatitis C virus RNA-dependent RNA polymerase benzothiadiazine inhibitor.

Recently, a benzo-1,2,4-thiadiazine antiviral agent (C(21)H(21)N(3)O(4)S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 microM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

Intracellular hepatitis C virus RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) infection represents a significant health concern in over 170 million individuals worldwide. Recently, Huh7 cell-based hepatitis C virus replicon systems, which rely upon the expression and cooperation of viral nonstructural proteins to mediate replication of the entire hepatitis C virus genome, were shown to be useful for studying viral replication and antiviral agents. We report that expression of the viral RNA-dependent RNA polymerase (RdRp) in yeast cells, independent of other viral proteins, is necessary and sufficient for initiation of RNA synthesis in cis from 3'-nontranslated hepatitis C virus RNA. Furthermore, expression of the polymerase alone appears incapable of transcribing across the entire viral genome, most likely due to the secondary structure of the RNA. Other viral polypeptides, such as helicase, which are presumed to be present in the functional replicase complex, are predicted to facilitate RNA synthesis across highly structured regions.

Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase.

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.

Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C(21)H(21)N(3)O(4)S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.

Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.