The human viral challenge model: accelerating the evaluation of respiratory antivirals, vaccines and novel diagnostics.

The Human Viral Challenge (HVC) model has, for many decades, helped in the understanding of respiratory viruses and their role in disease pathogenesis. In a controlled setting using small numbers of volunteers removed from community exposure to other infections, this experimental model enables proof of concept work to be undertaken on novel therapeutics, including vaccines, immunomodulators and antivirals, as well as new diagnostics.Crucially, unlike conventional phase 1 studies, challenge studies include evaluable efficacy endpoints that then guide decisions on how to optimise subsequent field studies, as recommended by the FDA and thus licensing studies that follow. Such a strategy optimises the benefit of the studies and identifies possible threats early on, minimising the risk to subsequent volunteers but also maximising the benefit of scarce resources available to the research group investing in the research. Inspired by the principles of the 3Rs (Replacement, Reduction and Refinement) now commonly applied in the preclinical phase, HVC studies allow refinement and reduction of the subsequent development phase, accelerating progress towards further statistically powered phase 2b studies. The breadth of data generated from challenge studies allows for exploration of a wide range of variables and endpoints that can then be taken through to pivotal phase 3 studies.We describe the disease burden for acute respiratory viral infections for which current conventional development strategies have failed to produce therapeutics that meet clinical need. The Authors describe the HVC model's utility in increasing scientific understanding and in progressing promising therapeutics through development.The contribution of the model to the elucidation of the virus-host interaction, both regarding viral pathogenicity and the body's immunological response is discussed, along with its utility to assist in the development of novel diagnostics.Future applications of the model are also explored.

Ficus religiosa L. bark extracts inhibit human rhinovirus and respiratory syncytial virus infection in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Ficus religiosa L. is one of the most relevant members of the family of Moraceae. It is the most sacred tree of South Asia, and it is used in traditional Ayurvedic and Unani medicine to cure respiratory disorders like cough, wheezing and asthma. Some studies were performed to investigate the anti-asthmatic potential of F. religiosa bark, leaves and fruit extracts but none of them tested their antiviral activity against viruses responsible for the exacerbation of wheezing and asthma. AIM OF THE STUDY: The present study was undertaken to investigate the antiviral activity of F. religiosa L. extracts against respiratory viruses such as human respiratory syncytial virus (RSV) and human rhinovirus (HRV). MATERIALS AND METHODS: The antiviral activity of F. religiosa L. was tested in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virus inactivation and time-of-addition assays. RESULTS: F. religiosa L. methanol bark extract was the most active against HRV with an EC50 of 5.52 µg/mL. This extract likely inhibited late steps of replicative cycle. Water bark extract was the most active against RSV with an EC50 between 2.23 and 4.37 µg/mL. Partial virus inactivation and interference with virus attachment were both found to contribute to the anti-RSV activity. Replication of both viruses was inhibited in viral yield reduction assays. CONCLUSIONS: The results of the present study demonstrate that F. religiosa L. is endowed with antiviral activity against RSV and HRV in vitro. Further work remains to be done to identify the active components and to assess the therapeutic potential in vivo.

Antiviral activity and possible mechanism of action of constituents identified in Paeonia lactiflora root toward human rhinoviruses.

Human rhinoviruses (HRVs) are responsible for more than half of all cases of the common cold and cost billions of USD annually in medical visits and missed school and work. An assessment was made of the antiviral activities and mechanisms of action of paeonol (PA) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) from Paeonia lactiflora root toward HRV-2 and HRV-4 in MRC5 cells using a tetrazolium method and real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results were compared with those of a reference control ribavirin. Based on 50% inhibitory concentration values, PGG was 13.4 and 18.0 times more active toward HRV-2 (17.89 µM) and HRV-4 (17.33 µM) in MRC5 cells, respectively, than ribavirin. The constituents had relatively high selective index values (3.3->8.5). The 100 µg/mL PA and 20 µg/mL PGG did not interact with the HRV-4 particles. These constituents inhibited HRV-4 infection only when they were added during the virus inoculation (0 h), the adsorption period of HRVs, but not after 1 h or later. Moreover, the RNA replication levels of HRVs were remarkably reduced in the MRC5 cultures treated with these constituents. These findings suggest that PGG and PA may block or reduce the entry of the viruses into the cells to protect the cells from the virus destruction and abate virus replication, which may play an important role in interfering with expressions of rhinovirus receptors (intercellular adhesion molecule-1 and low-density lipoprotein receptor), inflammatory cytokines (interleukin (IL)-6, IL-8, tumor necrosis factor, interferon beta, and IL-1ß), and Toll-like receptor, which resulted in diminishing symptoms induced by HRV. Global efforts to reduce the level of synthetic drugs justify further studies on P. lactiflora root-derived materials as potential anti-HRV products or lead molecules for the prevention or treatment of HRV.

Effects of vitamin D on airway epithelial cell morphology and rhinovirus replication.

Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)2D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0-69.0] vs. 30 µm [24.5-34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1ß) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells.

The efficacy of Echinacea in a 3-D tissue model of human airway epithelium.

We evaluated the antirhinovirus efficacy of a standardized preparation of Echinacea purpurea (Echinaforce) in a 3-dimensional organotypic model of normal human airway epithelium (EpiAirway tissue). Individual replicate tissue samples, maintained as inserts in culture for 3 days or 3 weeks, were infected with rhinovirus type 1A (RV1A), Echinacea alone, a combination of the two, or medium only. None of the treatments affected the histological appearance or integrity of the tissues, all of which maintained a high level of cell viability and preservation of cilia. RV infection resulted in increased mucopolysaccharide inclusions in the goblet cells, but this feature was reversed by Echinacea treatment. This result was confirmed by measurements of mucin secretion, which was stimulated by RV but reversed by Echinacea, suggesting that mucus production during colds could be ameliorated by Echinacea. We did not find evidence of virus replication, although the RV-infected tissues secreted substantial amounts of the pro-inflammatory cytokines IL-6 and IL-8 (CXCL8), and this response was reversed by Echinacea treatment. These results confirmed previous findings derived from studies of bronchial and lung epithelial cell lines, namely, that RV infection results in a substantial inflammatory response in the absence of virus replication.

Hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells.

BACKGROUND AND PURPOSE: A traditional Japanese herbal medicine, hochu-ekki-to, has been used for the symptomatic treatment of the common cold and to reduce the frequency of colds in patients with chronic obstructive pulmonary disease. However, the inhibitory effects of hochu-ekki-to on infection by rhinovirus (RV), the major cause of common colds, have not been studied. EXPERIMENTAL APPROACH: Human tracheal epithelial cells in culture were infected with a major group rhinovirus-RV14. Virus output and viral RNA were measured along with interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha), mRNA for intercellular adhesion molecule (ICAM)-1 and acidic endosomes in cells. KEY RESULTS: RV14 infection increased virus titers, the content of cytokines in supernatants and RV14 RNA in the cells. Hochu-ekki-to decreased virus output, RV14 RNA in the cells, susceptibility to RV infection and supernatant cytokine concentrations after RV14 infection. Hochu-ekki-to reduced mRNA for ICAM-1, the receptor for RV14, the concentration of the soluble form of ICAM-1 and the number and fluorescence intensity of acidic endosomes in the cells, from which RV RNA enters into the cytoplasm, at RV14 infection. Glycyrrhizin, one of the chemical constituents of hochu-ekki-to, reduced supernatant virus titers dose-dependently. CONCLUSION AND IMPLICATIONS: Hochu-ekki-to inhibited RV14 infection by decreasing ICAM-1 and by blocking entry of viral RNA into the cytoplasm from the endosomes, in airway epithelial cells. Glycyrrhizin may be partly responsible for inhibition of RV infection by hochu-ekki-to. Hochu-ekki-to could modulate airway inflammation by reducing production of cytokines in RV infections.

Echinacea extracts modulate the production of multiple transcription factors in uninfected cells and rhinovirus-infected cells.

Extracts of Echinacea purpurea are widely used for the prevention and treatment of common colds, coughs, bronchitis and other upper respiratory infections, many of which are caused by rhinoviruses (RVs). Recent reports have indicated that rhinoviruses can stimulate the release of various pro-inflammatory cytokines and chemokines from cultured nasal and bronchial human epithelial cells, and several transcription factors (TFs) have been implicated in this process. The effects of Echinacea treatment and rhinovirus infection on the activation of a range of transcription factors were evaluated by means of a protein/DNA array analysis. The BEAS-2B cell line was used as the model, and nuclear extracts of uninfected cells and rhinovirus-14 infected cells were examined with and without treatment with one of two chemically different Echinacea extracts. It was found that both Echinacea extracts increased the nuclear content of more than 30 transcription factors, including the 12 pro-inflammatory factors examined, such as NFkB, AP-1, AP-2 and STATs 1-6. Virus infection resulted in a more dramatic increase in these same TFs. However, when RV-infected cells were treated with either of the two Echinacea extracts, TF levels were reduced to low levels, although the pattern of the reductions was different for the two extracts. These results indicate that rhinovirus infection of epithelial cells, and treatment with Echinacea extracts, led to profound effects on numerous transcription factors, which could explain the previously observed modulation of secreted cytokines and chemokines, as well as other signaling pathways. In addition, the results could help to explain the beneficial effects of Echinacea consumption.

Rhinovirus chemotherapy.

Human rhinoviruses (HRV), members of the Picornaviridae family, are comprised of over 100 different virus serotypes. HRV represent the single most important etiological agents of the common cold [Arruda, E., Pitkaranta, A., Witek Jr., T.J., Doyle, C.A., Hayden, F.G., 1997. Frequency and natural history of rhinovirus infections in adults during autumn. J. Clin. Microbiol. 35, 2864-2868; Couch, R.B., 1990. Rhinoviruses. In: Fields, B.N., Knipe, D.M. (Eds.), Virology. Raven Press, New York, pp. 607-629; Turner, R.B., 2001. The treatment of rhinovirus infections: progress and potential. Antivir. Res. 49 (1), 1-14]. Although HRV-induced upper respiratory illness is often mild and self-limiting, the socioeconomic impact caused by missed school or work is enormous and the degree of inappropriate antibiotic use is significant. It has been estimated that upper respiratory disease accounts for at least 25 million absences from work and 23 million absences of school annually in the United States [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Rotbart, H.A., 2002. Treatment of picornavirus infections. Antivir. Res. 53, 83-98]. Increasing evidences also describe the link between HRV infection and more serious medical complications. HRV-induced colds are the important predisposing factors to acute otitis media, sinusitis, and are the major factors in the induction of exacerbations of asthma in adults and children. HRV infections are also associated with lower respiratory tract syndromes in individuals with cystic fibrosis, bronchitis, and other underlying respiratory disorders [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Gern, J.E., Busse, W.W., 1999. Association of rhinovirus infections with asthma. Clin. Microbiol. Rev. 12 (1), 9-18; Pitkaranta, A., Arruda, E., Malmberg, H., Hayden, F.G., 1997. Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-PCR. J. Clin. Microbiol. 35, 1791-1793; Pitkaranta, A., Virolainen, A., Jero, J., Arruda, E., Hayden, F.G., 1998. Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction. Pediatrics 102, 291-295; Rotbart, H.A., 2002. Treatment of picornavirus infections. Antivir. Res. 53, 83-98]. To date, no effective antiviral therapies have been approved for either the prevention or treatment of diseases caused by HRV infection. Thus, there still exists a significant unmet medical need to find agents that can shorten the duration of HRV-induced illness, lessen the severity of symptoms, minimize secondary bacterial infections and exacerbations of underlying disease and reduce virus transmission. Although effective over-the-counter products have been described that alleviate symptoms associated with the common cold [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Gwaltney, J.M., 2002a. Viral respiratory infection therapy: historical perspectives and current trials. Am. J. Med. 22 (112 Suppl. 6A), 33S-41S; Turner, R.B., 2001. The treatment of rhinovirus infections: progress and potential. Antivir. Res. 49 (1), 1-14; Sperber, S.J., Hayden, F.G., 1988. Chemotherapy of rhinovirus colds. Antimicrob. Agents Chemother. 32, 409-419], this review will primarily focus on the discovery and development of those agents that directly or indirectly impact virus replication specifically highlighting new advances and/or specific challenges with their development.

Isolation of a peptide inhibitor of human rhinovirus.

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.

Expression of intercellular adhesion molecule-1 (ICAM-1) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection?

Since clinical experimental studies indicate that upper respiratory tract viral infections may exacerbate acute asthma symptoms in atopic/asthmatic individuals, we have investigated the expression and modulation of ICAM-1 on human nasal epithelial cells (HNEC) from normal and atopic subjects. ICAM-1 is the attachment molecule for the majority of serotypes of human rhinovirus (HRV), including HRV-14, and is also critical for the migration and activation of immune effector cells. Basal ICAM-1 expression was significantly higher in HNEC obtained by brushings from atopic compared with non-atopic subjects (P = 0.031), and was also significantly increased on atopic HNEC harvested in season compared with out of season (P < 0.05). Atopic HNEC showed further up-regulation in ICAM-1 expression when cultured with clinically relevant allergen (P = 0.032). ICAM-1 levels on normal HNEC were also increased by infection with HRV-14 (P < 0.05). Basal expression of ICAM-1 on atopic nasal polyp epithelial cells (EC) was significantly higher than on both normal and atopic nasal HNEC. This elevated nasal polyp ICAM-1 level was not increased further by allergen, although HRV infection resulted in a small significant increase. Recovered viral titres from HRV-infected nasal polyp EC were 1.5-fold higher than from infected normal nasal HNEC. The data are consistent with the hypothesis that allergen, by enhancing expression of the HRV attachment target on host cells, facilitates viral infection in atopic subjects; simultaneously HRV-induced increases in ICAM-1 levels would favour migration and activation of immune effector cells to the airway, resulting in enhanced atopic inflammation.

Antiviral effect of hyperthermic treatment in rhinovirus infection.

Human rhinoviruses (HRV) are recognized as the major etiologic agents for the common cold. Starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (HT) on HRV replication in HeLa cells. We report that a 20-min HT at 45 degrees C is effective in suppressing HRV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins is not affected by HT, indicating that the target for treatment is a posttranslational event. The antiviral effect is a transient cell-mediated event and is associated with the synthesis of the 70-kDa heat shock protein hsp70. Unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat. The possibility that hsp70 could play a role in the control of rhinovirus replication is suggested by the fact that a different class of HSP inducers, the cyclopentenone prostaglandins PGA1 and delta 12-PGJ2, were also effective in inhibiting HRV replication in HeLa cells. Inhibition of hsp70 expression by actinomycin D prevented the antiviral activity of prostaglandins in HRV-infected cells. These results indicate that the beneficial effect of respiratory hyperthermia may be mediated by the induction of a cytoprotective heat shock response in rhinovirus-infected cells.

Antiviral activity of constituents of Tamus communis.

The antiviral activity of the phenanthrene derivatives 1-6, of the spyrostane triglycosides dioscin (7) and gracillin (8), of the furostanol tetraglycosides methylprotodioscin (9), its (25S) epimer methylprotoneodioscin (10), and methylprotogracillin 11, have been tested towards two RNA viruses: vesicular stomatitis virus and human rhinovirus type 1B. All these products were extracted from the rizomes of Tamus communis L; compound 11 was isolated also from Asparagus cochinchinesis, together with pseudoprotodioscin (12), a 20 (22)-unsaturated furostanoside, which was also investigated for antiviral activity. The results were of some interest mainly for the phenanthrene derivatives.