Respiratory Syncytial Virus Infection: From Biology to Therapy A Perspective
© World Allergy Organization; licensee BioMed Central Ltd. 2008
Received: 3 October 2007
Accepted: 13 December 2007
Published: 15 February 2008
Respiratory syncytial virus (RSV) is responsible for significant morbidity and mortality, particularly in infants younger than 18 months and in the elderly. To date, there are few effective treatment options available to prevent or treat RSV infections. Attractive therapeutic strategies include targeting host epithelial adhesion molecules required for RSV infection, enhancing localized cell-mediated immunity, interfering with RSV viral gene expression and developing a multigene DNA vaccine. The most recent data supporting the advantages and limitations of each of these approaches are discussed in detail. Several promising strategies offer hope for safe and effective prophylaxis and treatment of RSV infection.
Respiratory syncytial virus (RSV) is one of the most important respiratory pathogens targeting all age groups; however, infants (younger than 18 months) and the elderly experience the most severe aspects of the disease, which results in lower respiratory tract illnesses (ie, bronchiolitis and pneumonia) . Around 90% of infants are infected for the first time by the age of 2 years [1, 2]. Worldwide, about 5 million infants are hospitalized because of severe RSV infection. The first is usually the most severe, and previous findings indicate that infants with a history of premature birth, bronchopulmonary dysplasia, congenital heart disease, cystic fibrosis, or immunosuppression are more likely to develop the most severe clinical courses of bronchiolitis and pneumonia, which have the highest risk of death[1, 2].
However, an analysis of a comprehensive study done between 1979 and 1997 about RSV-associated deaths in US children suggests that most RSV-related deaths do not occur among children who are presumed to be at high risk for severe RSV lower respiratory tract illnesses . The leading cause in infant hospitalization is RSV bronchiolitis,  which imposes a severe burden upon health services. Costs related to emergency department visits between 1997 and 2000 amount to approximately 202 million US dollars . Complete immunity to RSV never develops, and reinfection throughout life is common. Although the major clinical manifestation of RSV in older children and adults is upper respiratory tract illness (rhinitis and acute otitis media), it may also cause up to 2.4% of community-acquired pneumonia in these population groups . In older adults, RSV was identified as responsible for 10% of winter hospital admissions and has a case-fatality rate that approaches 10%. In addition, 78% of RSV-associated deaths occur in individuals aged 65 years or older who have underlying cardiac and pulmonary pathology . In particular, RSV infection in adults with strong immunosuppression, for example, patients undergoing bone marrow transplantation is of great medical importance .
In the past 8 years, our research has identified both cellular and viral targets that may be useful for the prevention of RSV infection and its accompanying pathology. Differential microarray analysis was used to pinpoint gene expression changes in RSV-infected cells, and expression of candidate therapeutic genes was tested both in cultured lung epithelial cells in vitro and in animal models in vivo. Characterization of these gene expression changes includes immune modulation, signal transduction, and apoptosis. In this report, the biology of RSV and how these studies contribute to the basic mechanistic studies of RSV infection and have led to new targets to manage RSV infection will be discussed.
State of the Art in Treatment and Prophylaxis of RSV Infection
There is no treatment to protect against RSV infection, and the current treatment, Ribavirin, only produces modest short-term improvement in respiratory tract infection . Moreover, it is now restricted to a highly selected group of patients with T-cell immunodeficiency . Passive immunoprophylaxis, involving the administration of either a polyclonal antibody (Synagis) preparation or a humanized version of a monoclonal anti-RSV-F antibody (Palivizumab), is successful for protection of high-risk individuals against RSV infection. However, these approaches are only partially effective, expensive, and could generate resistant mutant RSV strains. Development of new and highly effective antibodies to modulate RSV infection remains a major medical and pharmaceutical goal.
To date, there is no licensed vaccine for the prevention of human RSV disease. Efforts have been made to develop active prophylaxis measures (vaccines), and both subunit and attenuated live vaccines are being pursed in clinical studies. Vaccine development has been limited after the testing of initial vaccines in the 1960s, which exacerbated the RSV disease [10, 11]. Some of the reasons for the lack of success in developing previous vaccines include the inadequate response to vaccination, the existence of 2 antigenically distinct RSV groups, and the history of disease enhancement after administration of a formalin-inactivated vaccine [12, 13].
Developing active or passive prophylaxis is important as they are expected to decrease the incidence of severe infections and thus may reduce or attenuate asthma pathogenesis. Recent advances in the vaccine area include research with plasmid-based DNA vaccines and small-interfering RNA (siRNA)-based approaches. To deliver these antiviral plasmids in the most effective way to target cells, a novel carrier system has been produced based on modified polysaccharide nanoparticles that protect the DNA and facilitate its introduction into the lungs. The advances in this field are reviewed in the following sections.
RSV Genome and Structure
Human RSV is in the genus Pneumovirus, subfamily Pneumovirinae, family Paramyxoviridae, order Mononegavirales, whose members consist of nonsegmented, negative-sense, single-stranded RNA viruses. In addition to human RSV, the genus Pneumovirus includes bovine RSV, ovine RSV, and pneumonia virus of mice. The RSV virions consist of a nucleocapsid contained within a lipid envelope of irregular spherical shape with sizes of 150 to 300 nm. Both infected cultures and viral preparations can also include filamentous forms of the virions that are 60 to 100 nm in diameter and up to 10 μm in length . The viral envelope is a lipid bilayer acquired from the host plasmatic membrane. The viral transmembrane glycoproteins--the fusion protein F, the attachment protein G, and the small hydrophobic protein SH--organize themselves to form spikes, which are visible under electron microscopy. Host lipid raft-derived proteins are also incorporated into the envelope of mature viral particles [15–17]. The envelope connects to the nucleocapsid through the viral matrix M protein. Using electron microscopy, the nucleocapsid is seen as an internal electrodense material with a diameter of 15 nm inside the round and filamentous forms of the virions . The nucleocapsids consist of the RNA genome and the associated nucleocapsid protein N, the phosphoprotein P, the large polymerase subunit L, and the antitermination factor M2-1. The viral RNA genome and the associated proteins in the nucleocapsid together form a very tight ribonucleoprotein complex, which is resistant to RNAse activity.
The genome for most of the virions is a negative-sense strand of RNA of 15,222 nucleotides in length. However, some virions are also found to have incorporated the positive-sense replicative intermediate (antigenomic RNA), which is synthesized during viral replication. Thus, this implies that during the viral assembly, there is no mechanism that allows discrimination in packaging. The viral genes are ordered from 3' to 5' in the following way: NS1-NS2-N-P-M-SH-G-F-M2-L. Glycoprotein G and F (and SH), respectively, mediate virus attachment and fusion to the host cell . In addition to fusion, protein F has also been postulated to participate in the attachment of the virus to the host cell membrane. Intercellular adhesion molecule 1 (ICAM-1), annexin-II, and Toll-like receptor 4 are receptors for protein F [19–21]. The matrix (M) protein forms a layer on the inner face of the viral envelope, and it plays an essential role in viral assembly through its interactions with the cell membrane, virus envelope, and virus nucleocapsid [22, 23]. The nucleocapsid-associated proteins N, P, M2-1, and L play essential roles at different stages for efficient viral transcription and replication. The nonstructural proteins NS1 and NS2 are thought to be antagonists of the interferon (IFN)-type I system. They seem to target the transcription factor IRF-3. Thus, the expression of these proteins helps the virus to reduce IFN-γ expression by infected cells [24, 25].
Prophylaxis and Treatment of RSV Infection
Summary of Studies Relating to RSV Infection
Ab, antisense RNA
F, G, SH, NS1, NS2, P, N
Development of Chitosan-Based Nanoparticles as a Platform for Gene and Drug Delivery
Numerous investigators, including those in our laboratory, have extensively studied chitosan, which we believe has the potential to be useful for the delivery of genes and drugs, as it has very low immunogenicity while having strong immunostimulatory properties . Moreover, as a carrier, it can most adequately provide heat stability to encapsulated or adsorbed vaccines. Chitosan, a natural biocompatible cationic polysaccharide extracted from crustacean shells, is capable of efficient drug and gene delivery [37–41]. Chitosan has many beneficial effects, including anticoagulant activity,  wound-healing properties,  and antimicrobial properties . In addition, chitosan is nontoxic, nonhemolytic, slowly biodegradable, and nuclease resistant, and it has been widely used in controlled drug delivery [37, 43–47]. Chitosan also increases transcellular and paracellular transport across the mucosal epithelium and, thus, may facilitate mucosal drug delivery and modulate immunity of the mucosal and bronchus-associated lymphoid tissues. Chitosan apparently binds to macrophages and myeloid cells via CD14.[49, 50]
The toxicity of mucosally administered chitosan has been studied in rodents. N-trimethyl chitosan and chitosan hydrochloride given intranasally do not alter the ciliary beat frequency of the rat nasal epithelium, and hence, both are considered to be nontoxic . In addition, the subacute oral toxicity of chitosan oligosaccharides was investigated in Sprague-Dawley rats of both sexes . The chitosan is metabolized and secreted through the viliary system. Thirty-six male and female rats were administered by gavage 500, 1000, and 2000 mg/kg per day of chitosan for 4 weeks (7 days per week), and their clinical signs, body weights, hematologic and biochemical parameters, and histopathology were examined. There were no significant differences in behavior, external appearance, body weight or food consumption between control and treated rats. In addition, no significant differences in urinalysis, hematology, blood biochemistry, relative organ weights, and histopathological findings were found in either control or treated rats. These results suggest that the acute toxicity of chitosan oligosaccharides is low and that the detection limit of toxicity is greater than 2000 mg/kg in rats. Furthermore, chlorophyllin-chitosan, an insoluble form of chlorophyllin, inhibits DNA adduct formation and mutagenesis by a heterocyclic food mutagen-carcinogen, 3-amino-1-methyl-5H-pyridoindole (Trp-P-2), in mice carrying the Escherichia coli rpsL gene as a mutagenesis reporter, this suggests that chlorophyllin-chitosan may be a candidate chemopreventive agent against the genotoxic action of Trp-P-2 and possibly other aromatic carcinogens in the diet.
The Environmental Protection Agency has ruled chitosan exempt from its tolerance guidelines because of its nontoxicity as evidenced by the: (1) literature search done for chitin, chitosan, N-acetyl-D-glucosamine, and D-glucosamine toxicity in humans using the databases PubMed, Hazardous Substances Data Bank, Integrated Risk Information System, Gene-Tox, Environmental Mutagen Information Center, Toxic Release Inventory, the Food and Drug Administration, the United States Department of Agriculture and ChemIDplus; (2) animal feeding studies, in which up to 5% of the diet is chitosan, that failed to show any adverse effects; and (3) the lack of reported complaints of toxicity against the database of 2700 complaints despite years of chitosan use in food and nutritional supplements.
A Nanoparticle Gene Expression Vaccine for RSV
The potential of vaccines has been intensely investigated since the discovery of the virus. All RSV proteins, except L, have been tested for immunogenicity and protective efficacy in rodents using recombinant vaccinia viruses [59–61]. A number of approaches, including recombinant live, attenuated, subunit vaccines, and DNA vaccines, are under intense investigation, [62–64] but none have crossed the clinical-phase hurdles and been licensed thus far. The development of RSV vaccines is complicated by the need to administer the vaccine at a very young age, between 6 weeks and 6 months, in the face of a premature immune system. In addition, because RSV is a mucosal pathogen, an effective vaccine must generate secreted mucosal antibodies, such as immunoglobulin A (IgA) and mucosal cytotoxic lymphocytes (CTLs) [65, 66]. The RSV-induced CTL response at mucosal sites is inadequate. Although evidence suggests the potential of a gene expression vaccine for RSV infection, the number of studies is limited. Previous reports using systemic injections of pDNA show variable results. The quantity of DNA used per unit body mass, as much as 10 mg/kg, and the route of administration chosen are inconvenient for infants and are suboptimal for inducing mucosal immunity against a pulmonary infection.
Our laboratory developed a nanoparticle multigene vaccination strategy against RSV infection using a complementary DNA cocktail produced by cloning 9 RSV antigens (NS1, NS2, M, SH, F, M2, N, G, or P in a pVAX plasmid) complexed with chitosan nanoparticles, referred to as nanoparticle gene expression vaccine (NGXV). The NGXV was administered to mice by the intranasal route. The rationale for developing this vaccine is based on the following reports. All of the RSV proteins, except L, have been tested individually (and in some cases, in combination) for immunogenicity and protective efficacy in rodents using recombinant vaccinia viruses [59–63]. The F and G proteins are the antigens that induce most of the the neutralizing antibodies against RSV [68–70]. The CTL repertoire in humans revealed that the N, SH, F, M, M2, and NS2 proteins were strong target antigens. In BALB/c mice, the F, N, and M2 proteins are major target antigens [61, 71–73]. Protection against and recovery from RSV infection are mediated largely by the immune system, with the specific direct effectors being secretory antibodies, serum antibodies, and major histocompatibility complex class I-restricted CTLs.
The results demonstrate that a single vaccination of about 1 mg/kg body weight of NGXV decreases viral titers by 2 orders of magnitude (100-fold) upon primary infection. In addition, NGXV significantly decreases pulmonary inflammation and does not alter airway hyperresponsiveness, thus making it a potentially safe vaccine. This may represent a major breakthrough in RSV vaccine development.
Host Gene Expression
Prophylactic IFN-γ gene transfer in BALB/c mice decreases viral replication and induces a Th1-like (increased production of IFN-γ and interleukin-12), instead of a Th2-like (decreased interleukin-5) immune response against RSV infection [74–76]. Viral infections induce IFN-γ, which in turn facilitates the resolution of viral infection . Levels of IFN-γ have been compared in bronchoalveolar lavage fluids after infection with RSV in control and pIFN-γ-treated mice. A 3-to 6-fold increase in IFN-γ production was found in RSV-infected mice compared with uninfected mice. Such increases are considered to be relatively low compared with other viral infections [74–76]. The finding that a natural live virus infection is cleared by elevated IFN-γ production, a response similar to that seen after live viral infection in mice, suggests that the results from this animal model may be applicable to human RSV disease.
A new prophylactic approach consists of taking advantage of the RNA interference mechanism initially discovered in plant cells and that is present in all species including mammals. RNA interference is triggered by double-stranded RNA that is cleaved by an RNAse III-like enzyme, Dicer, into 21-25-nucleotide fragments (siRNAs) with characteristic 5' and 3' termini [77, 78]. These siRNAs act as guides for a multiprotein complex, including a PAZ/PIWI domain, containing the protein Argonaute2, which cleaves the target messenger RNA (mRNA) . These gene-silencing mechanisms are highly specific and induce inhibition of gene expression throughout an organism. RNA interference is a known phenomenon that has been proven effective in silencing a number of genes of different viruses [80–82]. The siRNA to viral P or NS-1 mRNAs prevents RSV infection in cellular and animal model studies [83, 84]. Prophylactic intranasal administration of an siRNA formulation specific for RSV-P mRNA is able to significantly reduce the viral load and the disease parameters in RSV-infected mice . A carrier in the formulation is not required. In addition, a very low dose is effective in showing a protective effect. Moreover, siRNA-resistant virus did not appear after using this formulation . Although intranasal administration of naked siRNA to humans was found to be safe in a phase I study, other studies have shown toxicity.
Because the synthesis of RNA oligonucleotide-based siRNA is expensive, our laboratory engineered DNAvector-based approaches to introduce siNS1 into RSV-infected human cells and animal models. This is based on the principle of the intracellular transcription of small RNA molecules that are synthesized from a DNA template under the control of RNA polymerase III promoters, such as U6 . NS-1 was selected as the target because the NS1 protein interferes with type-1 IFN-mediated host antiviral responses [24, 86]. Silencing of the NS-1 gene attenuated RSV replication and boosted the immune response through an increase in IFN-γ production . The prophylactic intranasal administration of this formulation, combined with chitosan, significantly reduced the viral load and ameliorated the pulmonary pathology in RSV-infected mice . In addition, mice treated with this formulation develop protection against reinfection.
Moreover, this formulation also drives human dendritic cells to promote a Th1-like profile . Overall, siRNA-mediated silencing of the NS1 gene up-regulates host-antiviral genes and suppresses RSV replication compared with control groups. Studies confirm the role of siNS1 in a rat model of RSV infection. A phase I study is currently under development using the nanoparticle-incorporated siNS1, and it may represent a novel prophylaxis/therapy that can be used in a global population.
Summary and Conclusion
The RSV is the major pathogen responsible for serious upper and lower respiratory tract infections, primarily in infants, but also in the elderly worldwide. The precise molecular and cellular mechanisms are unclear, and satisfactory prophylaxis or treatment strategies are yet to emerge. This research has resulted in the understanding of the pathology and complexity of signaling pathways involved in successful infection; the role of host defense molecules such as ICAM-1, IFN-γ, and related pathways; and how they can be exploited to develop less costly prophylaxis and treatments for RSV infection. Finally, the potential to develop safe and effective prophylaxis and/or treatment by targeting important RSV genes is under investigation.
S. S. Mohapatra is supported by grants from the Veterans' Affairs Merit Review and VA Career Scientist Awards, and by US National Institutes of Health grant no. 5RO1HL71101-01A2. The support from the Joy McCann Culverhouse and Mabel and Ellsworth Simmons Endowments to the Division of Allergy and Immunology, Department of Internal Medicine, College of Medicine, and the Veterans' Affairs Hospital is also gratefully acknowledged.
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