Biochemical prevention and treatment of viral infections – A new paradigm in medicine for infectious diseases
Virology Journalvolume 1, Article number: 12 (2004)
For two centuries, vaccination has been the dominating approach to develop prophylaxis against viral infections through immunological prevention. However, vaccines are not always possible to make, are ineffective for many viral infections, and also carry certain risk for a small, yet significant portion of the population. In the recent years, FDA's approval and subsequent market acceptance of Synagis, a monoclonal antibody indicated for prevention and treatment of respiratory syncytial virus (RSV) has heralded a new era for viral infection prevention and treatment. This emerging paradigm, herein designated "Biochemical Prevention and Treatment", currently involves two aspects: (1) preventing viral entry via passive transfer of specific protein-based anti-viral molecules or host cell receptor blockers; (2) inhibiting viral amplification by targeting the viral mRNA with anti-sense DNA, ribozyme, or RNA interference (RNAi). This article summarizes the current status of this field.
A landmark in the battle against viral infectious diseases was made in 1798 when Jenner first inoculated humans against smallpox with the less virulent cowpox. For about two centuries since then, humans relied almost exclusively on vaccines for protection against viruses. Only in the recent years, new strategies for controlling viral infectious diseases have emerged, which have so far led to a couple of viral prophylaxis/therapeutics on the market. These strategies are fundamentally different from vaccines in that they attempt to directly interrupt viral infectious life cycle at molecular level by using proteins or oligonucleotides. To differentiate them from the conventional vaccines that prevent viral infection by boosting immune system, we refer the new antiviral approaches as "Biochemical Prevention and Treatment" (see figure 1). Biochemical Prevention and Treatment, as an alternative to vaccines and chemical compound based antiviral drugs, may prove to be particularly valuable in the areas where vaccines and/or chemical drugs can not be generated or have not been successful in human, including diseases caused by some common pathogenic viruses, such as HIV, hepatitis C virus (HCV), RSV and human rhinovirus (HRV). In this review, we will discuss various molecular intervention approaches.
1. Biochemical Prevention and Treatment via Protein targeting
Among the biochemical therapeutics currently in clinical trials, the majority consists of monoclonal antibodies (MAbs). Soluble receptor drug candidates have gradually lost favor over the past several years due to issues relating to low potency and cost. Peptide-based drug candidates are limited by insufficient efficacy and unfavorable pharmacokinetics. MAbs have increasingly gained favor in large part because of the development of chimeric, humanized, and human antibodies have reduced the immunogenicity of antibody therapies. The MAbs that are currently in clinical trials for viral infection prophylaxis and treatment are listed in Table 1.
Biochemical Prevention and Treatment of Respiratory Syncytial Virus Infection
The respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and young children producing bronchiolitis and pneumonia worldwide. RSV infection leads to more than 90,000 hospitalizations and a 2% mortality rate among infants nationwide [2–5]. Approximately two-thirds of infants are infected with RSV during the first year of life and approximately 95% of children test seropositive for RSV by the age of two . Unfortunately, even natural RSV infection produces limited immunity at best. In fact, an inactivated RSV vaccine paradoxically resulted in more severe disease instead of protection .
The most successful approach to date has been Biochemical Prevention and Treatment with anti-viral antibodies. In 1996, RespiGam™ (respiratory syncytial virus immune globulin or RSV-IG) became available for use in children less than two years of age with high-risk factors [8–10]. The use of RespiGam™ was largely supplanted with the approval of Synagis™ (Palivizumab) in 1998. Palivizumab is an IgG1 MAb administered IM monthly that selectively binds to the RSV surface glycoprotein F [1, 51]. The drug specifically inhibits RSV replication by preventing the virus from fusing with the respiratory endothelial cell membrane. Palivizumab has been shown to reduce the rate of hospitalization of at-risk infants by about 55% in clinical studies and now serves as the primary medical means of RSV prevention [11–13].
Prevention of Human Rhinovirus infections
Human rhinovirus (HRV) causes over 80% of the common cold in the fall . Developing vaccines against HRV is unfeasible because HRVs have at least 115 antigenically distinct serotypes [15, 16]. One of the proven methods to prevent and inhibit viral infections is to block host cell receptors that are used by viruses to gain cell entry. Receptor blockage is commonly achieved via application of MAbs that bind to specific epitopes on the receptor molecules. A plethora of in vitro studies have reported effective viral inhibition by receptor-blocking MAbs. However, these works have not yielded yet any approved drug on the market.
In HRV infection, about 90% of HRV serotypes utilize a single cell surface receptor exclusively, which is the intercellular adhesion molecule-1 (ICAM-1), for viral attachment and subsequent viral entry [17, 18]. As such, ICAM-1 has become a very promising target for biochemical prevention. A receptor blocking approach has shown that the soluble ICAM-1 and an anti-ICAM-1 monoclonal antibody, Mab 1A6, could prevent infections by a broad spectrum of rhinovirus serotypes in human cells in vitro [19–21]. Administration of soluble ICAM-1 and MAbs in human clinical trials had indeed achieved reduction in symptoms, but did not prevent the incidence of the disease [22–24]. For the MAbs, the limited efficacy is most likely due to its low functional affinity (or avidity) for ICAM-1 when compared to that of the multivalent HRV particles .
High avidity is achieved by multivalency. To improve avidity of HRV receptor blocking antibody, a novel tetravalent recombinant antibody, CFY196, has been generated against ICAM-1 . CFY196 is composed of Fab fragment of a humanized version of MAb 1A6 fused with a linker derived from human immunoglobulin D (IgD) hinge and a tetramerization domain derived from the coiled-coil sequence of human transcription factor ATFα. CFY196 is expressed in bacteria and purified as a homogenous tetrameric molecular complex. CFY196 exhibited almost two-orders-of-magnitude improvement in functional affinity compared with its bivalent counterpart based on the kinetic parameters measured by BIAcore analysis. Such kinetic improvement also directly leads to functional superiorities of CFY196. In in vitro assays, CFY196 consistently and significantly outpaced the best commercial anti-ICAM-1 MAbs in preventing HRV infection as measured by reduction of cytopathic effects and HRV viral titers . The preclinical findings of CFY196 bode well its efficacy in human since MAb 1A6, from which CFY196 is derived, has already exhibited positive effects in a human trial. Moreover, to prevent possible immunogenicity, CFY196 is humanized . Further pre-clinical and clinical development of CFY196 is warranted to fully evaluate its potential as a prophylaxis and therapeutics for the HRV induced common colds.
2. Biochemical Prevention and Treatment via targeting on viral mRNA
Targeting viral mRNA is one of the most active areas of research and development. Several strategies have emerged over the years and are being tested pre-clinically and clinically. They include: antisense-oligonucleotides (AS-ONs), ribozymes, and recently, RNA interference (RNAi). All these strategies share the features of conceptual simplicity, straightforward drug design and quick route to identify drug leads. However, the challenges have been to improve potency, pharmacokinetics and, most importantly, intracellular delivery of the drug candidates. As the oldest strategy, AS-ON technology has produced to date one drug in the market place, Vitravene®. A number of clinical trials of drug candidates from these technologies are currently ongoing.
Antisense-oligonucleotides (AS-ONs) are short synthetic oligonucleotides that form complementary pair with specific viral mRNA targets. AS-ONs inhibit viral protein production by both blocking viral mRNA translation and triggering its degradation. Since the discovery of viral inhibition effect of AS-ONs by Zamecnik and Stephenson in 1978 , antisense technology has been developed as a powerful tool for target validation and therapeutic purposes.
Vitravene is the first AS-ON based drug approved by FDA. Vitravene, or fomivirsen sodium, is a 21-base phosphorothioate oligodeoxynucleotide complementary to the messenger RNA of the major immediate-early region proteins of human cytomegalovirus, and is a potent and selective antiviral agent for cytomegalovirus retinitis, a herpes-like eye disease that afflicts the immune-suppressed [29, 30]. A number of clinical trials as well as one approved therapy based on AS-ON technologies are summarized in Table 2.
Phosphorothioate (PS) oligodeoxynucleotides are the 'first generation' DNA analogs. The 'second generation' ONs contain nucleotides with alkyl modifications at the 2' position of the ribose. They are less toxic than PS-DNAs and have a slightly enhanced affinity. DNA and RNA analogs with modified phosphate linkages, or different sugar residues substituting the furanose ring have been referred as 'third generation' . For instance, peptide nucleic acids and their analogs display superior sequence specificity and are resistant to nuclease degradation. These third generation AS-ON have limited non-specific interactions with other genes and, therefore, have shown great potentials in clinical trials.
Ribozymes (Rz) are catalytically active ONs that both bind and cleave target RNAs. They were discovered after the AS-ON technology. Initial findings on ribozymes raised the hope that they may offer a more potent alternative to AS-ONs. Many cell based and animal tests have performed on anti-viral effects of ribozymes, including HIV, hepatitis B, hepatitis C, influenza, etc. Results from these tests have shown that ribozymes are promising viral inhibitors [35–38]. However, further progress in the field has been hampered by difficulties to achieve satisfactory potency and efficient intracellular delivery of ribozymes in vivo. HEPTAZYME is a modified ribozyme that cleaves the internal ribosome entry site of the Hepatitis C virus. The Rz was demonstrated to inhibit viral replication up to 90% in cell culture . HEPTAZYME was tested in a Phase II clinical trial, but was later withdrawn from further clinical trials due to insufficient efficacy. So far, there is no anti-viral ribozymes that are being actively tested in advanced clinical trials.
RNA Interference (RNAi)
RNA interference, or RNAi, is the inhibition of expression of specific genes by double-stranded RNAs (dsRNAs). It is becoming the method of choice to knockdown gene expression rapidly and robustly in mammalian cells. Comparing to the traditional antisense method, RNAi technology has the advantage of significantly enhanced potency; therefore, only lower concentrations may be needed to achieve same level of gene knockdown. RNAi gained rapid acceptance by researchers after Tuschl and coworkers discovered that in vitro synthesized small interfering RNAs (siRNAs) of 21 to 23 nucleotides in length can effectively silence targeted genes in mammalian cells without triggering interferon production [40, 41]. In mammalian cells, the level of gene inhibition mediated by siRNA routinely reaches an impressive 90% .
Several initial studies, which test the potential application of synthetic siRNAs as antiviral agents, have shown very promising results. To date, RNAi has been used effectively to inhibit the replication of several different pathogenic viruses in culture, including: RSV (respiratory syncytial virus) , influenza virus , poliovirus  and HIV-1 [46–48]. In the case of HIV-1, several specific mRNAs have been successfully targeted for siRNA-mediated silencing, including those that encode Gag, Pol, Vif and the small regulatory proteins Tat and Rev. These studies show that RNAi can effectively trigger the degradation of not only viral mRNAs, but also genomic RNAs at both the pre- and post-integration stages of the viral lifecycle. In addition to targeting viruses directly, alternative strategies have employed siRNAs that silence the expression of essential host factors including Tsg101, required for vacuolar sorting and efficient budding of HIV-1 progeny , and the chemokine receptor CCR5, required as a co-receptor for HIV-1 cell entry .
Currently, our understanding of the biological mechanisms underlying RNAi lags behind the movement to apply this technology to human diseases such as viral infections. Some major technical hurdles need to be overcome before siRNA-based anti-viral prophylaxis and treatments move into the clinics. Especially, intracellular delivery of siRNA needs to be greatly improved. The next few years of research will indicate whether RNAi technology will realize its potential as the next wave of Biochemical Prevention and Treatment.
Anderson LJ, Bingham P, Hierholzer J: Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. J Virol 1988, 62: 4232-4238.
Chanock RM, Kim HW, Vargosko AJ, Deleva A, Johnson KM, Cumming C, Parrot RH: Respiratory syncytial virus: I. Virus recovery and other observations during 1960 outbreak of bronchiolitis, pneumonia, and minor respiratory diseases in children. JAMA 1961, 176: 647-653.
Parrott RH, Vargosko AJ, Kim HW, Cumming C, Turner H, Huebner RJ, Chanock RM: Respiratory syncytial virus. II. Serologic studies over a 34-month period of children with bronchiolitis, pneumonia, and minor respiratory diseases. JAMA 1961, 176: 653-657.
Prober CG, Wang EE: Reducing the morbidity of lower respiratory tract infections caused by respiratory syncytial virus: still no answer. Pediatrics 1997, 99: 454-61. 10.1542/peds.99.3.454
Simoes EAF, Rieger CHL: RSV infection in developed and developing countries. Infect Med 1999, 16: 11-17.
Parrott RH, Kim HW, Arrobio JO, Hodes DS, Murphy BR, Brandt CD, Camargo E, Chanock RM: Epidemiology of respiratory syncytial virus infection in Washington D.C. II. Infection and disease with respect to age, immunological status, race and sex. J Epidemiol 1973, 98: 289-300.
Levin MJ: Treatment and prevention options for respiratory syncytial virus infections. J Pediatrics 1994, 125: S22-S27.
Groothuis JR, Levin NJ, Rodriguez W, Hall CB, Long CE, Kim HW, Lauer BA, Hemming VG: Use of intravenous gamma globulin to passively immunize high-risk children against RSV: safety and pharmacokinetics. Antimicrob Agents Chemother 1991,35(7):1469-1473.
Meissner HC, Fulton DR, Groothuis JR, Geggel RL, Marx GR, Hemming VG, Hougen T, Snydman DR: Controlled trial to evaluate protection of high-risk infants against RSV disease by using standard intravenous immune globulin. Antimicrob Agents Chemother 1993, 37: 1655-1658.
MedImmune Inc: RespiGam™: Respiratory Syncytial Virus Immune Globulin Intravenous (Human), [RSV-IGIV]. Gaithersburg, MD 1996.
The Impact-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants Pediatrics 1998, 102: 531-537. 10.1542/peds.102.3.531
Cohen AH, Sorrentino M, Powers T: Effectiveness of palivizumab for preventing serious RSV disease. J Resp Dis 2000, 2: S30-S32.
MedImmune Inc: Synagis™: Palivizumab for intramuscular administration. Gaithersburg, MD 1996.
Arruda E, Pitkaranta A, Witek TJ Jr, Doyle CA, Hayden FG: Frequency and natural history of rhinovirus infections in adults during autumn. J Clin Microb 1997, 35: 2864-2868.
Stanway G: Rhinoviruses. In: Webster RG ed. In Encyclopedia of Virology. New York: Academic Press; 1994:1253-1259.
Skern T, Duechler M, Sommergruber W, Blaas D, Kuechler E: The molecular biology of human rhinoviruses. Biochem Soc Symp 1987, 53: 63-73.
Staunton DE, Merluzzi VJ, Rothlein R, Barton R, Marlin SD, Springer TA: A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cel 1989, 56: 849-853. 10.1016/0092-8674(89)90689-2
Uncapher CR, Dewitt CM, Colonno RJ: The major and minor group receptor families contain all but one human rhinovirus serotype. Virology 1991, 180: 814-817. 10.1016/0042-6822(91)90098-V
Marlin SD, Ltaunton DE, Springer TA: A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection. Nature 1990, 344: 70-72.
Huguenel ED, Cohn D, Dockum DP, Greve JM, Fournel MA, Hammond L, Irwin R, Mahoney J, McClelland A, Muchmore E, Ohlin AC, Scuderi P: Prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule-1. Am J Resp Critical Care Med 1997, 155: 1206-1210.
Colonno RJ, Callahan PL, Long WJ: Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses. J Virol 1986, 57: 7-12.
Turner RB, Wecker MT, Pohl G, Witek TJ, McNally E, St George R, Winther B, Hayden FG: Efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial. JAMA 1999, 281: 1797-1804. 10.1001/jama.281.19.1797
Colonno RJ: Virus receptors: the Achilles' heel of human rhinoviruses. Adv Exp Med Biol 1992, 312: 61-70.
Hayden FG, Gwaltney JM, Colonno RJ: Modification of experimental rhinovirus colds by receptor blockade. Antiviral Res 1988, 9: 233-247. 10.1016/0166-3542(88)90055-1
Casasnovas JM, Springer TA: Kinetics and thermodynamics of virus binding to receptor. Studies with rhinovirus, intercellular adhesion molecule-1 (ICAM-1), and surface plasmon resonance. J Biol Chem 1995, 270: 13216-13224. 10.1074/jbc.270.22.13216
Charles CH, Luo GX, Kohlstaedt LA, Gorfain E, Morantte I, Williams JH, Fang F: Prevention of Human Rhinovirus Infection by Multivalent Fab Molecules Directed against ICAM-1. Antimicrobial Agents and Chemotherapy 2003, 47: 1503-1508. 10.1128/AAC.47.5.1503-1508.2003
Luo GX, Kohlstaedt LA, Charles CH, Gorfain E, Morantte I, Williams JH, Fang F: Humanization of an anti-ICAM-1 antibody with over 50-fold affinity and functional improvement. J Immunol Methods 2003, 275: 31-40. 10.1016/S0022-1759(02)00542-2
Zamecnik PC, Stephenson ML: Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA 1978, 75: 280-284.
Orr RM: Technology evaluation: fomivirsen. Isis Pharmaceuticals Inc/CIBA vision. Curr Opin Mol Ther 2001, 3: 288-294.
Roehr B: Fomivirsen approved for CMV retinitis. J Int Assoc Physicians AIDS Care 1998, 4: 14-16.
Dove A: Antisense and sensibility. Nat Biotechnol 2002, 20: 121-124. 10.1038/nbt0202-121
Braasch DA, Corey DR: Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry 2002, 41: 4503-4509. 10.1021/bi0122112
Opalinska JB, Gewirtz AM: Nucleic acids therapeutics: Basic principles and recent applications. Nat Rev Drug Discov 2002, 1: 503-514. 10.1038/nrd837
Kurreck J: Antisense technologies: Improvement through novel chemical modifications. Eur J Biochem 2003, 270: 1628-1644.
Yu M, Ojwang J, Yamada O, Hampel A, Rappaport J, Looney D, Wong-Staal F: A Hairpin Ribozyme Inhibits Expression of Diverse Strains of HIV-1. Proc Natl Acad Sci USA 1993, 90: 6341.
Welch P, Tritz R, Yei S, Barber JR, Yu M: Intracellular Application of Hairpin Ribozyme Genes Against Hepatitis B Virus. Gene Therapy 1997, 4: 736. 10.1038/sj.gt.3300441
Welch PJ, Tritz R, Yei S, Leavitt M, Yu M, Barber J: Apotential therapeutic application of hairpin ribozymes: In vitro and in vivo studies of gene therapy for hepatitis C virus infection. Gene Ther 1996, 3: 994.
Tang XB, Hobom G, Luo D: Ribozyme mediated destruction of influenza A virus in vitro and in vivo . J Med Virol 1994, 42: 385.
Macejak D, Jensen KL, Jamison S, Domenico K, Roberts EC, Chaudhary N, von Carlowitz I, Bellon L, Tong MJ, Conrad A, Pavco PA, Blatt LM: Inhibition of Hepatitis C Virus (HCV)-RNA-dependent translation and replication of a chimeric HCV Poliovirus using synthetic stabilized ribozymes. Hepatology 2000, 31: 769-776. 10.1002/hep.510310331
McManus MT, Sharp PA: Gene silencing in mammals by small interfering RNAs. Nature Rev 2002, 3: 737-747. 10.1038/nrg908
Thompson JD: Applications of antisense and siRNAs during preclinical drug development. Drug Discovery Today 2002, 7: 912-917. 10.1016/S1359-6446(02)02410-8
Shi Y: Mammalian RNAi for the masses. Trends in Genetics 2003, 19: 9-12. 10.1016/S0168-9525(02)00005-7
Bitko V, Barik S: Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiology 2001, 1: 34-46. 10.1186/1471-2180-1-34
Ge O, McManus MT, Nguyen T, Shen CH, Sharp PA, Eisen HN: RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA 2003, 100: 2718-2723. 10.1073/pnas.0437841100
Gitlin L, Karelsky S, Andino R: Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 2002, 418: 430-434. 10.1038/nature00873
Coburn GA, Cullen BR: Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. Journal of Virology 2002, 76: 9225-9231. 10.1128/JVI.76.18.9225-9231.2002
Jacque JM, Triques K, Stevenson M: Modulation of HIV-1 replication by RNA interference. Nature 2002, 418: 435-438. 10.1038/nature00896
Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA: siRNA-directed inhibition of HIV-1 infection. Nature Medicine 2002, 8: 681-686.
Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, Myszka DG, Sundquist WI: Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001, 107: 55-65. 10.1016/S0092-8674(01)00506-2
Martinez MA, Gutierrez A, Armand-Ugon M, Blanco J, Parera M, Gomez J, Clotet B, Este JA: Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication. AIDS 2002, 16: 2385-2390. 10.1097/00002030-200212060-00002
Johnson S, Oliver C, Prince GA, Hemming VG, Pfarr DS, Wang SC, Dormitzer M, O'Grady J, Koenig S, Tamura JK, Woods R, Bansal G, Couchenour D, Tsao E, Hall WC, Young JF: Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis 1997, 176: 1215-1224.
The authors wish to thank Kosi Gramatikoff for graphic assistance and helpful discussions. They are grateful to Libby Weber for the critical assistance on the completion of this manuscript.
Dr. Hervé Le Calvez declares that he has no competing interest. Dr. Mang Yu and Dr. Fang Fang are the co-founders and current share holders of Perlan Therapeutics who has developed CFY196.
About this article
- viral mRNA
- anti-sense oligonucleotide
- RNA interference
- viral infectious disease
- blocking antibody
- soluble receptor