Pharmacologic Advances in the Treatment and Prevention of Respiratory Syncytial Virus

Abstract

There are currently only two drugs approved for respiratory syncytial virus (RSV). Palivizumab is a monoclonal antibody for the prevention of RSV in high-risk children and ribavirin is approved for treatment of severe RSV disease, however its effectiveness in improving outcomes is questionable. Over the past 40 years, many obstacles have delayed the development of safe and effective vaccines and treatment regimens. This article reviews these obstacles and presents the novel development strategies used to overcome many of them. Also discussed are promising new antiviral treatment candidates and their associated mechanism of action, the significant advances made in vaccine development, and exciting, new studies directed at improving outcomes through pharmacologic manipulation of the host response to RSV disease.

Introduction

RSV is the leading cause of pediatric viral respiratory tract infections. The World Health Organization estimates an annual mortality rate of ∼160,000 worldwide[1]; more inclusive all-cause mortality rates related to RSV approach 600,000[2]. RSV is also the second leading cause of viral death in the elderly[3]. By 18 months of age, 87% of children have developed RSV-specific antibodies[4]; by 3 years of age, virtually all children have been infected. In the United States alone, RSV infection results in over 120,000 childhood hospitalizations and up to 500 deaths[5]. Compared to influenza, RSV accounts for over nine times more deaths in children under the age of 1 year[6-11].

Only two FDA approved drugs are currently available for RSV disease. Palivizumab is indicated for prevention in high risk infants, including those with chronic lung disease, congenital heart disease (CHD) and those born prematurely[12]. This indication is based on hospitalization rates that are ∼5 times greater in high versus non-high risk infants. However, among all infants hospitalized with severe RSV disease, about 70% are term infants with no underlying risk factors compared to 10-20% of high risk infants[13]. Thus, previously healthy infants make up the majority of RSV hospital admissions[14, 15] but are not considered candidates for palivizumab therapy. Ribavirin has been used for treatment of severe infections despite limited evidence of benefit[16, 17], potential for toxicity to health care workers[18], and high cost[19]. The American Academy of Pediatrics does not generally recommend ribavirin treatment for RSV infections[20]. There is an urgent need for safe and effective drugs to treat and prevent RSV disease.

This review begins with a description of the RSV structure and mechanism of cellular invasion. It then summarizes the pharmacology and innovative strategies used to develop RSV treatment and prevention drugs. Concluding remarks emphasize promising anti-RSV drug candidates and their current development status.

Viral Drug Targets

RSV is a negative sense, single-stranded, enveloped RNA paramyxovirus. The RSV genome encodes a total of 11 proteins, many which are under investigation as possible drug targets (Figure 1; Table 1)[21-23]. There are two major antigenic subgroups, A and B, which are defined by different envelope proteins and co-circulate each year. Infectivity of the virus is determined by the surface glycoproteins, F and G, which also serve as targets for neutralizing antibodies[24]. The F glcyoprotein exists only on the surface membrane and is highly conserved across both major antigenic subgroups of RSV. The G glycoprotein exists in both a membrane-bound and secreted form, whereby the secreted form serves as a decoy to neutralizing antibody. Much of the antigenic diversity between and within RSV subgroups is due to variations in the G glycoprotein. Antibody responses to the G glycoprotein are subgroup specific; as low as 35% homology between subgroups A and B has been reported[25]. Antibody responses to the F glycoprotein are generally cross-reactive between RSV A and B subgroups (∼85% homology)[26]. Viral proteins are produced by RSV-specific RNA-dependent RNA polymerase (RdRp)[27]. The RdRp is comprised of at least five viral components, including genomic RNA, and the L, N, P, and M2-1 proteins[28]. The genomic RNA is encapsidated by the nucleocapsid (N) protein[29], which, together with the P and L proteins, forms the necessary unit for RNA replication (Figure 1)[22, 30-33]. These components form a ribonucleoprotein (RNP) complex whose activity results in the production of progeny virus particles.

Figure 1. A cartoon showing the RSV structure and pharmacologic targets.

Five of the eleven RSV proteins have been evaluated in preclinical or clinical trials as potential drug targets for anti-RSV therapy. These include the G and F glycoproteins responsible for viral attachment and fusion to host cells and the genome associated N, P, and L proteins required for RSV replication; together they are termed, the RNP complex.

Table 1. RSV proteins and targeted drug candidates.

Protein/Function		Drug Candidates	Mechanism	Development Status
Envelope glycoproteins
F	Mediates fusion and entry of the virion into the host cell and promotes fusion of infected host cells to facilitate cell-to-cell transmission (syncytial formation) [24]	Motavizumab, MEDI-524 [107]	mAba	Phase 1-III, complete; BLAb submitted February, 2008; pending approval
		TMC-353121 [56, 108, 109]	Fusion inhibitor	Preclinical, ongoing
		BMS-433771 [110]	Fusion inhibitor	Phase I/II, discontinued
		RFI-641 [62]	Fusion inhibitor	Phase I/II, discontinued
		VP-14637 [111]	Fusion inhibitor	Phase I, discontinued
		BTA9881 [36]	Fusion inhibitor	Phase I, ongoing
G	Mediates viral attachment [24]	MBX-300 [60, 61]	Attachment inhibitor	Preclinical, ongoing
		mAb 131-2G [46, 112]	mAb	Preclinical, ongoing
SH	Structural component; believed to inhibit c TNFα signaling [ 113 ]
Nucleocapsid proteins [28]
N	Major nucleocapsid protein [29]	ALN-RSV01 [114]	d siRNA	Phase I, complete; Phase II, ongoing
		RSV-604 [65]	N-protein inhibitor	Phase I, complete; Phase II, ongoing
P	Phosphoprotein
L	Large polymerase subunit	YM-53403 [64]	Benzazepine derivative	Preclinical, ongoing
		AUG-2 [115]	Peptide-conjugated e PMO	Preclinical, ongoing
Nucleocapsid-associated proteins [116]
M2-1	Transcription elongation factor
M2-2	Regulator of transcription
Matrix Protein
M1	Mediates virus assembly [117, 118]
Nonstructural proteins
NS1	Antagonize interferon-induced antiviral responses [119]
NS2

^amAb = monoclonal antibody;

^bBLA = Biologic License Agreement

^cTNFα = tumor necrosis factor alpha;

^dsiRNA = small interfering ribonucleic acid;

^ePMO = phosphorodiamidate morpholino oligomers.

Drug Candidates Targeting RSV

Polyclonal antibodies

In 1996 MedImmune's RSV Immune Globulin Intravenous (RSV-IGIV), consisting of a high concentration of polyclonal, anti-RSV IgG antibodies purified from the plasma of healthy individuals[34], became the first FDA approved agent for RSV disease prevention. The subsequent approval of palivuzmab, a monoclonal antibody (mAb) directed at the RSV F glycoprotein (discussed below), led many to question the superiority of one product over the other; see reference[35] for an excellent comparative meta-analysis. Despite qualitatively similar reductions in RSV hospitalizations and ICU admissions[35], RSV-IGIV had major disadvantages. These included significant adverse events in children with CHD, the need for IV access, and the risk of infectious disease transmission associated with human plasma-derived products. The availability of palivizumab, a safe and effective alternative, in 1998 led to the removal of RSV-IGIV from the US market that same year[35]. Despite this, a new candidate hyperimmune IVIG product, RI-001, (ADMA Biologicals, Inc.) (personal communication, Dr. Mark Sorrentino; Oct 20, 2009) is now being evaluated in Phase II clinical trials in immunosuppressed, RSV infected patients at risk for lower respiratory tract (LRT) illness[36]. Although human plasma products harbor certain risks, stringent purification requirements have been implemented for all human plasma-derived products to significantly minimize the risk of infection transmission[37]. Moreover, polyclonal antibodies contain a mixed population of antibodies targeting multiple viral epitopes, thus overcoming the mutagenic potential intrinsic among viruses. Results of these studies are ongoing and have not yet been published.

Monoclonal antibodies

Monoclonal anti-RSV antibodies target a single viral epitope. Palivizumab, the only FDA approved mAb for RSV, targets the highly conserved RSV F glycoprotein, inhibiting viral entry into host cells[38]. It has demonstrated no clinical benefit for the treatment of RSV disease, thus is indicated only for RSV prevention. Motavizumab (MEDI-524, MedImmune, Gaithersburgh, MD) is a second-generation humanized IgG1 monoclonal antibody, developed from palivizumab[39], with approximately 70-fold higher affinity for the RSV F glycoprotein and 20-fold greater neutralizing capacity[40]. In a rat model, motavizumab had 50 to 100 times greater anti-RSV activity in the lower respiratory tract compared to palivizumab[41] and reduced RSV viral load in the upper airways where palivizumab has minimal effect[40]. In a large phase III non-inferiority study comparing motavizumab to palivizumab for RSV prevention in high-risk children, motavizumab demonstrated 26% fewer RSV hospitalizations (p<0.01) and a 50% reduction in the incidence of RSV-specific outpatient LRIs (p=0.005) [42]. Moreover, motavizumab significantly reduced viral load by day 1 post-treatment in children hospitalized with RSV, suggesting it may be beneficial for RSV treatment as well as prevention[43]. Motavizumab is currently pending FDA approval.

The majority of RSV mAb candidates target the more conserved F glycoprotein; however, recent evidence suggests that mAbs targeting the G glycoprotein may impart dual anti-RSV activity. The RSV G glycoprotein has been shown to induce lung inflammation by binding to the chemokine receptor CXC3R1, and initiating a cascade of inflammatory mediators[44, 45]. Although still in early preclinical studies, a mAb targeting the CXC3 motif on the RSV G glycoprotein (mAb 131-2G) was shown to reduce both lung inflammation and RSV titers in BALB/c mice[46].

Antisense anti-RSV Drugs

Antisense technology, introduced in the 1980s, involves the targeting of mRNA and viral RNA by oligonucleotides. First generation oligodeoxyribonucleotides (ODNs) were comprised of synthetic DNA molecules 15-20 nucleotides in length that were complementary to small segments of target mRNA. To improve stability against cellular nucleases, phosphodiester bonds were later substituted with phosphorothioate, however, this occurred at the expense of lower sequence specificity and higher toxicity[47]. More recently, a variety of other mechanisms of oligonucleotide-induced antiviral activity have emerged. Those showing the most promise include 2-5A antisense compounds, phosphorodiamidate morpholino oligomers (PMOs), and small interfering RNAs (siRNAs). Of these, only siRNAs have advanced to clinical trials and will be discussed herein. For a thorough review of antisense approaches targeting RSV, see reference[48].

RNA interference (RNAi) is a posttranscriptional mechanism of gene silencing that occurs in plants, animals, and humans[49, 50]. It is important for the regulation of gene expression and participates in host defense against viral infections. The discovery that synthetic double-stranded, small interfering RNAs (siRNAs) could be used to inhibit protein synthesis by targeting mRNA transcripts in mammalian cells led to the emergence of a new field of drug discovery[51] spanning a variety of human diseases including cancer, metabolic diseases, and viral infections[52]. For RSV, siRNAs targeting the P protein[53], NS1 protein[54], and N protein genes[29] have been evaluated. Of these, a siRNA targeting the N protein (ALN-RSV01; Alnylam Pharmaceuticals, Cambridge, MA) is currently being studied in humans. In a phase II clinical trial, ALN-RSV01 or placebo was administered intranasally to 85 healthy adult volunteers two days prior to and three days following viral inoculation. Subjects receiving ALN-RSV01 experienced a 38.1% reduction in RSV infection (p < 0.01) and a 95% increase in the number of subjects who remained free from infection compared to placebo-treated subjects. In a second, recently completed phase II study, safety and tolerability of ALN-RSV01 among adult lung transplant patients naturally infected with RSV was demonstrated. Although not powered to study efficacy, results showed improvement in lung function at the 90 day endpoint[55]. Larger clinical trials in infants are needed to evaluate safety and efficacy; however ALN-RSV01 offers a promising targeted approach to treating infant RSV disease.

Fusion inhibitors

Fusion is a critical step in the life cycle of RSV. Inhibition of this step leads to reduction in viral load and syncytia formation[56, 57]. Upon viral coalescence with target cell membranes, the F glycoprotein undergoes a conformational change exposing hydrophobic pockets or epitopes (Figure 2)[58]. Binding of these exposed targets by RSV fusion inhibitors prevents viral entry in the host cell[59]. Several small-molecule fusion inhibitors have been screened, each targeting a slightly different epitope within the F glycoprotein (Table 2). Despite this, only two remain under investigation (Table 1). BTA9881 is currently in phase I clinical trials and preclinical studies in rats suggest that TMC-353121 is a highly potent (up to 90% inhibition of virus replication) anti-RSV drug candidate. A comprehensive review of RSV fusion inhibitors was recently published by Bonfanti and Roymans[56].

Figure 2.

The RSV F protein is a trimeric class I fusion protein in which each F1 monomer contains two heptad repeats, HR1 and HR2. The fusion peptides are adjacent to the HR1 segments at the amino terminus end of the F1 subunit. These peptides are responsible for fusion to target cell membranes. The HR2 segments located at the F1 carboxy terminus anchor the F glycoprotein to the viral membrane. Of the fusion inhibitors with known binding sites, all bind to the HR1 portion of the fusion glycoprotein (Table 2). Following fusion initiation, conformational changes occur in which the trimeric coiled-coil structure of three HR1 repeats is created. Three HR2 repeats collapse upon the coiled-coil structure to form a “six-helix bundle” (6HB)-complex (also referred to as a “hairpin structure” or “coiled coil heptad repeat”).[58] Modified from Drugs of the Future 2000; 25(3): 287-294. Copyright 2000 Prous Science, S.A. All rights reserved.

Table 2. RSV fusion inhibitors.

Compound	Company	a F1 Interaction	Structure	Route of administration	Sub-group	Model	Reference
TMC-353121	Johnson & Johnson/Tibotec	Hydrophobic cavity on bHR1 surface of c6HB	Benzimidazole derivatives	Inhaled/oral	A/B	Cotton rat	[56, 108, 109]
VP-14637	ViroPharma Inc/RSVCO	Hydrophobic cavity on HR1 surface of 6HB; similar mechanism to TMC-353121	Triphenolic compound	Inhaled	A/B	Cotton rat/humans	[56, 110, 111, 120]
BMS-433771	Bristol-Myers Squibb	Hydrophobic cavity on surface of HR1 trimeric coiled-coil	Benzimidazole derivatives	Oral	A/B	Cotton rat/mice	[110, 121]
RFI-641	Wyeth Research	Interacts with F-protein in its native state	Disulfonated stilbene	Inhaled	A/B	Mice/African green monkeys/humans	[122]
BTA9881	Biota Holdings Limited/AstraZeneca	Inhibition of F protein assumed based on inhibition of syncytium formation	Imidazoiso-indolone derivative	Oral	A/B	Rodents/humans	[123, 124]

^aF1 - Fusion 1 protein; HR – Heptad repeat; 6HB – Six-helix bundle.

Other small-molecule compounds

Other small-molecule compounds have been engineered to inhibit RSV by binding RSV-specific epitopes, including G, L, and N proteins (Figure 1; Table 1). MBX-300 (Microbiotix, Worcester, MA, US) targets the RSV G glycoprotein, resulting in inhibition of viral attachment to host cells [60]. As previously discussed, binding of the G glycoprotein also alters RSV-induced inflammatory responses[44]. In preclinical studies, MBX-300 was found to be safe in both rats and monkeys and demonstrated specific and potent anti-RSV activity[60-62].

The majority of anti-RSV compounds being actively pursued disrupt viral entry into the cell either through the F or G glycoproteins. YM-53403 (Yamanouchi Pharmaceutical Co., Deerfield, Illinois) is a novel compound targeting the RSV L protein, which together with the P and N proteins make up viral RNA polymerase (Figure 1)[63]. Sudo and colleagues demonstrated potent anti-RSV activity against both subgroups A and B, presumably by interfering with RNA synthesis[64]. YM-53403 EC₅₀ values against all RSV strains studied were 76-105-fold more potent than ribavirin. Preclinical studies are ongoing for YM-53403.

RSV604 is an oral benzodiazepine targeting the RSV N protein (Arrow Therapeutics, London, England and Novartis, East Hanover, NJ). Like YM-53403, its putative mechanism of anti-RSV activity is inhibition of viral RNA polymerase. It displays submicromolar activity against many clinical isolates of A and B RSV antigenic subgroups. In contrast to most fusion inhibitors, RSV604 was shown to be active when administered post-infection. Moreover, it significantly reduced viral spread in vitro when given up to 24h post-infection[65]. RSV604 is in ongoing phase II clinical trials.

Vaccines

Numerous obstacles have prevented the development of an effective RSV vaccine. The population most vulnerable to severe RSV disease, children less than 6 months of age[66, 67], may respond inadequately to vaccination due to immunologic immaturity or immune suppression caused by maternal antibodies[5, 68, 69]. In the 1960's, a formalin inactivated (FI)RSV vaccine was studied in human infants for the first time. Not only did it fail to protect against subsequent wild-type (wt) RSV disease, it induced an exaggerated clinical response causing as many as 80% of vaccinated children to be hospitalized and two infant deaths[70-73]. Infiltration of excess eosinophils into the peribronchial spaces[70, 73] and deposits of nonprotective antibodies complexed with virus in affected tissue were blamed for the vaccine-enhanced disease[73-75].

Live vaccines

RSV vaccine development is currently focused on live attenuated strains for intranasal administration. This strategy accomplishes several goals: it induces local mucosal and systemic immunity; the intranasal route partially escapes the suppressive effects of maternal serum antibodies[76]; compared to inactivated vaccines, live intranasal vaccines are more immunogenic and provide broader protection[77]; and live, attenuated vaccines are not associated with enhanced disease[78]. It is unlikely that a single vaccination will impart complete protection against RSV disease as evidenced by natural reinfection occurring throughout life[5, 79, 80]. Thus, the goal for a successful vaccine should be to prevent serious RSV-associated lower respiratory tract infections in those at risk.

The balance between attenuation and immunogenicity is critical in vaccine development. Live vaccine candidates have been developed using serial passages at decreasing temperatures (cold passage[cp]) and chemical mutagenesis to produce temperature-sensitive (ts) mutants. These cpts viruses will replicate at the low temperatures of the upper respiratory tract, but not at the high temperatures of the lower respiratory tract[78]. Initial vaccine candidates developed using these attenuation methods were found to be either too reactive or over-attenuated and mutations were often unstable[81, 82]. The latest strategy to safely and effectively attenuate RSV is through reverse genetics[78], which involves producing infectious virus in cell culture completely from cloned cDNAs[83, 84]. This method introduces targeted mutations to achieve more precise levels of attenuation while maintaining sufficient immunogenicity. rRSVA2cpts248/404/1030/ΔSH (MEDI-559; MedImmune/National Institute of Allergy and Infectious Diseases, Bethesda, MD) is a recombinant, tsRSV with a deletion of the SH gene[85, 86]. The SH protein has been shown to decrease Th1 responses, thereby inhibiting the host anti-viral response. A virus lacking the SH protein would thus impart greater immunogenicity[87]. It is the first vaccine candidate to be sufficiently attenuated for young infants (1-2 months of age). A phase 1/2a study is currently recruiting healthy children between the age of 1 and 24 months to evaluate immunogenicity, viral shedding, safety, and tolerability[36]. Other vaccine candidates under development using these attenuation strategies include rRSVA2cpts248/404/ΔNS2 and rRSVA2cpts530/1009ΔNS2, which include a deletion in the NS genes. The NS protein decreases type I IFN signaling, thus inhibiting host response[88]. Similar to SH deletions, virus lacking the NS proteins will be more immunogenic. Despite often having up to 5 mutations to protect against reversion to wtRSV, there is still concern regarding genetic stability with these vaccine candidates. To address this concern, highly attenuating gene deletion vaccines were developed, including ΔNS1, ΔM2-2, and ΔM2-2NS2[89, 90]. These vaccine candidates maintained a high level of immunogenicity when evaluated in chimpanzees and induced protection following wtRSV challenge; further evaluation in humans is needed[89-91].

Vector vaccines

An alternative method for overcoming genetic instability, while maintain immunogenicity is through the delivery of RSV proteins using viruses with substantially greater growth and stability[78]. The vector vaccine candidate rb/h PIV3/RSV F2 (MEDI-534) delivers RSV F using a bovine/human chimeric parainfluenza type 3 genome. rb/h PIV3/RSVF2 protected monkeys against challenge with wtRSV and generated high titers of RSV and hPIV3 neutralizing antibodies[92]. Safety was demonstrated in a Phase I study of RSV seropositive adults; further studies are needed to determine safety and immunogenicity in children[93]. Other viruses engineered to express RSV F and/or G glycoproteins include Newcastle disease and Sendai viruses, both of which demonstrated immune protection in rodent models[94, 95].

Subunit vaccines

Purified RSV F, G, and M proteins have been evaluated for their potential to induce neutralizing and protective antibodies. The following subunit vaccines have advanced to clinical trials: a) three RSV F subunit vaccines (purified F protein 1 [PFP-1], PFP-2, and PFP-3)[96, 97] b) a combined subunit vaccine containing F, G, and M proteins(Sanofi Pasteur, Swiftwater, PA)[98] and c) BBG2Na, a G peptide conjugated to streptococcal protein G[99]. Only modest rises in antibody titers have been observed in seropositive populations. Safety and efficacy in RSV naïve infants and young children have not been determined. Drawbacks to this vaccine approach include poor immunogenicity, immunosuppressive effects of maternally acquired antibodies, and potential for vaccine-enhanced disease.

Drugs targeting RSV disease pathophysiology

Despite over 50 years of RSV research, the immunopathology and incomplete immunity associated with infant RSV disease remain problematic in the development of effective vaccines and treatments. Novel approaches for altering the host response to RSV, rather than directly targeting the virus, are in the early stages of investigation. Some of these include MBX-300, fosfomycin, and the active metabolite of leflunomide (A77-1726). MBX-300, as previously discussed, targets the RSV G glycoprotein directly, but also competes with the potent chemokine, fractalkine, for binding to CX3CR1 in host cells resulting in reduction of the RSV-induced inflammatory response[44].

Fosfomycin is a structurally unique antibiotic shown to possess in vitro and in vivo immunomodulatory activity[100-102]. Initial studies performed in airway epithelial cells demonstrated that fosfomycin suppressed the RSV-induced transcription of RANTES[103], a chemokine shown to play an important role in RSV lung inflammation[104].

Davis and colleagues demonstrated that RSV is associated with reduced alveolar fluid clearance (AFC), a process that is crucial for efficient gas exchange in the lungs[105]. They went on to show that intranasal administration of A77-1726 to RSV-infected BALB/c mice prevents the RSV-induced decrease of AFC and the onset of arterial hypoxemia[106].

Concluding remarks

Palivizumab remains, perhaps the greatest advancement in RSV pharmacotherapy. Motavizumab, its more potent successor, demonstrates activity in both the upper and lower airways. Despite its pending FDA approval for RSV prevention in high-risk children, evidence suggests it may also play a role in RSV treatment[43]. Of the many RSV treatment candidates evaluated, three have advanced to clinical trials and remain ongoing (Table 1). The siRNA, ALN-RSV01, has received a great deal of attention for its innovative mechanism of action and promising clinical data, however, safety and efficacy in infants remains to be determined.

Vaccine development has made considerable progress over the past 50 years. rRSVA2cpts248/404/1030/ΔSH (MEDI-559) remains the first and only vaccine candidate to be tested in the target infant population since the 1960s FI-RSV trials. Its immunogenicity among infants is currently being evaluated in ongoing clinical trials. The vectored vaccine candidate, rb/hPIV3/RSVF2, takes advantage of the substantially greater growth and stability of the HPIVs compared to RSV. Clinical studies will be needed to determine safety and immunogenicity in infants.

Lastly, it is likely that immunomodulating agents will have a significant impact on RSV disease. Although still in early, preclinical stages, immunomodulatory agents will likely play an important role in combination with direct anti-viral agents.