Respiratory Syncytial Virus Bronchiolitis

Abstract

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness in infants and children worldwide and is responsible for over 120 000 annual hospitalizations in infants in the US alone. RSV is also recognized as a major respiratory viral pathogen in the elderly and other high-risk populations. Bronchiolitis, pneumonia, apnea, respiratory failure, and death are well known manifestations of severe acute RSV disease. RSV infection has also been associated with recurrent wheezing in children, but the mechanisms involved in this association are not completely understood. The host immune response plays a significant role in controlling the infection but is likely also involved in augmenting the disease through pathways that have not been completely identified. The treatment options for RSV infection are very limited. Ribavirin, corticosteroids, and bronchodilators are not used routinely because they have not proven to be sufficiently effective. Education of caregivers, strict handwashing, and avoidance of exposure to environmental factors associated with severe forms of RSV infection are among the most effective preventive means. Passive immunization with monoclonal antibodies provides protection against severe RSV disease in high-risk children. Clinical trials to evaluate the safety and efficacy of a second-generation monoclonal antibody are underway. Efforts to develop a safe and effective RSV vaccine have continued despite the poor outcomes observed following the administration of formalin-inactivated formulations in the 1960s. In the last decade, live attenuated vaccines (including those developed by recombinant techniques) and purified subunit vaccines have all been evaluated in humans. Results of clinical trials have been encouraging, but the availability of a safe and effective RSV vaccine is not a reality yet. Better prevention strategies will have an impact, not only on acute morbidity caused by RSV, but will also likely have an effect on ameliorating the chronic consequences of this disease.

Respiratory syncytial virus (RSV) is an enveloped, negative-sense, single-stranded RNA virus that belongs to the family Paramyxoviridae.[1] Its genome contains 15 222 nucleotides that encode ten proteins (variations of the M2 protein are referred to as M2-1 and M2-2). The two glycosylated surface proteins, the F and G proteins, play a significant role in the infectivity of the virus. The fusion (F) protein initiates viral penetration by fusing viral and cellular membranes. This protein also promotes direct cell-to-cell spread of RSV, thereby inducing the production of the characteristic syncytia. The attachment (G) protein of the virus mediates adherence of the virus to the host cells via interaction with heparan sulfate molecules (but not via a known, specific receptor). However, recombinant RSV particles with mutations that alter the putative heparin-binding domain, or that do not express protein G at all, are able to produce infection and limited replication both in vitro and in animal models.[2] Antigenic variations in the G protein have been used traditionally to differentiate the two RSV subtypes, A and B. Genetic analysis, especially of the G protein gene, allows classification of RSV isolates into different genotypes that can be used for molecular epidemiologic studies.[3,4] The F and G proteins carry the antigenic determinants that elicit the production of neutralizing antibodies by the host, and function as T-cell epitopes. The M protein is also a major T-cell epitope, at least in mice.[5,6]

This review summarizes the role of the humoral and cellular immunity in the pathogenesis of acute RSV disease in children, identifies strategies for treatment of RSV disease among high-risk infants and children, and reviews the preventive strategies available (passive and active immunization) to decrease the consequences of RSV infection.

1. Epidemiology

RSV is the most frequent etiologic agent of acute lower respiratory tract infection (LRTI) in infants (children younger than 1 year of age), causing more than 120 000 hospitalizations per year in this age group in the US alone.[7] Complications during RSV hospitalization may include: respiratory failure, apnea, pulmonary infiltration, and atelectasis; otitis media and bacterial pneumonia; and cardiac failure. These complications have been associated with increased hospital intensive care unit length of stay,[8] especially in patients with gestational age ≤35 weeks.[9] Deaths may exceed 1 million annually worldwide. Mortality attributed to RSV infection is <0.5% in developed countries, but may be as high as 5% in patients with risk factors predisposing to severe RSV infection, and in developing countries.[10] RSV infects essentially all children by age 3 years and is also an increasingly recognized pathogen in the adult population, especially the elderly.[11–13]

In temperate climates, RSV occurs in outbreaks that last 4–6 months starting from late fall through early spring. In other regions, RSV epidemics occur with a different pattern depending on the climate, intensity of exposure, and degree of herd immunity.[10] In the Northern US and in Canada the RSV season usually starts in December-January. In many equatorial regions RSV appears to be present year round. In Europe, Africa, Asia, and other regions worldwide, the timing of the RSV epidemics also varies according to factors mentioned above.[10]

Transmission occurs through contact with respiratory secretions of infected patients, and by large droplet aerosol. The virus can remain infective on human hands for as long as 1 hour, and it may survive on non-porous surfaces for up to 30 hours at room temperature.[14]

Clinical manifestations of RSV disease range from mild upper respiratory tract infection to bronchiolitis, pneumonia, respiratory failure, and death. RSV LRTI has also been associated (directly or indirectly) with an increased risk of long-term recurrent wheezing in children.[15,16] Despite the induction of neutralizing antibodies after infection, reinfections throughout life are commonly observed.[17]

The peak incidence of severe RSV disease occurs between 2 and 3 months of age. Risk factors for severe RSV disease in children include prematurity, chronic lung disease, congenital heart disease (CHD), male gender, birth during the first half of the RSV season, younger age at RSV acquisition, lack of breastfeeding, and malnutrition.[18] Also, environmental factors associated with increased RSV disease severity include crowding, passive exposure to tobacco smoke, daycare attendance, and interaction with older school-age siblings.[19–21]

2. Immunopathogenesis

The nature of the host immune response to RSV is not well understood. Both innate and adaptive responses are induced following infection, and overlap over time. Toll-like receptors and surfactant proteins are involved in early detection of the presence of RSV. The release of numerous cytokines and chemokines by epithelial cells stimulates the recruitment of innate immune cells, such as granulocytes, monocytes, and natural killer cells in an attempt to control and eradicate the infection. T cells are then recruited and become activated, proliferate, and differentiate to determine the nature of the long-lived humoral and cellular adaptive immune response. An exaggerated and unbalanced immune response induced by RSV could possibly explain the increased disease severity observed in some patients, but this remains speculative. Individuals with defects in cell-mediated immune responses may develop prolonged viral shedding and delayed clinical recovery.[22,23] Studies in mice have shown that RSV RNA can be detected by polymerase chain reaction (PCR) in the lungs for months after experimental infection,[24,25] and immunosuppressive interventions allowed the recovery of infective virus in this model of RSV infection.[25]

2.1 Humoral Immunity

IgG RSV-specific antibodies acquired transplacentally confer partial immunity to term infants but their levels decrease during the first few months of life. Maternal IgG antibody concentrations against RSV were significantly higher in mothers whose infants remained uninfected by RSV until after 6 months of age than in those whose children became infected before this time.[26] The levels of RSV antibodies in cord blood of infants correlated directly with their age at the time of initial RSV infection.[27] Antibodies against RSV found in infants older than 6 months of age are presumed to be the result of natural infection.[28] In infants less than 6 months of age, RSV induces a humoral response consisting primarily of mucosal IgA rather than serum IgG.[29] Studies have shown that resistance to RSV infection correlates better with serum IgG levels than with secretory antibody titers. In adults challenged with RSV after natural infection, higher neutralizing antibody levels before challenge correlated significantly with protection against infection. Mucosal IgA levels did not correlate significantly with protection and even the increased, high titer serum antibodies were not completely protective.[30] This suggests that induction of serum-neutralizing antibody may be a reasonable goal of passive and active immunization programs, although complete resistance to infection may not be achievable.

2.2 Cell-Mediated Immunity

Patients with primary cell-mediated immunodeficiency diseases or secondary immunodeficiencies related to chemotherapeutic regimens develop a severe form of RSV infection with prolonged viral shedding and progressive pneumonia, usually without wheezing as a predominant manifestation.[22,23] These findings underscore the possible importance of T cell-mediated immunity in controlling RSV infection. An overactive CD4+ T-cell response to the virus can be detrimental to the host by mediating immunopathology, at least in mice. Recombinant vaccinia viruses expressing the G, F, or 22K RSV proteins were used in mice to produce specific T-cell lines that varied in protein specificity according the type of antigen used for priming.[31] Priming with the 22K protein resulted in a predominance of class I-restricted cytolytic CD8+ cells. Protein F-specific T cells consisted of a mixture of cytolytic CD8+ cells and CD4+ cells with a T helper-1 cell (T_h1) cytokine secretion profile, whereas protein G-specific cells were predominantly CD4+ cells secreting T_h2 cytokines. When the G-specific CD4+ cell line was introduced into naïve mice followed by challenge with RSV, marked pulmonary eosinophilia and increased disease severity were observed. Following intranasal RSV challenge in mice primed with the other two proteins, eosinophilia was less, and illness was milder.[32] Later studies showed that elimination of only the subset of protein G-specific CD4+ cells, expressing the Vβ14 T-cell receptor (TCR), abolished the T_h2 type pulmonary injury.[33] These studies suggest a role for CD4+ cells in certain types of pathologic responses. The relevance of these findings to humans, who are never exposed to the G protein in isolation, is not clear.

Other studies have highlighted the role of CD8+ cells in the development and severity of RSV disease in mice. Infusion of CD8+ cells with a T_h1 profile into naïve mice followed by RSV challenge resulted in a different type of enhanced pathology resembling shock lung.[34] Again, the relevance to human disease, which does not always mimic shock lung, is uncertain.

Several RSV proteins are targets for cytotoxic T-lymphocyte (CTL) responses in humans.[35] Studies in BALB/c and DBA/2 mice have shown that an epitope in the M2 protein appears to be one of the major targets of the CD8+ T cell response to RSV infection.[36] This response is not long-lived, and may simulate the propensity for recurrent infection seen in humans.[37] Other studies have demonstrated that RSV interferes with the effector activity and TCR signaling of M2-specific CD8+ cells in the lungs.[38]

Immunologic factors contributing to the severity of RSV disease in humans have been studied by measuring relevant cytokines in nasopharyngeal secretions. Disease severity could be related to an imbalance in T_h1 and T_h2 responses, but there is no evidence that T_h2 cytokines are increased in human disease. This may or may not relate to inabilities to measure T_h2 cytokines at the site of disease. In children with mild (nonhypoxic) RSV bronchiolitis, levels of interferon-γ were higher than in children with RSV upper respiratory tract infection or in those with more severe (hypoxic) forms of bronchiolitis, suggesting a protective effect of this cytokine. Levels of interleukin (IL)-4 were low in patients with hypoxic bronchiolitis. Quantities of IL-5 and IL-13 were rarely detected, regardless of the form and severity of illness.[39] The findings of this study are against a role for T_h2 cytokines in severe forms of RSV bronchiolitis. In another study, IL-9 mRNA expression in the lower respiratory tract was significantly greater in 35 infants with RSV bronchiolitis compared with noninfected controls. IL-9 protein levels were significantly higher in the term infants compared with both preterm and control infants. The main cellular source of IL-9 was found to be neutrophils and not eosinophils.[40] Three independent studies performed by the authors using different assays for IL-9 have not replicated these results. It is likely that a T_h1/T_h2 cytokine imbalance is not the only factor, and perhaps is not an important factor, in determining disease severity in humans with RSV bronchiolitis. Chemokines and other factors produced locally after RSV infection are also likely to be involved.[41]

Other mediators have been associated with RSV disease severity in children. Expression of tumor necrosis factor-α (TNFα) and IL-6 mRNA in the lower respiratory tract of infants with RSV bronchiolitis was significantly greater than in control patients without infection.[42] These two proteins have been found in the nasopharyngeal secretions of children infected with RSV,[43] and the IL-6/TNFα ratio has been found to correlate inversely with disease severity in children with RSV LRTI.[44]

3. Treatment of Respiratory Syncytial Virus (RSV) Disease

Close monitoring of the clinical evolution and supportive care of patients are the mainstays of treatment of RSV disease. Bronchodilators such as nebulized albuterol (salbutamol) at a dose of 0.15 mg/kg per treatment, and epinephrine at different dosage regimens, have demonstrated modest short-term improvement in some studies, but they have not proven to have a definitive benefit in infants and young children with acute RSV bronchiolitis.[45–48] A double-blind, placebo-controlled study in children with RSV and respiratory failure showed that parenteral administration of dexamethasone had no effect on inflammatory markers or clinical outcomes such as the number of days of mechanical ventilation, intensive care, or hospital stay.[49] Viral clearance was delayed in patients who received dexamethasone compared with the placebo-treated group. The American Academy of Pediatrics does not recommend the use of corticosteroids for the management of RSV infection in children.[50,51]

Ribavirin, a guanosine analog, is the only antiviral approved for therapy of RSV. Double-blind, placebo-controlled studies involving small patient numbers have shown a modest beneficial effect on oxygenation in infants treated with aerosolized ribavirin.[52,53] The authors of a systematic review of 12 randomized, controlled trials comparing ribavirin, at a dose of 6g administered over 18–21 hours per day in most of these studies, with placebo in infants with RSV LRTI concluded that these trials lacked sufficient power to provide reliable estimates of the drugs’ effects on mortality, respiratory deterioration, and long-term pulmonary function.[54] At present, the American Academy of Pediatrics states that “ribavirin aerosol treatment for RSV infection is generally not recommended.”[50] Larger, prospective, randomized trials would help to clarify the controversy about the effects of ribavirin therapy on clinically important outcome measures. However, given the high cost and limited efficacy of the drug, such studies are unlikely to be initiated.

Cysteinyl leukotrienes are released during RSV bronchiolitis in infants. These proinflammatory molecules are known to cause mucosal edema, bronchial obstruction, and increased bronchial hyper-responsiveness. Different studies with leukotriene antagonists have shown contrasting results.[55,56] The only study conducted in humans was carried out in infants 3–36 months old (median age 9 months) hospitalized with acute RSV bronchiolitis. Sixty-one patients versus 55 control individuals were randomized into a double-blind, parallel comparison of montelukast 5mg, a cysteinyl leukotriene receptor antagonist, or matching placebo given for 28 days starting within 7 days of the initiation of symptoms. The effects of montelukast were evident after 2 weeks of therapy. A greater number of infants treated with montelukast were free of symptoms after the acute episode of bronchiolitis compared with infants receiving placebo (6/28 vs 1/28, p = 0.015). Daytime cough was significantly reduced during treatment, and exacerbations were significantly delayed with montelukast (23 days) compared with placebo (8 days). During follow-up, starting 4 weeks after finishing the intervention, there were no significant differences between treatment groups in any of the outcomes, and the trends seemed randomly directed.[55] The role of leukotriene antagonists in RSV disease needs to be further assessed. Larger prospective, randomized trials in younger infants are ongoing.

4. Prevention

The lack of effective therapy against RSV infection makes prophylactic interventions the best means to avoid the acute and chronic complications of the disease.

4.1 Avoidance of Exposure

Education of parents and other caregivers about methods to decrease RSV transmission is the mainstay of any RSV prophylaxis program. Hand hygiene is crucial, since transmission by hands contaminated with respiratory secretions is believed to be the major mode of spread. Exposure of high-risk infants and children to individuals who have respiratory infections should be avoided. Childcare centers and other places where many people gather represent an increased opportunity for such exposures. Exposure to tobacco smoke should be eliminated because it may be associated with more severe RSV disease.

RSV nosocomial transmission is common; the virus is highly infectious and healthcare workers can be a major source of infection.[57] Strict adherence to infection control measures is essential. The combination of cohort nursing and the wearing of gowns and gloves reduced the risk of nosocomial RSV infection in one study, although the effect of these more extreme measures could not be differentiated from that of reducing spread by hand contact.[50,58]

4.2 Passive Immunization

Studies in animal models showed that parenteral administration of screened, high-titer anti-RSV polyclonal antibodies had a 10-fold greater protective activity against RSV challenge relative to conventional immunoglobulin preparations.[59] Intravenous administration of RSV hyperimmune immunoglobulin (RSV-IG) significantly reduced the incidence and duration of hospitalization due to RSV in high-risk children.[60,61] Although the US FDA approved RSV-IG in 1996 for the prevention of RSV infection in high-risk infants, the product is no longer marketed in the US. RSV-IG recipients who had CHD experienced an increased number of deaths, probably related to increased plasma viscosity following RSV-IG infusions.[62] Monoclonal anti-RSV antibodies have replaced polyclonal preparations as the major prophylactic strategy against RSV disease in children.

4.3 Monoclonal Antibodies

A humanized monoclonal antibody, palivizumab, was approved by the US FDA in 1998 for prevention of severe RSV disease in preterm infants and children with chronic lung disease. Palivizumab is a neutralizing IgG1 antibody directed against one of the two most antigenically stable sites of the F surface glycoprotein of the virus, site A.[63] Due to the more conserved nature of these epitopes within the F protein, palivizumab shows a high degree of cross-reactivity among RSV subtypes A and B and is significantly more potent than polyclonal preparations.[64]

The safety and efficacy of palivizumab in high-risk infants and children was demonstrated in two large randomized, placebo-controlled trials. In the IMpact-RSV trial, 1502 infants were enrolled in a randomized, double-blind, multicenter clinical trial of palivizumab versus placebo.[65] Children eligible for the study included those younger than 24 months with chronic lung disease (CLD) requiring continuing medical therapy within the previous 6 months, and children born at ≤35 weeks’ gestation who were younger than 6 months at the start of the RSV season. Participants received five monthly intramuscular injections of palivizumab, each of 15 mg/kg, at the beginning of the RSV season. Controls received saline injections. The primary endpoint was the incidence of hospitalization for RSV infections. Overall, palivizumab prophylaxis resulted in a 55% reduction in RSV-related hospitalization compared with placebo (p < 0.00004). Treatment with palivizumab resulted in significant reduction in RSV-related hospitalization, compared with placebo, in all the subgroups analyzed:[65] premature infants with CLD (7.9% vs 12.8%, p < 0.038); premature infants without CLD (1.8% vs 8.1%, p < 0.001); infants born at 32–35 weeks gestational age (2% vs 9.8%, p < 0.001); and infants born at <32 weeks gestational age (4.8% vs 11%, p < 0.0026). Secondary efficacy endpoints that also demonstrated clinical benefit of palivizumab prophylaxis included a reduction of number of days of hospitalization for RSV infection, decreased requirement for supplemental oxygen, decreased number of days of moderate or severe lower respiratory tract illness, and decreased requirement for hospitalization in an intensive care unit. Palivizumab did not prevent RSV infection compared with placebo recipients.[66] Palivizumab injections were well tolerated, with only extremely rare occurrences of anaphylactoid reactions, and no actual anaphylaxis. There were no significant differences in the proportion of children reporting adverse events in the placebo and palivizumab groups.

Another randomized, double-blind, placebo-controlled trial with palivizumab to evaluate the safety, tolerance and efficacy of the antibody in children with hemodynamically significant CHD was conducted during four consecutive RSV seasons, from 1998 to 2002.[67] Eligible children were ≤24 months old and were stratified at entry to cyanotic or acyanotic subgroups. Palivizumab was administered intramuscularly at a dose of 15 mg/kg every 30 days for a total of five doses versus saline placebo. The most common cyanotic lesions among the 1287 children enrolled in this trial included hypoplastic left or right heart (21.9%), and tetralogy of Fallot (11.4%); the most common acyanotic lesions were ventricular septal defect (18%) and atrioventricular septal defect (7.2%). RSV-related hospitalization was decreased by 45% in palivizumab recipients compared with placebo (p = 0.003). In the cyanotic subgroup, RSV-related hospitalization was reduced by 29% (p = 0.285) whereas in the acyanotic group it was reduced by 58% (p = 0.003). There was also a 56% decrease in total days of RSV-associated hospitalization per 100 children (p = 0.003) and a 73% decrease in total hospital RSV-associated hospital days with supplemental oxygen per 100 children (p = 0.014). The death rate was 4.2% in the placebo recipients and 3.3% in the palivizumab recipients. No deaths were attributed to the study drug. No serious adverse events were related to palivizumab administration. Palivizumab serum concentrations decreased by 58% after cardiopulmonary bypass; it is therefore recommended that a postoperative dose be administered to those children who continue to require prophylaxis after such surgery. The results of this study led to approval of palivizumab by the US FDA for prophylaxis in this high-risk group of children.

Studies in other countries have confirmed the efficacy of palivizumab in preventing RSV hospitalization. A meta-analysis of several studies performed in the US, Canada, and Europe classified patients into three groups according to their risk factors for severe RSV disease. Hospitalization rates were considerably lower in each subpopulation of patients who had been treated with palivizumab than in those who had not.[68]

The Palivizumab Outcomes Registry was initiated during the 2000–1 RSV season to prospectively characterize the population of infants who received palivizumab for RSV prophylaxis at 63 different sites across the US.[69] RSV hospitalization outcomes were also described. The total RSV hospitalization rate in the 2049 children enrolled in this study was 2.9%, which is lower than the 4.8% reported in the Impact-RSV trial. Seventy-five percent of the hospitalizations observed in the Palivizumab Outcomes Registry occurred between the first and second palivizumab injections. A multicenter, open-label, dose-escalating trial in 65 children showed that the percentage of children with trough palivizumab serum concentrations >40 μg/mL increased with each subsequent 15 mg/kg intramuscular monthly dose.[70] Another study in infants born at or before 30 weeks of gestation showed that serum palivizumab trough concentrations were <40 μg/mL in 17 of 22 (77%) of infants tested before the second dose.[71] Although there is no evidence that RSV infection occurs more frequently near the end of the month between palivizumab injections, it is important to avoid delaying administration of the second injection dose, in order to achieve adequate serum concentrations.

The American Academy of Pediatrics (AAP) has issued a policy statement for the use of palivizumab for prophylaxis of RSV infections in high-risk children (table I).[72] Palivizumab is recommended at a dose of 15 mg/kg administered intramuscularly every 30 days during the RSV season. Once the prophylaxis regimen has been started it should not be interrupted even if the child reaches the age of 12 months while on therapy, or if the child develops an RSV infection while receiving palivizumab. The cost of this prophylactic strategy is high and several economic analyses have shown that it exceeds the anticipated savings from reduced RSV hospitalizations. Identification of infants at highest risk for severe RSV disease will likely help to improve the cost effectiveness of palivizumab prophylaxis.[73,74] Palivizumab is now available in a preservative-free liquid formulation with either 50mg or 100mg of palivizumab (Synagis®,¹ MedImmune Inc.) in 0.5mL or 1mL vials, respectively.[75] The liquid form is stable at refrigerator temperatures, and for reasonable periods at room temperature. The lyophilized form continues to be available. Palivizumab can be given with routine childhood immunizations, since it does not interfere with the immunologic response to vaccines. RSV mutants resistant to palivizumab have been derived in the laboratory,[76] but when tested in vitro and in cotton rats these mutations were not capable of causing infection in the face of RSV prophylaxis.[77] No palivizumab-resistant mutants were identified among 371 RSV isolates obtained from hospitalized children, including those from 25 children who were receiving palivizumab prophylaxis.[78] Acute hypersensitivity reactions have been described on initial and subsequent exposure to palivizumab since its approval in the US, and very rare cases of anaphylaxis (<1 in 100 000) have also been reported after re-exposure.[75]

Table I

High-risk populations for whom palivizumab prophylaxis is recommended[72]

Monoclonal antibody variants derived from palivizumab have been generated by exchanging constituent amino acids individually and in groups. This has resulted in second-generation monoclonal antibodies with increased binding avidity and viral neutralization activity.[79] One of these antibodies, motavizumab (Medi-524), has shown to decrease upper and lower airway RSV loads in cotton rats by 50- to 110-fold more effectively compared with palivizumab. An investigational new drug application was submitted to the US FDA, and two phase 3 clinical studies are ongoing to evaluate safety and efficacy of motavizumab versus palivizumab in reducing severe RSV disease in high-risk children. Results of a phase 1 clinical trial presented recently in abstract form showed dose-dependent increase in antibody concentrations in the nasal secretions of children with RSV bronchiolitis treated with a single intravenous dose of motavizumab 3, 5, or 30 mg/kg. Nasal RSV loads, measured by viral culture and by reverse transcription-PCR, were significantly lower in antibody-treated children compared with placebo.[80] Thus, motavizumab may be capable of reducing nasal replication of RSV and, therefore, may prevent even mild upper respiratory tract infection during natural infection.

The effect of RSV prophylaxis on the long-term consequences of RSV infection in humans is unknown. Studies in animals showed that airway hyper-responsiveness was decreased in those animals that received monoclonal antibodies before innoculation with RSV compared with placebo recipients.[81,82] In humans, preliminary results of a case-cohort, multicenter study showed that preterm infants treated with prophylacatic palivizumab during their first RSV season had a significantly lower risk of subsequent recurrent wheezing during a 12-month follow-up compared with age-matched control individuals.[83]

5. Active Immunization

5.1 Vaccines

The history of RSV vaccine development is illuminating in many ways. Studies using a formalin-inactivated RSV vaccine (FI-RSV) were conducted in the 1960s. Infants aged 2–7 months were given two intramuscular injections of vaccine 1 month apart and a third dose 3 months later. The majority of infants developed an antibody response after the vaccination regimen, but 80% of vaccinees required hospitalization after subsequent infection with RSV. More than half of those who completed the three-dose vaccine regimen developed bronchiolitis with or without pneumonia. Similar illnesses were also seen in one patient who received one dose and in eight patients who received two doses of FI-RSV. Two of the infants who received three doses of the vaccine died.[84]

The adverse effects of FI-RSV have profoundly retarded the development of an RSV vaccine, and have prompted the study of the pathophysiology of vaccine-enhanced RSV disease. Recipients of FI-RSV developed anti-RSV antibodies that were non-neutralizing.[84,85] Some authors have suggested, as a possible explanation for the lack of protective response, that formalin inactivation of RSV altered epitopes of the F or G glycoproteins that stimulated neutralizing antibodies.[86] However, mechanisms explaining enhanced disease following formalin-inactivated vaccination are still not clear. Lung tissue samples obtained at autopsy from two fatal cases of FI-RSV recipients showed lymphocytic and eosinophilic infiltration of the lung – characteristics that are not seen in the lungs of unvaccinated infants dying of RSV bronchiolitis.[84] Interestingly, formalin-inactivated parainfluenza vaccines prepared similarly did not enhance disease severity in infants subsequently undergoing parainfluenza virus infection.[84]

Another important factor that has interfered with the development of an ideal RSV vaccine is the variable immune response according to age. RSV disease is most severe in very young infants, in whom weak immune responses are typical. Antibody responses in these children may be suppressed by circulating maternally derived antibody. It is possible that different vaccines will be needed for various target populations: non-replicating vaccines may be useful in populations of older children and adults, and live virus vaccines would likely be required for RSV-naive infants.[87] Intranasal formulations offer the potential advantage of inducing appropriate local and systemic immune responses and a painless route of administration.[88]

The main potential goal of an RSV vaccination program is to prevent lower respiratory tract illness, thereby decreasing RSV-related hospitalizations and deaths. Secondary benefits would include a reduction of complications such as otitis media, the infrequent bacterial superinfections of the lungs, the inappropriate use of antibacterials,[89] and the eventual development of reactive airway disease. Some of the characteristics of an ideal RSV vaccine are mentioned in table II. The following sections summarize the most relevant developments related to clinical RSV vaccine trials in adults and children.

Table II

Proposed characteristics for an ideal respiratory syncytial virus (RSV) vaccine

5.1.1 Live Attenuated RSV Vaccines

One of the major challenges of the live attenuated RSV vaccines is to achieve an appropriate balance between attenuation and immunogenicity. Biologically derived as well as genetically engineered live attenuated RSV vaccines have been developed and tested in clinical trials in children and adults (figure 1).

Fig. 1

A schematic presentation of selected vaccines used in clinical trials over the last 4 decades against respiratory syncytial virus (RSV) infection. cp = cold-passaged; F = fusion protein; FI-RSV = formalin-inactivated RSV vaccine; G = virus attachment protein; M = matrix protein; PFP = purified F protein; PIV = parainfluenza; ts = temperature sensitive.

Initial studies in the late 1960s and in the 1970s using live virus mutants that were cold-passaged (cp) or temperature-sensitive (ts) RSV subgroup A intranasal strains failed to provide adequate protection to vaccine recipients.[90,91] Transmission of these viral mutants from vaccinees to placebo recipients was observed. These initial viruses also exhibited potential problems with genetic stability, as observed by reversion to wild-type phenotype.[91] The next generation of biologically derived live attenuated RSV vaccines was created based on the principle that viruses with multiple attenuating mutations are more genetically stable than those with single mutations. Chemical mutagenesis was used to obtain ts mutant derivatives from a cp RSV A2 mutant, resulting in cpts RSV mutants that were attenuated and genetically stable. Phase I studies were performed using two of these mutants, cpts 248/955 and cpts 530/1009, in both seropositive and seronegative children 6–60 months of age, and in adults. The two mutants were attenuated in seropositive children and adults but neither was sufficiently attenuated in seronegative children to permit studies in very young infants. Eighty-eight percent of seronegative infants and children shed vaccine virus, and vaccine virus was transmitted to placebo recipients.[92] Another attenuated mutant, cpts 248/404, had very limited infectivity in adults and was therefore tested only in children.[93] Another phase I trial in 114 children that included infants aged 1–2 months showed that the cpts 248/404 mutant did not produce disease enhancement when infants were subsequently infected with wild-type RSV. These young infants developed a predominant serum and mucosal IgA response with very poor serum neutralizing antibody formation. The vaccine also caused nasal congestion that in some cases interfered with feeding and sleeping.[94]

Infectious virus can be created completely from cloned cDNA;[95] with the use of recombinant technology, genetically engineered vaccines have been generated. Combination of attenuating mutations from biologically derived RSV vaccines allowed the production of further attenuated vaccine candidates.[96] This technique also allows the deletion of nonessential genes, such as SH or NS2 (ΔSH or ΔNS2, respectively), and potentially to insert G genes from RSV A and B in order to create bivalent vaccine candidates.[97] The first recombinant (r) vaccine candidate was made by combining the biologically derived mutations in cpts 248/404 (cp, 248 and 404) with deletion of the SH gene, resulting in the rcp248/404ΔSH vaccine. When this recombinant vaccine was tested in adults, seropositive children and seronegative children, it showed a similar level of replication as its cpts 248/404 parent, and it was therefore not further tested in infants.[98] The rcp248/404ΔSH was then modified by the insertion of the 1030 point mutation in the L gene to produce the derivative rcp248/404/1030ΔSH. This virus proved to be more attenuated than its predecessor and, when it was evaluated in 1- to 2-month-old infants, it proved to be well tolerated. Although the serum antibody response to this virus was better in infants aged 6–24 months than in those aged 1–2 months, replication of a second dose was highly restricted in the youngest population.[98] This suggests that non-antibody mechanisms may contribute to vaccine-induced immunity. This virus, however, showed evidence of genetic instability in some vaccinees due to reversion of a single mutation, either 248 or 1030.

Other recombinant candidates that will likely need to be evaluated clinically include viruses with the following modifications: (i) 248 and 1030 amino acid point mutations that will be more stable; (ii) point mutations in the RNA synthetic machinery; (iii) ts and non-ts mutations; and (iv) vectored vaccines.[99]

5.1.2 Subunit Vaccines

Purification and characterization of the RSV fusion (F) glycoprotein[100] allowed the creation of several subunit vaccines that have been tested in clinical trials. These vaccines have not been evaluated in infants; they are most likely to be useful in high-risk children, the elderly and in pregnant women. Purified F protein (PFP) vaccines include the incrementally more purified PFP-1, PFP-2 and PFP-3.[101–106] Because most infants hospitalized for RSV are younger than 5 months of age, maternal immunization could potentially prevent the morbidity associated with LRTIs in early infancy. Evaluation of a purified PFP-2 subunit vaccine was performed in a phase II randomized, double-blind, placebo-controlled trial. Thirty-five healthy pregnant women in weeks 30–34 of gestation received either vaccine or placebo intramuscularly in a 4 : 3 ratio. The vaccine was well tolerated and immunogenic in these women, and the transmission of neutralizing antibodies to their infants was virtually complete.[107] All infants born to the mothers who participated in this trial were healthy at delivery. It is not known whether the vaccine could exert a protective effect on those infants born before 32 weeks’ gestation who have the highest risk of infection and in whom the placental transference of antibodies occurs late during gestation.

A phase II, multicenter, adjuvant-controlled trial in 1- to 12-year-old children with cystic fibrosis immunized with PFP-3 vaccine showed that the vaccine was safe, well tolerated and immunogenic. A 4-fold increase in neutralizing antibodies to RSV A and RSV B was observed in 67% and 55% of vaccinees compared with 2% and 3%, respectively, of the adjuvant-control cohort.[108] Overall morbidity was reduced in these patients, although the total number of RSV infections was not reduced.

In a meta-analysis focused on randomized, double-blind, controlled phase I trials with PFP-1 or PFP-2, these vaccines were found to reduce the overall incidence of all RSV infections but the effect of vaccination on RSV LRTI did not reach statistical significance. The authors emphasized that the purpose of the phase I trials included in their study was to establish safety and not efficacy of these vaccines, and thus actual field trials were necessary.[109]

A subunit G protein vaccine, BBG2Na, was tested in 108 healthy volunteers aged 18–45 years who were randomized to receive one, two or three doses of 10, 100, or 300µg of vaccine or one to three placebo doses, administered intramuscularly, at 4-week intervals.[110] After only one vaccine dose, 33% and 71% of vaccine recipients treated with the 100 and 300µg doses, respectively, showed a ≥2-fold increase in neutralizing antibodies.[110] The vaccine was safe and well tolerated in this adult population; however, it is no longer being tested in clinical trials.

5.1.3 RSV Vaccines: the Future

The use of the reverse genetics methods will likely allow the development and testing of new recombinant vaccine candidates. Bivalent vaccine virus using attenuated human parainfluenza virus can be used as vectors by adding transcriptional units to express RSV F and G protective antigens.[99] Vectored vaccines have the advantage of growing robustly in cell culture and maintaining infectivity. A potential disadvantage of this strategy is the delivery of only a subset of RSV antigens. The potential safety and efficacy of these newer vaccine candidates will need to be determined.

6. Conclusion

RSV disease is responsible for significant morbidity in all ages, especially in well identified groups of high-risk patients. Current treatment modalities do not modify the course of the disease effectively. Newer approaches to antiviral therapy include fusion inhibitors, short interfering RNA particles, and compounds with unexplained anti-RSV activity identified in mass screening programs. Inhibitors of leukotriene synthesis may be effective in reversing symptoms. Effective prophylaxis for select groups of children is now available through the use of monoclonal antibodies, and the development and clinical testing of similar, improved molecules continues. Simple and less expensive infection control measures will likely benefit the majority of people at risk. Intensive research into a candidate vaccine has yielded live attenuated vaccines and subunit vaccines which have been studied in humans with encouraging results. However, a safe and effective RSV vaccine will likely not be available in the near future.