As with other acute respiratory viruses, RSV infection usually is completely resolved by innate and adaptive immunity (21
). As with many viruses, RSV infection or uptake by respiratory epithelial cells and resident macrophages results in widespread changes in cellular gene expression and upregulates expression of a variety of factors, including surfactants, cytokines, chemokines, and cell-surface molecules. Some of these factors have direct antiviral properties; others stimulate the influx and activation of natural killer (NK) cells, granulocytes, monocytes, macrophages, dendritric cells, and T lymphocytes that provide direct antiviral activities and initiate an effective adaptive immune response.
Virus-neutralizing antibodies in the respiratory tract likely contribute to viral clearance and certainly play an important role in protection against reinfection (21
). They include secretory immunoglobulin A (IgA) and transudated, serum-derived IgG. Secretory IgA is particularly important in protecting the upper respiratory tract, which is accessed only very inefficiently by serum IgG (124
). The IgA response is short-lived following primary infection but can increase in duration following reinfection (109
). Serum IgG antibodies are somewhat more efficient in accessing the lower respiratory tract and can provide substantial protection in that compartment. In RSV-naïve infants, the maternal serum antibody titer is positively correlated with a reduced level of severe RSV disease. The clinical experience with palivizumab also shows that serum antibodies alone can provide substantial protection from severe disease. However, protection from passive antibodies quickly wanes, because they decay with a half-life of approximately 21 to 24 days. CD8+
T lymphocytes are important for clearing virus-infected cells as well as for contributing cytokines, notably gamma IFN (IFN-γ), that promote a protective Th1 response (53
Protective immunity to RSV induced by natural infection is generally described in the literature as weak and short-lived. This is based mainly on the frequent incidence of reinfection of humans in nature and under experimental conditions. However, as discussed later, viral immune evasion strategies also may contribute to reinfection. Typical RSV-neutralizing serum antibody titers in adults are quite high (mean reciprocal titer of 1,450 in a 50%-infected-well-reduction assay), and following natural infection, they were increased fourfold or more in 64% of young adult and 79% of frail elderly patients, suggestive of good responses (41
). While postinfection increases in antibody titers wane in most individuals within a year, this decay might not be unique to RSV (40
), and the residual titers remain quite high. Brisk serum antibody responses also have been noted in children with primary and secondary infections (66
), and even young infants of 2 months of age can have substantial neutralizing serum antibody responses when the titer of immunosuppressive maternal antibody is low (R. A. Karron, personal communication). Primary RSV infection of seronegative experimental animals, including the chimpanzee, results in robust protective immune responses, at least in the short term (23
). In addition, in clinical studies, experimental live RSV vaccines do not seem to be obviously reduced in immunogenicity compared to live human parainfluenza virus type 3 (HPIV3) and influenza A virus vaccines, although these studies were not designed for virus-to-virus comparisons (R. A. Karron, personal communication). There are reports of effects on cellular immunity. RSV-specific T-helper cell responses, as measured by in vitro lymphoproliferation, appeared to be deficient during reinfection in infancy (12
). Increased apoptosis of CD4+
lymphocytes resulting in lymphopenia also has been described for RSV-infected infants compared to uninfected controls, with the effect being greater with younger age and more severe illness (130
). Mitogen-induced proliferation of peripheral blood lymphocytes in vitro was inhibited by contact with RSV-infected cell monolayers, an effect that did not prevent the expression of T-cell activation markers but impeded the cell cycle (135
). This effect appeared to be mediated by the viral F protein and was augmented by G. Studies of mice suggested that the pulmonary CD8+
CTL response to RSV was less functional and shorter lived than that to influenza virus (18
). However, this difference between viruses has not been confirmed and, as noted later, functional impairment to the pulmonary CTL response might be a feature of the tissue rather than the virus (153
). In summary, it remains unclear to what extent frequent reinfection by RSV reflects inadequate or inappropriate responses by the host versus viral immune evasion strategies.
Contribution of the host response to pathogenesis.
The most dramatic demonstration of the potential of host immunity to contribute to disease associated with an RSV infection was the experience with a formalin-inactivated RSV (FI-RSV) vaccine that was administered intramuscularly to infants and children in the 1960s. This vaccine was poorly protective and, in RSV-naïve individuals, it primed for enhanced disease upon subsequent natural infection with RSV, with up to 80% of vaccinees hospitalized with RSV-like disease, resulting in two deaths (21
). Retrospective studies showed that FI-RSV induced serum antibodies that bound efficiently to viral antigen but did not efficiently neutralize infectivity, contributing to the poor vaccine efficacy (111
). This atypical antibody response probably reflected denaturation of the antigen as well as a possible deficiency in antibody affinity maturation. An analysis of lung tissue from the fatal vaccine cases and from experimental animal models of enchanced disease provided evidence of antibody-antigen complex deposition and complement activation in the lung occurring during subsequent RSV infection (120
In addition, peripheral blood lymphocytes from FI-RSV vaccinees exhibited an exaggerated proliferative response to RSV antigens in vitro compared to lymphocytes isolated following natural infection (82
). Subsequent studies with experimental animals confirmed that, compared to natural infection, FI-RSV induced a heightened response of virus-specific CD4+
T lymphocytes biased toward the Th2 subset (54
). Th1 and Th2 CD4+
T lymphocytes play important roles in immune regulation and function and to some extent are self-stimulatory and reciprocally inhibitory. Th1 responses (signature cytokines IFN-γ and IL-12) tend to promote cell-mediated immunity important for protection against intracellular pathogens, such as viruses. Th2 responses (signature cytokines IL-4, IL-5, IL-10, and IL-13) can be associated with eosinophilia, goblet cell hyperplasia, mucus overproduction, IgE production, and airway hypersensitivity. The Th2-biased response to FI-RSV appears to be a consequence of inefficient induction of IFN-γ-secreting NK cells and CD8+
T lymphocytes by this nonreplicating vaccine (70
). In FI-RSV-immunized animals, depletion studies confirmed that the Th2 cells and cytokines were important in vaccine-enhanced pathology (24
). This experience amply demonstrated immune-mediated (particularly Th2-mediated) pathology associated with an inactivated vaccine and subsequent RSV infection. However, comparable disease enhancement does not occur with natural RSV infections and reinfections, and the relevance of FI-RSV-associated pathogenesis to natural infection is unclear.
As often is the case for acute infections, host immunity appears to contribute to pathogenesis during natural RSV infection, although not as dramatically as with FI-RSV. However, the relative contributions to RSV pathogenesis of direct viral cytopathology versus the host immune response, and the host factors that are responsible, remain controversial.
Several observations suggest a substantial contribution of host immunity to RSV disease. For example, clinical observations (as noted above) and in vitro studies (described later) showed that RSV is not highly cytopathic or invasive. In infants coinfected with human immunodeficiency virus type 1, prolonged clinical shedding occurred for more than 199 days without substantial disease (83
). When cotton rats with an established RSV infection were administered a neutralizing antibody that reduced pulmonary virus replication more than 1,000-fold, there was little effect on pulmonary pathology; the addition of anti-inflammatory glucocorticoid therapy was necessary to reduce pathology (122
). A similar lack of clinical improvement was observed for intubated children with an established infection, for whom antibody therapy reduced viral shedding 30-fold compared to controls (97
). The inability to block disease progression by sharply reducing virus replication is suggestive of immunopathology rather than direct viral cytopathology. Finally, genetic polymorphisms that increase expression of the IL-4, IL-8, and (tentatively) CCR5 genes were associated with an increased frequency of severe pediatric RSV disease, suggesting that these host factors can contribute to pathogenesis (69
Conversely, other observations indicate a contribution of viral cytopathology to RSV disease. RSV disease often is more frequent and more severe in highly immunosuppressed or immunocompromised individuals, which seems inconsistent with disease being primarily immune mediated (43
). In humans, immunity to RSV due to maternal antibodies or prior infection ameliorates rather than enhances disease upon reinfection. While it is not as cytopathic as influenza virus, RSV perturbs ciliary action and leads to cell shedding, effects that would contribute to airway obstruction. There generally is a positive correlation between the magnitude of virus replication and clinical disease in natural and experimental infections with wild-type or attenuated RSV (30
), although findings to the contrary also have been reported (163
). However, this does not argue solely for viral cytopathology, since diminishing the viral load will reduce the antigenic stimulation driving immunopathology. In experimental infections of chimpanzees and human adults, children, and infants with wild-type or attenuated RSV, symptoms began 1 to 4 days following the onset of viral shedding and ended either coincident with the cessation of shedding or continued for several days, depending on the individual (8
). This result suggests that both viral cytopathology and host immunity contribute to disease and that there is variation among different individuals. A recent evaluation of lung specimens from young infants with rapidly fatal, untreated RSV infection provided evidence of extensive viral replication with few CD8+
T lymphocytes or NK cells and minimal lymphocyte-derived cytokines (159
). This suggested that these cases of fatal disease involved an inadequate rather than excessive adaptive immune response. However, another recent study involving a case of RSV disease in a 15-month-old patient, in which death was due to a vehicular accident rather than the virus, provided evidence of substantial immune infiltrate, including monocytes, T lymphocytes, and neutrophils (72
). The controversy over the contribution of immunopathology versus viral cytopathology continues because the natural human host is not amenable to pathogenesis studies and animal models are poor surrogates.
Excessive T-lymphocyte cytotoxicity is one potential mechanism of immune-mediated pathogenesis. Studies of mice showed that the T-cell response helps resolve RSV infection but can make a substantial contribution to disease. For example, depletion of CD4+
T cells reduced disease, and depletion of both resulted in long-term infection without illness (53
). In a patient with severe combined immunodeficiency and a high level of persistent RSV shedding, T-cell reconstitution dramatically reduced viral shedding but also resulted in a dramatic increase in pulmonary disease (35
). Conversely, there also is evidence arguing against a prominent role of T-lymphocyte cytotoxicity in RSV pathogenesis. Among infants with immunodeficiencies, those with cell-mediated deficits have more difficulty controlling the virus and have more severe outcomes, suggesting that the net effect of T cells is protective rather than pathogenic (43
). As already noted, one study of lung autopsy specimens provided evidence of a deficient rather than overly robust CD8+
T-cell response associated with fatal RSV disease (159
), although a substantial response was observed in a second study (72
). In infants hospitalized for RSV bronchiolitis, RSV-specific CTLs were not detected until at least 6 days later (20
) and therefore did not correlate temporally with severe disease. Thus, T cells are important for clearing RSV infection, but their contribution to pathogenesis may depend on the situation.
A role for Th2-biased responses in RSV pathogenesis was suggested by (i) the Th2-mediated disease associated with FI-RSV discussed above, (ii) the Th2 bias of the young infant (discussed later), in whom severe disease is more frequent, and (iii) the association of Th2 responses with asthma, which involves small airway constriction, mucus plugging, and wheezing similar to patterns seen with RSV disease. A number of clinical studies have documented elevated ratios of Th2/Th1 cytokines or their mRNAs, measured in nasal secretions or in stimulated or nonstimulated peripheral blood mononuclear cells, in association with severe pediatric RSV disease (80
). In some cases, the overall Th response actually was decreased, but the relative Th2 component was increased. In addition, two studies demonstrated a positive association between RSV disease and a genetic polymorphism in the Th2 cytokine IL-4 gene that increases gene expression (69
). Conversely, other groups have reported a Th1-biased response or mixed responses associated with severe pediatric RSV disease (13
). Thus, there is suggestive but inconsistent evidence of a link between an increased Th2/Th1 ratio and RSV pathogenesis. In some studies, patients with severe disease fell into subgroups with respect to Th responses, suggestive of substantial host variability (80
The Th2 cytokines IL-4 and IL-13 promote isotype switching to IgE. IgE is bound by receptors on mast cells and basophils and, upon contact with antigen, induces cell activation and the release of mediators, including histamine and leukotrienes. These can mediate neural, vascular, and muscular responses, including rhinorrhoea, cough, and wheeze, which are disease signs associated with RSV. Indeed, some studies have found persistent increased levels of free RSV-specific IgE and histamine in secretions of infants experiencing an RSV infection with wheezing (158
). However, other studies have not confirmed these findings (28
), and the association of IgE to RSV disease remains unclear.
Eosinophils can be involved in either Th2-mediated or inflammatory responses (see below) and have been suspected to have a role in RSV pathogenesis based on several observations. Increased numbers of eosinophils were reported in autopsy lung tissues from two infants who died of enhanced RSV disease subsequent to vaccination with FI-RSV, although a reanalysis of these specimens indicated that they were a minor population (123
). Pulmonary eosinophilia is observed in connection with Th2 responses to RSV antigens in BALB/c mice, as already noted. Also, increased levels of eosinophil degranulation proteins were found in respiratory secretions from patients with severe cases of RSV disease (46
). The prominent association of eosinophils in asthma also makes them of particular interest. Eosinophils are recruited by Th2 or inflammatory chemoattractants, including IL-5, eotaxin/CCL11, and RANTES, and are activated to release cytotoxic proteins and antiviral RNase as well as Th2 cytokines and inflammatory chemokines and cytokines. However, the prevalence of eosinophils among airway leukocytes from RSV-infected infants, 1 to 3%, was low and approximately the same as for influenza virus, and thus does not seem to represent a prominent or unique feature of RSV pathogenesis (101
). Furthermore, the net effects of eosinophils are not necessarily pathogenic: hypereosinophilia in a transgenic mouse that overexpresses IL-5 was associated with protective and disease-sparing effects against RSV rather than increased disease (118
). Thus, while eosinophilia sometimes is prominent in animal models of RSV pathogenesis, its contribution to authentic human disease is unclear.
Overly robust inflammatory responses also have been suggested to contribute to RSV pathogenesis. These initiate when the virus interacts with respiratory epithelial cells and macrophages and is detected by TLRs and other pattern-recognition proteins, leading to upregulation of the expression of inflammatory factors, such as chemokines and surface proteins involved in cell interactions. The further activation of NF-κB by the RSV F and NS2 proteins (described later) likely augments the response (90
). This leads to the influx and activation of leukocytes, which add to the production of inflammatory factors and can help resolve infection but also contribute to pathogenesis through tissue damage and other effects. Clinical studies have documented increased expression of inflammatory mediators or their mRNAs in respiratory secretions of infants and children hospitalized for RSV disease compared to controls, including IL-6, tumor necrosis factor α, IL-8, RANTES, macrophage inflammatory protein 1α/CCL3, eotaxin, and monocyte chemotactic protein 1/CCL2, among others (47
). As is typical for acute inflammation, neutrophils are the predominant airway leukocyte in infants with RSV bronchiolitis, accounting for 84% or more of the cells, compared to 66% for influenza virus (101
). IL-8 is the major chemoattractant for neutrophils. Its concentration in respiratory secretions is elevated in infants with severe RSV disease (1
) and in some reports appeared to be somewhat increased for RSV versus other respiratory virus infections (48
). Activation of neutrophils occurs in response to inflammatory mediators and possibly to RSV itself and results in the release of cytotoxic enzymes in addition to inflammatory cytokines and chemokines (1
). Neutrophils presumably can have antiviral activity due to the destruction of RSV-infected cells, to which they may be attracted by the virus-induced expression of surface adhesion molecules. However, the extent to which RSV neutrophilia and other aspects of the inflammatory response are protective versus pathogenic, and whether this is particular to RSV compared to other respiratory viruses, is unclear.
As noted, genetic polymorphisms that increase IL-8 and CCR5 expression have been associated with increased RSV disease, consistent with a role of inflammatory chemokines in RSV disease (69
). However, a strong inflammatory response does not seem to be essential for the development of viral respiratory tract disease: in one study, the level of inflammatory cytokines and cytokines in nasal wash samples from infected infants was substantially lower for HMPV than for RSV despite similar disease signs (91
). In most clinical studies, anti-inflammatory therapy (oral or inhaled corticosteroid) has not provided significant improvement of short- or long-term outcomes of RSV infection (14
Thus, a number of host immune factors involved in restricting and clearing the virus likely also contribute to pathogenesis, at least under some conditions. However, it is unclear whether one or more factors are particularly responsible for RSV disease, and whether this is different for RSV than for other respiratory viruses.