These data suggest that the presence of the secreted form of RSV G during initial antigen presentation (and not solely the primary or secondary antigenic structure) affects the composition of immune responses to RSV infection. Previous work has shown that immunization with RSV G results in more severe disease and lung pathology following RSV challenge (25
). The molecular basis for this alteration in the immune response has not been defined. The unique nature of G provides several attributes which may generate the altered immune response to RSV challenge. First, G is extensively glycosylated: >50% of the weight of the mature protein is contributed by carbohydrate residues, attached predominantly via O-glycosidic bonds (70
). Also, G contains a high proportion (30.6%) of serine and threonine residues which serve as acceptors for the O-linked oligosaccharides. Third, G protein lacks homology to any other known paramyxovirus protein. The high proline content of 10.1% has more in common with mucinous glycoproteins than viral glycoproteins (31
). G exhibits neither hemagglutinating nor neuraminidase activities (22
) and is also unusual in that it lacks both a hydrophobic NH2
-terminal signal sequence and a hydrophobic COOH-terminal domain, features present in RSV F and other paramyxovirus proteins (70
In contrast to previously published studies using recombinant vaccinia virus expressing wild-type RSV G (25
), mice primed with recombinant vaccinia virus expressing only membrane-anchored G are partially protected during RSV challenge, with reduced viral titers observed, although illness patterns are similar. However, in the absence of membrane-anchored G, secreted G increases illness and decreases viral clearance. We have shown that priming with secreted G (for both vvWT G and vvM48) is associated with increased IL-5 production and more severe immunopathology, including increased tissue eosinophilia. However, illness correlates with alveolar infiltration of eosinophils, as determined by their presence in BAL, and suggests that secreted G induces factors other than IL-5 which may be responsible for a second step in the activation and migration of eosinophils. These data suggest the secreted form of RSV G modulates the composition of the immune response by inducing IL-5 production and altering patterns of leukocyte trafficking, while the membrane-anchored form of G may have a separate and dominant immunoregulatory effect on factors producing illness.
Several immunoregulatory mechanisms have been described for soluble antigen. Soluble antigen has been shown to regulate the development of germinal center B cells by selectively inducing apoptosis of high-affinity antigen-specific B lymphocytes (50
). This ability to direct B-cell differentiation is hypothesized to indicate clonal deletion of self-reactive B cells and, thus, be a mechanism of avoiding autoimmune disease. Soluble antigen will be processed and presented in the context of major histocompatibility (MHC) class II molecules. MHC context of antigen presentation (41
), the type of antigen-presenting cell used (11
), and the presence of different costimulatory molecules (13
) have been shown to regulate the development and maturation of T-cell subsets. Antigen dose may also be a determinant in T-cell differentiation. In most systems, immunization with large doses of antigen induces effector cells exhibiting a type 1 cytokine profile, whereas lower concentrations of priming antigen tend to generate type 2 effector cells (12
). However, some antigens have the reversed profile, with high doses favoring development of Th2 cells and low doses inducing Th1 cells (51
). Thus, the induction of severe disease by the presence of soluble G during priming suggests that either the concentration or processing of G or its subsequent recognition by selected T-cell subsets may be a key determinant regulating immune responses following subsequent RSV challenge.
Infectious organisms ensure their survival by developing mechanisms to avoid detection or subvert immune responses. Some strategies include production of cytokine receptors by the organism (30
) or blocking induction of the immune response at the level of antigen presentation (14
). A body of evidence which suggests that induction of Th2-mediated immune responses provides a survival advantage for many intracellular pathogens, including viruses, is also accumulating (13
). For example, measles virus suppresses cell-mediated immunity by down-regulation of IL-12 expression through signals mediated by measles virus binding CD46 (33
). Also, disease progression and infection with human immunodeficiency virus have been associated with Th2-dominated responses (16
). Human immunodeficiency virus infection has been shown to suppress IL-2, IL-12, and IFN-γ, but not tumor necrosis factor alpha or IL-6, induction by Toxoplasma gondii
). Thus, the induction of a type 2 T-cell response by secreted RSV G may serve as a defense mechanism the virus has evolved to favorably modulate immune responses.
Previous work has established priming or infection with G-expressing vectors results in increased illness and pathology (61
). The ability of RSV G to produce cytokines associated with a Th2 response has been clearly demonstrated (2
). In this and other studies, FI-RSV priming has also been shown to induce Th2-type cytokine profiles in mice (19
), thereby associating this type of response with the RSV vaccine-enhanced illness seen in children in FI-RSV vaccine studies in the 1960s (32
). In this study, we have addressed whether it is the primary or secondary antigen structure of G, or the presence of secreted G or antigens with obligate processing through the endocytic pathway and MHC class II expression, which increases disease severity. The IgG2a isotype antibodies and disease protection observed in mice primed with membrane-anchored G and the IgG1 isotype antibodies and more severe illness induced by immunization with secreted G imply that different Th1- and Th2-associated cytokine profiles may be induced by priming without and with secreted G, respectively. We therefore propose that the link between RSV G immunization, FI-RSV-induced vaccine-enhanced illness, and induction of type 2 cytokine profiles is not induced by G itself but rather results from the initial presentation of G to the immune system in secreted form. This might then promote the induction of Th2 CD4+
T-cell differentiation in part by precluding intracellular antigen processing, presentation of antigen by MHC class I molecules, and loss of the early contribution of CD8+
T cells to the cytokine milieu (17
Evidence suggests that full activation of eosinophils requires a two-step process involving IL-5, eotaxin, or other cofactors (42
). Extracellular matrix proteins (36
) and VLA-4 on eosinophils (44
) may also be involved in eosinophil recruitment and infiltration. These observations may be invoked to explain the differential patterns of eosinophilia and disease severity induced by priming with membrane-anchored and secreted G. Our data suggest the hypothesis that immunization with any form of G (membrane anchored or secreted) may recruit eosinophils upon challenge via the induction of IL-5, but the presence of secreted G is required for inducing the activation associated with movement of eosinophils into alveolar spaces to produce more severe disease.
The data presented in this paper suggest that priming with secreted RSV G promotes eosinophil recruitment associated with IL-5 production and that illness is associated with the movement of eosinophils from the interstitial compartment in the lung to the alveoli. We propose the secretion of G from RSV-infected cells represents a strategy to modulate and evade effective immune responses. The presence of a small number of alveolar eosinophils in mice primed with membrane-anchored G suggests a direct effect of the primary or secondary antigenic structure of G on the induction of type 2 immune responses that is compounded by its secretion as a soluble protein, resulting in increased eosinophilia. Understanding how secreted G interacts with different elements of the innate and adaptive immune system might provide insight into basic mechanisms of clearance for RSV and other viruses.