RSV remains the leading viral agent of serious pediatric respiratory tract disease worldwide as well as a major cause of disease in individuals of all ages, and the development of a safe and effective vaccine remains a human health priority. The need to immunize very young infants and the previous experience with FI-RSV vaccine-enhanced illness emphasize the importance of ensuring vaccine safety. The RSV G glycoprotein is one of the two neutralization antigens and one of the major protective antigens and thus would seem an obvious candidate for inclusion in a vaccine. On the other hand, numerous studies have suggested that immunization with RSV G is associated with the induction of aberrant immune responses that are manifest upon subsequent exposure to RSV and result in severe disease. Therefore, the exclusion of RSV G from a vaccine has also been suggested.
In the present study we compared the disease-priming capabilities of FI-RSV vaccines prepared either from rRSV wt or from derivatives lacking RSV G, its immunodominant region, or RSV SH. Deletion of RSV G or its immunodominant region did not diminish the ability of FI-RSV to prime for enhanced disease, whereas deletion of RSV SH resulted in only a modest diminution of the vaccine-enhanced disease induced by FI-RSV priming. These results clearly demonstrate that neither RSV G nor its immunodominant region is essential for disease priming by FI-RSV and indeed they do not make a discernible additive contribution under these conditions. RSV SH appeared to contribute to priming for increased disease but was not essential. In addition, the protective component of the immune response to FI-RSV was diminished by removal of RSV G or, unexpectedly, RSV SH. These results indicate that RSV G in the context of FI-RSV contributes protective epitopes as well as those that may be associated with disease enhancement.
While disease in FI-RSV- and RSV G-immunized animals may appear to be analogous, there are several indications that they have distinct pathogenic mechanisms, leading to a common final pathway. It has been demonstrated that immunization with FI-RSV (8
) or with RSV G (1
) predisposes for severe RSV-induced disease typified by enhanced illness, pulmonary eosinophilia, and type 2 cytokine production in the BALB/c mouse model. While the end points may be similar, it is apparent that the cytokine requirements for these two immunogens are distinct. For example, while illness and type 2 cytokine production are reduced in RSV-challenged FI-RSV-immunized mice with IL-4 depletion (24
), the enhanced disease is unaltered in vvGs-primed mice when IL-4 function is inhibited either by antibody depletion or in IL-4-deficient mice (24
). A similar pattern is seen for IL-13. Inhibition of IL-13 alone alters disease in FI-RSV-primed mice but not in vvGs-primed mice (26
). Thus, disease associated with FI-RSV can be modulated by blocking IL-4 or IL-13 alone, whereas both IL-4 and IL-13 function must be blocked to modulate disease in mice immunized with RSV G (26
Vaccine-enhanced disease was observed in 80% of FI-RSV vaccinees in the 1960s trial (27
) and occurs in many animal models (4
) and thus does not appear to be dependent upon a specific genetic background. In contrast, some elements of the immune responses associated with RSV G-induced illness appear to be genetically restricted, such as pulmonary eosinophilia, which is absent or dramatically reduced in mouse strains other than BALB/c and other H-2d
-restricted mouse strains (20
). Immunization with peptides from the immunodominant region of RSV G is sufficient to elicit pulmonary eosinophilia and both type 1 and type 2 cytokine production in BALB/c mice (52
) and is largely restricted to a subset of CD4+
T cells expressing the Vβ14 T-cell receptor (66
). The existence of this immunodominant region that is sufficient by itself to induce those immune responses associated with vaccine-enhanced disease underscores the phenomenon of genetically restricted RSV G immunogenicity. This is very different from the nearly universal induction of vaccine-enhanced illness generated by the FI-RSV vaccine in children less than 6 months of age, where the frequency of 80% indicated a lack of dependence on a specific genetic background (30
The ability of RSV G to predispose for eosinophilia and type 2 cytokine production is not unique among the RSV proteins. When administered as a purified protein in the context of alum, immunization with RSV F or with an FG chimeric protein also induces immune responses resulting in eosinophilia and IL-4 and IL-5 production following RSV challenge (7
). However, in contrast to G-specific responses, F-specific immune responses may be modified by Th1-modulating adjuvants such as monophosphoryl lipid A or QS-21 (16
). Thus, the ability to induce disease-enhancing immune responses is not restricted to RSV G but instead may be characteristic of an antigen that is administered parenterally and presented to the immune system as a soluble protein that cannot be processed via the major histocompatibility complex (MHC) class I processing pathway.
These data are the first to suggest a role for RSV SH in disease pathogenesis. Although the functional role(s) of SH remains unknown, its status as a viral surface glycoprotein may influence early events of virus infection as well as host cell integrity later in infection. A second mechanism may be the alteration of membrane permeability. Expression of SH proteins from several viruses including RSV (42
), human immunodeficiency virus type 1 (33
), and influenza virus (33
) has been shown to increase the permeability of the host cell membrane. Our data suggest that the removal of SH from a candidate RSV vaccine may improve safety.
In the present study the RSV G component of FI-RSV clearly functioned as a protective antigen as evidenced by the reduced protective efficacy of FI-RSV ΔG. A similar effect was observed with FI-RSV Gep−
, consistent with the idea that aa 187 to 197 represent the major antigenic site within RSV G in the BALB/c mouse model. Unexpectedly, removal of the SH component from FI-RSV also resulted in a reduction in protective efficacy. RSV SH is not generally considered to be a protective antigen because it did not function as an independent protective antigen when expressed in rodents by a recombinant vaccinia virus (6
). However, RSV SH has been shown to contain epitopes for B cells and T-helper T lymphocytes in the BALB/c mouse model (39
). Thus, RSV SH may function directly as a protective antigen or may contribute indirectly, possibly by contributing T-helper cell epitopes or by maintaining the integrity of RSV G and F.
There is precedence for exacerbated type 2 cytokine production and pulmonary eosinophilia upon aerosol challenge when initial antigen exposure is via the parenteral route. During primary RSV infection mice sensitized to ovalbumin exhibit enhanced airway hyperresponsiveness, increased production of type 2 cytokines, and more severe illness (41
). Similarly, initial exposure to antigens by cutaneous routes results in Th2 responses following subsequent inhalation contact with the same substance (10
). Additionally, obligate MHC class II processing of particulate antigens can bias immune responses toward a Th2-restricted pattern while the same protein processed by the MHC class I pathway generates more-Th1-like responses (32
). These patterns of antigen presentation are similar to those described in this study and may suggest common pathogenic mechanisms.
During final preparation of the manuscript Haynes et al. published data demonstrating reduced cellular infiltration in the BAL compartment of mice immunized with FI preparations of RSV expressing either wt or mutant forms of RSV G after RSV challenge (17
) and concluded that FI-RSV-enhanced disease may be reduced by removal of RSV G or by inhibition of the CX3C-CX3CR1 interaction. These data appear to contradict the data presented here. However, there are several points to consider that may account for the apparent discrepancies. Much of the data reported are in mice primed with FI-A2, a wt RSV, and challenged with mutant RSV or with antibody treatment at RSV challenge, thus inhibiting recall of the memory response. This is in contrast to the system used in our experiments in which rRSV was utilized during immunization, affecting induction of immune responses. In contrast to the targeted removal of defined and specific genetic sequences in the rRSV that we used, the mutant viruses used for challenge in the studies by Haynes et al. have multiple mutations, with cp52
having deletions of RSV G and SH and mutations in F and L proteins (9
) and with R10C7G having 10 nucleotide changes resulting in six amino acid changes in RSV G (48
). While in vitro replication of these viruses appears to be intact, these alterations have been shown to result in reduced in vivo replication (9
) or altered electrophoretic mobility suggestive of altered protein structure and, potentially, disruption of RSV G epitopes other than the CX3C fractalkine domain (48
). Anti-CX3CR1 antibody treatment was shown to reduce RSV infectivity (63
). Thus, an alternative explanation for the reduced BAL cell numbers and eosinophilia observed in FI-A2 anti-CX3CR1-treated mice and in cp52
-challenged mice is altered cell trafficking due to reduced viral burden and not merely the absence of RSV G or inhibition of CX3CR1 interaction. Additionally, most of the analyses were conducted at 40 h after challenge, which may not reflect the peak of cellular recruitment, previously shown to be around day 6 postchallenge for BAL cells (40
) and 7 to 9 days postinfection for lung tissue (13
). This may be of particular importance in comparing mice challenged with wt RSV to mice challenged with mutant viruses producing lower viral burdens where the kinetics of cell recruitment may be different. These combined factors make it difficult to compare the data described by Haynes et al. to the data that we now report.
The data reported here demonstrate that the factor involved in the induction of disease-enhancing immune responses during immunization with FI-RSV is not RSV G or its immunodominant region. Rather, this study suggests that it is the pathway of initial antigen presentation, specifically obligate processing of exogenous antigen through the MHC class II pathway, that is the critical determining factor in the induction of immune responses that result in severe disease upon intranasal challenge with live RSV. This hypothesis is strengthened by previous studies from our lab (24
) that demonstrated that mice immunized with vaccinia virus expressing only the membrane-anchored form of RSV G are protected against severe disease following RSV challenge. Thus, the presentation of FI-RSV antigens in the context of alum is sufficient to induce those immune responses that result in type 2 cytokine production and pulmonary eosinophilia following subsequent infection with live RSV. Further studies are required to determine the basic molecular and cellular mechanisms that are involved in the programming for vaccine-enhanced immune responses during immunization with exogenous RSV antigens that require antigen processing and presentation via endocytic pathways. Taken together, these and previous studies from our lab (24
) suggest that the basis of RSV vaccine-enhanced disease is not antigenic content but is rather the pathway of antigen processing and, therefore, that RSV G should not necessarily be excluded from potential vaccine products that target endogenous antigen processing and presentation by MHC class I molecules.