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Immunization with native fusion (F) protein from respiratory syncytial virus (RSV) adsorbed to alum adjuvant generates greater than fourfold rises in serum neutralizing antibody titers in approximately 50% of seropositive humans. Using BALB/c mice we demonstrate herein that enhanced neutralization titers and accelerated clearance of virus from the lungs after challenge are possible if the attachment (G) glycoprotein is added to F protein-based vaccines. We further reveal for the first time that polarized type 2 T cell responses and immunopathology associated with G protein are inhibited by adjuvants recognized by toll-like receptors (TLR). Co-formulation with compounds that targeted TLR-2, TLR-3, TLR-4, or TLR-9 elicited significantly diminished type 2 T cell responses that caused granulocytic inflammation and eosinophilia in the airways after challenge. These results were not observed with recombinant IL-12 or QS-21. The data are important for improving combination vaccines for RSV.

Development of subunit vaccines against respiratory syncytial virus (RSV) for naive human infants is hindered by concerns that immunization with the fusion or attachment (G) proteins will elicit polarized Type 2 T cell responses and cause immunopotentiation upon subsequent natural infection. We investigated the regions of G protein responsible for inducing a Type 2 T cell phenotype in inbred mice of different MHC haplotype toward development of vaccines with improved safety. As demonstrated by IL-5-dependent pulmonary eosinophilia after challenge and serum anti-G protein IgG1 to IgG2 ratios, highly purified native G protein sensitized all strains for a Type 2 T cell phenotype. Stimulation of G protein-primed splenocytes with synthetic overlapping peptides indicated that the nonglycosylated ectodomain was primarily responsible. Respectively the recall responses of BALB/c (H2(d)), C57BL/6 (H-2(b)), SJL (H-2(s)), and C3H/HeJ (H-2(k)) mice were directed against epitopes within peptides spanning amino acids 184-198 (pep(184-198)), 168-181 (pep(168-181)) or 171-185 (pep(171-185)), 176-190 (pep(176-190)), and 104-118 (pep(104-118)) or 159-173 (pep(159-173)). Injection of pep(184-198) conjugated to KLH (pep(184-198)-KLH) primed H2(d) [BALB/c, B6.C-H2(d)/bBy], but not H-2(b) [C57Bl/6, C.B10-H2(b)/LiMcd] mice for pulmonary eosinophilia. Sensitization with a peptide-KLH conjugate encompassing amino acids 149-200 (pep(149-200)-KLH) further confirmed that Type 2 T cell responses in BALB/c, C57BL/6 and SJL, but not C3H/HeJ mice were induced by the nonglycosylated ectodomain of G protein. These data are important for design of safe and efficacious subunit and attenuated vaccines for RSV.

In the murine model of respiratory syncytial virus (RSV) infection, recombinant interleukin-12 (rIL-12) was previously demonstrated to be most effective as an adjuvant for fusion (F) protein-based vaccines after adsorption to alum adjuvant. Alum, however, is a potent inducer of polarized T-cell responses and the type 2 phenotype. In addition, studies in human cancer patients demonstrated that rIL-12 was toxic when administered systemically at high doses. Because of these issues, we investigated whether the saponin QS-21 could facilitate a reduction in dose of rIL-12 when F protein was prepared in the absence of alum (F/rIL-12). The results demonstrated that a suboptimal dose (0.8 microg) of QS-21 enhanced the capacity of F/rIL-2 to elicit antigen-dependent killer cell precursors and complement-assisted neutralizing antibodies in spleens and sera respectively of BALB/c mice. The killer cell activity of mice vaccinated with F/rIL-12 (0.1 microg) plus 0.8 microg QS-21 was equivalent to that elicited after vaccination by experimental infection, or with an optimal dose of QS-21. The neutralizing titers generated by F/rIL-12 (0.01 to 1.0 microg) were also significantly elevated in the presence of 0.8 microg QS-21. Thus, rIL-12 and QS-21 together form a potent adjuvant for eliciting functional cell-mediated and humoral immune responses against F protein.

Subunit vaccines formulated with purified fusion proteins from the A2 (PFP-2) or attenuated 248/404 (PFP-3) strains of respiratory syncytial virus (RSV) were evaluated, either alone or in combination with native attachment (G) protein, for their ability to augment serum neutralizing antibody titers in seropositive chimpanzees. The results suggested that combination vaccines enhanced serum neutralizing antibody titers against both laboratory strains and clinical isolates of RSV. When compared with PFP-2 alone, the resultant neutralizing antibody titers after vaccination with PFP-2+Ga protein were significantly elevated against 71% of A strains tested. In a confirmatory experiment, immunization with PFP-3+Ga+Gb proteins resulted in elevated serum neutralizing antibody titers against 86% of A and 50% of B strains tested versus injection with PFP-3 alone. The results suggest that subunit vaccines composed of both PFP and G proteins have more potential than PFP alone to augment neutralizing antibody titers in seropositive recipients.

The effects of recombinant interleukin-12 (rIL-12) on immune responses generated by subunit vaccines for respiratory syncytial virus (RSV) were evaluated in BALB/c mice. Parenteral co-administration of rIL-12 with F/AlOH or F/PBS resulted in accelerated clearance of infectious virus from the lungs 4 days after challenge. The immune responses elicited by 0.03 microg of F protein plus 10 ng of rIL-12 adsorbed to AlOH were more efficacious than those induced by 3 microg of F protein co-formulated with 1,000 ng of rIL-12 in PBS alone. Adsorption to AIOH prolonged the presence of rIL-12 in the sera. The resultant systemic humoral immune responses after vaccination with F/AlOH or G/AlOH were dependent on the dose of rIL-12 and characterized by heightened serum immunoglobulin G2a (IgG2a) antibody titers. Co-administration of rIL-12 with F/AlOH was also associated with diminished protein-specific IgE titers, elevated neutralizing antibody titers, and interferon-gamma and (IFN-gamma) in the sera, and enhanced antigen-dependent killer cell activity in the lungs after challenge. For maximum benefit, the data suggested that rIL-12 must be co-administered with F/AlOH. Collectively, the results indicated that rIL-12 directed immune responses toward a type 1 phenotype. However, examination of pulmonary inflammatory cells after challenge suggested that the type 1 phenotype was not absolute. Co-formulation with rIL-12 did not diminish pulmonary eosinophilia upon challenge of naive mice primed with F/AlOH, G/AlOH, or FI-RSV, and CD4+ T cells were expanded relative to the CD8+ T-cell compartment. These results are important for the future design of subunit vaccines against RSV.

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpΔG150-222 or rA2cpΔG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37°C, but pinpoint morphology was observed at 39°C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log 10 -unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpΔG150-222 or rA2cpΔG177-220. These findings are important for the development of attenuated RSV vaccines.

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpΔG118, rA2cpΔG174, rA2cpΔG193, and rA2cpΔG213. All rA2cpΔG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37°C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpΔG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpΔG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts 248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpΔG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

The feasibility of using oligodeoxynucleotides (ODN) containing unmethylated CpG motifs as parenteral adjuvants for subunit vaccines against RSV was tested in BALB/c mice. Compared with immunization with natural F protein adsorbed to aluminum hydroxide (F/AlOH) adjuvant alone, coadministration of F/AlOH with CpG ODN resulted in statistically significant increases in serum neutralization titers, an enhanced generation of splenic antigen-dependent killer cell precursors, and accelerated clearance of infectious virus from lungs 4 days after challenge. The statistically significant increases in serum IFNgamma and anti-F protein IgG2a titers, and significantly diminished pulmonary IL-5 and eosinophilia after challenge indicated that CpG ODN enhanced the ability of F/AlOH to elicit type 1 immune responses. F protein-specific serum IgE titers were also reduced. Further analysis of pulmonary inflammatory cells demonstrated an expansion of CD8(+) T cells, relative to the CD4(+) T cell compartment. The potency of CpG ODN was not adversely affected in gene knockout mice devoid of the p35 chain of the IL-12 heterodimer. Taken together, the results suggest a novel formulation for naive recipients of F protein-based subunit vaccines that does not result in a type 2 phenotype.