Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory tract disease worldwide and also is important in the elderly, bone marrow transplant recipients, and adults in general. A licensed vaccine or effective antiviral therapy is unavailable. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We developed a method for producing infectious RSV entirely from cDNA clones. This makes possible the direct introduction of defined changes into infectious virus via the cDNA intermediate, providing a method for expedited development and characterization of mutants for basic biological studies and for evaluation as an intranasally-administered live-attenuated vaccine. We previously showed that RSV encodes ten mRNAs and eleven unique proteins. We found that the coding sequences for five RSV proteins, namely NS1, NS2, SH, M2-2 and G, could be deleted singly and in some cases in combination without greatly compromising replication in vitro. The mutants exhibited a spectrum of attenuation phenotypes in vivo. Thus, these deletions are excellent candidates for inclusion in a live-attenuated vaccine. Certain deletions provide novel characteristics that might be advantageous for a vaccine. For example, the M2-2 deletion virus exhibited a shift favoring transcription over replication. This implicates this protein as a regulatory factor in RNA synthesis. More importantly, this deletion confers the desirable property of increased antigen expression in the context of decreased replicative capacity. Fully-viable chimeric viruses were constructed between human RSVs representing the two antigenic subgroups, making it possible to use a single attenuated backbone to express the major antigenic determinants of each of the subgroups. Fully-viable chimeras also were constructed between human RSV and bovine RSV, a virus that has a host range restriction that renders it highly attenuated in primates and thus represents a new method of attenuating an RSV vaccine. As another approach to making an RSV vaccine """"""""better than nature"""""""", recombinant RSV was engineered to express various cytokines and chemokines in order to enhance immunogenicity and, in some cases, attenuate the virus. Finally, we tested the novel strategy of using a recombinant parainfluenza virus type 3 (PIV3) as a vector to express the major RSV protective antigens, the G and F proteins, from added, supernumerary genes inserted individually or together into the promoter-proximal position. The PIV3 vector was rB/HPIV3, a recombinant bovine (B) PIV3 in which the major protective PIV3 HN and F antigen genes have been replaced by those of human (H) PIV3. This chimera thus consists of the BPIV3 backbone, which is attenuated in primates due to a natural host range restriction, bearing the major antigenic determinants of HPIV3. The rB/HPIV3 vector is a promising candidate as a vaccine against HPIV3, and the inclusion of the antigenic determinants of RSV offers an approach to make a combined HPIV3/RSV vaccine. rB/HPIV3/RSV chimeras were made that represented both RSV antigenic subgroups. Evaluation in rodents and rhesus monkeys showed that the rB/HPIV3/RSV viruses were somewhat more attenuated than the rB/HPIV3 parent, presumably due to the added genes, but were equivalent to rB/HPIV3 and RSV in immunogenicity against HPIV3 and RSV, respectively. The use of PIV3 as a vector for RSV antigens provides an approach that overcomes difficulties inherent in working with RSV and provides a combined vaccine against HPIV3 and both RSV antigenic subgroups.
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