Human Respiratory Syncytial Virus Biology And Vaccine Development

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Abstract

Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory tract disease worldwide. RSV also is a significant cause of morbidity and mortality in the elderly, with an impact approaching that of non-pandemic influenza virus. RSV readily infects severely immunocompromised individuals, most notably allogeneic bone marrow transplant recipients, causing high mortality. RSV also makes a substantial contribution to upper respiratory tract disease in individuals of all ages. Globally, the World Health Organization estimates that RSV causes 64 million infections and 160,000 deaths annually. Neither a vaccine nor an effective antiviral therapy is available against RSV.[unreadable] We previously developed a method for producing infectious RSV entirely from cloned cDNAs (reverse genetics), whereby defined changes can be introduced into infectious virus via the cDNA intermediate. We have used this technique extensively to characterize the molecular biology and pathogenesis of the virus and to develop live attenuated viruses for use as a live intranasal pediatric vaccine. Vaccine development for HRSV is difficult because immunization should begin by 1 to 2 months of age, a time in life when immune responses are reduced due to immunologic immaturity and the immunosuppressive effects of maternally-derived serum antibodies.[unreadable] We developed several recombinant vaccine candidates that have been evaluated in clinical trials. One virus, called rA2cp2484041030delSH, was safe and immunogenic in young infants. However, approximately one-third of the vaccine virus isolates recovered from vaccinees had evidence of a partial loss of the temperature-sensitive (ts) phenotype. Sequence analysis indicated that this was associated with reversion and loss of either the 248 or the 1030 point mutation present in the large polymerase L protein. Reversion can readily occur because each amino acid substitution is based on a single nucleotide substitution relative to wild type. Amino acid substitutions based on two or three nucleotide changes differences relative to wild type would be correspondingly less susceptible to reversion. We presently have constructed panels of viruses in which either the 248 or the 1030 locus has been modified so as to represent each of the 20 possible amino acid assignments, resulting in 20 viruses per locus. We are in the process of analyzing each of these to determine the ts phenotype in vitro and the attenuation phenotype in mice. With this information, and taking advantage of the degeneracy of the genetic code, we will choose a codon for each locus that encodes an attenuating amino acid assignment and differs by as many nucleotides as possible from codons for non-attenuating assignments. This should enable us to construct an improved version of rA2cp2484041030delSH with increased genetic and phenotypic stability. We also have been preparing additional attenuated versions of RSV under conditions suitable for making clinical material for evaluation in humans. One virus involves deletion of the interferon antagonist NS1 protein, and a second involves deletion of the M2-2 protein that is involved in regulating viral RNA synthesis. [unreadable] Pneumonia virus of mice (PVM) is a murine relative of RSV that has essentially the same genome organization and array of encoded viral RNAs and proteins. It replicates efficiently in the respiratory tract of mice and causes severe respiratory tract disease. Thus, it provides a potential surrogate for studying an RSV-like virus in a natural host that is easily manipulated and has extensive genetic and immunological reagents. This contrasts with RSV, which does not replicate efficiently in experimental animals other than the chimpanzee and thus is not amenable to pathogenesis studies. We have developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express the green fluorescent protein replicated as efficiently as the biological parent in vitro and replicated nearly as efficiently in mice. The G proteins of RSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant PVM mutants bearing a deletion of the entire G gene (delG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent but both were non-pathogenic in mice at doses that otherwise would be lethal. We could not detect replication of the delG mutant in mice, indicating that its attenuation is based on a severe reduction in virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant was not due to a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild type-like efficiency but does not cause disease defines optimal properties for vaccine development.