New generation live vaccines against human respiratory syncytial virus designed by reverse genetics

Abstract

Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non-temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN alpha/beta antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use.

Figure 1.

Respiratory syncytial virus (RSV) genome and gene products. The electron micrographs (50) show an RSV virion budding from the plasma membrane of an infected cell (left) and a free virion (right), with the viral proteins indicated. The viral genome is diagramed at the bottom. Each large shaded rectangle indicates a gene encoding a separate mRNA. The two overlapping open reading frames (ORF) of the M2 mRNA are indicated. “Le” and “tr” refer to leader and trailer, respectively, which are small extragenic regions at each end of the genome. The leader region contains the genome promoter. The amino acid lengths of the RSV proteins are: NS1, 139; NS2, 124; N, 391; P, 241; M, 256; SH, 64; G, 298; F, 574; M2-1, 194; M2-2, 90; L, 2,165.

Figure 2.

Recovery of infectious recombinant RSV entirely from cloned cDNAs. The five cDNAs are indicated by shaded rectangles and encode a positive-sense copy of the viral genome (antigenome) and mRNAs for the N, P, L, and M2-1 proteins of the nucleocapsid/polymerase complex. Expression of the cDNAs is under the control of a promoter for bacteriophage T7 RNA polymerase (T7pr). The plasmid backbones that bear the cDNAs are not shown. The indicated ribozyme encoded at the downstream end of the antigenome executes self-cleavage to produce a correct 3′ end. The T7 RNA polymerase can be provided intracellularly using one of the three indicated strategies.

Figure 3.

Mean peak virus titers present in nasopharyngeal swabs (left panel) and tracheal lavages (right panel) of chimpanzees that had been infected simultaneously by the intranasal and intratracheal routes with the indicated recombinant wild-type or attenuated RSVs. The cp248/404 virus is a recombinant version of the biologically derived cpts248/404 virus that retained mild residual virulence in RSV-naive infants and serves as a benchmark. The dotted line indicates the limit of detectability.

Figure 4.

Current live recombinant RSV vaccine candidates based on attenuated RSV (A–D) or on the attenuated bovine/human parainfluenza virus (B/HPIV) type 3 virus expressing RSV F from an inserted gene (drawn immediately over its site of insertion). RSV genes and ORFs are represented by lightly shaded rectangles, BPIV3 genes are represented by open rectangles, and HPIV3 genes are represented by darkly shaded rectangles. Gene deletions are demarcated with dotted vertical lines. The locations of attenuating cp, 248, 404, and 1030 mutations are indicated.