Recombinant respiratory syncytial viruses lacking the C-terminal third of the attachment (G) protein are immunogenic and attenuated in vivo and in vitro

Abstract

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 rA2cpDeltaG118, rA2cpDeltaG174, rA2cpDeltaG193, and rA2cpDeltaG213. All rA2cpDeltaG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37 degrees C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpDeltaG 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 rA2cpDeltaG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpDeltaG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

FIG. 1.

Construction of rRSV antigenomic cDNA with altered G protein. Large cDNA fragments of total RSV genomic RNA (denoted le-P, M-M2, L, and tr) were sequentially cloned into plasmid pFL. A clone of the M-M2 fragment, containing the M, SH, G, F, and M2 genes of RSV, was genetically altered by PCR mutagenesis such that G protein gene sequences encoding amino acids C terminal of amino acid (aa) 118, 174, 193, or 213 were excluded from PCR amplification. These truncations are denoted below the schematic representation of G protein. The mutated M-M2 fragment was used to replace the corresponding fragment in the full-length cDNA clone and along with helper RSV plasmids was transfected into Vero cells for virus rescue and purification. CT, cytoplasmic tail; TM, transmembrane region; P, proline residues; C, cystine residues; stalks with circles, potential O-linked carbohydrate acceptor sites; N, potential N-linked carbohydrate acceptor sites.

FIG. 2.

MAb mapping of rRSV strains with altered G protein. Correct truncations were confirmed by using MAbs 131-2G, L9, and 130-2G, which are reactive with epitopes within regions respectively spanned by amino acids (aa) 1 to 118, 174 to 213, and 213 to 298 of G protein. Antibody binding was detected by plaque assay as described in Materials and Methods. 100% denotes amino acids 164 to 176 conserved among all RSV strains. Vertical bars, potential O-linked carbohydrate acceptor sites; “bells,” potential N-linked carbohydrate acceptor sites.

FIG. 3.

Plaque morphology of rRSV strains with altered G protein. Monolayers of HEp-2 cells were infected with the indicated virus and cultured for 3 to 4 days at the denoted temperature. The plaques were visualized by immunostaining for F protein as described in Materials and Methods.

FIG. 4.

Ratios of total RSV genome copy number to total shed virus. A549 cell monolayers were infected with RSV (MOI of 0.09), and 72 h thereafter, genome copy numbers were determined by qPCR. Infectious virus titers in the A549 culture supernatants were ascertained on HEp-2 cell monolayers by plaque assay. (A) RSV genome copy per PFU in cells infected with A2, cp-RSV, rRSV, or cpts248/404 and cultured at 37°C. (B) RSV genome copy per PFU in cells infected with cp-RSV, rA2cpΔG213, cpts248/404, or rA2cpts248/404ΔSH and cultured at 32°C.

FIG. 5.

Replication of rRSV strains in the respiratory tract of BALB/c mice. Naïve BALB/c mice were infected (∼10⁶ PFU) with the indicated viruses. Lung and nasal tissues were collected 4 (A) and 7 (B) days thereafter for the determination of infectious virus titer (log₁₀ per gram of tissue) by plaque assay. Infectious virus was not detected in nasal tissues on day 7 and so is not shown. There were five mice per group. The lower limit of detection for both data sets was approximately1.5 log₁₀.