Replacement of the ectodomains of the hemagglutinin-neuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates

Abstract

We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955-2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503-510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.

FIG. 1

Construction of chimeric antigenomic cDNAs pFLC.PIV32TM and pFLC.PIV32TMcp45, which encode F and HN proteins containing PIV2-derived ectodomains and PIV3-derived transmembrane and cytoplasmic domains. The region of the PIV3 F ORF, in pLit.PIV3.F3a (A1), encoding the ectodomain was deleted (C1) by PCR using a PIV3 F-specific primer pair. The region of the PIV2 F ORF encoding the ectodomain was amplified from pLit.PIV32Fhc (B1) by PCR using a PIV2 F-specific primer pair. The two resulting fragments (C1 and D1) were ligated to generate pLit.PIV32FTM (E1). In parallel, the region of the PIV3 HN ORF, in pLit.PIV3.HN4 (A2), encoding the ectodomain was deleted (C2) by PCR using a PIV3 HN-specific primer pair. The region of the PIV2 HN ORF encoding the ectodomain was amplified from pLit.PIV32HNhc (B2) by PCR with a PIV2 HN-specific primer pair. Those two DNA fragments (C2 and D2) were ligated together to generate pLit.PIV32HNTM (E2). pLit.PIV32FTM and pLit.PIV32HNTM were digested with PpuMI and SpeI and assembled to generate pLit.PIV32TM (F), whose PIV insert was sequenced and confirmed in its entirety. The BspEI-SpeI fragment from pLit.PIV32TM was ligated to the BspEI-SpeI window of p38′ΔPIV31hc (G) to generate p38′ΔPIV32TM (H). The insert containing chimeric PIV3-PIV2 F and HN was introduced as a 6.5-kb BspEI-SphI fragment into the BspEI-SphI window of the full-length wild-type PIV3-PIV1 antigenomic cDNA, pFLC.2G+.hc, and the full-length cp45 PIV3 antigenomic cDNA, pFLCcp45, to generate pFLC.PIV32TM and pFLC.PIV32TMcp45 (I), respectively.

FIG. 2

Structures of the genomic RNAs of PIV3-PIV2 chimeric viruses, and junction sequences within the chimeric glycoprotein ORFs of rPIV3-2CT and rPIV3-2TM. (A) Structures of the genomic RNAs of the PIV3-PIV2 chimeric viruses (middle three schemes) are compared with that of rPIV3 (top), representing wild-type PIV3, and rPIV3-1 in which the PIV3 F and HN ORFs were replaced with those of PIV1 (bottom). The cp45 derivative of each virus which contains the 12 amino acid or nucleotide mutations are marked with arrows. For the cp45 derivatives, only the F and HN genes were different; the remaining genes, all from PIV3, remained identical. From top to bottom, the three PIV3-PIV2 chimeras carry an increasing amount of each PIV2 glycoprotein ORF. Note that rPIV3-2, carrying the complete PIV2 F and HN ORFs, was not recoverable. (B) Nucleotide sequences within the junctions of the chimeric F and HN glycoprotein ORFs for rPIV3-2TM, along with protein translation. The shaded portions represent sequences from PIV2. The amino acids are numbered with respect to their positions in the corresponding wild-type glycoproteins. Three extra nucleotides were inserted in PIV3-PIV2 HN TM as indicated to make the construct conform to the rule of six. (C) Nucleotide sequences of the junctions within the chimeric F and HN glycoprotein ORFs for rPIV3-2CT, along with protein translation. The shaded portions represent sequences from PIV2. The amino acids are numbered with respect to their positions in the corresponding wild-type glycoproteins. GE, gene end; I, intergenic; GS, gene start; TM, transmembrane domain; CT, cytoplasmic domain; *, stop codon; ntr, nontranslated region.

FIG. 3

Multicycle replication of chimeric PIV3-PIV2 compared with that of the wild-type parents rPIV3/JS and PIV2/V94. (A) rPIV3-2TM, rPIV3-2TMcp45, rPIV3/JS, and PIV2/V94 were used to infect LLC-MK2 cells in six-well plates, each in triplicate, at a multiplicity of infection of 0.01. All cultures were incubated at 32°C. After a 1-h adsorption period, the inocula were removed, and the cells were washed three times with serum-free OptiMEM I. The cultures were overlaid with 2 ml of the same medium per well. For rPIV3-2TM- and rPIV3-2TMcp45-infected cells, p-trypsin (0.5 μg/ml) was included. Aliquots of 0.5 ml were taken from each well at 24-h intervals for 6 days, flash-frozen on dry ice, and stored at −80°C. Each aliquot was replaced with 0.5 ml of fresh medium with or without p-trypsin as appropriate. The virus present in the aliquots was titered on LLC-MK2 plates by terminal dilution at 32°C for 6 days, and the endpoints were identified with hemadsorption. Virus titers are expressed as means ± standard errors. (B) rPIV3-2CT and rPIV3-2CTcp45, along with the wild-type parents rPIV3/JS and PIV2/V94 were analyzed as described for panel A.