Mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups A and B and human parainfluenza virus type 3 by using a live cDNA-derived vaccine based on a host range-attenuated bovine parainfluenza virus type 3 vector backbone

Abstract

Reverse genetics was used to develop a two-component, trivalent live attenuated vaccine against human parainfluenza virus type 3 (HPIV3) and respiratory syncytial virus (RSV) subgroups A and B. The backbone for each of the two components of this vaccine was the attenuated recombinant bovine/human PIV3 (rB/HPIV3), a recombinant BPIV3 in which the bovine HN and F protective antigens are replaced by their HPIV3 counterparts (48). This chimera retains the well-characterized host range attenuation phenotype of BPIV3, which appears to be appropriate for immunization of young infants. The open reading frames (ORFs) for the G and F major protective antigens of RSV subgroup A and B were each placed under the control of PIV3 transcription signals and inserted individually or in homologous pairs as supernumerary genes in the promoter proximal position of rB/HPIV3. The level of replication of rB/HPIV3-RSV chimeric viruses in the respiratory tract of rhesus monkeys was similar to that of their parent virus rB/HPIV3, and each of the chimeras induced a robust immune response to both RSV and HPIV3. RSV-neutralizing antibody titers induced by rB/HPIV3-RSV chimeric viruses were equivalent to those induced by infection with wild-type RSV, and HPIV3-specific antibody responses were similar to, or slightly less than, after infection with the rB/HPIV3 vector itself. This study describes a novel vaccine strategy against RSV in which vaccine viruses with a common attenuated backbone, specifically rB/HPIV3 derivatives expressing the G and/or F major protective antigens of RSV subgroup A and of RSV subgroup B, are used to immunize by the intranasal route against RSV and HPIV3, which are the first and second most important viral agents of pediatric respiratory tract disease worldwide.

FIG. 1.

Insertion of the G and F ORFs of RSV subgroup A or B into rB/HPIV3 as additional, promoter-proximal genes. The rB/HPIV3 antigenomic cDNA was modified previously by introduction of a unique BlpI site (boldface italic type) in the nontranslated region of the N gene preceding the start codon of the N ORF (49). PCR mutagenesis was used to add a PIV3 gene end (GE) (5′-AAGTAAGAAAAA), intergenic region (5′-CTT), and gene start (GS) (5′-AGGATTAAAG) signal immediately downstream of the stop codon of the G and F ORFs of subgroups A and B. An NheI site was added to the downstream end of each G ORF and on either side of each F ORF, and BlpI sites were added on either side of each insert. The modifications to the subgroup A ORFs were performed in previous work (49), and the subgroup B ORFs were modified in the same way in the present study. Each insert was cloned into the BlpI site in the rB/HPIV3 cDNA to create constructs containing the four single-gene insert viruses, namely, rB/HPIV3-G_A, rB/HPIV3-F_A, rB/HPIV3-G_B, and rB/HPIV3-F_B. To create the double-insert constructs rB/HPIV3-G_AF_Aand rB/HPIV3-G_BF_B the NheI fragment bearing each F ORF was excised from the rB/HPIV3-F_A and rB/HPIV3-F_B antigenomic cDNA and inserted into the NheI site of the rB/HPIV3-G_A and rB/HPIV3-G_B cDNAs, respectively. Elements of the viral promoter that, by analogy to Sendai virus (56), are present in the N gene nontranslated region are indicated by the three boxed hexamers, with important G residues shown in boldface type. Nucleotides that differ between the subgroup A and subgroup B constructs are shown on separate lines (top line for subgroup A constructs and bottom line for subgroup B constructs).

FIG. 2.

Transcription and translation of RSV G and F glycoprotein genes in PIV-RSV subgroup A chimeric viruses and in RSV. Accumulation of RSV G and F mRNAs (A) and proteins (B and C) was examined in HEp-2 cells infected with rB/HPIV3 (lanes 1), rB/HPIV3-G_A (lanes 2), rB/HPIV3-F_A (lanes 3), rB/HPIV3-G_AF_A (lanes 4), and RSV (lanes 5) at an MOI of 5, and cells were harvested at the indicated time points postinfection. mRNAs were detected with a mixture of RSV G and F specific ³²P-labeled DNA probes (A). The F mRNA transcribed from rB/HPIV3-F_A and rB/HPIV3-G_AF_A is shorter than that transcribed from RSV because the 5′ nontranslated region of F was deleted in the chimeric viruses. G and F glycoproteins were detected by a mixture of antisera raised against the cytoplasmic domain of RSV A2 F protein and against the ectodomain of the RSV A2 G protein (B). Glycoproteins were visualized by incubation with horseradish peroxidase-labeled goat anti-rabbit IgG antibodies. Since the F₁ subunit of the fusion glycoprotein and the partially glycosylated G glycoprotein have similar electrophoretic mobilities, an additional Western blot was stained with F-specific antiserum only (C).

FIG. 3.

Replication of rB/HPIV3-RSV subgroup A (panels A) and subgroup B (panels B) chimeras in the respiratory tract of rhesus monkeys. Monkeys were inoculated with one of the indicated rB/HPIV3-RSV chimeras, rB/HPIV3, or rHPIV3, as described in the text. The number (n) of animals in each group is indicated in parentheses. Mean daily titers in NP swab and TL fluid were determined by serial dilution on LLC-MK2 cell cultures, and the presence of virus in the culture was determined by hemadsorption. Titers are expressed as mean TCID₅₀ per milliliter, and the detection limit was 10 TCID₅₀/ml. Titers for rB/HPIV3-RSV subgroup A chimeras are shown in panels A, and those for subgroup B chimeras are shown in panels B. Top panels represent NP swabs (upper respiratory tract [URT]), and bottom panels represent TLs (lower respiratory tract [LRT]). The titers for the rHPIV3 and rB/HPIV3 viruses are included in both graphs for comparison. Specimens in which virus was not detected were assigned a value of 10^0.5 TCID₅₀/ml.