Human parainfluenza viruses 1, 2, and 3 are significant causes of severe pediatric respiratory tract disease worldwide. The HPIVs are enveloped, non-segmented, negative strand RNA viruses of the family Paramyxoviridae. The broad outlines of their biology and molecular genetics have been defined in previous studies by this laboratory and others. The HPIV genome encodes a nucleoprotein N, phosphoprotein P, large polymerase protein L, internal matrix protein M, and fusion F and hemagglutinin-neuraminidase HN transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and the major protective antigens. In addition, the P gene encodes various accessory protein(s)from one or more additional ORFs: C (HPIV1), V (HPIV2), and C, D, and possibly V (HPIV3). These accessory proteins have a number of functions that antagonize the host response to viral infection, as described in previous years. We are developing attenuated versions of HPIV1, 2, and 3 that also express the fusion F protein of human respiratory syncytial virus (RSV). RSV is the most important viral agent of severe pediatric respiratory tract disease, with a contribution to human disease comparable to that of the HPIVs combined, and the F protein is the major RSV neutralization and protective antigen. HPIV1, 2, and 3 expressing the RSV F protein would provide bivalent vaccines against each respective HPIV and RSV. Compared to RSV strains, the HPIVs replicate more efficiently in cell culture and have much greater physical stability. They also form spherical particles compared to the large filaments of RSV, making them more amenable to filtration and other steps in manufacture. These attributes make HPIV vectors much easier to manufacture, distribute, store, and use compared to attenuated RSV strains. The greater physical stability in particular may be essential for extending RSV vaccines to resource-challenged countries. Furthermore, in experimental animals, boosting RSV responses was more efficient using HPIV/RSV vectors as opposed to attenuated RSV strains, since the latter are subject to greater restriction by prior RSV-specific immunity. We have been evaluating a number of parameters of vaccine vector design using, as proof of principle, an attenuated HPIV3 virus called B/HPIV3 This consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3, yielding a chimeric virus that is attenuated in primates due to the bovine backbone and bears the neutralization and major protective F and HN antigens of HPIV3. Previously, B/HPIV3 has been evaluated in clinical phase 1 studies, both as an empty vector (LID/NIAID study) and as a vector for RSV-F (MedImmune study), and was shown to be well-tolerated in either role in infants and young children. In the initial clinical study of B/HPIV3-RSV-F, the RSV F insert exhibited substantial instability and was not as immunogenic as hoped. Our goal therefore has been to increase the immunogenicity and stability of the RSV F insert. We previously evaluated the effects of the position of insertion of the RSV F gene into the B/HPIV3 backbone, and found that the first (pre-N) and second (N-P) gene positions readily accommodated the RSV F insert. This resulted in greatly increased expression of RSV F protein compared to downstream locations (up to 69-fold increase) and greatly increased fusion. Surprisingly, this did not appear to interfere with vector replication in vitro or in hamsters. In the past year, we found that expression of the RSV F protein was further enhanced 5-fold by codon-optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypo-fusogenic phenotype that presumably reflects the original clinical isolate, and suggests that this strain of RSV may have mutated to acquire a hyper-fusogenic phenotype during passage in vitro. We then compared vectors expressing stabilized pre- and post-fusion versions of RSV F protein. In a hamster model, pre-fusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wt RSV challenge, compared to native F. In contrast, vector expressing the post-fusion F was more immunogenic and protective than native RSV F, but less than pre-fusion F. Use of a double-staining immunofluorescence assay showed that the stability of expression of the RSV F protein was high and was not affected by enhanced expression or the pre- or post-fusion conformations of RSV F. These studies provide an improved version of the rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. HPIV1 also was developed as a vector for RSV F during the past year. The RSV F gene was inserted individually into three different genome locations (pre-N (F1), N-P (F2), or P-M (F3)) in each of two attenuated rHPIV1 backbones. Each backbone contained a single previously-described attenuating mutation that was stabilized against de-attenuation: (1) a non-temperature-sensitivity deletion mutation involving six nucleotides in the overlapping P/C ORFs (Cdel170), or (2) a temperature-sensitivity missense mutation in the L ORF (LY942A). In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as wt HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-Cdel170-F1, -F2, and -F3, and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-Cdel170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates and highly restricted in lungs, and were genetically stable in vivo. Among the Cdel170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of over-attenuation. The Cdel170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. We also investigated regulation of gene expression in HPIV3. The gene end (GE) transcription signals of the HPIV3 genes are highly conserved except that the M GE signal contains an apparent 8-nucleotide insert. This is associated with increased synthesis of a read-through transcript of the M gene plus the downstream F protein gene. We hypothesized that this insert may function to down-regulate expression of F protein by interfering with termination/re-initiation at the M-F gene junction, thus promoting the production of M-F read-through mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses were generated from which this insert in the M-GE signal was removed. The M-GE mutants exhibited a reduction in M-F read-through mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in the infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wt HPIV3 both in vitro and in vivo. However, the F protein-specific serum antibody response in hamsters was increased for the mutants as compared to wt HPIV3. This study identifies a novel viral mechanism for reducing stimulation of the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation.