Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine

Abstract

Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously described attenuating mutation that was stabilized against deattenuation, specifically, a non-temperature-sensitive deletion mutation involving 6 nucleotides in the overlapping P/C open reading frames (ORFs) (C ⌬170 ) or a temperature-sensitive missense mutation in the L ORF (L Y942A ). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as the final titer of wild-type (wt) HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-C ⌬170 -F1, -F2, and -F3 and rHPIV1-L Y942A -F1. In hamsters, the rHPIV1-C ⌬170 -F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates, highly restricted in lungs, and genetically stable in vivo. Among the C ⌬170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-L Y942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of overattenuation. The C ⌬170 -F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. H uman respiratory syncytial virus (RSV) is the leading viral cause of severe acute respiratory infection (ARI) in infants and young children worldwide. RSV is an enveloped, nonsegmented, negative-strand RNA virus of the family Paramyxoviridae. RSV infects early in life and is responsible globally for an estimated 34 million annual pediatric cases of acute bronchiolitis and pneumonia, 4 million hospitalizations, and up to 199,000 pediatric deaths, 99% of which occur in the developing world (1). The human parainfluenza viruses (HPIVs) are also enveloped nonsegmented negative-strand RNA viruses of the family Paramyxoviridae. HPIV serotype 1 (HPIV1), HPIV2, and HPIV3 in particular are important agents of pediatric ARI and in aggregate are second in importance only to RSV (2). No vaccines are currently available for RSV or any of the HPIVs. Here we describe the development and evaluation of attenuated strains of HPIV1 as vectors to express the fusion (F) protein of RSV as a bivalent HPIV1/RSV vaccine.A formalin-inactivated RSV vaccine evaluated in infants and children in the 1960s was poorly protective and, paradoxically, primed for greatly enhanced RSV disease (3, 4). Markers for RSV disease enhancement have also been observed with purified RSV subunit vaccines in experimental animals (5, 6). Therefore, inactivated and subunit vaccines are considered contraindic...

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys

“…This similarity in expression between the pre-N and N-P positions suggests that the expected polar gradient in transcription was not observed, which also is consistent with our previous findings for RSV F protein expressed from HPIV1 (17). In contrast, Western blot analysis of sucrose-purified virions did show a gradient effect, with 6.3-fold more EBOV GP incorporated in the pre-N versus N-P viruses (Fig.…”

“…EBOV GP was detected with mouse anti-GP and anti-mouse IRDye 680 RD antibodies. Individual HPIV1 proteins were detected with rabbit polyclonal hyperimmune sera raised individually against peptides derived from HPIV1 N, P, F, and HN proteins, as previously described (17). Tubulin was detected on all blots, as a loading control, using mouse antitubulin and anti-mouse IRDye 680RD antibodies; a representative blot is shown.…”

“…The GP and GP-TMCT inserts in pre-N and N-P sites had the hexamer phasing of the N and P genes, respectively. All inserts were synthetically derived (Genscript) and cloned into the rHPIV1-C Δ170 antigenomic plasmid containing the unique restriction sites MluI and AscI in the pre-N and N-P positions, respectively, using published methods (17). This resulted in four constructs: the two with inserts in the pre-N position were called GP1 and GP-TMCT1, and the two with inserts in the N-P position were called GP2 and GP-TMCT2.…”

“…The P gene carries an additional overlapping open reading frame expressing the C proteins, which antagonize host interferon and apoptosis responses (15). We previously developed a non-temperature-sensitive attenuating mutation, called C Δ170 , that consists of a 6-nucleotide deletion in the overlapping P and C ORFs (16,17). This mutation reduces the ability of C proteins to inhibit the host type I interferon response and apoptosis (15,18,19), resulting in viral attenuation.…”

“…This mutation reduces the ability of C proteins to inhibit the host type I interferon response and apoptosis (15,18,19), resulting in viral attenuation. We previously evaluated HPIV1 as a vaccine vector for expressing the respiratory syncytial virus (RSV) fusion (F) glycoprotein and showed that the HPIV1-C Δ170 backbone was the better of two attenuated backbones (17).…”