Chimeric Bovine/Human Parainfluenza Virus Type 3 Expressing Respiratory Syncytial Virus (RSV) F Glycoprotein: Effect of Insert Position on Expression, Replication, Immunogenicity, Stability, and Protection against RSV Infection

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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...

Section: ⌬170supporting

confidence: 80%

Section: ⌬170mentioning

confidence: 99%

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

Mackow

Amaro-Carambot

Liang

et al. 2015

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“…Similarly, subcutaneous vaccination of macaques with 10 4 infectious units of Moratenbased rMVs found that positions 3 and 6 induced equivalent immune responses for the parameters examined (34). However, another recent study showed that insertion of an ATU into positions 3 and 6 of a human parainfluenza virus 3 vaccine candidate caused a temperature-sensitive phenotype, resulting in attenuation of the virus in vivo (35). The lack of ability of rMV EZ EGFP(1) to induce comparable immune responses is in contrast to the situation with wild-type MVs, which tolerate insertion of an ATU in the same position upstream of the N ORF (6)(7)(8) and are pathogenic in macaques.…”

Section: Fig 3 Target Cells Of Rmv Ez Egfp(3) Egfp Distribution In Mmentioning

confidence: 99%

Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

Rennick

Vries

Carsillo

et al. 2015

Although live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb ( IMPORTANCE Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicates in vivo.This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

“…To determine the serum virus-neutralizing activity, sera from immunized hamsters were analyzed by a 60% plaque reduction neutralization test (PRNT 60 ) using green fluorescent protein (GFP)-expressing HPIV3 in the presence of guinea pig complement, as previously described (20). No significant differences in neutralization activity were detected for the three viruses (data not shown), which may reflect the dominance of HN as the major neutralization antigen.…”

Section: Effect Of Deletion Of the 8-nt M-ge Insert On Virus Replicatmentioning

confidence: 99%

The Aberrant Gene-End Transcription Signal of the Matrix M Gene of Human Parainfluenza Virus Type 3 Downregulates Fusion F Protein Expression and the F-Specific Antibody Response In Vivo

Lingemann

Surman

Amaro-Carambot

et al. 2015

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Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is a major viral cause of severe lower respiratory tract disease in infants and children. The gene-end (GE) transcription signal of the HPIV3 matrix (M) protein gene is identical to those of the nucleoprotein and phosphoprotein genes except that it contains an apparent 8-nucleotide insert. This was associated with an increased synthesis of a readthrough transcript of the M gene and the downstream fusion (F) protein gene. We hypothesized that this insert may function to downregulate the expression of F protein by interfering with termination/reinitiation at the M-F gene junction, thus promoting the production of M-F readthrough mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses from which this insert in the M-GE signal was removed were generated. The M-GE mutants exhibited a reduction in M-F readthrough mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wild-type (wt) HPIV3 both in vitro and in vivo. However, the F-proteinspecific serum antibody response in hamsters was increased for the mutants compared to wt HPIV3. This study identifies a previously undescribed viral mechanism for attenuating 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. IMPORTANCEThe HPIV3 M-GE signal was previously shown to contain an apparent 8-nucleotide insert that was associated with increased synthesis of a readthrough mRNA of the M gene and the downstream F gene. However, whether this had any significant effect on the synthesis of monocistronic F mRNA or F protein, virus replication, virion morphogenesis, and immunogenicity was unknown. Here, we show that the removal of this insert shifts F gene transcription from readthrough M-F mRNA to monocistronic F mRNA. This resulted in a substantial increase in the amount of F protein expressed in the cell and packaged in the virus particle. This did not affect virus replication but increased the F-specific antibody response in hamsters. Thus, in wild-type HPIV3, the aberrant M-GE signal operates a previously undescribed mechanism that reduces the expression of a major neutralization and protective antigen, resulting in reduced immunogenicity. This has implications for the design of live attenuated HPIV3 vaccines; specifically, the antibody response against F can be elevated by "repairing" the M-GE signal to achieve higher-level F antigen expression, with no effect on attenuation.H uman parainfluenza viruses (HPIVs) are human respiratory tract pathogens present as four serotypes, namely, HPIV1, HPIV2, HPIV3, and HPIV4. HPIVs infect and cause respiratory illness in humans of all ages worldwide, but their greatest impact...