A Single-Dose Recombinant Parainfluenza Virus 5-Vectored Vaccine Expressing Respiratory Syncytial Virus (RSV) F or G Protein Protected Cotton Rats and African Green Monkeys from RSV Challenge

Abstract

Human respiratory syncytial virus (RSV) is a common cause of severe respiratory disease among infants, immunocompromised individuals, and the elderly. No licensed vaccine is currently available. In this study, we evaluated two parainfluenza virus 5 (PIV5)-vectored vaccines expressing RSV F (PIV5/F) or G (PIV5/G) protein in the cotton rat and African green monkey models for their replication, immunogenicity, and efficacy of protection against RSV challenge. Following a single intranasal inoculation, both animal species shed the vaccine viruses for a limited time but without noticeable clinical symptoms. In cotton rats, the vaccines elicited RSV F- or G-specific serum antibodies and conferred complete lung protection against RSV challenge at doses as low as 10³ PFU. Neither vaccine produced the enhanced lung pathology observed in animals immunized with formalin-inactivated RSV. In African green monkeys, vaccine-induced serum and mucosal antibody responses were readily detected, as well. PIV5/F provided nearly complete protection against RSV infection in the upper and lower respiratory tract at a dose of 10⁶ PFU of vaccine. At the same dose levels, PIV5/G was less efficacious. Both PIV5/F and PIV5/G were also able to boost neutralization titers in RSV-preexposed African green monkeys. Overall, our data indicated that PIV5/F is a promising RSV vaccine candidate.IMPORTANCE A safe and efficacious respiratory syncytial virus (RSV) vaccine remains elusive. We tested the recombinant parainfluenza virus 5 (PIV5) vectors expressing RSV glycoproteins for their immunogenicity and protective efficacy in cotton rats and African green monkeys, which are among the best available animal models to study RSV infection. In both species, a single dose of intranasal immunization with PIV5-vectored vaccines was able to produce systemic and local immunity and to protect animals from RSV challenge. The vaccines could also boost RSV neutralization antibody titers in African green monkeys that had been infected previously. Our data suggest that PIV5-vectored vaccines could potentially protect both the pediatric and elderly populations and support continued development of the vector platform.

Keywords: PIV5 vectors; respiratory syncytial virus; vaccines.

FIG 1

PIV5 replication in cotton rats. Cotton rats were infected intranasally with 1 × 10⁵ PFU of PIV5 in 10-μl or 100-μl volumes. At 4 and 6 days postchallenge, noses and lungs were harvested, and viral loads were determined by plaque assay. Each group consisted of 4 cotton rats. The bars represent the GMT of each group. The dotted line represents the limit of detection. The error bars indicate standard deviations.

FIG 2

Serum antibody titers of cotton rats vaccinated with PIV5/F or PIV5/G. Cotton rats were immunized intranasally with 10 μl of vaccines containing 1 × 10³, 1 × 10⁴, 1 × 10⁵, or 1 × 10⁶ PFU of PIV5/F, PIV5/G, or PBS. Sera were collected 28 days postimmunization, and IgG endpoint titers were determined by ELISA. Functional antibody activity was measured by a microneutralization assay. Each group consisted of 4 cotton rats. The bars represent the GMT of each group. The dotted line represents the limit of detection. The error bars indicate standard deviations. Each circle represents an individual animal.

FIG 3

Mucosal IgA responses in cotton rats immunized with PIV5/F or PIV5/G. Cotton rats were immunized intranasally with 1 × 10³, 1 × 10⁴, 1 × 10⁵, or 1 × 10⁶ PFU of PIV5/F, PIV5/G, or PBS. Lungs were collected at 21 days postimmunization. Titers of IgA specific to RSV F (A) and RSV G (B) in the lung homogenates were measured by ELISA. Each group consisted of 4 animals. The dotted line represents the limit of detection. The error bars indicate standard deviations.

FIG 4

Immunization with PIV5/F or PIV5/G protected cotton rats against RSV challenge. Cotton rats were immunized intranasally with 1 × 10³, 1 × 10⁴, 1 × 10⁵, or 1 × 10⁶ PFU of PIV5/F, PIV5/G, or PBS. At 21 days postimmunization, the animals were challenged with 10^5.5 PFU of RSV A2. Four days postchallenge, noses (A) and lungs (B) were harvested, and viral loads were determined by plaque assay. Each group consisted of 4 cotton rats. The bars represent the GMT of each group. The dotted line represents the limit of detection. The error bars indicate standard deviations.

FIG 5

PIV5, PIV5/F, and PIV5/G replication in African green monkeys. (A and B) African green monkeys were infected intranasally with 1 × 10², 1 × 10⁴, 1 × 10⁶, or 1 × 10⁸ PFU of PIV5. BAL (A) and nasopharyngeal swab (B) samples were collected on days 3, 5, 7, 10, and 14 postimmunization. (C and D) African green monkeys were immunized intranasally with 1 × 10⁴ or 1 × 10⁶ PFU of PIV5/F or PIV5/G or 1 × 10⁶ PFU of PIV5. BAL (C) and nasopharyngeal swab (D) samples were collected on days 3, 5, 7, 10, and 14 postimmunization. Viral loads from each animal at each time point were determined by plaque assay. Each group consisted of 4 monkeys. All the monkeys were PIV5 seronegative and RSV seronegative. Each bar represents the GMT of the group. The dotted line represents the limit of detection. The error bars indicate standard deviations.

FIG 6

Serum and mucosal antibody responses in RSV-naive African green monkeys immunized with PIV5/F or PIV5/G. African green monkeys were immunized intranasally with 1 × 10⁴ or 1 × 10⁶ PFU of PIV5/F, PIV5/G, or 10⁶ PFU of PIV5. (A to C) Sera were collected 3 days prior to immunization [d(−3)] and 21 days postimmunization (d21). The immunogenicity of the vaccine was determined by antibody titers against serum neutralization titers (A), RSV F protein (B), and RSV G protein (C). (D and E) Mucosal secretions were collected 3 days prior to immunization and 21 days postimmunization. IgA titers specific to RSV F (D) and RSV G (E) were measured by ELISA. Each group consisted of 4 PIV5- and RSV-seronegative monkeys. The dotted line represents the limit of detection. The error bars indicate standard deviations.

FIG 7

Cell-mediated immune responses in PIV5/F- or PIV5/G-vaccinated African green monkeys. PBMCs were collected from African green monkeys 21 days after intranasal immunization with 1 × 10⁶ PFU of PIV5/F, PIV5/G, or PIV5. The cells were mock stimulated or stimulated with pools of synthetic peptides representing RSV proteins F, G, and N or concanavalin A (ConA). The F peptides were divided into two pools, F1 (aa 1 to 295) and F2 (aa 285 to 574). Each group consisted of 4 PIV5- and RSV-seronegative monkeys. The solid horizontal lines represent the geometric means within each group. Values above the dotted lines represent positive responses.

FIG 8

Protection against RSV challenge by PIV5/F or PIV5/G in vaccinated African green monkeys. African green monkeys were immunized intranasally with 10⁴ or 10⁶ PFU of PIV5/F or PIV5/G or 10⁶ PFU of PIV5. At 28 days postimmunization, animals were challenged with 10^5.5 PFU of RSV A2 intranasally. Nasopharyngeal swabs and BAL samples were collected 3, 5, 7, 10, and 14 days postchallenge, and viral loads were determined by plaque assay. Each group consisted of 4 PIV5- and RSV-seronegative monkeys. The dotted line represents the limit of detection. The error bars indicate standard deviations.

FIG 9

Serum neutralizing antibody responses in RSV-exposed African green monkeys immunized with PIV5/F or PIV5/G. RSV-seropositive African green monkeys were immunized intranasally with 1 × 10⁶ PFU of PIV5/F, PIV5/G, or PIV5. Sera were collected 28 days postimmunization, and endpoint neutralizing antibody titers were determined. Each group consisted of 3 PIV5-seronegative, RSV-seropositive monkeys.

FIG 10

Cotton rat lung histopathology. Cotton rats were immunized intranasally with a single dose of 1 × 10⁶ PFU of PIV5/F or PIV5/G or two doses of FI-RSV at day 0 and day 21. Control animals were immunized with PBS. The animals were then challenged with 10^5.5 PFU of RSV A2 at day 49. Lung tissues were harvested 5 days postchallenge, fixed, and stained with hematoxylin and eosin. Lung pathological changes—alveolitis (A), interstitial pneumonitis (IP), perivasculitis (PV), and peribronchiolitis (PB)—are indicated by arrows.