Neonatal Genetic Delivery of Anti-Respiratory Syncytial Virus (RSV) Antibody by Non-Human Primate-Based Adenoviral Vector to Provide Protection against RSV

Abstract

Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infection in infants. Immunoprophylaxis with the anti-RSV monoclonal antibody, palivizumab, reduces the risk for RSV-related hospitalizations, but its use is restricted to high-risk infants due to the high costs. In this study, we investigated if genetic delivery of anti-RSV antibody to neonatal mice by chimpanzee adenovirus type 7 expressing the murine form of palivizumab (AdC7αRSV) can provide protection against RSV. Intranasal and intramuscular administration of AdC7αRSV to adult mice resulted in similar levels of anti-RSV IgG in the serum. However, only intranasal administration resulted in detectable levels of anti-RSV IgG in the bronchoalveolar lavage fluid. Intranasal administration of AdC7αRSV provided protection against subsequent RSV challenge. Expression of the anti-RSV antibody was prolonged following intranasal administration of AdC7αRSV to neonatal mice. Protection against RSV was confirmed at 6 weeks of age. These data suggest that neonatal genetic delivery of anti-RSV antibody by AdC7αRSV can provide protection against RSV.

Keywords: chimpanzee adenovirus type 7 (AdC7); genetic gene delivery; immunoprophylaxis; neonate; palivizumab; passive immunization; respiratory syncytial virus (RSV).

Figure 1

Schema of the chimpanzee adenovirus type 7 vector expressing murine anti-respiratory syncytial virus antibody (AdC7αRSV). The E1/E3 genes of AdC7 are deleted (ΔE1/ΔE3) and replaced by the expression cassette of the anti-RSV antibody cDNAs using the restriction enzyme sites I-CeuI and PI-SceI. The expression cassette includes the cytomegalovirus promoter (P CMV), followed by cDNAs encoding the anti-RSV light chain (LC), the poliovirus internal ribosome entry site (IRES), the anti-RSV heavy chain (HC), and SV40 polyadenylation signal (SV40 pA).

Figure 2

Expression of murine anti-respiratory syncytial virus (anti-RSV) IgG in vitro. Anti-RSV IgG in supernatants of A549 cells infected with chimpanzee adenovirus type 7 vector expressing murine anti-RSV IgG (AdC7αRSV) was detected by Western Blot analysis. (A) Expression of the full-length murine IgG under non-reducing conditions. (B) Expression of the heavy chain (HC) and light chain (LC) of murine IgG under reducing conditions. The supernatant of mock-infected cells was used as a negative control (lanes 2, 5). Mouse serum 8 weeks post infection with RSV was used as a positive control (lanes 3, 6). Detection was with a horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG and HRP chemiluminescence substrate.

Figure 3

Assessment of anti-respiratory syncytial virus (anti-RSV) IgG expression in vitro. Supernatants of HEK-293 cells infected with AdC7αRSV were assessed for the presence of functional anti-RSV IgG. (A) Binding to RSV. Supernatants were incubated with RSV Line19 or Ad5 (control) immobilized on a polyvinylidene difluoride (PVDF) membrane followed by an HRP-conjugated sheep anti-mouse IgG. Mouse serum 8 weeks post infection with RSV was used as a positive control (lane 4). (B) Plaque-reduction assay. Serial dilutions of supernatants were incubated with RSV Line19 (5 × 10³ pfu/mL) for 1 h, followed by infection of Vero cells. Data are shown as % reduction of plaques after 4 days, with mean ± SEM of 4 replicates.

Figure 4

Assessment of anti-respiratory syncytial virus (anti-RSV) IgG expression in adult mice. (A) Kinetics of anti-RSV IgG in the serum following intranasal (i.n.) or intramuscular (i.m.) administration of AdC7αRSV (5 × 10¹⁰ pu). Titers were measured by ELISA. Data are shown as mean ± SEM of 4 mice per group. (B) Anti-RSV IgG in the bronchoalveolar lavage (BAL) 1 week following administration of AdC7αRSV (5 × 10¹⁰ pu). Titers were measured by ELISA. Data are shown with mean ± SEM.

Figure 5

Protection against RSV infection following AdC7αRSV administration to adult mice. AdC7αRSV, AdC7GFP (5 × 10¹⁰ pu) or PBS (No AdC7 control) were intranasally administered to 8-week-old BALB/c mice, followed by RSV Line19 (10⁶ pfu) challenge 3 days later. (A) RSV viral loads in the lungs 4 days after the RSV challenge by plaque assay. (B) RSV genome expression in the lungs 4 days after the challenge by RT-qPCR. Data are shown with mean ± SEM. * and ** denote p < 0.05 and p < 0.01, respectively.

Figure 6

Protection against RSV infection following AdC7αRSV administration to neonatal mice. AdC7αRSV or AdC7GFP (6 × 10¹⁰ pu) were intranasally administered to 1-day-old BALB/c mice, followed by RSV A2 (10⁶ pfu) challenge at 6 weeks of age. (A) Anti-RSV IgG titer in serum before the RSV challenge. Serum was collected at 4 and 6 weeks, and titers were measured by ELISA. Data are shown with mean ± SEM. (B) RSV viral loads in the lungs 4 days after the RSV challenge by plaque assay. Data are shown with mean ± SEM. * and *** denote p < 0.05 and p < 0.005, respectively.