Robust IgA and IgG-producing antibody forming cells in the diffuse-NALT and lungs of Sendai virus-vaccinated cotton rats associate with rapid protection against human parainfluenza virus-type 1

Abstract

Sendai virus (SeV), a natural mouse pathogen, shows considerable promise as a candidate vaccine for human parainfluenza virus-type 1 (hPIV-1), and also as a vaccine vector for other serious pathogens of infants including respiratory syncytial virus (RSV). In an effort to define correlates of immunity, we examined the virus-specific serum antibody of cotton rats inoculated intranasally (I.N.) with SeV. Virus-specific antibody forming cells (AFCs) were also measured in the bone marrow, because these are considered responsible for durable serum antibody levels in other viral systems. Results showed that a single SeV inoculation was sufficient to induce virus-specific serum antibodies and bone marrow-resident AFCs that persisted for as many as 8 months post-vaccination. Given that the predominant SeV-specific serum antibody isotype was IgG, an isotype that traffics poorly to the upper respiratory tract (URT), we asked if local nasal and lung-associated antibodies and AFCs were also present. Studies showed that: (i) SeV-specific antibodies appeared in the URT and lower respiratory tract (LRT) within 7 days after immunization, (ii) corresponding AFCs were present in the diffuse-NALT (d-NALT) and lung, (iii) AFCs in the d-NALT and lung peaked at approximately 6 weeks and persisted for the lifetime of the animal, reaching a level exceeding that of the bone marrow by an order of magnitude, (iv) IgA was the dominant isotype among AFCs in the d-NALT and lung at 4-weeks post-vaccination and thereafter, and (v) antibody and AFC responses associated with the prevention of lung infection when animals were challenged with hPIV-1 just 1 week after vaccination.

Figure 1. Long-sustained virus-specific IgG, but not IgA in the serum of SeV-primed cotton rats

Cotton rats were inoculated I.N. with 2 × 10⁶ EID₅₀ SeV. At various time points during an eight month period, groups of animals were tested for SeV-specific serum antibodies by ELISA. Values represent the mean and standard error for replicates from 2 animals per group.

Figure 2. Sustained virus-specific AFCs in the bone marrow but not spleen of SeV-primed cotton rats

Cotton rats were inoculated I.N. with 2 × 10⁶ EID₅₀ SeV and monitored for eight months thereafter for SeV-specific ELISPOTS in the bone marrow and spleen. The Y axis shows the ELISPOT number per 10⁵ plated lymphocytes. Values represent the mean and standard error for replicates from 2 animals per group.

Figure 3. IgA and IgG in the nasal wash of SeV-primed animals

Cotton rats were inoculated I.N. with 2 × 10⁶ EID₅₀ SeV and monitored for eight months thereafter for SeV-specific antibodies in the nasal wash by ELISA. Values represent the mean and standard error for replicates from 4 (IgG) and 2 (IgA) animals per group.

Figure 4. Sustained IgA and IgG AFC in the d-NALT

Cotton rats were inoculated I.N. with 2 × 10⁶ EID₅₀ SeV and monitored for eight months to measure AFC responses in the CLN and d-NALT. The Y axis shows the ELISPOT number per 10⁵ plated lymphocytes. Values represent the mean and standard error for replicates of 2 animals per group.

Figure 5. AFCs in d-NALT following SeV inoculation

Eight months following the inoculation of cotton rats with 2 × 10⁶ EID₅₀ SeV, AFCs were measured in respiratory and systemic tissues. Shown are representative results for (A) Bone marrow, (B) spleen, (C) CLN and (D) d-NALT.

Figure 6. IgA and IgG in the LRT

Cotton rats were inoculated I.N. with 2 × 10⁶ EID₅₀ SeV and monitored for eight months thereafter for SeV-specific antibodies in the BAL. Values represent the mean and standard error for replicates from 2 animals per set.

Figure 7. AFCs in the lung reflect virus-specific antibody in the BAL

Groups of cotton rats were inoculated I.N. with 2 × 10⁶ EID₅₀ SeV and monitored for eight months to measure AFC responses in the BAL and lung. The Y axis shows the ELISPOT number per 10⁵ plated lymphocytes. Values represent the mean and standard error for replicates of 2 animals per set.

Figure 8. Protection against challenge with hPIV-1 within seven days of vaccination

Cotton rat groups were inoculated I.N. with 3 × 10⁶ EID₅₀ SeV. After seven days, animals were challenged I.N. with hPIV-1 (C35). On day three, animals were sacrificed for lung titer. Each point represents the viral load in an individual animal with 4 animals per group. As shown, there was no lung infection in vaccinated animals, but all four PBS control animals were infected.