Development of mucosal vaccines for avian influenza A virus composed of recombinant Lactococcus lactis and Streptococcus gordonii

Novel mucosal vaccines (LL-M2e, LL-HAe, SG-HAe) were constructed from live, non-pathogenic Lactococcus lactis or Streptococcus gordonii that express conserved regions of HA or M2 antigens from avian influenza virus (AIV) A. All three vaccines evoked antigen-specific serum immunoglobulin G (IgG) responses in vaccinated chickens. The addition of the adjuvant cholera toxin B (CTB) increased the antigen specific IgG response. Fecal IgA and cell mediated immune responses were not detectable in the chickens. LL-M2e provided significant protection against lethal challenge compared to mock-vaccinated chickens. None of the chickens vaccinated with the control strain (LL-PIP) or mock-vaccine survived lethal challenge past 5 days, whereas 2 of the 12 chickens vaccinated with LL-M2e survived at least 7 days post-infection. An M2e-specific serum IgG concentration of 1100 ng/ml was found to be the minimum required for increased protection against AIV infection. SG-HAe, but not LL-HAe, provided a significant increase in the number of chickens that survived lethal challenge compared to control-vaccinated birds. However, no correlation between HAe-specific serum IgG and survival was found

A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface of Lactococcus lactis (LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLHM2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly ( < 0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG

Bacterial Resistance to Antisense Peptide-Phosphorodiamidate Morpholino Oligomers

Peptide phosphorodiamidate morpholino oligomers (PPMO) are synthetic DNA mimics that bind complementary RNA and inhibit bacterial gene expression. (RFF)₃RXB- AcpP PPMO (R, arginine; F, phenylalanine; X, 6-aminohexanoic acid; B, β-alanine) is complementary to 11 bases of the essential gene acpP (encodes acyl carrier protein). The MIC of (RFF)₃RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)₃RXB-AcpP was 4 x 10⁻⁷ mutations/cell division. A spontaneous (RFF)₃RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)₃RXB-AcpP was 40 μM (224 μg/ml) in PR200.1. The MICs of standard antibiotics were identical in PR200.1 and W3110. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)₃RXB or peptides with a similar composition or pattern of cationic and non-polar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)₃RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)₃RXB-AcpP. Deletion of sbmA caused resistance to (RFF)₃RXB-AcpP. We conclude that resistance to (RFF)₃RXB-AcpP was linked to the peptide and not the PMO, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)₃R-XB PPMOs may be transported across the plasma membrane by SbmA