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. 2015 Apr 2:11:85.
doi: 10.1186/s12917-015-0399-4.

Protective immunity against influenza H5N1 virus challenge in chickens by oral administration of recombinant Lactococcus lactis expressing neuraminidase

Han Lei  1   2   3 , Xiaojue Peng  4 , Jiexiu Ouyang  5 , Daxian Zhao  6 , Huifeng Jiao  7 , Handing Shu  8 , Xinqi Ge  9
Affiliations

Protective immunity against influenza H5N1 virus challenge in chickens by oral administration of recombinant Lactococcus lactis expressing neuraminidase

Han Lei et al. BMC Vet Res. .

Abstract

Background: Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat in poultry. Current influenza vaccines predominantly focus on hemagglutinin (HA) which anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. However, Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. The aim of this study was to evaluate the protective immunity of NA based on Lactococcus lactis (L.lactis) expression system against homologous H5N1 virus challenge in a chicken model.

Results: L.lactis/pNZ2103-NA which NA is derived from A/Vietnam/1203/2004 (H5N1) (VN/1203/04) was constructed based on L.lactis constitutive expression system in this study. Chickens vaccinated orally with 10(12) colony-forming unit (CFU) of L.lactis/pNZ2103-NA could elicit significant NA-specific serum IgG and mucosa IgA antibodies, as well as neuraminidase inhibition (NI) titer compared with chickens administered orally with saline or L.lactis/pNZ2103 control. Most importantly, the results revealed that chickens administered orally with L.lactis/pNZ2103-NA were completely protected from a lethal H5N1 virus challenge.

Conclusions: The data obtained in the present study indicate that recombinant L.lactis/pNZ2103-NA in the absence of adjuvant can be considered an effective mucosal vaccine against H5N1 infection in chickens via oral administration. Further, these findings support that recombinant L.lactis/pNZ2103-NA can be used to perform mass vaccination in poultry during A/H5N1 pandemic.

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Figures

Figure 1
Figure 1
Construction of pNZ2103-NA and expression of NA protein on L.lactis . (A) A schematic diagram of constitutive plasmid pNZ2103-NA. (B) Western blot analysis of recombinant L.lactis/pNZ2103-NA expression. Lane 1: negative control L.lactis/pNZ2103; Lane 2: MagicMark™ XP Western Protein Standard; Lane 3: L.lactis/pNZ2103-NA. A specific protein band of around 54 kDa corresponding to NA was detected using anti-NA monoclonal antibody.
Figure 2
Figure 2
Oral administration of recombinant L.lactis /pNZ2103-NA induces NA-specific immune responses in chickens. Chickens were immunized orally with saline, L.lactis/pNZ2103 or L.lactis/pNZ2103-NA at day 0, 1, 2, 3 and day 17, 18, 19, 20. Sera (n = 16/group), intestine (n = 3/group) and upper respiratory (n = 3/group) washes were collected at day 15 and day 34 after the prime immunization. (A) NA-specific IgG antibody was measured by ELISA in the sera. (B) NA-specific IgA antibody was assessed in the intestinal washes. (C) NA-specific IgA antibody was assessed in the upper respiratory washes. (D) NI titers were determined using rNA protein. Data are presented as the means ± standard deviations (S.D.). The asterisk indicates a significant difference between L.lactis/pNZ2103-NA and other groups (saline or L.lactis/pNZ2103) (* p < 0.05).
Figure 3
Figure 3
Protection efficacy of L.lactis /pNZ2103-NA against H5N1 virus challenge. (A) Weight changes as a percentage. (B) Survival rate. (n = 10 per group).
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