Recombinant Lactococcus Lactis Expressing M1-HA2 Fusion Protein Provides Protective Mucosal Immunity Against H9N2 Avian Influenza Virus in Chickens

Abstract

H9N2 subtype low pathogenicity avian influenza virus (LPAIV) is distributed worldwide and causes enormous economic losses in the poultry industry. Despite immunization of almost all chickens with inactivated vaccines, the disease still remains widespread. We speculated that improving mucosal or cellular immune responses could contribute to improved control of H9N2 viruses. In this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the M1 and HA2 proteins derived from an antigenically conserved endemic H9N2 virus strain. The M1-HA2 fusion protein was cloned downstream of a gene encoding a secretory peptide, and we subsequently confirmed that the fusion protein was secreted from L. lactis by Western blotting. We assessed the immunogenicity and protective effects of this recombinant L. lactis strain. Eighty 1-day-old chickens were divided into four groups, and the experimental groups were orally vaccinated twice with the recombinant L. lactis strain. Fecal and intestinal samples, sera, and bronchoalveolar lavage fluid were collected at 7, 14, and 21 days post-vaccination (dpv). Chickens vaccinated with the recombinant L. lactis strain showed significantly increased levels of serum antibodies, T cell-mediated immune responses, and mucosal secretory IgA (SIgA). Following challenge with H9N2 virus at 21 dpv, chickens vaccinated with the recombinant L. lactis strain showed decreased weight loss, lower viral titers in the lung, and reduced lung pathological damage. In summary, our results demonstrated that a recombinant L. lactis strain expressing an H9N2 M1-HA2 fusion protein could induce protective mucosal and systemic immunity. This oral vaccine is H9N2 virus-specific and represents a significant design improvement compared with previous studies. Our study provides a theoretical basis for improving mucosal immune responses to prevent and control H9N2 virus infection.

Probiotics and probiotic-based vaccines: A novel approach for improving vaccine efficacy

“…The serum of animals that received L. lactis with cell wall-anchored RV VP8 antigen could inhibit viral infection in vitro by 100% and vaccinated mice developed significant levels of intestinal IgA antibodies in vivo (129). The oral vaccine with L. Lactis expressing a recombinant fusion protein of M1 and HA2 proteins derived from the H9N2 virus successfully induces protective mucosal and systemic immunity in eighty 1-day-old chickens (130). Mohseni et al employed L. lactis as a vector for expressing the codon-optimized human papillomavirus (HPV) -16 E7 oncogenes, and it showed cytotoxic T lymphocytes (CTL), and humoral responses after vaccination in healthy women volunteers with this probiotic-based vaccine (131).…”

Recent Progress in Recombinant Influenza Vaccine Development Toward Heterosubtypic Immune Response

“…M1 combination with other internal antigens such as NP and PB1 showed higher immune response and full protection ( 117 ). The fusion of M1 and HA2 (HA monomer subunit) in the L. lactis vector for oral vaccination in chickens induced antibodies, cytokines, and T-cell mediated immune response with comparable protective efficacy to that of H9N2 WVVs ( 235 ). However, M1 VLPs using adenoviral dodecahedron structures were able to efficiently enter and activate myeloid dendritic cells, facilitate antigen presentation, and induce CD8 + T cells ( 236 ).…”

Comparison of Immune Effects Between Brucella Recombinant Omp10-Omp28-L7/L12 Proteins Expressed in Eukaryotic and Prokaryotic Systems

“…Then added the FITC antimouse CD4 + and APC anti-mouse CD8 + dyes to samples to be tested, and placed them in a 4 • C refrigerator for 20 min after taking measures to prevent light. Measured the percentages of CD4 + and CD8 + T lymphocytes using flow cytometry (Guava Easy Cyte Mini, USA) (45).…”