Equine Influenza vaccines based on conserved regions of the HA glycoprotein

IBAÑEZ LORENA ITATI; PAREDES ROJAS YESICA; CALDEVILLA CECILIA; MATTION NORA

Buenos Aires

Congreso; 10 th International equine Infectious Diseases Conference.; 2016

IEIDCX 2016

Current equine influenza vaccines elicit a strong humoral immune response against surface glycoproteins, in particular haemagglutinin (HA). However, the virus undergoes continuous evolution by mutation of sequences encoding amino acids, mostly those present in the exposed head domain of HA, which results in poor induction of cross-reactive antibodies against non-matching strains. In contrast, it has been reported that epitopes in the stem region of HA are highly conserved and can be used to stimulate a broader immune response. This work describes the generation of two immunogens based on the stem region of HA from the A/Equine/Argentina/93(H3N8) influenza strain: a recombinant protein expressed in prokaryotic cells (ΔHAp), and a DNA vaccine (ΔHAe). A DNA fragment encoding ΔHAp was cloned into the pET22b vector, and the recombinant protein was purified from inclusion bodies. The ΔHAe coding sequence was cloned into eukaryotic vectors that were transfected in HEK 293T cells. The expression of proteins was determined by Western blot assay. The immunogenicity of both vaccine candidates was studied using different combination of antigens in mice. Immunizations were done two times with a three-week interval. The DNA vaccine was given intramuscularly and the recombinant protein was given intraperitoneally in incomplete Freund?s adjuvant (Sigma). A group of five mice received two immunizations with 50 ug of DNA vaccine; a second group received two immunizations with 10 ug of recombinant protein; a third group was primed with 50 µg of DNA vaccine and boosted with 10 ug of recombinant protein; a fourth group was primed with 10 ug of protein and boosted with 50 µg of DNA vaccine; and a fifth group received only PBS. We found that immunization with two doses of recombinant protein was more immunogenic that vaccination with DNA only or combination of DNA and protein. In order to analyze cross-reactivity of the immune sera, ELISA plates were coated with 4 HA units of influenza strains belonging to groups 1 (H1N1) and 2 (H3N2). Plates coated with recombinant protein were used as a positive control. Higher antibody titers against viruses belonging to the same phylogenetic group were observed when mice were immunized with recombinant protein. However antibody titers were broader and reacted against viruses belonging to different phylogenetic groups when mice were immunized with DNA. Conclusions: Recombinant proteins were expressed correctly in prokaryotic and eukaryotic systems. Homologous antibody titers were higher in animals immunized with recombinant proteins, while DNA immunization induced a heterotypic immune response.