Expression of recombinant rotavirus proteins harboring antigenic epitopes of the hepatitis a virus polyprotein in insect cells

Abstract

Rotavirus and hepatitis A virus (HAV) spread by the fecal-oral route and infections are important in public health, especially in developing countries. Here, two antigenic epitopes of the HAV polyprotein, domain 2 (D2) and domain 3 (D3), were recombined with rotavirus VP7, generating D2/VP7 and D3/VP7, cloned in a baculovirus expression system, and expressed in Spodoptera frugiperda 9 (Sf9) insect cells. All were highly expressed, with peak expression 2 days post-infection. Western blotting and ELISA revealed that two chimeric proteins were antigenic, but only D2/VP7 was immunogenic and elicited neutralizing antibody responses against rotavirus and HAV by neutralization assay, implicating D2/VP7 as a multivalent subunit-vaccine Candidate for preventing both rotavirus and HAV infections.

Keywords: Hepatitis A virus; Recombinant chimera protein; Rotavirus.

Fig. 1.. (A) Schematic representation of the two recombinant rotavirus proteins carrying antigenic epitopes of the HAV polyprotein. Recombinant gene cassettes were recombined in frame with a short peptide linker of Leu-Glu-Pro-Gly (LEPG). DNA regions of the vector plasmid are indicated by the filled circles (Օ ). (B) Expression of two recombinant rotavirus proteins using a baculovirus expression system in Sf9 cells. Whole cell lysates of infected Sf9 cells with individual recombinant baculoviruses were resolved by 10% SDS-PAGE. (C) Western blot analysis was performed using anti-V5 antibody and HRP-conjugated goat ani-mouse antibody.

Fig. 2.. Kinetics of recombinant baculovirus production of D2/VP7. Cells were harvested every 24 hrs during the experimental period of 10 days after the initial viral infection. Aliquots of infected Sf9 cells were clarified by low-speed centrifugation, lysed with RIPA buffer, and analyzed by Western blot. Lanes 1-10: Cells from 1 to 10 days post-infection.

Fig. 3.. Western blot analyses using anti-sera from either (A) rotavirus-infected rabbits or (B) HAV-infected human patients. The Western XP marker (Invitrogen, USA) is shown at the left. Numbers (21, 22 and 23) are patient serum numbers.

Fig. 4.. Production of the rotavirus-specific antibodies after experimental immunization of D2/VP7 against rabbits. Rabbit serum antibody response to rotavirus Wa strain was examined using ELISA. Rabbit serum antibody response to rotavirus Wa strain (A) and hepatitis a HM175 strain (B) was examined using ELISA (see Materials and Methods). The sera were serially diluted from 1:10 to 1:640 and analyzed at 492 nm. Negative rabbit serum (♦); Immunized rabbit serum (■); Rabbit serum against rotavirus Wa or goat anti HAV HM175 (▲). OD values are indicated at the right.