Evaluation of a recombinant measles virus expressing hepatitis C virus envelope proteins by infection of human PBL-NOD/Scid/Jak3null mouse

Abstract

In this study, we infected NOD/Scid/Jak3null mice engrafted human peripheral blood leukocytes (hu-PBL-NOJ) with measles virus Edmonston B strain (MV-Edm) expressing hepatitis C virus (HCV) envelope proteins (rMV-E1E2) to evaluate the immunogenicity as a vaccine candidate. Although human leukocytes could be isolated from the spleen of mock-infected mice during the 2-weeks experiment, the proportion of engrafted human leukocytes in mice infected with MV (10(3)-10(5)pfu) or rMV-E1E2 (10(4)pfu) was decreased. Viral infection of the splenocytes was confirmed by the development of cytopathic effects (CPEs) in co-cultures of splenocytes and B95a cells and verified using RT-PCR. Finally, human antibodies against MV were more frequently observed than E2-specific antibodies in serum from mice infected with a low dose of virus (MV, 10(0)-10(1)pfu, and rMV-E1E2, 10(1)-10(2)pfu). These results showed the possibility of hu-PBL-NOJ mice for the evaluation of the immunogenicity of viral proteins.

Fig. 1

Construction of the recombinant MV vectors. (A) The rMV full genome vector derived from the MV-Ed strain is illustrated in the upper panel and is labelled with letters as follows: N, nucleocapsid; P, phosphoprotein; M, matrix; F, fusion; H, hemagglutinin; and L, large. T7 indicates the T7 RNA polymerase promoter. The cDNA encoding the HCV envelope glycoproteins (E1 and E2) containing the signal peptide sequence (SP) and the transmembrane domain (TMD, underlined) regions, the N gene end signal (E), the P gene start signal (S), and the intercistronic region of the H protein genes at the 5′ end, which was flanked by Fse I sites at both ends, was introduced into the unique Fse I site in between the N and P genes in the pMV vector. The resulting plasmid was designated pMV-E1E2. (B) The HCV E1 and E2 proteins were detected in rMV-E1E2-, rMV-Ed- and mock-infected B95a cells by western blot with MoAb 384 and 544 (arrows). (C) rMV-E1E2-infected B95a cells were stained with MoAb 299 (anti-E1) or MoAb 187 (anti-E2) and analysed by immunofluorescence. Nuclei were stained with Topro-3 and the bright field and merged images are indicated (400×).

Fig. 2

Infection of hu-PBL-NOD/Scid mice with rMV and rMV-E1E2. (A) Course of infection of hu-PBL-NOD/SCID mice with MV and rMV-E1E2. (B) CPE formation in co-cultures of splenocytes isolated from MV- (MV1, 2), rMV-E1E2-, or mock-infected hu-PBL-NOD/SCID and B95a cells (40× magnification). (C) Detection of viral RNA by RT-PCR. Detection of MV in MV-1- or 2-infected mouse splenocyte co-cultures (820 bp) and rMV-E1E2-infected splenocyte co-cultures (564 bp), and HCV E1 (263 bp) and E2 (360 bp) in rMV-E1E2 (10⁴ pfu)-infected splenocyte co-cultures (arrows).

Fig. 3

Flow cytometric analysis of splenocytes isolated from hu-PBL-NOJ mice inoculated with medium, MV-Ed (10⁰–10⁴ pfu), or rMV-E1E2 (10⁰–10² or 10⁴ pfu). Splenocytes, consisting of both human and murine cells, were stained with antibodies against human or mouse CD45. Representative flow cytometric profiles of each group of infected mice are shown. The percentages of mouse and human leukocytes are shown.

Fig. 4

Detection of human MV-specific antibodies in the serum of rMV- or rMV-E1E2-infected mice. (A) Serum (1:100) from MV-infected mice (10⁰–10¹ pfu) was analysed by ELISA using an MV-infected B95a cell lysate as the target. An anti-MV-NP antibody was used as a positive control and NMS indicates normal mouse serum. The asterisk (*) indicates a significant reaction (p < 0.01) compared to the medium alone control. (B) Serum (1:100) from rMV-E1E2-infected mice (10¹–10² pfu) was analysed by ELISA. An anti-MV-NP antibody was used as a positive control and NMS indicates normal mouse serum. The double asterisk (**) indicates a highly significant reaction (p < 0.001) compared to NMS and a single asterisk (*) indicates a significant reaction (p < 0.05) compared to NMS.

Fig. 5

Detection of E2-specific antibodies using ELISA and western blot. (A) Baculovirus-expressed E2 protein was used as the ELISA antigen and serum (diluted 1:100) from rMV-E1E2-infected mice (10¹–10² pfu) was analysed. Anti-E2 monoclonal antibody (MoAb 544) was used as positive control. The asterisk (*) indicates a significant reaction (p < 0.05) compared to NMS. (B) An anti-E2 monoclonal antibody (MoAb 544), serum from rMV-E1E2 infected mice (1:100), or normal mouse serum (1:100) was used as primary antibodies in a western blot to detect baculovirus-expressed E2 protein. The triangles indicate bands that correspond to HCV E2.