Lentivector expressing HBsAg and immunoglobulin Fc fusion antigen induces potent immune responses and results in seroconversion in HBsAg transgenic mice

Abstract

Even though hepatitis B virus(HBV) vaccines effectively prevent new cases of HBV infection, with approximately 350 million patients worldwide, chronic HBV infection remains a major health problem because of the associated complications (such as liver cirrhosis and hepatocellular carcinoma) and the limited treatment options. Immunotherapy has the potential to effectively control HBV replication. In this current study, we found that recombinant lentivectors could induce potent HBV surface antigen (HBsAg) specific T cell responses and humoral immune responses. Tagging the HBsAg with immunoglobulin Fc fragment further substantially increased the HBsAg specific immune responses. Remarkably, the HBS-Fc-lv lentivector could effectively break immune tolerance and induce potent HBsAg specific adaptive immune responses in HBsAg transgenic (Tg) mice with low serum level of HBsAg. More importantly, the induction of HBsAg specific immune responses in Tg mice accompanied seroconversion from HBsAg to anti-HBsAg antibody (anti-HBsAb). Our study demonstrated the potential of utilizing lentivector to treat chronic HBV infection following reduction of viral load with antiviral drug therapy.

Fig.1

Tagging of HBsAg with the Fc fragment of mouse IgG2a increases the HBsAg level. A. Schematic structure of recombinant lentivector HBS-lv and HBS-Fc-lv. HBsAg gene and HBS-Fc fusion gene were inserted behind the CMV promoter in the recombinant lentivector. B. Increase of the expression and secretion of HBsAg after transduction. 293T cells were transduced with either HBS-lv or HBS-Fc-lv lentivector. Untransduced 293T cells were used as control. Two days after transduction, HBsAg level in the supernatant and cell lysate was examined by ELISA. The data value was presented as OD450 in the total cell lysate or supernatant. The mean±SEM was indicated on the figure by the central value and error bar (95% confidence limit). Statistiacal anlysis was done using unpaired t-test. The experiment was repeated three times with similar data.

Fig.2

Lentivector HBS-Fc-lv immunization stimulates potent CD8 T cell responses. Female C57BL/6 mice (5 mice in each group) were immunized with lentivector HBS-lv or HBS-Fc-lv on footpad. For DNA immunization, intramuscular injection was performed. Two weeks later, immune responses were examined. A. Intracellular staining of the IFNγ among CD8 T cells in the peripheral blood after brief (3hr) ex vivo stimulation with HBS peptide. The numbers on the upper right quadrant represent the percentage of IFNγ producing CD8 T cells out of total CD8 T cells. B. The in vivo CTL activity was assayed on day 14 by in vivo killing assay. Only the CFSE+ cells were shown in the histogram. The numbers represent the percentage of each CFSE+ cell population. The experiment was reproduced at least 5 times with similar observation. C. A summary of the IFNγ+ CD8 from five mice in each group is presented. The mean±SEM was indicated on the figure by the central value and error bar (95% confidence limit). Unpaired t-test was used for statistical analysis.

Fig.3

Lentivector HBS-Fc-lv immunization elicits CD4 T cell responses and humoral immune responses. A. To examine the CD4 T cell responses, peripheral blood cells were stimulated with HBsAg whole protein before intracellular staining of IFNγ and surface staining of CD4. Only the Thy1.2+CD4+ T cells were shown. The numbers indicate the percentage of each cell population. A summarized data from 5 mice was also presented. B. The humoral immune response was determined by the anti-HBsAb level using ELISA. The data value was presented as OD450/ml of serum. A summary of 10 immunized mice was presented. The mean±SEM was indicated on the figure by the central value and error bar (95% confidence limit). Unpaired t-test was used for analysis.

Fig.4

The CD4 and humoral immune responses but not CD8 T cell responses are affected by the Fc receptor. To determine if the HBS-Fc-lv induced immune responses were mediated by Fc receptor, five wt C57BL/6 mice and FcRγ KO (also on C57BL/6 background) mice were immunized with HBS-Fc-lv. A. The CD8 T cell response was not affected by Fc receptor knockout. B and C. CD4 T cell response and humoral immune response were severely compromised in the Fc receptor KO mice. Only the indicated cell population was shown in the dot plot. The anti-HBs Ab value was presented as OD450/ml of serum. A summary of data from 5 mice was presented. The mean±SEM was indicated on the figure by the central value and error bar (95% confidence limit). Unpaired t-test was used for statistical analysis.

Fig.5

HBS-Fc-lv immunization can break immune tolerance in Tg mice expressing low level of HBsAg. A. Offspring of HBsAg Tg mice from Jackson Laboratory could be divided into two groups based on the serum level of HBsAg (upper figure 5A): HBsAg^high (OD450>1.0) and HBsAg^low (0.1>OD450>0.05) mice; the cutoff value of OD450 is 0.0405. The HBsAg level in the liver of HBsAg Tg mice was also determined. Although significantly lower than HBsAg^high, Tg-lo mouse liver contained a definitive level of HBsAg (lower part of Fig 5A). B. Following lentivector HBS-Fc-lv immunization, CD8 T cell immune responses were detected in peripheral blood and liver of both WT (normal C57BL/6) mice and HBsAg^low Tg mice. However, no CD8 response was detected in the HBsAg^hi Tg mice. A summary of 5 mice was also presented. Unpaired t-test was utilized for analysis. C. In vivo CTL activity was determined by in vivo killing assay. The Ctrl mice (normal C57BL/6 without treatment) and the HBsAg^hi Tg mice did not show any in vivo killing activity), while immunized WT mice had high level of CTL activity. A lower level of in vivo killing activity was detected in the HBsAg^low Tg mice. A summary of 4 mice was also shown. The mean±SEM was indicated on the figure by the central value and error bar (95% confidence limit). Unpaired t-test was used for analysis.

Fig.6

HBS-Fc-lv immunization results in seroconversion of HBsAg to anti-HBsAb. A. HBS-Fc-lv immunization reduced the serum HBsAg in the HBsAg^low Tg mice to become negative (left column). Although it was unable to fully convert liver HBsAg to negative, immunization also decreased the HBsAg level compared to untreated HBsAg^low Tg mice. B. Anti-HBs Ab could be detected in the HBsAg^low Tg mouse serum after but not before HBS-Fc-lv immunization. Data from 7 mice was presented. The ELISA data value was presented as OD450/ml of serum. C. Compared to control (non-immunized) HBsAg^low Tg mice, no significantly increase of serum ALT was found in the immunized HBsAg^low Tg mice. The mean±SEM was indicated on the figure by the central value and error bar (95% confidence limit). Statistical analysis was performed using unpaired t-test.