Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver

Abstract

Background & aims: Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear.

Methods: We performed studies with BALB/c Rag2^-/-Il2rg^-/-Sirpa^NODAlb-uPA^tg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells.

Results: Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation.

Conclusion: In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

Keywords: CXCL10; HBe; IL10; Mouse Model; NK Cell.

Figure 1

Viral progression is controlled in immunocompetent HIS-HUHEP mice. (A and B) HBV viremia (black line) and hAlbumin (grey bars) were measured in the plasma of HBV-infected HUHEP (A) and HIS-HUHEP (B) mice inoculated at 10e9. Means of HUHEP (n=4) and HIS-HUHEP (n=12) mice. (C and D) Analysis of viral load: for each plasma sample viremia was plotted against the hAlbumin concentration from HUHEP (C) (n=4) or HIS-HUHEP mice (D) (n=12). (E) Analysis of viral progression over time by linear regression analysis of the ratio of HBV DNA over hAlbumin concentration, plotted against time post infection, from HUHEP (dotted line) or HIS-HUHEP mice (filled line). The P value determines whether the slopes of each linear regression are significantly different from each other. (F and G) Plasma viral antigen loads (HBeAg and HBsAg) correlated with HBV viremia in HBV-infected HIS-HUHEP mice. (H, I, and J) Quantification of cccDNA and total HBV DNA in the liver of infected HIS-HUHEP mice. The ratio of total liver HBV DNA/cccDNA indicates the virus’ replicative activity (n=3). Bars show mininum to maximum, line at the mean. (K) Human hepatotoxicity (M65) was normalized to the degree of human liver chimerism (hAlbumin) per mouse. The fold change of M65/hAlbumin pre- and post-infection (19 ±4 wpi) are shown for each group (control n=8; HBV 10e7, n=7; HBV 10e9, n=4) Histograms show means and SEM. Statistical significance: Mann Whitney U test. Correlation analysis: r² and P values calculated using 2-tailed Pearson's χ² test.

Figure 2

Human immune cells are recruited to the liver in chronically infected HIS-HUHEP mice. Immunofluorescence analysis of liver sections from control (top panels) and HBV-infected (bottom panels) mice co-stained for hAlbumin (blue) and HBc (green), with either hCD45 (red), or hCD3 (red), or hCD68 (red). DAPI-stained nuclei are shown in grey. Scale bar represents 100 μm.

Figure 3

NK-cell mediated immune response in HBV-infected HIS-HUHEP mice. (A) Absolute numbers of total human leukocytes (hCD45+) and (B) NK cells (CD3^-NKp46⁺) from the liver and spleen of control and HBV-infected mice. Representative FACS plots show the percent positive cells in each gate. (C) Frequency of CD69⁺ cells among NK cells in the liver and spleen from control (grey filled line) and HBV-infected (black open line) HIS-HUHEP mice. The percentage of positive cells from HBV-infected mice is shown. (D) Expression of CD56 and CD16 in NK cells from the liver of control or HBV-infected HIS-HUHEP mice. (E) Liver leukocytes or splenocytes were restimulated ex vivo overnight and analyzed by FACS for the expression of IFN-γ or TNF-α by NK cells. Histograms show the mean and SEM. In plots each dot represents a mouse, data obtained at 14–20 wpi. Statistical analysis in A–D: Mann Whitney U test, E: 2 way ANOVA.

Figure 4

Characterization of human T-cell subsets during chronic HBV infection. (A) Total numbers of human T lymphocytes (hCD45⁺CD3⁺) and (B) CD4⁺ or CD8⁺ T cells isolated from the liver and spleen. (C) Representative FACS plots of CD4⁺ or CD8⁺ T cells from the liver of control or HBV-infected samples, histograms show normalized frequencies of naïve (CD45RA⁺ [white bar]) or memory (CD45RO⁺ [black bar]) cells among CD4⁺ or CD8⁺ T cells. Mean and SEM are shown. (D) Frequency of PD-1⁺ cells among the CD4⁺CD45RO⁺ or CD8⁺ CD45RO⁺ memory T cells from a representative HIS-HUHEP control (grey filled line) and HBV 10e9 inoculated (black empty line) mouse liver. Each dot represents a mouse, data obtained at 14–20 wpi. A–D: Mann Whiteny U test. (E) Analysis of an exhaustion marker (PD-1⁺) on intrahepatic CD4⁺CD45RO⁺ T cells as a function of viral load with Pearson’s correlation test.

Figure 5

Humoral immune responses in HBV-infected HIS-HUHEP mice. (A) Total human IgM and IgG concentrations in the plasma of HIS-HUHEP control (white bar; n=11) or HBV-infected mice (10e7 inoculum: grey bar; n=5, 10e9 inoculum: black bar, n=9) plotted against weeks post infection (wpi). Bars show the mean with SEM. Statistics used unpaired two-tailed t test. (B) Analysis of anti-HBV antibodies in HIS-HUHEP mice. The frequency of mice positive for HBcAb IgG or HBsAb IgG from serially diluted plasma at the indicated concentration of total human IgG was analyzed by nonlinear regression. HIS-HUHEP control (n=19, dotted line), HBV-inoculated at 10e7 (n=4, grey line) or 10e9 (n=9, black line). P values indicate the differences between the slopes compared with the control data. (C) Representative images of IHC for anti-HBsAb.

Figure 6

Biomarker analysis in HBV-infected HIS-HUHEP mice. (A) Plasma from HIS-HUHEP control (n=11) and HBV-infected mice (inoculum 10e7, n=5; or 10e9, n=10) at endpoint were analyzed using human cytokine multiplex assay. No cross-reactivity with mouse cytokines was detected. Plasma levels of IL-1β, IL-1Ra, IL-2, IL-2R, IL-4, IL-12, IL-13, IL-15, IFN-γ, GM-CSF, and RANTES were similar between control and HBV-infected mice; while IL-5, IL-17, and Eotaxin were not detected (data not shown). (B) Correlation of PD-1⁺ memory CD4⁺ CD45RO⁺ T cells with either IFN-γ, or IP-10/CXCL10, or IL-10 plasma cytokines quantified in (A). Each dot represents a mouse: grey or black inoculated with, respectively, HBV 10e7 or HBV 10e9. (C) RT-qPCR analysis of liver samples from HIS-HUHEP control (n=14) and HBV-infected mice (inoculum 10e7, n=5; or 10e9, n=9). Fold changes in gene expression of HBV-infected compared with control mice are shown. Data was normalized to the internal control human GAPDH (hGAPDH) to account for differences in humanization levels on triplicate samples. Dotted line indicates fold change of 1. Histograms show the mean and SEM. Data from 14–20 wpi. Statistical significance: Mann Whitney U tests.

Figure 7

ETV treatment of HBV chronically infected HIS-HUHEP mice reverses hepatitis. (A) Liver engraftment (hAlbumin: grey bars) and viremia (HBV DNA: black line) in the plasma of mice previously infected for 3 months undergoing ETV treatment (n=4 mice). (B to F) FACS analysis of intrahepatic leukocytes and splenocytes from control, HBV-infected, and HBV-infected post-ETV treated mice. Absolute numbers of (B) total human leukocytes (hCD45⁺), (C) monocytes (HLA-DR⁺CD14⁺CD33⁺), (D) NK cells (CD3^-NKp46⁺), and the frequency of CD56⁺CD16⁺ cells among NK cells, (E) CD4+, CD8+ (F), and Foxp3+ T cells. The number of intrahepatic Foxp3+ T_REG cells was plotted against the viral load at end point for each mouse and analyzed with Pearson’s correlation test.

Supplementary Figure 1

Longitudinal follow-up of HBV viremia (black line) and hAlbumin (grey bars) in the plasma of individual HUHEP and HIS-HUHEP mice inoculated with high-dose HBV 10e9.

Supplementary Figure 2

Follow-up of viral loads and liver chimerism in HIS-HUHEP mice inoculated at low-dose HBV 10e7. (A and B) HBV viremia (black line) and hAlbumin (grey bars) were measured longitudinally in the plasma of HBV-infected HUHEP (A) and HIS-HUHEP (B) mice. Graphs show the mean from 6 HUHEP and 6 HIS-HUHEP mice. (C and D) Correlative analysis of the viral load vs liver humanization: for each plasma sample viremia was plotted against hAlbumin concentration from HUHEP (C) (n=6) or HIS-HUHEP mice (D) (n=6). Correlation was evaluated using a 2-tailed Pearson's χ² test. (E and F) Analysis of viral progression over time: linear regression analysis of the ratio of HBV viral load over hAlbumin concentration plotted against time post infection, from (E) HUHEP (dotted line) or HIS-HUHEP mice inoculated at 10e7 or (F) from HIS-HUHEP mice inoculated at 10e7 vs 10e9 (respectively, grey or black filled line). The P value determines whether the slopes are significantly different. (G) Fold change of M65/hAlbumin pre- and post-infection (17 ±6 wpi) in HUHEP mice (control n=4 HBV 10e7; and 10e9 n=6).

Supplementary Figure 3

Longitudinal follow-up of HBV viremia (black line) and hAlbumin (grey bars) in the plasma of individual HUHEP and HIS-HUHEP mice inoculated with low-dose HBV 10e7.

Supplementary Figure 4

Immunofluorescence analysis of liver sections from HIS-HUHEP control or low-dose (HBV 10e7) infected mice co-stained for hAlbumin (blue) and HBc (green), with either hCD45 (red), or hCD3 (red), or hCD68 (red). DAPI-stained nuclei shown in grey. Scale bar represents 100 μm.

Supplementary Figure 5

Characterization of human T-cell subsets in HIS-HUHEP–infected mice by FACS analysis. Percentage of HLA-DR–positive cells in memory CD4⁺CD45RO⁺ or CD8⁺CD45RO⁺ T cells from control or HBV-infected mice at low (HBV 10e7) or high (HBV 10e9) doses.

Supplementary Figure 6

Immunofluorescence analysis of liver sections from HIS-HUHEP control or HBV-inoculated mice at either low dose (HBV 10e7) or high dose (HBV 10e9) co-stained for hAlbumin (blue), HBc (green), and PD-L1 (red) with DAPI-stained nuclei shown in grey. Scale bar represents 100 μm. In the second, third, and fourth vertical panels 1 color channel has been removed to visualize the staining patterns by pairs. White arrowheads: double-stained cells for Albumin and PD-L1; white open arrows: triple-stained cells for Albumin, HBc, and PD-L1; white filled arrows: cells mono-stained cells for PD-L1.

Supplementary Figure 7

Biomarker expression patterns in HBV-infected and control HUHEP mice. Plasma from control (n=4) and HBV-infected (n=10) mice at the time of sacrifice (20 ±2 wpi) was analyzed by multiplex assay for human cytokines. IL-1b, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, CCL2, CCL3, CCL5, GM-CSF, IFN-γ, and TNF-α were not detected. Dotted lines indicate the lower limit of quantification for each assay.

Supplementary Figure 8

HBV-infected liver progenitor cells in highly viremic HIS-HUHEP mice. Immunofluorescence analysis of liver sections from (A) control, or HBV-infected mice inoculated with (B) HBV 10e7 or (C) HBV 10e9 for Albumin (blue), HBcAg (green) and either (left column) EpCAM (red) or (right column) CK7 (red). White open arrows show mono-stained cells for either EpCAM or CK7; white arrowheads show double-stained cells co-expressing HBc and CK7; white filled arrows show triple-stained cells co-expressing Albumin, HBc, and either EpCAM or CK7. (D) HBV-infected liver co-stained for Albumin (blue), EpCAM (green) and CD3 (red). Scale bar represents 100 μm.

Supplementary Figure 9

(A) Longitudinal analysis of viremia and liver chimerism in HBV-infected (10e7) ETV-treated HIS-HUHEP mice. The dotted line indicates start of treatment. (B–D) FACS analysis of T-cell phenotypes in control, HBV-infected (10e7), and HBV-infected (10e7) ETV-treated, HIS-HUHEP mice. (A) The frequency of naïve (CD45RA⁺) vs memory (CD45RO⁺) CD4⁺ and CD8⁺ T cells was evaluated in the liver and spleen of each cohort. (B) The frequency of PD-1⁺ cells in memory (CD45RO⁺) CD4⁺ or CD8⁺ T cells from the liver and spleen. (C) Correlation analysis of the viral load (HBV DNA) relative to the frequency of memory CD4⁺CD45RO⁺PD-1⁺ T cells in the liver using Pearson’s correlation test.