Distinct Cytokine Profiles Correlate with Disease Severity and Outcome in Longitudinal Studies of Acute Hepatitis B Virus and Hepatitis D Virus Infection in Chimpanzees

Abstract

Historical studies conducted in chimpanzees gave us the opportunity to investigate the basis for the different severities of liver damage and disease outcome associated with infection with wild-type hepatitis B virus (HBV) versus a precore HBV mutant, HBV/hepatitis D virus (HDV) coinfection, and HDV superinfection. Weekly samples from 9 chimpanzees were studied for immune responses by measuring plasma levels of 29 cytokines in parallel with alanine aminotransferase (ALT) levels and viral kinetics. Comparison of classic acute hepatitis B (AHB) with severe or progressive AHB and HBV/HDV coinfection or superinfection identified distinct cytokine profiles. Classic AHB (mean ALT peak, 362 IU/liter) correlated with an early and significant induction of interferon alpha-2 (IFN-α2), IFN-γ, interleukin-12 p70 (IL-12 p70), and IL-17A. In contrast, these cytokines were virtually undetectable in severe AHB (mean ALT peak, 1,335 IU/liter), characterized by significant elevations of IL-10, tumor necrosis factor alpha (TNF-α), and MIP-1β. In progressive AHB (mean ALT peak, 166 IU/liter), there was a delayed and lower-magnitude induction of cytokines. The ALT peak was also delayed (mean, 23.5 weeks) compared to those of classic (13.5 weeks) and severe AHB (7.5 weeks). HBV/HDV coinfection correlated with significantly lower levels of IFN-α2, IFN-γ, and IL-17A, associated with the presence of multiple proinflammatory cytokines, including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-15. Conversely, HDV superinfection induced the highest ALT peak (1,910 IU/liter) and was associated with a general suppression of cytokines. Our data demonstrate that the most severe liver damage, caused by an HBV precore mutant and HDV, correlated with restricted cytokine expression and lack of Th1 response, raising the question of whether these viruses are directly cytopathic.IMPORTANCE Studies performed in chimpanzees at the National Institutes of Health (NIH) demonstrated a significant difference in ALT levels during acute hepatitis of different viral etiologies, with a hierarchy in the extent of liver damage according to the infecting virus: the highest level was in HDV superinfection, followed by infection with a precore HBV mutant, HBV/HDV coinfection, and, lastly, wild-type HBV infection. Our study demonstrates that both the virus and host are important in disease pathogenesis and offers new insights into their roles. We found that distinct cytokine profiles were associated with disease severity and clinical outcome. In particular, resolution of classic acute hepatitis B (AHB) correlated with a predominant Th1 response, whereas HBV/HDV coinfection showed a predominant proinflammatory response. Severe AHB and HDV superinfection showed a restricted cytokine profile and no evidence of Th1 response. The lack of cytokines associated with adaptive T-cell responses toward the precore HBV mutant and HDV superinfection argues in favor of a direct cytopathic effect of these viruses.

Keywords: chimpanzees; cytokines; hepatitis; hepatitis B virus; hepatitis D virus; pathogenesis; precore HBV mutant; viral kinetics; wild-type HBV.

FIG 1

Alanine aminotransferase (ALT) levels in chimpanzees acutely infected with HBV alone, with HBV/HDV coinfection, and with HDV superinfection. AHB, acute hepatitis B.

FIG 2

Biochemical and virological course in four chimpanzees with acute resolving hepatitis B and in two chimpanzees with acute progressing hepatitis B. AHB, acute hepatitis B; HBsAg, hepatitis B surface antigen; anti-HBs, antibodies to hepatitis B surface antigen.

FIG 3

Biochemical and virological course of representative chimpanzees with acute classic or severe acute resolving hepatitis B or progressive acute hepatitis B. AHB, acute hepatitis B; HBeAg, hepatitis B e antigen; anti-HBe, antibodies to hepatitis B e antigen; anti-HBc, antibodies to hepatitis B core antigen; HBsAg, hepatitis B surface antigen; anti-HBs, antibodies to hepatitis B surface antigen.

FIG 4

Clinical, serological, and virological course of acute resolving HBV/HDV infection in two chimpanzees and HDV superinfection in a chimpanzee chronically infected by HBV. HBeAg, hepatitis B e antigen; Anti-HBe, antibodies to hepatitis B e antigen; anti-HBc, antibodies to hepatitis B core antigen; HBsAg, hepatitis B surface antigen; anti-HBs, antibodies to hepatitis B surface antigen; anti-HD, antibodies to hepatitis delta antigen (HDAg).

FIG 5

Distinct cytokine profiles correlated with disease severity and clinical outcome. Cytokine profiles in chimpanzees with classic or severe acute resolving or progressive hepatitis B, and acute resolving HBV/HDV coinfection, are shown. The circulating cytokine levels were quantified at weekly or biweekly intervals in each of the 8 animals after inoculation using a multiplex assay. The data, expressed in picograms per milliliter, represent the results from 3 sequential weeks at each time point (t1-3, t4-6, t7-9, t10-12, t13-15, t16-18, and t19-21) and are shown in a box-and-whisker plot, in which the median of the values for each time point is indicated by a horizontal line, with the 25th and 75th percentiles shown at the top and bottom of each box; the maximum and the minimum values are indicated by the whiskers. In each graph, the gray triangle represents the time point of the ALT peak, and the height is proportional to the mean value of the peak in each group. In progressive AHB, the archived samples available for weeks 4 to 6 were insufficient for cytokine profiling. AHB, acute hepatitis B.

FIG 5

Distinct cytokine profiles correlated with disease severity and clinical outcome. Cytokine profiles in chimpanzees with classic or severe acute resolving or progressive hepatitis B, and acute resolving HBV/HDV coinfection, are shown. The circulating cytokine levels were quantified at weekly or biweekly intervals in each of the 8 animals after inoculation using a multiplex assay. The data, expressed in picograms per milliliter, represent the results from 3 sequential weeks at each time point (t1-3, t4-6, t7-9, t10-12, t13-15, t16-18, and t19-21) and are shown in a box-and-whisker plot, in which the median of the values for each time point is indicated by a horizontal line, with the 25th and 75th percentiles shown at the top and bottom of each box; the maximum and the minimum values are indicated by the whiskers. In each graph, the gray triangle represents the time point of the ALT peak, and the height is proportional to the mean value of the peak in each group. In progressive AHB, the archived samples available for weeks 4 to 6 were insufficient for cytokine profiling. AHB, acute hepatitis B.

FIG 5

Distinct cytokine profiles correlated with disease severity and clinical outcome. Cytokine profiles in chimpanzees with classic or severe acute resolving or progressive hepatitis B, and acute resolving HBV/HDV coinfection, are shown. The circulating cytokine levels were quantified at weekly or biweekly intervals in each of the 8 animals after inoculation using a multiplex assay. The data, expressed in picograms per milliliter, represent the results from 3 sequential weeks at each time point (t1-3, t4-6, t7-9, t10-12, t13-15, t16-18, and t19-21) and are shown in a box-and-whisker plot, in which the median of the values for each time point is indicated by a horizontal line, with the 25th and 75th percentiles shown at the top and bottom of each box; the maximum and the minimum values are indicated by the whiskers. In each graph, the gray triangle represents the time point of the ALT peak, and the height is proportional to the mean value of the peak in each group. In progressive AHB, the archived samples available for weeks 4 to 6 were insufficient for cytokine profiling. AHB, acute hepatitis B.

FIG 6

Cytokine profiles in HDV superinfection of a chimpanzee chronically infected by HBV. The graphs represent the longitudinal analysis of circulating cytokine/chemokine levels. The red line represents the plasma/serum values of each cytokine, while the gray areas represent the ALT levels.

FIG 6

Cytokine profiles in HDV superinfection of a chimpanzee chronically infected by HBV. The graphs represent the longitudinal analysis of circulating cytokine/chemokine levels. The red line represents the plasma/serum values of each cytokine, while the gray areas represent the ALT levels.

FIG 7

Levels of circulating IP-10 (CXCL10) in chimpanzees with acute classic or severe acute resolving hepatitis B or progressive acute hepatitis B, HBV/HDV coinfection, and HDV superinfection. In each graph, the red line represents the plasma/serum IP-10 values of each animal at different time points, while the gray area shows the ALT levels. AHB, acute hepatitis B.