Hepatology highlights†
Authors: Harvey J. Alter
Journal: Hepatology (2006)

Using the chimpanzee model, Puig and coworkers analyzed the protective effect of a vaccine-induced T cell response in the absence of neutralizing antibody. Two animals, previously recovered from HCV infection, were immunized with an NS3, NS5A, or NS5B DNA primer followed by a recombinant vaccinia virus-HCV protein boost containing only nonstructural proteins. One HCV-naïve chimpanzee received the same construct as a primary immunization. Each animal was challenged with the HCV1a infectious clone. Vaccination did not prevent infection in any of the animals, but the infection/disease in the 2 animals that had spontaneously recovered prior to reimmunization was markedly delayed and attenuated compared to that in the HCV-naïve animal. The attenuated infection coincided with enhanced postchallenge T cell responses that remained strong even after viral clearance. Of primary interest in this study is that the naïve-immunized animal initially contained infection with an approximate 4 log decline in HCV RNA levels, only to have rebound viremia at week 10 that then became persistent. Importantly, in this animal, compared to those who cleared infection, CD4+ T cell responses did not increase with the onset of viremia and showed decreased proliferative capacity in both the acute and chronic phases. Sequence analysis demonstrated 2 nonsynonymous mutations in the samples at 8 and 13 weeks after challenge. T cells recognized wild-type peptides, but did not recognize the mutant epitopes and thus the virus had developed critical escape mutations in both the NS3 and NS5 regions. Further, over an interval up to 53 weeks, T cell responses to the wild-type virus declined and no T cell response to the mutant virus emerged. Interestingly, the escape mutations occurred in CD4 epitopes whereas most previously described escape mutants have been in CD8+ epitopes. What can we take away from this? First, it would appear that subjects who recover from prior HCV infection are better positioned to control and clear a second infection by virtue of T cell memory and early T cell effector responses. Second, viral clearance can be achieved in the absence of neutralizing antibodies to envelope proteins. Third, persistent infection can be the result of viral escape mutants and that these critical mutations can occur in CD4+ epitopes as well as CD8+ epitopes. Fourth, a DNA prime-protein boost vaccination strategy did not achieve sterilizing immunity in any animal, but did appear to attenuate infection in those previously exposed. Fifth, an apparent successful early attenuation of infection following primary infection/immunization can be overcome by the emergence of T cell escape mutants and hence that vaccine-induced T cell responses before challenge do not necessarily prevent persistent HCV infection. Most HCV vaccine programs have retreated from the prospect of inducing sterilizing immunity and are basing success on the prevention of chronic infection. It appears that even this more limited goal will not always be achieved. This is one tough virus. (See HEPATOLOGY 2006;44:736–745.) After entry into hepatocytes, the HBV viral genome is translocated to the nucleus and converted to supercoiled covalently closed circular DNA (cccDNA) that exists in episomal form and serves as template for HBV mRNA transcripts and for transcription of pregenomic RNA (pgRNA), the primary step in HBV replication. Thus, cccDNA is not only circular, but also central to HCV persistence and replication. Laras et al. developed sensitive and specific real-time PCR assays to measure both cccDNA (lower limit of detection is 100 copies per assay in serum and 2 × 103 copies per liver cell) and pgRNA in the liver of 34 untreated patients with chronic active hepatitis B (12 HBeAg+, 21% with cirrhosis). The cccDNA in serum was detected in 7 of 9 patients tested and ranged from 9.4 × 102 to 4.1 × 105 copies/mL; levels in the liver ranged from 1.8 × 104 to 1.4 × 107 per million cells. Total serum HBV DNA was 1,000-fold higher than cccDNA and hence cccDNA represented 0.1% of serum HBV DNA. In contrast, cccDNA represented 6% of intrahepatic HBV DNA. Intrahepatic cccDNA levels were approximately 1 log higher in subjects positive for hepatitis B e-antigen (HBeAg+) than in those negative for the antigen (HBeAg−). A very strong correlation was seen between intrahepatic HBV DNA and cccDNA (r = 0.945). The median level of pgRNA was 6.5 × 106 copies per million cells (6 copies per cell) (see Fig.). The levels of pgRNA were significantly higher than the corresponding levels of cccDNA, being 90-fold higher in HBeAg+ and 5-fold higher in HBeAg− subjects. The pgRNA in liver correlated well with both cccDNA (r = 0.863) and total intrahepatic HBV DNA (r = 0.964). Further, serum HBV DNA showed significant correlation with intrahepatic cccDNA (r = 0.744), pgRNA (r = 0.874), and HBV DNA (r = 0.840). These precise quantitations and correlations add a new dimension to our understanding of HBV infection. Further, the simultaneous measurement of cccDNA and pgRNA now allows for determination of cccDNA's replicative activity. In addition, the finding that 3 HBeAg-negative patients had relatively high replicative activity in the absence of increased cccDNA suggests modulatory factors for replication other than cccDNA. Overall, measurement of intrahepatic pgRNA was a better reflection of replicative activity than measurement of cccDNA alone. It appears we have come full circle on the measurement of HBV replication, and thus, this highlight is covalently closed. (See HEPATOLOGY 2006;44:694–702.) Hwang et al. at the CDC investigated the infectious risk of what was termed “cosmetic procedures.” These included body piercing, tattooing, intranasal drug use (IDU), and manicuring, but not the cosmetic procedures that have made me what I am today. To minimize known parenteral risks, they studied students attending 8 colleges and junior colleges in Texas (who knew that Texas had so many educated people?) and administered anonymous risk questionnaires and obtained blood samples. The study analyzed 5,282 U.S. or Canadian-born subjects (average age 21, 62% female, 42% white, 26% black, 22% Hispanic, and 10% Asian). As anticipated, reported IDU was only 2% and transfusion history was similarly low, thus allowing for a clearer evaluation of other percutaneous or mucous membrane exposures. Anti-HCV prevalence was only 0.9% (85% HCV RNA+) and anti-HBc prevalence was 9.5% (13% HBsAg+). In this population, 14% reported IDU, 21% body piercing, 25% tattooing, 54% professional manicures, and 42% the sharing of toothbrushes or razors. With all these extracurricular percutaneous activities, it is amazing they found time to study. In the final multivariate analysis, HCV infection was independently associated only with increased age and a history of IDU, blood transfusion, or incarceration. No increase in HCV prevalence was noted in those who reported snorting (0.8%), tattoos (0.6%), body piercing (0.4%), professional manicure, or sharing toothbrushes or razors, even after separate stepwise logistic regression models were employed. A comparable multivariate analysis for HBV showed that the factors independently associated with high HBV prevalence were increasing age, black or Asian race, MSM, 5 or more heterosexual partners in the previous 4 months, and a history of STD. This is an excellent epidemiologic study, but there are pitfalls that the authors frankly state. First, there is the potential for selection bias among the 10% of eligible persons who volunteered for study. Second, college students are themselves a highly select population. Third, by including only young persons, the study does not address exposures that occurred prior to the improved hygienic practices that now accompany tattoos and piercings. Thus, this study does not address whether older persons who have never used intravenous drugs might have been infected by tattoos or other percutaneous penetrations when shared, unsterilized needle use was common. The authors draw three important conclusions from this study: 1) that there is not sufficient demonstrated risk among young persons who undergo tattoos or body piercing in the absence of IDU to warrant routine HCV screening; 2) that intravenous drug use carries significant risk even when that use is experimental and sporadic; 3) that a disappointingly low proportion of young educated adults (20% in this study) have been vaccinated for HBV. Routine vaccination of children and adolescents will ultimately rectify this gap, but at present most persons older than 20 remain at risk for HBV infection and many continue to engage in high risk activities. Overall, hepatitis transmission remains a sticky and a sticking issue. (See HEPATOLOGY 2006;44:341–351.) Eight HBV genotypes and multiple subtypes have been defined and shown to have characteristic geographic distributions. Genotype A has been subtyped as Aa/A1 (Asian/African type) and genotype B as Bj/B1 (Japanese type) and Ba/B2 (Asian type). Evidence has accumulated that HBV genotypes have clinical relevance in that they influence the rate of viral replication and the frequency of mutations in the precore region and hence affect the expression of HBeAg. Ozasa et al. describe the outcome of 301 patients hospitalized for acute hepatitis B between 1982 and 2004. This long study makes several key observations in comparing recovered subjects (87%) with those who had fulminant hepatitis (13%). The high proportion of fulminant cases reflects a study bias wherein only hospitalized severe acute hepatitis cases were entered into the cohort. Although the observations may not characterize the full spectrum of HBV infections, the findings were quite striking. Patients with fulminant hepatitis compared to acute resolving cases were: significantly older (44.7 years vs. 36.0 years; P < .001); female (57% vs. 29%; P = .005); HBeAg negative (77% vs. 40%; P < .0001); less infected with genotype Ae (0% vs. 13%; P < .05), and; more likely to be genotype Bj (30% vs. 4%; P < .0001); have precore (G1896A) mutations (53% vs. 9%) and core promoter (A1762T/G1764A) mutations (50% vs. 17%, P < .0001). In a multivariate analysis, genotype HBV/Bj had a strong association with fulminant hepatitis (odds ratio 7.0; P = .006) as did the precore mutation, G1896A (odds ratio 4.16; P =.019). To explore the impact of precore and core promoter mutations, the authors established an HBV culture system and showed that HBV DNA mutations at these positions caused increased HBV replication compared to wild-type virus. It is implied, but not proved, that a highly replicating mutant virus would infect a larger number of hepatocytes and thus would be more likely to cause fulminant hepatitis. HBV DNA levels were lower in those with fulminant hepatitis, presumably because viral replication had diminished as the result of massive liver cell death. Thus, in Japan, fulminant hepatitis is relatively common (13%) among patients with overt acute hepatitis B and is associated with subtype Bj, precore mutation, HBeAg negativity, and higher levels of viral replication in in vitro assays. (See HEPATOLOGY 2006;44:326–334.) An independent, complementary study by Sugiyama et al. examined the characteristics of diverse HBV isolates in a Huh 7 transfection system. Subgenotype Aa showed the lowest expression of HBV DNA, genotype C showed the highest, and Bj showed the second highest level of expression. The intracellular expression of core protein was highest for Bj, the strain associated with fulminant hepatitis in the Ozasa study. Similarly, the extracellular expression of HBV DNA and HBeAg was 3-fold to 5-fold higher for genotype Bj than for any of the others. Genotype Bj also had the highest level of pregenome/precore mRNA, but the lowest expression of HBsAg and preS/S mRNA. A conclusion of this study was that Bj had low intracellular virion retention and that high virion secretion may endow Bj with high infectivity and cell-to-cell spread, inducing a strong host immune response, perhaps accounting for its association with fulminant hepatitis. In contrast genotype A has the lowest replicative capacity which might enhance its ability to evade the immune system and persist in up to 10% of infected individuals. The bottom line of both these studies is that HBV genotypes are not simply surface markers, but rather play an important functional role in determining the outcome of HBV infection. (See HEPATOLOGY 2006;44:915–924.) In the never-ending quest to explain the impaired immune response that characterizes HCV persistence, Nattermann and coworkers explored interesting and informative aspects of dendritic cell (DC) trafficking. The investigators provide convincing data to support the hypothesis that within the HCV-infected liver, HCV E2 protein binds to CD81 on CD8+ T cells. This results in the release of RANTES and other chemokines that attract immature DCs to the site of hepatic inflammation with resultant binding to chemokine receptors such as CCR5. Having trafficked to the liver, DCs then take up HCV antigens and begin a maturation process in which the receptors that bind inflammatory cytokines (e.g., CCR5) are internalized and new receptors, such as CCR7, are expressed and then attracted to chemokines, such as CCL21, expressed in lymphoid organs. Thus, in the proper course of events, viral antigens would be processed in DC and then make their way to lymphoid organs where appropriate immune responses would be generated (see Fig.). Natterman et al. provide evidence that when the HCV E2 surface protein interacts with CD81 on DCs, it down-regulates CCR7 expression and makes these cells unresponsive to the chemokine CCL21 and thus inhibits trafficking to lymphoid tissue. In essence, dendritic cells traffic to the liver, but then get trapped there impairing their function as antigen-presenting cells. In vitro studies support this hypothesis showing cross-linking of HCV E2 to CD81 and impaired migration of DCs to CCL21. In control experiments, it was shown that this DC dysfunction could be blocked by antibodies to CD81, that inhibition did not occur in response to HCV core protein, and that impaired trafficking did not occur in patients with chronic hepatitis B or sclerosing cholangitis. The suggestion was that inhibition of DC migration toward lymphocyte tissues was relatively specific for HCV infection. I found this a convincing argument for yet another mechanism wherein HCV fosters its own survival by impairing multiple arms of the HCV-directed immune response. (See HEPATOLOGY 2006;44:945–954.) Kawamura et al. suggest still another mechanism for HCV persistence. Using a model of concanavalin A (ConA)-induced liver injury in transgenic mice, they showed that mice expressing HCV core antigen in the liver were protected from Con A liver injury, whereas mice expressing core only in lymphoid tissue were not protected. Similarly, core transgenic hepatocytes were relatively resistant to death induced by Fas and TNFα. The investigators traced the mechanism of protection from ConA to inhibition of STAT1 and stimulation of STAT3. Further, they showed that core transgenic mice undergo accelerated liver regeneration after partial (70%) hepatectomy, again thought to be related to activation of STAT3. Viewed teleologically, an efficient virus will protect the cells it lives in from immune or apoptotic damage and rapidly repair those cells that do get injured. In HCV's insistence on persistence, it has evolved many mechanisms to ensure its survival and also that of the cells in which it has taken up residence. The HCV core protein seems central to this survival strategy, in part through its ability to modulate the STAT transcription pathways, particularly through inhibition of STAT1 and activation of STAT3. These mechanisms may also confer some resistance to interferon. HCV has become a formidable enemy by ensuring that it doesn't do too much damage too much of the time. Like a child, it gets away with what it can, but doesn't want to be thrown out of the house. (See HEPATOLOGY 2006;44:936–944.) This is my last highlight for HEPATOLOGY I will now concentrate on my aging biology These articles have consumed many of my nights For what I like to call my Andy Blei-lights With mixed joy and sadness, I turn this over to my successor With a warning that your personal time will be significantly lesser And repeated deadlines will become your oppressor But in the end, of greater knowledge, you'll be the possessor On this my final Hep highlight What in reality is my goodbye write I wander off into the twilight Murrowing you all, good luck and good night