Folgori A, Capone S, Ruggeri L, Meola A, Sporeno E, Bruni Ercole B, Pezzanera M, et al. A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nat Med 2006;12:190-197. (Reprinted by permission from Macmillan Publishers Ltd: www.nature.com.)
Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8+ T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.
Hepatitis C virus (HCV) infection is a disease of high prevalence around the world, and continues to spread rapidly in countries with untested blood supplies. Why is no effective vaccine available so far?
A key feature of most vaccines is the induction of neutralizing antibodies. In many cases, infusion of neutralizing antibodies is also used as passive post-exposure prophylaxis. Although antibody-based neutralization has already been described years ago in the chimpanzee model of HCV infection,1 efficient screening for neutralizing antibodies, that inhibit HCV entry into hepatocytes, has only recently become feasible when appropriate in vitro infection systems were developed.2
In the natural course of HCV infection, however, the humoral immune response seems to fail because high titers of in vitro-neutralizing antibodies coexist with high HCV titers in persistently infected patients.3 Thus, these antibodies, albeit neutralizing in vitro, appear to be insufficient to completely block HCV entry into cells in vivo and to clear the infection. Moreover, HCV antibody titers have been shown to decline and are often undetectable in long-term recovered persons,4 raising the question of the longevity of an antibody-based vaccine response against HCV.
Although it remains possible that a panel of antibodies can be engineered in vitro and used as post-exposure prophylaxis,5 recent studies have emphasized the role of the cellular immune response in protection against HCV. In particular, a series of rechallenge studies of spontaneously HCV-recovered chimpanzees demonstrated a crucial role of CD4 and CD8 T cells in protective immunity.6–10 Thus, most preclinical vaccination trials are now focused on the induction of robust T cell responses, which via induction of CD4 T cell help for B cells, may ultimately also aid the humoral immune response.
In a recent study,11 Folgori et al. explored a T cell-based vaccine in the chimpanzee model of HCV infection. Five chimpanzees were vaccinated at weeks 0 and 4 with a replication-deficient serotype 6 adenoviral vector encoding the NS3-NS5B region of the HCV BK strain (genotype 1b), followed by vaccination at week 25 with a replication-deficient serotype 24 adenoviral vector encoding the same HCV antigens. At weeks 35, 37 and 39, the immune response was boosted by intramuscular injection of a recombinant DNA plasmid. Five control chimpanzees received equivalent doses of control adenovirus and DNA vectors encoding the HIV gag antigen. Forty-nine weeks after the start of the study, all chimpanzees were then intravenously challenged with 100 chimpanzee 50% infectious doses (CID50) of the heterologous HCV H77 (genotype 1a) inoculum. Given that about 50% of chimpanzees are able to clear HCV spontaneously,12 the outcome of infection did not significantly differ between both groups: four of five vaccinated chimpanzees cleared HCV, as compared to three of five control chimpanzees. However, the virological and clinical course of infection differed considerably between both groups. Whereas all control chimpanzees developed high serum HCV-RNA titers and elevated ALT levels, the four vaccinated chimpanzees displayed significantly lower and delayed RNA titers and maintained normal ALT values. This course of infection is reminiscent of the low HCV RNA titers and normal ALT values that are characteristic for the rechallenge of spontaneously recovered chimpanzees with strong cellular immune responses.6–10 Indeed, the effect of the vaccine became most evident when the authors analyzed the induced T cell responses: the sole chimpanzee in the vaccinated group that did not clear the virus was the animal that did not display potent T cell responses and developed HCV escape mutations. In other words, all chimpanzees that showed a good vaccine “take” with induction of robust T cell responses were able to clear the challenge virus.
To fully characterize this protective immune response, the authors chose flow cytometry and analyzed T cell function at the single cell level. In particular, stimulation with pools of overlapping HCV peptides and intracellular staining of cytokines were employed to separately analyze the generation of memory CD4 and CD8 T cells during the course of immunization. HCV-specific, IFN-γ-producing CD8 T cells were already detectable after the initial adenoviral immunization in four of the five vaccinated chimpanzees. In contrast, HCV-specific IFN-γ-producing CD4 T cells were not observed until after the DNA boost.
After challenge with the heterologous HCV strain, vaccine-induced CD4 and CD8 T cell populations started to proliferate as early as 2-4 weeks after the infection resulting in potent and broad IFN-γ responses and cytotoxicity. Vigorous memory T cell responses were also detectable in the liver, thus indicating a direct correlation between immune responses and eradication of HCV at the site of infection.
The data are consistent with previous vaccine trials in smaller groups of chimpanzees. Rollier et al.13 immunized two chimpanzees with DNA plasmids expressing HCV E1, E2, core and NS3, followed by a boost with recombinant protein. T cell proliferation in response to E1 and NS3 and production of IFN-γ and IL-2 became detectable only after the boost and correlated to the clinical outcome upon the challenge with a heterologous HCV strain. However, intrahepatic T cell responses were not analyzed in this study. Youn et al.14 vaccinated six chimpanzees with a mixture of DNA plasmids encoding NS3-NS5B and envelope proteins followed by a boost with recombinant adenovirus. Strong proliferative T cell responses in addition to HCV envelope-specific antibodies were observed after heterologous HCV challenge in the two chimpanzees that recovered. Thus, although vaccine-induced HCV-specific memory T cells cannot prevent HCV infection upon rechallenge, they appear to ameliorate disease activation (increase in serum alanine aminotransferase levels) during the period of viremia, mediate rapid HCV clearance and protect from development of chronic infection.
None of the studies had fully characterized the type of memory T cells that the vaccine induced. So what are the typical characteristics of memory T cells? Based mostly on murine models of infection with the liver-trophic lymphocytic choriomeningitis virus, it is known that a large number of T cells proliferate during the acute phase of infection, but more than 90% of them die by apoptosis when antigen levels decline. A subpopulation of virus-specific cells with high levels of IL-7 receptor preferentially survives and differentiates into long-lived memory T cells.15 Signaling via the IL-7 receptor maintains T cell viability by increasing the expression of Bcl-2 and Bcl-XL, suppressing the proapoptotic BAX molecule and regulating glucose metabolism to prevent cellular atrophy and death.16, 17
Generation of effective CD8 T cell memory depends on “CD4 T cell help”. If CD4 T cell help is lacking, the primary infection may be cleared but the resulting “helpless” CD8 cells are defective in their ability to generate a secondary response upon reinfection.18–22 Protective immunity against HCV requires CD4 T cell help not only in the primary infection, but also after recovery, at the time of secondary rechallenge.10 How CD4 help is administered in this scenario is not known — CD4 cells may either directly activate dendritic cells and CD8 T cells via CD40-dependent costimulation or indirectly potentiate B cell and CD8 T cell responses via secretion of IL-2 and IL-4.
Successful generation of memory T cells results in two memory T cell subpopulations, effector memory cells and central memory cells, which have been detected in healthy humans.23 Both populations produce antiviral cytokines such as IFN-γ and TNF-α, but can be distinguished by means of their trafficking pattern. Effector memory cells do not express the lymph node homing markers CD62L and CCR7 and therefore traffic to peripheral organs. Central memory cells express CD62L and CCR7 for homing and entry into lymphoid tissues. In contrast to effector memory cells, central memory cells produce IL-2 and are highly responsive to IL-715 and IL-15.24 This cytokine responsiveness enables central memory cells to proliferate efficiently, and thereby maintain the memory T cell population even in the absence of antigens.25 Human vaccinia virus-specific CD8 T cells, for example, display a half-life of 8-15 years and the vaccinia-virus-specific memory T cell population has been shown to persist for up to 75 years after infection.26
The publication by Folgori et al.11 also poses interesting questions for further studies. First, how long does protection by the vaccine last? Does the induced vaccine-induced memory T cell population contain central memory T cells with the capacity to proliferate in the absence of antigen and to maintain long-lived responses?
Second, how can memory T cells recognize and conquer mutated or heterologous HCV strains? Although Folgori et al.11 used a heterologous HCV 1a strain for challenge after vaccination with HCV 1b sequences, both strains are still relatively closely related, when compared to other HCV genotypes. Induction of T cells against multiple epitopes, including some in conserved sequence regions may partially resolve this issue.
Third, can the same vaccine be used therapeutically in chronic HCV infection? Chronic viral infections tend to induce exhaustion of memory T cells ranging from loss of proliferation, cytotoxicity and cytokine production to physical deletion.27–29 The reduced proliferative potential of T cells in a chronic infection appears to be one of the largest obstacles for the development of successful therapeutic vaccinations. As shown in a recent study on SIV-infected macaques, the proliferative potential of virus-specific T cells in a chronic infection may be enhanced by lowering viral load with antivirals (e.g., providing rest from antigen) prior to therapeutic vaccination.30 Thus, a combination of vaccines with effective and, in the case of HCV, yet to be developed antivirals, appears to be necessary to achieve this goal.
The authors are supported by the NIDDK intramural research program.