Photo of hepatitis C virus
Courtesy of Alfred M. Prince, MD


Hepatitis C Background:

In 1974, Prince and colleagues documented the importance of a form of transfusion associated hepatitis not due to hepatitis A or B viruses. This virus was tentatively termed hepatitis non-A, non-B virus, however after the cloning and sequencing of its genes, it was called hepatitis C virus (HCV). Cloning of the viral genes made it possible to develop tests for the diagnosis of HCV infection, revealing for the first time the enormous magnitude of the prevalence of this infection, and of the disease burden. It is now known that about 200- 400 million persons worldwide are chronically infected with HCV, about 3- 5 million of these are in the U.S.

HCV is transmitted by transfusion and use of unsterile injection practices, medically, and by drug users. In parts of the developing world traditional practices such as acupuncture, scarification, male and female circumcision also must play a role. Only about 5% of HCV infections are transmitted by blood transfusion, even less in the developing world, thus improved blood screening, while important, will by no means solve the problem of the increasing prevalence of HCV infection. The WHO program to improve injection practices by use of disposable non-reusable syringes and needles is valuable, but will likely take decades to reach all parts of the developing world, and in particular drug users. Thus development of a vaccine is essential to the control of HCV infection.

The chronic infection, which affects about 80% of those infected, lasts life long and predisposes to the development of cirrhosis and liver cancer. In the U.S. HCV is the most common cause of liver cirrhosis leading to liver transplantation. WHO has stated that liver cancer is one of the most common cancers in the world, about half of the cases are due to chronic HCV infection, with most of the remainder being due to hepatitis B infection.

HCV Facts:

According to David Satcher, the current surgeon general, and C. Everett Koop, the former surgeon general, hepatitis C is one of the most significant public health threats facing Americans. There are an estimated 3.9 million (1.8%) Americans infected with HCV of whom 2.7 million have chronic hepatitis C. This makes the virus 4 times more prevalent than HIV.

According to the 1996 estimates, there are 36,000 new cases each year. 2/3 of those infected are baby-boomers between the ages of 30 and 49.

Hepatitis C causes chronic liver disease in 20% of those infected. This often leads to cirrhosis of the liver, cancer of the liver and liver failure. Each year Hepatitis C causes 8,000 - 10,000 deaths. Hepatitis C is particularly fatal in patients with HIV.

It is estimated that hepatitis C costs the country $600 million in medical costs and lost work (excluding liver transplants). For patients who do not undergo liver transplants, the average lifetime cost of hepatitis C is $100,000.

Hepatitis C is the leading cause of liver transplantation (about 1,000 liver transplants in the US) and has caused the waiting list for donors to triple in the past 5 years.

The incidence of hepatitis C in veterans is 8 - 10% or 4 or 5 times that of the population in general with Vietnam veterans particularly at risk. The past Miss America, Heather French, chose hepatitis C awareness for veterans to be her national campaign.

Most people with hepatitis C do not know they are infected because about 2/3 of those infected never experience symptoms. Among those who do experience effects of the disease, often take the form of flu-like symptoms, i.e., fatigue, muscle aches, and nausea.

Symptoms can appear up to 20 to 30 years after infection, often after significant liver damage has already occurred. Because of the long, latency period of the disease, the death rate from hepatitis C is expected to triple in the next 10 to 20 years.

If caught early, hepatitis C can be treated with alpha interferon and ribavirin with a 20 - 50% effectiveness rate. With early warning, damage to the liver can also be reduced by eliminating drinking alcohol.

People at high risk for hepatitis C include: those who received blood transfusion or organ transplant before 1992 or were given clotting factors before 1987, those who have undergone hemodialysis, those who have used illegal injectable drugs (even if only once), those who work in health care, those whose mothers were infected with hepatitis C, those with tattoos or body piercing and, those who have had multiple sex partners or have been the partner of someone with hepatitis C.

CURRENT STATUS OF PROPHYLAXIS:

The first attempt to produce an HCV vaccine was reported almost a decade ago (Choo et al., 1994). This candidate vaccine was composed of HCV surface proteins expressed by vaccinia virus in mammalian cells. It induced high antibody titers, which were however short lasting. When immunized chimpanzees were challenged with a low dose of homologous virus, at the peak of the antibody titer, a high proportion were protected. This vaccine, however, was deficient in a number of respects: protection was mediated mainly by antibody, which is known to be relatively ineffective against higher dose challenges due to the coating of virions by host lipoproteins (Hijikata et al., 1993); the short duration of high antibody titers cast doubt on the duration of protection; use of surface proteins alone would be unlikely to produce cross genotype protection; and lastly, the production process was costly, making this vaccine inherently unaffordable in the developing world.

During the years since the development of the above subunit vaccine candidate, investigators have explored alternative approaches to HCV immunization in mice and in the chimpanzee model. Most approaches have attempted to induce strong cell-mediated immunity, since this was considered more likely to achieve prevention of chronic infections, which are likely to be avoided by broad and strong cell-mediated immune responses (Guidotti et al., 1996; Cooper et al., 1999) Such responses were considered more likely to protect against multiple genotypes and quasispecies, due to the large number of available CTL epitopes associated with each of the viral proteins. These newer approaches to development of an HCV vaccine have utilized DNA-based immunization (Major et al., 1995; Arichi et al., 2000; Gordon et al., 2000; Forns et al., 2000), prime boost strategies usually consisting of DNA priming followed by a recombinant viral vector boosting (Pancholi et al., 2000; Pancholi et al., 2003), DNA priming followed by HCV protein boosting (Rollier et al., 2004), HCV virus-like particles synthesized in insect cells (Baumert et al., 1999), and more recently use of heterologous prime-boost with recombinant viral vectors alone ( Folgari et al, 2005, and see below). A major impediment to progress has been the high cost of research with chimpanzees and their limited availability (see below), Chimpanzees are unfortunately the only established animal model for HCV infection.

DNA immunization alone has been found to be relatively weakly immunogenic, and requires high doses of DNA, which would make it totally unaffordable in the developing world. The prime boost strategies have been found to be more immunogenic, however protection in the limited chimpanzee evaluations reported so far carried out has been disappointing: acute phase viral loads have been reduced in immunized animals after challenge with live virus, and viral loads in chronic infection have been reduced, but effective prevention of chronic infection (the goal of CMI-based vaccines) has not been demonstrated. However, in recent studies J.W.Youn and A.M, Prince at the New York Blood Center have observed protection against the development of chronic infection in 4/4 immunized chimpanzees after challenge with a recombinant vaccinia vector (see below). They now need to search for a safer poxvirus vector than the vaccinia (WR) used in this study. Such a vector has now been developed in a study by B. Jacobs, J.W.Youn, A.M. Prince et al.

An additional problem is that preliminary findings showing development of chronic infection after challenge with different HCV isolates of unknown genotype (Farci et al., 1992), as well as data from the Prince laboratory on heterologous challenge with diverse genotypes (Prince et al 2005). These results suggest that protection of animals immunized with one genotype may not extend to other genotypes, thus a multi-genotypic vaccine may be necessary. However, recently Lanford et al. (2004) reported in a small study that heterologous genotype challenge of 3 animals did not result in chronic infection. It has also been reported that 2 chimpanzees immunized with HCV 1a and challenged with HCV 1b did not become chronically infected (Rollier et al., 2004).

The goal of future studies is to develop a stronger, long-lasting immune response, using as vectors for priming and for boosting attenuated replicating vaccinia viruses (VV), possibly containing genes or epitopes from multiple HCV genotypes, and multiple pathogenic viruses e.g. HBV and HIV.

This approach has two major advantages over presently pursued strategies: (1) it would be affordable in the developing world where an HCV vaccine is particularly needed; and (2) the large amount of foreign DNA (25-30 kb) that can be inserted into pox viruses would permit inclusion of genes or epitopes from multiple genotypes or from other pathogens requiring affordable immunization strategies, e.g. HBV, HIV, etc.

Vaccinia virus (VV) is one of the most effective vaccines ever used, having eliminated smallpox globally for less than 10 cents/dose. No cold chain is needed, and protection lasts for at least 10 years, and probably much longer. Recombinant VV, in which genes from other pathogens are introduced into the genome of VV, have been shown to protect against HBV (Moss et al., 1984), Japanese encephalitis virus (Konishi et al., 1992), canine distemper (Stephenson et al., 1997), Influenza (Bennink et al., 1984; Yewdell et al., 1985), vesicular stomatitis virus (Mackett et al., 1985), and rabies virus (Wiktor et al., 1984; Rupprecht et al., 1986).

It may be asked why this strategy was not pursued further. There are several reasons:

(1) VV vaccines were not used after the eradication of smallpox, however, now the threat of bioterrorism mandates their reintroduction),

(2) Present VV vaccines have significant side effects: most importantly generalized vaccinia in immunosuppressed individuals, which is frequently fatal. As immunosuppression due to AIDS is now widely prevalent, this is a serious problem. Fortunately, introduction of additional genes into a vaccine strain of HCV has yielded a safe attenuated vector which is entirely noon-pathogenic even in immunodeficient SCID mice (Jacob, Youn, Prince et al, In preparation, 2006) This research was funded under NIH grant # UO1 57303-01 to develop safe and highly immunogenic VV vectors for use in defense against use of smallpox as an agent of bioterrorism, using this or similar attenuation strategies. These attenuated vectors, engineered to express HCV or other viral antigens, will be used in the further development of an HCV vaccine.

An alternate approach, which is currently popular, is the use of recombinant non-replicating, and thus attenuated, poxviruses such as MVA, NYVAC, fowlpox and canary poxviruses. These have been shown to be safe, immunogenic, and protective in experimental test systems (Belyakov et al., 2004), however, as large doses must be used for boosting, these non-replicating vector are more expensive to produce than replicating vectors, which do not require large doses for boosting. Thus, the replicating attenuated vaccinia approach will be more affordable in the developing world.

Overall Strategy and Use of Chimpanzees

The Prince laboratory approach to vaccine development is first to evaluate and optimize immunogenicity in mice, with emphasis on all aspects of cell-mediated immunity. However, they believe that prior to embarking on clinical trials it is desirable to evaluate the optimized vaccine in the chimpanzee model. It is only in this model that one can confidently determine whether a vaccine protects against chronic infection. If 8 chimpanzees are immunized and challenged with live HCV, and none become chronically infected this would indicate statistically significant successful prevention of chronic infection.

The Prince laboratory's chimpanzee research facility in Liberia, Vilab II, presently houses 74 chimpanzees, of which 12 are unexposed to HCV and seronegative. An average of 12 additional seronegative animals can be obtained each year by breeding and adoption of unwanted pets. This laboratory can be maintained in Africa at a much lower cost than would be possible in the U.S. The availability of sufficient chimpanzees for HCV vaccine research is a unique advantage.

It is important to recognize that, for reasons that are not yet well understood, chimpanzees do not suffer or develop significant disease as a result of infection with hepatitis viruses, In contrast to humans, these animals do not develop the chronic diseases, cirrhosis and hepatocellular carcinoma, that affect humans infected with these viruses.<

The Prince laboratory was asked by the New York Blood Center to explore the possibility that this research could be done in the U.S. There are now only three centers in which chimpanzee research can be done in the world: the Southwest Foundation for Biomedical Research (SFBR), New Iberia, and the MD Anderson Cancer Center in Texas. We queried these centers regarding the availability of chimpanzees for this study. Southwest Foundation for Biomedical Research informed us that, because of the moratorium on chimpanzee breeding in the U.S., they did not have sufficient non-immune animals to support this study. The other centers did not reply, presumably for the same reason.

Liberia has recently emerged from 12 years of civil conflict. During this period our research was interrupted for only 2 weeks. Presently the conflict has ended and security is enforced by 15,000 UN peacekeepers. We do not anticipate any problem with the performance of the proposed study as the UN forces are expected to remain for 5-10 years. Vilab II is located at the Liberian Institute for Biomedical Research (LIBR), a division of the Liberian Ministry of Health. Vilab II, and its research is strongly supported by the Director of LIBR, and by the Minister of Health.

Vilab II is directed by an outstanding veterinarian/virologist, Wolfram Pfahler, DVM, PhD who supervises a full time staff of about 40 well-trained and dedicated technicians, animal handlers and support staff. The laboratory is well equipped for laboratory work, including real time PCR, and surgical procedures. All samples are stored and shipped in liquid N2. All procedures are carried out in accordance with GLP.

TREATMENT OF CHRONIC HCV INFECTIONS WITH IMMUNOTHERAPY

As drug therapy is totally unaffordable in the developing world, a very exciting possibility is that effective immunization could allow the host to control the infection through his/her own immune response. Vilab II has conducted 8 studies to investigate this approach. So far none has proven effective, however the search continues.

SCIENTIFIC COLLABORATIONS AND COMMUNICATION

An essential collaboration for this project is the laboratory of Bertram Jacobs, PhD at Arizona State University. Dr. Jacobs and his 15 postdoctoral collaborators are outstanding vaccinia geneticists. In collaboration with the Prince laboratory, they were awarded an NIH Bioterrorism grant (UO1 AI 5753-01) to develop an absolutely safe replicating smallpox vaccine. A safe and effective attenuated vaccinia virus vector has been developed, as briefly summarized above This will make an ideal vector for HCV immunization.

In addition, the Prince laboratory plans to hold an annual 3-day meeting for its collaborators, and invited outside experts, following the format of the Symposia on Molecular Biology and Immunology of HCV, which it have sponsored, with the support of the Hepatitis Research foundation, in previous years. As these meetings are limited to a relatively small number of experts, they proved to be outstandingly stimulating.