Animal Models

A small animal model for hepatitis C would offer unprecedented opportunities for the research into the pathogenesis of the virus and offer a platform for drug and vaccine testing. The following paragraph will show the possible positive impact of a small

animal model on the example of the polio mouse model as well as the current state of research on the hepatitis model.

A good example for the success of small animal models is the TgPVR mouse model for poliovirus. It had been know that the poliovirus enters the human cells through the poliovirus receptor CD155 [84] a protein markedly different to his rodent homolog.

After the human gene of this poliovirus receptor (PVR) was transferred into now transgenic mice the virus was able to replicate in the neuronal tissue and animals displayed symptoms of polio comparable to humans [85]. But this model was still lacking the normal entry of viral particles into the body of the animals, since only an intravenous or intracranial injection would lead to the desired infection. It was demonstrated that after blunting of the immune response in the gut of these mice by further knocking out the interferon-/-receptor on oral infection with subsequent viral replication and clinical symptoms could be recapitulated [86]. These models have accelerated the development and the approval of new oral vaccines immensely.

They have been used not only in the phase of development, but also as a tool for safety testing reducing the need for studies in monkeys [87]. The mouse model for polio provides only example of how the hurdles of transspecies transmission can be overcome and the subsequent benefits that can be obtained from a small animal model. This process also generates immense knowledge acquired through the intensive research on the virus itself.

There are three main approaches in creating a small animal model for hepatitis C virus: 1. The adaptation of HCV to non-human hosts, 2. The xenotransplantation of human tissue into animals and 3. The genetic host adaptation [88]. As already described, HCV uses a minimal set of four entry factors to penetrate the human cell:

SCARB1, CD81, hOCLN and hCLD1 [46]. Two of these, CD81 and OCLN, cannot be used by HCV to enter murine cells. The idea of the first system is to adapt the virus to the rodent factors in order to allow entry. It has been shown recently that a laboratory strain of HCV could be adapted to murine CD81 using selection pressure [53]. During this process mutations in the E1 and E2 envelope proteins appeared to change not only the behaviour towards CD81, but also towards the other entry factors. It is unclear how close this process mimics the naturally occurring entry and it remains to be shown that these viruses work in vivo [89]. The aims of this approach include the gain in knowledge about the entry factors and the viral glycoproteins and the

prospect of finding new therapeutic targets, as well as a system needing only little modifications in the virus. It has yet to be shown, if results obtained with an engineered virus would allow deduction to a human strain. Until now, only the process of entry has been addressed leaving the question, if the virus would overcome hurdles in replication and assembly in the murine liver [88].

The second approach is a xenotransplanted model in which mouse cells replace the human liver cells. With these models up to 99% chimerism can be achieved [90]. Two requirements have to be fulfilled to allow successful engraftment: The recipient animals must be immunocompromised and a liver injury must be induced to destroy the endogenous liver cells. Two well-characterized models are used for this purpose:

the urokinase plasminogen activator transgenic mouse [91] and the fumarylacetoacetate hydrolase (FAH) gene deficient mouse of which the latter one allows a more robust model and a higher throughput [92, 93]. FAH is the last step in the tyrosine breakdown. A knockout leads to hypertyrosinemia, which is lethal in humans and leads to liver failure in mice. Treatment with 2-(2-nitro-4-trifluoromethylbenzyol)-cyclohexane-1,3-dione (NTBC) prevents liver toxicity and offers the option of inducing the liver injury at any point by withdrawing the drug (NTBC). These mice have been crossed with immunocompromised background models and could subsequently be engrafted to high levels with human hepatocytes [90]. Human liver chimeric mice are susceptible to HCV and HBV and can be used for drug testing [88]. Unfortunately the generation of these animals is expensive and complicated. Furthermore they do not display any of the long term complictions of chronic HCV infection such as cirrhosis or hepatocellular carcinoma. Another disadvantage is the lack of a working immune system, which limits its utility for the study of the pathogenisis of HCV and the inflammatory response to the virus [90].

Although there are attempts to transplant parts of the human immune system into these mice, this manipulation and the necessary surgery further decreases the survival rate of the animals [89].

The third way of rendering rodents susceptible to HCV is the generation of inbred genetically modified model with inherited susceptibility to human hepatitic C virus. To achieve this one has to identify the barriers in the murine organism that hinder HCV from successfully multiplying. This could be either human specific genes whose murine orthologs are non-functional and need to be expressed in mice or obstacles

that have to be removed e.g. by targeted gene disruption [89]. At the level of entry it has been shown that human CD81 and OCLN constitute the minimal set of genes required for viral uptake in vitro [55] and in vivo [54]. The model presented by Dorner et al is only limited to entry of the virus, but since these animals have a fully competent immune system they can already be used for the study of vaccines and drugs inhibiting the entry process. Research in recent years has tried to dissect the remaining life cycle of HCV in order to identify the crucial steps which are blocked in mice. Translation of viral RNA into proteins is known to be successful, but only under high selection pressure HCV is able to replicate in these mouse derived cells [94].

The replication can be further increased by blunting of interferon type 1 response [95]. This suggests that an overactivation of the cellular antiviral response plays a key role in the decreased RNA replication. It is encouraging that Frentzen et al were able to show that no contrutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in murine cells [96]. The later stages of the life cycle appear to be supported in rodents if all the required apolipoproteins are provided [97].

The aim of my research work is to unravel and fit all the pieces of the puzzle together and demonstrate that the entire HCV life cycle can be recapitulated in one cell. I hope that my findings will significantly contribute to the translation and construction of an in vivo model.