Contribución de los modelos murino y primate al estudio de las enfermedades por arenavirus y fiebres hemorrágicas

Contribuição dos modelos murinos e primatas ao estudo de doenças por arenavirus e febre hemorrágica

Juan D Rodas¹, MV, MSc, PhD; Roger Hewson², PhD; María Salvato³, PhD.

¹ Grupo de Investigaciones en Ciencias Veterinarias “Centauro”, Facultad de Ciencias Agrarias, Universidad de Antioquia, Medellín, Colombia AA1226,juandavid.rodas@gmail.com. ² Novel and Dangerous Pathogens, Virology, Centre for Emergency Preparedness and Response, Health Protection Agency - Porton Down, Salisbury, Wilts SP4 0JG, UK roger.hewson@hpa.org.uk. ³ Institute of Human Virology; University of Maryland School of Medicine,725 West Lombard St., Baltimore, MD 21201, msalvato@ihv.umaryland.edu

(Recibido: 2 junio, 2009 ; aceptado: 14 julio, 2009)

Summary

This manuscript is an inedited part of my PhD dissertation, based on historical and recent findings on animal models, that was presented as part of the requirements to fulfill the conditions to become a philosophical doctor on Veterinary Sciences at the University of Wisconsin on October of 2003.The current mini-review written on a free-version style, underlines some of the cornerstones of immunology as a science, understood thanks to the use of the Lymphocytic Choriomeningitis virus (LCMV) experimentally and naturally infected mouse model. It should suffice to say that there have been two Nobel prices of Medicine for discoveries made through the employment of this animal model, in order to recognize the right importance to it. In addition, several laboratories, Dr. Salvato´s among them, have also employed the LCMV-infected Rhesus monkey model as a tool to unravel the mysteries of arenaviral hemorrhagic fever, and particularly the physiopathology of Lassa disease in humans. Here I show some of the knowledge generated through the study of both animal infections.

Resumen

El siguiente manuscrito, es un capítulo inédito de mi tesis doctoral, basado en hallazgos históricos y recientes sobre modelos animales, que fue presentado como parte de los requisitos para obtener el título de Ph.D. en Ciencias veterinarias en la Universidad de Wisconsin, en Octubre de 2003.La actual mini-revisión escrita en estilo de versión libre, subraya algunas de las piedras angulares de la inmunología como ciencia, entendidas gracias al uso del modelo murino infectado natural y experimentalmente con el virus de Coriomeningitis Linfocítica (LCMV). Sería suficiente mencionar que han existido dos premios Nóbel de Medicina por descubrimientos realizados a través del empleo de este modelo animal, para reconocer la real importancia del mismo. Adicionalmente, varios laboratorios, el de la Dra. Salvato entre ellos, también han empleado el modelo del mono Rhesus como un instrumento para desvelar los misterios de las fiebres hemorrágicas por arenavirus, y particularmente la fisiopatología de la enfermedad de Lassa en humanos. Aquí yo muestro alo del conocimiento generado a través del estudio de ambas infecciones animales.

Palabras clave: arenavirus, inmunología, modelos animales, LCMV.

Resumo

O seguinte manuscrito é um capítulo inédito da minha tese doutoral, baseado em casos históricos e recentes sobre modelos animais, que foram apresentados na defesa da tese de Ph.D. em Ciências Veterinárias da Universidade de Wisconsin, em outubro de 2003. A atual mini revisão escrita em estilo de versão livre, enfatiza em algumas pedras angulares da inmunologìa como ciência. Graças ao uso do modelo murino infectado natural e experimentalmente com o vírus da Coriomeningitis Linfocítica (LCMV). Seria suficiente mencionar que foram outorgados dois prêmios Nobel de Medicina pelos descobrimentos realizados a través do uso deste modelo, para reconhecer a real importância do mesmo. Adicionalmente, vários laboratórios, entre eles o da Dra. Salvato, tem utilizado o modelo macaco Rhesus como um instrumento para desvelar os mistérios das febres hemorrágicas por arenavirus, e particularmente a fisiologia e patologia da doença de Lassa em humanos. Aqui eu indico algo do conhecimento gerado a través do estudo das duas infecções animais.

Palavras chave: arenavirus, inmunologia, modelos animais, LCMV.

The Arenaviridae are a family of enveloped viruses with bi-segmented, negative-stranded RNA genomes. Previous research has established arenaviruses as excellent probes of cell-mediated immunity in mice and as possible bio-threat agents in primates. Several laboratories have investigated the acute disease in primates by comparing virulent and avirulent strains of virus, with the goal of determining the molecular basis of virulence. Five years ago we practiced infections of rhesus macaques with the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV) using the WE strain that has been known to cause both encephalopathy and multifocal hemorrhage, and serve to describe some of the fundamental aspects of the viral human hemorrhagic diseases. This discussion is inspired on some of those studies, but before that, is fair to give recognition to all the scientific data that made of LCMV in the mouse model, one of the best described and developed animal models for the study of both, the basic immune response and the understanding of the immune-mediated disease.

Contribution of the LCMV-infected mouse to immunology

Until the HIV era, the LCMV-infected mouse was the best-described model for cell-mediated immunity. This model was instrumental in the discovery of some of the most important concepts in immunology. In the 50's and 60's Rowe and Hotchin introduced the concept of immunopathogenesis, in other words the capacity of the immune system to cause disease (Hotchin, 1962; Rowe, 1954). Meanwhile, Rowe described how the immune system sometimes exerted only partial control of virus infection resulting in virus persistence as a chronic infection (Rowe, 1954). In the 60's Hotchin and in the 80's Jacobson and Ahmed, showed that different isolates of LCMV had different tropisms and displayed various degrees of immuno-pathological disease (Ahmed et al, 1984; Hotchin, 1962; Jacobson and Pfau, 1980).

During the 70's Cole explained how cytotoxic T cells cause lethal pathology in the form of T cell-mediated choriomeningitis (Cole et al, 1972) but very soon Mims, Doherty and Zinkernagel, independently described how T cells also mediate anti-viral protection against LCMV (Doherty, 1976; Mims and Blanden, 1972; Zinkernagel, 1976). In 1974, again Zinkernagel and Doherty, using LCMVinfected cultures, described a fundamental tenet of cell-mediated immunity, MHC-restriction of the cytolytic reaction between effector T cells and their infected targets. They demonstrated how MHC class I regulates cytotoxic T cell immune response (Zinkernagel and Doherty, 1974), which brought them the Nobel prize in Medicine more than 20 years later (1996).

In 1977, Riviere described the detrimental effects of interferon on suckling mice and Welsh showed that natural killer cells, a source of interferon, were important effectors of innate immune responses after systemic virus infection (Riviere et al, 1977; Welsh Jr and. Zinkernagel, 1977). In the 80's Oldstone showed the feasibility of using cytotherapy with CD8+ cells to clear infected virus carriers (Oldstone et al, 1986). In the 90's Planz and coworkers treated infected carriers with CD4⁺ T cells plus neutralizing antibody-producing B cells (Planz et al, 1997) based on older experiments from Volkert (1963). Also in1982, Oldstone and Buchmeier described the selective down-modulation of some viral transcripts during virus persistence in vitro and in vivo (Oldstone and Buchmeier, 1982).

Between 1968 and 1991, four labs (Leist et al, 1988; Mims and Wainwright, 1968; Odermatt et al, 1991; Silberman et al, 1978) described immunosupression by T cell-mediated antiviral immunity; this mechanism also contributes to destruction of antigen presentation in AIDS (AlthageA et al, 1992; Borrow et al, 1995).

In the 1990's several experiments demonstrated the role of cytotoxic T cells in selecting virus, in causing immunological exhaustion or in evading immune detection. Pircher et al (1990) described the selection of LCMV mutants that escape CTL activity. In the next year Ohashi and Oldstone, independently, found that viral antigen on cells strictly outside of lymphoid tissue, such as pancreatic B-islet cells, are immunologically ignored. In contrast to infection with LCMV, immunization with a vaccinia-recombinant expressing the LCMV-Gp is not sufficient to cause disease, although it induces a potent, primed CTL response, thus suggesting that there is a high threshold for causing lymphocytic choriomeningitis (Ohashi et al, 1991; Oldstone et al, 1991).

In 1993, Moskophidis described exhaustion of precursor T cells by virus that spread throughout the lymphohemopoietic system, particularly LCMVvariants that replicate quickly and to high titers (Moskophidis et al, 1993). In 1994 Kagi and Walsh discover that perforin is essential for effi cient, rapid CD8⁺ T cell-dependent LCMV elimination (Kagi et al, 1994; Walsh et al, 1994). Four years later Klenerman described a case of “original antigenic sin” at the CD8⁺ T cell level. This means that a CTL escape mutant virus may still induce a CTL response against the original wild-type virus that is not active against the mutant (Klenerman and Zinkernagel, 1998).

Still some issues remain unsolved in the mouse model for arenavirus research:

1. What is the reason behind the delay of neutralizing anti-LCMV responses? It is well known that neutralizing antibody responses against LCMV are delayed (Hotchin, 1962; Rowe, 1974). The delay suggests that viral antigen exhibiting the correct neutralizing epitope must still be available or even increased after day 50-100 at the time when neutralizing antibodies appear. CD8 elimination during the early phase of virus infection enhances generation of neutralizing antibody responses (Battegay et al, 1993; Planz et al, 1996). Both T cell-mediated general immuno-pathology and specific elimination of neutralizing antibody producing B cells may be involved.

2. What is the mechanism for survival of Cytotoxic T cell memory? While it is generally accepted that increased T (or B) cell frequencies of primed mice are antigen-independent, protective immunity against challenge with viruses (or tumors) outside of lymphoid organs is antigen-dependent (Bachmann et al, 1997; Kundig et al, 1996; Lau et al, 1994; Ochsenbein et al, 1999).

Could viral immuno-pathology described in the mouse contribute to primate hemorrhagic fever disease?

Immuno-pathology is a disease mediated by the immune response to a pathogen rather than by direct effect of the pathogen. Since many immune functions are controlled and restricted by host genetic factors, every individual responds uniquely and the outcome may vary greatly.

Immune-complexes that accumulate and deposit on organs such as kidney or spleen, are one source of immuno-pathology. Murine LCMV infection is considered a classical example of virus-induced immune complex disease. In this persistent LCMV infection non-neutralizing antibodies circulate in ratios and quantities that favor binding of complement and deposition in tissues that results in immunopathology. In a state that is affected by host and viral genetic factors (Oldstone et al, 1983), large quantities of immune-complexes along with IFN-γ contribute to glomerulo-nephritis (Walker and Murphy, 1987).

Innate immune responses represent another source of immuno-pathology. The induction of INF α/β by virus-infected cells occurs very rapidly (within a few hours of the infection) and represents an early and nonspecific antiviral response of the host. These molecules can inhibit the replication of many viruses through the induction of an “antiviral” state in the surrounding cells through the expression of: 1) 2'5' -oligoadenylate synthetases that mediates antiviral activities mainly by the induction of RNAse L, a cellular RNAse that degrades viral transcripts (Dong and Silverman, 1999; Terenzi et al, 1999), 2) the Mx proteins such as MxA protein (interferoninduced GTPase) that selectively inhibits influenza and bunyaviruses (Haller et al, 1998), 3) the double-stranded RNA-activated protein kinase (PKR), that apparently suppresses viral infections such as encephalomyocarditis virus and reovirus (Kalvakolanu and Borden, 1996).

Importantly, the early, virus-induced cytokine response may not be limited to INF α/β. Certain viruses such as LCMV and HIV have the potential to infect and activate macrophages, which secrete cytokines such as TNF-α (Guidotti et al, 1996) and Il-12 that induce IFN-γ (that also triggers other antiviral activities).

Another source of immuno-pathology is the response of antiviral lymphocytes, whose functions are not only to destroy infected cells expressing viral components presented in the MHC class I complex but also to secrete cytokines and chemokines that amplify the cellular immune response and have direct antiviral effects on infected cells (Von Herrath, 2002). Cytotoxic T lymphocytes effect some of the quickest and most specific antiviral immune response. There are at least 2 ways through which CD8 cells can be involved in immune-mediated viral diseases:

1. Cytolytically, they release perforins and induce the expression of Fas ligand that will result in apoptosis of virally infected cells (Kagi et al, 1994). A good example of immune-mediated pathogenesis where CD8+ cells are the key players, is the hepatitis induced in the mouse model after the ip inoculation with a viscerotropic strain of LCMV WE. Virus infection within the liver is predominantly confined to Kupffer cells from where it spreads to other cell types including hepatocytes. Apparently, the cellular damage is carried out by direct antiviral activity that is usually no cytotoxic. The number of infected hepatocytes is reduced, with less than 5% cellular destruction and this clearance is helped by the great regenerating capacity of the liver (Guidotti et al, 1996; Zinkernagel et al, 1986). In the HBV transgenic mouse model, there is an additional example of cell-mediated immune-pathology, where inefficient (or immature) CMI seems to lead to a serious condition known as cirrhosis. Cirrhosis is caused by progressive destruction and regeneration of hepatocytes, cirrhosis compromises the function of the organ and predisposes to liver cancer. In patients that develop cirrhosis cellular immune responses are restricted and weak in comparison to patients that overcome the acute HBV infection via high levels of IFN-γ and TNF-α that control the viral infection in a non- cytopathic way (Chisari, 1996).

2. Non-cytolitically, cytotoxic T cells secrete cytokines such as IFN-γ that not only increases antigen presentation by inducing expression of MHC class 1 and 2 but also induces TNF-α expression on macrophages (Guidotti and Chisari, 1996). IFN-γ also induces the release of NO (Guidotti et al, 2000), which affects viral targets such as the protease of Coxsackie virus (Saura et al, 1999; Zaragoza et al, 1997). TNF-α, in turn, increases expression of acute phase proteins such as IL-6 on Kupffer cells as part of the overall inflammatory response and proliferation after liver injury (Michalopoulos and DeFrances, 1997). More specifically, it has been shown that TNF-α also mediates downregulation of hepatic HBV messengers (2.1-Kb mRNA) induced by IL-2 (Guilhot et al, 1993). A good example of cytokine-mediated disease is the mouse model for insulin-dependent diabetes mellitus (type I diabetes) where inflammatory cytokines such as IFN-γ, TNF-α and IL-1β are secreted by CTL and/or antiviral CD4 lymphocytes that mediate death of islet b cells. Apparently perforin-mediated killing of b cells by autoreactive CTL is not sufficient to produce clinically overt diabetes in vivo; a direct effect of IFN-γ produced by islet-infi ltrating CD4⁺ and CD8⁺ lymphocytes is also required. Conversely, regulatory cells characterized by their ability to secrete cytokines such as IL-4, IL-10 and TGF-β can act in a suppressive regulatory manner if they home to a site targeted for autoimmune attack and prevent disease (Bach, 1994).

Very few examples have illustrated better the delicate balance between cell-mediated immunity and immune-mediated pathogenesis, than the transgenic HBV mouse (Ando et al, 1993) and the intra-cerebral LCMV infected mouse (Cole et al, 1971; Cole and Nathanson, 1974; Cole et al, 1972), repectivelly. In the first one the presence of CD8+ competent cells favor recovery, while in the second one the same cells induce lethal inflammation.

In a series of experiments with the hepatitis B virus (HBV) transgenic mouse, Chisari and Guidoti have demonstrated how IFN-γ and TNF-α/β secreted by activated T lymphocytes are the chief mechanisms for eliminating virus from liver in the absence of cell death (Guidotti et al, 1994; Guidotti and Chisari, 1999; Guidotti et al, 2002) . The rationale behind these studies is that viruses such as HBV outnumber the CTL by several orders of magnitude, yet the clearance in more than 95% of acutely infected adults could only be explained by the presence of cell mediators released by local macrophages and activated T cells. Viral clearance occurs in the absence of massive destruction or regeneration of hepatocytes and in the presence of inflammatory cells and Kupffer cell hyperplasia (Guidotti and Chisari, 1996). Their results suggest that HBV specific CTLs recognize and kill a small fraction of infected hepatocytes and later on, they secrete inflammatory cytokines that directly or indirectly (via macrophage activation) “cure” most of the infected hepatocytes by non-lytic intracellular inactivation pathways. Other authors have previously introduced the concept of cytokinedependent “intracellular inactivation” for viruses such as herpes simplex (Martz and Howell et al, 1989).

A growing literature attributes further examples of cytokines controlling viral infections, and viruses encoding proteins that control cytokine activities, e.g. blocking the transcriptional activation of the IFN-activatable genes (Foster et al, 1991; Kalvakolanu et al, 1991), down-regulating TNF-α/β production in vitro (Adler et al, 1996) or presenting receptor analogues for IFN-α/β, IFN-γ and TNF-α/β (Alcami and Smith, 1995; McFadden et al, 1995; Guidotti and Chisari, 2000; Guidotti and Chisari, 2001; Schreiber and McFadden, 1994; Guidotti et al, 1999; Symons et al, 1995; Upton et al, 1992) , apparently as a strategy to blunt the antiviral activity of these cytokines.

In summary, if cytokines do play an effector role in the antiviral immune response, each virus can be expected to display its own cytokine sensitivity profile, and some viruses (including different subtypes of the same virus) may simply be insensitive to cytokine-mediated control. For example LCMV can establish a persistent infection in vivo in the presence of concentrations of TNFα/β and IFN-α/β that abolish HBV replication, thus LCMV is resistant to these cytokines in comparison with HBV (Guidotti and Chisari, 1996).

On the other hand, it has been suggested that the lethal choriomeningitis observed in the murine model intracerebraly inoculated with LCMV could be related to the deleterious effects of the inflammation caused by the presence of infiltrating cells. Even though the mechanism of death has not been completely elucidated, it is thought that besides the direct disruption of blood-brain barrier by lysis of infected cells and the increase of pressure in the skull produced by inflammatory cells, cytokines and other mediators of inflammation such as nitric oxide that are produced by infiltrating CD8+ T cells, could also be important effectors of the immune-mediated tissue damage (Borrow and Oldstone, 1997).

Evidence against immune-mediated pathology in the arenaviral hemorrhagic fever

Clinical studies of disease in the LCMV-infected mouse focused on the role of host-immune response to viral antigens as the actual cause of the disease. In contrast there is no evidence that the immune response exerts an adverse effect in the arenaviral hemorrhagic fever (HF). It has been shown, for instance, that immunosuppression with cyclosporin A or cyclophosphamide does not ameliorate disease of guinea pigs infected with the Junin virus. In fact these drugs affect the immune response to attenuated Junin viruses converting benign to lethal infections (Kenyon et al, 1985; Kenyon and Peters, 1986). The same results have been corroborated by others in different models: Machupo-infected monkeys (Eddy et al, 1975), Pichinde-infected guinea pigs (Johnson unpublished), Pichinde-infected hamsters (Murphy et al, 1977), LCMV-infected guinea pigs (Buchmeier et al, unpublished) and LCMV-infected hamsters (Genovesi and Peters, 1987).

Another evidence such as the close correlation between the level of viral replication in vivo and the burden of clinical symptoms (Peters et al, 1987), also disputes the concept of immune-mediated pathogenesis by arenaviruses in different models of human HF. Even though in the mouse model, LCMV-WE can produce lethal immune-pathology of the liver (Bonilla et al, 2002) the disease differs from the one observed in primates in the low level of lymphocytic infiltration. In addition the major virulence determinants of LCMV WE (Riviere et al, 1985) and Lassa (Lukashevich, 1992; Lukashevich et al, 1991) are encoded on the large genomic segment and since this segment carries the genes that determine the level of virus replication, the amount of replication is likely to be the primary determinant of virulence.

Of course there are some other important factors that determine viral pathogenesis, for example the capacity to regulate mediators of infl ammation such as IL-8. This capacity has been well characterized and mapped to the S segment of Lassa by comparing the in vitro induction levels of this cytokine after infection with the reassortant Lassa / Mopeia and the parental viruses (Lukashevich et al, 1999).

High circulating levels of IL-6, IL-6 receptor, and TNF receptors 1 and 2 are found in clinical cases of acute hepatitis in rhesus iv or ig inoculated with the WE strain of LCMV (Lukashevich et al, 2003) and they could be interpreted as signs of immune-mediated pathogenesis. However these factors seem to be more beneficial than detrimental since they help to regenerate injured hepatocytes (Michalopoulos and DeFrances, 1997). So it would be interesting to pursue additional and more conclusive experiments showing whether the inhibition of these specific cytokines or the depletion of other limbs of the immune response such as CD8, CD4, CD20 or even unspecific immune defense mechanisms such as complement, play a critical role in pathogenesis or immunity. Examples of these elegant approaches have been already performed on other models such as the chimpanzee infected with HBV or HCV (Thimme R, et al., 2003) and SIV-infected rhesus monkeys (Schmitz et al, 1999; Schmitz et al, 2003; Schmitz et al, 1999).

Therefore, in spite of the negative experiments with immunosuppressive agents, the participation of the immune system in host damage has not been completely ruled out. Based on the studies from Chisari et al in the transgenic mouse model and the chimpanzee model for HBV it is plausible to speculate that we still do not know enough about the local and circulating level of apoptosis potentially induced by the population of CD8+ cells during the different stages of the LCMV-mediated hepatitis, nor do we have enough information about the role played by different important cytokines such as IFN-α, β and γ and TNF-α. As it has been described before, there is a precedent for the non-cythopatic viral clearance mediated by cytokines released by CD8+ cells activated during the acute infection with HBV.

Until the time comes when as we can manipulate the monkey model for hemorrhagic fever in such a way that we can explore the impact of different immunological effectors, as it has been done for the SIV- infected rhesus and the HBV-infected chimpanzee, we will rely on the indirect evidence that has already been presented. Future research should examine functional knock out of different arms of the immune system in order to determine the roles of immuno-pathology on hemorrhagic fever disease.

After having studied the LCMV-WE strain and its effects on the monkey model as a way to understand the pathogenesis of arenaviral hemorrhagic fevers, still some questions remain to be answered, among those are: What are the viral epitopes that elicit immune responses? A systematic study of Np and Gp peptides known from mouse studies, and using haplotyped monkey PBMC in immune-assays must be done in the future; the tools for doing CTL assays in the monkey could be greatly improved by identifying those. Interferon gamma secretion in response to peptides would also help to identify important CTL epitopes.

Finally, we need to reveal virus dissemination and gene expression changes that occur during the course of acute infection. In that sense and as an anecdotic comment, by the end of my academic training, I had the chance to participate in the necropsy of 10 monkeys that were inoculated with a lethal dose (10³ PFU) of LCMV WE virus and euthanized each day for a week. The stored tissues were and have been studied to prove our hypothesis, what is that the liver is the main reservoir for virus replication, and that gene expression changes in the liver contribute to the multi-organ disaster that is hemorrhagic fever. Some of the results of these studies have been published during the course of the last three years after my departure from my advisor´s lab, and some more are still to come and are already accepted for publication in the Memorias of the Institute Oswaldo Cruz from Brazil.

In summary, unlike the aberrant disease immune response-mediated to the artificial intracranial inoculation in the mouse model, the response to a viral infection in which the disease is proportional to dose in the monkey model, must be closely examined in the infected organs and use to learn the sequence of events leading to death and to determine the possible targets for therapeutic interventions or best suited response to prevent fatal outcomes.

Acknowledgments

Many thanks to my mentor Maria Salvato, for her ideas and constructive discussions, that are the inspiration of this article.

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