The following paper was commissioned by the Committee on the Use of Chimpanzees in Biomedical and Behavioral Research. The responsibility for the content of this paper rests with the authors and does not necessarily represent the views of the Institute of Medicine or its committees and convening bodies.
By: Nancy L. Haigwood, Ph.D.
Professor of Microbiology and Immunology Director
Oregon National Primate Research Center
Christopher M. Walker, Ph.D.
Professor of Pediatrics
Nationwide Children’s Hospital
The Ohio State University
The purpose of this white paper is to compare genetic and functional features of immunity and the response to infection in humans and major nonhuman primate species currently used in biomedical research. The search for appropriate disease models has been stimulated by the need to understand the most intractable of the persistent and lethal pathogens, as well as chronic diseases and conditions that are determined by the genetic makeup of the individual. Because the outcome of infection is governed by carefully coordinated innate and adaptive immune responses, and pathogens have evolved strategies to evade these defenses, use of animal models that recapitulate key features of human infection is critical. Successful nonhuman primate models closely emulate human immunity, inflammation, and disease sequelae. They can also provide a critical pathway to clinical testing of risky prevention or treatment strategies for serious human diseases.
Some past successes of infectious diseases research in nonhuman primates are described. However, the primary objective of the paper is to identify conditions that either support or limit use of these animals for the study of human viral, bacterial, or parasitic infections. A survey of the published literature reveals that the common chimpanzee (Pan troglodytes) is the only great ape used in infectious disease research. With few exceptions there is usually no alternative, because lower species are not permissive for infection or fail to replicate key features of disease. Most studies involve very small numbers of chimpanzees to ensure safe translation of vaccines or therapeutics to humans, or provide incontrovertible evidence for basic mechanisms of immune control and evasion that cannot be obtained in human subjects. Various monkey species, primarily the Asian macaques (Macaca species), have served as models for infection with human viruses and microbes. Alternatively, monkey pathogens like the simian immunodeficiency viruses (SIV) provide a reliable model of human infection with closely related viruses like human immunodeficiency virus (HIV). Infection studies with human and monkey viruses have facilitated advances in vaccine development and studies of immunity and pathogenesis relevant to humans.
Sequencing of the human, chimpanzee, and macaque genomes has provided unprecedented insight into the evolutionary relationship between these species, especially for genes that regulate host defense and susceptibility to infection. Here we also provide examples of gene families involved in immunity that have been largely conserved since speciation,
and others that have undergone rapid evolution because of selective pressure by infectious diseases. How these adaptive changes might affect modeling of human infectious diseases in monkeys and great apes is discussed. Contemporary examples of primate infectious disease models that replicate most if not all features of human infection and immunity are provided. Where infection models are not perfectly matched in humans and nonhuman primates, differences have provided insight into key features of the relationship between the pathogen and its human host.
Finally, several practical advantages of nonhuman primate models are also reviewed. The include the ability to (1) infect with clonal or genetically modified pathogens, (2) modify the immune response to identify protective mechanisms, (3) sample at the earliest times after infection, often before symptoms are apparent in humans, and (4) access organs or tissues that are the primary site of infection. The latter is particularly important because blood, which is often the only compartment available for human sampling, may not adequately reflect immunity at the site of infection. Advances in adapting the most sophisticated technologies to nonhuman primates, including methods to monitor immunity, and understand molecular aspects of infection using genomic and proteomic approaches, has the potential to provide new insight into vaccination and infection with known and emerging pathogens.
Historical and Current Examples of Human Infectious Disease
Research in Chimpanzees
Chimpanzees have been used for over 100 years to model human viral, bacterial, and parasitic infections. This long history has revealed that chimpanzees are often uniquely permissive for infection with some medically important human pathogens. These animals can also provide a more faithful model of human disease than lower nonhuman primates. Studies in chimpanzees, particularly with hepatotropic viruses, have provided critical insight into host defense mechanisms and facilitated development of vaccines that have changed global public health. Yet for other pathogens key features of immunity and infection outcome differ between humans and chimpanzees. In these instances, the host-pathogen interaction is influenced by adaptations that are species-specific despite a strikingly close genetic relationship.
The promise and limitations of chimpanzees as an infectious disease model were first recognized in a 1904 publication from Albert Grunbaum who transmitted the Eberth-Gaffky bacillus (Salmonella typhi) to two animals (Grunbaum, 1904). The bacillus, isolated 10 years earlier, was the suspected cause of enteric fever. Efforts to satisfy Koch’s third postulate by transmission of disease to rats, rabbits, and monkeys had failed. Infection of chimpanzees was successful, but with much milder disease symptoms than expected. The author noted “the virulence of my cultures was not sufficient to produce a fatal result in the two instances in which they were given the opportunity to do so” (Grunbaum, 1904). That the chimpanzee might not be suitable for S. typhi vaccine development was highlighted in a 1914 publication (Nichols, 1914). The author, a proponent of a killed vaccine, critiqued an earlier study where such an approach had failed. “The authors found that a whole killed vaccine did not protect chimpanzees. But they used tremendous infecting doses—the contents of a whole Kolle flask. The problems must be settled, as some of them already have been settled, by actual experience with large numbers of men kept under close observation” (Nichols, 1914).
Infectious disease research involving chimpanzees published in the last 30-40 years fits into three broad categories. They include (1) identification and characterization of infectious agents that are serious public health threats; (2) characterization of protective immunity and how it is subverted; and (3) development of strategies to prevent or treat human infections. All published experimental infection studies used human pathogens and not closely related (and thus potentially different) chimpanzee pathogens as a model. Here, factors that determine the suitability of chimpanzees for research on infectious diseases are reviewed. Malaria, respiratory syncytial virus (RSV), HIV, and the hepatitis viruses are used as case studies.
Malaria vaccine research is made difficult by complexities of the parasite life cycle and selection of antigens to either interrupt transmission of infection or protect from disease after a mosquito bite (Good and Doolan, 2010). That irradiated P. falciparum sporozoites prevent disease was established several decades ago, but this approach is not easily scaled for human vaccination (Hoffman and Doolan, 2000). A small pilot study demonstrating protection of chimpanzees by a recombinant liver stage antigen derived from the sporozoites (Daubersies et al., 2000,
2008) laid the foundation for a recent human clinical trial of this concept (ClinicalTrials.gov NCT00509158). Nevertheless, malaria is exceptional because human challenge studies are permissible and studies in lowerorder species can provide guidance for vaccine development. Many malaria trials that are either underway or completed involved parasite challenge of vaccinated human volunteers followed by co-artemether eradication therapy if necessary (as an example see Porter et al., 2011). Recent studies have also included sophisticated analyses of humoral and cellular immune responses with goal of identifying protective correlates in human volunteers (Good, 2011). While chimpanzees have been used sparingly to date, there is increasing concern that no successful vaccine has emerged from the concepts tested to date. If progress requires identification of new antigens and a better understanding of immunity, especially in the liver (Good, 2011), the place of the chimpanzee in malaria research may be reconsidered.
Chimpanzees have also been critically important for the in vivo generation of malaria parasites that are recombinants between drug-resistant and drug-sensitive strains in order to map drug resistance genes and thereby better understand the metabolism of these pathogens and to develop improved drugs. Several studies that used parasites generated by this approach have been published recently (Hayton et al., 2008; Nguitragool et al., 2011; Sa et al., 2009).
Respiratory Syncytial Virus
RSV was first isolated from captive chimpanzees with upper respiratory tract disease (Blount et al., 1956) but was quickly identified as a human virus (Chanock and Finberg, 1957; Chanock et al., 1957). It is now recognized as the most important viral agent of severe respiratory tract disease in infants and children worldwide (Hall et al., 2009; Nair et al., 2010). RSV is also an important cause of morbidity and mortality in the elderly and in profoundly immunosuppressed individuals. Protection of vulnerable infants and young children from severe airway disease by a licensed monoclonal antibody against the RSV F protein, as well as the protection in the general population afforded by prior infection, suggests that active vaccination is also feasible (Graham, 2011). Progress over the past 4 decades was slowed by an unfortunate clinical trial of a formalin inactivated RSV vaccine that worsened disease and resulted in two deaths upon natural infection with the virus (Kapikian et al., 1969). There is a general consensus that the vaccine failed to induce potent neutralizing
antibody responses, provoked heightened lymphoproliferative responses, and was associated with eosinophilia and immune complex deposition in airways. Rodent and monkey models have demonstrated Th2 responses and eosinophilia using formalin-inactivated vaccines, but the precise mechanisms of immunopathogenesis remain undefined (reviewed in Graham, 2011). With the failure of this killed vaccine, development of live-attenuated RSV vaccines was initiated.
Chimpanzees are the only experimental animal in which RSV replication and pathogenicity approach that of humans. Small numbers of chimpanzees were used to demonstrate the safety of live-attenuated vaccines as well as identifying candidates that were sufficiently attenuated to move forward into clinical trials (e.g., Clinicaltrials.gov NCT00767416) (Crowe et al., 1993, 1994; Teng et al., 2000; Whitehead et al., 1999). Importantly, the body temperature of the chimpanzee is the same as that of humans. Other available nonhuman primates have higher body temperatures and so chimpanzees are uniquely suited for pre-clinical evaluation of temperature-sensitive vaccine candidates, which comprise all of the candidates evaluated in clinical trials to date. In addition, the chimpanzee experiments added to a body of evidence that both live attenuated vaccines and vectored vaccines are not associated with enhanced disease. A series of clinical trials have been initiated in infants and young children to evaluate safety, attenuation, and immunogenicity of several live RSV vaccines (for instance, see ClinicalTrials.gov NCT00767416). It is too soon to know if live RSV vaccines that are sufficiently attenuated to be well tolerated in young infants (Karron et al., 2005) will be sufficiently immunogenic to prevent severe RSV disease. Alternate approaches involving recombinant viral vectors (such as attenuated parainfluenza virus type 3; see ClinicalTrials.gov NCT00686075), virus-like particles (ClinicalTrials.gov NCT01290419), and subunit proteins are at various stages of development and evaluation. RSV subunit vaccines are considered unsuitable for use in RSV-naïve individuals, based on studies in mice, cotton rats, and African green monkeys. However, the evolutionary distance relative to humans, reduced permissiveness to RSV replication, and lack of disease may limit the predictive value of these models (Graham, 2011). Until the significant medical need for an RSV vaccine is fully met, it is difficult to exclude the possibility that chimpanzees will be required to answer questions about mechanisms and duration of immune protection and disease potentiation.
The Human Immunodeficiency Virus
Susceptibility of chimpanzees to persistent HIV infection was first reported in 1984, a little more than 1 year after discovery of the virus (Alter et al., 1984). Successful infection of two animals, and persistence of lymphadenopathy for several weeks in one of them, suggested that the chimpanzee would be valuable for further studies of acquired immune deficiency syndrome (AIDS) (Alter et al., 1985). This study, and all other early studies of immunity and vaccine development, used viruses like HIVIIIb and HIVSF2 that adapted in culture to use CXCR4 as a co-receptor for cell entry. These viruses did initiate infection in chimpanzees, but viremia was usually low or short-lived and immunodeficiency was not observed (with one notable exception described below). Several studies of the early studies with CXCR4 adapted viruses nonetheless provided insight into the nature of antiviral immunity in infected chimpanzees (Nara et al., 1987), including the development of neutralizing antibodies (Prince et al., 1987), proliferative responses, and lack of CD4+ T cell impairment (Eichberg et al., 1987). Important advances were made in understanding mucosal routes of HIV-1 transmission using chimpanzees (Fultz et al., 1986), as well as the now better-appreciated issue of superinfection (Fultz et al., 1987). Some success in protecting animals from infection with the CXCR4-dependent HIV strains was achieved by active and passive vaccination. Sterilizing immunity was induced by immunization with recombinant subunit envelope glycoproteins, but only with the CXCR4-utilizing virus HIVIIIB matched to the envelope immunogen (Berman et al., 1988). The vaccine was not fully protective when a different strain belonging to the same subtype, HIVSF2, was used (El-Amad et al., 1995). Passive transfer of HIVIG at a higher dose eventually showed protection against HIVIIIB (Prince et al., 1991), as did one of the first neutralizing monoclonal antibodies directed against the HIV envelope (Emini et al., 1990). The interpretation of vaccine and antibody-based protection work in chimpanzees was complicated by the observation that chimpanzees were mostly resistant to infection with primary human HIV isolate that required the CCR5 chemokine receptor for cell entry. This discovery brought pause to the vaccine field, since none of the vaccines in testing could elicit neutralizing antibodies against the primary isolates that required CCR5 for infection. Later attempts to block infection using a human monoclonal against a primary HIV-1 challenge were also less successful, though they showed some effect in reducing acute phase viremia (Conley et al., 1996).
Notably, one chimpanzee did develop immunodeficiency after more than a decade of subclinical infection with two prototype strains of CXCR4-adapted HIV (Novembre et al., 1997). Isolation of a pathogenic virus from this animal sparked a debate on the role of chimpanzees in HIV vaccine research. Specifically, it was proposed that a pathogenic virus could facilitate a direct test of vaccines designed to slow CD4+ T cell loss and immunodeficiency, if not infection (Cohen, 1999; Letvin, 1998). Vaccinated chimpanzees were never challenged with this virus in the face of persuasive ethical and scientific arguments (Cohen, 1999; Prince and Andrus, 1998). From the scientific perspective, there was considerable doubt about whether very rapid CD4+ T cell loss observed after transmission of the virus to new animals was representative of human disease. Moreover, because immunodeficiency was not a consistent finding, there were also practical concerns with design of a study involving two or three animals per group. These experimental infections with the pathogenic HIV strain were perhaps the last conducted at a primate research facility in the United States. No new HIV infection studies in chimpanzees have been published in the past decade.
Studies on the origin of human HIV infection are beginning to yield insight into a host-virus relationship so finely tuned that it cannot be recapitulated in an animal with 99 percent genetic identity. It is now apparent that HIV originated from a chimpanzee simian immunodeficiency virus (SIVcpz) introduced into human populations by zoonotic infection at least three times since the beginning of the 20th century (Keele et al., 2009a). Most simian retroviruses, including those from monkeys, are restricted from growth in human cells by species-specific factors (see section below on Monkey-human models of infection). SIVcpz is no exception, as Vpu and Nef proteins had to adapt to neutralize human tetherin, a protein that is induced by interferon and restricts virus release from infected cells (Lim et al., 2010; Sauter et al., 2009). Adaptations like this one may explain attenuation of HIV infectivity for chimpanzees and perhaps limit its value as a model for vaccine development.
The Hepatitis Viruses
Chimpanzees are currently used to study the host response to four hepatitis viruses (HAV, HBV, HCV, and HEV) and to develop or refine approaches for prevention and treatment of the liver disease that they cause. Prevention and treatment of transmissible hepatitis in humans has been a public health priority for over 60 years. Progress toward isolation
of the agent(s) responsible for the disease burden was slow, in part because hepatotropic viruses are fastidious and not easily propogated in cell culture. By the 1960s there was strong clinical, epidemiological, and immunological evidence for two distinct forms of transmissible hepatitis in humans (Krugman and Giles, 1972). Type A (or infectious) hepatitis had a short incubation period and was self-limited. Type B (or serum) hepatitis had a longer incubation period and was characterized by the prolonged presence of the Australia antigen (hepatitis B surface antigen; HBsAg) in serum. Use of chimpanzees in hepatitis research predated the discovery of the viruses that caused type A and B hepatitis. Sporadic outbreaks of liver disease in chimpanzee colonies with occasional zoonotic transmission indicated that the animals might be susceptible to infection with human viruses (Maynard et al., 1972a). Transmission of type A (Dienstag et al., 1975; Maynard et al., 1975) and B (Barker et al., 1973; Maynard et al., 1972b) hepatitis to chimpanzees facilitated characterization of both viruses and rapid development of diagnostics and highly effective vaccines. Chimpanzee research provided critical proof that HBV infection was preventable by vaccination with HBsAg purified from the serum of human carriers (Buynak et al., 1976a, 1976b; Purcell and Gerin, 1975), and for the transition to a safer recombinant subunit vaccine (McAleer et al., 1984). Attenuated and inactivated vaccines were also shown to prevent HAV infection of chimpanzees (Feinstone et al., 1983; Provost et al., 1983; Purcell et al., 1992). The principle that a vaccine can prevent disease when administered as post-exposure prophylaxis was also established using HAV-infected chimpanzees (Purcell et al., 1992). Universal childhood vaccination against HAV and HBV is now recommended in the United States.
Chimpanzees were also critically important to the discovery of the agent causing a third major form of human hepatitis. Studies published in 1975 concluded that unidentified type C hepatitis virus(es) were responsible for post-transfusion hepatitis in subjects not infected with HAV or HBV (Feinstone et al., 1975; Prince et al., 1974). The infectious nature of non-A, non-B hepatitis was not established by experimental transmission of liver disease to uninfected humans, an approach used to define features of type A and B hepatitis in the highly controversial Willowbrook experiments (Krugman, 1986). Instead, evidence that the disease was caused by a small, enveloped RNA virus was obtained by physico-chemical analysis of patient serum that transmitted persistent hepatitis to chimpanzees (Alter et al., 1978; Bradley et al., 1983, 1985; Tabor et al., 1978). Serum that was titrated and serially passaged in
chimpanzees provided the pedigreed material from which the HCV genome was eventually cloned as described in 1989 (Choo et al., 1989).
All four hepatitis viruses remain significant public health problems today. A box summarizing research objectives of contemporary hepatitis virus research using chimpanzees is provided (Box 1). As described in detail below, HAV, HBV, HCV, and HEV all cause robust infection in chimpanzees. These viruses can cause the same spectrum of liver disease observed in humans, although in both species most infections tend to clinically mild and/or slowly progressive.
Major Uses of Chimpanzees in Infectious Disease Research
• Characterize and identify new infectious agents, especially those that cannot be propagated in lower species or cell culture.
• Define mechanisms of protective innate and adaptive immunity and pathogen evasion strategies. This is particularly important in settings where early phases of acute infection are not easily identified in humans, or infected tissues are not accessibly for studies of immunity.
• Establish that new concepts for vaccination or therapy of infection are safe and effective before translation to humans.
• Determine if reagents critical to development of therapeutics like clonal viruses or parasites replicate in a host closely related to humans.
Enteric Hepatitis Viruses
HAV and HEV cause acute hepatitis and self-limited infection in humans and chimpanzees. Although liver disease may be somewhat milder in chimpanzees, the kinetics and magnitude of virus replication, onset of liver disease, and histopathological changes in the liver are similar to those in HAV-infected humans (Dienstag et al., 1975, 1976). The course of HEV infection in chimpanzees is variable, ranging from low viremia with no obvious liver disease to high viremia with biochemical and histological evidence of hepatitis (Li et al., 2006; McCaustland et al., 2000). This may be similar to the spectrum of disease in HEV-infected humans (McCaustland et al., 2000). HAV and HEV infections are preventable by vaccination. The efficacy of a subunit HEV vaccine was approximately
90 percent in two large human trials in Nepal and China, but there is uncertainty about the durability of protective immunity as currently formulated and how (or if) it will be deployed where needed (Shrestha et al., 2007; Wedemeyer and Pischke, 2011; Zhu et al., 2010). Thus it is likely that endemic and epidemic HEV will remain a cause of serious liver disease in developing countries (Aggarwal, 2011). HEV immunity and pathogenesis are still very poorly understood (Aggarwal, 2011). For HAV, socioeconomic development accompanied by improved sanitation and opportunity for vaccination has changed epidemiology in regions where the virus is still endemic, as illustrated by a recent outbreak in South Korea (Kim and Lee, 2010; Kwon, 2009). Under these circumstances, HAV infection shifts from the first to the second and third decades of life with an associated increase in the severity of disease. This situation has highlighted a gap in knowledge about mechanisms of immunity and hepatocellular injury caused by HAV. Very recent studies in chimpanzees provided insight into patterns of innate immunity and host gene expression immediately after infection with HAV and HEV, with the goal of understanding the pathogenesis of these infections and how they compare to responses elicited by HCV that often establishes a persistent infection (Lanford et al., 2011; Yu et al., 2010a). Follow up studies of adaptive immunity to these viruses in animals should be anticipated. Similar studies in humans will be difficult, if not impossible, because infections with these small RNA viruses are often not symptomatic for several weeks and access to liver may be challenging as there is typically no medical need for liver biopsy.
HBV Worldwide, approximately 500 million people are infected with HBV. Hepadnaviruses are widespread in nature and chimpanzees do harbor indigenous strains of HBV that can be distinguished from human viruses based on genomic signatures despite overall identity of about 90 percent (Barker et al., 1975a, 1975b; Dienstag et al., 1976; Guidotti et al., 1999; Hu et al., 2000; Rizzetto et al., 1981). Chimpanzees are nevertheless highly susceptible to challenge with human HBV. Chimpanzees develop persistent and resolved infections after challenge with the virus (Barker et al., 1975a, 1975b). The incubation period preceding symptoms is long and biochemical evidence of acute hepatitis is associated with parenchymal inflammation, as in man. The magnitude and general pattern of viremia and antigenemia during the acute and chronic phases of infection are also similar between the species (Barker et al., 1975a, 1975b; Kwon and Lok, 2011). Severe progressive hepatitis and cirrhosis
observed in some humans appears to be uncommon in chimpanzees. Implementation of universal HBV immunization will gradually reduce the number of human infections in future decades, but there is a current need for therapies to control this chronic condition. Nucleoside analog inhibitors of the HBV polymerase that suppress production of infectious virus have been available for years but do not cure infection. Because the HBV genome cannot be eradicated from the liver, most individuals require life-long therapy (Kwon and Lok, 2011). The problem of HBVresistance to direct-acting antivirals is increasing, and will probably accelerate in regions where treatment practice and availability of highquality pharmaceuticals of required potency are inadequate (Kwon and Lok, 2011). Immunotherapy to reactivate effective immunity against HBV is an alternative (Rijckborst et al., 2011). A finite course of type I interferon can reverse immune tolerance in about 30 percent of chronically infected patients, conferring long-term control of HBV infection (Rijckborst et al., 2011). As discussed below, new and perhaps more effective approaches to reverse immune tolerance are being considered for chronic viral hepatitis.
Contemporary research in HBV-infected chimpanzees addresses questions that are highly translational to humans. As for HCV, the animals provide a way to develop titered pools of monoclonal HBV for vaccine and related studies (Asabe et al., 2009). These animals were also used to determine if resistance mutations that arise during antiviral therapy facilitate escape from vaccine protection (Kamili et al., 2009). In this study, chimpanzees vaccinated with a commercial HBV vaccine were challenged with a virus containing mutations in key neutralization epitopes of HBsAg caused by development of lamivudine in the viral polymerase gene that is encoded in an overlapping but alternate reading frame (Kamili et al., 2009). Lamuvidine-resistant variants now circulate in some human populations, so this experiment addressed an important public health problem.
HCV For HCV, there is no vaccine to prevent infection and therapies remain inadequate despite recent progress in developing direct-acting antivirals that target key replicative enzymes of the virus. It should be emphasized that acute infections sometimes resolve spontaneously and chronic infections can be cured. For these reasons, vaccination to prevent persistence is more realistic than for HIV, even though both viruses present similar challenges in their adaptability to immune pressure. Similarly, the goal of effective therapy is to eradicate the virus rather than
simply control chronic infection as in HIV and HBV. Chimpanzees are the only species other than humans with known susceptibility to HCV infection. No chimpanzee homolog of HCV has been found and closely related viruses that consistently cause a similar pattern of resolving and persistent infection have not been described in other species. Only chimpanzees have the correct combination of four entry receptors and other cellular co-factors required to recapitulate key features of human infection. Woodchucks and old- and new-world monkeys tested to date are not susceptible to infection (Bukh et al., 2001). HCV infection of a tree shrew (Tupaia belangeri) has been reported, but viremia was intermittent and several orders of magnitude less than that measured in chimpanzees and humans (Amako et al., 2010). HCV infection can either resolve spontaneously or persist in humans and chimpanzees. The typical pattern of virus replication is identical, with high levels of viremia for at least 7-12 weeks followed by a decline that is usually associated with a spike in serum transaminases (Abe et al., 1992; Thimme et al., 2002; Walker, 2010). Virus can fluctuate at low levels for several weeks or months before the infection resolves or persists (Walker, 2010). One study reported a lower rate of chronic infection in chimpanzees (40 percent) than humans (60-70 percent) (Bassett et al., 1998), although there is not unanimity on this point. It is difficult in a retrospective chart review to exclude the possibility that some animals thought to be naïve at the time of HCV challenge were already immune because of prior exposure to unscreened human blood products. At least some of the difference may also be explained by over-estimation of virus persistence in humans because acute resolving infections that are clinically silent or mild are missed. Differences related to the young of age of infection in most chimpanzees, and the dose or strain of virus used for experimental challenge, are also possible. If the rate of persistence is lower, it has had no apparent impact on interpretation of studies on immunity to HCV in chimpanzees, or relevance of the findings to human infection (reviewed below). Liver disease is typically mild in persistently infected chimpanzees, as it is in most humans with chronic hepatitis C. More serious liver disease may become evident after several decades. Late-stage disease, including hepatocellular carcinoma, has been observed in some animals more than 30 years after HCV or HBV infection. It should be emphasized that sub-clinical hepatitis over a course of many years falls within the spectrum of hepatitis observed in many humans, and is not uncommon in those without risk factors for rapid progression like male sex, older age, and alcohol intake. Objectives of current chimpanzee research are directly relevant to human
health. Highly translatable studies include the first evidence that interferon-free control of HCV infection is possible with combinations of directacting antivirals (Olsen et al., 2011), and that interference with a cellular microRNA can prevent HCV replication (Lanford et al., 2010). The latter represents an entirely new approach to control of virus infections. More basic studies focused on the balance between innate and adaptive immunity in HCV infection outcome (Barth et al., 2011), and patterns of host gene expression in liver before infection is clinically evident in humans (Yu et al., 2010b).
In summary, whether chimpanzees are required for progress in understanding and controlling human infectious diseases is highly dependent on several factors, including the availability of valid alternatives, intricacies of the relationship between the host and each pathogen, and the objective of the research. Here, malaria, RSV, HIV and the hepatitis viruses were used as case studies to illustrate these points. To summarize:
• Malaria provides an example where there are alternatives to chimpanzee research, including experimental infection of humans, lower primates, and rodents. The animal model may retain value for testing new vaccine concepts, identification of candidate antigens, and characterization intrahepatic immunity, especially if current strategies to protect humans from infection are inadequate.
• For other pathogens like RSV, the chimpanzee provides the only faithful model of human disease even though lower species are permissive for infection. This has been critical for development of candidate RSV vaccines that have the potential to cause harm.
• The example of HIV illustrates how an animal model can fail despite a very close genetic relationship to humans. Adaptation of SIVcpz to humans after it crossed the species barrier apparently attenuated its replication and pathogenicity for chimpanzees, the species from which it originated. A second very important point that emerged from the HIV experience is that the objectives of vaccination will determine utility of the animal model. If the goal of vaccination is to prevent serious progressive disease (rather than infection), it must be carefully balanced against ethical considerations and any scientific limitations of the model.
• Hepatitis virus research in the chimpanzee has a track record of success that began almost half a century ago. It continues to the present day. As described in more detail later in this paper, proof
that humoral and/or cellular immunity protect against HCV infection was generated from chimpanzee studies before the initiation of vaccine trials in humans. As an example, chimpanzees studies within the past decade documented the critical need for T lymphocytes to control HCV infection (Grakoui et al., 2003; Shoukry et al., 2003), and that a vaccine based on this principle could dramatically alter primary viremia (Folgori et al., 2006). These experiments were direct antecedents of current human clinical trials (ClinicalTrials.gov NCT01070407). Publications within the last 18 months addressed important public health concerns surrounding HBV escape variants and vaccination, and tested new concepts for control of chronic hepatitis C virus infection. As noted above, liver disease caused by chronic hepatitis B and C is usually at the mild end of the spectrum observed in humans, but to date this has not been a barrier to successful development of vaccines or therapeutics that target the viruses.
Comparative and Evolutionary Immunology in Humans
Humans and chimpanzees diverged approximately 5-7 million years ago. In the 19th century paleontology and comparative anatomy were used to study kinship between the species, but the advent of serology provided a new avenue for investigation. Landsteiner and Miller summarized these studies in a 1925 publication that documented distinct differences between humans, chimpanzees, and orangutans in patterns of hemagglutination by anti-erythrocyte sera (Landsteiner and Miller, 1925). It was nonetheless concluded that chimpanzees and humans were closely related as serology revealed that “the arthropoid apes do not rank in the genealogical tree between lower monkeys and man.” Evolution of the immune system remains an important approach to probe the relationship between the species. Publication of the draft genome sequence of a common chimpanzee (Pan troglodytes) 80 years after the Landsteiner study facilitated a comparative analysis with the human genome (Mikkelsen et al., 2005). The genomes diverged by approximately 1 percent when estimated polymorphism was excluded. A total of 13,454 pairs of human and chimpanzee genes with unambiguous 1:1 orthology were identified (Mikkelsen et al., 2005). Alignment revealed that the most rapidly diverging gene clusters in both species were associated with taste,
olfaction, reproduction, and immunity. With regard to immunity, rapid diversification of chemokine ligands, cytokine biosynthesis, human leukocyte antigen (HLA), and immunoglobulin-like receptors could be discerned even at the relatively close evolutionary distance of humanchimpanzee divergence. Over the past 30 years much has been learned about chimpanzee and human immunogenetics through studies of rapidly evolving genes in the major histocompatibility complex (MHC) and killer cell immunoglobulin-like receptor (KIR) family. Similarities and differences in genes that regulate immunity are reviewed in this section. How these studies of gene evolution in chimpanzees have facilitated and provided insight into infectious disease research is also discussed.
The Major Histocompatibility Complex
Immunogenetic differences between humans and chimpanzees were first explored in the 1960s when mixed leukocyte cultures (Bach et al., 1972) and isoantisera (Balner et al., 1967) were used to define antigenicity of chimpanzee leukocytes. This interest in transplantation biology led to initial characterization of the chimpanzee histocompatibility complex that is now designated Patr (Pan troglodytes). During this era experimental transplantation of chimpanzee liver to pediatric patients suffering from biliary artersia was undertaken (Giles et al., 1970). Contemporary immunogenetic research involving the chimpanzee has focused on the evolutionary relationship with man (Lienert and Parham, 1996). The HLA and Patr gene complexes are remarkably similar considering the genetic polymorphism at class I and II loci (Lienert and Parham, 1996). Humans and chimpanzees have orthologous MHC class I A, B, and C loci. Remarkably, there are no species-defining characteristics amongst the highly polymorphic alleles at these loci (Lienert and Parham, 1996). For instance, it is not possible to distinguish HLA-A from Patr-A alleles based on genetic signature. Class II loci are similarly conserved, as humans and chimpanzees express DP, DQ, and DR gene products (de Groot et al., 2009). Chimpanzee and human class I genes are functionally identical. Detailed studies of peptide binding to chimpanzee and human class I molecules demonstrated remarkable overlap in the pool of viral epitopes presented to T cells (Mizukoshi et al., 2002; Sidney et al., 2006). As noted below, complete characterization of Patr haplotype in virus-infected chimpanzees has facilitated adaptation of state-of-the-art reagents for monitoring T cell immunity. Finally, specific class I MHC alleles have been associated with infection outcome in HIV- and HCV-infected
humans. Some of these protective alleles appear to have functional orthologs in the chimpanzee, as a subset of Patr class I alleles were shown to bind highly conserved HIV gag epitopes associated with protection from AIDS (de Groot et al., 2010).
The KIR Gene Family
Natural killer (NK) cells are involved in regulation of pregnancy and host defense. With regard to host defense, cytolytic activity and production of effector cytokines by NK cells is tightly controlled by the interaction of activating and inhibitory receptors with their ligands, the class I MHC molecules (Jamil and Khakoo, 2011; Lienert and Parham, 1996; Parham, 2008). Comparison of human and chimpanzee class I ligands over the past 3 decades has stimulated recent interest in evolution of NK receptors that might influence the outcome of infection. Two primary groups of NK receptors have been described. The NKG2 family is relatively non-polymorphic and conserved between humans and chimpanzees. The other family, comprised of KIRs, is highly polymorphic and rapidly evolving as observed in the draft genome sequence of the chimpanzee (Mikkelsen et al., 2005). Humans and chimpanzees each have 10 variable KIR genes but only two, designated 2DL5 and 2DS4, are common between the species. In humans, but not chimpanzees, KIR genes are organized into two haplotypes proposed to roughly correlate with host defense (haplotype A) and reproduction (haplotype B) functions (Abi-Rached et al., 2010). Evolution may have also altered the functional profile of human KIR gene products, as species-specific mutations that reduce avidity of activating KIR for HLA class I, while retaining highavidity inhibitory KIR, have been found (Abi-Rached et al., 2010). It is apparent that these evolutionary changes over the past 7 million years were driven by selection pressure from infectious diseases and possibly the physiological demands of reproduction in humans versus chimpanzees. KIR gene diversity between the species may influence the outcome of chimpanzee infections with human pathogens. Resolution of human HCV infections has been associated with homozygous expression of the KIR2DL3 receptor and its specific HLA-C ligand (Khakoo et al., 2004). A KIR haplotype association with HBV infection, and a specific protective effect of KIR2DL3, has also been reported (Gao et al., 2010). It is important to emphasize that these associations were identified in large population studies and the effect is not sufficiently strong to have practical
predictive value for individuals. Overall, inhibitory and activating functions of the KIR genes are conserved in humans and chimpanzees. Given the complexity and redundancy of compound KIR:HLA genotypes on NK responsiveness, it is unlikely that KIR genetics have a material impact on typical studies of immunity or vaccine protection involving small populations of humans or chimpanzees.
T Cell Receptor Genetics
T cell recognition is mediated by the heterdimeric T cell αβ receptor (TCRαβ) that recognizes antigens presented by MHC class I or II complexes. TcRβ chain diversity is generated by the rearrangement of V, D, J, and C regions. The random insertion of non-germline-encoded nucleotides at the junctions of these rearranged segments provides additional diversity and is the main site of Ag recognition (complementarity determining region [CDR3]). The human TcRVβ repertoire consists of 54 functional TcRVβ genes belonging to approximately 25 families based on DNA sequence similarities. Partial characterization of the chimpanzee TCR repertoire revealed 42 TcRVβ genes that could be aligned with known human genes (Jaeger et al., 1998; Meyer-Olson et al., 2003, 2004). All functionally rearranged human TcRVβ families were represented in the chimpanzee TcRVβ repertoire. No evidence of new TcRVβ families was found in the chimpanzee, and some genes were identical between the species (Meyer-Olson et al., 2003, 2004). These data indicate a high degree conservation of the TcRVβ repertoire in humans and chimpanzees, and suggest complexity of the T cell repertoire responding to highly mutable viruses like HCV is similar.
Innate immune defenses are a potentially important determinant of infection outcome in humans and chimpanzees. This was recently documented in human HCV infection, where a chronic outcome of infection and response to therapy was strongly influenced by a polymorphism in the non-coding region of the IL-28β gene. Whether the same IL-28β polymorphisms exist in the chimpanzee is not yet known, but seems likely given that the range of infection outcomes is identical with humans. Most information on coding sequence differences in innate genes has derived from comparison of evolutionary pressures on key gene families since separation of humans and chimpanzees from a common ancestor
(Barreiro and Quintana-Murci, 2010). These studies have provided insight into natural selection of human and chimpanzee defense genes by infectious diseases. Loss of genes that regulate innate immunity from the chimpanzee but not the human genome has been described. For instance, three genes (IL1F7, IL1F8, and ICEBERG) that appear to be deleted from the chimpanzee genome are involved with regulation of proinflammatory responses. ICEBERG is an inhibitor of IL-1β and its loss may indicate a species-specific modulation of inflammasome function, perhaps to reduce sepsis risk (Mikkelsen et al., 2005). Relatively little is known about how deletions or even coding sequence differences in innate genes (which are usually minor) alter immune responsiveness in these species. Using the toll-like receptors as an example, natural selection studies have documented that six chimpanzee TLR genes fit within the range of haplotypes found in European-American, African-American, and Indian human populations (Mukherjee et al., 2009). Despite this similarity, the modal human haplotypes are many mutational steps away from the chimpanzee haplotypes indicating species-specific adaptation to pathogens (Mukherjee et al., 2009). From a practical standpoint, the TLR genes from both species are very close in sequence. For instance, chimpanzee and human TLR4 gene sequences differ at only three amino positions (Smirnova et al., 2000). Differences in patterns of gene expression were observed in primary monocytes stimulated with the TLR agonist lipopolysaccharide (Barreiro et al., 2010). A difference in the number of responding genes in human (335) versus chimpanzee (273) monocytes was observed. Many of the activated genes common to both species were regulated by the transcription factor NFκB and involved in host defense (Barreiro et al., 2010). Others were species-specific, and fell into gene families related to apoptosis (for humans) or SIV control (chimpanzees) (Barreiro et al., 2010). The impact of differences in innate gene coding sequences to the study of specific pathogens in the chimpanzee is unclear, but might be greatest for viruses that originated in the animals and adapted to a new human host. As noted above, HIV strains that adapted to interfere with human tetherin may lose their ability to replicate efficiently in chimpanzee CD4+ T cells, or to infect via the CCR5 coreceptor because of species-specific differences in regulation of gene expression (Wooding et al., 2005).
In summary, comparison of the chimpanzee and human genomes has revealed remarkable conservation of genes; about 30 percent are identical and single base pair substitutions account for about half of the genetic change (Mikkelsen et al., 2005). At the same time, selective pressure
against genes associated with immunity is apparent, and almost certainly attributable to infectious diseases that uniquely afflict each species. It is likely that most of these coding differences have limited impact on the value of the chimpanzee as a model for most infectious diseases because of functional redundancies common to immune pathways. An important exception may be a virus like HIV that targets the immune system and only recently adapted to humans after zoonotic transmission from chimpanzees, the intended animal model.
The high degree of protein sequence homology between the species has practical significance for studies of immunity and evaluation of therapeutics like monoclonal antibodies. A body of published literature has documented that most antibodies against cluster of differentiation (CD) antigens that define lymphocyte subsets, differentiation status, and function are fully cross-reactive for human and chimpanzee mononuclear cells. Most importantly, some of these molecules (and others not associated with immunity) are considered targets for monoclonal antibody therapy of human diseases. Examples are provided below. Advantages of chimpanzees as a pre-clinical model for monoclonal antibody development have been summarized elsewhere (VandeBerg et al., 2006), but include increased probability of detecting unintended effects against proteins that are orthologous to the primary target, similar binding affinities that might alter cellular responses to a therapeutic antibody, and identical pharmacokinetics of human and humanized antibodies in humans and chimpanzees but not lower primate species.
Functional Immunology and Vaccine Research in Humans
Evolutionary studies have revealed similarities and differences in immune response genes between the species. How different coding sequences in immune response genes alters infection and immunity in a chimpanzee versus a human is difficult to predict and probably pathogenspecific. Infection of chimpanzees with the hepatitis C virus illustrates the strengths and limitations of studying immunity to a human virus in chimpanzees. Non-A, non-B hepatitis (hepatitis C) was first studied in chimpanzees to ask fundamentally important questions unrelated to immunity. For instance, the ability to transmit non-A, non-B hepatitis from humans to chimpanzees indicated an infectious etiology of disease. It also facilitated physico-chemical characterization of the agent as a small,
enveloped RNA virus and provided a pedigreed stock of infectious serum for molecular cloning of the HCV genome as noted above. The observation that some animals, like humans, developed chronic hepatitis C while others spontaneously cleared the virus provided a unique opportunity to identify protective immune responses that might be relevant to humans. In this section, functional adaptive immune responses elicited by infection with HBV and HCV in chimpanzees and humans are compared. Value of the animals for vaccine development is also highlighted. Limitations of the model, and examples of experimental approaches that can be taken in chimpanzees but not humans are described.
Detailed studies of cellular immunity to HBV and HCV have provided insight into mechanisms of protection from persistence and how these responses fail.
Chimpanzees offer at least three distinct advantages for this research: (1) The first few weeks of HBV and HCV infection are clinically silent and so critical events that shape the adaptive immune response and infection outcome are very difficult to study in humans; (2) animals can be challenged with well-defined HCV quasispecies and even clonal HCV genomes to facilitate studies of virus adaptation to the host and immune selection pressure; and (3) the liver can be sampled by percutaneous needle biopsy from the earliest times after infection, so that patterns of innate and adaptive gene expression can be studied.
It is important to emphasize that the tools for measuring cellular immunity in chimpanzees are as sophisticated as those available for human studies. Antibodies to key differentiation, regulatory, and effector molecules expressed by human T cells cross-react with the equivalent chimpanzee molecules. Virus-specific T cell responses in chimpanzees can be very precisely quantified by functional assays that measure production of effector cytokines or killing. As noted above, evolutionary studies of the chimpanzee Patr complex provided insight into MHC class I and II restriction of the T cell response to hepatitis viruses in chimpanzees. This work on the Patr complex also facilitated development of soluble class I and II molecules (tetramers) for direct visualization of virus-specific T cells in the blood and liver of chimpanzees infected with HCV and HBV. These chimpanzee reagents are produced by an NIH-funded facility that established to provide human and murine class I and II tetramers. In summary, there are no technical disadvantages, and several distinct advantages,
to the study of antiviral T cell immunity in chimpanzees versus humans.
Patterns of virus replication and T cell immunity are identical in humans and chimpanzees infected with HCV and HBV (Cooper et al., 1996; Guidotti et al., 1999; Rehermann, 2009; Thimme et al., 2001, 2002, 2003; Walker, 2010). For instance, HCV replicates at high levels for 8-12 weeks before the onset of CD4+ helper and CD8+ cytotoxic T cell responses that are associated with a spike in biochemical markers of hepatitis and initial control of viremia (Rehermann, 2009; Walker, 2010). In some humans and chimpanzees, the T cell response is sustained and the infection is terminated within a few days or weeks. In others, the CD4+ T cell response fails and the virus persists. Failure of the HCVspecific CD4+ T cell response before apparent resolution of infection is the best predicator of a chronic course of infection. Importantly, mechanisms of acute phase CD4+ T cell failure remain unknown (Rehermann, 2009; Walker, 2010). HCV-specific CD8+ T cells are present in the liver at high frequency for decades but provide no apparent control of virus replication. Infection of chimpanzees with viruses of known sequence was essential to show that some of the HCV epitopes targeted by these CD8+ T cells acquired escape mutations that prevent recognition of infected cells (Bowen and Walker, 2005). Other long-lived intrahepatic CD8+ T cells target intact epitopes but lack effector functions (Rehermann, 2009; Walker, 2010). Based on these observations, current research in chimpanzees has two goals. The first goal is to facilitate development of vaccines that skew the outcome of HCV infection from persistence to resolution. The second goal is to determine the defect that underlies CD4+ and CD8+ T cell failure in chronic hepatitis C (and B), and to test approaches to reverse exhaustion.
Very early studies demonstrated that spontaneous resolution of HCV infection in a chimpanzee did not protect from liver disease when the animal was re-exposed to the same infectious inoculum (Farci et al., 1992; Prince et al., 1992). It was concluded that anti-HCV immunity was weak and that vaccine development would be difficult. Subsequent studies revealed that this was not necessarily the case. While some second infections in naturally immune animals do persist, the majority of infections are very rapidly controlled (Walker, 2010). As noted above, primary HCV infections typically do not resolve for 3-4 months, but most second
infections clear within days, and are associated with an accelerated memory T cell response. The chimpanzee model was essential to prove the importance of memory CD4+ and CD8+ T cells to protection from persistence. Animals that had successfully resolved two infections were treated with monoclonal antibodies directed against CD4 or CD8 to temporarily deplete these subsets before a third challenge with HCV. In the absence of CD4+ T cells, the virus persisted and CD8+ T cells (that were not depleted) selected for virus variants with escape mutations in class I epitopes (Grakoui et al., 2003). Depletion of CD8+ T cells in a second set of immune chimpanzees prolonged a subsequent infection; termination of the infection coincided with recovery of these effector cells (Shoukry et al., 2003).
Together, these studies demonstrated that sterilizing immunity provided by antibodies is not necessarily required for HCV protection. Instead, they indicated that induction of T cell immunity to prevent persistence (but not infection) may be a realistic goal for vaccination. Indeed, the T cell depletion studies in chimpanzees led directly to the design of a recombinant adenovirus vector that expressed the nonstructural proteins of HCV that are predominantly targeted by the cellular immune response (Folgori et al., 2006). Structural proteins, including envelope glycoproteins that are the targets of neutralizing antibodies, were not incorporated into the vaccine. Chimpanzees vaccinated with this vector had dramatically lower levels of primary HCV viremia than mock-vaccinated controls, and all cleared the infection (Folgori et al., 2006). This vaccine is now in human clinical testing for prevention and treatment of HCV infection (see ClinicalTrials.gov NCT01070407, NCT01094873, and NCT01296451). It should be noted that these vaccines developed in Europe by IRBM (Merck) and Okairos were evaluated in chimpanzees at primate facilities in the United States.
The contribution of antibodies to vaccine-mediated protection against HCV also cannot be minimized, as indicated by a recent metaanalysis of chimpanzee vaccine studies (Dahari et al., 2010). Active vaccination with recombinant subunit vaccines comprised of the HCV E2 envelope glycoprotein protected chimpanzees from virus challenge. Proof of protection by these vaccines was obtained in chimpanzees before initiation of phase I and II testing in humans. Finally, chimpanzees were used to test post-exposure prophylaxis with anti-HCV antibodies, a potentially important approach to infection control in the setting of needlestick injury (Krawczynski et al., 1996). These antibodies substantially
modified the course of acute hepatitis C, but did not prevent persistence of the virus.
The last decade has brought tremendous progress in understanding mechanisms of T cell evasion by persistent viruses. Studies in murine models of virus (LCMV) persistence have demonstrated that functionally impaired virus-specific T cells express the inhibitory molecule PD-1. Antibody-mediated interruption of the PD-1 interaction with its ligand at least partially restores T cell function and leads to accelerated control of virus replication (Barber et al., 2004). It is now clear that exhausted T cells in humans and chimpanzees persistently infected with HCV and HBV express multiple inhibitory receptors including PD-1 that put a brake on effector function (Boni et al., 2007; Golden-Mason et al., 2007; Penna et al., 2007; Raziorrouh et al., 2010). Interruption of ligand binding by inhibitory PD-1, CTLA-4, and TIM-3 receptors on HCV-specific CD8+ T cells restores function in cell culture assays (Boni et al., 2007; Golden-Mason et al., 2007; Penna et al., 2007; Raziorrouh et al., 2010). Initial studies in persistently infected chimpanzees indicate that PD-1 blockade can have a dramatic effect on viremia in some but not all animals (C. Walker, unpublished). Similar studies have been completed in humans (ClinicalTrials.gov NCT00703469) but the results have not been released. Based on studies in the animal model, it might be predicted that the human trial had some successes but more failures.
There is a continuing need for chimpanzees to develop nextgeneration therapeutics against persistent viruses, especially those that cannot be eradicated from infected cells (Callendret and Walker, 2011). Very recent cell culture studies have indicated that blockade of one inhibitory receptor may not be adequate to fully restore function the HBV or HCV specific T cells (McMahan et al., 2010; Nakamoto et al., 2009). Blockade of multiple pathways is feasible, but the approach carries risk as these pathways were designed to temper unwanted or dangerous immune responses (Callendret and Walker, 2011). Also, studies in mice indicate blockade of one of more than inhibitory pathway in combination with vaccination might potentiate activity against persistent viruses (Ha et al., 2008). In considering these strategies for human use, two considerations are paramount:
(1) Most humans with chronic hepatitis are relatively healthy and so combinations of blocking antibodies and vaccines carry more risk than studies in patients with advanced stages of cancer. Under these circumstances, a nonhuman primate model like the chimpanzee is important for progress.
(2) Antiviral therapy for chronic hepatitis C will become more effective with the advent of small molecules antivirals. It is too soon to know if there is a place for immunotherapy with vaccines and/or blocking antibodies in hepatitis C, although the propensity of the virus to develop resistance is remarkable and a perhaps a significant barrier to availability and use of the multiple drug cocktails in developing countries. There is a significant need for this type of therapy in chronic hepatitis B, where direct-acting antivirals do not eradicate the infection and it is necessary to break immune tolerance for long-term control of virus replication. Chimpanzees will remain important to advance this concept.
The Chimpanzee in an Era of New and Emerging Technologies for Studying Immunity
Chimpanzee infection studies continue to have value beyond proof-of-concept studies to validate new preventive or therapeutic strategies. New technologies offer great promise in unraveling molecular mechanisms underlying the failure of immunity in acute and persistent infections with viruses like HAV, HBV, and HCV. It is now possible to probe the innate and adaptive immune responses, and how they are coordinated, a level of resolution not possible a few short years ago. Genes that are expressed in virus-specific T cells that successfully control infection or become exhausted can be identified with new technologies (Haining and Wherry, 2010). Chimpanzees may be integral to the future of this work that could provide new targets for intervention in acute and persistent infections. Human genomic and proteomic technologies are directly adaptable to the chimpanzee, and isolation of antiviral T cells to high purity is possible. Perhaps most importantly, the chimpanzees provide unique access to paired blood and liver specimens at very early time points after virus exposure when there are no symptoms but the outcome of infection is probably determined. It is likely that molecular characterization of antiviral T cells in liver at the earliest stages of infection will identify and validate therapeutic targets relevant to humans.
MACAQUES AS MODELS FOR HUMAN DISEASE
In this section, we address host factors in macaque models for human diseases, macaque immunogenetics, and understanding the roles of innate and adaptive responses in macaque models for the development of vaccines and immunotherapies. With 93 percent sequence identity with humans, Macaca species that predominate in Asia are the most widely utilized nonhuman primates in biomedical research. The three most commonly utilized in biomedical research today are M. mulatta (rhesus), M. nemestrina (pigtailed) and M. fascicularis (crab-eating or longtailed); and within the genus the rhesus macaque is by far the most frequently studied. The landmark work by Landsteiner and Wiener in 1937 to define Rh factors in blood, allowing blood typing, was an early example of the contributions that this species has made to medical research (Landsteiner and Wiener, 1937, 1941). Efforts since then have been focused on developing multiple models for understanding human disease states. Macaques share many similarities with humans and chimpanzees in their hematology, reproductive biology, neurological development, behavior, immunogenetics, and immune responses to pathogens. Much of this subsequent research has focused on models in the rhesus macaque, due to their relative ease of breeding in captivity and high adaptability to novel environments. These models include (references are representative examples and not comprehensive in nature): reproduction, including stem cell research (Ben-Yehudah et al., 2010; Schatten and Mitalipov, 2009; Tachibana et al., 2009), bone marrow transplantation and hematopoietic stem cell gene therapy (Donahue and Dunbar, 2001), aging (Messaoudi et al., 2006), metabolic diseases and their sequelae such as diabetes (Grove et al., 2005), brain and neurological development (Sarma et al., 2010; Soderstrom et al., 2006; Voytko and Tinkler, 2004), behavioral (Bethea et al., 2004; Sabatini et al., 2007; Stevens et al., 2009) including addiction (Barr et al., 2010), and infectious diseases (Daniel et al., 1985; Haigwood, 2009; Hansen et al., 2010; Messaoudi et al., 2009) including virus-associated malignancies (Messaoudi et al., 2008). There has also been some work to understand autoimmunity and arthritis (collageninduced arthritis and spontaneous arthritis) in macaques, reviewed in Vierboom and ’t Hart, 2008. With the advent of array technologies to examine multiplex responses to disease, it will be increasingly possible to identify the roles of innate and adaptive immunity in the macaque models for human disease models. Models for aging and immune senescence have been developed using rhesus macaques that are 18 years and
older. These animals are characterized by a progressive loss of naïve T cells and an accumulation of memory type T cells with age. The models can be used to examine therapeutic approaches to reinstating naïve T cells, such as IL-7 therapy (Aspinall et al., 2007) and caloric restriction (Messaoudi et al., 2006). At this time, one of the best probes for understanding host immunity is to utilize pathogens that elicit similar responses to infection in humans and macaques. To introduce this subject, we provide a brief review of host restriction factors and infectious disease models for human disease in the macaque.
Host Factors in Macaque Models for Human Diseases
Host Interactions with Pathogens at the Cellular Level
A major distinction for macaques compared with chimpanzees is that their greater genetic distance from humans results in lack of susceptibility to certain human pathogens. Viral agents and intracellular pathogens utilize host cellular receptors and intracellular molecules such as the nucleic acid polymerases and transcription machinery for replication and propagation. In order to assure priority treatment when inside the cells, these organisms or viruses utilize specific proteins encoded in their genomes to interfere with basic cellular activities such as host protein production. In contrast, extracellular bacterial and protozoan parasites (at least in some stages of their life cycles) replicate independently of the human cell and thus are more capable of establishing infection in a wider range of hosts. Typically pathogens are adapted to certain hosts, a property termed “host range” that is conferred by a number of factors. For viruses, it has become clear that there are specific restriction elements that interact with portions of the virus to limit replication or assembly. Concomitantly, viral proteins can confer resistance to restriction elements in “spy versus spy” interplay at the molecular level, such as Nef from HIV-1 or SIV (Schmokel et al., 2009). No fewer than four different host-pathogen mechanisms have been identified to limit the cytopathic effects of HIV and SIV alone, summarized in a recent review (Lifson and Haigwood, in press) and publication on the latest factor to be discovered (Laguette et al., 2011; St Gelais and Wu, 2011). Generally speaking, viruses with larger genomes such as those from the herpesvirus family are better equipped to stave off the antiviral effects of the host, with multiple pathways for downregulation of various host proteins, including those
that are critical for immune responses such as MHC Class I proteins (Hansen et al., 2010). The process of adaptation of viruses during zoonoses is one of accruing sufficient mutations to overcome these host factors, and one of the best examples of recent zoonoses that have had a major impact upon human health is the transmission of HIV-1 and HIV-2 to humans (Gao et al., 1999; Hahn et al., 2000). A well-adapted virus is one that can peacefully coexist with the host in the absence of pathogenic effects, an example being SIV in African nonhuman primates (sooty mangabeys, African green monkeys, and mandrills) (Pandrea et al., 2006; Sodora et al., 2009). Understanding the immunological differences between pathogens that are recently acquired and poorly adapted, compared with those that are established and benign, may yield important information about both the host and the virus (Silvestri, 2009). The consequence of this host range restriction is that certain types of infectious disease and immune-based research can only be performed in chimpanzees or in humans, and not in macaques. In this section, we provide examples of macaque models for major human diseases that utilize either the same microbe or species-specific pathogens that are related to the human pathogens.
Pathogenic Models for Human Diseases Using the Same Organism
Due to the physiological and genetic similarities of humans and macaques, many human pathogens can infect and cause disease in macaques, and the degree of adoption for experimental usage is dependent upon several factors such as length and severity of the disease, as well as similar pathologic outcomes to humans. An example is tuberculosis, which is highly infectious in macaques and represents a good model for acute and latent infection as well as re-activation and progressive disease (Chen et al., 2009; Lewinsohn et al., 2006). Other models include acute infections that are typically resolved in humans, such as measles (McChesney et al., 1997; Permar et al., 2007; Zhu et al., 1997), potentially severe acute respiratory syndrome (SARS), (Miyoshi-Akiyama et al., 2011), Ebola (Sullivan et al., 2006), monkeypox (Estep et al., 2011), and dengue virus (Onlamoon et al., 2010; Smits et al., 2010). The considerable work that goes into model development for these agents includes the production and in vivo titration of infectious challenge stocks, in some cases necessitating testing many different sources to find the right balance of infectiousness and pathogenicity, the development of key assays for monitoring disease sequelae and the progression of infection short of
necropsy, and the development of reagents that are appropriate for monitoring innate and adaptive immunity in vivo (discussed below). In addition, experiments using these agents require special containment to reduce biohazardous exposure to humans and other nonhuman primates. However, significant progress has been made not only in understanding correlates of protection from challenge in some cases but also in developing human vaccines that show some promise, discussed with examples in the section below on vaccines. Obvious advantages of these models include the ability to identify virulence genes and to perform more frequent and more invasive sampling, with the judicious use of serial sacrifice studies to examine tissues in more depth.
Pathogenic Models for Human Diseases Using Closely Related But Distinct Organisms
When host range limits the infectivity of certain agents, closely related pathogens that are adapted for Macaca species can sometimes be found. These include: simian adenoviruses, simian varicella virus (SVV) as a model for varicella zoster virus (Messaoudi et al., 2009), rhesus cytomegalovirus (CMV) as a model for human CMV (Hansen et al., 2010), and SIV and chimeric simian/human immunodeficiency virus (SHIV) as a model for HIV-1 (Lifson and Haigwood, in press). Because the HIV-1 and SIV Envelope proteins do not elicit cross-protective neutralizing antibodies, testing of human monoclonal antibodies or HIV Env-based vaccines for protection was not possible with SIV. Therefore chimeric viruses were made consisting of the SIV backbone and a “swapped” HIV-1 env gene and these viruses were passaged in vivo in macaques to obtain more pathogenic strains. Originally, SHIVs were tropic for CXCR4 and caused rapid loss of CD4+ T cells in the periphery (Reimann et al., 1996), but then CCR5-utilizing isolates with slower pathogenesis were successfully constructed and shown to be transmitted by mucosal routes in adult (Harouse et al., 2001) and newborn macaques (Jayaraman et al., 2007). Despite their appeal, these models have potentially more limitations that must be kept in mind in interpreting the results. The specific agent (genetically or phenotypically characterized), the host, or that particular combination can each contribute to differences in disease outcomes. All of the caveats noted in the section above hold in this case, and in addition there is the potential for difficulty in translating the findings from macaque-specific pathogens to their human homologs. Nonetheless, these models can be and have been highly instructive in
establishing certain basic principles that would have been difficult or impossible to determine by experimentation in humans or in chimpanzees, as summarized in Table 1. Although many of these concepts have since been proven in clinical studies, it can be argued that the timing of the discovery of the concept in the macaque accelerated understanding and exploration in human studies. Furthermore, many of the findings could not be directly tested in humans for ethical or safety reasons.
TABLE 1 Examples of Lessons Learned from Macaque Models for Human Diseases
|Human Pathogen||Macaque Pathogen||Concept Revealed||References|
|HIV-1||SIV||Mucosal routes of infection require greater doses than parenteral for productive infection after a single challenge; influenced by hormonal status||(Hirsch and Lifson, 2000; Sodora et al., 1997)|
|SIV||Timing of tissue distribution using serial sacrifice; earliest events in infection||(Milush et al., 2004)|
|SIV||Multiple low dose mucosal challenge with can mimic the transmission dose and composition, similar to sexual mucosal acquisition of HIV-1 in humans of a few variants to seed the infection||(Keele et al., 2009b)|
|Relatively more rapid pathogenesis in newborns compared with adults||(Baba et al., 1995, Jayaraman et al., 2007; Marthas et al., 1995)|
|SIV||Early loss of GALT||(Smit-McBride et al., 1998)|
|SIV||Neurotropic strains of SIV and HIV-2||(Van Rompay and Haigwood, 2008)|
|Superinfection definitively Demonstrated||(Yeh et al., 2009)|
|Differential pathogenesis conferred by the same viruses in different macaque species||(Polacino et al., 2008; Sodora et al., 2009)|
|Human Pathogen||Macaque Pathogen||Concept Revealed||References|
|Human CMV||Mechanisms of superinfection revealed||(Hansen et al., 2010)|
|Ebola virus||Ebola virus||Demonstration that antibodies are a correlate of protection but are not sufficient for protection||(Sullivan et al., 2009)|
Due to the more extensive use of the Indian-origin rhesus macaque, this was the first of the macaque genomes to be sequenced. The draft DNA sequence of an Indian-origin female rhesus was completed in 2007 and compared with the human and chimpanzee genomes (Gibbs et al., 2007); the Chinese rhesus macaque genome was just published in 2011 (Fang et al., 2011). This three-way comparison study gave some of the first insight to inform an understanding of mutational mechanisms that have, during the last 25 million years, shaped the biology of the three species. Prior work had focused on specific regions of the genome that encode gene members of the immune system, as noted below.
Major Histocompatibility Complex
MHC loci in rhesus macaques have been explored and compared, with a great deal of progress in the rhesus macaque. Class I alleles are termed Mamu (for Macaca mulatta) (Doxiadis et al., 2007; Otting et al., 2007); in contrast to humans only two MHC class I loci are found, A and B, with at least two expressed B loci, indicating a duplication of the B locus. Macaques have three MHC class II loci: DP, DQ, and DR. Haplotype diversity can result from crossing over events, since rhesus macaques have several class I alleles on each chromosome. Comparison of rhesus and human class I and class II evolution shows that the class I alleles are not shared between the species (Boyson et al., 1996), although similar epitope binding motifs are shared by macaque and human MHC class I molecules (Loffredo et al., 2009). MHC class I haplotype can clearly affect disease progression in SIV infection, in that certain MHC haplotypes affect the ability to control viral replication in vivo (Bontrop and Watkins, 2005; Goulder and Watkins, 2008). Mamu-A*01 animals typically show better control of infection in vaccine studies (Pal et al., 2002), and haplotypes B*08 and B*17 are associated with elite control of
viremia (Loffredo et al., 2008; Yant et al., 2006). There also appears to be an effect of class I haplotype upon antiretroviral drug effectiveness (Wilson et al., 2008). Certain MHC class I alleles in humans also appear to be associated with better control of viremia and disease outcomes, reviewed in Goulder and Watkins, 2008.
The KIR Gene Family
As noted above in the section on chimpanzees, MHC class I molecules are ligands for the KIRs, which are expressed by natural killer cells and T cells. As with chimpanzee KIRs, the interactions between these molecules contribute to both innate and adaptive immunity, and combinations of MHC class I and KIR variants influence resistance to infections, susceptibility to autoimmune diseases and complications of pregnancy, and outcomes of transplantation (Parham, 2005). The genes encoding the KIRs all arose recently from a single-copy gene during the evolutions of simian primates, after which the KIR and MHC class I genes co-evolved. The genes have been sequenced in rhesus, humans, chimpanzees, gorillas, gibbons, orangutans, and marmosets (Sambrook et al., 2006), and in Mauritian cynomolgous macaques (Bimber et al., 2008). There has been a recent comprehensive rhesus macaque KIR data set developed (Moreland et al., 2011) and an overview of MHC/KIR coevolution (Parham et al., 2010), emphasizing rapid evolution of KIR sequences, in large part due to evolutionary pressure from infectious pathogens. Of note, the old-world monkeys have been recently described as being the species most likely to provide useful and informative models for human disease.
T Cell Receptor Genetics
The diversity of T cell receptor (TCR) alpha and beta chains is created by somatic recombination of germ-line genes, as described above. Several studies have examined TCRs in chimpanzees and macaques (Jaeger et al., 1993). Macaques that are not infected display a diverse T cell repertoire characterized by a Gaussian distribution of betaCDR3 lengths (Currier et al., 1999). T cell repertoire analysis has revealed a dominance of T cells expressing specific V-beta segments during chronic infection (such as SIV infection). Rhesus CMV infection led to polyclonal CD4+ T cells that changed over time and chronic infection to reveal a skewed hierarchy dominated by two or three clonotypes (Price et al.,
2008). These kinds of comparisons aid in understanding the role of T cells in controlling infection and how these types of changes compare with natural progression of aging, for example (summarized in Messaoudi et al., 2011).
Antibody Gene Families
In macaques as in humans, the diversity of B cell receptors (BCR) and the soluble forms, or antibodies, that result, is generated by somatic recombination as with the TCR. The immunoglobulin loci for antibodies are similarly arranged in the rhesus macaque as in humans, and the antibodies have the same structures, with clear homologs identified for IgG and IgA classes and for subclasses of IgG (Scinicariello et al., 2004). There are differences, as would be anticipated with the time since speciation, but remarkable functional and genetic conservation.
Elegant comparative studies examining the relative proportions and cell surface markers of human versus macaque cells have been described and are summarized in a recent review (Messaoudi et al., 2011). The strong conservation of these molecules has allowed detailed studies on immune cell ontogeny, homing, survival, proliferation, and death that also have opened the field of immune senescence in the macaque. As noted in the sections below, the roles of specific cells in disease have been studied in vivo by transient depletion of subsets.
CD16+ and CD56+ NK subsets are largely similar in function and distribution in humans and macaques. The distribution of NK cells in blood and tissues differs somewhat in macaques, where CD16 predominates in the blood, and CD16 negative cells positive for CD56 can be found in tissues (Reeves et al., 2010). Prior studies had suggested that NK cells might not play a strong role in the containment of SIV; the role of NK cells in control of SIV still uncertain but certainly cannot be discounted. Macaques are appropriate models in which to address questions in acute infection, which is a phase of the infection that is very difficult to identify and thus study in humans. There are new data that have identified the macaque counterpart of mucosal NK cells producing IL-22
(Reeves et al., 2011), as previously identified in humans. These studies emphasize the importance of performing studies in macaques where greater access to mucosal samples is possible.
Monitoring and Understanding the Roles of Innate and Adaptive
Responses in Macaque Models for the Development
of Vaccines and Immunotherapies
Flow cytometric analysis of macaque immune-related cells using murine anti-human CD antibodies that were developed to bind to human cell surface markers demonstrates the strong immunological conservation of the vast majority of these surface molecules. This attribute of shared receptor recognition is not surprising knowing the genetic conservation of the species, and it has meant that the nuances of antigen presentation and the respective roles of B and T cells in macaque immunity is now a well-established area of research. A recent review provides an overview and examples of the staining and separation of these subsets in rhesus macaques (Messaoudi et al., 2011). The relative contribution of specific types of cells in innate and adaptive immune responses in the outbred Macaca species has been made possible by infusion studies using murine monoclonals directed at human CD8, CD4, CD16, CD20 (or other cell surface markers of interest) to transiently deplete specific subsets of lymphocytes in vivo. These studies have demonstrated the relative contribution of CD4+ and CD8+ T cells, B cells, and NK cells to the control of specific pathogens at different time points during infection, with examples in the sections that follow.
Stimulating Innate Responses
As in humans, the mediation of innate responses includes neutrophils, NK cells, dendritic cells (DC), and macrophages. Recognition of microbes depends upon the detection of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors, which fall into two families, toll-like receptors (TLR) and RIG-I-like RNA helicases (RLH). DCs and NKs have been well characterized in the rhesus. The major two subsets, myeloid (mDC) and plasmacytoid (pDC) types, are identified with the same surface markers and share functionality and cytokine response following viral infection. These two types of DCs express the same TLRs as human DCs, which differ drastically from murine DCs,
and thus help establish the macaque as an excellent species in which to evaluate TLR ligands as adjuvants (Rhee and Barouch, 2009).
Magnitude and Quality of T Cell Responses
In the T cell realm, there has been significant development work to understand and to quantify specific T cell subsets in the blood and effector sites (Pitcher et al., 2002; Walker et al., 2004), aided by advances in sampling methodologies that provide repeated sampling opportunities at effector sites such as the lung, for example. The functional diversity of the T cell response in macaques, as in humans, can now be measured via “staining” for multiple cytokines simultaneously. Intracellular cytokine staining (ICS) is a commonly utilized technique, where cells are stimulated with antigens (virus, lysates, purified antigens, or peptides) and then treated to block cytokine secretion, then labeled for each cytokine with a different-colored tag, and enumerated on a flow cytometer. Much of this work has been stimulated by interest in HIV pathogenesis and subsequently SIV infections, as these viruses destroy CD4+ T cells in the gut and peripheral blood compartments, with concomitant negative effects on T cell help; the rapidity of destruction varies with the strain. The time course of longitudinal development of T cell help (CD4+ T cells) and cytotoxic cells (CD8+ T cells) in response to SIV or SHIV infection is similar to that of HIV-1 in humans. In acute SIV infection, CD8+ T cells were shown to be very important for viral control (Schmitz et al., 1999). More recent studies have further elucidated the impact of CD4 and CD8 T cells, in experiments that clearly show that CD8+ T cell removal during this timeframe results in rapid disease progression due to unchecked viremia (Okoye et al., 2009); the depletion also resulted in proliferation of CD4 T cells, particularly effector memory cells. Antiviral CD8+ T cells have also been implicated in controlling vaginal infection upon exposure to a highly virulent SIVmac239 after vaccination with SHIV89.6, an infectious but non-pathogenic strain (Genesca et al., 2009).
Antibodies and Affinity Maturation
The development of antibody responses is dependent upon the effective antigen presentation and, for some diseases, persistence of antigens. Antibody response kinetics are indistinguishable in macaques and in humans, but it has been easier to perform longitudinal studies to examine the maturation of the response with more frequent time points (Cole et
al., 1997). Antibodies increase in avidity with time of exposure to antigen and the quality of the response depends upon the form of the antigen. Many persistent pathogens utilize various methods for immune evasion, including antigenic variation of their surface proteins to elude recognition B cells and thus to prevent containment by antibodies. SIV and SHIV have served as excellent models for longitudinal studies of antibody development, including neutralizing antibodies, which typically increase in magnitude and breadth with time (Hirsch et al., 1998; Kraft et al., 2007), as is seen in humans infected with HIV-1 (Mahalanabis et al., 2009; Sather et al., 2009). Antibody responses directed to HIV or SIV Envelope typically are robust and directed to hydrophilic, hypervariable regions; responses to conserved regions (such as those required for receptor and coreceptor binding) arise later and do not appear in all subjects (Li et al., 2007). Variation and post-translational modifications such as N-linked glycosylation in the SIV Envelope protein have been shown to lead to escape (Rudensey et al., 1998). There is now extensive molecular and sequence data of both viral variants and antibodies that HIV and SIV infection leads to similar B cell responses in humans and macaques, respectively. The development of binding and neutralizing antibodies occurs over the same time frame and requires extensive affinity maturation from the germ line antibody genes (Moore et al., 2011). Monoclonal antibodies from SIV-infected macaques have been instructive in understanding the neutralizing epitopes targeted (Cole et al., 2001; Glamann et al., 1998; Robinson et al., 1998), although no extraordinarily powerful monoclonal antibodies have yet been isolated with properties similar to those found in HIV+ subjects who are elite neutralizers (Doria-Rose et al., 2009; Walker et al., 2010; Wu et al., 2010). Contributions by CD20-positive cells in SIV infection were evaluated and shown to be limited to the chronic phase, with no apparent effect on early viremia (Schmitz et al., 1999, 2003). A recent clinical report showed that an HIV-positive subject with lymphoma who was treated with anti-CD20 (Rituximab) to reduce his B cells had a transient increase in plasma virus and a reduction in the level of neutralizing antibodies. These data were interpreted as evidence for contributions to viral control by neutralizing antibodies during the chronic stage of HIV-1 infection, consistent with the data on SIV-infected macaques treated with anti-CD20 (Huang et al., 2010). Depletion studies have been highly informative in other diseases, such as measles (Permar et al., 2003, 2004). A comparison of B cell depletion during the acute phase of infection in this model alone or in combination with CD8+ T cell depletion clearly showed that the antibodies
played a limited role in the control of measles viremia, while the CD8 effector T cells were critically important for limiting viremia and rash production.
Vaccine Approaches in Macaques
Macaque vaccine experiments performed over the last 25 plus years have accrued a large body of data about relative immunogenicity of various vaccine approaches, and these experiments have also allowed correlates of vaccine protection to be determined in many cases. In the sections below, we have attempted to summarize briefly the status of research in five major pathogens, to introduce the major concepts under investigation and the importance of these models to the discovery of effective vaccines for diseases that are emerging, or re-emerging due to persistence in the host, such as tuberculosis. Approaches for vaccines depend upon the desired type of immunity required for protection. Protein formulations are often processed through endocytic pathways that stimulate CD4+ T helper 2 (TH2) cell responses and promote antibody production. By contrast, vaccines that allow synthesis of foreign proteins within cells lead to processing of antigens through the proteasome, a process that more effectively elicits CD8+ T cell responses, while also eliciting antibody responses. Some gene-based vaccines have the potential to generate broad responses because of their ability to target antigenpresenting cells (APCs) directly, which is a property of certain viral vectors. The quality and range of vaccine-induced immune responses can therefore be influenced by the specificity of viral vectors for different APC targets.
With the advent of molecular tools for genetic manipulation and the identification of virulence factors, it has been possible to utilize this knowledge to build recombinant vectors to precisely excise unwanted genes and to express one or more vaccine antigens in their place. Advances in the development of safer, more attenuated viral vectors utilized for one major disease, smallpox, were the genesis of recombinant pox-virus-based vectors that persist in the human vaccine armamentarium today. These viral vectors are attractive because they stimulate innate and adaptive immunity and persist long enough to provide a strong antigen pool to boost B cells, much as the currently licensed live attenuated vaccines. The ability to challenge in macaques prior to testing in humans provides a measure of confidence that the immune responses elicited by
the new vaccines are effective against diseases that closely model human pathologies.
As with model development to study pathogenesis, it is also true that the use of related pathogens as “vectors” for vaccine delivery has also necessitated understanding the host range of the various vectors for humans and for macaques. Thus there has been an intensive search for nonpathogenic viruses that can replicate equally and stimulate similar levels of immunity directed to the foreign antigen(s) in both species. Current investigations with viral vectors include Vesicular stomatitis virus, Sendai virus, adenoviruses (chimpanzee, human, and macaque), poxviruses (various levels of attenuation and species specificity) and herpesviruses (including cytomegalovirus, and adenovirus-associated virus vectors), to name a few. Due to cellular targeting via receptors, replication capacity, and the inclusion of cofactors such as cytokine genes, some vectors are better at stimulating specific arms of the immune response; combinations can work additively and possibly synergistically.
In addition to viral vectors, other promising approaches in use include DNA delivered by a variety of routes (intramuscular, intradermal, via microneedles, and with electroporation to enhance uptake) (Yin et al., 2010). DNA vaccines showed impressive protection in a mouse influenza model (Ulmer et al., 1993) but have been much less immunogenic in macaques and humans with SIV or SHIV immunogens and viral challenge (Doria-Rose et al., 2003; Rosati et al., 2005; Yin et al., 2010), albeit there was significant improvement in immunogenicity with added cytokine genes (Barouch and Letvin, 2000) and with electroporation to enhance intramuscular uptake (Patel et al., 2010) and with improved vector design (Kulkarni et al., 2010). DNA vaccine immunogenicity is also greatly enhanced by protein boosting (Malherbe et al., 2011; Vaine et al., 2008). DNA vaccines have recently shown some promise for influenza vaccines in humans (Smith et al., 2010). They are attractive because they can deliver multiple antigens that can express genes in vivo to assemble noninfectious virus-like particles or native proteins that are difficult or impossible to produce by recombinant methodologies. They can be delivered multiple times without inducing anti-vector immunity that limits the effectiveness of viral vectors.
Another noteworthy area of active research in nonhuman primate models is the development of protein immunogens and improved adjuvants. Novel adjuvants are needed to stimulate both innate and adaptive responses, and with a better understanding of TLR binding it may be possible to direct responses more effectively, while increasing the magnitude
of the response. In addition to whole virion approaches to preserve native structures (Frank et al., 2002; Johnson et al., 1992; Lifson et al., 2002; Warfield et al., 2007b; Willey et al., 2003), novel and native scaffold approaches are being modeled to present epitopes out of context on the surface of heterologous proteins (Guenaga et al., 2011; van Montfort et al., 2011; Zolla-Pazner et al., 2011), often in multimeric arrays that B cells prefer (De Berardinis et al., 1999).
The overwhelming body of literature on HIV vaccines in macaques has been summarized a number of times in several recent reviews (Haigwood, 2009) and chapters and thus the committee is encouraged to avail themselves of the detailed information summarized in a recent review (Lifson and Haigwood, in press). Many vaccines have focused on SIV only, while some have also included HIV Envelope for SHIV challenge. These studies have provided key observations about the immune responses elicited and how these have correlated with protection from infection. A critical example of this is the T cell-focused vaccines based on a non-replicating recombinant human adenovirus (Ad5) expressing SIV Gag, Pol, and Nef. A simiar SIV Gag vaccine had an effect on plasma viremia, reducing it 1 or 2 log10 using a less pathogenic X4 SHIV challenge virus (Shiver et al., 2002). When these SIV vaccines expressing Gag, Pol, and Nef were tested using the most pathogenic challenge (SIVmac239), there was no evidence of control of viremia post infection (Casimiro et al., 2010), the result found in the STEP clinical trial (Buchbinder et al., 2008). However, until there is a definitively positive human vaccine trial with correlates of immunity, we will not know for certain which of these macaque infection models and/or which immune responses are predictive of protection (Haigwood, 2009; Morgan et al., 2008). At this stage in vaccine development, the field awaits with great interest the ongoing immunological analysis of the RV144 trial, which showed modest, transient efficacy in a subset of the trial participants, those with lowest risk of exposure (Rerks-Ngarm et al., 2009). A few key lessons from this field are highlighted below:
Combination vaccines show more promise than single entities Combination vaccines—prime-boost—with multiple antigens that stimulate T-cell and B-cell responses generally have been found to be consistently more immunogenic and more effective in resisting challenge or
controlling viremia than single approaches, but even these experiments had different outcomes depending upon the challenge virus. Early success with highly immunogenic yet risky vaccinia virus led to the testing of attenuated poxviruses such as Modified Vaccinia Ankara (MVA) and the avian poxvirus ALVAC. For SIV and SHIV, there are many examples of combination poxvirus vaccines that include one or more vectors with or without DNA or a recombinant protein, and these have shown different degrees of viral control upon challenge, depending upon the virus used for the challenge (Doria-Rose et al., 2003; Hel et al., 2002; Pal et al., 2006; Patterson et al., 2003). The recent RV144 trial was designed with an avian poxvirus prime, followed by a ALVAC plus Env protein boost. This vaccine has shown some modest and transient protection in humans (Rerks-Ngarm et al., 2009)—was it predicted by macaque experiments? Because no exact replica of the RV144 design was tested, a closely similar experiment based on SIV immunogens is in progress in macaques at this writing so that comparisons can be made.
New vectors to stimulate effector T cell memory Current promising SIV vaccines based on rhCMV have demonstrated an impressive ability to control viremia to nearly undetectable levels in approximately half of the macaques challenged with SIVmac239 (Hansen et al., 2009, 2010, 2011). The mechanism of this vaccine is the persistence of the vector, which is apparently years at least, and the very strong induction of effector T cells against the SIV gene products. Further research is underway to understand the bimodal effect of the vaccine (all-or-none effects on virus loads) as well as to combine this approach with other vaccines that could stimulate central memory, such as adenovirus vectors, and antibodies, such as recombinant adjuvanted proteins. To move this approach into the clinic, it will require making HIV antigens in an attenuated hCMV vector, since hCMV is a high-risk infection for fetuses and immunecompromised humans.
Immunity to adapted viruses may provide clues for non-adapted infection Because both HIV-1 and HIV-2 are derived from naturally occurring SIVs, understanding the immune responses in the host to which they are adapted could yield clues as to whether immunity in the non-adapted host contributes to pathogenesis (Silvestri, 2009). SIV not only replicates well in its natural host (sooty mangabeys, Cercocebus atys) (Sodora et al., 2009), but there is also evidence for loss of gut CD4 T cells; thus the idea has been proposed that these cells may not be the
major source of plasma virus, even though they are an early target for destruction (Lay et al., 2009). These studies suggest that inflammation induces CCR5 expression as a co-factor for pathogenesis, as CD4+ cells that are also CCR5+ are greatly reduced in blood and tissue in the naturally infected hosts (Pandrea et al., 2007). Immune activation in pathogenic infection with SIV and HIV has been shown to be related to enhanced microbial translocation, which is related to CCR5 expression and can be partially controlled by antiretroviral treatment (Brenchley et al., 2006); translocation is reduced in the sooty mangabeys and is likely to be a similar story with the other highly adapted viruses in African green monkeys (Pandrea et al., 2006).
Improved challenge models to better mimic low-dose mucosal exposure Studies in macaques have extended early findings in chimpanzee models for HIV-1 that passive transfer of antibodies (monoclonals or IgG from infected animals) can be delivered by intravenous, intramuscular, or subcutaneous routes to directly demonstrate a role for antibodies in blocking infection (reviewed in Lifson and Haigwood, in press). These studies have informed the magnitude and breadth of antibodies that may be necessary for a fully effective prophylactic vaccine, but much of this work was based on high-dose mucosal challenges, in order to achieve infection of all control animals after a limited number (1-2) of challenges. A very important advance to our understanding has been furthered by the use of low-dose challenge models that more closely resemble human transmission (Keele et al., 2008). These newer studies indicate that much lower doses than previously tested can be effective in preventing viral acquisition upon repeated low-dose challenge (Hessell et al., 2009a, 2009b). The use of repeated low-dose challenge in vaccine studies increases the expense and time needed to obtain answers but may provide more realistic assessment of the types and magnitude of immunity needed for more typical human sexual exposure to HIV-1.
Gene therapy as proof of principle for antibody-mediated protection One of the most innovative uses of the SIV/macaque model was to directly prove that neutralizing antibodies expressed in vivo could prevent infection. This question was critically important to address because work prior to this point had suggested that only extraordinarily high levels of antibodies would be effective in preventing infection. Gene therapy and adenovirus associated virus (AAV) expressing an SIV neutralizing monoclonal sFv were utilized to prevent intravenous infection by SIV
(Johnson et al., 2009). Not all macaques were able to resist infection, and this appeared to correlate with the persistent expression of the antibody in vivo. This experiment could have failed completely, or could have caused major pathogenic consequences in vivo, and thus was an excellent use of the SIV macaque system to test proof of principle. If the problems can be overcome, this may see further development.
Tuberculosis (TB) is a major threat worldwide, particularly with ~2 billion people latently infected. Identifying immune mechanisms that control the initial infection and prevent reactivation remain critical goals (summarized in Lin and Flynn, 2010). Examples of the contributions of macaques to understanding these issues includes a demonstration of the role of T cells in disease control via a CD8 depletion study in BCGvaccinated macaques that were infected with M. tuberculosis (Chen et al., 2009). Diedrich et al. (2010) used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection (Diedrich et al., 2010). Further studies on SIV and TB co-infection indicate that events during acute HIV infection are likely to include distortions in proinflammatory and anti-inflammatory T cell responses within the granuloma that have significant effects on reactivation of latent TB. In this study, mycobacteriaspecific multifunctional T cells were better correlates of Ag load (i.e., disease status) than of protection (Mattila et al., 2011).
Smallpox and Monkeypox
Smallpox and monkeypox are closely related orthopoxviruses that differ in their pathogenicity for humans. Although less infectious and the cause of less mortality, monkeypox is still a problem for humans when zoonotic transmissions take place. Due to the eradication of smallpox, vaccines can no longer be tested in humans for the prevention of the infection and thus nonhuman primate (macaque) studies are necessary for licensing. MVA has shown strong protective effects in M. fasicularis (Earl et al., 2004) and was shown to be more effective than the standard
smallpox vaccine. Protection correlated with the more rapid immune response to MVA, potentially related to the higher dose of MVA that can be tolerated safely (Earl et al., 2008). Another group showed by depletion studies that B cells are essential for protection in this model (Edghill-Smith et al., 2005), and went on to develop a DNA/protein approach that shows promise of being a safe and effective vaccine (Heraud et al., 2006).
Yellow Fever Virus
Although there is a vaccine for yellow fever virus (YFV-17D) in use since 1945 that was originally developed in the macaque, it is not a fully efficacious vaccine, and severe adverse events have been reported that may be related in some cases to impaired innate responses (Pulendran et al., 2008). The vaccine elicits long-lived persistent T (Akondy et al., 2009) and B cell responses (Poland et al., 1981), and systems biology has been applied to determine correlates of protective immunity (Querec et al., 2009). To test for improved vaccines, there is currently a model for yellow fever using the YFV-Dakar strain of virus that has previously been characterized as viscerotropic and capable of being lethal in rhesus macaques (Monath et al., 1981). Following challenge with YFV-Dakar, unvaccinated animals demonstrated fever, lymphocytopenia, and fulminant viscerotropic disease with multi-organ failure, resulting in death within 4-6 days after infection. Histological analysis of the liver demonstrated widespread neutrophil infiltration, councilman bodies, and severe tissue necrosis, which correlated well with ALT (alanine aminotransferase). In contrast, animals that were vaccinated can show protection against lethal challenge and there is evidence that novel inactivated vaccines may protect against viremia, at least below detection (<50 genome copies/mL of serum) (M. Slifka, personal communication). Whether these new vaccines will offer improved safety profiles along with broad efficacy in humans remains to be determined.
The acute hemorrhagic filoviruses Ebola and Marburg cause infections with very high mortality rates in humans and nonhuman primates. Several approaches have been tested in macaques, with a primary focus on Ebola virus (EBOV). These have included DNA-prime-adenovirus boost with glycoprotein and nucleoprotein, either in a prolonged
(Sullivan et al., 2000) or shortened regimen (Sullivan et al., 2003). Although effective, preexisting adenovirus immunity may limit the utility of this approach. Other approaches tested have included live attenuated vaccines based on vesicular stomatitis virus (VSV) (Jones et al., 2005), as well as parainfluenzavirus (Bukreyev et al., 2010) and virus-like particles (Warfield et al., 2007a, 2007b). Discordant results of vaccine efficacy studies between mice and nonhuman primates have been observed with Ebola vaccines and have underscored the importance of examining this question. Although IgG titers are correlated with protection from EBOV challenge, passive antibody transfer was not fully effective in protection in macaques, demonstrating that protection is multifaceted. This subject is summarized in an excellent recent review that lays out the argument for using the “animal rule” for vaccine approval (Sullivan et al., 2009). Ultimately, the development of a vaccine for humans based on a replication defective Ad5 platform has shown significant promise and good immunogenicity (Ledgerwood et al., 2010).
CONCLUSIONS AND FUTURE USES OF NONHUMAN
PRIMATE MODELS, INCLUDING THE CHIMPANZEE
• Chimpanzees have been essential for the study of human pathogens that do not infect lower species or reproduce key features of human disease. It is difficult to exclude the possibility that emerging infectious diseases will have a similar highly-restricted host range and thus be difficult to model in lower primates.
• There are as yet no vaccines for many of the human infectious diseases that have benefited from chimpanzee studies, including HCV, RSV, and malaria. All three remain important public health problems and there are high hurdles to successful vaccine development. These hurdles include a poor understanding of how to (1) vaccinate against highly mutable viruses that establish persistence (e.g., HCV), (2) safely balance vaccine immunogenicity with attenuation (e.g., RSV), and (3) select antigens for vaccination against a parasite with a complex life cycle and poorly understood strategies for immune evasion (e.g., malaria). It should be emphasized that even for an existing successful vaccine, unexpected adaptation of a virus like HBV can create vaccine escape variants. The chimpanzee model has in the recent
past, and may again in the future, be important to test the threat that such adaptations present to public health.
• Monoclonal antibodies and other biologicals designed to modulate inflammation and immunity in infectious and non-infectious diseases are routinely assessed for off-target effects in chimpanzees. The chimpanzee is valuable for these pre-clinical studies because unexpected cross-reactivity with orthologous proteins is more likely to be revealed. Humanized monoclonal antibodies are also less likely to elicit a neutralizing humoral response in chimpanzees when compared with more distant non-human primate species. This use of the animal model should accelerate as new targets for intervention are identified. Antibodies against T cell activating and inhibitory receptors that might modulate immune function in autoimmunity, cancer, and infectious diseases provide a prime example. For instance, combinations of inhibitory receptor blocking antibodies may be useful to restore immunity in chronic hepatitis B, but the animal model will be important to assess effectiveness and risk in a disease that is often subclinical and slowly progressive.
• The very close genetic relationship between humans and chimpanzees affords the opportunity to define the molecular pathogenesis of infections caused by viruses, microbes, and parasites that afflict humans. Genomic and proteomic technologies can be applied to understand host responses over the course of acute and (where relevant) persistent infection in primary target tissues. These tissues are often not available from humans because biopsies are not medically indicated. Also, critical aspects of innate and adaptive immune responses may be missed in humans because early stages of the infection are asymptomatic. Thus, chimpanzees provide a means to define immune responses at time points and locations that are inaccessible in humans.
• The close similarity of the macaque and human immune systems and the susceptibility of Macaca species to many human pathogens have together afforded opportunities to explore systematic comparison of multiple approaches for vaccine design, delivery, and comparative analyses of immunogenicity and responses to challenge. Furthermore, the relative availability of macaques for experimentation has allowed the discrimination of the contributions of different arms of the immune response by passive transfer
of antibodies or transient depletion of specific subsets of cells.
• Having access to a broad range, or “full spectrum” of nonhuman primates has been enormously useful in order to understand how pathogens affect different species, in order to gain an understanding of the interplay between the host and the pathogen. Disease models differ in robustness depending on which species is used, so it is critical to have multiple species available. Understanding control in a species that has adapted to a virus may yield insights into novel therapeutics.
• “Failure” of an imperfect model can lead to a better understanding of the host-pathogen relationship. Mismatched outcomes between humans and animals, once understood, can provide insight into the pathogenesis of infection in humans. They can also lead to important refinements in a nonhuman primate model so that it better reflects the situation in humans. An example is high- versus low-dose mucosal challenge with SIV and SHIV in macaques. There was very strong resistance to changing challenge modalities based on the cost of requiring many more animals per group, time, manpower and virus stocks needed to deliver daily challenges for weeks or months, and statistical complications due to different times of acquisition. When it was demonstrated that typical sexual transmission of HIV generally results in a single or few founder viruses, then there was a stronger scientific rationale for the lower-dose challenges that are typically used today. This has led to encouraging news that protection from this type of challenge and indeed protection in humans may be more attainable, based on the amounts of antibodies needed for protection in macaques.
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