Nonhuman primates are relevant models for research in hematology, immunology and virology

ARTICLE

Auteur(s) :, F Hérodin^*, P Thullier, D Garin, M Drouet

Centre de recherches du service de santé des armées, 24 avenue des Maquis du Grésivaudan, BP 87, 38702 La Tronche, France

From basic to preclinical research: baboons, macaques and marmosets are models pertinent to humans for studies in hematology, immunology and virology

The convention on international trade in endangered species of wild fauna and flora (CITES) distinguishes animals the trade of which is prohibited (in annex I) and animals for which trade is regulated (www.cites.org/eng/disc/text.shtml). According to the CITES, experimental research with Hominidae (chimpanzee, gorilla) primates is prohibited except for special, applied research in virology [2].

The two Cercopithecidae (baboons and macaques from the Old World) and Ceboidae (marmosets and squirrel monkeys from the New World, see table 1( Table 1 )) families provide most of the primates used for biomedical research, in particular in hematology, immunology and virology. There is a wide diversity in body weight according to genera and species (table 2)( Table 2 ).

NHPs are models relevant to humans because of the high level of gene homology which underlies physiological and biochemical similarities. Historically, the Rhesus monkey (Macaca mulatta) model was used by Wiener to describe the Rh system [3]. A high level of homology has been reported between humans and baboons for MHC class I molecules (90%) [4] and for transcriptosome genes of BM CD34⁺ cells [5]. Nucleotidic and proteic sequences of numerous cytokines are quite homologous in humans and macaques (93-99%) [6]. The similarities between the sequences of macaque and human antibodies (Abs) are equivalent to the similarities between human Abs originating from different individuals. As for marmosets, which are always natural hematopoietic chimera resulting from a double fertilization associated with placental anastomoses, they provide a highly original graft model due to the tolerance status between twins [7, 8].

Rhesus/cynomolgus monkeys and baboons have specific advantages in research. Adult baboons are large primates (25 kg on average), allowing repetitive blood and BM sampling and leukapheresis-driven, mobilized hematopoietic stem and progenitor cell (HSPC) collection [9], without the arterial catheterising required for macaques [10]. Owing to their venous system, baboons and macaques are relevant models for studying the infusion and recirculation of hematopoietic cell grafts of variable maturity. The intramedullary route is also easily accessible. The ABO system [3], hemoglobin, coagulation and fibrinolysis physiology [11] are similar in humans and baboons.

Yet these models have several disadvantages: low breeding efficiency, high cost of purchase and cytokine treatments, the need for general anaesthesia with ketamin for almost all manipulations, low availability of umbilical cord blood HSPC due to difficulties in pregnancy follow-up, reduction of animal cohorts related to ethical considerations, etc. The indeterminate viral status is also a major concern. Thus, although baboons as well as cynomolgus macaques from Mauritius are considered to be Herpes B- negative, most Cercopithecidae, apart from African green monkeys (vervets) from Barbados, host viruses such as spumavirus that are involved in in vitro cytopathogenic effects on adherent stromal cells which are potentially useful in cell therapy. Moreover, NHPs exhibit a certain degree of species-specificity relative to humans: it has been shown that only 45% and 63% of murine Abs directed against human cytokine receptors and lymphocyte cell antigens cross-react with rhesus monkey and chimpanzee antigen counterparts respectively [12]. Recombinant macaque cytokines are available to assess ligand-receptor binding in homospecific conditions [13], but these studies must always be completed in monkeys by evaluating the recombinant human (rhu) molecules to assess specific toxicity and pharmacological effects before administration to patients.

Of the teams which have developed NHP models (table 3)( Table 3 ), Berenson et al. showed, 17 years ago, that CD34 antigen was expressed at primate HSPC level, by using an Ab raised against human CD34 [14, 15]. The existence of peripheral blood (PB) HSPC and their engrafting potential had previously been demonstrated by Storb et al. in non-irradiated and irradiated parabiotic animals [16]. NHP studies include healthy and myelo- or immunosuppressed (using total body irradiation [TBI], immunosuppressors, chemotherapeutic drugs) animal models. The relevance of the latter is particularly related to the easy use of drugs, materials and protocols derived from human clinical practice (table 4)( Table 4 )[17, 18].
Table 1 Order of Primates

Nonhuman primate models in hematology

In vitro studies

Pathophysiological studies

Myelosuppression following TBI or chemotherapy has been well documented and has proved to be similar in monkeys and humans. The pharmacokinetics of chemotherapy agents has been studied in NHPs [29]. As a model of a reduced intensity conditioning regimen, it has recently been shown that low dose TBI causes clonal fluctuation of NHP HSPC, without impairing long-term recapitulation of stem cell polyclonality [30]. Studies regarding the efficacy of hematopoietic growth factor (HGF) in healthy monkeys provide good models of medullar hyperactivity. The knowledge of possible pathological consequences of hyperleucocytosis over 100x10⁹ white blood cells (WBC)/L or thrombocytosis over 1x10¹² platelets (PLT)/L appears crucial to determine the therapeutic index of HGFs [31]. Moreover, long-term treatment with G-CSF or G-CSF + SCF has been evaluated in rhesus macaques and does not seem to result in stem cell exhaustion or clonal dominance [32]. Rhesus monkeys and baboons have also proved to be good models for exploring vascular biology and hemostasis [33, 34].

Numerous radiobiological studies have been performed with primates and show that radiation exposure results in early, systemic inflammatory reaction and hemostasis disorders preceding BM aplasia. Baboons are very pertinent models in radiopathology since they mimic the intrinsic heterogeneity of accidental irradiations, characterized by an absorbed radiation dose gradient, mainly related to body thickness [35]. Residual HSPC can be collected in monkeys and then enumerated, using blood sampling and multiple-site BM aspirations. Thus, the FLT-3 ligand serum level has recently been proposed as a prognostic bioindicator of BM damage [36]. Chemotherapeutic drugs have also been tested in primates. Unfortunately, most of the toxicopharmacological studies performed by pharmaceutical companies have remained unpublished. Monkeys appear to be very sensitive to specific drugs such as cyclophosphamide which must be administered at reduced doses in comparison to humans (FH & MD, unpublished data).

Therapeutic models

The safety and efficacy of cytokines in healthy and myelosuppressed NHPs

Finally, NHP models which are closer to humans than mice and dogs, have clearly shown that the extent of BM damage is the limiting factor for HGF efficacy. In fact, high grade myelosuppression is a major indication for stem cell therapy.
Table 6 Examples of cytokines evaluated in myeloablated primates

Transplantation models of unmanipulated or ex vivo, expanded hematopoietic stem cells

Several agents mobilizing CD34⁺ cells have been evaluated in monkeys and have shown a variable efficacy: HGF, combined or not with, myelosuppressive drugs, chemokines, glycans, Abs directed against adhesion molecules (table 7)( Table 7 ). However, these normal animal models imperfectly mimic the situation of a reduced HSPC pool of “poor mobilizers” in oncology.

Numerous autologous but very few allogeneic [18] HSCT models have been developed so far in monkeys. In fact, the dog is the best animal model for allogeneic graft studies, as abundant litters provide related animals in both DLA-matched and haploidentical situations [60]. The canine model is also the gold standard for non-myeloablative conditioning regimen evaluation. However, the relevance to humans of dog and NHP models, respectively, remains to be determined in terms of MHC allelic polymorphism.

Transplantation models of ex vivo, expanded autologous CD34⁺ cells have been developed in NHPs (table 8)( Table 8 ). Interestingly, graft efficacy can be ethically assessed in monkeys without co-administration of an unmanipulated rescue graft [9]. To date, the benefit of ex vivo expansion remains under debate as it depends on different parameters such as cytokine combination (early-acting versus late-acting cytokines), culture media (liquid culture without or with stromal cell layers), and culture duration. Thus, several preclinical models [9](table 8) have shown that ex vivo expansion using cytokines permits wide amplification of progenitors and precursors, but achieves only low amplification of LTC-IC and NOD-SCID repopulating cells [66]. NHP models could also be used to evaluate the safety and efficacy of grafts resulting from co-cultures performed on allogeneic or xenogeneic stromal cell layers [64, 67]. Moreover, the ability of mesenchymal stem cells (MSC) to modulate alloreactivity in vivo in an allogeneic skin graft model has been studied [68]. Finally, optimisation of ex vivo stem cell expansion may be achieved through other strategies based on activation of stem cell regulatory pathways [69]. In this context, the overexpression of transcription factors (TF) such as HoxB4 or the use of recombinant proteins such as TAT-HoxB4 has been proposed. Ex vivo expansion of human hematopoietic stem cells can also be achieved in a co-culture system using stromal cells genetically engineered to secrete HoxB4 [70]. The activation of Wnt signalling in HSCs has been shown to increase expression of HoxB4 and Notch1. The efficacy and safety of such approaches could usefully be assessed in NHPs.
Table 7 CSPH mobilization models in primates

Monkey models of in utero HSPC transplantation

This strategy was used to induce tolerance in a context of solid organ grafts. In utero HSPC transplantation has been shown to extend the survival of postnatal kidney transplants in monkeys [73]. However, immunosuppressive therapy remained necessary in this model to counteract long-term host versus graft reaction.

The induction of tolerance to donor could make organ transplantation safer and more uniformly successful. One of the most promising approaches currently being investigated involves the induction of deletional tolerance through the establishment of mixed chimerism [74].

Gene marking and gene therapy

NHP models are used to study host tolerance of transduced HSPCs. Xenogenes that are efficient in gene marking could induce high immunological conflicts leading to transduced cell elimination mainly due to cell lysis [81]. Autoimmune anemia has recently been reported in macaques following homologous Epo gene therapy using a skeletal muscle route, in correlation with the appearance of neutralizing Abs against the endogenous Epo [82]. However, prolonged murine or human adenosine deaminase expression was observed after transduction of macaque HSPCs using retroviruses. Moreover, macaque CD34⁺ cells have been successfully transduced by different teams using an EGFP xenogene [83]. In fact, reduced HSPC transduction efficacy could be counterbalanced by the in vivo expansion of immature cells. Such a goal could be achieved in NHP models using a cell growth gene co-transfection strategy. Efficient and durable gene marking of HSPCs in monkeys can be achieved after non-myeloablative conditioning [84]. Moreover, gene transfer into fetal hematopoietic progenitor cells can be performed in baboons, providing an efficient model for human studies, since both species exhibit similar, terminal, hematopoietic differentiation during the last three months of pregnancy [85]. An in vivo transduction strategy targeting BM stromal cells using adenoviruses has also been tested in NHPs and could be useful in stimulating hematopoietic recovery, post-myelosuppression through transient cytokine gene expression [86].

Furthermore, it has been shown in baboons that autologous transplantation of genetically modified HSPCs is more pertinent for the evaluation of long-term repopulating cells than xenotransplantation to NOD/SCID mice [87]. Linear amplification-mediated PCR has been developed in NHPs to identify and track vector-genomic DNA junctions. Each individual junction is a unique tag for a stem or progenitor cell and all its progeny. This strategy can be used to study proviral insertion sites, and to look for potential carcinogenic side effects, the knowledge of which is mandatory from a therapeutic perspective. In conclusion, NHP models are very useful to check the safety of viral constructs. To date, gene marking studies are far more common in research than in preclinical settings.

NHP models and pluripotent stem cells

Stem cell plasticity. Recent studies have demonstrated the hematopoietic potential of non-hematopoietic tissues, mainly in mouse models. This may be related to the so-called pluripotency of somatic stem cells. Previous studies in baboons have focused on fetal/adult liver stem cells. The putative interest in extrahematopoietic stem cells is currently being explored by many teams. Interestingly, preliminary studies seem to indicate that stem cell plasticity would be more restricted in NHPs than has been seen in mice. Thus, our team recently evaluated the ability of mobilized stem cells to repair cardiac tissue injury in a baboon model of acute myocardial infarction [94]. In this model, stem cell mobilization was carried out, with SCF and G-CSF administered 4 hours after circumflex coronary artery ligation. This study suggests a mobilization of endothelial progenitors that promote angiogenesis in the infarcted myocardium without significant myocardial repair. As for the presence of hematopoietic stem cells in monkey and human muscle, it appears highly hypothetical [95]. MSCs can be considered to be a source of multipotent stem cells, since they are capable of producing osteoblasts, chondrocytes, adipocytes and neurons. Biodistribution of MSCs after systemic or intramedullar injection has been studied in NHPs in order to evaluate their level of engraftment and their possible contribution to injured tissue regeneration [96].

NHP models in immunology and virology

Obtaining antibodies for prophylactic or therapeutic use

Abs chimerization and humanisation have been performed because the production of Abs of entirely human origin has frequently given unsatisfactory results, such as the instability of human hybridomas or the poor affinity of Abs isolated from naïv libraries. Certain mouse strains (Xenomice^®), whose immunoglobulin genes have been replaced by some of their human counterparts, have been developed to produce human IgGs using standard hybridoma technology, but they are available on a commercial basis only. Several Abs with the desired activities have, however, been isolated following human immunization, using phage technology to produce and screen immune libraries of human scFvs or Fabs (fragments of IgG that include the V regions), obtained from plasmocytes of immunized donors using molecular techniques [98]. The necessity of immunizing humans and sampling their plasmocytes (in peripheral blood, but more often in bone marrow or spleen) is indeed a major disadvantage of this technique, for practical and ethical reasons.

Immunization of NHPs has been proposed as a means of obtaining Abs sharing many similarities with their human counterparts, as it has even been assumed from somatic gene sequences that the differences between macaque and human IgGs are no greater than those found between the human IgGs from different individuals [99]. For various reasons, NHPs are more easily immunized, and their plasmocytes are more easily sampled, than humans. NHPs can also be very useful for the production of Abs against epitopes or antigens that do not elicit a humoral response in humans, for instance those of human origin. V domains of NHP origin can be fused to constant regions of human IgGs to produce primatized Ab [100], three of which (IDEC 112 directed against human CD4 to be used as immunosuppressive agents, IDEC 114 directed against human CD80/B7-1 to participate in treating autoimmune diseases such as rheumatoid arthritis and psoriasis and IDEC 152 directed against human CD53/RFcεII to counteract asthma) are currently undergoing clinical trials (Biogen Idec Inc., Cambridge, MA, USA). Preliminary results show good tolerances of these primatized IgG [101, 102].

However, not much information about the generation of these primatized antibodies has been reported, and only one article [103] had, until recently, described the isolation of an NHP Fab (Fragment antigen-binding, including the V regions). This scarcity has been discussed [104]. We have described in detail how an immune library was constructed using peripheral blood as a template [105], after immunizing an NHP with a model antigen (tetanus toxoid, TT). Amplicons were obtained only on the 4^th day after the final boost, and none were obtained on days 7 and 12. This result led us to consider that these amplicons specifically coded for anti-TT Fabs, and we constructed and screened a small phage-displayed immune library (5x10⁵ clones). A Fab, which was named 6-ATT, was successfully isolated from this library and it showed a high affinity for its antigen (Kd = 4x10^–10 M).

The 6-ATT sequence was recognized as originating from a gene similar to human germline genes, using on-line computational analysis with IMGT/V-QUEST and IMGT/JunctionAnalysis tools [106]. The match in identity between 6-ATT V regions and their most similar germline human counterparts was 89%, reaching 93% for the frameworks sub-regions, which are the most frequently involved in clinical tolerance of antibodies. Similar features have been obtained with another NHP Fab, which very efficiently neutralizes the anthrax toxin (data not shown), and they are the basis for good clinical tolerance of primatized IgGs. NHP may therefore be regarded as a rich source of an, as yet, almost untapped source of therapeutic molecules.

IL-7: the case of an immunostimulatory cytokine

In NHPs and humans, IL-7 has a specific immune response limited to T-cells, not involving major B-cell proliferation, in contrast to the situation in rodents. Recombinant simian IL-7 has a similar proliferative activity as rhIL-7 on human CD4⁺ T cells. The efficacy of IL-7 immunostimulatory activity is currently under investigation in T-cell-depleted monkeys [107], in which naïve T-cell production can be assessed by the quantification of PB T-cell receptor excision circles (TREC). Interestingly, in simian immunodeficiency virus-(SIV) infected macaques, IL-7 treatment induced a significant increase in PB CD4⁺ and CD8⁺ T cells, even though declines in the frequency and absolute number of TREC⁺ cells in the PB reflects effects on mature cells. Furthermore, in these SIV-infected animals that received continuing antiretroviral therapy during IL-7 treatment, no change in viral load was observed [108].

Models of vaccine

Preclinical studies in NHPs play key roles in AIDS vaccine development efforts. In addition to their traditional application to gauge vaccine safety and immunogenicity, NHP models are currently employed to explore fundamental mechanisms of primate immune system regulation, to investigate pathogenic AIDS mechanisms, and to optimize immunization [116-118]. Macaques infected with pathogenic strains of SIV or related chimeras expressing the envelope of HIV-1 (SHIV), constitute a powerful model for studies of the fundamental mechanisms of HIV pathogenesis. Indeed, SIV/SHIV and HIV have similar biological properties, using similar cellular receptors (CD4) and co-receptors (CCR5 for SIV, and both CCR and CXCR4 for several SHIV) and having identical target cells in vivo. In addition, infection of macaques with pathogenic SIV/SHIV isolates reproducibly induces an immunodeficiency strikingly similar to that observed in human AIDS [119]. In this context, it has recently been shown that severe BM damage commonly observed in macaques infected with SIV, as in HIV-seropositive patients, results in impaired T-cell production, which may contribute to the disruption of T-lymphocyte homeostasis [120]. These models have been of particular interest for understanding viral/host interactions including the installation of the immune response during early steps of infection and establishment of viral reservoirs. Major advances have been made in the description of viral transmission mechanisms, particularly at the mucosal level, emphasizing, for instance, the role of dendritic cells of the vagina [121]. However, to date there is no ideal animal model of human HIV-1 infection and, although considered as the most relevant, primate models of AIDS may have some limitations: 1) a model of pathogenic infection with HIV-1 is still lacking, chimpanzees could be chronically infected with HIV-1 but do not develop AIDS in most cases, and reliable infection of macaques with HIV-1 cannot be achieved although this species is susceptible to experimental infection with SIVmac or the almost identical HIV-2 and do develop AIDS; 2) host factor-driven resistance of macaques to HIV-1 infection are not fully understood. The contribution of TRIM5α factors has been recently suggested. Indeed, primate genomes encode a variety of innate immune strategies to defend themselves against retroviruses. Thus, if HIV-1 efficiently enters the cells of Old World monkeys, it encounters a block before reverse transcription. This species-specific restriction is mediated by a dominant repressive factor, TRIM5α a member of the tripartite motif (TRIM) family of proteins containing RING domains, B boxes, coiled coils and carboxyterminal B30.2 (SPRY) domains [122]. Small differences in the TRIM5α sequence between monkey species may account for the specificity for viral capsid proteins and subsequent breadth of the restriction. However, other cellular factors such as those of the APOBEC family [123] that restrict viral replication in different cell types, seem to have similar activities against HIV and SIV. In this way, comparison of lentiviral/host interactions in different primate species may help us find the clue to HIV-1 pathogenesis in humans. Indeed, natural infection of primates in Africa did not result in the development of AIDS, although chronic infection could be characterized by high viral load and replication rate [124]. African green monkey cells are able to restrict murine leukaemia virus N tropic (MLV-N), HIV-1, HIV-2, SIVmac and equine infectious anemia virus (EIAV), whereas macaque cells strongly restrict HIV-1 only [125]. The role of the SPRY domain sequence in TRIM5α specificity is currently under investigation. In fact, the SPRY domain, also found in the immunoglobulin superfamily, is a hot spot for insertions/deletions and positive selection, which have occurred throughout primate evolution [126]. As it has been demonstrated, using chimeras between the human and rhesus monkey TRIM5 genes [127], that a single amino acid residue controls HIV restriction, NHPs could be valuable models with which to examine the role of TRIM5α polymorphism in intraspecies variability of viral permissivity. Conversely, in addition to potentially providing resistance to animal retrovirus infection, human TRIM5α may limit the usefulness of certain retroviruses, particularly EIAV, as vectors for gene therapy. The use of experimental challenges of immunized NHPs with either SIV or chimeric SIV/HIV to generate preclinical vaccine efficacy data has emerged as an important criterion for facilitating the entry of a given vaccine candidate into early phase, clinical evaluation in humans. Primate models also represent important tools for the study of AIDS and viral pathogenesis, although some limitations should be pointed out. Additional efforts need to be devoted to generate challenge models that more closely recapitulate HIV-1 infection in humans.

For naturally occurring microbial threats, even those related to emerging diseases due to changes in human demographics and behavior, the last stage of vaccine evaluation is to perform human clinical trials from phases I to IV. However, another factor in microbial emergence has attracted an increasing degree of attention: the possible deliberate and criminal release of pathogenic microbes. Of the microbial agents that could be used for bioterrorism, most of them are very rarely (anthrax, plague…) or even no longer observed in humans (smallpox…). This threat requires the protective efficacies of former or new vaccines against highly infectious aerosol exposure, to be evaluated. This prompted the US Food and Drug Administration (FDA) to adopt a regulation that permits the demonstration of efficacy using animal models when efficacy trials in humans are impossible [128]. This is the case for smallpox, which disappeared in the 1980s thanks to the WHO eradication program. The cynomolgus monkey model infected by means of intravenous injection or aerosols of monkeypox virus was used to determine the efficacy of available vaccinia virus vaccines [129], and the screening of new, safer vaccine candidates such as the highly-attenuated, modified vaccinia Ankara (MVA) [130].

Ethical rules regarding the experimental use of NHPs

• – substitute animals of other orders for NHPs as often as possible,
• – only use the species of NHP appropriate to the experimental aim,
• – primate cohorts should be restricted in quantity consistently with statistical analysis.

Primates for experimental research must not be captured from the wild, but must come from accredited breeding facilities. Each primate enrolled in a protocol should be followed as an individual (identification with chips, personal file), and every effort made to enrich its own environment. Researchers must comply with official guidelines for animal care and use. Any protocol must receive the approval of both a scientific review board and an accredited ethics committee.

Conclusion

Acknowledgements

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