Special Issue on the Pathobiology of Laboratory Nonhuman Primates: A Review of Species,Substrain,Geographical Origin,Age,and Modality-Related Factors

Abstract

Nonhuman primates (NHPs) are utilized in nonclinical safety testing due to their phylogenetic proximity to humans and similarity in physiology and anatomy. However, ethical considerations and the increased demand for NHPs, coupled with the current shortage in their supply, have increased the calls to minimize their use. In addition, the increased demand and supply shortage of NHPs have increased the use of animals sourced from different geographical origins, and animals of different ages, which can complicate the interpretation of study results. Coupled with the relative uniqueness of findings induced by novel therapeutic modalities, there is an increasing need for a deeper understanding of the systemic pathobiology of NHPs. Here we provide a brief preview of the two main themes discussed in this special issue, which include the influence of geographical origin, age, and sex on background pathology, clinical pathology reference values, other relevant toxicology endpoints, and organ system pathology.

The laboratory nonhuman primate (NHP) continues to play a crucial role in the discovery and development of biopharmaceutical and vaccines, and model development for many human disease conditions in basic research. ¹ In nonclinical development of biopharmaceuticals such as monoclonal antibodies, NHPs are often the only pharmacologically relevant species, and the rapidly increasing number of these molecules in development has resulted in increased use of NHP. ² However, ethical considerations and recent efforts to reduce the use of NHPs, coupled with the current shortage in the supply of laboratory NHPs, brought about by increased NHP usage for vaccine and drugs development during the COVID-19 pandemic, have amplified calls to explore and implement science-driven ways for more effective use of animals and to maximize the knowledge gained from animal studies using the fewest numbers of animals possible. ² Among the proposed opportunities to reduce NHP use in nonclinical toxicity assessment are using fewer animals per dose group, re-use of recovery animals, combining studies on a drug development program (e.g., introducing toxicology endpoints in pharmacology studies), and reducing the use of or reliance on concurrent control animals (particularly in the recovery studies), which can be accomplished by sharing of historical control data (HCD) or the development of a control database of NHP biological data and background pathology. ² These measures, though ethical and principled, may complicate the interpretation of study results on occasion, and coupled with the relative uniqueness of findings associated with novel engineered biotherapeutic compounds, and the increasing demand for the characterization of immuno-toxicologic effects associated with biologics, toxicologic pathologists are faced with a greater need for deeper understanding of background pathobiology (normal organ weights, anatomy, and common background lesions) and clinical pathology of laboratory NHPs and how to identify and characterize test article-induced lesions.

In addition, the shortage of NHPs largely created by the cessation or reduction of NHP exports from mainland China in 2020 has led importers of NHPs to expand and diversify their supply chain, by increased utilization of cynomolgus macaques from other locations, such as Cambodia, Vietnam, and Mauritius, among others. This in turn increases the likelihood of using animals sourced from various geographical regions, or animals of varying age groups on a particular study or during the course of a development program, which can further complicate the interpretation and integration of results across studies, as considerable genetic and environmental variability associated with an animal’s geographic origin has the potential to affect pre-study baseline and reference data.

In an attempt to address some of these challenges, the NHP Special Interest Group of the Society of Toxicologic Pathology coordinated an effort to publish a special issue of Toxicologic Pathology focused on the pathobiology of the laboratory NHP. The primary objective of this publication is to address this topical issue of the possible influence of geographical origin or substrain, age and sex of laboratory NHP on the biology, background pathology, clinical pathology reference values, organ weights, and other relevant toxicological endpoints. It is envisaged that this effort will serve as a basis for the creation of comprehensive and up-to-date HCD databases for NHPs that are sensitive to the animal’s geographical origin, sex, and age. A second objective of this endeavor is to increase the depth of available knowledge on the systemic pathobiology of laboratory NHPs, by incorporating subject expert–authored review articles on spontaneous and modality-related organ system pathology. It is anticipated that an in-depth knowledge and understanding of the normal biology, background changes, and test article-induced pathology in the NHP will enhance the more efficient and responsible use of NHPs, by reducing animal wastage by maximizing and facilitating study data interpretation from the fewest number of animals possible. This Special Issue is organized into two sections based on the two main themes encompassed in these objectives: the influence of origin, age, and sex variability in toxicity studies with NHPs, and organ system pathology.

The first part of the special issue discusses the topic of genetic diversity and geographical origin of cynomolgus macaques, the most commonly used laboratory NHP in nonclinical safety testing, and presents results from several surveys and investigations on the influence of geographical origin on background pathology, clinical pathology reference values, and other toxicological endpoints, such as organ weights and reproductive parameters. It is now well-recognized that captive stocks of cynomolgus macaques are derived from at least one of the three following subpopulations: (1) Native Indochinese or mainland macaques, alternatively referred as “continental,” which originate from Thailand, Cambodia, or Vietnam, but are frequently captive-bred in China (cynomolgus macaques are not native to China), and have higher genetic variability; (2) insular Asian macaques from the region south of the Isthmus of Kra into insular Asia and Philippines, which are more homogeneous compared with Indochinese macaques; and (3) non-native cynomolgus macaques from the Island of Mauritius, which have a very low genetic diversity when compared with native Asian macaques.^3-6 The comparatively poor genetic polymorphism in macaques from Mauritius is believed to represent a more inbred population of macaques, while the high genetic diversity in Indochinese or mainland macaques is believed to be due, at least in part, to the gene flow from rhesus to cynomolgus macaques. Hence, cynomolgus macaques from China, Vietnam, or Cambodia share certain genetic variations with rhesus macaques, while cynomolgus macaques derived from Insular Malaysia and Philippines, and Mauritian macaques are free of the genetic influx from rhesus.^3,4,7 As a result of these differences in genetic variation, animals derived from each of these geographical origins have been reported to demonstrate distinct differences in clinical pathology parameters, response to pathogens, response to vaccines, differences in microbial flora, as well as differences in spontaneous pathology findings.^6,8

An investigation into the influence of geographical origin on several selected toxicology study endpoints indicated that mainland Asian animals from different countries such as China, Cambodia, and Vietnam do not appear to demonstrate any substantive differences in toxicologic endpoints, while NHPs from Mauritius have consistent differences from Asian animals in several of the typical study endpoints.^8,9,10,11 The absence of major relevant baseline differences between cynomolgus macaques sourced from the mainland Asia (also known as continental) provides evidence to support the interchangeable use of animals from these geographical regions within a development program. However, while animals from Mauritius and mainland Asia were shown to be comparable for developmental and reproductive parameters, NHPs from Mauritius appeared to mature faster, reaching sexual maturity earlier than Asia mainland animals, ¹¹ and attaining comparably higher body weights ¹⁰ relative to their mainland Asia counterparts. The higher body weight in Mauritian animals correlated with higher absolute organ weights in this subpopulation, when compared with Asian mainland macaques. A further analysis of body and organ weights in relation to age and sex in cynomolgus monkeys demonstrated that males tend to have higher body weight gains than females that lasted into adulthood (defined as 9.5-20 years), whereas females have even less body weight gain after young adulthood (4-9.5 years). ¹² For most organs, males also had higher absolute organ weights than female, whereas females had higher relative ratios of organ weights to body weights.

Origin-associated differences were also observed in the incidence of spontaneous pathology and clinical pathology reference values between Mauritian Island cynomolgus macaques and mainland Asian animals. ¹³ Chamanza et al. (2022) ¹⁴ carried out what is as of to date the largest, multisite and multi-continental, comparative analysis of the influence of geographical origin, age, sex, and animal husbandry practices on the spontaneous pathology of Asian (Chinese, Vietnamese, and Cambodian) and Mauritian macaques. Their findings were largely in alignment with those reported for other toxicology endpoints. Differences in the incidence of background inflammatory lesions were observed between Chinese cynomolgus macaques and those from Mauritius, while those from Vietnam and Cambodia were comparable to those from China. However, contrary to previous reports of overall higher incidences of background pathology findings in Asian macaques, when compared with those from Mauritius,^8,15 the authors observed organ-dependent increases in both groups, with Mauritian macaques showing higher incidences of inflammatory findings in the liver, brain, lung, eye, and skin. Conversely, Chinese macaques had higher incidences of inflammatory cell infiltrates in the gastrointestinal tract (GIT), gall bladder, and some salivary glands. ¹⁴ The authors concluded that the overall incidence of background pathology findings, the nature of the most common findings, and most commonly affected tissues were comparable among animals from the different geographical regions and that animals from all the geographical regions were suitable for use in toxicity testing. Routine clinical pathology parameters from cynomolgus macaques of mainland Asia origin (China, Vietnam, and Cambodia) and those from Mauritius were also compared. ¹³ In this study, which included animals from regulated toxicology studies conducted in North American and European Union testing facilities, authors identified and reported a few notable differences in hematology endpoints and several minor differences in serum biochemistry parameters and coagulation test results. The small magnitude of these differences provides support for the inclusion of different origins of macaques in a drug development program, as long as the testing facilities maintain separate, origin-specific clinical pathology reference intervals. Similar recommendations were presented for histopathology HCD. The overall conclusions and recommendations from the first section suggest that while animals from different geographical origins can be used within a drug development program, origin-related differences in toxicological and anatomic and clinical pathology endpoints need to be considered when interpreting individual study data to avoid erroneous data interpretation and that distinct geographical origin-based and age-specified HCD or reference values also be used. Within individual studies, it is strongly recommended to use macaques of the same origin and ideally across a drug development program, recognizing that the latter consideration is not always possible. Part 1 ends with a comprehensive presentation of frequently encountered spontaneous diseases in the common marmoset as well as aspects of basic husbandry and biology along with animal model applications of the marmoset in biomedical research. ¹⁶

The second part of the special issue discusses organ system toxicity in various species of NHPs, in particular, organ system responses to biotherapeutics. One of the consequences of the increased demand for NHPs for SARS-CoV-19 vaccine research and development has been the incorporation of more varied cohorts of macaques in studies, including animals from different sources, wider age ranges, and, in some cases, increased exposure to naturally occurring pathogens, leading to an increased frequency of clinical problems, particularly gastrointestinal (GI) diseases. Diarrhea and the attendant GI diseases are the most frequent spontaneous findings in both rhesus and cynomolgus macaques of all origins and ages. As such, pathologists are frequently called upon to differentiate spontaneously occurring diarrhea from a test article-related effect. ¹⁷ Bringing together a wealth of experience and expertise from the national primate centers, other captive macaque colonies, and commercial toxicologic pathology research centers, this review provides pathologists, toxicologists, and researchers with a comprehensive overview of the main features of GI lesions, which can help determine the etiology, and include or exclude any potential relationship to a test article. Spontaneous findings in the male reproductive system of sexually mature cynomolgus macaques may also present a challenge to pathologists unfamiliar with this age group as there is a paucity of information on this subject. The review by Vidal et al. ¹¹ provides comprehensive descriptions of common microscopic findings in the male reproductive system of sexually mature NHPs, with a brief discussion on the comparative incidence of these findings based on body weights and geographic origin. Skeletal abnormalities in laboratory macaques are discussed in a report based on a series of cases investigating coxofemoral joint abnormalities that have a similar presentation to idiopathic femoral head chondrolysis, ¹⁸ while a case of pseudoreticulocytosis in a cynomolgus macaque with malaria draws attention to the clinical pathology presentation of this plasmodial disease, demonstrating that erroneously high reticulocyte counts may occur in cases with high parasitic burden when evaluated by hematology analyzers utilizing fluorescence measurement. ¹⁹

Two articles then discuss organ system responses of NHPs to emerging biotherapeutic modalities. In the first from the Special Interest Group in Neuropathology (SIGN) of the Society of Toxicologic Pathology (STP), an in-depth discussion of neuropathologic findings associated with the administration of candidate biotherapeutics (nucleic acids, proteins, viral gene therapy vectors, etc.) for treatment of nervous system disorders is presented. ²⁰ Pathologists evaluating and interpreting neuropathology findings related to biomolecule-based test articles must be proficient in recognizing tissue alterations common to administration procedures of biomolecules, and to also be able to distinguish direct test article–induced effects from any procedure-related changes and incidental background findings. The authors provide a practical guide to diagnosing and interpreting common biomolecule-induced findings in neural tissues of NHPs, as well as key new information on the neuropathology associated by some novel and innovative classes of neurotherapeutics. ²⁰ The next paper in this section on organ system response to biopharmaceuticals is an original research article that describes differential immunosuppressive effects induced by a monoclonal antibody construct associated with secondary infections in rhesus macaques. ²¹ The challenges associated with separating primary findings related to the intended pharmacology of an immunomodulating therapeutic antibody in lymphoid organs from alterations associated with secondary infections due to the expected immunosuppression are outlined. The final article in this section is a pathology-focused compilation of current Internet and print resources that may be used to assist in the pathology evaluation of NHPs utilized in nonclinical safety testing. ²² This mini review presents a readily available list of essential books, journal articles, and websites that provide context to safety assessment and research scientists working with NHP models.

The Special Issue editors hope that this compilation of articles will prove to be a valuable addition to the existing knowledge on the pathobiology of NHP and enhance our understanding of the influence of geographical origin, age, and sex on pathology, clinical pathology, and other study endpoints. Finally, although the present compilation of articles was not specifically intended to address the issue of NHP shortages, it is hoped that they will stimulate meaningful discussion toward a more efficient and responsible use of NHPs and serve as a catalyst for the creation of HCD databases for NHPs, which are specific to the animal’s geographical origin, sex, and age.