Primates Research Articles

Evaluating Pupillometry as a Tool for Assessing Facial and Emotional Processing in Nonhuman Primates

Non-human primates (NHPs) are extensively utilized to investigate the neural mechanisms underlying face processing; however, measuring their brain activity necessitates a diverse array of technologies. Pupillometry emerges as a convenient, cost-effective, and non-invasive alternative for indirectly assessing brain activity. To evaluate the efficacy of pupillometry in assessing facial and emotional processing in NHPs, this study designed a face fixation task for experimental monkeys (Rhesus macaque) and recorded variations in their pupil size in response to face images with differing characteristics, such as species, emotional expression, viewing angles, and orientation (upright vs. inverted). All face images were balanced with luminance and spatial frequency. A sophisticated eye-tracking system (Eye-link 1000 plus) was employed to observe the pupils and track the viewing trajectories of monkeys as they examined images of faces. Our findings reveal that monkeys exhibited larger pupil sizes in response to carnivore faces (versus human faces, p = 0.035), negative conspecific faces (versus human faces, p = 0.018), and profile viewing angles (versus frontal view angles, p = 0.010). Notably, pupil size recorded during the 500–1000 ms post-stimulus interval was negatively correlated with their gaze durations directed at those images (r = −0.357, p = 0.016). Overall, this study demonstrates that pupillometry effectively captures subtle differences in facial and emotional processing, underscoring its potential as a valuable tool in future cognitive research and the diagnosis of disorders.

Immunogenicity and Safety Profile of Two Adjuvanted-PD-L1-Based Vaccine Candidates in Mice, Rats, Rabbits, and Cynomolgus Monkeys

Background: The therapeutic blockade of the PD1/PD-L1 axis with monoclonal antibodies has led to a breakthrough in cancer treatment, as it plays a key role in the immune evasion of tumors. Nevertheless, treating patients with cancer with vaccines that stimulate a targeted immune response is another attractive approach for which few side effects have been observed in combination immunotherapy clinical trials. In this sense, our group has recently developed a therapeutic cancer vaccine candidate called PKPD-L1Vac which contains as an antigen the extracellular domain of human PD-L1 fused to a 47 amino-terminal, part of the LpdA gene of N. meningitides, which is produced in E. coli. The investigation of potential toxicities associated with PD-L1 blockade by a new therapy in preclinical studies is critical to optimizing the efficacy and safety of that new therapy. Methods: Here, we describe immunogenicity and preliminary safety studies in mice, rats, rabbits, and non-human primates that make use of a 200 μg dose of PKPD-L1 in combination with VSSPs or alum phosphate to contribute to the assessment of potential adverse events that are relevant to the future clinical development program of this novel candidate. Results: The administration of PKPD-L1Vac to the four species at the doses studied was immunogenic and did not result in behavioral, clinical, hematological, or serum biochemical changes. Conclusions: Therefore, PKPD-L1Vac could be considered suitable for further complex toxicological studies and the way for its clinical evaluation in humans has been opened.

A Metric-Based, Meta-Analytic Appraisal of Environmental Enrichment Efficacy in Captive Primates

Non-human primates (hereafter ‘primates’) constitute a common group of animals in captivity but their captive maintenance involves ethical and husbandry issues, many of which can be addressed through environmental enrichment (hereafter ‘enrichment’). Enrichment is often applied in a trial-and-error fashion or based on subjective assessments of efficacy. Thus, a predictive framework for enrichment is necessary to ensure it achieves the desired outcomes. As one of the initial steps towards the development of a predictive enrichment science, we aimed to identify the most effective enrichment approaches for captive primates. We conducted a comprehensive meta-analysis of the peer-reviewed literature and extracted information on methodological approaches, experimental design, species and environmental context. We developed and applied a novel efficacy index score to each study protocol as a comparable metric of enrichment efficacy in order to appraise current enrichment practices. Our findings suggest that captive primate enrichment approaches vary in their efficacy, with training-based enrichment being the most effective. Furthermore, the social context of subjects appears to influence efficacy, with primates housed alone deriving the most benefit from enrichment. A species’ natural minimum group size may also influence enrichment efficacy but this relationship requires further investigation. Testing and reporting trends for captive primate enrichment are presented and discussed, highlighting important gaps in the literature and avenues of future research while identifying the beneficial effects of different enrichment practices.

A Novel Model of Traumatic Optic Neuropathy Under Direct Vision Through the Anterior Orbital Approach in Non-human Primates

A Novel Model of Traumatic Optic Neuropathy Under Direct Vision Through the Anterior Orbital Approach in Non-human Primates

Activation of endogenous full-length utrophin by MyoAAV-UA as a therapeutic approach for Duchenne muscular dystrophy

Activation of endogenous full-length utrophin, a dystrophin homolog, presents an attractive therapeutic strategy for Duchenne muscular dystrophy (DMD), regardless of mutation types and loci. However, current dCas9-based activators are too large for efficient adeno-associated virus delivery, and the feasibility and durability of such treatments remain unclear. Here, we develop a muscle-targeted utrophin activation system using the compact dCasMINI-VPR system, termed MyoAAV-UA. Systemic administration of MyoAAV-UA in male mdx mice leads to substantial upregulation of utrophin at the sarcolemma, resulting in significant improvements in skeletal muscle function and a slowing of heart function deterioration. These benefits remain observable at six months post-treatment. In male nonhuman primates, systemic administration of MyoAAV-UA increases utrophin expression by twofold in skeletal muscle, with no significant side effects observed. Furthermore, MyoAAV-UA upregulates utrophin and utrophin-glycoprotein complexes in induced pluripotent stem cell-derived myotubes from DMD patients. In conclusion, these findings demonstrate the potential of MyoAAV-UA as a therapeutic approach for DMD.

Frontostriatal Networks Undergo Functional Specialization During Adolescence that Follows a Ventral-Dorsal Gradient: Developmental Trajectories and Longitudinal Associations

Seminal studies in animal neuroscience demonstrate that frontostriatal circuits exhibit a ventral-dorsal functional gradient to integrate neural functions related to reward processing and cognitive control. Prominent neurodevelopmental models posit that heightened reward-seeking and risk-taking during adolescence result from maturational imbalances between frontostriatal neural systems underlying reward processing and cognitive control. The present study investigated whether the development of ventral (VS) and dorsal (DS) striatal resting-state connectivity (rsFC) networks along this proposed functional gradient relates to putative imbalances between reward and executive systems posited by a dual neural systems theory of adolescent development. 163 participants aged 11-25 years (54% female, 90% white) underwent resting scans at baseline and biennially thereafter, yielding 339 scans across four assessment waves. We observed developmental increases in VS rsFC with brain areas implicated in reward processing (e.g., subgenual cingulate gyrus and medial orbitofrontal cortex) and concurrent decreases with areas implicated in executive function (e.g., ventrolateral and dorsolateral prefrontal cortices). DS rsFC exhibited the opposite pattern. More rapid developmental increases in VS rsFC with reward areas were associated with developmental improvements in reward-based decision making, whereas increases in DS rsFC with executive function areas were associated with improved executive function, though each network exhibited some crossover in function. Collectively, these findings suggest that typical adolescent neurodevelopment is characterized by a divergence in ventral and dorsal frontostriatal connectivity that may relate to developmental improvements in affective decision-making and executive function.Significance StatementAnatomical studies in nonhuman primates demonstrate that frontostriatal circuits are essential for integration of neural functions underlying reward processing and cognition, with human neuroimaging studies linking alterations in these circuits to psychopathology. The present study characterized the developmental trajectories of frontostriatal resting state networks from childhood to young adulthood. We demonstrate that ventral and dorsal aspects of the striatum exhibit distinct age-related changes that predicted developmental improvements in reward-related decision making and executive function. These results highlight that adolescence is characterized by distinct changes in frontostriatal networks that may relate to normative increases in risk-taking. Atypical developmental trajectories of frontostriatal networks may contribute to adolescent-onset psychopathology.

Haemato-biochemical Profile Assessment of Immobilized Wild Rhesus Macaques (Macaca mulatta) in Jabalpur, India

Indian rhesus macaque has been extensively used in biomedical research, particularly in disease studies and drug discovery. Accurate assessment of clinical pathology parameters is crucial for evaluating their health status, particularly in studies involving disease modeling and drug development. However, comprehensive data on hematological and biochemical parameters, especially in wild rhesus macaques, remain limited. Due to increased use of non-human primates in research studies, it is necessary to understand the effect of immobilizing drugs on haemato-biochemical values in wild rhesus macaques. The study focuses on the immobilization of wild adult male rhesus macaques using anesthetic drug combination viz. Inj. Xylazine at the rate of 2mg/kg body weight combined with Inj. Ketamine hydrochloride at the rate of 5mg/kg body weight and their impact on blood parameter estimation. This study has aimed to present an accurate hematological and biochemical values of anesthetized wild Indian rhesus macaques. These findings provide essential baseline reference values for the species and highlight the influence of immobilization on blood parameters on wild adult male rhesus macaques. The mean values of TEC, TLC, PCV and Hb observed in the present study were towards lower side of the normal reference range, however, the mean values of DLC, MCV, MCH and MCHC remained within the normal reference range. This study contributes to the refinement of biomedical research protocols by ensuring accurate health assessments and improving animal welfare in both laboratory and field settings.

Persistent Postirradiation Skeletal Muscle Protein and Insulin Sensitivity Changes in Nonhuman Primates.

Increased incidence of diabetes has been reported after whole-body irradiation in cancer survivors and in the years after exposure in research studies of nonhuman primates. Type 2 diabetes presents in the absence of obesity and suggests that skeletal muscle, the predominant organ responsible for minute-to-minute glucose disposal, is persistently dysfunctional. We evaluated skeletal muscle (SkM) from control (CTL, n = 8) and irradiated (IRRAD, n = 16) male rhesus macaques (Macaca mulatta) that had been exposed to an average whole-body dose of 6.5 Gy after an average of 4 years of follow-up. Irradiated animals had deficient SkM basal and insulin-stimulated receptor activation that was unrelated to histologically assessed fiber size, extracellular matrix and endothelial components. Protein extracted from irradiated muscle showed that Akt2, downstream of insulin receptor activation, was sulfenyl-modified and thus a target for radiation-related glycemic dysregulation. Shotgun proteomics identified upregulation of many mitochondrial and peroxisome-associated proteins, and increases were confirmed by immunoblotting of select protein targets. Proteomic pathway enrichment mapping showed distinct protein clustering between CTL and IRRAD groups. Mitochondrial proteins were surveyed and confirm that mitochondrial turnover may be increased after irradiation with higher fission and fusion markers. The results indicate that irradiated muscle is persistently insulin resistant, with evidence of intracellular protein oxidation and shifts in mitochondrial dynamics and function.

DREDge: robust motion correction for high-density extracellular recordings across species.

High-density microelectrode arrays have opened new possibilities for systems neuroscience, but brain motion relative to the array poses challenges for downstream analyses. We introduce DREDge (Decentralized Registration of Electrophysiology Data), a robust algorithm for the registration of noisy, nonstationary extracellular electrophysiology recordings. In addition to estimating motion from action potential data, DREDge enables automated, high-temporal-resolution motion tracking in local field potential data. In human intraoperative recordings, DREDge's local field potential-based tracking reliably recovered evoked potentials and single-unit spike sorting. In recordings of deep probe insertions in nonhuman primates, DREDge tracked motion across centimeters of tissue and several brain regions while mapping single-unit electrophysiological features. DREDge reliably improved motion correction in acute mouse recordings, especially in those made with a recent ultrahigh-density probe. Applying DREDge to recordings from chronic implantations in mice yielded stable motion tracking despite changes in neural activity between experimental sessions. These advances enable automated, scalable registration of electrophysiological data across species, probes and drift types, providing a foundation for downstream analyses of these rich datasets.

Inhibition of mTORC1 by rapamycin results in feedback activation of Akt S473 and aggravates hallmarks of osteoarthritis in female mice and non-human primates.

Genetic deletion of mTOR has protected against post-traumatic osteoarthritis (OA) in male mice, however, effects of pharmacological mTOR-inhibition are equivocal and have not been tested in aging models nor in female subjects. Therefore, the goal of this study was to determine if mTOR-inhibition by rapamycin can modify OA pathology in aging non-human primates and female mice. Common marmosets were administered oral rapamycin (1mg/kg/day) or vehicle starting near mid-life until death. Five-month-old, female C57BL/6J mice were treated with vehicle or rapamycin (IP, 2mg/kg, 3x/week) for 8-weeks following non-invasive ACL rupture. Knee OA pathology was assessed via microCT and histology. Phosphorylation of mTORC1 (p-RPS6S235/36) and mTORC2 (p-AktS473, p-NDRG1T638, p-PKCαT348) substrates were evaluated via western blot in articular cartilage, meniscus, and/or infrapatellar fat pad. ATDC5 cells were cultured with rapamycin to determine time and dose effects on mTORC1/2 signaling. In marmosets, rapamycin did not impact age-related radiographic OA severity or cartilage pathology but increased medial meniscus calcification and lowered lateral tibia subchondral thickness, particularly in females. In female mice, rapamycin worsened ACLR-induced meniscus calcification and cartilage pathology. In marmoset and mouse joint tissues, rapamycin inhibited mTORC1 and increased p-AktS473 but not p-NDRG1T638 or p-PKCαT348. This mTOR signaling pattern was replicated in ATDC5 cells during exposure to low concentrations of rapamycin. Rapamycin attenuated mTORC1 signaling with feedback activation of AktS473 in articular cartilage, meniscus, and/or infrapatellar fat pad and was accompanied by deleterious effects on meniscus calcification and/or cartilage pathology in female mice and common marmosets. marmosets.

Monitoring mRNA vaccine antigen expression in vivo using PET/CT

Noninvasive visualization of the distribution and persistence of mRNA vaccine antigen expression in mammalian systems has implications for the development and evaluation of future mRNA vaccines. Here, we genetically fuse E. coli dihydrofolate reductase (eDHFR) to the delta furin diproline modified SARS-CoV-2 spike glycoprotein (S2P∆f) mRNA vaccine and image its expression in female mice and male non-human primates using [18F]fluoropropyl-trimethoprim ([18F]FP-TMP). Whole body positron emission tomography (PET) imaging revealed transient expression of the vaccine antigen in the injection site and draining lymph nodes (dLNs). Fusion of eDHFR did not impact S2P immunogenicity and no humoral or cellular immune response was detected against eDHFR in either species. In this work, we show that eDHFR can be used as an mRNA-encoded PET reporter gene to monitor the spatiotemporal dynamics of mRNA vaccine antigen expression in vivo. This technique could be applied in clinical translation of future mRNA vaccines or therapeutics.

Characterizing the Rates and Patterns of De Novo Germline Mutations in the Aye-Aye (Daubentonia madagascariensis).

Given the many levels of biological variation in mutation rates observed to date in primates-spanning from species to individuals to genomic regions-future steps in our understanding of mutation rate evolution will not only be aided by a greater breadth of species coverage across the primate clade but also by a greater depth as afforded by an evaluation of multiple trios within individual species. In order to help bridge these gaps, we here present an analysis of a species representing one of the most basal splits on the primate tree (aye-ayes), combining whole-genome sequencing of seven parent-offspring trios from a three-generation pedigree with a novel computational pipeline that takes advantage of recently developed pan-genome graphs, thereby circumventing the application of (highly subjective) quality metrics that has previously been shown to result in notable differences in the detection of de novo mutations and ultimately estimates of mutation rates. This deep sampling has enabled both a detailed picture of parental age effects and sex dependency in mutation rates, which we here compare with previously studied primates, but has also provided unique insights into the nature of genetic variation in one of the most endangered primates on the planet.

Safety and biocompatibility of a novel biodegradable aflibercept-drug delivery system in rhesus macaques

A clinical need exists for more effective intravitreal (IVT) drug delivery systems (DDS). This study tested the hypothesis that a novel biodegradable, injectable microsphere-hydrogel drug delivery system loaded with aflibercept (aflibercept-DDS) would exhibit long-term safety and biocompatibility in a non-human primate (NHP) model. We generated aflibercept-loaded poly (lactic-co-glycolic acid) microparticles with a modified double emulsion technique then embedded them into a biodegradable, thermo-responsive poly (ethylene glycol)-co-(L-lactic-acid) diacrylate/N-isopropylacrylamide hydrogel. Aflibercept-DDS (50 µL, 15 µg) was injected into the right eye of 23 healthy rhesus macaques. A complete ophthalmic examination, intraocular pressure (IOP), corneal pachymetry, specular microscopy, A-scan biometry, streak retinoscopy, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography (FA), and electroretinography (ERG) were performed monthly. Globes from 7 NHPs were histologically examined. Aflibercept-DDS was visualized in the vitreous up to 9 months post-IVT injection, slightly impeding fundoscopy in 4 of 23 eyes; no other consistent abnormalities were appreciated during ophthalmic examination. The IOP and total retinal thickness remained normal in all animals over all timepoints. Central corneal thickness, endothelial cell density, axial globe length, and refractive error did not significantly differ from baseline. Scotopic mixed rod-cone implicit times and amplitudes along with photopic cone response implicit times and amplitudes did not significantly differ from control values. No retinal or choroidal vascular abnormalities were detected with FA and normal retinal architecture was preserved using SD-OCT. Intravitreal injection of a biodegradable aflibercept-DDS was safe and well tolerated in NHPs up to 24 months.

Development and Assessment of a Multiple-Analysis System for Diagnosing Malaria and Other Blood Parasite Infections in Humans and Non-Human Primates

Background/Objectives: Many tropical diseases such as malaria, Chagas, human African Trypanosomiasis, and Lymphatic filariasis coexist in endemic countries, affecting more than 1 billion people worldwide, and are recognised as major global vector-borne diseases. Tackling this disease requires an accurate diagnosis that is sensitive, specific, and rapid. This study aimed to describe and validate a new highly sensitive and specific multiple-analysis system that can effectively detect numerous etiological agents in a single test. Methods: A total of 230 human blood samples were assessed retrospectively for parasite characterisation, as well as 58 stool samples from non-human primates. Primers and probes were designed in the small subunit ribosomal RNA gene, except for Plasmodium spp., for which the novel target was Cytochrome Oxidase Subunit 1. Results: The analytical specificity of the presented method was 100%, with no unspecific amplifications or cross-reactions with other blood parasitic diseases. The detection limit obtained was between 0.6 and 3.01 parasites/µL for Plasmodium species, 1.8 parasites/mL for Trypanosomatidae, and 2 microfilariae/mL in the case of Filariae. The sensitivity, specificity, predictive values, and kappa coefficient reached almost 100%, except for Filariae, whose sensitivity dropped to 93.9% and whose negative predicted value dropped to 89.5%. The operational features described a turnaround and a hands-on time shorter than the compared methods with a lower cost per essay. Conclusions: This work presents a cost-effective and highly sensitive multiplexed tool (RT-PCR-bp) capable of performing simultaneous detection for blood parasitic diseases using specific fluorescence probes, enabling the diagnosis of low parasite loads and coinfections.

The saponin monophosphoryl lipid A nanoparticle adjuvant induces dose-dependent HIV vaccine responses in non-human primates.

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play a role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (TMem and BMem) and long-lived plasma cells in non-human primates (NHPs). Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L+OX40+ CD4 TMem (11-fold), IFN-γ+ CD4 TMem (15-fold), IL21+ CD4 TMem (9-fold), circulating TFH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in NHPs, which are relevant for human use and next-generation vaccines.

A novel long-acting relaxin-2 fusion, AZD3427, improves cardiac performance in non-human primates with cardiac dysfunction.

Relaxin-2, a well-known human hormone primarily associated with pregnancy, has shown promising cardiovascular benefits in both preclinical models and clinical trials. However, its therapeutic potential has been limited due to the short half-life and the short duration of treatment. To address this, we developed AZD3427, a novel long-acting relaxin-2 analogue and assessed its efficacy during prolonged treatment in a large animal model with cardiac dysfunction. Extensive protein engineering resulted in AZD3427, a novel fusion protein, which closely mimics the natural hormone's structure and consists of a single relaxin-2 and the Fc fragment of human IgG1 to extend its half-life. AZD3427 exhibits an improved pharmacokinetic profile, allowing for weekly or less frequent, subcutaneous dosing, and maintains the pharmacology profile of relaxin-2 with signalling via RXFP1 in cell systems. The effects of chronic RXFP1 agonism with AZD3427 were investigated in a non-human primate (NHP) model with systolic dysfunction and metabolic syndrome. Administration of AZD3427 over a twenty-one-week period led to significant improvements in cardiac function, as evidenced by increased ejection fraction (EF), cardiac output (CO) and stroke volume (SV), as well as reduced systemic vascular resistance (SVR). Importantly, no adverse events related to treatments were observed and there were no concomitant changes in heart rate (HR) or blood pressure (BP). During the eighteen-week washout period, the observed effects gradually disappeared. Prolonged administration of AZD3427, a long-acting relaxin receptor RXFP1 agonist, resulted in remarkable improvement in cardiac function in a NHP model. Findings of this study are an important translational step to developing future therapies and support further clinical development of AZD3427 as a novel treatment for patients with heart failure (HF).

Cortical processing of discrete prosodic patterns in continuous speech

Prosody has a vital function in speech, structuring a speaker’s intended message for the listener. The superior temporal gyrus (STG) is considered a critical hub for prosody, but the role of earlier auditory regions like Heschl’s gyrus (HG), associated with pitch processing, remains unclear. Using intracerebral recordings in humans and non-human primate models, we investigated prosody processing in narrative speech, focusing on pitch accents—abstract phonological units that signal word prominence and communicative intent. In humans, HG encoded pitch accents as abstract representations beyond spectrotemporal features, distinct from segmental speech processing, and outperforms STG in disambiguating pitch accents. Multivariate models confirm HG’s unique representation of pitch accent categories. In the non-human primate, pitch accents were not abstractly encoded, despite robust spectrotemporal processing, highlighting the role of experience in shaping abstract representations. These findings emphasize a key role for the HG in early prosodic abstraction and advance our understanding of human speech processing.

Experimental basis for generating nonhuman primate models of frontotemporal dementia and Alzheimer's disease.

To our knowledge, no reports have described nonhuman primate (NHP) models of frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) that do not depend on an overexpression paradigm. Based on our recent success in generating single human MAPT knock-in mouse models of FTDP-17, we describe the experimental basis for generating knock-in marmoset models of FTDP-17. In addition, successful generation of mutant PSEN1 knock-in marmoset models lacking exon 9 (PSEN1-Δ9) of Alzheimer's disease (AD) indicates that we will be able to reconstitute two major pathological features of AD, i.e., amyloid plaques and neurofibrillary tangles, in an accelerated manner by combining these models.

Comparing a BCI Communication System in a Patient with Multiple System Atrophy, with an animal model.

Comparing a BCI Communication System in a Patient with Multiple System Atrophy, with an animal model.

Systemic delivery of AAV5, AAV8, and AAV9 packaging a C5-12-microdystrophin-FLAG expression cassette in non-human primates.

Systemic delivery of AAV5, AAV8, and AAV9 packaging a C5-12-microdystrophin-FLAG expression cassette in non-human primates.