Preclinical evaluation of PHH-1V vaccine candidate against SARS-CoV-2 in non-human primates.

SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programs, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralizing antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques.

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.

Comparison of high-resolution magnetic resonance imaging and micro-computed tomography arthrography for in-vivo assessment of cartilage in non-human primate models.

BACKGROUND: Non-human primate (NHP) could be an interesting model for osteoarthritis (OA) longitudinal studies but standard medical imaging protocols are not able to acquire sufficiently high-resolution images to depict the thinner cartilage (compared to human) in an in vivo context. The aim of this study was thus to develop and validate the acquisition protocols for knee joint examination of NHP using magnetic resonance imaging (MRI) at 1.5 T and X-ray micro-computed tomography arthrography (µCTA). METHODS: The first phase of the study focused on developing dedicated in vivo HR-MRI and µCTA protocols for simultaneous acquisitions of both knee joints on NHP. For MR, a dedicated two-channel receiver array coil and acquisition sequence were developed on a 1.5 T Siemens Sonata system and tuned to respect safety issues and reasonable examination time. For µCTA, an experimental setup was devised so as to fulfill similar requirements. The two imaging protocols were used during a longitudinal study so as to confirm that repeated injections of loxaglic acid (contrast agent used for µCTA) didn't induce any bias in cartilage assessment and to compare segmentation results from the two modalities. Lateral and medial cartilage tibial plateaus were assessed using a common image processing protocol leading to a 3D estimation of the cartilage thickness. RESULTS: From HR-MRI and µCTA images, thickness distributions were extracted allowing for proper evaluation of knee cartilage thickness of the primates. Results obtained in vivo indicated that the µCTA protocol did not induce any bias in the measured cartilage parameters and moreover, segmentation results obtained from the two imaging modalities were consistent. CONCLUSIONS: MR and µCTA are valuable imaging tools for the morphological evaluation of cartilage in NHP models which in turn can be used for OA studies.

Cortical inflammation and brain signs of high-risk atherosclerosis in a non-human primate model.

Atherosclerosis is a chronic systemic inflammatory disease, inducing cardiovascular and cerebrovascular acute events. A role of neuroinflammation is suspected, but not yet investigated in the gyrencephalic brain and the related activity at blood-brain interfaces is unknown. A non-human primate model of advanced atherosclerosis was first established using longitudinal blood samples, multimodal imaging and gene analysis in aged animals. Non-human primate carotid lesions were compared with human carotid endarterectomy samples. During the whole-body imaging session, imaging of neuroinflammation and choroid plexus function was performed. Advanced plaques were present in multiple sites, premature deaths occurred and downstream lesions (myocardial fibrosis, lacunar stroke) were present in this model. Vascular lesions were similar to in humans: high plaque activity on PET and MRI imaging and systemic inflammation (high plasma C-reactive protein levels: 42 ± 14 µg/ml). We also found the same gene association (metabolic, inflammatory and anti-inflammatory markers) as in patients with similar histological features. Metabolic imaging localized abnormal brain glucose metabolism in the frontal cortex. It corresponded to cortical neuro-inflammation (PET imaging) that correlated with C-reactive protein level. Multimodal imaging also revealed pronounced choroid plexus function impairment in aging atherosclerotic non-human primates. In conclusion, multimodal whole-body inflammation exploration at the vascular level and blood-brain interfaces identified high-risk aging atherosclerosis. These results open the way for systemic and central inflammation targeting in atherosclerosis in the new era of immunotherapy.

Repair of full-thickness articular cartilage defects using IEIK13 self-assembling peptide hydrogel in a non-human primate model.

Articular cartilage is built by chondrocytes which become less active with age. This declining function of the chondrocytes, together with the avascular nature of the cartilage, impedes the spontaneous healing of chondral injuries. These lesions can progress to more serious degenerative articular conditions as in the case of osteoarthritis. As no efficient cure for cartilage lesions exist yet, cartilage tissue engineering has emerged as a promising method aiming at repairing joint defects and restoring articular function. In the present work, we investigated if a new self-assembling peptide (referred as IEIK13), combined with articular chondrocytes treated with a chondrogenic cocktail (BMP-2, insulin and T3, designated BIT) could be efficient to restore full-thickness cartilage defects induced in the femoral condyles of a non-human primate model, the cynomolgus monkey. First, in vitro molecular studies indicated that IEIK13 was efficient to support production of cartilage by monkey articular chondrocytes treated with BIT. In vivo, cartilage implant integration was monitored non-invasively by contrast-enhanced micro-computed tomography, and then by post-mortem histological analysis and immunohistochemical staining of the condyles collected 3 months post-implantation. Our results revealed that the full-thickness cartilage injuries treated with either IEIK13 implants loaded with or devoid of chondrocytes showed similar cartilage-characteristic regeneration. This pilot study demonstrates that IEIK13 can be used as a valuable scaffold to support the in vitro activity of articular chondrocytes and the repair of articular cartilage defects, when implanted alone or with chondrocytes.

A non-human primate model of stroke reproducing endovascular thrombectomy and allowing long-term imaging and neurological read-outs.

Stroke is a devastating disease. Endovascular mechanical thrombectomy is dramatically changing the management of acute ischemic stroke, raising new challenges regarding brain outcome and opening up new avenues for brain protection. In this context, relevant experiment models are required for testing new therapies and addressing important questions about infarct progression despite successful recanalization, reversibility of ischemic lesions, blood-brain barrier disruption and reperfusion damage. Here, we developed a minimally invasive non-human primate model of cerebral ischemia (Macaca fascicularis) based on an endovascular transient occlusion and recanalization of the middle cerebral artery (MCA). We evaluated per-occlusion and post-recanalization impairment on PET-MRI, in addition to acute and chronic neuro-functional assessment. Voxel-based analyses between per-occlusion PET-MRI and day-7 MRI showed two different patterns of lesion evolution: "symptomatic salvaged tissue" (SST) and "asymptomatic infarcted tissue" (AIT). Extended SST was present in all cases. AIT, remote from the area at risk, represented 45% of the final lesion. This model also expresses both worsening of fine motor skills and dysexecutive behavior over the chronic post-stroke period, a result in agreement with cortical-subcortical lesions. We thus fully characterized an original translational model of ischemia-reperfusion damage after stroke, with consistent ischemia time, and thrombus retrieval for effective recanalization.

PET-MRI nanoparticles imaging of blood-brain barrier damage and modulation after stroke reperfusion.

In an acute ischaemic stroke, understanding the dynamics of blood-brain barrier injury is of particular importance for the prevention of symptomatic haemorrhagic transformation. However, the available techniques assessing blood-brain barrier permeability are not quantitative and are little used in the context of acute reperfusion therapy. Nanoparticles cross the healthy or impaired blood-brain barrier through combined passive and active processes. Imaging and quantifying their transfer rate could better characterize blood-brain barrier damage and refine the delivery of neuroprotective agents. We previously developed an original endovascular stroke model of acute ischaemic stroke treated by mechanical thrombectomy followed by positron emission tomography-magnetic resonance imaging. Cerebral capillary permeability was quantified for two molecule sizes: small clinical gadolinium Gd-DOTA (<1 nm) and AGuIX® nanoparticles (∼5 nm) used for brain theranostics. On dynamic contrast-enhanced magnetic resonance imaging, the baseline transfer constant K trans was 0.94 [0.48, 1.72] and 0.16 [0.08, 0.33] ×10-3 min-1, respectively, in the normal brain parenchyma, consistent with their respective sizes, and 1.90 [1.23, 3.95] and 2.86 [1.39, 4.52] ×10-3 min-1 in choroid plexus, confirming higher permeability than brain parenchyma. At early reperfusion, K trans for both Gd-DOTA and AGuIX® nanoparticles was significantly higher within the ischaemic area compared to the contralateral hemisphere; 2.23 [1.17, 4.13] and 0.82 [0.46, 1.87] ×10-3 min-1 for Gd-DOTA and AGuIX® nanoparticles, respectively. With AGuIX® nanoparticles, K trans also increased within the ischaemic growth areas, suggesting added value for AGuIX®. Finally, K trans was significantly lower in both the lesion and the choroid plexus in a drug-treated group (ciclosporin A, n = 7) compared to placebo (n = 5). K trans quantification with AGuIX® nanoparticles can monitor early blood-brain barrier damage and treatment effect in ischaemic stroke after reperfusion.

Regeneration of the cementum and periodontal ligament using local BDNF delivery in class II furcation defects.

Periodontal furcation defects are usually addressed by the placement of a physical barrier which may limit the regenerative potential of periodontal wounds. This study morphometrically quantified the regenerative effect of brain-derived neurotrophic factor (BDNF) in furcation defects in a non-human primate model. Grade II furcation defects (with and without induced inflammation prior to surgery) were created on the first and second molars of eight non-human primates. Defects were treated with open flap debridement and subsequently filled with either: Group A; BDNF (500 µg mL-1 ) in high-molecular weight-hyaluronic acid (HMW-HA), Group B; BDNF (50 µg mL-1 ) in HMW-HA, Group C; HMW-HA acid only, Group D; unfilled defect, or Group E; BDNF (500 µg mL-1 ) in saline. Periodontal wound healing was observed every 2 weeks by computed-tomography. At 11 weeks all animals were sacrificed and maxillary and mandibular block biopsies were referred for nondecalcified histology. Linear measurements of new cementum (cellular and acellular) and periodontal ligament (PDL) formation were performed. Computerized-tomography reconstruction and software quantification demonstrated successful bone fill for all groups. However, histometric assessment demonstrated significantly higher level of total periodontal regeneration for the 500 µg mL-1 BDNF HMW-HA relative to all other groups. No significant differences in cementogenesis were observed among groups. Significantly higher acellular cementum formation was observed for sites where inflammation was not induced prior to surgical procedures. While all groups experienced similar bone fill and cementogenesis, the 500 µg mL-1 BDNF HMW-HA appeared to most effectively repair PDL (minimum increase of ∼22% relative to all groups; over 200% relative to unfilled defects). © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1611-1617, 2018.

Microdialysis in awake macaque monkeys for central nervous system pharmacokinetics.

BACKGROUND: The brain bioavailability of novel small molecules developed to address central nervous system disease is classically documented through ex vivo or in vivo analyses conducted in rodent models. Data acquired in rodent models are, however, not easily transferrable to human as the pharmacokinetic and pharmacodynamics profiles of the species are quite different. METHODS: Using drugs selected for their differential transport across the blood-brain barrier, we here demonstrate the feasibility of brain microdialysis in normal vigil macaque monkey by measuring brain extracellular fluid bioavailability of carbamazepine, digoxin, oxycodone, and quinidine. RESULTS: All drugs, but digoxin, were found in dialysate samples. Drugs that are substrate of P-glycoprotein show a difference of bioavailability or brain pharmacokinetic parameters between rodents and primates. CONCLUSION: Data suggest that brain microdialysis in vigil macaque monkey, the species of choice for classic pharmacokinetic/pharmacodynamics studies could help predicting human brain bioavailability of a small molecule depending on the protein involved in the efflux transport from the brain.

Safety Evaluation and Imaging Properties of Gadolinium-Based Nanoparticles in nonhuman primates.

In this article, we report the safety evaluation of gadolinium-based nanoparticles in nonhuman primates (NHP) in the context of magnetic resonance imaging (MRI) studies in atherosclerosis bearing animals and healthy controls. In healthy NHP, the pharmacokinetics and toxicity profiles demonstrated the absence of dose, time, and sex-effects, as well as a suitable tolerance of intravenous administration of the nanoparticles. We investigated their imaging properties for arterial plaque imaging in a standard diet or a high cholesterol diet NHP, and compared their characteristics with clinically applied Gd-chelate. This preliminary investigation reports the efficient and safe imaging of atherosclerotic plaques.

Permeability of blood-brain barrier in macaque model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson disease.

Brain bioavailability of drugs developed to address central nervous system diseases is classically documented through cerebrospinal fluid collected in normal animals, i.e., through an approximation as there are fundamental differences between cerebrospinal fluid and tissue contents. The fact that disease might affect brain availability of drugs is almost never considered at this stage although several conditions are associated with blood-brain barrier damage. Building upon our expertise in Parkinson's disease translational research, the present study addressed this gap comparing plasma and cerebrospinal fluid bioavailability of l-3,4-dihydroxyphenylalanine, carbamazepine, quinidine, lovastatin, and simvastatin, in healthy and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated macaques, the gold standard model of Parkinson's disease. The drugs were selected based upon their differential transport across the blood-brain barrier. Interestingly, brain bioavailability of quinidine was decreased while others were unaffected. Pharmacokinetics and pharmacodynamics experiments of drugs addressing Parkinson's disease might thus be performed in healthy animals unless the drugs are known to interact with the organic cation transporter.

Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors.

There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections.

Differential Interaction of the Staphylococcal Toxins Panton-Valentine Leukocidin and γ-Hemolysin CB with Human C5a Receptors.

Staphylococcus aureus is well adapted to the human host. Evasion of the host phagocyte response is critical for successful infection. The staphylococcal bicomponent pore-forming toxins Panton-Valentine leukocidin LukSF-PV (PVL) and γ-hemolysin CB (HlgCB) target human phagocytes through interaction with the complement receptors C5aR1 and C5aR2. Currently, the apparent redundancy of both toxins cannot be adequately addressed in experimental models of infection because mice are resistant to PVL and HlgCB. The molecular basis for species specificity of the two toxins in animal models is not completely understood. We show that PVL and HlgCB feature distinct activity toward neutrophils of different mammalian species, where activity of PVL is found to be restricted to fewer species than that of HlgCB. Overexpression of various mammalian C5a receptors in HEK cells confirms that cytotoxicity toward neutrophils is driven by species-specific interactions of the toxins with C5aR1. By taking advantage of the species-specific engagement of the toxins with their receptors, we demonstrate that PVL and HlgCB differentially interact with human C5aR1 and C5aR2. In addition, binding studies illustrate that different parts of the receptor are involved in the initial binding of the toxin and the subsequent formation of lytic pores. These findings allow a better understanding of the molecular mechanism of pore formation. Finally, we show that the toxicity of PVL, but not of HlgCB, is neutralized by various C5aR1 antagonists. This study offers directions for the development of improved preclinical models for infection, as well as for the design of drugs antagonizing leukocidin toxicity.

Evidence for an intranasal immune response to human respiratory syncytial virus infection in cynomolgus macaques.

There is no large-scale therapy available against human respiratory syncytial virus (hRSV), a major pathogen responsible for acute respiratory diseases. Macaques represent an interesting animal model to evaluate potential treatments because of their genetic, anatomical and immunological proximity with humans. However, the parameters that influence hRSV growth and control in this model are still poorly understood. We have documented in the following study the influence of age as well as repeated infections on the virological, clinical and immunological parameters of this animal model. Following intranasal inoculation, hRSV replicated in the upper respiratory tract for less than 15 days with no clinical signs regardless of age. Interestingly, we observed the induction of a local immune response at the nasal mucosa as assessed by expression profiles of inflammatory and IFN-stimulated genes. Animals also developed specific antibodies and were immune to reinfection. Thus, we showed that even in infant macaques, intranasal hRSV infection induced both local and systemic immune responses to efficiently control the virus.

Repeated intravenous injections in non-human primates demonstrate preclinical safety of an anti-inflammatory phosphorus-based dendrimer.

Dendrimers are nanosized hyperbranched polymers synthesized through an iterative step-by-step process; their size and structure are perfectly controlled, and they are widely used for biomedical purposes. Previously, we showed that a phosphorous-based dendrimer capped with anionic AzaBisPhosphonate groups (so-called ABP dendrimer) has immunomodulatory and anti-inflammatory properties toward the human immune system. It dramatically inhibits the onset and development of experimental arthritis in a mouse model relevant for human rheumatoid arthritis, a chronic inflammatory disease of auto-immune origin. In this article, we demonstrate in an unprecedented study that cynomolgus macaques repeatedly injected with the ABP dendrimer displayed no adverse response. Indeed, biochemical, haematological, clotting and immunological parameters remained with a normal physiological range during the study. Moreover, quantification of serum cytokines and histopathological analyses failed to reveal any noticeable lesion or noteworthy non-physiological occurrence. These results strengthen the potential of the ABP dendrimer as an innovative drug-candidate for the treatment of inflammatory diseases and favor the regulatory preclinical development of the molecule.

Measles vaccine expressing the secreted form of West Nile virus envelope glycoprotein induces protective immunity in squirrel monkeys, a new model of West Nile virus infection.

West Nile virus (WNV) is a mosquito-borne flavivirus that emerged in North America and caused numerous cases of human encephalitis, thus urging the development of a vaccine. We previously demonstrated the efficacy of a recombinant measles vaccine (MV) expressing the secreted form of the envelope glycoprotein from WNV to prevent WNV encephalitis in mice. In the present study, we investigated the capacity of this vaccine candidate to control WNV infection in a primate model. We first established experimental WNV infection of squirrel monkeys (Saimiri sciureus). A high titer of virus was detected in plasma on day 2 after infection, and viremia persisted for 5 days. A single immunization of recombinant MV-WNV vaccine elicited anti-WNV neutralizing antibodies that strongly reduced WNV viremia at challenge. This study demonstrates for the first time the capacity of a recombinant live attenuated measles vector to protect nonhuman primates from a heterologous infectious challenge.

A minimally-invasive closed chest myocardial occlusion-reperfusion model in rhesus monkeys (Macaca mulatta): monitoring by contrast-enhanced ultrasound imaging.

Myocardial infarction is frequently developed in canine and porcine models but exceptionally in non-human primates. The aim of this study was to develop a minimally invasive myocardial ischemic/reperfusion model in the monkey intended to be combined with imaging techniques, in particular myocardial contrast echocardiography (MCE). A balloon-tipped catheter was advanced via the femoral artery into the left anterior descending artery (LAD) under fluoroscopic guidance in ten anaesthetized male rhesus monkeys (Macaca mulatta). The balloon was inflated to completely occlude the vessel. Coronary angiography (CA) was performed to control the reality of the LAD occlusion/reperfusion. The ischemia period was followed by 3-6 h of reperfusion. Myocardial perfusion was evaluated during ischemia and at reperfusion by MCE using a novel ultrasound contrast agent (BR38). Occlusion was successfully induced during 18-50 min in nine out of the ten evaluated monkeys. ST segment elevation indicated myocardial ischemia. MCE showed complete transmural arrest of myocardial blood flow during the ischemia period and no persistent microvascular perfusion defects during reperfusion. A minimally invasive closed-chest model was successfully developed for creating myocardial ischemia in the rhesus monkey (Macaca mulatta). This technique could have an important role in mimicking acute coronary syndrome under physiologically and ethically-acceptable conditions. MCE provides non-invasively information on myocardial perfusion status, information not available from CA.

Neuroanatomical study of the A11 diencephalospinal pathway in the non-human primate.

BACKGROUND: The A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP. METHODS AND FINDINGS: In situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group. CONCLUSION: The diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells.

Experimental infection of squirrel monkeys with nipah virus.

We infected squirrel monkeys (Saimiri sciureus) with Nipah virus to determine the monkeys' suitability for use as primate models in preclinical testing of preventive and therapeutic treatments. Infection of squirrel monkeys through intravenous injection was followed by high death rates associated with acute neurologic and respiratory illness and viral RNA and antigen production.

Phylogeny and phylogeography of squirrel monkeys (genus Saimiri) based on cytochrome b genetic analysis.

Squirrel monkeys (genus Saimiri) are distributed over a wide area encompassing the Amazon Basin: French Guiana, Suriname, and Guyana, together with Western Panama and Western Costa Rica. The genus Saimiri includes a complex of species and subspecies displaying considerable morphological variation. Taxonomic and systematic studies have identified, in this genus, one to seven species comprising up to 16 subspecies. The phylogenetic relationships between these taxa are poorly understood. Molecular markers have yielded a consistent framework for the systematics of Central and South American Saimiri, identifying four distinct clades: S. oerstedii, S. sciureus, S. boliviensis, and S. ustus. Here, we reconsider the phylogenetic and biogeographic history of Saimiri on the basis of mitochondrial (mtDNA) sequence data, focusing mostly on individuals originating from the Amazon Basin. We studied 32 monkeys with well-defined geographic origins and inferred the phylogenetic relationships between them on the basis of full-length cytochrome b gene nucleotide sequences. The high level of gene diversity observed (0.966) is consistent with the high level of behavioral and morphological variation observed across the geographic range of the genus: 20 mtDNA haplotypes were identified with a maximum divergence of 4.81% between S. b. boliviensis and S. ustus. In addition to confirming the existence of the four clades previously identified on the basis of molecular characters, we suggest several new lineages, including S. s. macrodon, S. s. albigena, S. s. cassiquiarensis, and S. s. collinsi. We also propose new patterns of dispersion and diversification for the genus Saimiri, and discuss the contribution of certain rivers and forest refuges to its structuring.