Early administration of tecovirimat shortens the time to mpox clearance in a model of human infection.

Despite use of tecovirimat since the beginning of the 2022 outbreak, few data have been published on its antiviral effect in humans. We here predict tecovirimat efficacy using a unique set of data in nonhuman primates (NHPs) and humans. We analyzed tecovirimat antiviral activity on viral kinetics in NHP to characterize its concentration-effect relationship in vivo. Next, we used a pharmacological model developed in healthy volunteers to project its antiviral efficacy in humans. Finally, a viral dynamic model was applied to characterize mpox kinetics in skin lesions from 54 untreated patients, and we used this modeling framework to predict the impact of tecovirimat on viral clearance in skin lesions. At human-recommended doses, tecovirimat could inhibit viral replication from infected cells by more than 90% after 3 to 5 days of drug administration and achieved over 97% efficacy at drug steady state. With an estimated mpox within-host basic reproduction number, R0, equal to 5.6, tecovirimat could therefore shorten the time to viral clearance if given before viral peak. We predicted that initiating treatment at symptom onset, which on average occurred 2 days before viral peak, could reduce the time to viral clearance by about 6 days. Immediate postexposure prophylaxis could not only reduce time to clearance but also lower peak viral load by more than 1.0 log10 copies/mL and shorten the duration of positive viral culture by about 7 to 10 days. These findings support the early administration of tecovirimat against mpox infection, ideally starting from the infection day as a postexposure prophylaxis.

Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates.

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.

Modeling the kinetics of the neutralizing antibody response against SARS-CoV-2 variants after several administrations of Bnt162b2.

Because SARS-CoV-2 constantly mutates to escape from the immune response, there is a reduction of neutralizing capacity of antibodies initially targeting the historical strain against emerging Variants of Concern (VoC)s. That is why the measure of the protection conferred by vaccination cannot solely rely on the antibody levels, but also requires to measure their neutralization capacity. Here we used a mathematical model to follow the humoral response in 26 individuals that received up to three vaccination doses of Bnt162b2 vaccine, and for whom both anti-S IgG and neutralization capacity was measured longitudinally against all main VoCs. Our model could identify two independent mechanisms that led to a marked increase in measured humoral response over the successive vaccination doses. In addition to the already known increase in IgG levels after each dose, we identified that the neutralization capacity was significantly increased after the third vaccine administration against all VoCs, despite large inter-individual variability. Consequently, the model projects that the mean duration of detectable neutralizing capacity against non-Omicron VoC is between 348 days (Beta variant, 95% Prediction Intervals PI [307; 389]) and 587 days (Alpha variant, 95% PI [537; 636]). Despite the low neutralization levels after three doses, the mean duration of detectable neutralizing capacity against Omicron variants varies between 173 days (BA.5 variant, 95% PI [142; 200]) and 256 days (BA.1 variant, 95% PI [227; 286]). Our model shows the benefit of incorporating the neutralization capacity in the follow-up of patients to better inform on their level of protection against the different SARS-CoV-2 variants. Trial registration: This clinical trial is registered with ClinicalTrials.gov, Trial IDs NCT04750720 and NCT05315583.

Impact of variants of concern on SARS-CoV-2 viral dynamics in non-human primates.

The impact of variants of concern (VoC) on SARS-CoV-2 viral dynamics remains poorly understood and essentially relies on observational studies subject to various sorts of biases. In contrast, experimental models of infection constitute a powerful model to perform controlled comparisons of the viral dynamics observed with VoC and better quantify how VoC escape from the immune response. Here we used molecular and infectious viral load of 78 cynomolgus macaques to characterize in detail the effects of VoC on viral dynamics. We first developed a mathematical model that recapitulate the observed dynamics, and we found that the best model describing the data assumed a rapid antigen-dependent stimulation of the immune response leading to a rapid reduction of viral infectivity. When compared with the historical variant, all VoC except beta were associated with an escape from this immune response, and this effect was particularly sensitive for delta and omicron variant (p<10-6 for both). Interestingly, delta variant was associated with a 1.8-fold increased viral production rate (p = 0.046), while conversely omicron variant was associated with a 14-fold reduction in viral production rate (p<10-6). During a natural infection, our models predict that delta variant is associated with a higher peak viral RNA than omicron variant (7.6 log10 copies/mL 95% CI 6.8-8 for delta; 5.6 log10 copies/mL 95% CI 4.8-6.3 for omicron) while having similar peak infectious titers (3.7 log10 PFU/mL 95% CI 2.4-4.6 for delta; 2.8 log10 PFU/mL 95% CI 1.9-3.8 for omicron). These results provide a detailed picture of the effects of VoC on total and infectious viral load and may help understand some differences observed in the patterns of viral transmission of these viruses.

Modeling SARS-CoV-2 viral kinetics and association with mortality in hospitalized patients from the French COVID cohort.

The characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral kinetics in hospitalized patients and its association with mortality is unknown. We analyzed death and nasopharyngeal viral kinetics in 655 hospitalized patients from the prospective French COVID cohort. The model predicted a median peak viral load that coincided with symptom onset. Patients with age ≥65 y had a smaller loss rate of infected cells, leading to a delayed median time to viral clearance occurring 16 d after symptom onset as compared to 13 d in younger patients (P < 10-4). In multivariate analysis, the risk factors associated with mortality were age ≥65 y, male gender, and presence of chronic pulmonary disease (hazard ratio [HR] > 2.0). Using a joint model, viral dynamics after hospital admission was an independent predictor of mortality (HR = 1.31, P < 10-3). Finally, we used our model to simulate the effects of effective pharmacological interventions on time to viral clearance and mortality. A treatment able to reduce viral production by 90% upon hospital admission would shorten the time to viral clearance by 2.0 and 2.9 d in patients of age <65 y and ≥65 y, respectively. Assuming that the association between viral dynamics and mortality would remain similar to that observed in our population, this could translate into a reduction of mortality from 19 to 14% in patients of age ≥65 y with risk factors. Our results show that viral dynamics is associated with mortality in hospitalized patients. Strategies aiming to reduce viral load could have an effect on mortality rate in this population.

Support to early clinical decisions in drug development and personalised medicine with checkpoint inhibitors using dynamic biomarker-overall survival models.

Longitudinal models of biomarkers such as tumour size dynamics capture treatment efficacy and predict treatment outcome (overall survival) of a variety of anticancer therapies, including chemotherapies, targeted therapies, immunotherapies and their combinations. These pharmacological endpoints like tumour dynamic (tumour growth inhibition) metrics have been proposed as alternative endpoints to complement the classical RECIST endpoints (objective response rate, progression-free survival) to support early decisions both at the study level in drug development as well as at the patients level in personalised therapy with checkpoint inhibitors. This perspective paper presents recent developments and future directions to enable wider and robust use of model-based decision frameworks based on pharmacological endpoints.

High sera levels of SARS-CoV-2 N antigen are associated with death in hospitalized COVID-19 patients.

The presence of free severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid-antigen in sera (N-antigenemia) has been shown in COVID-19 patients. However, the link between the quantitative levels of N-antigenemia and COVID-19 disease severity is not entirely understood. To assess the dynamics and clinical association of N-antigen sera levels with disease severity in COVID-19 patients, we analyzed data from patients included in the French COVID cohort, with at least one sera sample between January and September 2020. We assessed N-antigenemia levels and anti-N IgG titers, and patient outcomes was classified in two groups, survival or death. In samples collected within 8 days since symptom onset, we observed that deceased patients had a higher positivity rate (93% vs. 81%; p < 0.001) and higher median levels of predicted N-antigenemia (2500 vs. 1200 pg/mL; p < 0.001) than surviving patients. Predicted time to N-antigen clearance in sera was prolonged in deceased patients compared to survivors (23.3 vs 19.3 days; p < 0.0001). In a subset of patients with both sera and nasopharyngeal (NP) swabs, predicted time to N-antigen clearance in sera was prolonged in deceased patients (p < 0.001), whereas NP viral load clearance did not differ between the groups (p = 0.07). Our results demonstrate a strong relationship between N-antigenemia levels and COVID-19 severity on a prospective cohort.

Nonlinear multilevel joint model for individual lesion kinetics and survival to characterize intra-individual heterogeneity in patients with advanced cancer.

In advanced cancer patients, tumor burden is calculated using the sum of the longest diameters (SLD) of the target lesions, a measure that lumps all lesions together and ignores intra-patient heterogeneity. Here, we used a rich dataset of 342 metastatic bladder cancer patients treated with a novel immunotherapy agent to develop a Bayesian multilevel joint model that can quantify heterogeneity in lesion dynamics and measure their impact on survival. Using a nonlinear model of tumor growth inhibition, we estimated that dynamics differed greatly among lesions, and inter-lesion variability accounted for 21% and 28% of the total variance in tumor shrinkage and treatment effect duration, respectively. Next, we investigated the impact of individual lesion dynamics on survival. Lesions located in the liver and in the bladder had twice as much impact on the instantaneous risk of death compared to those located in the lung or the lymph nodes. Finally, we evaluated the utility of individual lesion follow-up for dynamic predictions. Consistent with results at the population level, the individual lesion model outperformed a model relying only on SLD, especially at early landmark times and in patients with liver or bladder target lesions. Our results show that an individual lesion model can characterize the heterogeneity in tumor dynamics and its impact on survival in advanced cancer patients.

Effect of remdesivir on viral dynamics in COVID-19 hospitalized patients: a modelling analysis of the randomized, controlled, open-label DisCoVeRy trial.

BACKGROUND: The antiviral efficacy of remdesivir in COVID-19 hospitalized patients remains controversial. OBJECTIVES: To estimate the effect of remdesivir in blocking viral replication. METHODS: We analysed nasopharyngeal normalized viral loads from 665 hospitalized patients included in the DisCoVeRy trial (NCT04315948; EudraCT 2020-000936-23), randomized to either standard of care (SoC) or SoC + remdesivir. We used a mathematical model to reconstruct viral kinetic profiles and estimate the antiviral efficacy of remdesivir in blocking viral replication. Additional analyses were conducted stratified on time of treatment initiation (≤7 or >7 days since symptom onset) or viral load at randomization (< or ≥3.5 log10 copies/104 cells). RESULTS: In our model, remdesivir reduced viral production by infected cells by 2-fold on average (95% CI: 1.5-3.2-fold). Model-based simulations predict that remdesivir reduced time to viral clearance by 0.7 days compared with SoC, with large inter-individual variabilities (IQR: 0.0-1.3 days). Remdesivir had a larger impact in patients with high viral load at randomization, reducing viral production by 5-fold on average (95% CI: 2.8-25-fold) and the median time to viral clearance by 2.4 days (IQR: 0.9-4.5 days). CONCLUSIONS: Remdesivir halved viral production, leading to a median reduction of 0.7 days in the time to viral clearance compared with SoC. The efficacy was larger in patients with high viral load at randomization.

Variant-specific SARS-CoV-2 within-host kinetics.

Since early 2021, SARS-CoV-2 variants of concern (VOCs) have been causing epidemic rebounds in many countries. Their properties are well characterized at the epidemiological level but the potential underlying within-host determinants remain poorly understood. We analyze a longitudinal cohort of 6944 individuals with 14 304 cycle threshold (Ct) values of reverse-transcription quantitative polymerase chain reaction (RT-qPCR) VOC screening tests performed in the general population and hospitals in France between February 6 and August 21, 2021. To convert Ct values into numbers of virus copies, we performed an additional analysis using droplet digital PCR (ddPCR). We find that the number of viral genome copies reaches a higher peak value and has a slower decay rate in infections caused by Alpha variant compared to that caused by historical lineages. Following the evidence that viral genome copies in upper respiratory tract swabs are informative on contagiousness, we show that the kinetics of the Alpha variant translate into significantly higher transmission potentials, especially in older populations. Finally, comparing infections caused by the Alpha and Delta variants, we find no significant difference in the peak viral copy number. These results highlight that some of the differences between variants may be detected in virus load variations.

Lassa viral dynamics in non-human primates treated with favipiravir or ribavirin.

Lassa fever is an haemorrhagic fever caused by Lassa virus (LASV). There is no vaccine approved against LASV and the only recommended antiviral treatment relies on ribavirin, despite limited evidence of efficacy. Recently, the nucleotide analogue favipiravir showed a high antiviral efficacy, with 100% survival obtained in an otherwise fully lethal non-human primate (NHP) model of Lassa fever. However the mechanism of action of the drug is not known and the absence of pharmacokinetic data limits the translation of these results to the human setting. Here we aimed to better understand the antiviral effect of favipiravir by developping the first mathematical model recapitulating Lassa viral dynamics and treatment. We analyzed the viral dynamics in 24 NHPs left untreated or treated with ribavirin or favipiravir, and we put the results in perspective with those obtained with the same drugs in the context of Ebola infection. Our model estimates favipiravir EC50 in vivo to 2.89 µg.mL-1, which is much lower than what was found against Ebola virus. The main mechanism of action of favipiravir was to decrease virus infectivity, with an efficacy of 91% at the highest dose. Based on our knowledge acquired on the drug pharmacokinetics in humans, our model predicts that favipiravir doses larger than 1200 mg twice a day should have the capability to strongly reduce the production infectious virus and provide a milestone towards a future use in humans.

Zika virus dynamics: Effects of inoculum dose, the innate immune response and viral interference.

Experimental Zika virus infection in non-human primates results in acute viral load dynamics that can be well-described by mathematical models. The inoculum dose that would be received in a natural infection setting is likely lower than the experimental infections and how this difference affects the viral dynamics and immune response is unclear. Here we study a dataset of experimental infection of non-human primates with a range of doses of Zika virus. We develop new models of infection incorporating both an innate immune response and viral interference with that response. We find that such a model explains the data better than models with no interaction between virus and the immune response. We also find that larger inoculum doses lead to faster dynamics of infection, but approximately the same total amount of viral production.

Success of prophylactic antiviral therapy for SARS-CoV-2: Predicted critical efficacies and impact of different drug-specific mechanisms of action.

Repurposed drugs that are safe and immediately available constitute a first line of defense against new viral infections. Despite limited antiviral activity against SARS-CoV-2, several drugs are being tested as medication or as prophylaxis to prevent infection. Using a stochastic model of early phase infection, we evaluate the success of prophylactic treatment with different drug types to prevent viral infection. We find that there exists a critical efficacy that a treatment must reach in order to block viral establishment. Treatment by a combination of drugs reduces the critical efficacy, most effectively by the combination of a drug blocking viral entry into cells and a drug increasing viral clearance. Below the critical efficacy, the risk of infection can nonetheless be reduced. Drugs blocking viral entry into cells or enhancing viral clearance reduce the risk of infection more than drugs that reduce viral production in infected cells. The larger the initial inoculum of infectious virus, the less likely is prevention of an infection. In our model, we find that as long as the viral inoculum is smaller than 10 infectious virus particles, viral infection can be prevented almost certainly with drugs of 90% efficacy (or more). Even when a viral infection cannot be prevented, antivirals delay the time to detectable viral loads. The largest delay of viral infection is achieved by drugs reducing viral production in infected cells. A delay of virus infection flattens the within-host viral dynamic curve, possibly reducing transmission and symptom severity. Thus, antiviral prophylaxis, even with reduced efficacy, could be efficiently used to prevent or alleviate infection in people at high risk.

SARS-CoV-2 viral dynamics in non-human primates.

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>104 virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.

Modelling the association between biomarkers and clinical outcome: An introduction to nonlinear joint models.

Nonlinear joint models are a powerful tool to precisely analyse the association between a nonlinear biomarker and a time-to-event process, such as death. Here, we review the main methodological techniques required to build these models and to make inferences and predictions. We describe the main clinical applications and discuss the future developments of such models.

What drives the dynamics of HBV RNA during treatment?

Hepatitis B virus RNA (HBV RNA)-containing particles are encapsidated pre-genomic RNA (pgRNA) detectable in chronically infected patients in addition to virions (HBV DNA) that have been suggested as a marker of the treatment efficacy. This makes promising the use of core protein allosteric modulators, such as RG7907, which disrupt the nucleocapsid assembly and profoundly reduce HBV RNA. Here, we developed a multiscale model of HBV extending the standard viral dynamic models to analyse the kinetics of HBV DNA and HBV RNA in 35 patients treated with RG7907 for 28 days. We compare the predictions with those obtained in patients treated with the nucleotide analog tenofovir. RG7907 blocked 99.3% of pgRNA encapsidation (range: 92.1%-99.9%) which led to a decline of both HBV DNA and HBV RNA. As a consequence of its mode of action, the first phase of decline of HBV RNA was rapid, uncovering the clearance of viral particles with half-life of 45 min. In contrast, HBV DNA decline was predicted to be less rapid, due to the continuous secretion of already formed viral capsids (t1/2  = 17 ± 6 h). After few days, both markers declined at the same rate, which was attributed to the loss of infected cells (t1/2  â‰… 6 ± 0.8 days). By blocking efficiently RNA reverse transcription but not its encapsidation, nucleotide analog in contrast was predicted to lead to a transient accumulation of HBV RNA both intracellularly and extracellularly. The model brings a conceptual framework for understanding the differences between HBV DNA and HBV RNA dynamics. Integration of HBV RNA in viral dynamic models may be helpful to better quantify the treatment effect, especially in viral-suppressed patients where HBV DNA is no longer detectable.

Rationale of a loading dose initiation for hydroxychloroquine treatment in COVID-19 infection in the DisCoVeRy trial.

Around the world, several dose regimens of hydroxychloroquine have been used for COVID-19 infection treatment, with the objective of identifying a short-term course. Hydroxychloroquine was found to decrease the viral replication in a concentration-dependent manner in vitro and to be more active when added prior to the viral challenge. A loading dose is used to rapidly attain a target drug concentration, which is usually considered as approximately the steady-state concentration. With a loading dose, the minimum effective concentration is reached much more rapidly than when using only the maintenance dose from the start. Thus, we propose a hydroxychloroquine sulphate dose regimen of 400 mg twice daily at Day 1 then 400 mg once daily from Day 2 to Day 10. We aim to evaluate this in the C-20-15 DisCoVeRy trial.

Bayesian inference using Hamiltonian Monte-Carlo algorithm for nonlinear joint modeling in the context of cancer immunotherapy.

Treatment evaluation in advanced cancer mainly relies on overall survival and tumor size dynamics. Both markers and their association can be simultaneously analyzed by using joint models, and these approaches are supported by many softwares or packages. However, these approaches are essentially limited to linear models for the longitudinal part, which limit their biological interpretation. More biological models of tumor dynamics can be obtained by using nonlinear models, but they are limited by the fact that parameter identifiability require rich dataset. In that context Bayesian approaches are particularly suited to incorporate the biological knowledge and increase the information available, but they are limited by the high computing cost of Monte-Carlo by Markov Chains algorithms. Here, we aimed to assess the performances of the Hamiltonian Monte-Carlo (HMC) algorithm implemented in Stan for inference in a nonlinear joint model. The method was validated on simulated data where HMC provided proper posterior distributions and credibility intervals in a reasonable computational time. Then the association between tumor size dynamics and survival was assessed in patients with advanced or metastatic bladder cancer treated with atezolizumab, an immunotherapy agent. HMC confirmed limited sensitivity to prior distributions. A cross-validation approach was developed and identified the current slope of tumor size dynamics as the most relevant driver of survival. In summary, HMC is an efficient approach to perform nonlinear joint models in a Bayesian framework, and opens the way for the use of nonlinear models to characterize both the rapid dynamics and the intersubject variability observed during cancer immunotherapy treatment.

Longitudinal analysis of organ-specific tumor lesion sizes in metastatic colorectal cancer patients receiving first line standard chemotherapy in combination with anti-angiogenic treatment.

The purpose of this work is to assess the heterogeneity across organs of response to treatment in metastatic colorectal patient based on longitudinal individual target lesion diameters (ILD) in comparison to sum of tumor lesion diameters (SLD). Data were from the McCAVE trial, in which 189 previously untreated patients with metastatic colorectal carcinoma (mCRC) received either bevacizumab (control, C) or vanucizumab (experimental, E), on top of standard chemotherapy. Bayesian hierarchical longitudinal non-linear mixed effect models were fitted to the data using Hamilton Monte Carlo algorithm to characterize the time dynamics of the tumor burden, and to obtain estimates of the tumor shrinkage and regrowth rates. The ILD model brought more nuanced results than to the SLD model. Besides substantial differences in tumor size at baseline (with lesions located in liver more than twice as large as the ones in lungs), it revealed a more durable response in lesions located in lymph nodes and 'other organs' compared to liver and lungs. Specifically, in lymph nodes and 'other organs', the projected time to nadir was doubled in group E (2.12 and 2.44 years respectively) compared to group C (1.07 and 1.20 years respectively). This long period of tumor shrinkage associated with a slightly larger change from baseline at nadir (- 51.4% in lymph nodes and - 62.6% in 'other organs' in the group E, compared to - 46.2% and - 46.9% in group C) resulted in a clinically meaningful difference in the tumor dynamics of patients in group E compared to the group C. The proportion of variance explained by the inter-lesion variability for each model parameter was large (ranging between 10 and 56%), reflecting the heterogeneity in tumor dynamics across organs. These findings suggest that there is value in understanding both within- and between-patient variability in tumor size's time dynamics using an appropriate modeling framework, as this information may help in pairing the right treatment with individual patient profile.

Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies.

The recent outbreak of Zika virus (ZIKV) has been associated with fetal abnormalities and neurological complications, prompting global concern. Here we present a mathematical analysis of the within-host dynamics of plasma ZIKV burden in a nonhuman primate model, allowing for characterization of the growth and clearance of ZIKV within individual macaques. We estimate that the eclipse phase for ZIKV, the time between cell infection and viral production, is most likely short (∼4 h), the median within-host basic reproductive number R0 is 10.7, the rate of viral production is rapid (>25,000 virions d-1), and the lifetime of an infected cell while producing virus is ∼5 h. We also estimate that the minimum number of virions produced by an infected cell over its lifetime is ∼5,500. We assess the potential effect of an antiviral treatment that blocks viral replication, showing that the median time to undetectable plasma viral load (VL) can be reduced from ∼5 d to ∼3 d with a drug concentration ∼15 times the drug's EC50 when treatment is given prophylactically starting at the time of infection. In the case of favipiravir, a polymerase inhibitor with activity against ZIKV, we predict a dose of 150 mg/kg given twice a day initiated at the time of infection can reduce the peak median VL by ∼3 logs and shorten the time to undetectable median VL by ∼2 d, whereas treatment given 2 d postinfection is mostly ineffective in accelerating plasma VL loss in macaques.