Behavioral and Cognitive Outcomes of Rhesus Macaques Following Neonatal Exposure to Antiseizure Medications.

OBJECTIVE: Exposure of neonatal macaques to the antiseizure medications phenobarbital and midazolam (PbM) causes widespread apoptotic death of neurons and oligodendrocytes. We studied behavior and neurocognitive performance in 12 to 24 month-old macaques treated as neonates with PbM. METHODS: A total of 14 monkeys received phenobarbital and midazolam over 24 hours under normothermia (n = 8) or mild hypothermia (n = 6). Controls (n = 8) received no treatment. Animals underwent testing in the human intruder paradigm at ages 12 and 18 months, and a 3-step stimulus discrimination task at ages 12, 18, and 24 months. RESULTS: Animals treated with PbM displayed lower scores for environmental exploration, and higher scores for locomotion and vocalizations compared with controls. Combined PbM and hypothermia resulted in lower scores for aggression and vigilance at 12 months compared with controls and normothermic PbM animals. A mixed-effects generalized linear model was used to test for differences in neurocognitive performance between the control and PbM groups in the first step of the stimulus discrimination task battery (shape center baited to shape center non-baited). The odds of passing this step differed by group (p = 0.044). At any given age, the odds of passing for a control animal were 9.53-fold (95% CI 1.06-85) the odds for a PbM animal. There was also evidence suggesting a higher learning rate in the shape center non-baited for the control relative to the PbM group (Cox model HR 2.13, 95% CI 1.02-4.43; p = 0.044). INTERPRETATION: These findings demonstrate that a 24-hour-long neonatal treatment with a clinically relevant combination of antiseizure medications can have long-lasting effects on behavior and cognition in nonhuman primates. ANN NEUROL 2023.

Mild hypothermia fails to protect infant macaques from brain injury caused by prolonged exposure to Antiseizure drugs.

Barbiturates and benzodiazepines are GABAA-receptor agonists and potent antiseizure medications. We reported that exposure of neonatal macaques to combination of phenobarbital and midazolam (Pb/M) for 24 h, at clinically relevant doses and plasma levels, causes widespread apoptosis affecting neurons and oligodendrocytes. Notably, the extent of injury was markedly more severe compared to shorter (8 h) exposure to these drugs. We also reported that, in the infant macaque, mild hypothermia ameliorates the apoptosis response to the anesthetic sevoflurane. These findings prompted us explore whether mild hypothermia might protect infant nonhuman primates from neuro- and gliotoxicity of Pb/M. Since human infants with seizures may receive combinations of benzodiazepines and barbiturates for days, we opted for 24 h treatment with Pb/M. Neonatal rhesus monkeys received phenobarbital intravenously, followed by midazolam infusion over 24 h under normothermia (T > 36.5 °C-37.5 °C; n = 4) or mild hypothermia (T = 35 °C-36.5 °C; n = 5). Medication doses and blood levels measured were comparable to those in human infants. Animals were euthanized at 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated. Extensive degeneration of neurons and oligodendrocytes was seen at 36 h in both groups within neocortex, basal ganglia, hippocampus and brainstem. Mild hypothermia over 36 h (maintained until terminal perfusion) conferred no protection against the neurotoxic and gliotoxic effects of Pb/M. This is in marked contrast to our previous findings that mild hypothermia is protective in the context of a 5 h-long exposure to sevoflurane in infant macaques. These findings demonstrate that brain injury caused by prolonged exposure to Pb/M in the neonatal primate cannot be ameliorated by mild hypothermia.

Non-neutralizing Antibodies May Contribute to Suppression of SIVmac239 Viremia in Indian Rhesus Macaques.

The antiviral properties of broadly neutralizing antibodies against HIV are well-documented but no vaccine is currently able to elicit protective titers of these responses in primates. While current vaccine modalities can readily induce non-neutralizing antibodies against simian immunodeficiency virus (SIV) and HIV, the ability of these responses to restrict lentivirus transmission and replication remains controversial. Here, we investigated the antiviral properties of non-neutralizing antibodies in a group of Indian rhesus macaques (RMs) that were vaccinated with vif, rev, tat, nef, and env, as part of a previous study conducted by our group. These animals manifested rapid and durable control of viral replication to below detection limits shortly after SIVmac239 infection. Although these animals had no serological neutralizing activity against SIVmac239 prior to infection, their pre-challenge titers of Env-binding antibodies correlated with control of viral replication. To assess the contribution of anti-Env humoral immune responses to virologic control in two of these animals, we transiently depleted their circulating antibodies via extracorporeal plasma immunoadsorption and inhibition of IgG recycling through antibody-mediated blockade of the neonatal Fc receptor. These procedures reduced Ig serum concentrations by up to 80% and temporarily induced SIVmac239 replication in these animals. Next, we transferred purified total Ig from the rapid controllers into six vaccinated RMs one day before intrarectal challenge with SIVmac239. Although recipients of the hyperimmune anti-SIV Ig fraction were not protected from infection, their peak and chronic phase viral loads were significantly lower than those in concurrent unvaccinated control animals. Together, our results suggest that non-neutralizing Abs may play a role in the suppression of SIVmac239 viremia.

Brain pathology caused in the neonatal macaque by short and prolonged exposures to anticonvulsant drugs.

Barbiturates and benzodiazepines are potent GABAA receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life. Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain. Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naïve controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners. These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored.

Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo.

Mild hypothermia ameliorates anesthesia toxicity in the neonatal macaque brain.

Sedatives and anesthetics can injure the developing brain. They cause apoptosis of neurons and oligodendrocytes, impair synaptic plasticity, inhibit neurogenesis and trigger long-term neurocognitive deficits. The projected vulnerable period in humans extends from the third trimester of pregnancy to the third year of life. Despite all concerns, there is no ethically and medically acceptable alternative to the use of sedatives and anesthetics for surgeries and painful interventions. Development of measures that prevent injury while allowing the medications to exert their desired actions has enormous translational value. Here we investigated protective potential of hypothermia against histological toxicity of the anesthetic sevoflurane in the developing nonhuman primate brain. Neonatal rhesus monkeys underwent sevoflurane anesthesia over 5 h. Body temperature was regulated in the normothermic (>36.5 °C), mild hypothermic (35-36.5 °C) and moderately hypothermic (<35 °C) range. Animals were euthanized at 8 h and brains examined immunohistochemically (activated caspase 3) and stereologically to quantify apoptotic neuronal and oligodendroglial death. Sevoflurane anesthesia was well tolerated at all temperatures, with oxygen saturations, end tidal CO2 and blood gases remaining at optimal levels. Compared to controls, sevoflurane exposed brains displayed significant apoptosis in gray and white matter affecting neurons and oligodendrocytes. Mild hypothermia (35-36.5 °C) conferred significant protection from apoptotic brain injury, whereas moderate hypothermia (<35 °C) did not. Hypothermia ameliorates anesthesia-induced apoptosis in the neonatal primate brain within a narrow temperature window (35-36.5 °C). Protection is lost at temperatures below 35 °C. Given the mild degree of cooling needed to achieve significant brain protection, application of our findings to humans should be explored further.

Quantitative ultrasound and apoptotic death in the neonatal primate brain.

Apoptosis is triggered in the developing mammalian brain by sedative, anesthetic or antiepileptic drugs during late gestation and early life. Whether human children are vulnerable to this toxicity mechanism remains unknown, as there are no imaging techniques to capture it. Apoptosis is characterized by distinct structural features, which affect the way damaged tissue scatters ultrasound compared to healthy tissue. We evaluated whether apoptosis, triggered by the anesthetic sevoflurane in the brains of neonatal rhesus macaques, can be detected using quantitative ultrasound (QUS). Neonatal (n = 15) rhesus macaques underwent 5 h of sevoflurane anesthesia. QUS images were obtained through the sagittal suture at 0.5 and 6 h. Brains were collected at 8 h and examined immunohistochemically to analyze apoptotic neuronal and oligodendroglial death. Significant apoptosis was detected in white and gray matter throughout the brain, including the thalamus. We measured a change in the effective scatterer size (ESS), a QUS biomarker derived from ultrasound echo signals obtained with clinical scanners, after sevoflurane-anesthesia in the thalamus. Although initial inclusion of all measurements did not reveal a significant correlation, when outliers were excluded, the change in the ESS between the pre- and post-anesthesia measurements correlated strongly and proportionally with the severity of apoptotic death. We report for the first time in vivo changes in QUS parameters, which may reflect severity of apoptosis in the brains of infant nonhuman primates. These findings suggest that QUS may enable in vivo studies of apoptosis in the brains of human infants following exposure to anesthetics, antiepileptics and other brain injury mechanisms.

KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques.

Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFα) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFα upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.

Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication.

Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute "pre-peak" phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque 'elite controllers' with a short-course of dextran sulfate sodium (DSS)-stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host's capacity to contain infection.

Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection.

Human pegivirus (HPgV)-formerly known as GB virus C and hepatitis G virus-is a poorly characterized RNA virus that infects about one-sixth of the global human population and is transmitted frequently in the blood supply. We create an animal model of HPgV infection by infecting macaque monkeys with a new simian pegivirus (SPgV) discovered in wild baboons. Using this model, we provide a high-resolution, longitudinal picture of SPgV viremia where the dose, route, and timing of infection are known. We detail the highly variable acute phase of SPgV infection, showing that the viral load trajectory early in infection is dependent on the infecting dose, whereas the chronic-phase viremic set point is not. We also show that SPgV has an extremely low propensity for accumulating sequence variation, with no consensus-level variants detected during the acute phase of infection and an average of only 1.5 variants generated per 100 infection-days. Finally, we show that SPgV RNA is highly concentrated in only two tissues: spleen and bone marrow, with bone marrow likely producing most of the virus detected in plasma. Together, these results reconcile several paradoxical observations from cross-sectional analyses of HPgV in humans and provide an animal model for studying pegivirus biology.

Rapid, repeated, low-dose challenges with SIVmac239 infect animals in a condensed challenge window.

BACKGROUND: Simian immunodeficiency virus (SIV) infection of nonhuman primates is the predominant model for preclinical evaluation of human immunodeficiency virus (HIV) vaccines. These studies frequently utilize high-doses of SIV that ensure infection after a single challenge but do not recapitulate critical facets of sexual HIV transmission. Investigators are increasingly using low-dose challenges in which animals are challenged once every week or every two weeks in order to better replicate sexual HIV transmission. Using this protocol, some animals require over ten challenges before SIV infection is detectable, potentially inducing localized immunity. Moreover, the lack of certainty over which challenge will lead to productive infection prevents tissue sampling immediately surrounding the time of infection. FINDINGS: Here we challenged Mauritian cynomolgus macaques with 100 50% tissue culture infectious doses (TCID50) of SIVmac239 intrarectally three times a day for three consecutive days. Ten of twelve animals had positive plasma viral loads after this challenge regimen. CONCLUSIONS: This approach represents a straightforward advance in SIV challenge protocols that may avoid induction of local immunity, avoid inconsistent timing between last immunization and infection, and allow sampling immediately after infection using low-dose challenge protocols.