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Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 ( 1 ), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C -nucleoside GS-646089 ( 2 ) with broad antiviral activity against RSV (EC 50 = 3-46 nM), human metapneumovirus (EC 50 = 210 nM), human rhinovirus (EC 50 = 54-61 nM), and enterovirus (EC 50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [( S )-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo . 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO 2 /FiO 2 ), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO 2 /FiO 2 , but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.

Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) are each leading causes of mortality and morbidity worldwide, and present additional treatment considerations when they are comorbid (TBI+HS) as a result of competing pathophysiological responses. The current study rigorously quantified injury biomechanics with high precision sensors and examined whether blood-based surrogate markers were altered in general trauma as well as post-neurotrauma. Eighty-nine sexually mature male and female Yucatan swine were subjected to a closed-head TBI+HS (40% of circulating blood volume; n  = 68), HS only ( n  = 9), or sham trauma ( n  = 12). Markers of systemic (e.g., glucose, lactate) and neural functioning were obtained at baseline, and at 35 and 295 min post-trauma. Opposite and approximately twofold differences existed for both magnitude (device > head) and duration (head > device) of quantified injury biomechanics. Circulating levels of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and ubiquitin C-terminal hydrolase L1 (UCH-L1) demonstrated differential sensitivity for both general trauma (HS) and neurotrauma (TBI+HS) relative to shams in a temporally dynamic fashion. GFAP and NfL were both strongly associated with changes in systemic markers during general trauma and exhibited consistent time-dependent changes in individual sham animals. Finally, circulating GFAP was associated with histopathological markers of diffuse axonal injury and blood-brain barrier breach, as well as variations in device kinematics following TBI+HS. Current findings therefore highlight the need to directly quantify injury biomechanics with head mounted sensors and suggest that GFAP, NfL, and UCH-L1 are sensitive to multiple forms of trauma rather than having a single pathological indication (e.g., GFAP = astrogliosis).

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 ( 2 ) into lung cells, thereby forming the bioactive triphosphate 2-NTP . 2-NTP , an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP . Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3 ) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP , leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2 , which supports development of 3 as a promising COVID-19 treatment.

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host-specific factors that predict disease severity and progression to the neurologic syndrome. EV-D68 infection of various animal models has served as an important platform for characterization and comparison of disease pathogenesis between historic and contemporary isolates. Still, there are significant gaps in our knowledge of EV-D68 pathogenesis that constrain the development and evaluation of targeted vaccines and antiviral therapies. Continued refinement and characterization of animal models that faithfully reproduce key elements of EV-D68 infection and disease is essential for ensuring public health preparedness for future EV-D68 outbreaks.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.

Background: Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) represent leading causes of trauma-induced mortality, especially when co-occurring in pre-hospital settings where standard therapies are not readily available. The primary objective of this study was to determine if 17α-ethinyl estradiol-3-sulfate (EE-3-SO 4 ) increases survival, promotes more rapid cardiovascular recovery, or confers neuroprotection relative to Placebo following TBI + HS., Methods: All methods were approved by required regulatory agencies prior to study initiation. In this fully randomized, blinded preclinical study, eighty (50% females) sexually mature (190.64 ± 21.04 days old; 28.18 ± 2.72 kg) Yucatan swine were used. Sixty-eight animals received a closed-head, accelerative TBI followed by removal of approximately 40% of circulating blood volume. Animals were then intravenously administered EE-3-SO 4 formulated in the vehicle at 5.0 mg/mL (dosed at 0.2 mL/kg) or Placebo (0.45% sodium chloride solution) via a continuous pump (0.2 mL/kg over 5 min). Twelve swine were included as uninjured Shams to further characterize model pathology and replicate previous findings. All animals were monitored for up to 5 h in the absence of any other life-saving measures (e.g., mechanical ventilation, fluid resuscitation)., Results: A comparison of Placebo-treated relative to Sham animals indicated evidence of acidosis, decreased arterial pressure, increased heart rate, diffuse axonal injury and blood-brain barrier breach. The percentage of animals surviving to 295 min post-injury was significantly higher for the EE-3-SO 4 (28/31; 90.3%) relative to Placebo (24/33; 72.7%) cohort. EE-3-SO 4 also restored pulse pressure more rapidly post-drug administration, but did not confer any benefits in terms of shock index. Primary blood-based measurements of neuroinflammation and blood brain breach were also null, whereas secondary measurements of diffuse axonal injury suggested a more rapid return to baseline for the EE-3-SO 4 group. Survival status was associated with biological sex (female > male), as well as evidence of increased acidosis and neurotrauma independent of EE-3-SO 4 or Placebo administration., Conclusions: EE-3-SO 4 is efficacious in promoting survival and more rapidly restoring cardiovascular homeostasis following polytraumatic injuries in pre-hospital environments (rural and military) in the absence of standard therapies. Poly-therapeutic approaches targeting additional mechanisms (increased hemostasis, oxygen-carrying capacity, etc.) should be considered in future studies., (© 2021. The Author(s).)

Acceleration parameters have been utilized for the last six decades to investigate pathology in both human and animal models of traumatic brain injury (TBI), design safety equipment, and develop injury thresholds. Previous large animal models have quantified acceleration from impulsive loading forces (i.e., machine/object kinematics) rather than directly measuring head kinematics. No study has evaluated the reproducibility of head kinematics in large animal models. Nine (five males) sexually mature Yucatan swine were exposed to head rotation at a targeted peak angular velocity of 250 rad/s in the coronal plane. The results indicated that the measured peak angular velocity of the skull was 51% of the impulsive load, was experienced over 91% longer duration, and was multi- rather than uni-planar. These findings were replicated in a second experiment with a smaller cohort ( N = 4). The reproducibility of skull kinematics data was mostly within acceptable ranges based on published industry standards, although the coefficients of variation (8.9% for peak angular velocity or 12.3% for duration) were higher than the impulsive loading parameters produced by the machine (1.1 vs. 2.5%, respectively). Immunohistochemical markers of diffuse axonal injury and blood-brain barrier breach were not associated with variation in either skull or machine kinematics, suggesting that the observed levels of variance in skull kinematics may not be biologically meaningful with the current sample sizes. The findings highlight the reproducibility of a large animal acceleration model of TBI and the importance of direct measurements of skull kinematics to determine the magnitude of angular velocity, refine injury criteria, and determine critical thresholds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mayer, Ling, Dodd, Rannou-Latella, Stephenson, Dodd, Mehos, Patton, Cullen, Johnson, Pabbathi Reddy, Robertson-Benta, Gigliotti, Meier, Vermillion, Smith and Kinsler.)

Introduction: The pathology resulting from concurrent traumatic brain injury (TBI) and hemorrhagic shock (HS; TBI+HS) are leading causes of mortality and morbidity worldwide following trauma. However, the majority of large animal models of TBI+HS have utilized focal/contusional injuries rather than incorporating the types of brain trauma (closed-head injury caused by dynamic acceleration) that typify human injury., Objective: To examine survival rates and effects on biomarkers from rotational TBI with two levels of HS., Methods: Twenty-two sexually mature Yucatan swine (30.39 ± 2.25 kg; 11 females) therefore underwent either Sham trauma procedures (n = 6) or a dynamic acceleration TBI combined with either 55% (n = 8) or 40% (n = 8) blood loss in this serial study., Results: Survival rates were significantly higher for the TBI+40% (87.5%) relative to TBI+55% (12.5%) cohort, with the majority of TBI+55% animals expiring within 2 h post-trauma from apnea. Blood-based neural biomarkers and immunohistochemistry indicated evidence of diffuse axonal injury (increased NFL/Aβ42), blood-brain barrier breach (increased immunoglobulin G) and inflammation (increased glial fibrillary acidic protein/ionized calcium-binding adaptor molecule 1) in the injured cohorts relative to Shams. Invasive hemodynamic measurements indicated increased shock index and decreased pulse pressure in both injury cohorts, with evidence of partial recovery for invasive hemodynamic measurements in the TBI+40% cohort. Similarly, although both injury groups demonstrated ionic and blood gas abnormalities immediately postinjury, metabolic acidosis continued to increase in the TBI+55% group ∼85 min postinjury. Somewhat surprisingly, both neural and physiological biomarkers showed significant changes within the Sham cohort across the multi-hour experimental procedure, most likely associated with prolonged anesthesia., Conclusion: Current results suggest the TBI+55% model may be more appropriate for severe trauma requiring immediate medical attention/standard fluid resuscitation protocols whereas the TBI+40% model may be useful for studies of prolonged field care., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 by the Shock Society.)

Traumatic brain injury (TBI) and severe blood loss (SBL) frequently co-occur in human trauma, resulting in high levels of mortality and morbidity. Importantly, each of the individual post-injury cascades is characterized by complex and potentially opposing pathophysiological responses, complicating optimal resuscitation and therapeutic approaches. Large animal models of poly-neurotrauma closely mimic human physiology, but a systematic literature review of published models has been lacking. The current review suggests a relative paucity of large animal poly-neurotrauma studies (N = 52), with meta-statistics revealing trends for animal species (exclusively swine), characteristics (use of single biological sex, use of juveniles) and TBI models. Although most studies have targeted blood loss volumes of 35-45%, the associated mortality rates are much lower relative to Class III/IV human trauma. This discrepancy may result from potentially mitigating experimental factors (e.g., mechanical ventilation prior to or during injury, pausing/resuming blood loss based on physiological parameters, administration of small volume fluid resuscitation) that are rarely associated with human trauma, highlighting the need for additional work in this area., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Vaccine-induced immunity declines with age, which may differ between males and females. Using human sera collected before and 21 days after receipt of the monovalent A/Cal/09 H1N1 vaccine, we evaluated cytokine and antibody responses in adult (18-45 years) and aged (65+ years) individuals. After vaccination, adult females developed greater IL-6 and antibody responses than either adult males or aged females, with female antibody responses being positively associated with concentrations of estradiol. To test whether protection against influenza virus challenge was greater in females than males, we primed and boosted adult (8-10 weeks) and aged (68-70 weeks) male and female mice with an inactivated A/Cal/09 H1N1 vaccine or no vaccine and challenged with a drift variant A/Cal/09 virus. As compared with unvaccinated mice, vaccinated adult, but not aged, mice experienced less morbidity and better pulmonary viral clearance following challenge, regardless of sex. Vaccinated adult female mice developed antibody responses that were of greater quantity and quality and more protective than vaccinated adult males. Sex differences in vaccine efficacy diminished with age in mice. To determine the role of sex steroids in vaccine-induced immune responses, adult mice were gonadectomized and hormones (estradiol in females and testosterone in males) were replaced in subsets of animals before vaccination. Vaccine-induced antibody responses were increased in females by estradiol and decreased in males by testosterone. The benefit of elevated estradiol on antibody responses and protection against influenza in females is diminished with age in both mice and humans., Competing Interests: Competing interestsThe authors declare no competing interests.

Zika virus (ZIKV) infection during pregnancy causes significant adverse sequelae in the developing fetus, and results in long-term structural and neurologic defects. Most preventive and therapeutic efforts have focused on the development of vaccines, antivirals, and antibodies. The placental immunologic response to ZIKV, however, has been largely overlooked as a target for therapeutic intervention. The placental inflammatory response, specifically IL-1β secretion and signaling, is induced by ZIKV infection and represents an environmental factor that is known to increase the risk of perinatal developmental abnormalities. We show in a mouse model that maternally administrated IL-1 receptor antagonist (IRA; Kineret, or anakinra), following ZIKV exposure, can preserve placental function (by improving trophoblast invasion and placental vasculature), increase fetal viability, and reduce neurobehavioral deficits in the offspring. We further demonstrate that while ZIKV RNA is highly detectable in placentas, it is not correlated with fetal viability. Beyond its effects in the placenta, we show that IL-1 blockade may also directly decrease fetal neuroinflammation by mitigating fetal microglial activation in a dose-dependent manner. Our studies distinguish the role of placental inflammation during ZIKV-infected pregnancies, and demonstrate that maternal IRA may attenuate fetal neuroinflammation and improve perinatal outcomes.

Vaccines are commonly used to control Foot-and-Mouth Disease (FMD) in endemic regions and form an important part of contingency plans for FMD-free countries. Conventional FMD vaccines have numerous limitations, and the U.S. government supports the development of next-generation vaccines. In the U.S., vaccine efficacy is typically demonstrated through experimental vaccination and challenge of animals using the World Organization for Animal Health (OIE) standards. Although conventional challenge and immunogenicity studies provide useful information, they have limitations and results do not always accurately predict field performance. Consequently, there is a need to test next-generation vaccines under field conditions to gain a better understanding of field performance to inform policy decisions and support their viability as a commercial product. In June 2017, an expert consultation was organised to discuss and define an optimal field study design for novel FMD vaccines. Cattle were the primary species considered, although parallel strategies for swine and small ruminants were also discussed. Many methodological and logistical considerations in the study design were identified, including: (1) study site selection and the importance of baseline studies to understand exposure risk, (2) ethics of using a placebo and assessing equivalence with conventional vaccines, (3) merits of using individual randomised versus cluster randomised trials, (4) preventive versus reactive vaccination, and (5) methods of randomisation and blinding. The proposed optimal study design was a multicentre (i.e. farm), three-arm, double-blind randomised controlled trial comparing groups receiving the novel vaccine to a conventional vaccine group and a placebo group. Large farms in areas of high exposure risk were identified as ideal study sites, and the primary study outcome was susceptibility to disease or infection, during a six-month observation period, following a single dose of vaccine. This report provides a summary of the important issues to consider when designing a field efficacy study in livestock and proposes a study design that could be utilised for novel FMD vaccines., (Copyright © 2019.)

Pregnancy is associated with significant anatomic and functional changes to the cardiopulmonary system. Using pregnant C57BL/6 mice, we characterized changes in pulmonary structure and function during pregnancy in healthy animals and following infection with influenza A virus (IAV). We hypothesized that pregnancy-associated alterations in pulmonary physiology would contribute to the more severe outcome of IAV infection. Nonpregnant and pregnant females (at embryonic day 10.5) were either mock-infected or infected with 2009 H1N1 IAV for assessment of pulmonary function, structure, and inflammation at 8 days postinoculation. There were baseline differences in pulmonary function, with pregnant females having greater lung compliance, total lung capacity, and fixed lung volume than nonpregnant females. Following IAV infection, both pregnant and nonpregnant females exhibited reduced circulating progesterone, which in nonpregnant females was associated with increased pulmonary resistance and decreased lung compliance, minute ventilation, and oxygen diffusing capacity compared with uninfected nonpregnant females. In pregnant females, reduced concentrations of progesterone were associated with adverse pregnancy outcomes, but measures of pulmonary function were preserved following IAV infection and were not significantly different from uninfected pregnant mice. Following IAV infection, infectious virus titers and total numbers of pulmonary leukocytes were similar between pregnant and nonpregnant females, but the histological density of pulmonary inflammation was reduced in pregnant animals. These data suggest that pregnancy in mice is associated with significant alterations in pulmonary physiology but that these changes served to preserve lung function during IAV infection. Pregnancy-associated alterations in pulmonary physiology may serve to protect females during severe influenza.

Estriol (E3) is an endogenous estrogen in females with broad biological activity within diverse tissue types. In the context of certain T-cell-mediated autoimmune inflammatory diseases, E3 can ameliorate disease severity through immunomodulatory mechanisms that decrease tissue inflammation. Severe disease caused by influenza A virus (IAV) infection is also characterized by aberrant inflammation and immunopathology. How E3 might affect the pathogenesis of IAV infection, however, has not been explored. Gonadally intact female C57BL/6 mice that were treated with exogenous E3 during infection with mouse-adapted 2009 H1N1 had reduced total pulmonary inflammation and improved disease outcomes compared with females that received no hormone. Furthermore, compared with no hormone treatment, E3 treatment reduced the induction of genes associated with proinflammatory cytokine and chemokine responses in the lungs, which preceded clinical disease, reductions in innate immune cell recruitment, altered pulmonary T-cell skewing, and reduced antibody titers during IAV infection. Although E3 treatment was associated with reduced local and systemic anti-influenza adaptive immune responses, there was no effect of E3 on viral replication or clearance. Together, these data suggest that exogenous E3 confers protection during IAV infection through immunomodulatory mechanisms and that E3 may have broad therapeutic potential in the context of both infectious and noninfectious inflammatory diseases.

Background: Amphiregulin (AREG) is an epidermal growth factor that is a significant mediator of tissue repair at mucosal sites, including in the lungs during influenza A virus (IAV) infection. Previous research illustrates that males of reproductive ages experience less severe disease and recover faster than females following infection with IAV., Methods: Whether males and females differentially produce and utilize AREG for pulmonary repair after IAV infection was investigated using murine models on a C57BL/6 background and primary mouse and human epithelial cell culture systems., Results: Following sublethal infection with 2009 H1N1 IAV, adult female mice experienced greater morbidity and pulmonary inflammation during the acute phase of infection as well as worse pulmonary function during the recovery phase of infection than males, despite having similar virus clearance kinetics. As compared with females, AREG expression was greater in the lungs of male mice as well as in primary respiratory epithelial cells derived from mouse and human male donors, in response to H1N1 IAVs. Internalization of the epidermal growth factor receptor (EGFR) was also greater in respiratory epithelial cells derived from male than female mice. IAV infection of Areg knock-out (Areg -/- ) mice eliminated sex differences in IAV pathogenesis, with a more significant role for AREG in infection of male compared to female mice. Deletion of Areg had no effect on virus replication kinetics in either sex. Gonadectomy and treatment of either wild-type or Areg -/- males with testosterone improved the outcome of IAV as compared with their placebo-treated conspecifics., Conclusions: Taken together, these data show that elevated levels of testosterone and AREG, either independently or in combination, improve resilience (i.e., repair and recovery of damaged tissue) and contribute to better influenza outcomes in males compared with females.

Vaccination is the mainstay of preventative medicine for many infectious diseases. Pregnant women, unborn fetuses, and neonates represent three at-risk populations that can be simultaneously protected by strategic vaccination protocols. Because the pathogenesis of different infectious microbes varies based on tissue tropism, timing of infection, and host susceptibility, the goals of immunization are not uniform across all vaccines. Mechanistic understanding of infectious disease pathogenesis and immune responses is therefore essential to inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses, and neonates., Competing Interests: The authors declare no competing financial interests.

Blood pressure is a critical parameter for evaluating cardiovascular health, assessing effects of drugs and procedures, monitoring physiologic status during anesthesia, and making clinical decisions. The placement of an arterial catheter is the most direct and accurate method for measuring blood pressure; however, this approach is invasive and of limited use during brief sedated examinations. The objective of this study was to determine which method of indirect blood pressure monitoring was most accurate compared with measurement by direct arterial catheterization. In addition, we sought to determine the relative accuracy of each indirect method (compared with direct arterial measurement) at a given body location and to assess whether the accuracy of each indirect method was dependent on body location. We compared direct blood pressure measurements by means of catheterization of the saphenous artery with oscillometric and ultrasonic Doppler flow detection measurements at 3 body locations (forearm, distal leg, and tail base) in 16 anesthetized, male rhesus macaques. The results indicate that oscillometry at the forearm is the best indirect method and location for accurately and consistently measuring blood pressure in healthy male rhesus macaques.

Serial phlebotomy is a common sampling practice for repeated-measures studies in biomedical research. In NHP, the effect of serial blood collection on RBC parameters has been characterized, but the effects on platelet parameters and other aspects of the hemogram have not been well studied. We sought to characterize the circulating platelet phenotype throughout the course of 7 serial phlebotomies spanning 30 d in pigtailed macaques (Macaca nemestrina). Phlebotomy was performed on 23 animals at days 0, 2, 4, 7, 10, 21, and 30 to quantify the circulating platelet count and markers of both hemostatic and immune platelet activation. Platelet immune activation was characterized by increases in surface MHC class I and II expression and increases in circulating platelet-leukocyte aggregates. These changes occurred in the absence of increases in the prohemostatic markers P-selectin and CD40L and without evidence of adverse clinical effects. Mild increases in platelet count, mean platelet volume, and immune activation occurred early in the study. After day 21, mean platelet volume and other hematologic parameters returned to baseline while changes in platelet count and immune activation were greater than during the first 10 d of the study. These data demonstrate that serial phlebotomy in NHP has delayed effects on platelet parameters, which may be a source of clinically silent, immunologic and physiologic variability within repeated measures studies. The impact of these effects on research aims should be considered when designing protocols requiring serial phlebotomy in NHP.

In addition to their intended use, progesterone (P4)-based contraceptives promote anti-inflammatory immune responses, yet their effects on the outcome of infectious diseases, including influenza A virus (IAV) infection, are rarely evaluated. To evaluate their impact on immune responses to sequential IAV infections, adult female mice were treated with placebo or one of two progestins, P4 or levonorgestrel (LNG), and infected with a mouse-adapted H1N1 (maH1N1) virus. Treatment with P4 or LNG reduced morbidity but had no effect on pulmonary virus titers during primary H1N1 infection compared to placebo treatment. In serum and bronchoalveolar lavage fluid, total anti-IAV IgG and IgA titers and virus-neutralizing antibody titers but not hemagglutinin stalk antibody titers were lower in progestin-treated mice than placebo-treated mice. Females were challenged 6 weeks later with either an maH1N1 drift variant (maH1N1dv) or maH3N2 IAV. The level of protection following infection with the maH1N1dv was similar among all groups. In contrast, following challenge with maH3N2, progestin treatment reduced survival as well as the numbers and activity of H1N1- and H3N2-specific memory CD8 + T cells, including tissue-resident cells, compared with placebo treatment. In contrast to primary IAV infection, progestin treatment increased the titers of neutralizing and IgG antibodies against both challenge viruses compared with those achieved with placebo treatment. While the immunomodulatory properties of progestins protected immunologically naive female mice from the severe outcomes from IAV infection, it made them more susceptible to secondary challenge with a heterologous IAV, despite improving their antibody responses against a secondary IAV infection. Taken together, the immunomodulatory effects of progestins differentially regulate the outcome of infection depending on exposure history. IMPORTANCE The impact of hormone-based contraceptives on the outcome of infectious diseases outside the reproductive tract is rarely considered. Using a mouse model, we have made the novel observation that treatment with either progesterone or a synthetic analog found in hormonal contraceptives, levonorgestrel, impacts sequential influenza A virus infection by modulating antibody responses and decreasing the numbers and activity of memory CD8 + T cells. Progestins reduced the antibody responses during primary H1N1 virus infection but increased antibody titers following a sequential infection with either an H1N1 drift variant or an H3N2 virus. Following challenge with an H3N2 virus, female mice treated with progestins experienced greater mortality with increased pulmonary inflammation and reduced numbers and activity of CD8 + T cells. This study suggests that progestins significantly affect adaptive immune responses to influenza A virus infection, with their effect on the outcome of infection depending on exposure history., (Copyright © 2017 American Society for Microbiology.)

Zika virus (ZIKV) crosses the placenta and causes congenital disease. Here we develop an animal model utilizing direct ZIKV inoculation into the uterine wall of pregnant, immunocompetent mice to evaluate transplacental transmission. Intrauterine inoculation at embryonic day (E) 10, but not E14, with African, Asian or American strains of ZIKV reduces fetal viability and increases infection of placental and fetal tissues. ZIKV inoculation at E10 causes placental inflammation, placental dysfunction and reduces neonatal brain cortical thickness, which is associated with increased activation of microglia. Viral antigen localizes in trophoblast and endothelial cells in the placenta, and endothelial, microglial and neural progenitor cells in the fetal brain. ZIKV infection of the placenta increases production of IFNβ and expression of IFN-stimulated genes 48 h after infection. This mouse model provides a platform for identifying factors at the maternal-fetal interface that contribute to adverse perinatal outcomes in a host with an intact immune system.

Influenza severity increases with age, with hospitalization and mortality rates during seasonal influenza epidemics being higher in older men than age-matched women. As it is known that with age, circulating testosterone levels decline in males, we hypothesized that reduced testosterone contributes to age-associated increases in influenza severity. A murine model was used to test this hypothesis. As in men, testosterone concentrations were lower in aged (18 mo) than young (2 mo) male C57BL/6 mice. Following inoculation with influenza A virus (IAV), aged males experienced greater morbidity, clinical disease, and pulmonary inflammation than young males, and had lower neutralizing and total anti-influenza IgG antibody responses. Peak titers of virus in the lungs did not differ between aged and young males, but virus clearance was delayed in aged males. In young males, removal of the gonads increased-whereas treatment of gonadectomized males with testosterone reduced-morbidity, clinical illness, and pulmonary pathology, but viral replication was not altered by hormone manipulation in young males. Treatment of aged males with testosterone improved survival following infection but did not alter either virus replication or pulmonary pathology. These results indicate that low concentrations of testosterone, whether induced surgically in young males or naturally occurring in aged males, negatively impact the outcome of influenza., (Copyright © 2016 the American Physiological Society.)

Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women's health., Competing Interests: The authors have declared that no competing interests exist.

Background: Sensitive assays are needed for detection of residual human immunodeficiency virus (HIV) in patients with undetectable plasma viral loads to determine whether eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir. We sought to determine whether xenograft of leukocytes from HIV type 1 (HIV)-infected patients with undetectable plasma viral loads into immunocompromised mice would result in viral amplification., Methods: Peripheral blood mononuclear cells or purified CD4(+) T cells from HIV or simian immunodeficiency virus (SIV)-infected subjects with undetectable plasma viral loads were adoptively transferred into NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. The mice were monitored for viremia following depletion of human CD8(+) T cells to minimize antiviral activity. In some cases, humanized mice were also treated with activating anti-CD3 antibody., Results: With this murine viral outgrowth assay (MVOA), we successfully amplified replication-competent HIV or SIV from all subjects tested, including 5 HIV-positive patients receiving suppressive antiretroviral therapy (ART) and 6 elite controllers or suppressors who were maintaining undetectable viral loads without ART, including an elite suppressor from whom we were unable to recover virus by QVOA., Conclusions: Our results suggest that the MVOA has the potential to serve as a powerful tool to identify residual HIV in patients with undetectable viral loads., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)