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Summary The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.

Effective therapeutics have been developed against acute Ebola virus disease (EVD) in both humans and experimentally infected nonhuman primates. However, the risk of viral persistence and associated disease recrudescence in survivors receiving these therapeutics remains unclear. In contrast to rhesus macaques that survived Ebola virus (EBOV) exposure in the absence of treatment, we discovered that EBOV, despite being cleared from all other organs, persisted in the brain ventricular system of rhesus macaque survivors that had received monoclonal antibody (mAb) treatment. In mAb-treated macaque survivors, EBOV persisted in macrophages infiltrating the brain ventricular system, including the choroid plexuses. This macrophage infiltration was accompanied by severe tissue damage, including ventriculitis, choroid plexitis, and meningoencephalitis. Specifically, choroid plexus endothelium-derived EBOV infection led to viral persistence in the macaque brain ventricular system. This resulted in apoptosis of ependymal cells, which constitute the blood–cerebrospinal fluid barrier of the choroid plexuses. Fatal brain-confined recrudescence of EBOV infection manifested as severe inflammation, local pathology, and widespread infection of the ventricular system and adjacent neuropil in some of the mAb-treated macaque survivors. This study highlights organ-specific EBOV persistence and fatal recrudescent disease in rhesus macaque survivors after therapeutic treatment and has implications for the long-term follow-up of human survivors of EVD.

Filoviruses (Family Filoviridae genera Ebolavirus and Marburgvirus) are negative-stranded RNA viruses that cause severe health effects in humans and non-human primates, including death. Except in outbreak settings, vaccines and other medical countermeasures against Ebola virus (EBOV) will require testing under the FDA Animal Rule. Multiple vaccine candidates have been evaluated using cynomolgus monkeys (CM) exposed to EBOV Kikwit strain. To the best of our knowledge, however, animal model development data supporting the use of CM in vaccine research have not been submitted to the FDA. This study describes a large CM database (122 CM, 62 female and 60 male, age 2 to 9 years) and demonstrates the consistency of the CM model through time to death models and descriptive statistics. CMs were exposed to EBOV doses of 0.1 to 100,000 PFU in 33 studies conducted at three Animal Biosafety Level 4 facilities, by three exposure routes. Time to death was modeled using Cox proportional hazards models with a frailty term that incorporated study-to-study variability. Despite significant differences attributed to exposure variables, all CMs exposed to the 100 to 1,000 pfu doses commonly used in vaccine studies died or met euthanasia criteria within 21 days of exposure, median 7 days, 93% between 5 and 12 days of exposure. Moderate clinical signs were observed 4 to 5 days after exposure and preceded death or euthanasia by approximately one day. Viremia was detected within a few days of infection. Hematology indices were indicative of viremia and the propensity for hemorrhage with progression of Ebola viremia. Changes associated with coagulation parameters and platelets were consistent with coagulation disruption. Changes in leukocyte profiles were indicative of an acute inflammatory response. Increased liver enzymes were observed shortly after exposure. Taken together, these factors suggest that the cynomolgus monkey is a reliable animal model for human disease.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30–90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0–4.2°C), respiration rate (+56–128%), activity (-15-76% daytime and +5–22% nighttime), heart rate (+67–190%), systolic (+44–67%) and diastolic blood pressure (+45–80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (~12–24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106–140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics., Author summary In North America, EEEV causes the most severe mosquito-borne disease in humans highlighted by fatal encephalitis and permeant debilitating neurological sequelae in survivors. The first confirmed human cases were reported more than 80 years ago and since then multiple sporadic outbreaks have occurred including one of the largest in 2019. Unfortunately, most human infections are diagnosed at the on-set of severe neurological symptoms and consequently a detailed disease course in humans is lacking. This gap in knowledge is a significant obstacle in the development of appropriate animal models to evaluate countermeasures. Here, we performed a cutting-edge study by utilizing a new telemetry technology to understand the course of EEEV infection in a susceptible macaque model by measuring multiple physiological parameters relevant to human disease. Our study demonstrates that the infection rapidly produces considerable alterations in many critical parameters including the electrical activity of the heart and the brain leading to severe disease. The study also highlights the extraordinary potential of new telemetry technology to develop the next generation of animal models to comprehensively investigate pathogenesis as well as evaluate countermeasures to treat and/or prevent EEEV disease.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30-90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0-4.2 °C), respiration rate (+56-128%), activity (+15-76% daytime and +5-22% nighttime), heart rate (+67-190%), systolic (+44-67%) and diastolic blood pressure (+45-80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (12-24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ∼99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ∼106-140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.Author SummaryIn North America, EEEV causes the most severe mosquito-borne disease in humans highlighted by fatal encephalitis and permeant debilitating neurological sequelae in survivors. The first confirmed human cases were reported more than 80 years ago and since then multiple sporadic outbreaks have occurred including one of the largest in 2019. Unfortunately, most human infections are diagnosed at the on-set of severe neurological symptoms and consequently a detailed disease course in humans is lacking. This gap in knowledge is a significant obstacle in the development of appropriate animal models to evaluate countermeasures. Here, we performed a cutting-edge study by utilizing a new telemetry technology to understand the course of EEEV infection in a susceptible macaque model by measuring multiple physiological parameters relevant to human disease. Our study demonstrates that the infection rapidly produces considerable alterations in many critical parameters including the electrical activity of the heart and the brain leading to severe disease. The study also highlights the extraordinary potential of new telemetry technology to develop the next generation of animal models in order to comprehensively investigate pathogenesis as well as evaluate countermeasures to treat and/or prevent EEEV disease.

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state of the art telemetry to measure critical physiological parameters. Following challenge, all parameters were rapidly and profoundly altered, and all nonhuman primates (NHPs) met the euthanasia criteria. In this study, we performed the first comprehensive pathology investigation of tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation of EEEV clinical disease course and pathogenesis in a NHP model and will provide significant insights into the evaluation of countermeasure.Author SummaryEEEV is an arbovirus endemic in parts of North America and is able to produce fatal encephalitis in humans and domesticated animals. Despite multiple human outbreaks during the last 80 years, there are still no therapeutic or vaccines to treat or prevent human disease. One critical obstacle in the development of effective countermeasure is the lack of insights into EEEV pathogenesis in a susceptible animal host. We recently conducted a study in cynomolgus macaques to investigate the disease course by measuring clinical parameters relevant to humans. Following infection, these parameters were rapidly and profoundly altered leading to severe disease. In this study, we examined the potential mechanisms that underlie pathogenesis to cause severe disease. The virus was present in many parts of the brain and spinal cord, however, little or no pathological lesions as well as active virus replication were observed. Additionally, neurons were the predominant target of EEEV and virus transport was facilitated by axonal transport system to spread neuron-to-neuron throughout the brain and spinal cord. These data show that EEEV likely hijacks host cell transport system to rapidly spread in the brain and local/global neuronal dysfunction rather than death is the principal cause of severe disease.

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61–68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comprehensive and comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to define parameters critical to disease progression and the extent to which they correlate with human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.One Sentence SummaryNonhuman primates develop COVID-19 following airborne virus exposure.

Background For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

Zika virus (ZIKV) is a mosquito-borne member of the Spondweni complex of the genus Flavivirus, family Flaviviridae. The genus Flavivirus comprises many important human pathogens such as Saint Louis encephalitis, yellow fever virus, dengue virus (DENV1–DENV4), West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus.1 The ZIKV genome comprises a single-strand, positive-sense RNA of approximately 11 kb in length. The genome consists of a 5′ untranslated region (UTR) (∼107 nt), an open reading frame (ORF) ∼10.2 kb in length, and a 3′ UTR (∼428 nt). The single ORF encodes three structural proteins: capsid (C), premembrane/membrane (prM), envelope (E), and seven non–structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).1 Similar to other flaviviruses, ZIKV enters cells through receptor-mediated endocytosis, and the low endosomal pH induces fusion to release the genomic RNA into the cytoplasm. The genomic RNA is translated into a single polyprotein that is processed to facilitate replication, virion assembly, and release of mature virions. The mature ZIKV virion structure is similar to that of other flaviviruses, comprising a nucleocapsid core consisting of C protein and genomic RNA and an icosahedral shell consisting of 180 copies of E and M proteins (or prM) embedded in a host-derived lipid bilayer.2 ZIKV was first isolated from the blood of a sentinel rhesus macaque in 1947 in the Zika Forest in Uganda.3 In the following year, ZIKV was isolated from pools of Aedes africanus mosquitoes, and subsequent studies demonstrated virus transmission by Aedes aegypti to both mice and rhesus macaques.4 In the following decades (1950s–1980s), ZIKV was isolated from humans and multiple mosquito species in sub-Saharan Africa and Southeast Asia, and suggesting maintenance in both sylvatic (primarily Aedes spp. mosquitoes and nonhuman primates [NHPs]) and urban cycles (mainly A. aegypti and humans).5–19 ZIKV consists of a single serotype, with isolates comprising two geographic lineages (African and Asian) that have caused sporadic or underreported human outbreaks.20,21 However, starting in 2007, outbreaks were reported in the Yap island in the Federated States of Micronesia and in Gabon, and by 2013 ZIKV was imported from French Polynesia into northeast Brazil resulting in the largest reported epidemic to date and its subsequent expansion to many parts of Americas including mainland USA (Florida and Texas).22–27 Since the start of the 2007 epidemic, ZIKV has spread to or been detected in 84 countries and territories in the Americas, Asia, and Africa, with reported evidence of autochthonous transmission (http://apps.who.int/iris/bitstream/10665/254714/1/zikasitrep10Mar17-eng.pdf?ua=1). Most human ZIKV infections are asymptomatic.28,29 Symptomatic infections typically present as a self-limiting acute febrile illness with symptoms ranging from fever, headache, myalgia, and rash; however, severe clinical manifestations including congenital microcephaly and other fetal defects, Guillain–Barre syndrome (GBS), and death have been reported.30,31 Although ZIKV is transmitted primarily through mosquito/human cycle, the virus can also be transmitted through sexual contact and blood transfusions.32–34 Before its arrival in the Americas, ZIKV was an obscure and understudied pathogen; as a consequence, few virus isolates were accessible for research and vaccine/therapeutic development. In addition, little or no basic in vitro and/or in vivo characterization data were available on accessible isolates. In the present study, we investigated the potential phenotypic differences between low-passage African (ArD 41525) and Asian isolates (CPC-0740 and SV0127-14) in vitro and in vivo.

Venezuelan equine encephalitis virus (VEEV) is an important pathogen of medical and veterinary importance in the Americas. In this report, we present the complete genome sequences of five VEEV isolates obtained from pools of Culex (Melanoconion) gnomatos (4) or Culex (Melanoconion) pedroi (1) from Iquitos, Peru. Genetic and phylogenetic analyses showed that all five isolates grouped within the VEEV complex sister to VEEV IIIC and are members of subtype IIID. This is the first report of full-length genomic sequences of VEEV IIID.

Most alphaviruses are mosquito-borne and can cause severe disease in domesticated animals and humans. The most notable recent outbreak in the Americas was the 2014 chikungunya virus (CHIKV) outbreak affecting millions and producing disease highlighted by rash and arthralgia. Chikungunya virus is a member of the Semliki Forest (SF) serocomplex, and before its arrival in the Americas, two other member of the SF complex, Una (UNAV) and Mayaro (MAYV) viruses, were circulating in Central and South America. This study examined whether antibodies from convalescent CHIKV patients could cross-neutralize UNAV and MAYV. Considerable cross-neutralization of both viruses was observed, suggesting that exposure to CHIKV can produce antibodies that may mitigate infection with UNAV or MAYV. Understanding the impact of CHIKV exposure on population susceptibility to other emerging viruses may help predict outbreaks; moreover, identification of cross-reactive immune responses among alphaviruses may lead to the development of vaccines targeting multiple viruses.

A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs. IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

A well-characterized exposure chamber is necessary to generate reproducible atmospheres for inhalation toxicology studies. The aim of the present study was to characterize a head-only exposure chamber for non-human primates. Aerosols containing bovine serum albumin (BSA) were used to characterize a 16-L dynamic airflow head-only exposure chamber. A 250-ml plastic bottle with a respirator attached located inside the chamber was used to simulate a breathing head. Chamber leak rate, mixing, and aerosol spatial distributions were quantified. The chamber concentration profile was measured at the chamber exhaust using an aerodynamic particle sizer. Aerosol spatial distribution was determined by collecting filter samples at several chamber locations. The particle size distribution was determined by collecting cascade impactor samples at several chamber locations. The estimated chamber leak rate was within standards suggested in the literature. The measured average aerosol residence time was similar to theoretical aerosol residence time, suggesting that the chamber was mixing well. Additionally, the average concentration measured at each of the sampling locations within the chamber was similar, and the within-run coefficients of variation (CV) across all sampling locations was similar to those reported in previously published studies, again suggesting that the aerosol concentration throughout the chamber was uniform. The particle size distribution was similar throughout the exposure chamber. Additionally, the BSA concentration and particle size distributions measured in the breathing zone of the simulated head were not significantly different from measurements made elsewhere in the chamber, suggesting that respiration does not affect the average aerosol concentration or particle size distribution at the mouth.

In these studies, a serological correlate of protection against anthrax was identified in New Zealand white (NZW) rabbits that had been given one or two injections of various amounts of recombinant protective antigen (rPA) combined with aluminum hydroxide adjuvant (Alhydrogel). Rabbits were subsequently challenged by the aerosol route with spores of the Ames isolate of Bacillus anthracis. Results suggested that the antibody response, as determined by the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay, were significant predictors (P

The current human plague vaccine, a killed Yersinia pestis whole-cell preparation, does not protect against aerosol challenge and is reactogenic and antigenically undefined. Live attenuated Y. pestis, such as pigmentation-deficient (Pgm-) strains, have been used frequently as vaccines and are efficacious. They are used widely in plague research and assumed to be safe. However, they can cause serious adverse reactions, and their aerosol infectivity is not known. We tested the virulence of a defined Pgm- variant of the C092 strain of Y. pestis in mouse and non-human primate models of pneumonic plague. The ten-fold lower median lethal dose by the aerosol compared to the subcutaneous (s.c.) routes of the Pgm- strain in mice suggested that the Pgm- strain might be less attenuated by the former than by the latter route. After exposure of 16 African green monkeys to inhaled doses ranging from 1.1 x 10(4) to 8.1 x 10(7)cfu, eight died and eight survived. The terminal cultures collected from five of the non-survivors were all positive for Y. pestis. Two of the remaining three non-survivors were culture-negative but had pathologic and immunologic evidence of infection with Y. pestis, specimens could not be obtained nor the cause of death determined for the third one. The deaths were not dose-related, and there were some differences in the pathology associated with infection by the Pgm- strain compared to the wild-type (wt) strain. However, the Pgm- derivative was clearly virulent for monkeys by the aerosol route. A mutant of the Pgm- strain, which has a deletion in the plasminogen activator (Pla) virulence locus (pla), appeared to be more attenuated than was either the Pgm- single mutant (in NHPs and mice) or the Pla- single mutant strain (in mice) and has potential as a live vaccine.

The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin. Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B. anthracis. Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups. There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified. New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs. AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates. Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol. AVA protected 80 and 100% of the animals from these two isolates. These studies demonstrated that, although AVA confers variable protection against different B. anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques.

In this paper we describe the principles that have guided our design and the experience we have gained building multi-wire proportional chambers detectors for the ultra-cold neutron (UCN) source at the Los Alamos Neutron Science Center (LANSCE). Simple robust detectors with 50 cm2 of active area have been designed. These have been used both in ion chamber and proportional mode for the detection of UCN.

A mouse model was developed to evaluate the efficacy of antibiotic treatment of pneumonic plague; streptomycin was compared to antibiotics with which there is little or no clinical experience. Infection was induced by inhalation of aerosolized Yersinia pestis organisms. Antibiotics were administered by intraperitoneal injection every 6 hours for 5 days, at doses that produced levels of drug in serum comparable to those observed in humans treated for other serious infections. These studies compared in vitro to in vivo activity and evaluated the efficacy of antibiotics started at different times after exposure. Early treatment (started 24 h after challenge, when 0 of 10 mice tested had positive blood cultures) with netilmicin, ciprofloxacin, ofloxacin, ceftriaxone, ceftazidime, aztreonam, ampicillin, and rifampin (but not cefazolin, cefotetan, or ceftizoxime) demonstrated efficacy comparable to streptomycin. Late treatment (started 42 h after exposure, when five of five mice tested had positive blood cultures) with netilmicin, ciprofloxacin, ofloxacin, and a high dose (20 mg/kg of body weight every 6 h) of gentamicin produced survival rates comparable to that with streptomycin, while all of the beta-lactam antibiotics (cefazolin, cefotetan, ceftriaxone, ceftazidime, aztreonam, and ampicillin) and rifampin were significantly inferior to streptomycin. In fact, all groups of mice treated late with beta-lactam antibiotics experienced accelerated mortality rates compared to normal-saline-treated control mice. These studies indicate that netilmicin, gentamicin, ciprofloxacin, and ofloxacin may be alternatives for the treatment of pneumonic plague in humans. However, the beta-lactam antibiotics are not recommended, based upon poor efficacy in this mouse model of pneumonic plague, particularly when pneumonic plague may be associated with bacteremia.

Parenteral vaccination with ricin toxoid, although protective against death after a lethal aerosol ricin challenge, only partially protects against lung lesions. Therefore, we tested whether passive protection with aerosolized specific anti-ricin IgG (goat polyclonal, affinity-purified) could protect against both lethality and lung lesions in unvaccinated mice. Healthy CD-l mice were administered antibody (Ab) by small particle aerosol. Group 1 received non-specific control Ab (2160 mg/min/m3), and groups 2 and 3 received anti-ricin IgG (960 and 3280 mg/min/m3, respectively). Each group was challenged with a lethal dose of aerosolized ricin 1 hr after Ab exposure. All group 1 (control Ab) mice developed diffuse airway epithelial necrosis, with severe interstitial edema and inflammation involving all lung lobes, and died 48-96 hr post-challenge (PC). In contrast, in groups 2 and 3 at 24 hr PC, lung lesions were absent to very mild although there was rare epithelial necrosis in the upper airways in both groups. By 48 hr PC, necrosis of the tracheal epithelium and peritracheal inflammation were noted in some group 3 mice only. By 4 days PC, lungs and airways did not differ from cage controls in most group 2 and 3 mice. Weight gain in group 2 and 3 mice paralleled that of control mice. At 14 days PC, lungs were no different in controls than in group 3 mice. However, two non-survivors in group 3 had obstructions due to proximal airway epithelial damage. All group 2 mice survived, although a mild lymphoplasmacytic perivasculitis was present at 14 days PC which was not noted in the group 3 mice.

Yersinia pestis, the causative agent of plague, possesses multiple virulence determinants encoded on its three plasmids and on its chromosome. We evaluated the role of the protein capsule F1 in virulence an immunity against plague. Strains lacking F1, either those that are naturally occurring or those with genetically defined nonpolar mutations in the structural gene, retained their virulence for mice and nonhuman primates. However, both active immunization with F1, from either a recombinant vector or Y. pestis, and passive immunization with F1 monoclonal antibody protected mice from experimental infection with wild-type F1-positive organisms. These results suggest that protective immunogens like F1 need not be essential for virulence. The rare isolation of virulent F1-negative organisms from F1-immunized animals infected with F1-positive strains supports this conclusion and also suggests that, in addition to F1, an optimal vaccine against plague should include essential virulence factors as immunogens.

Previous studies have demonstrated that aerosol sampling devices can have deleterious effects on bacteria due to stresses intrinsic to the sampling processes. Although a significant amount of work has been carried out to develop animal models of inhalational melioidosis, little information has been reported on the effects of the aerosol sampling devices on the causative bacterium, Burkholderia pseudomallei. The aim of this study was to compare the efficiencies for collection of aerosolized bacteria in three sampling devices that have been used in studies utilizing aerosolized B. pseudomallei. The data from this study demonstrate the equivalence of the Mercer impactor, gelatin filter, and model 7541 AGI for sampling respirable aerosols containing B. pseudomallei across a range of aerosol concentrations. It was also determined that the retention efficiency of gelatin filters for culturable B. pseudomallei was near unity, suggesting that desiccation of collected material did not occur for the short sampling period tested. The retention efficiency of the model 7541 AGI for culturable B. pseudomallei was significantly less than unity, and it was determined that this decrease was likely due to the stresses associated with repetitive sampler bubbling. The results of this study also confirmed the results of previous studies on the deleterious effects of the Collison nebulizer on microorganisms and extended these data to include B. pseudomallei.

Long-term protection of rabbits that had been vaccinated with two doses of a recombinant protective antigen (rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival was 74.1% (20/27) with quantitative ELISA titer of 22.3g of anti-rPA IgG per millilitre and toxin neutralizing antibody (TNA) assay titer of 332. At 12 months after the primary injection, only 37.5% (9/24) of the rabbits were protected with quantitative ELISA titer of 19.8 g of anti-rPA IgG per millilitre and TNA assay titer of 286. There was a significant loss of protection ( p = 0.0117) and a significant difference in survival curves (p = 0.0157) between the 6- and 12-month groups. When ELISA or TNA assay titer, gender, and challenge dose were entered into a forward logistic regression model, week 26 ELISA titer (p = 0.0236) and week 13 TNA assay titer (p = 0.0147) for the 6-month group, and week 26 ELISA titer (p = 0.0326) and week 8 TNA assay titer (p = 0.0190) for the 12-month group, were significant predictors of survival. Neither gender nor challenge dose were identified as having a statistically significant effect on survival. Booster vaccinations with rPA may be required for the long-term protection of rabbits against anthrax. Published by Elsevier Ltd.

Preliminary evidence gathered in rodents and livestock suggested that a phase I chloroform:methanol residue (CMR) extracted vaccine was safe and efficacious in protecting these animals from challenge with the obligate phagolysosomal pathogen (Coxiella burnetii). Prior to the initiation of phase II studies in human volunteers, we compared, in non-human primates (Macaca fascicularis), the efficacy of CMR vaccine with Q-Vax, a licensed cellular Australian Q fever vaccine that has been demonstrated to provide complete protection in human volunteers. Vaccine efficacy was assessed by evaluating thoracic radiographs and the presence of fever and bacteremia in monkeys challenged by aerosol with Coxiella burnetii. Changes in blood chemistries, hematology, behavior and pulmonary function were also examined. CMR, whether administered in single 30 or 100 microg doses or two 30 microg subcutaneous doses, gave equivalent protection in vaccine recipients as a single 30 microg dose of Q-Vax. In addition, vaccination resulted in significant, although temporary, increases in specific antibody titers against C. burnetii phases I and II antigens. The C. burnetii CMR vaccine may be an efficacious alternative to cellular Q fever vaccines in humans.

A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals were inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed (AVA) ranging from a human dose to a 1:256 dilution in phosphate-buffered saline (PBS). At 6 and 10 weeks, both the quantitative anti-protective antigen (PA) IgG ELISA and the toxin-neutralizing antibody (TNA) assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose (84-133 LD(50)) of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P

The incidence of anthrax in humans is extremely low. Human vaccine efficacy studies for inhalational anthrax cannot be conducted. The identification of a correlate of protection that predicts vaccine efficacy is crucial for determining the immune status of immunized humans. This surrogate marker of immunity can only be established by using an appropriate animal model. Numerous studies showed that protective antigen (PA) is the principle protective antigen in naturally- or vaccine-induced immunity. However, attempts to correlate the quantity of anti-PA antibodies with protective immunity in the guinea pig model for anthrax and various vaccine formulations have failed. In these studies, we used the licensed anthrax vaccine adsorbed (AVA) in rabbits. Groups of New Zealand white rabbits, 10 or 20 per group, were immunized intramuscularly (two doses, 4 weeks apart) with varying doses of AVA, ranging from a human dose to 1:256 dilution in sterile phosphate-buffered saline (PBS). Control rabbits received PBS/Alhydrogel according to the same schedule. Each rabbit was bled 2 weeks after the second dose, and antibody levels to PA measured by both the quantitative anti-PA IgG ELISA and the toxin-neutralizing antibody (TNA) assay. Rabbits were aerosol-challenged 10 weeks from day 0 with a lethal dose of Ames spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. All the controls died. Rabbit survival was compared with the antibody response. Statistical models were used to test for significance of the peak antibody responses to predict survival. Results showed that both the amount of anti-PA IgG and TNA titres present in the sera at the time of the peak antibody response were significant (P0.0001) predictors of survival. These results demonstrate that the humoral immune response to AVA can predict protection in the rabbit model of inhalational anthrax.

The authors examined the efficacy of Bacillus anthracis protective antigen (PA) combined with adjuvants as vaccines against an aerosol challenge of virulent anthrax spores in rhesus macaques. Adjuvants tested included i) aluminum hydroxide (Alhydrogel), ii) saponin QS-21 and iii) monophosphoryl lipid A (MPL) in squalene/ lecithin/Tween 80 emulsion (SLT). Animals were immunized once with either 50 μg of recombinant PA plus adjuvant, or with Anthrax Vaccine Adsorbed (AVA), the licensed human anthrax vaccine. The serological response to PA was measured by enzymelinked immunosorbent assay. Lymphocyte proliferation and serum neutralization of in vitro lethal toxin cytotoxicity were also assayed. In all vaccine groups, anti-PA IgM and IgG titers peaked at 2 weeks and 4–5 weeks postimmunization, respectively. Five weeks postimmunization, animals in all vaccine groups demonstrated PA-specific lymphocyte proliferation and sera that neutralized in vitro cytotoxicity. Six weeks after immunization, the animals were challenged by aerosol with approximately 93 LD 50 of virulent anthrax spores. Animals were bled daily for 1 week to monitor bacteremia, and deaths were recorded. Anti-PA ELISA titers in all groups of immunized animals were substantially increased 2 weeks after challenge. One dose of each vaccine provided significant protection ( > 90%) against inhalation anthrax in the rhesus macaques.

Inhalation anthrax is a rare disease that is almost invariably fatal. This study determined whether a prolonged course of postexposure antibiotics with or without vaccination would protect monkeys exposed to a lethal aerosol dose of Bacillus anthracis when the antibiotic was discontinued. Beginning 1 day after exposure, groups of 10 animals were given penicillin, ciprofloxacin, doxycycline, doxycycline plus vaccination, vaccination alone, or saline. Antibiotics were administered for 30 days and then discontinued. Vaccine was given on days 1 and 15. Two animals died of causes other than anthrax and were not included in the statistical analysis. Nine of 10 controls and 8 of 10 animals given only vaccine died. Each antibiotic regimen completely protected animals while on therapy and provided significant long-term protection upon discontinuance of the drug (penicillin, 7 of 10 survived, P < .02; ciprofloxacin, 8 of 9 survived, P < .002; doxycycline, 9 of 10 survived, P < .002; doxycycline plus vaccination, 9 of 9 survived, P < .0002). Protection against rechallenge was provided by combining postexposure antibiotic treatment with vaccination.

Erratum Erratum to “Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin” [Vaccine 19 (2001) 3241–3247] P.F. Fellows a, M.K. Linscott a, B.E. Ivins a, M.L.M. Pitt b, C.A. Rossi c, P.H. Gibbs d, A.M. Friedlander a a Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA b Toxinology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA c Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA d Biometrics and Information Management Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA