[18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection.

Real-time bioimaging of infectious disease processes may aid countermeasure development and lead to an improved understanding of pathogenesis. However, few studies have identified biomarkers for monitoring infections using in vivo imaging. Previously, we demonstrated that positron emission tomography/computed tomography (PET/CT) imaging with [18F]-fluorodeoxyglucose (FDG) can monitor monkeypox disease progression in vivo in nonhuman primates (NHPs). In this study, we investigated [18F]-FDG-PET/CT imaging of immune processes in lymphoid tissues to identify patterns of inflammation in the monkepox NHP model and to determine the value of [18F]-FDG-PET/CT as a biomarker for disease and treatment outcomes. Quantitative analysis of [18F]-FDG-PET/CT images revealed differences between moribund and surviving animals at two sites vital to the immune response to viral infections, bone marrow and lymph nodes (LNs). Moribund NHPs demonstrated increased [18F]-FDG uptake in bone marrow 4 days postinfection compared to surviving NHPs. In surviving, treated NHPs, increase in LN volume correlated with [18F]-FDG uptake and peaked 10 days postinfection, while minimal lymphadenopathy and higher glycolytic activity were observed in moribund NHPs early in infection. Imaging data were supported by standard virology, pathology, and immunology findings. Even with the limited number of subjects, imaging was able to differentiate the difference between disease outcomes, warranting additional studies to demonstrate whether [18F]-FDG-PET/CT can identify other, subtler effects. Visualizing altered metabolic activity at sites involved in the immune response by [18F]-FDG-PET/CT imaging is a powerful tool for identifying key disease-specific time points and locations that are most relevant for pathogenesis and treatment.IMPORTANCE Positron emission tomography and computed tomography (PET/CT) imaging is a universal tool in oncology and neuroscience. The application of this technology to infectious diseases is far less developed. We used PET/CT imaging with [18F]-labeled fluorodeoxyglucose ([18F]-FDG) in monkeys after monkeypox virus exposure to monitor the immune response in lymphoid tissues. In lymph nodes of surviving monkeys, changes in [18F]-FDG uptake positively correlated with enlargement of the lymph nodes and peaked on day 10 postinfection. In contrast, the bone marrow and lymph nodes of nonsurvivors showed increased [18F]-FDG uptake by day 4 postinfection with minimal lymph node enlargement, indicating that elevated cell metabolic activity early after infection is predictive of disease outcome. [18F]-FDG-PET/CT imaging can provide real-time snapshots of metabolic activity changes in response to viral infections and identify key time points and locations most relevant for monitoring the development of pathogenesis and for potential treatment to be effective.

Animal Models of Ebolavirus Infection.

Ebola virus is a highly pathogenic member of the family Filoviridae that causes a severe hemorrhagic disease in humans and NHP. The 2013-2016 West African outbreak has increased interest in the development and refinement of animal models of Ebola virus disease. These models are used to test countermeasures and vaccines, gain scientific insights into the mechanisms of disease progression and transmission, and study key correlates of immunology. Ebola virus is classified as a BSL4 pathogen and Category A agent, for which the United States government requires preparedness in case of bioterrorism. Rodents, such as Syrian golden hamsters (Mesocricetus auratus), mice (Mus musculus), and guinea pigs (Cavia porcellus), are the most common research species. However, NHP, especially macaques, are favored for Ebola virus disease research due to similarities with humans regarding the pathogenesis, clinical presentation, laboratory findings, and causes of fatality. To satisfy the regulatory requirements for approval of countermeasures against high-consequence pathogens, the FDA instituted the Animal Rule, which permits efficacy studies in animal models in place of human clinical data when such studies are not feasible or ethical. This review provides a comprehensive summary of various animal models and their use in Ebola virus disease research.

Divergent Simian Arteriviruses Cause Simian Hemorrhagic Fever of Differing Severities in Macaques.

UNLABELLED: Simian hemorrhagic fever (SHF) is a highly lethal disease in captive macaques. Three distinct arteriviruses are known etiological agents of past SHF epizootics, but only one, simian hemorrhagic fever virus (SHFV), has been isolated in cell culture. The natural reservoir(s) of the three viruses have yet to be identified, but African nonhuman primates are suspected. Eleven additional divergent simian arteriviruses have been detected recently in diverse and apparently healthy African cercopithecid monkeys. Here, we report the successful isolation in MARC-145 cell culture of one of these viruses, Kibale red colobus virus 1 (KRCV-1), from serum of a naturally infected red colobus (Procolobus [Piliocolobus] rufomitratus tephrosceles) sampled in Kibale National Park, Uganda. Intramuscular (i.m.) injection of KRCV-1 into four cynomolgus macaques (Macaca fascicularis) resulted in a self-limiting nonlethal disease characterized by depressive behavioral changes, disturbance in coagulation parameters, and liver enzyme elevations. In contrast, i.m. injection of SHFV resulted in typical lethal SHF characterized by mild fever, lethargy, lymphoid depletion, lymphoid and hepatocellular necrosis, low platelet counts, increased liver enzyme concentrations, coagulation abnormalities, and increasing viral loads. As hypothesized based on the genetic and presumed antigenic distance between KRCV-1 and SHFV, all four macaques that had survived KRCV-1 injection died of SHF after subsequent SHFV injection, indicating a lack of protective heterotypic immunity. Our data indicate that SHF is a disease of macaques that in all likelihood can be caused by a number of distinct simian arteriviruses, although with different severity depending on the specific arterivirus involved. Consequently, we recommend that current screening procedures for SHFV in primate-holding facilities be modified to detect all known simian arteriviruses. IMPORTANCE: Outbreaks of simian hemorrhagic fever (SHF) have devastated captive Asian macaque colonies in the past. SHF is caused by at least three viruses of the family Arteriviridae: simian hemorrhagic fever virus (SHFV), simian hemorrhagic encephalitis virus (SHEV), and Pebjah virus (PBJV). Nine additional distant relatives of these three viruses were recently discovered in apparently healthy African nonhuman primates. We hypothesized that all simian arteriviruses are potential causes of SHF. To test this hypothesis, we inoculated cynomolgus macaques with a highly divergent simian arterivirus (Kibale red colobus virus 1 [KRCV-1]) from a wild Ugandan red colobus. Despite being only distantly related to red colobuses, all of the macaques developed disease. In contrast to SHFV-infected animals, KRCV-1-infected animals survived after a mild disease presentation. Our study advances the understanding of an important primate disease. Furthermore, our data indicate a need to include the full diversity of simian arteriviruses in nonhuman primate SHF screening assays.

Evaluation of monkeypox disease progression by molecular imaging.

Infection of nonhuman primates (NHPs) with monkeypox virus (MPXV) is currently being developed as an animal model of variola infection in humans. We used positron emission tomography and computed tomography (PET/CT) to identify inflammatory patterns as predictors for the outcome of MPXV disease in NHPs. Two NHPs were sublethally inoculated by the intravenous (IV) or intrabronchial (IB) routes and imaged sequentially using fluorine-18 fluorodeoxyglucose ((18)FDG) uptake as a nonspecific marker of inflammation/immune activation. Inflammation was observed in the lungs of IB-infected NHPs, and bilobular involvement was associated with morbidity. Lymphadenopathy and immune activation in the axillary lymph nodes were evident in IV- and IB-infected NHPs. Interestingly, the surviving NHPs had significant (18)FDG uptake in the axillary lymph nodes at the time of MPXV challenge with no clinical signs of illness, suggesting an association between preexisting immune activation and survival. Molecular imaging identified patterns of inflammation/immune activation that may allow risk assessment of monkeypox disease.

Simian hemorrhagic fever virus infection of rhesus macaques as a model of viral hemorrhagic fever: clinical characterization and risk factors for severe disease.

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.

Intracardiac thrombosis and aortic dissecting aneurysms in mustached tamarins (Saguinus mystax) with cardiomyopathy.

Spontaneous intracardiac thrombosis is rarely reported in animals, particularly nonhuman primates. The finding of 2 cases of intracardiac thrombi in mustached tamarins (Saguinus mystax) that died as a consequence of congestive heart failure prompted us to do a retrospective study to determine the frequency of this condition. Clinical records, necropsy reports, and tissues from 60 mustached tamarins that died or were euthanized between 1996 and 2009 were reviewed. Of the 60 monkeys whose cases were reviewed, 10 (16.6%) had intracardiac thrombi, and 4 (6.6%) had dissecting aortic aneurysms. Of the 10 animals with intracardiac thrombosis, 3 had left ventricular involvement alone; 4 monkeys had thrombi only in the right ventricle, and the remaining 3 animals exhibited thrombi in both ventricles. Myocardial fibrosis and chronic renal disease were common findings in affected animals. The causes of the intracardiac thrombosis in the tamarins in the present study are not known, but the clinical signs and gross and microscopic lesions suggest that congestive heart failure secondary to cardiomyopathy is the primary contributor. In addition, the cause of the aortic dissecting aneurysms in the tamarins in this study is not known. Further studies are required to determine whether factors including aortic curvature, genetic background, or hypertension-alone or in combination-play a role. To our knowledge, the current retrospective study is the first report of intracardiac thrombosis and aortic aneurysms in mustached tamarins.

Comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route.

Monkeypox virus (MPXV) infection has recently expanded in geographic distribution and can be fatal in up to 10% of cases. The intravenous (i.v.) inoculation of nonhuman primates (NHPs) results in an accelerated fulminant disease course compared to that of naturally occurring MPXV infection in humans. Alternative routes of inoculation are being investigated to define an NHP model of infection that more closely resembles natural disease progression. Our goal was to determine if the intrabronchial (i.b.) exposure of NHPs to MPXV results in a systemic disease that better resembles the progression of human MPXV infection. Here, we compared the disease course following an i.v. or i.b. inoculation of NHPs with 10-fold serial doses of MPXV Zaire. Classical pox-like disease was observed in NHPs administered a high virus dose by either route. Several key events were delayed in the highest doses tested of the i.b. model compared to the timing of the i.v. model, including the onset of fever, lesion appearance, peak viremia, viral shedding in nasal and oral swabs, peak cytokine levels, and time to reach endpoint criteria. Virus distribution across 19 tissues was largely unaffected by the inoculation route at the highest doses tested. The NHPs inoculated by the i.b. route developed a viral pneumonia that likely exacerbated disease progression. Based on the observations of the delayed onset of clinical and virological parameters and endpoint criteria that may more closely resemble those of human MPXV infection, the i.b. MPXV model should be considered for the further investigation of viral pathogenesis and countermeasures.

Pretargeted radioimmunotherapy of pancreatic cancer xenografts: TF10-90Y-IMP-288 alone and combined with gemcitabine.

UNLABELLED: Pancreatic cancer is a silent disease that most commonly presents in an already metastatic form. Current treatment options provide little survival benefit. Radiolabeled PAM4 IgG, a monoclonal antibody that recognizes a unique epitope associated with a mucin found almost exclusively in pancreatic cancer, has shown encouraging therapeutic effects in animal models and in early clinical testing ((90)Y-humanized PAM4 IgG, (90)Y-clivatuzumab tetraxetan). The studies reported herein examine a new pretargeting procedure for delivering therapeutic radionuclides. METHODS: We prepared a humanized, recombinant tri-Fab bispecific monoclonal antibody (bsmAb) (TF10) using specificity for targeting pancreatic cancer of PAM4 and another Fab binding to a hapten (histamine-succinyl-glycine [HSG]) and tested this in a pretargeting setting with a (90)Y-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-di-HSG-peptide (pretargeted radioimmunotherapy [PT-RAIT]). Nude mice bearing established Capan-1 human pancreatic cancer xenografts were given TF10 and then received the (90)Y peptide as a single bolus dose 19 h later, or the therapy cycle was fractionated weekly. Other studies examined different combinations with gemcitabine. RESULTS: PT-RAIT of 18.5 MBq ( approximately 50% of its maximum tolerated dose [MTD]) was as effective as the MTD of (90)Y-PAM4 IgG (5.55 MBq). Three monthly doses of 9.25 MBq of PT-RAIT combined with a monthly cycle of gemcitabine (3 weekly, 6-mg doses) significantly enhanced survival, compared with PT-RAIT alone. Adding gemcitabine as a radiosensitizer to 9.25 MBq of PT-RAIT enhanced objective responses. Weekly fractionation of the PT-RAIT, as compared with a single treatment, improved responses. CONCLUSION: PAM4-based PT-RAIT with (90)Y hapten peptide is an effective treatment for pancreatic cancer, with less toxicity than (90)Y-PAM4 IgG, in this model. Combinations with gemcitabine and dose fractionation of the PT-RAIT enhanced therapeutic responses.

Therapeutic advantage of pretargeted radioimmunotherapy using a recombinant bispecific antibody in a human colon cancer xenograft.

PURPOSE: To assess if pretargeting, using a combination of a recombinant bispecific antibody (bsMAb) that binds divalently to carcinoembryonic antigen (CEA) and monovalently to the hapten histamine-succinyl-glycine and a (90)Y-peptide, improves therapeutic efficacy in a human colon cancer-nude mouse xenograft compared with control animals given (90)Y-humanized anti-CEA immunoglobulin G (IgG). EXPERIMENTAL DESIGN: Clearance and biodistribution were monitored by whole-body readings and necropsy. Animals were monitored for 34 weeks with a determination of residual disease and renal pathology in survivors. Hematologic toxicity was assessed separately in non-tumor-bearing NIH Swiss mice. RESULTS: Hematologic toxicity was severe at doses of 100 to 200 microCi of (90)Y-IgG, yet mild in the pretargeted animals given 500 or 700 microCi of the (90)Y-peptide. Evidence of end-stage renal disease was found at 900 microCi of the pretargeted (90)Y-peptide whereas animals given 700 microCi showed only mild renal pathology, similar to that seen in control animals given (90)Y-IgG. Biodistribution data indicated that the average amount of tumor radioactivity by a 700-microCi dose of the pretargeted peptide over a 96-hour period was increased 2.5-fold (48 microCi/g) compared with 150 microCi of (90)Y-IgG (18.9 microCi/g). At these doses, survival (i.e., time to progression to 2.5 cm(3)) was significantly improved (P < 0.04) compared with (90)Y-IgG, with ablation of about one third of the tumors, whereas viable tumor was present in all of the (90)Y-IgG-treated animals. CONCLUSION: Pretargeting increases the amount of radioactivity delivered to colorectal tumors sufficiently to improve the therapeutic index and responses as compared with conventional radioimmunotherapy.