Lassa antiviral LHF-535 protects guinea pigs from lethal challenge.

LHF-535 is a small molecule antiviral currently in development for the treatment of Lassa fever, a zoonotic disease endemic in West Africa that generates significant morbidity and mortality. Current treatment options are inadequate, and there are no approved therapeutics or vaccines for Lassa fever. LHF-535 was evaluated in a lethal guinea pig model of Lassa pathogenesis, using once-daily administration of a fixed dose (50 mg/kg/day) initiating either 1 or 3 days after inoculation with a lethal dose of Lassa virus. LHF-535 reduced viremia and clinical signs and protected all animals from lethality. A subset of surviving animals was rechallenged four months later with a second lethal challenge of Lassa virus and were found to be protected from disease. LHF-535 pharmacokinetics at the protective dose in guinea pigs showed plasma concentrations well within the range observed in clinical trials in healthy volunteers, supporting the continued development of LHF-535 as a Lassa therapeutic.

Immune-Mediated Systemic Vasculitis as the Proposed Cause of Sudden-Onset Sensorineural Hearing Loss following Lassa Virus Exposure in Cynomolgus Macaques.

Lassa virus (LASV) causes a severe, often fatal hemorrhagic disease in regions in Africa where the disease is endemic, and approximately 30% of patients develop sudden-onset sensorineural hearing loss after recovering from acute disease. The causal mechanism of hearing loss in LASV-infected patients remains elusive. Here, we report findings after closely examining the chronic disease experienced by surviving macaques assigned to LASV exposure control groups in two different studies. All nonhuman primates (NHPs) developed typical signs and symptoms of Lassa fever, and seven succumbed during the acute phase of disease. Three NHPs survived beyond the acute phase and became chronically ill but survived to the study endpoint, 45 days postexposure. All three of these survivors displayed continuous disease symptoms, and apparent hearing loss was observed using daily subjective measurements, including response to auditory stimulation and tuning fork tests. Objective measurements of profound unilateral or bilateral sensorineural hearing loss were confirmed for two of the survivors by brainstem auditory evoked response (BAER) analysis. Histologic examination of inner ear structures and other tissues revealed the presence of severe vascular lesions consistent with systemic vasculitides. These systemic immune-mediated vascular disorders have been associated with sudden hearing loss. Other vascular-specific damage was also observed to be present in many of the sampled tissues, and we were able to identify persistent virus in the perivascular tissues in the brain tissue of survivors. Serological analyses of two of the three survivors revealed the presence of autoimmune disease markers. Our findings point toward an immune-mediated etiology for Lassa fever-associated sudden-onset hearing loss and lay the foundation for developing potential therapies to prevent and/or cure Lassa fever-associated sudden-onset hearing loss.IMPORTANCE Lassa virus is one of the most common causes of viral hemorrhagic fever. A frequent, but as yet unexplained, consequence of infection with Lassa virus is acute, sudden-onset sensorineural hearing loss in one or both ears. Deafness is observed in approximately 30% of surviving Lassa fever patients, an attack rate that is approximately 300% higher than mumps virus infection, which was previously thought to be the most common cause of virus-induced deafness. Here, we provide evidence from Lassa virus-infected cynomolgus macaques implicating an immune-mediated vasculitis syndrome underlying the pathology of Lassa fever-associated deafness. These findings could change the way human Lassa fever patients are medically managed in order to prevent deafness by including diagnostic monitoring of human survivors for onset of vasculitides via available imaging methods and/or other diagnostic markers of immune-mediated vascular disease.

DNA vaccines elicit durable protective immunity against individual or simultaneous infections with Lassa and Ebola viruses in guinea pigs.

We previously developed optimized DNA vaccines against both Lassa fever and Ebola hemorrhagic fever viruses and demonstrated that they were protective individually in guinea pig and nonhuman primate models. In this study, we vaccinated groups of strain 13 guinea pigs two times, four weeks apart with 50 µg of each DNA vaccine or a mock vaccine at discrete sites by intradermal electroporation. Five weeks following the second vaccinations, guinea pigs were exposed to lethal doses of Lassa virus, Ebola virus, or a combination of both viruses simultaneously. None of the vaccinated guinea pigs, regardless of challenge virus and including the coinfected group, displayed weight loss, fever or other disease signs, and all survived to the study endpoint. All of the mock-vaccinated guinea pigs that were infected with Lassa virus, and all but one of the EBOV-infected mock-vaccinated guinea pigs succumbed. In order to determine if the dual-agent vaccination strategy could protect against both viruses if exposures were temporally separated, we held the surviving vaccinates in BSL-4 for approximately 120 days to perform a cross-challenge experiment in which guinea pigs originally infected with Lassa virus received a lethal dose of Ebola virus and those originally infected with Ebola virus were infected with a lethal dose of Lassa virus. All guinea pigs remained healthy and survived to the study endpoint. This study clearly demonstrates that DNA vaccines against Lassa and Ebola viruses can elicit protective immunity against both individual virus exposures as well as in a mixed-infection environment.

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever.

Lassa virus (LASV) is an ambisense RNA virus in the Arenaviridae family and is the etiological agent of Lassa fever, a severe hemorrhagic disease endemic to West and Central Africa. 1,2 There are no US Food and Drug Administration (FDA)-licensed vaccines available to prevent Lassa fever. 1,2 in our previous studies, we developed a gene-optimized DNA vaccine that encodes the glycoprotein precursor gene of LASV (Josiah strain) and demonstrated that 3 vaccinations accompanied by dermal electroporation protected guinea pigs from LASV-associated illness and death. Here, we describe an initial efficacy experiment in cynomolgus macaque nonhuman primates (NHPs) in which we followed an identical 3-dose vaccine schedule that was successful in guinea pigs, and a follow-on experiment in which we used an accelerated vaccination strategy consisting of 2 administrations, spaced 4 weeks apart. In both studies, all of the LASV DNA-vaccinated NHPs survived challenge and none of them had measureable, sustained viremia or displayed weight loss or other disease signs post-exposure. Three of 10 mock-vaccinates survived exposure to LASV, but all of them became acutely ill post-exposure and remained chronically ill to the study end point (45 d post-exposure). Two of the 3 survivors experienced sensorineural hearing loss (described elsewhere). These results clearly demonstrate that the LASV DNA vaccine combined with dermal electroporation is a highly effective candidate for eventual use in humans.

Dose Response of MARV/Angola Infection in Cynomolgus Macaques following IM or Aerosol Exposure.

Marburg virus infection in humans causes a hemorrhagic disease with a high case fatality rate. Countermeasure development requires the use of well-characterized animal models that mimic human disease. To further characterize the cynomolgus macaque model of MARV/Angola, two independent dose response studies were performed using the intramuscular or aerosol routes of exposure. All animals succumbed at the lowest target dose; therefore, a dose effect could not be determined. For intramuscular-exposed animals, 100 PFU was the first target dose that was not significantly different than higher target doses in terms of time to disposition, clinical pathology, and histopathology. Although a significant difference was not observed between aerosol-exposed animals in the 10 PFU and 100 PFU target dose groups, 100 PFU was determined to be the lowest target dose that could be consistently obtained and accurately titrated in aerosol studies.

Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation.

Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development.

Evaluation of urothelial stretch-induced cyclooxygenase-2 expression in a mouse ureteral obstruction model.

Prostanoids play a major role in the nociceptive response to ureteral obstruction. Cyclooxygenases (COXs) 1 and 2 catalyze the rate-limiting step in prostanoid synthesis; COX-2 is the more inducible isoform. Previous studies in human and animal models have shown that COX-2 is highly induced during ureteral obstruction. Our objective was to characterize acute COX-2 induction in a reproducible mouse model. Unilateral ureteral ligation was performed, and obstruction was maintained for 2, 4, 6, 8, or 12 hours. We evaluated COX-2 protein expression using Western immunoblotting, and found that ureteral obstruction induced COX-2 expression ninefold within 6 hours. This is the first report to characterize in vivo temporal stretch-induced COX-2 expression in a mouse model. This model will be critical for elucidation of COX-2 signaling pathways and may eventually help to identify novel therapeutic targets for treating ureteral obstruction.

A novel murine model for the study of human renal cryoablation.

OBJECTIVE: to develop a reproducible, non-debilitating in vivo murine model of human renal cryoablation using a standard closed argon-delivery system. MATERIALS AND METHODS: Custom engineered 2-mm conical tip cryoprobes for use on the standard argon-based cryoablation unit (Endocare, Inc. Irvine, CA, USA) were used to create small controllable iceballs (−160 °C) in the mouse kidney. The time to create a 4-mm cryolesion was compared using a contact vs puncture technique in 10 mice. To show consistency of the induced-freeze injury, a 4-mm iceball was created in 20 murine renal units and the time to creation and the size of the resultant cryolesion measured. To investigate lesion regression and histological changes, we created a 4-mm renal cryolesion in 28 mice and killed four each at 1, 3, 7, 14, 21, 28, and 35 days. The measured coronal cross-sectional area of the cryoablation site at necroscopy was compared to the initial calculated area as a percentage. To assess renal preservation, blood urea nitrogen (BUN) and creatinine levels at 1 week after cryoablation or sham ablation was compared (10 mononephric mice in each group). RESULTS: The time to create the desired iceball was 1.9 times quicker using the puncture vs the contact technique. The mean (sd) time to forming a 4-mm iceball was 35.3 (4.8) s with a mean maximum length of the resultant post-thaw injury of 5.7 (0.5) mm and a 9% coefficient of variance. Regression analysis of the two-dimensional cross-sectional coronal area of the cryolesion showed a statistically significant linear pattern of regression over time (P = 0.037) and classic histological findings. There was no significant difference in the BUN or creatinine levels in mononephric mice 1 week after cryotherapy compared with the sham-ablated controls. CONCLUSIONS: We describe a reproducible, non-debilitating, easily manipulated murine model for the study of human renal cryoablation.

Elastographic measurements of in-vivo radiofrequency ablation lesions of the kidney.

BACKGROUND AND PURPOSE: Elastography may prove useful for monitoring radiofrequency ablative (RFA) therapy because heat-ablated tissues are more elastic than untreated tissues. Herein, we report our initial evaluations of the reliability of elastography for delineating thermal-lesion boundaries at the time of RFA of porcine kidneys. MATERIALS AND METHODS: In-vivo RFA was performed on 20 kidneys from 10 40-kg female pigs. Elastography was performed at the time of surgery and after 48 hours. The imaging plane was perpendicular to the axis of the RF electrode so that the ablated region was around the center of the plane. Measurements of the sections representing the same image plane used for elastography were taken at pathologic examination and compared with the measurements obtained from the elastograms. RESULTS: We found a statistically significant correlation between elastography and pathology measurements with respect to the area and volume estimates (r = 0.9302 and r = 0.953, respectively). Overall, elastography slightly underestimated the lesion size, as judged by the digitalized pathologic images, a finding consistent with previous reports. CONCLUSION: We found a correlation between the area and volume estimates of thermal lesions that were based on elastographic images and the measurements from gross pathologic dimensions. A significant limitation of renal RFA is the inaccuracy of current imaging modalities to provide real-time monitoring, and elastography may prove to be reliable for delineating the resulting thermal lesions.

Third prize: Prostaglandin E(2)-3 receptor is involved in ureteral contractility in obstruction.

BACKGROUND AND PURPOSE: We previously found that prostaglandin (PG) E2 contracts acutely obstructed ureters while relaxing normal ureters. This study investigated the procontractile effects of the PG EP3 receptor in PGE(2)-mediated contractility in obstructed and normal porcine ureters. MATERIALS AND METHODS: We created unilateral ureteral obstruction laparoscopically using titanium clips in farm pigs; the contralateral ureters were dissected as sham controls. Ureters were harvested 48 hours post-obstruction, cut into 5-mm segments, and suspended in water-jacketed tissue baths in Krebs buffer. Tissues were equilibrated for 1 hour, and spontaneous contractile rates were recorded. After 2 hours of incubation in Krebs (controls) or pertussis toxin (G(alpha)i signaling-protein inhibitor [EP-3 blockade]) 500 ng/mL, a concentration- response curve (10(-9) M-10(-5) M) to PGE(2), PGF(2), sulprostone (EP 3 agonist), or 0.01% ethanol (vehicle) was created (N = 4). RESULTS: In the normal ureters, PGE(2) relaxed both pertussis toxin-treated and control tissues. In obstructed segments, PGE(2) increased contractions by 60%; this was reversed by pertussis toxin to a 67% reduction in contractile rate. In both obstructed and contralateral segments, sulprostone induced contractility in the controls; this was attenuated by pertussis toxin. The PGF(2) produced a contractile effect in both the controls and the pertussis toxin-treated segments, demonstrating the selectivity of pertussis toxin for EP3 receptors. CONCLUSION: Our data indicate that the EP3 receptor is involved in hypercontractility during ureteral obstruction. However, it may not be the sole factor behind the condition-dependent effect of PGE(2).

Haemostatic partial nephrectomy using bipolar radiofrequency ablation.

OBJECTIVE: To determine whether an electrode array with a bipolar radiofrequency ablation (RFA) energy source can be used to perform a haemostatic partial nephrectomy by simultaneously ablating and coagulating renal tissue. MATERIALS AND METHODS: Lower-pole partial nephrectomy was performed in 12 porcine kidneys using a bipolar RFA system. Intraoperative ultrasonography was used to identify and avoid the collecting system. Tissues were positioned between opposing electrodes and tissue impedance monitored using a proprietary feedback and control algorithm. Ablation time and power, lesion width and length, and tissue thickness were recorded. The kidneys were assessed in vivo to show haemostasis of the remaining renal unit. Collecting system integrity was assessed with methylene blue injection, and the resected tissue analysed histologically. RESULTS: Partial nephrectomies were successful in all 12 porcine kidneys; the mean nephrectomy specimen was 3.2 x 2.6 cm. The total ablation time (sem) per lesion was 211 (15) s and the mean power was 23 W. Methylene blue injection showed an intact collecting system in 11 of the 12 kidneys, and haematoxylin and eosin staining showed a mean zone of necrosis of 9 mm at the resection margin. Ultrasonography revealed flow to the remaining kidneys after RFA and the in vivo assessment of haemostasis revealed no abnormal bleeding or haemorrhage from the kidneys. CONCLUSIONS: Applying bipolar RF energy to an electrode array can enable transmural excision of renal parenchyma in vivo in a bloodless fashion without collecting system injury.