Comparison of Two Different Alfaxalone Concentrations Combined with Midazolam to Anesthetize Cynomolgus Macaques (Macaca fascicularis) for Plethysmography.

Plethysmography is employed in nonhuman primates (NHPs) to calculate respiratory minute volume and determine the exposure time required to deliver an aerosol at the target dose. Anesthetic drugs can impact breathing parameters like steady-state minute volume (SSMV) central to aerosol dosing. Alfaxalone-midazolam mixtures (AM) provide superior parameters for plethysmography in cynomolgus macaques. An obstacle to the use of AM is the volume required to anesthetize via intramuscular injection. A more concentrated formulation of alfaxalone will reduce injection volumes and refine AM protocols. The purpose of this study was to compare AM using the Indexed 10-mg/mL (AM10) formulation compared with an investigational 40-mg/mL (AM40) formulation for IM administration in cynomolgus macaques undergoing plethysmography. We hypothesized that AM10 and AM40 would show no difference in quality of anesthesia (QA), duration of anesthesia, SSMV, accumulated minute volume (AMV), and side effects. We also hypothesized that female macaques would have a longer duration of anesthesia compared with males using both formulations. The study used 15 cynomolgus macaques comprised of 8 females and 7 males. NHPs were compared between 2 separate and randomized anesthetic events no less than one week apart. Each animal served as its own control and animals were randomized by random number generation. Anesthetized NHPs were placed in a sealed plethysmography chamber, and minute volume measurements were calculated every 10 s to determine SSMV. Once SSMV was achieved for 20 min, the trial ended. There were no statistically significant differences between AM10 and AM40 for duration of anesthesia, SSMV, AMV, side effects, or QA. AM40 had a significantly smaller injection volume. Females did not show a significantly longer median duration of anesthesia using either of the alfaxalone formulations. Overall, AM40 offers a more humane anesthetic than AM10 for plethysmography in cynomolgus macaques.

Defining the Cynomolgus Macaque (Macaca fascicularis) Animal Model for Aerosolized Venezuelan Equine Encephalitis: Importance of Challenge Dose and Viral Subtype.

Venezuelan equine encephalitis virus (VEEV) outbreaks occur sporadically. Additionally, VEEV has a history of development as a biothreat agent. Yet, no FDA-approved vaccine or therapeutic exists for VEEV disease. The sporadic outbreaks present a challenge for testing medical countermeasures (MCMs) in humans; therefore, well-defined animal models are needed for FDA Animal Rule licensure. The cynomolgus macaque (CM) model has been studied extensively at high challenge doses of the VEEV Trinidad donkey strain (>1.0 × 108 plaque-forming units [PFU]), doses that are too high to be a representative human dose. Based on viremia of two subtypes of VEEV, IC, and IAB, we found the CM infectious dose fifty (ID50) to be low, 12 PFU, and 6.7 PFU, respectively. Additionally, we characterized the pattern of three clinical parameters (viremia, temperature, and lymphopenia) across a range of doses to identify a challenge dose producing consistent signs of infection. Based on these studies, we propose a shift to using a lower challenge dose of 1.0 × 103 PFU in the aerosol CM model of VEEV disease. At this dose, NHPs had the highest viremia, demonstrated a fever response, and had a measurable reduction in complete lymphocyte counts-biomarkers that can demonstrate MCM efficacy.

Efficacy of Western, Eastern, and Venezuelan Equine Encephalitis (WEVEE) Virus-Replicon Particle (VRP) Vaccine against WEEV in a Non-Human Primate Animal Model.

The purpose of this study was to evaluate the effects of the route of administration on the immunogenicity and efficacy of a combined western, eastern, and Venezuelan equine encephalitis (WEVEE) virus-like replicon particle (VRP) vaccine in cynomolgus macaques. The vaccine consisted of equal amounts of WEEV, EEEV, and VEEV VRPs. Thirty-three animals were randomly assigned to five treatment or control groups. Animals were vaccinated with two doses of WEVEE VRPs or the control 28 days apart. Blood was collected 28 days following primary vaccination and 21 days following boost vaccination for analysis of the immune response to the WEVEE VRP vaccine. NHPs were challenged by aerosol 28 or 29 days following second vaccination with WEEV CBA87. Vaccination with two doses of WEVEE VRP was immunogenic and resulted in neutralizing antibody responses specific for VEEV, EEEV and WEEV. None of the vaccinated animals met euthanasia criteria following aerosol exposure to WEEV CBA87. However, one NHP control (total of 11 controls) met euthanasia criteria after infection with WEEV CBA87. Statistically significant differences in median fever hours were noted in control NHPs compared to vaccinated NHPs, providing a quantitative measure of infection and efficacy of the vaccine against a WEEV challenge. Alterations in lymphocytes, monocytes, and neutrophils were observed. Lymphopenia was observed in control NHPs.

Comparison of Alfaxalone-Midazolam, Tiletamine-Zolazepam, and KetamineAcepromazine Anesthesia during Plethysmography in Cynomolgus Macaques (Macaca fascicularis) and Rhesus Macaques (Macaca mulatta).

Plethysmography is used in nonhuman primates (NHPs) to measure minute volume before aerosol exposure to an agent to calculate total time necessary in the exposure chamber. The consistency of respiratory parameters during the entire exposure time is paramount to ensuring dosing accuracy. Our study sought to validate an alfaxalone-midazolam (AM) anesthetic combination for use in aerosol studies. We hypothesized that AM would provide an adequate duration of anesthesia, achieve and maintain steady state minute volume (SSMV) for 20 min, and have anesthetic quality and side effects comparable to or better than either tiletamine-zolazepam (TZ) and ketamine-acepromazine (KA), the most common anesthetics used for this purpose currently. Two groups of NHPs, one consisting of 15 cynomolgus macaques and one of 15 rhesus macaques, received 3 intramuscular anesthetic combinations (AM, TZ, and KA), no less than one week apart. Anesthetized NHPs were placed in a plethysmograph chamber and their minute volumes were measured every 10 s to determine whether they had achieved SSMV and maintained it for at least 20 consecutive min. Achieving and reliably maintaining an SSMV for at least 20 min facilitates precise aerosol dosing of a challenge agent. Quality of anesthesia, based on the NHP's ability to achieve and maintain SSMV, was higher with AM compared with TZ and KA in both species, and AM had a longer duration of SSMV as compared with TZ and KA in cynomolgus macaques. Average SSMV was larger with AM compared with TZ in cynomolgus macaques, but larger with KA compared with AM in rhesus macaques. Duration of anesthesia was sufficient with all combinations but was longer for TZ than both AM and KA in both species. These results suggest that the AM anesthetic combination would produce the most accurate dosing for an aerosol challenge.

Therapeutic monoclonal antibody treatment protects nonhuman primates from severe Venezuelan equine encephalitis virus disease after aerosol exposure.

There are no FDA licensed vaccines or therapeutics for Venezuelan equine encephalitis virus (VEEV) which causes a debilitating acute febrile illness in humans that can progress to encephalitis. Previous studies demonstrated that murine and macaque monoclonal antibodies (mAbs) provide prophylactic and therapeutic efficacy against VEEV peripheral and aerosol challenge in mice. Additionally, humanized versions of two neutralizing mAbs specific for the E2 glycoprotein, 1A3B-7 and 1A4A-1, administered singly protected mice against aerosolized VEEV. However, no studies have demonstrated protection in nonhuman primate (NHP) models of VEEV infection. Here, we evaluated a chimeric antibody 1A3B-7 (c1A3B-7) containing mouse variable regions on a human IgG framework and a humanized antibody 1A4A-1 containing a serum half-life extension modification (Hu-1A4A-1-YTE) for their post-exposure efficacy in NHPs exposed to aerosolized VEEV. Approximately 24 hours after exposure, NHPs were administered a single bolus intravenous mAb. Control NHPs had typical biomarkers of VEEV infection including measurable viremia, fever, and lymphopenia. In contrast, c1A3B-7 treated NHPs had significant reductions in viremia and lymphopenia and on average approximately 50% reduction in fever. Although not statistically significant, Hu-1A4A-1-YTE administration did result in reductions in viremia and fever duration. Delay of treatment with c1A3B-7 to 48 hours post-exposure still provided NHPs protection from severe VEE disease through reductions in viremia and fever. These results demonstrate that post-exposure administration of c1A3B-7 protected macaques from development of severe VEE disease even when administered 48 hours following aerosol exposure and describe the first evaluations of VEEV-specific mAbs for post-exposure prophylactic use in NHPs. Viral mutations were identified in one NHP after c1A3B-7 treatment administered 24 hrs after virus exposure. This suggests that a cocktail-based therapy, or an alternative mAb against an epitope that cannot mutate without resulting in loss of viral fitness may be necessary for a highly effective therapeutic.

Impact of phlebotomine sand flies on U.S. Military operations at Tallil Air Base, Iraq: 2. Temporal and geographic distribution of sand flies.

CDC miniature light traps were used to evaluate the general biology of phlebotomine sand flies from April 2003 to November 2004 at Tallil Air Base, Iraq. Factors evaluated include species diversity and temporal (daily and seasonal) and geographic distribution of the sand flies. In addition, the abundance of sand flies inside and outside tents and buildings was observed. In total, 61,630 sand flies were collected during 1,174 trap nights (mean 52 per trap, range 0-1,161), with 90% of traps containing sand flies. Sand fly numbers were low in April, rose through May, were highest from mid-June to early September, and dropped rapidly in late September and October. More than 70% of the sand flies were female, and of these sand flies, 8% contained visible blood. Phlebotomus alexandri Sinton, Phlebotomus papatasi Scopoli, Phlebotomus sergenti Parrot, and Sergentomyia spp. accounted for 30, 24, 1, and 45% of the sand flies that were identified, respectively. P. alexandri was more abundant earlier in the season (April and May) than P. papatasi, whereas P. papatasi predominated later in the season (August and September). Studies on the nocturnal activity of sand flies indicated that they were most active early in the evening during the cooler months, whereas they were more active in the middle of the night during the hotter months. Light traps placed inside tents with and without air conditioners collected 83 and 70% fewer sand flies, respectively, than did light traps placed outside the tents. The implications of these findings to Leishmania transmission in the vicinity of Tallil Air Base are discussed.

Comparison of rectal, microchip transponder, and infrared thermometry techniques for obtaining body temperature in the laboratory rabbit (Oryctolagus cuniculus).

This study compared rabbit rectal thermometry with 4 other thermometry techniques: an implantable microchip temperature transponder, an environmental noncontact infrared thermometer, a tympanic infrared thermometer designed for use on humans, and a tympanic infrared thermometer designed for use on animals. The microchip transponder was implanted between the shoulder blades; the environmental noncontact infrared thermometer recorded results from the base of the right pinna and the left inner thigh, and the tympanic infrared thermometer temperatures were taken from the right ear. Results from each technique were compared to determine agreement between the test modality and the rectal temperature. The practicality and reliability of the modalities were reviewed also. According to this study, the implantable microchip transponder measurements agreed most closely with the rectal temperature.

A comparison of target-controlled infusion versus volatile inhalant anesthesia for heart rate, respiratory rate, and recovery time in a rat model.

We conducted this study to determine whether heart rate, respiratory rate, and recovery time differed significantly between rats receiving target-controlled infusion (TCI) and those under volatile inhalant anesthesia. TCI rats received intravenous propofol at an average effect site concentration of 11.3 microg/ml or propofol plus ketamine (5 mg/ml of propofol) at an average effect site concentration of 8.7 microg/ml. Inhalant anesthesia rats received isoflurane (average, 1.8%) delivered in medical-grade air. We used a tail-clamp response test to determine when a surgical plane of anesthesia was attained. Anesthesia was continued for 1 h from the first negative tail-clamp test. During this time the test was repeated every 10 min to confirm that a surgical plane of anesthesia was being maintained. Anesthesia then was discontinued, and the animals were monitored continuously until they recovered. Average heart rate was higher for rats during anesthesia with isoflurane compared with TCI propofol-ketamine (P =0.0053). Average respiratory rate was higher for TCI regimens compared with isoflurane anesthesia, with male rats having consistently faster respiratory rates than females (P <0.001). Recovery time was longer for both TCI regimens compared with isoflurane (P <0.001). Once venous access was accomplished, TCI anesthesia with propofol or propofol combined with a low dose of ketamine was comparable to an isoflurane inhalant regimen in ease of administration and control of the anesthetic event when used in rats for procedures of 1-h duration. Respiratory rate was increased and recovery time was longer for rats receiving the TCI regimens.

Production of the subdomains of the Plasmodium falciparum apical membrane antigen 1 ectodomain and analysis of the immune response.

The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.

Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.