A Phase 2 study of a purified, inactivated virus vaccine to prevent Japanese encephalitis.

Japanese encephalitis (JE) is a serious disease caused by the JE virus. New generation JE vaccines are needed to prevent this disease. We conducted this Phase 2 randomized, open label, unblinded, single center study of a new, cell-culture derived, purified inactivated virus (JE-PIV) vaccine. The JE-PIV vaccine was administered in either two or three intramuscular (IM) doses (6.0 or 12.0 mcg each) with observation over 8 weeks. All volunteers completed the protocol without serious adverse reactions. Headache and transient tenderness at the injection site were the most common complaints. There were no laboratory abnormalities believed to be related to vaccine during the study. JE-PIV was well tolerated, resulted in high seroconversion rates [Day 56 (primary endpoint); 95-100%] and induced enduring immune responses up to 2 years after vaccination. Expanded Phase 3 trials are planned.

Correlation between lethal toxin-neutralizing antibody titers and protection from intranasal challenge with Bacillus anthracis Ames strain spores in mice after transcutaneous immunization with recombinant anthrax protective antigen.

Transcutaneous immunization of mice with recombinant protective antigen (rPA) of Bacillus anthracis resulted in significantly higher lethal toxin-neutralizing antibody titers than did intramuscular injection of alum-adsorbed rPA. Immunized mice were partially protected against intranasal challenge with 235,000 (10 50% lethal doses) Ames strain B. anthracis spores. A highly significant correlation was observed between toxin-neutralizing antibody titer and survival after challenge. Future experiments with rabbits and nonhuman primates should confirm the significance of protection by this vaccine strategy.

Field-user acceptability of new camouflage face paint formulations in the Republic of Korea.

New formulations of camouflage face paint (CFP), one with 30% N,N-diethyl-3-methylbenzamide (DEET) and the other without DEET, were evaluated for soldier-user acceptability during a military field-training exercise in the Republic of Korea. Soldiers testing the CFP formulations were members of one of four U.S. Army infantry companies (A, B, C, or D). The formulations were evaluated while soldiers participated in simulated combat exercises for 5 days during hot, humid summer weather in Korea. Results showed that soldiers found both of the new formulations easier to apply (91.3% of respondents who used CFP without DEET and 87.9% of respondents who used CFP with DEET) and remove (82.6% without DEET and 81.2% with DEET) than the previous standard military-issue CFP. Soldier acceptability was higher for the new CFP formulation with 30% DEET (70.5%) than for the formulation without 30% DEET (52.9%). Soldiers recommended it more frequently (70.5%) than the formulation without 30% DEET (50.0%). The new CFP formulation with 30% DEET was rated more often (79.5%) as either good or excellent than the new formulation without 30% DEET (67.4%). Soldiers reported that the CFP formulation with 30% DEET more successfully camouflaged the face (92.7%) than the formulation without 30% DEET (80.0%).

Detection of antibodies to squalene: III. Naturally occurring antibodies to squalene in humans and mice.

An ELISA-based assay is described for the measurement of antibodies to squalene (SQE) in human serum and plasma. The assay was adapted from the previously described assay for murine antibodies to SQE (J. Immunol. Methods 267 (2002) 119). Like the murine SQE antibody assay, the human antibody assay used sterile cell culture 96-well plates coated with SQE (20 nmol/well). Phosphate-buffered saline (PBS)-0.5% casein was used as both a blocking agent and dilution buffer. The assay has a high through-put capacity and is reproducible and quantitative. This assay was used to evaluate samples from three different human cohorts. The first cohort was retired employees of the United States Army Medical Research Institute of Infectious Diseases (USAMRIID alumni). The mean age was 68 (N=40; range 58-82). Most were vaccinated with the U.S. licensed anthrax vaccine (AVA) and most had received several other vaccines through a USAMRIID special immunization program. The second cohort was of similar age (N=372; mean age 67; range 54-97) from the normal population of Frederick, MD and were not vaccinated with AVA. The third cohort (N=299) was from Camp Memorial Blood Center, United States Army Medical Department Activities, Fort Knox, KY. (No additional volunteer information is available.) Using this new ELISA method, antibodies to SQE were detected in all three of the cohorts. IgG antibodies to SQE were detected in 7.5% and 15.1% of the samples from the USAMRIID alumni and Frederick cohorts, respectively. These differences were not significantly different (chi((1))(2)=1.69, p=0.19). In contrast, no IgG antibodies to SQE were detected in the Fort Knox cohort which is significantly different than the Frederick cohort (chi((1))(2)=49.25, p<0.0001). IgM antibodies to SQE were detected in 37.5% and 32.3% of the samples from the USAMRIID and Frederick cohorts, respectively, but there was no significant difference between the cohorts. In the Fort Knox cohort, 19.4% of the samples were positive for IgM antibodies to SQE, which was significantly different from the Frederick cohort (chi((1))(2)=14.23, p=0.0002). Although the age of the volunteers from the Fort Knox cohort is unknown, the demographic of the donors at the blood bank volunteers is 85% 17-21 years of age. This suggested that the prevalence of antibodies to SQE may increase with age. This was confirmed with mouse studies in which the presence of antibodies was monitored as a function of time. No antibodies to SQE were detected in female BALB/c, B10.Br and C57BL/6 mice at 2 months of age, but they reached a maximum prevalence with 100% and 89% of animals testing positive for IgG and IgM antibodies to SQE, respectively, in the C57Bl/6 mice at 18 months of age. BALB/c and B10.Br mice also developed antibodies to SQE over time, but were at a lower prevalence than those observed in the C57BL/6 mice. Thirty-five of the 40 volunteers in the USAMRIID were vaccinated with AVA (mean no. doses=26; range 3-47). Comparison of the prevalence of antibodies to SQE from the AVA immunized group with the Frederick cohort revealed that there was no statistical differences for IgG (chi((1))(2)=2.3, p=0.13) or IgM (chi((1))(2)=0.33, p=0.56). When the data from the USAMRIID and Frederick cohorts were combined and analyzed for the presence of antibodies to SQE with respect to the sex of the volunteer, females (40.8%) were found to have a higher prevalence of IgM antibodies to SQE than men (28.4%) (chi((1))(2)=6.59, p=0.01). No significant difference was observed in the prevalence for IgG antibodies to SQE in females (17.7%) and males (12.5%). We conclude that antibodies to SQE occur naturally in humans; have an increased prevalence in females; are not correlated with vaccination with AVA; and appear to increase in prevalence with age.

Comparison of artificial membrane feeding with direct skin feeding to estimate the infectiousness of Plasmodium vivax gametocyte carriers to mosquitoes.

The efficacy of a membrane-feeding apparatus as a means of infecting Anopheles dirus mosquitoes with Plasmodium vivax was compared with direct feeding of mosquitoes on gametocyte carriers. Volunteers participating in the study were symptomatic patients reporting to malaria clinics in western Thailand. Direct mosquito feeds were conducted on 285 P. vivax-infected individuals. Four methods of preparing blood for the membrane-feeding apparatus were evaluated. They included 1) replacement of patient plasma with sera from a P. vivax-naive donor (n = 276), 2) replacement of patient plasma with plasma from a P. vivax-naive donor (n = 83), 3) replacement of patient plasma with that individual's own plasma (n = 80), and 4) whole blood added directly to the feeder (n = 221). Criteria used to compare the different methods included 1) number of feeds infecting mosquitoes, 2) percent of mosquitoes with oocysts, and 3) mean number of oocysts per positive mosquito. For most parameters, the direct- feeding method was not significantly different from methods that replaced patient plasma with sera/plasma from a P. vivax-naive donor. However, direct feeding was more effective than use of whole blood or blood that was reconstituted with the patient's own plasma. These data suggest a possible role of transmission-blocking antibody. The implications towards development of a membrane-feeding assay for the evaluation of candidate transmission-blocking malaria vaccines is discussed.

Evaluation of Injectable Anesthetics for Major Surgical Procedures in Guinea Pigs.

Anesthetizing guinea pigs is difficult with varying outcomes. The primary purpose of the study reported here was to evaluate six injectable anesthetic regimens for use in guinea pigs and assess the depth of anesthesia and, thus, their effectiveness in terms of their use for major surgical procedures. Other variables that were measured and evaluated included time from injection until onset of anesthesia, duration of anesthesia, depth of anesthesia, and vital signs (i. e., respiratory rate, heart rate, and body temperature). Female Dunkin Hartley guinea pigs that were 9 to 12 weeks old were randomly assigned to receive one of the following anesthetic regimens: ketamine/xylazine (KX), ketamine/detomidine (KD), ketamine/medetomidine (KM), 4) tiletaminezolazepam/ xylazine (TX), tiletamine-zolazepam/detomidine (TD), or tiletamine-zolazepam/medetomidine (TM). All anesthetics were administered intramuscularly. Anesthesia was assessed by attempting to perform an ovariohysterectomy. Surgery could not be performed on any guinea pigs in the groups given ketamine or TD. There was a high rate of adverse effects in guinea pigs receiving detomidine. Four of six guinea pigs in the TD group died during or after the anesthetic episode. Fourteen of 30 (46.7%) guinea pigs given TX underwent successful surgery, and 23 of 29 (79.3%) given TM underwent successful surgery. A combination of tiletamine-zolazepam and xylazine or medetomidine was effective for inducing anesthesia and providing sufficient analgesia to perform a major surgical procedure on guinea pigs. However, TM was the most reliable regimen.