Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines.

We describe the results of initial safety testing of 10 live-attenuated dengue virus (DENV) vaccine candidates modified by serial passage in primary dog kidney (PDK) cells at the Walter Reed Army Institute of Research. The Phase 1 studies, conducted in 65 volunteers, were designed to select an attenuated vaccine candidate for each DENV serotype. No recipient of the DENV candidate vaccines sustained serious injury or required treatment. Three vaccine candidates were associated with transient idiosyncratic reactions in one volunteer each, resulting in their withdrawal from further clinical development. Increasing PDK cell passage of DENV-1, DENV-2, and DENV-3 candidate vaccines increased attenuation for volunteers, yet also decreased infectivity and immunogenicity. This effect was less clear for DENV-4 candidate vaccines following 15 and 20 PDK cell passages. Only one passage level each of the tested DENV-2, -3, and -4 vaccine candidates was judged acceptably reactogenic and suitable for expanded clinical study. Subsequent studies with more recipients will further establish safety and immunogenicity of the four selected vaccine candidates: DENV-1 45AZ5 PDK 20, DENV-2 S16803 PDK 50, DENV-3 CH53489 PDK 20, and DENV-4 341750 PDK 20.

Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers.

A randomized, controlled, double-blinded study was conducted to determine safety and immunogenicity of five live attenuated dengue vaccines produced by Aventis Pasteur (AvP). The study was completed with 40 flavivirus non-immune volunteers: five recipients of each monovalent (dengue-1, dengue-2, dengue-3, or dengue-4) vaccine, ten recipients of tetravalent (dengue-1, dengue-2, dengue-3, and dengue-4) vaccine, and ten recipients of vaccine vehicle alone. All vaccines were administered in a single subcutaneous dose (range, 3.6-4.4 log(10) plaque forming units). No serious adverse reactions occurred in volunteers followed for 6 months after vaccination. Five vaccine recipients developed fever (T > or = 38.0 degrees C), including four tetravalent vaccinees between days 8 and 10 after vaccination. Dengue-1, dengue-2, dengue-3, or dengue-4 vaccine recipients reported similar frequency of mild symptoms of headache, malaise, and eye pain. Tetravalent vaccinees noted more moderate symptoms with onset from study days 8-11 and developed maculopapular rashes distributed over trunk and extremities. Transient neutropenia (white blood cells < 4000/mm3) was noted after vaccination but not thrombocytopenia (platelets < 100,000/mm3). All dengue-3, dengue-4, and tetravalent vaccine recipients were viremic between days 7 and 12 but viremia was rarely detected in dengue-1 or dengue-2 vaccinees. All dengue-2, dengue-3, and dengue-4, and 60% of dengue-1 vaccine recipients developed neutralizing and/or immunoglobulin M antibodies. All tetravalent vaccine recipients were viremic with dengue-3 virus and developed neutralizing antibodies to dengue-3 virus. Seven volunteers also had multivalent antibody responses, yet the highest antibody titers were against dengue-3 virus. The AvP live attenuated dengue virus vaccines are safe and tolerable in humans. The live attenuated tetravalent dengue vaccine was most reactogenic, and preferential replication of dengue-3 virus may have affected its infectivity and immunogenicity.

Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus--poxvirus vaccines in vaccinia-nonimmune and vaccinia-immune humans.

A controlled, randomized, double-blind clinical trial evaluated whether two attenuated recombinant poxviruses with identical Japanese encephalitis virus (JEV) gene insertions, NYVAC-JEV and ALVAC-JEV, were safe and immunogenic in volunteers. Groups of 10 volunteers distinguished by vaccinia immune status received two doses of each vaccine. The vaccines appeared to be equally safe and well tolerated in volunteers, but more reactogenic than licensed formalin-inactivated JE and placebo vaccines given as controls. NYVAC-JEV and ALVAC-JEV vaccine recipients had frequent occurrence of local warmth, erythema, tenderness, and/or arm pain after vaccination. There was no apparent effect of vaccinia immune status on frequency or magnitude of local and systemic reactions. NYVAC-JEV elicited antibody responses to JEV antigens in recipients but ALVAC-JEV vaccine poorly induced antibody responses. However, NYVAC-JEV vaccine induced neutralizing antibody responses only in vaccinia-nonimmune recipients while vaccinia-immune volunteers failed to develop protective antibodies (5/5 vs. 0/5 seroconversion, p<0.01). These data suggest that preexisting immunity to poxvirus vector may suppress antibody responses to recombinant gene products.

Precision and comparability of Abuscreen OnLine assays for drugs of abuse screening in urine on Hitachi 917 with other immunochemical tests and with GC/MS.

Abuscreen OnLine assays for drugs of abuse screening in urine have recently been developed for use on Hitachi 917 analyzers (Roche Diagnostics GmbH). The assays are based on the kinetic interaction of microparticles as measured by changes in light transmission. Drug in a sample inhibits the formation of particle aggregates and diminishes absorbance change increases. It was the goal of this study to evaluate precision and comparability of the new asssys with CEDIA drugs of abuse tests on Hitachi 917 in different laboratories (three European and three US). The assays were calibrated in the nonlinear mode with four to six standards (semiquantitative application). Initial within-run (21 replicates, four labs) and between-day (10 days, two labs) imprecision studies using Abuscreen OnLine tests and commercial negative (0.5 x cut-off) and positive (1.5 x cut-off) controls revealed the following median CVs [withinrun neg./pos. control/between-day neg./pos. control]: amphetamines 1.9/1.3/3.4/2.4, barbiturates 3.0/1.6/3.9/3.1, benzodiazepines 4.7/1.5/6.3/3.0, cocaine metabolite 1.8/0.9/2.4/1.7, methadone 5.4/1.6/5.5/2.2, opiates 5.5/2.8/5.3/2.7, THC 8.9/4.8/21.8/12.1. CVs < 10% were obtained for the THC test using controls with concentrations closer to the cut-off. An identical set of 170 GC/MS analyzed urine samples was distributed to the six laboratories and measured with Abuscreen OnLine tests on Hitachi 917. The median values for each individual sample were calculated and compared with the results obtained on individual Hitachi 917 analyzers by Passing-Bablok regression analysis. A good agreement between the laboratories was found with less than +/- 11% slope deviation and intercepts below 7% of the cut-off except for benzodiazepines (one slope 17%, one slope--26%) and THC (one slope 34%, one slope--18%). The comparability with CEDIA tests was analyzed by concordance plots using randomized routine samples in three laboratories. The following results were obtained in one of the participating laboratories [cut-off ng/mL] (No. of positive/negative/discrepant samples): amphetamines [500] 2/147/0, barbiturates [200] 1/148/0, benzodiazepines [100] 52/91/7, cocaine metabolite [300] 17/129/3, methadone [300] 113/34/2, opiates [300] 31/114/4, THC [50] 66/81/2. GC/MS was performed for clarification of the discrepant results. In summary, Abuscreen OnLine tests on Hitachi 917 give precise results which compare well when analyzed in different laboratories. They can be rated as convenient and flexible methods for drugs of abuse screening in the routine.

An intranasal challenge model for testing Japanese encephalitis vaccines in rhesus monkeys.

Placebo-controlled field efficacy trials of new Japanese encephalitis (JE) vaccines may be impractical. Therefore, an animal model to evaluate efficacy of candidate JE vaccines is sought. Previous work has shown that exposure of monkeys to JE virus (JEV) via the intranasal route results in encephalitis. Here we report the further development of this model and the availability of titered virus stocks to assess the protective efficacy of JE vaccines. To determine the effective dose of our JE challenge virus, dilutions of a stock JEV (KE-93 isolate) were inoculated into four groups of three rhesus monkeys. A dose-dependent response was observed and the 50% effective dose (ED50) was determined to be 6.0 x 10(7) plaque forming units (pfu). Among animals that developed encephalitis, clinical signs occurred 9-14 days postinoculation. Infection with JEV was confirmed by detection of JEV in nervous tissues and IgM to JEV in the cerebrospinal fluid. Viremia with JEV was also detected intermittently throughout infection. Validation of the model was performed using a known effective JE vaccine and saline control. One ED90 of virus (2.0 x 10(9) pfu) was used as a challenge dose. Four of four animals that received saline control developed encephalitis while one of four monkeys administered the JE vaccine did so. This study demonstrates that the virus strain, route of inoculation, dose, and the outcome measure (encephalitis) are suitable for assessment of protective efficacy of candidate JE vaccines.

Production of lethal infection that resembles fatal human disease by intranasal inoculation of macaques with Japanese encephalitis virus.

Twelve rhesus macaques (Macaca mulatta) challenged intranasally with a wild-type Japanese encephalitis virus (JEV) developed clinical signs 11-14 days later. Tissues from the cerebral cortex, cerebellum, brainstem, thalamus, meninges, and all levels of the spinal cord were stained for JEV antigen with hyperimmune mouse ascitic fluid and streptavidin-alkaline phosphatase; immunofluorescent staining was also done on frozen sections. Viral antigen was found in all cell layers of the cerebellum, the gray matter of the thalamus and brainstem, and the ventral horn of all levels of the spinal cord. Staining was limited to neurons and their processes. Histopathologic changes were limited to the nervous system and characterized by nonsuppurative meningoencephalitis. These results were comparable with those of previous studies done with human autopsy tissues. Intranasal inoculation of rhesus monkeys with JEV was effective in producing clinical disease comparable with natural disease in humans and may serve as a model to evaluate protective efficacy of candidate JEV vaccines.

Safety, immunogenicity, and protective efficacy of NYVAC-JEV and ALVAC-JEV recombinant Japanese encephalitis vaccines in rhesus monkeys.

Two poxvirus-vectored vaccines for Japanese encephalitis (JE), NYVAC-JEV and ALVAC-JEV, were evaluated in rhesus monkeys for safety, immunogenicity, and protective efficacy. The vaccines were given to four monkeys each on study days 0 and 28 along with saline placebo on day 7. For controls, the licensed BIKEN JE vaccine and a saline placebo were given to other groups of four monkeys on days 0, 7, and 28. No systemic effects were observed. All injection site reactions were mild. All vaccines elicited appreciable JE-specific neutralizing antibody responses. However, a more rapid increase and higher peak level of antibody were seen in the BIKEN group as compared with the NYVAC-JEV and ALVAC-JEV groups. The peak neutralizing antibody level in the NYVAC-JEV group was higher than that of the ALVAC-JEV group. Antibody persisted in all four BIKEN recipients through 273 days of follow-up, whereas, the antibody level decreased to the threshold of detection in two NYVAC-JEV and all four ALVAC-JEV recipients by day 120. On day 273, all monkeys were given a booster dose. A rapid increase in neutralizing antibody was seen in all vaccine recipients by seven days. Two months after the booster dose, all monkeys were challenged intranasally with one 90% effective dose of JE virus. Four recipients of saline, three of ALVAC-JEV, one of NYVAC-JEV, and one of BIKEN experienced encephalitis. This study suggests that the NYVAC-JEV and ALVAC-JEV vaccines are safe and immunogenic in monkeys and that the NYVAC-JEV and BIKEN vaccines are effective in protecting monkeys from encephalitis.

Japanese encephalitis vaccine (inactivated, BIKEN) in U.S. soldiers: immunogenicity and safety of vaccine administered in two dosing regimens.

The safety and immunogenicity of Japanese encephalitis (JE) vaccine (Nakayama strain, monovalent / BIKEN) was studied in 538 U.S. soldiers in 1990. Three doses of vaccine from three consecutively manufactured lots were given on days 0, 7, and either 14 or 30. Serum for antibody determination was drawn at months 0, 2, and 6. Japanese encephalitis plaque reduction neutralization tests were performed by three laboratories on each specimen. Five hundred twenty-eight (98%) participants completed the immunization series. All recipients without antibody before immunization developed neutralizing antibody against JE virus. There were no differences in geometric mean titer among the three test lots at months 2 and 6. Soldiers who received the third dose on day 30 had higher titers at both time points. Antibody to yellow fever had no significant effect on immune response to vaccine. Conclusions drawn from analysis of serologic data from the three labs were nearly identical. Symptoms were generally limited to mild local effects and were reduced in frequency with each subsequent does in the series (21% to 11%; P < 0.0001). Generalized symptoms were rare (e.g., fever = 5%) with no reported cases of anaphylaxis.

Leishmaniasis in the United States military.

Leishmaniasis is a recurrent health problem for the U.S. and other militaries. Health care workers may be unfamiliar with the risk factors, transmission, clinical features, diagnosis, and treatment of this disease. A team of highly trained specialists is required to properly manage service members with leishmaniasis. Such care is available only in a few medical centers. Although there are no prophylactic drugs to prevent this disease, control of insect populations and use of personal protection measures can minimize arthropod-related casualties. The impact of leishmaniasis on military operations and research initiatives to better prevent, diagnose, and treat infection are discussed.

Induction of Japanese encephalitis virus-specific cytotoxic T lymphocytes in humans by poxvirus-based JE vaccine candidates.

Poxvirus-based recombinant Japanese encephalitis (JE) vaccine candidates, NYVAC-JEV and ALVAC-JEV, were examined for their ability to induce JE virus-specific cytotoxic T lymphocytes (CTLs) in a phase I clinical trial. These vaccine candidates encoded the JE virus premembrane (prM), envelope (E) and non-structural 1 (NS1) proteins. The volunteers received subcutaneous inoculations with each of these candidates on days 0 and 28, and blood was drawn 2 days before vaccination and on day 58. Anti-E and anti-NS1 antibodies were elicited in most vaccinees inoculated with NYVAC-JEV and in some vaccinees inoculated with ALVAC-JEV. Peripheral blood mononuclear cells (PBMCs) obtained from approximately one half of vaccines showed positive proliferation in response to stimulation with live JE virus. Cytotoxic assays demonstrated the presence of JE virus-specific CTLs in in vitro-stimulated PBMCs obtained from two NYVAC-JEV and two ALVAC-JEV vaccinees. Cell depletion tests using PBMCs from one NYVAC-JEV recipient indicated that the phenotype of CTLs was CD8+CD4-.

Molecular and epidemiologic analysis of dengue virus isolates from Somalia.

Nucleotide sequence analysis was performed on 14 dengue virus isolates (13 dengue-2 viruses and 1 dengue-3 virus) recovered from febrile soldiers in Somalia in 1993. The dengue-2 viruses were most closely related to dengue-2 virus recovered in Somalia in 1984. However, differences in nucleotide sequence (0.35% to 1.35%) were evident among the 1993 isolates. These differences were closely associated with the geographic location of the infection as well as with different times of infection at the same location. Genetic difference between strains was not associated with differences in clinical features. Molecular analysis of dengue viruses is a useful adjunct to epidemiologic investigation of their distribution over distance and time.

Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis.

The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.

Evaluation and interference study of hemoglobin A1c measured by turbidimetric inhibition immunoassay.

The technical performance of the turbidimetric immunoinhibition (TI) assay for hemoglobin (Hb) A1c (Tina-quant Hb A1c, Boehringer Mannheim, Indianapolis, Ind) was evaluated by using the BM/Hitachi 911 analyzer. Intra-assay imprecision was less than 2.7%, and interassay imprecision was less than 2.8% as measured by coefficient of variation. In 93 subjects with diabetes who did not have hemoglobin variants, results of the TI assay for Hb A1c correlated strongly with those obtained by using a high-performance liquid chromatography analyzer (Diamat, BioRad Laboratories, Hercules, Calif). Among 241 subjects who had or did not have hemoglobin variants, the TI assay for Hb A1c correlated strongly with results of affinity chromatography for total glycated hemoglobin (Glyc-Affin GHb, IsoLab, Akron, Ohio). We also studied the effect of various percentages of hemoglobin S, C, E, and F on the accuracy of the TI Hb A1c assay. Only high hemoglobin F percentages caused interference. More than 14 times as many samples can be analyzed per hour by using the TI Hb A1c assay than can be analyzed by using the HPLC assay. For high-volume reference laboratories, using the fully automated TI Hb A1c assay to monitor glycemic control in patients with diabetes may be preferable to using the conventional ion-exchange high-performance liquid chromatography Hb A1c assay because the TI assay measures Hb A1c more accurately in patients with diabetes who have hemoglobin variants, and it requires less time.

A cluster of acute hepatitis E infection in United Nations Bangladeshi peacekeepers in Haiti.

In the fall of 1995, within a month of deployment to Haiti for peacekeeping duty, four Bangladeshi soldiers developed acute icteric hepatitis in rapid succession. Hepatitis E virus (HEV) was found to be the etiology by demonstrating HEV genomic sequences in serum samples by the polymerase chain reaction (PCR) and serologically by the detection of elevated IgM titers to HEV. No case had serologic evidence of acute hepatitis A or C infection. The soldiers had probably acquired their infection while living in a cantonment area outside Dhaka, Bangladesh for one month prior to deployment. Cloning and sequencing of amplified PCR products demonstrated a single strain suggestive of a common source of infection. Furthermore, high genomic identity with Asian strains of HEV and dissimilarity with the Mexican strain was demonstrated, verifying that the strain had indeed been imported. Human waste management from the Bangladesh camp in Haiti was strictly controlled and no secondary cases were observed. A convenience sample of 105 (12%) soldiers from the Bangladesh battalion (850 men) revealed anicteric or asymptomatic HEV infection in seven (7%) of 105. This report contains the first demonstration of acute hepatitis E in natives of Bangladesh and demonstrates the power of the PCR in the rapid diagnosis and epidemiologic analysis of HEV infection. More importantly, this cluster demonstrates the importation of an important infectious disease by multinational peacekeepers to a potentially susceptible host country.

Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney cells.

The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-O and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-O was derived. Twenty-five (0.23%) nucleotide and 10 (0.29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-O, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine. These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-O and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0.23%) nucleotide and 11 (0.32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-O strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-O and 27.

Dengue fever in US military personnel in Haiti.

OBJECTIVE: To describe clinical manifestations and public health implications of an outbreak of dengue fever (DF) during Operation Uphold Democracy, Haiti, 1994. DESIGN: Consecutive sample. SETTING: Military combat support hospital, Port-au-Prince, Haiti. PATIENTS: A total of 101 US military personnel with acute febrile illnesses. INTERVENTIONS: A disease surveillance team collected clinical and epidemiologic data from US military clinics throughout Haiti. Febrile patients admitted to the combat support hospital were evaluated with standardized clinical and laboratory procedures. The surveillance team followed patients daily. MAIN OUTCOME MEASURES: Arbovirus isolation and specific antibody determination and symptoms and physical findings. RESULTS: Febrile illnesses accounted for 103 (25%) of the 406 combat support hospital admissions during the first 6 weeks of deployment. All patients with febrile illness recovered. A total of 30 patients had DF; no patient had evidence of infection with malaria. Dengue virus serotypes 1, 2, and 4 were isolated from 22 patients, and 8 patients developed IgM antibody to dengue virus. Patients with DF could not be distinguished from other febrile patients on clinical grounds alone. No arboviruses other than dengue were identified. CONCLUSIONS: Active surveillance, with clinical and laboratory evaluation directed by an epidemiologic team, led to the timely recognition of an outbreak of febrile illness among US troops in Haiti. Viral isolation and serological studies were essential in confirming DF. During the surveillance period, DF accounted for at least 30% of the febrile illnesses among hospitalized US troops. Dengue fever is a significant threat to military personnel and civilian travelers in Haiti and has the potential for introduction to and transmission in the United States.

Identification of hepatitis E virus in clinical specimens: amplification of hydroxyapatite-purified virus RNA and restriction endonuclease analysis.

A multi-site nested reverse transcription and polymerase chain reaction (RT-PCR) followed by restriction endonuclease analysis (REA) was developed to identify hepatitis E virus (HEV) in clinical specimens. Four sets of primers were selected to amplify regions in the HEV genome supposed to encode the helicase, polymerase, and parts of the viral capsid protein. Digestion of the nested PCR products with HinfI, HaeII, AvaII, BglI, KpnI, SmaI, or EcoRI generated readily recognizable profiles that confirm the HEV sequences and/or distinguish the unique Mexico genotype (our positive control) from all other isolates (Asian genotype). In addition, the hydroxyapatite (HA) adsorption method was compared to other adsorption and extraction methods widely used to purify viral RNA from clinical specimens for RT-PCR. All methods presented the same sensitivity of recovery of HEV RNA, but only the adsorption methods efficiently removed fecal enzymatic inhibitors. The HA method gave the best results and was the most economic in terms of time, cost, manipulations and reagents. The method was validated by screening a small number of serum and fecal specimens available from patients with acute non-A,B,C hepatitis in Nepal. HEV RNA was identified in half (5/11) of the fecal specimens obtained from patients with evidence of recent HEV infection, but in none of the 14 patients without a serological marker for hepatitis E.