Leonard N. Binn

Phylogenetic analysis of hepatitis E virus isolates from Egypt.

Hepatitis E virus (HEV) genome was detected by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in fecal samples of two sporadic cases of hepatitis E in Cairo Egypt. Sequence of the complete putative structural region [open reading frame (ORF)‐2] and complete region of unknown function (ORF‐3) was determined for the two HEV isolates. Phylogenetic analysis of the nucleotide sequences was performed using neighbor joining or maximum parsimony methods of tree reconstruction. Direct correspondence between the HEV evolutionary trees and geographic origin of the HEV isolates was observed. Three genotypes of HEV were identified: genotype I (Asia‐Africa), genotype II (US), and genotype III (Mexico). Genotype I was further divided into two subgenotypes (Asia and Africa). In the Asian subgenotype, three smaller genetic clusters were observed (China‐like sequences, Burma‐like sequences, and sequence from a fulminant case of HEV). The segregation of all these genetic clusters was supported by the high level of bootstrap probabilities. Four regions of the HEV genome were used for phylogenetic analysis. In all four regions, Egyptian HEV isolates were grouped in a separate African clade. J. Med. Virol. 57:68–74, 1999.

Rapid Detection of Adenovirus in Throat Swab Specimens by PCR during Respiratory Disease Outbreaks among Military Recruits

ABSTRACT We evaluated the performance of a generic PCR test to detect adenoviruses (AdV) in throat swab specimens collected from asymptomatic and ill military recruits with acute respiratory disease. Samples (n = 210) were collected at entry to basic training and at the time of large outbreaks of AdV-associated acute respiratory disease among military recruits at Fort Jackson, South Carolina, from 1997 to 1998. Compared to cell culture, a sensitivity of 99% and a specificity of 98% were noted for the PCR method to detect AdV in throat swabs. Similar results were obtained with or without DNA extraction, suggesting the absence of significant inhibitors for the PCR method in throat swab samples. No AdV was detected by culture or PCR in throat swabs from healthy recruits, suggesting the absence of latency or asymptomatic shedding. Throat swab specimens proved to be adequate, noninvasive samples to rapidly diagnose respiratory disease in young adults. This generic direct PCR proved to be a useful test for the rapid diagnosis of AdV-associated respiratory disease, detecting all serotypes tested to date and furnishing results within 6 h of specimen arrival. The use of this direct, rapid, sensitive, and specific assay would assist health care providers and public health practitioners in the early diagnosis, management, and control of AdV-associated respiratory disease.

A double-blind, placebo-controlled study of the safety and immunogenicity of live, oral type 4 and type 7 adenovirus vaccines in adults ☆

Adenovirus serotypes 4 (ADV-4) and 7 (ADV-7) are important causes of febrile acute respiratory disease (ARD) in US military recruits. Previously licensed vaccines, which effectively controlled adenovirus-associated ARD, are no longer available. In the Fall of 2004 we conducted this Phase 1 randomized, double-blind, placebo-controlled trial of the live, oral ADV-4 and ADV-7 vaccines made by a new manufacturer to assess their safety and immunogenicity. The adenovirus vaccines were administered orally together in a single dose to thirty subjects. Twenty eight additional subjects received placebo. Subjects were then observed for 8 weeks. The most commonly reported adverse events were nasal congestion (33%), cough (33%), sore throat (27%), headache (20%), abdominal pain (17%), arthralgia (13%), nausea (13%) and diarrhea (13%). None of these rates differed significantly from placebo. The duration of vaccine virus fecal shedding was 7-21 days. Seventy three percent of vaccine recipients seroconverted to ADV-4 (GMT 23.3) while 63% seroconverted to ADV-7 (GMT 51.1) by Day 28. The new ADV-4 and ADV-7 vaccines were safe and induced a good immune response in the study population. Expanded trials for safety and efficacy are in progress.

Inactivated hepatitis A vaccine: active and passive immunoprophylaxis in chimpanzees

Studies of active and passive immunoprophylaxis were carried out in chimpanzees to determine whether a candidate hepatitis A virus (HAV) vaccine could stimulate antibody to HAV (anti-HAV) that was qualitatively similar to anti-HAV stimulated by natural infection. Normal immune globulin (Ig) was prepared from plasma obtained from human volunteers before and after vaccination with the HAV vaccine, and these preparations or commercially prepared Ig were administered to chimpanzees. Protective efficacy was compared to that obtained after vaccination of chimpanzees. As expected, pre-vaccination Ig did not protect chimpanzees against challenge with virulent hepatitis A. In contrast, chimpanzees were protected against hepatitis A by Ig prepared from volunteers who had received hepatitis A vaccine. The protection was qualitatively similar to that afforded by commercial normal Ig containing convalescent anti-HAV. The minimum protective dose of passively acquired anti-HAV was approximately the minimum dose detectable by serological means. This information will be useful in calculating minimum acceptable titres of anti-HAV in normal Ig. Whereas administration of Ig protected chimpanzees against hepatitis A pathology, it did not protect them from infection with HAV. Thus, these chimpanzees were protected by classical passive-active immunoprophylaxis. In contrast, chimpanzees actively immunized with HAV vaccine were apparently protected against both hepatitis A pathology and HAV infection. The mechanism of this complete protection is unknown but may simply represent the higher titre of anti-HAV in the vaccinated chimpanzees, compared to the passively protected animals.

Immunogenicity of an Inactivated Hepatitis A Vaccine

Although hepatitis A is a disease without chronic sequelae, it is associated with serious morbidity in adults. An inactivated hepatitis A vaccine prepared at the Walter Reed Army Institute of Resea...

Hepatitis A in the US Army: epidemiology and vaccine development

Control of hepatitis A has been an important concern for US military forces in war and peace. Immune serum globulin, although effective, is exceedingly cumbersome to use. The prevalence of antibody against hepatitis A is decreasing in young American soldiers, putting them at risk of hepatitis A during deployment. The US Army has been an active participant in development of hepatitis A vaccine. The first successful cell-culture-derived, formalin-inactivated hepatitis A vaccine was developed at the Walter Reed Army Institute of Research. This prototype vaccine was shown, in 1986, to be safe and immunogenic for humans. Since then we have evaluated the following issues related to the use of inactivated hepatitis A vaccines in military populations. Immunogenicity of vaccine derived from the CLF and HM175 strains; immunogenicity of hepatitis A vaccine given by jet injector; immunogenicity of hepatitis A vaccine when given with hepatitis B vaccine; immunogenicity when given in shortened schedules; safety and immunogenicity in Thai children; and efficacy under field conditions in the tropics. The hepatitis A vaccines which we tested are safe and highly immunogenic. Immunization by jet gun confers immunity equivalent to immunization by needle. Hepatitis A vaccine is equally potent when given with hepatitis B vaccine. Data on rapid immunization schedules and efficacy are under evaluation. We conclude that hepatitis A vaccine is a major improvement in our ability to prevent hepatitis A in soldiers.

Emergence of Adenovirus Type 14 in US Military Recruits—A New Challenge

In this issue of the Journal, Metzgar et al. [1] report a significant addition to the adenovirus (Ad) types infecting US military recruits with acute respiratory disease (ARD). Their ongoing epidemiological studies, which use rapid molecular diagnostic procedures, enabled the detection and identification of a species B2 Ad, type 14 (Ad14), that had not been previously observed in US military recruits. This virus was originally recovered 50 years ago from Dutch recruits [2], with only 2 subsequent reports of ARD associated with Ad14 [1, 3]. In contrast to the B1 Ads (i.e., types 3, 7, and 21) that often infect the respiratory tract, Ad14 respiratory infections have been very rarely observed in adult military and civilian populations [4]. At present, studies are under way at military training centers to field test replacements for the live oral Ad4 and Ad7 vaccines that had been used for 25 years but were lost when production ceased in the 1990s [5–7]. The recent recovery of Ad14 raises new questions as to whether Ad14 will take hold as a respiratory disease agent in civilian and military populations and whether the candidate Ad4 and Ad7 vaccines will protect against Ad14. The absence of identified Ad14 respiratory infections in the United States suggests that we have highly susceptible civilian and military populations. Review of past experience with the live oral Ad vaccines may be of value in assessing the long-term threat of Ad14 to recruits. Previous observation of responses to the Ad4 and Ad7 vaccines indicated that they were highly effective against homologous viruses and Ad3 [6, 7]. Vaccines responses to Ad3 were attributed to some level of Ad3 occurring naturally and to Ad3 being antigenically related to Ad7, both being species B1 viruses. Therefore, after exposure to the Ad7 vaccine virus, heterotypic responses to Ad3 occurred [8]. In contrast to Ad3, naturally occurring background infections with Ad21, also a species B1 virus, have been considered rare. Recent studies of recruits with ARD found only small numbers infected with Ad21 [1]. Only a few additional Ad21 infections were identified via antibody testing, suggesting that the virus currently is not highly communicable [9, 10]. However, in 1975–1976, Ad21 was recovered from large numbers of recruits with ARD who had been vaccinated against Ad4 and Ad7. ARD-associated Ad21 infections continued for months at most training facilities, resulting in the testing of an experimental live oral vaccine [11, 12]. However, long-term observation revealed that Ad21 did not persist as a significant cause of ARD, even though outbreaks occurred in the 1970s and again in 1985 [13]. Thus, there has been no interest in renewing studies of an Ad21 vaccine. It is possible that Ad14 and other Ads could cause significant ARD morbidity among military recruits for a limited period and then disappear as an important cause of respiratory disease. It is also possible that some protection against emerging Ads could be provided as a result of heterotypic antibody responses to other viruses, including the Ad7 vaccine virus. Past studies in The Netherlands found that Ad14-associated ARD did not persist in their recruits [2, 14]. Additionally, the development of heterotypic antibody to Ad14 after Ad7 immunization was reported in 1960 [15]. Contemporary serologic surveys of new US military recruits will provide important information on their susceptibility. Testing archival serum-bank specimens from recruits vaccinated against Ad4 and Ad7 and from recruits with ARD who had laboratory confirmation of a specific Ad type could provide data on the occurrence of heterotypic antibody reReceived 4 June 2007; accepted 4 June 2007; electronically published 31 October 2007. Potential conflicts of interest: none reported. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the true views of the Department of the Army or the Department of Defense. Reprints or correspondence: Dr. Leonard N. Binn, Div. of Viral Diseases, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910 (leonard.binn@na.amedd.army.mil). The Journal of Infectious Diseases 2007; 196:1436 –7 This article is in the public domain, and no copyright is claimed. 0022-1899/2007/19610-0003

Clinical and laboratory observations following oral or intramuscular administration of a live attenuated hepatitis A vaccine candidate.

15.00 DOI: 10.1086/522969 E D I T O R I A L C O M M E N T A R Y

Multiplexed Luminex xMAP Assay for Detection and Identification of Five Adenovirus Serotypes Associated with Epidemics of Respiratory Disease in Adults

Clinical observations made after immunising volunteers with a live attenuated hepatitis A vaccine are described. The candidate vaccine was prepared with the HM175 strain of hepatitis A virus and shown to be safe, immunogenic and efficacious in experimental animals. When the candidate vaccine was tested by oral administration in humans at increasing doses--10(4), 10(5), 10(6) and 10(7) median tissue culture infective doses (TCID50)--an antibody response was not observed at any dose. Volunteers who received similar doses by the intramuscular route developed antibody to hepatitis A three weeks after immunization with 10(6) or 10(7) TCID50. The antibody response was sustained for the 12 weeks of the observation period. All volunteers remained healthy with normal results from liver tests throughout the monitoring period. Further clinical observations of this product are in progress.

Hepatitis E virus DNA vaccine elicits immunologic memory in mice

ABSTRACT Several serotypes of human adenovirus (HAdV) cause acute respiratory disease (ARD) among healthy adults, sometimes generating broad outbreaks with high attack rates and occasional fatalities. Timely serotype identification provides valuable epidemiological information and significantly contributes to prevention (vaccination) strategies. The prevalence of specific serotypes causing ARD varies geographically. HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 are the serotypes most commonly found in adult populations in the Western Hemisphere. Unfortunately, conventional serotype identification is a tedious process which can take a week or longer. For this reason, new molecular methods for serotype identification are needed. Commercially available rapid antigen and PCR assays for the detection of HAdV are universal but do not distinguish between the different serotypes. We describe the development of a sensitive and specific multiplex assay capable of identifying serotypes 3, 4, 7, 14, and 21. Two sets of primers were used for nonspecific (universal) PCR amplification, and serotype-specific probes coupled to Luminex tags were used for target-specific extension (TSE). PCR and TSE primers were designed using known hexon gene sequences of HAdV. The TSE products of HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 were correctly identified using the Luminex xMAP fluid microsphere-based array system. No cross-reactivity with other respiratory pathogens or other HAdV serotypes was observed. This multiplexed assay can be expanded to include more serotypes and will allow broad and rapid detection and identification of adenoviral serotypes in a high-throughput environment.