Epidemic of adenovirus-induced respiratory illness among US military recruits: epidemiologic and immunologic risk factors in healthy, young adults.

Adenovirus (Ad)-induced acute respiratory illnesses resurged among civilian adults and selected military training populations in the United States during the late 1990s. We examined the epidemiologic and immunologic correlates of Ad-induced respiratory illnesses during a large outbreak at an Army basic training installation in southeast United States during a 9-day period in November 1997. A total of 79 recruits hospitalized with acute respiratory illnesses were evaluated during the outbreak period; confirmation of Ad infection by isolation of Ad-like cytopathic agents from throat cultures was detected in 71 (90%) of these patients. Serotyping of 19 (27%) of these 71 isolates identified the etiologic agent to be Ad type 4 (Ad4). In addition, 30 (81%) of 37 patients in whom paired sera were collected demonstrated significant increases (i.e., 4-fold or higher) in serum anti-Ad4 neutralizing antibodies. Anti-Ad4 immunity in new recruits was found to be very low (15 to 22%). A case-control study involving 66 of the 79 hospitalized cases and 189 non-ill controls from the same units was conducted. A lower risk of hospitalization for acute respiratory illnesses was documented for female recruits (odds ratio[OR] = 0.47, P <.05) whereas, a higher risk was noted for smokers (OR = 1.89, P <.05). Unit (training company) attack rates as high as 8 to 10% per week were documented and the outbreak quickly subsided after live, oral Ad types 4 and 7 vaccination was resumed in November 1997. Re-establishment of a military Ad vaccination program is critical for control of Ad-induced acute respiratory illnesses.

Hepatitis E virus DNA vaccine elicits immunologic memory in mice.

Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.

Detection of adenoviruses (AdV) in culture-negative environmental samples by PCR during an AdV-associated respiratory disease outbreak.

Since 1954, adenoviruses (AdV) have been recognized as an important cause of acute respiratory disease (ARD) among U.S. military recruits. Until recently, routine oral vaccination for AdV serotypes 4 and 7 eliminated epidemic AdV-associated ARD in this population. Now that the manufacturer has ceased production, vaccination has ended and AdV epidemics have reappeared. As part of a prospective epidemiological study during the high-risk ARD season, serial samples were obtained from ventilation system filters and tested for AdV by culture and PCR. An outbreak occurred during this surveillance. Of 59 air filters, 26 (44%) were AdV positive only by PCR. Sequence analysis confirmed the presence of AdV serotype 4, the implicated outbreak serotype. The number of AdV-related hospitalizations was directly correlated with the proportion of filters containing AdV; correlation coefficients were 0.86 (Pearson) and 0.90 (Spearman's rho). This is the first report describing a PCR method to detect airborne AdV during an ARD outbreak. It suggests that this technique can detect and quantify AdV-associated ARD exposure and may enable further definition of environmental effects on AdV-associated ARD spread.

Molecular characterization of a hepatitis E virus isolate from Namibia.

Hepatitis E virus (HEV) causes sporadic and epidemic acute viral hepatitis in many developing countries. In Africa, hepatitis E has been documented by virus detection (reverse transcriptase polymerase chain reaction, RT-PCR) in Egypt, Chad, Algeria, Morocco and Tunisia. Cases of presumptive hepatitis E also have been documented by detection of antibody to HEV in the Sudan, Kenya, Ethiopia, Somalia, Djibouti and South Africa. Recently, we reported the recovery of 9 isolates of HEV from feces collected during an outbreak of jaundice in Namibia. These specimens were stored frozen for many years at the South African Institute for Medical Research awaiting new methods to determine the etiology of jaundice. HEV genomic sequences were detected by antigen-capture RT-PCR with primers that amplified 2 independent regions of the HEV genome (ORF-2 and ORF-3). To further characterize the HEV 83-Namibia isolates, we determined the nucleotide (nt) sequence of the 3' end of the capsid gene (296 of 1, 980 nt in ORF-2) and ORF-3 for 1 isolate. The capsid gene sequence shared 86% identity with the prototype Burma strain and up to 96% identity with other African strains at the (nt) level, and 99% identity with Burma or other Africa strains at the amino acid level. A 188 (nt) fragment amplified from ORF-3 was also highly homologous to other HEV but was too short for meaningful comparison. Phylogenetic analysis indicated that HEV 83-Namibia is closely related to other African isolates, and differs from Burmese, Mexican and Chinese HEV. These data link the HEV causing the 1983 Namibia outbreak to more recent HEV transmission in northern and sub-Saharan Africa, suggesting this subgenotype of HEV is firmly established throughout the continent.

Antiserum generated by DNA vaccine binds to hepatitis E virus (HEV) as determined by PCR and immune electron microscopy (IEM): application for HEV detection by affinity-capture RT-PCR.

Previously, we have described that injection of an expression vector containing hepatitis E virus (HEV) open reading frame 2 (HEV-ORF-2) generated a strong antibody response in mice. To characterize the reaction of this antiserum with native HEV and to evaluate its potential diagnostic application, we tested the antiserum's ability to bind HEV using immune electron microscope (IEM) and affinity-capture reverse transcription polymerase chain reaction (RT-PCR) amplification. Antiserum to ORF-2 aggregated HEV virions as seen by electron microscopy, providing direct evidence that ORF-2 encodes a structural protein. Antiserum also captured HEV for RT-PCR amplification. This antiserum bound HEV from diverse origins (Asia, Africa, Mexico) at virus concentrations found in patient fecal specimens and bile from inoculated non-human primates. The specificity of the affinity binding was demonstrated when pre-immune sera or sera collected from mice injected with control DNA vector (lacking the HEV ORF-2 gene) failed to bind HEV for RT-PCR amplification and IEM. Specific RT-PCR amplification was confirmed by restriction enzyme digestion of PCR products. The sensitivity of the binding was evaluated by RT-PCR amplification of serially diluted bile containing a genetically divergent HEV, Mexico'86. HEV was detected in a 10(-8) dilution of this bile. This is the first report that antibodies elicited by a DNA vaccine recognize native HEV. Our results indicate that ORF-2 encodes a structural protein and that antiserum to this protein enables simple, sensitive, and specific HEV detection by affinity-capture RT-PCR.

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.

Experimental African HEV infection in cynomolgus macaques (Macaca fascicularis).

Experimental infection with hepatitis E virus (HEV) from Africa has not been investigated. Our purpose was to study hepatitis E produced by HEV from Chad (North Africa) and to analyze the genetic sequence of the HEV obtained after animal passage. An HEV-containing fecal sample from Chad was intravenously inoculated in four cynomolgus macaques. When serum Alanine Amino Transferase (ALT) levels rose, open liver biopsy and bile aspiration were performed. In all the monkeys, an ALT rise occurred 25 to 32 days after inoculation and new anti-HEV was detected by Enzyme Immuno Assay (EIA). Hepatic histopathology was consistent with acute viral hepatitis. HEV was detected by polymerase chain reaction (PCR) in bile (3/4 animals) and feces (2/4 animals) and by imunoelectron microscopy (IEM) in the inoculum and one bile specimen. A genetic variant HEV was identified in one monkey. The Chad HEV produced hepatitis E with pathophysiologic and histopathologic findings similar to those observed with HEV from other geographic origins. A genomic variant HEV population was produced after one passage in a macaque.

Pathogenesis of hepatitis A in orally inoculated owl monkeys (Aotus trivirgatus).

The pathogenesis of hepatitis A virus (HAV) infection was studied in owl monkeys following oral administration of the wild-type HM-175 strain of HAV. Stools were collected daily and blood and pharyngeal swabs twice weekly for viral isolation, and animals were necropsied at various intervals after inoculation. Organs were examined for the presence of virus by isolation in cell culture and for viral antigens by immunofluorescence. Monkeys excreted HAV in the stools for 1-4 days after inoculation, presumably due to the residual unabsorbed inoculum. No virus was found in stools for the next 2-3 days. HAV re-appeared on days 4-7 and then persisted through day 39. Viremia occurred on the 10th day and continued until day 35. Virus was isolated occasionally from throat swabs 1 or 2 weeks after it was detected in stools and blood, and there was no evidence that HAV replicated in the pharyngeal tissues. Animals acquired anti-HAV antibody by the 4th week, and alanine aminotransferase (ALT) was elevated 5-5.5 weeks after inoculation. HAV was isolated from liver 5 days after inoculation; however, viral antigens were first detected in Kupffer cells of the liver at 14 days and in hepatocytes at 21 days. HAV antigen was detected in epithelial cells of the intestinal crypts and in the cells of the lamina propria of the small intestine 3 days postinoculation and thereafter until the 5th week, suggesting that these cells might represent an additional site of HAV replication.

Immunization of US soldiers with a two-dose primary series of inactivated hepatitis A vaccine: early immune response, persistence of antibody, and response to a third dose at 1 year.

To study the feasibility of using inactivated hepatitis A vaccine for rapid immunization of US soldiers, 276 randomized seronegative volunteers received one of four regimens: two injections, on day 0 or one each on day 0 and 14, day 0 and 30, or day 0 and 180. A third dose was given on day 380. Among the 256 recipients of two doses, 99% responded with antibody (by ELISA) with few symptoms. A higher percentage of recipients of both doses on day 0 had antibody at day 14 (68% vs. 52% of all others, P < .03). The highest antibody concentrations (711 mIU/mL on day 240) were observed in subjects given a second dose on day 180. Recipients of the third injection developed a median 15-fold rise in antibody within 2 weeks. Virus-neutralizing antibody was detected in high titers after the third dose and neutralized strains of hepatitis A virus from several countries. Vaccines containing 1440 ELISA units of antigen may be useful for rapid immunization.

Administration of hepatitis A vaccine to a military population by needle and jet injector and with hepatitis B vaccine.

Military personnel are an important target population for hepatitis A immunization. Soldiers are often given vaccines by jet injector and may be required to receive multiple vaccines at one time. Formalin-inactivated hepatitis A vaccine containing 360 ELISA units of antigen was evaluated at Fort Campbell. Volunteers received vaccine at 0, 1, and 6 months as follows: group 1, hepatitis A vaccine by needle; group 2, hepatitis A vaccine by jet injector; group 3, hepatitis B vaccine by needle; and group 4, both hepatitis vaccines by needle in separate arms. Immune response and reactogenicity were evaluated. After two doses, recipients of vaccine administered by jet injector had a higher prevalence of antibody than those who received vaccine by needle (93% vs. 79%). By the 8th month, the vaccine was 100% immunogenic by either route or with hepatitis B vaccine. No interaction between hepatitis A and B vaccines was detected.

Immunohistochemical detection of cytokines in tissues of Aotus monkeys infected with hepatitis A virus.

Cytokines IL-1-beta, IL-2, and TNF alpha were detected in occasional cells within portal inflammatory infiltrates beginning 3 weeks after oral inoculation of monkeys with HAV. The number of cells secreting those cytokines did not increase, and they were not of importance in the pathogenesis. Production of cytokines IL-6 and IL-4 by T lymphocytes infiltrating portal areas started 4 weeks after inoculation, stimulating local expansion of B cells, probably secreting antibodies to HAV. IL-6 and IL-4 may also stimulate cytotoxic activity of a few CD8+ lymphocytes.

A trial of the reactogenicity and immunogenicity of an inactivated hepatitis A vaccine.

Purified, formaldehyde-inactivated and alum-adjuvanted hepatitis A virus (HAV) vaccines have recently become available for clinical trials. The vaccine is administered intramuscularly in a schedule of 0, 1, and 6 months. The aim of the study was to evaluate the reactogenicity and immunogenicity of an inactivated hepatitis A (HA) vaccine. Three groups of volunteers comprised the study population: 28 volunteers without antibody to HAV were given HA vaccine and, for comparison, 43 subjects received hepatitis B vaccine for possible adverse reactions to the HA vaccine; 12 other subjects received immunoglobulin alone. Each 1 ml dose of HA vaccine contained 720 enzyme units or about 100 ng of antigen. Anti-HAV was determined by means of a commercial assay (Abbott Laboratories: HAV-EIA), and by a more sensitive ELISA. No significant adverse reactions were reported. In the group that received HA vaccine, 4 weeks following the first dose all had detectable antibodies (> or = 20 mIU/ml) by the sensitive ELISA. By commercial HAV-EIA, at 20 weeks following the second dose 75.0% had detectable antibodies, and after the third vaccine all had detectable antibodies. This new inactivated HA vaccine is highly immunogenic and had no significant side effects.

Depression of the immune response to an inactivated hepatitis A vaccine administered concomitantly with immune globulin.

Inactivated hepatitis A virus (HAV) vaccine is given in a three-dose schedule. When rapid protection is needed, injection of immune globulin (IG) concomitantly with the first dose could provide passive protection until adequate active antibody response has developed. A possible effect of IG on the immune response to the vaccine was studied in healthy volunteers; 28 received vaccine alone, and 34 received the first dose simultaneously with 5 mL of IG. A control group received hepatitis B vaccine, and a fourth group received IG alone. Four weeks after the first vaccine dose, all subjects had detectable ELISA anti-HAV antibodies. Several weeks after each vaccine dose, the geometric mean titer of antibodies was significantly lower in those who received vaccine with IG but higher than in those who received IG alone. Results for neutralizing antibodies yielded a similar trend. If IG is given with HAV vaccine, a further booster vaccine dose may be required to ensure long-lasting immunity.

Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis).

The pathogenesis of experimental hepatitis E has not been thoroughly investigated. The purpose of this study was to more accurately document the events in this disease. Cynomolgus macaques were inoculated intravenously with bile or feces containing hepatitis E virus (HEV). Serum, bile, and liver specimens were evaluated with light microscopy, immune electron microscopy, immunofluorescence microscopy, EIA, and polymerase chain reaction. In the third week, there were histopathologic changes and HEV antigen (HEVAg) in liver, HEV in bile, and alanine aminotransferase (ALT) elevations. Widespread pathologic changes were detected during the fourth week and antibody to HEV (anti-HEV) and peak ALT values in the fifth or sixth week. By the sixth week, HEVAg had disappeared but pathologic changes persisted. This study supports the concept that experimental hepatitis E has an initial phase in which hepatic HEV replication is accompanied by the onset of hepatitis and a later phase in which the appearance of anti-HEV is accompanied by progression of the hepatitis.

Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies.

Laboratory tests and reference reagents employed in studies of inactivated hepatitis A vaccine.

Procedures to evaluate inactivated hepatitis A vaccines in volunteers have been examined. Solid-phase immunoassays were standardized with reference preparations and have been tested to measure antibody response to immunization and antigen content of vaccines. Following immunization, there was a good correlation between antibody response, determined with commercial immunoassays, and neutralization titres, as measured by the radioimmunofocus inhibition test. However, at lower titres of neutralizing antibody, the commercial immunoassay often yielded negative results. To improve the sensitivity of the immunoassay, the serum volume was increased. A fourfold increase of test serum resulted in greater sensitivity, increasing from 54 to 94%, while retaining 100% specificity. Further increases in the volume of test serum resulted in a loss of specificity. In a comparison of neutralization tests, similar titres of postvaccination sera were obtained by using the HM175/18f cytopathic strain of hepatitis A virus in a plaque reduction assay or the HM175 parental virus in the radioimmunofocus inhibition test. Use of the cytopathic virus obviates the need for radioactively labelled serum and reduces the time taken to conduct neutralization tests. The current laboratory procedures can meet the needs of large field trials of inactivated hepatitis A vaccines.

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.

Large-scale purification of inactivated hepatitis A virus by centrifugation in non-ionic gradients.

Formalin-inactivated hepatitis A virus (HAV) can be purified for vaccine preparation by centrifugation in Renografin-76 (diatrizoate meglumine and diatrizoate sodium) gradients. Both continuous-flow rate-zonal and isopycnic methods were used for the separation of a major antigen component from minor antigen and host protein. The major antigen component, which appeared to contain complete virions by electron microscopy, could be recovered from gradients and accounted for approximately one third of the total antigen in the starting material. The HAV-specific purified antigen could be enriched 200-300-fold by either centrifugation procedure. The purified HAV antigen, when adsorbed to alum and inoculated into mice, was found to be highly immunogenic.