Immune responses and protection obtained with rotavirus VP6 DNA vaccines given by intramuscular injection.

Intramuscular (i.m.) injection of murine VP6 DNA vaccines raised high titers of rotavirus-specific serum IgG and IgA antibodies in BALB/c mice. A Th1-like antibody response was generated based on the ratio of serum IgG2a to IgG1 antibodies. Rotavirus-specific serum IgA but not fecal IgA was detected in mice prior to rotavirus challenge. Partial protection against rotavirus challenge was achieved as measured by reduction of rotavirus antigen shedding in feces. A similar level of protection was found with a bovine rotavirus VP6 DNA vaccine against a murine rotavirus challenge, suggesting that heterologous protection can be obtained by immunizing with VP6 DNA vaccines. We did not directly test for cytotoxic T lymphocyte (CTL) activity, but in vivo depletion of CD8+ T cells in mice immunized with a murine VP6 DNA vaccine did not significantly change the duration of virus shedding or the pattern of protection obtained. This finding suggested that CD8+ CTL activity was not essential for the partial protection we obtained by i.m. immunization of mice with VP6 DNA vaccines.

Human astrovirus isolation and propagation in multiple cell lines.

Laboratory adapted human astrovirus serotypes 1 through 7 were tested for growth in 15 human, 7 simian, and 10 other non-primate mammalian cell lines. Propagation of all seven serotypes was successful in the human cell lines Caco-2, T84, HT-29, and in the African green monkey kidney cell line MA-104. Both primary and secondary African green monkey kidney cells were more effective than Rhesus monkey kidney cells for cultivation of astrovirus. Except for human foreskin cells, all of the other human and simian cell lines supported growth of at least one astrovirus serotype. The only non-primate cell line that permitted sustained passage of astroviruses was the BHK-21 (C13) cell line for astrovirus serotype 2. Seventeen human stool specimens that had previously been shown to be astrovirus positive by ELISA were cultured in Caco-2, T84, HT-29, SK-CO-1, PLC/PRF/5, MA-104, and VERO cells. Caco-2 cells (13 isolates), T84 cells (12 isolates) and PLC/PRF/5 cells (12 isolates) were the cell lines most effective for isolation of human astroviruses from clinical stool specimens. By immunofluorescent staining of infected cells, culturing of the same 17 specimens in shell vials for 18 h was positive for astroviruses in all 17 specimens in Caco-2 cells, 12 in T84 cells, and 7 in PLC/PRF/5 cells. Shell vial assay is suitable as a rapid and sensitive culture technique for detection of astroviruses in clinical specimens.

Immunity obtained by gene-gun inoculation of a rotavirus DNA vaccine to the abdominal epidermis or anorectal epithelium.

We have previously shown that gene-gun delivery of murine rotavirus DNA vaccines to the epidermis induced protection against rotavirus challenge in mice. In this study, we used a rotavirus group antigen (VP6)-specific DNA vaccine to compare epidermal immunization with immunization to the anorectal epithelium for efficacy in inducing protective immunity. The vaccine was administered into cells of the abdominal epidermis or anorectal epithelium of adult BALB/c mice with an Accell gene-gun (PowderJect, Inc). Vaccines administered by either route elicited rotavirus-specific ELISA antibodies and analysis of the IgG subtypes indicated Th2-type responses were generated by both routes of administration, in contrast to Th1-type responses generated by live rotavirus. Protection against virus challenge was obtained in mice inoculated by either route, as shown by significant reduction of virus excreted in stools. The protection obtained by immunization of the anorectal epithelium was greater than that for epidermal immunization at the same vaccine dose. These results suggest that mucosal immunization of DNA vaccines may be an effective means to generate protective immunity against mucosal pathogens.

Immunoglobulin M antibody test to detect genogroup II Norwalk-like virus infection.

Sera obtained from adult volunteers inoculated with genogroup II Norwalk-like viruses (NLVs), Hawaii virus, and Snow Mountain virus and from patients involved in outbreaks of gastroenteritis were tested for genogroup II NLV Mexico virus-specific immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant Mexico virus antigen (rMXV)-based IgM capture enzyme-linked immunosorbent assay (ELISA). Sera from genogroup I Norwalk virus (NV)-inoculated volunteers and from patients involved in a genogroup I NLV outbreak were also tested. In sera from those infected with genogroup I NV or NLVs in volunteer and outbreak studies, only 3 of 25 were rMXV IgM positive; in contrast, 24 of 25 were IgM positive for recombinant NV (rNV). In sera from those infected with genogroup II NLVs in volunteer and outbreak studies, 28 of 47 were rMXV IgM positive and none were IgM positive for rNV, showing the specificity of each IgM test for its respective genogroup. In an outbreak of gastroenteritis not characterized as being of viral etiology but suspected to be due to NV, 7 of 13 persons had IgM responses to rMXV, whereas none had IgM responses to rNV, thus establishing the diagnosis as genogroup II NLV infection. The rMXV-based IgM capture ELISA developed is specific for the diagnosis of genogroup II NLV infections.

Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles.

Protective immune responses in mice were obtained after oral immunization with rotavirus DNA vaccines encapsulated in poly(lactide-co-glycolide) (PLG) microparticles. The DNA vaccines used encoded outer capsid proteins VP4 and VP7; proteins that are the basis for rotavirus serotyping and the generation of virus neutralizing antibodies. One dose of vaccine was given to BALB/c mice by oral gavage (75 microg DNA/mouse). Rotavirus-specific serum antibodies and intestinal IgA antibodies were detectable by 6 weeks postimmunization. After challenge with homologous murine rotavirus at 12 weeks postimmunization, fecal rotavirus antigen was reduced significantly in immunized mice compared with controls. Protective immunity also was generated by oral delivery of unencapsulated VP 7 DNA vaccine but to a lesser degree. These results demonstrate that the oral route is effective for generating protective immune responses with rotavirus DNA vaccines targeting neutralization antigens.

Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles.

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.

Detection of Norwalk virus and other genogroup 1 human caliciviruses by a monoclonal antibody, recombinant-antigen-based immunoglobulin M capture enzyme immunoassay.

Sera obtained from two groups of adult volunteers infected with Norwalk virus (NV) and two groups of patients involved in two natural outbreaks were tested for NV-reactive immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant-antigen-based IgM capture enzyme immunoassay (EIA). No NV-reactive IgM was detected in the preinoculation sera of 15 volunteers, and 14 of 15 showed NV-reactive antibodies postinfection with NV. All of the volunteers showed IgG seroconversion to NV. In the outbreak studies, all 9 persons in one outbreak and 19 of 24 in another outbreak had NV-reactive IgM. In the first outbreak, only three of nine seroconverted to NV, which was likely due to late collection of acute-phase sera. In the second outbreak, 21 of 24 showed IgG seroconversion to NV. Sequencing of viruses isolated from five stool samples selected from those in the second outbreak showed that they were human calicivirus (HuCV) genogroup 1 viruses related, but not identical, to NV. In the volunteer studies, NV-reactive IgM was first detected 8 days postinoculation. The time of development of NV-reactive IgM antibodies in natural outbreaks was estimated to be similar to that found in the volunteer studies. Sera from three Hawaii virus-infected volunteers, four Snow Mountain virus patients, and 80 healthy individuals were negative for NV-reactive IgM, indicating test specificity for HuCV genogroup I infections. This capture IgM EIA is suitable for diagnosis of NV and other HuCV genogroup I infections and is especially useful when sera and fecal samples have not been collected early in the course of an outbreak.

Astrovirus infection in South Africa: a pilot study.

Astroviruses have been shown to be important aetiological agents associated with gastroenteritis in children, as have rotaviruses and the enteric adenoviruses. However, no inclusive studies have been conducted in South Africa to allow a comparison of the relative roles of these different viral agents. In this study, stool specimens were obtained between 1991 and 1993 from 225 young children with acute gastro-enteritis. These were examined for the presence of astroviruses using a monoclonal antibody-based ELISA, and for rotaviruses and enteric adenoviruses using commercially available kits. A control group of 56 infants and young children without symptoms of diarrhoeal illness was included in the study. Astroviruses were detected in 7% of the stools compared with 20% infected with rotaviruses and only 3% infected with enteric adenoviruses. In the control group, one specimen each had astrovirus or adenovirus and two shed rotaviruses. The astrovirus prevalence observed in this study is similar to that reported in other developing communities. Rotavirus and astrovirus infections were more prevalent in the autumn and early winter than in other seasons. Astrovirus and rotavirus infections predominated in children between 3 and 22 months of age.

Protective immunity induced by rotavirus DNA vaccines.

It is estimated that Group A rotavirus diarrhea causes as many as one million deaths per year in children worldwide, and effective vaccines will be essential for their control. Plasmid DNA vaccines encoding murine rotaviral proteins VP4, VP6, or VP7 were tested in adult BALB/c mice for their ability to induce immune responses and provide protection against rotavirus challenge. The vaccines were administered by inoculation into cells of the epidermis with an Accell gene gun. (Auragen, Inc., Middleton, WI, USA). Each vaccine elicited rotavirus-specific serum antibodies as measured by ELISA. Virus neutralizing antibodies were detected in mice receiving plasmid DNAs encoding for outer capsid proteins VP4 and VP7, but not for VP6, an inner capsid protein, and all of the vaccines generated virus-specific CTL responses. Each vaccine was effective in protecting mice against infection after homotypic rotavirus (100 ID50) challenge, showing reductions (P < 0.0002) in viral excretion measured over a 9 day period. Increased rotavirus-specific intestinal IgA antibodies were seen in vaccinated mice after rotavirus challenge, particularly in mice that received the VP6 DNA vaccine. This suggests that intracellular IgA-mediated neutralization may be involved in protective immunity induced by the VP6 DNA vaccine, and may represent a new mechanism for protection by DNA vaccines.

Protection against rotavirus infections by DNA vaccination.

DNA vaccines encoding for murine rotavirus proteins VP4, VP6, or VP7 were tested in adult BALB/c mice for their ability to induce immune responses and protect against rotavirus challenge. A gene gun was used to inoculate vaccines into the epidermis. Rotavirus-specific serum antibodies, as measured by ELISA, and virus-specific cytotoxic T lymphocyte responses were generated by each of the three vaccines, but virus-neutralizing antibodies were detected only in mice that were inoculated with DNA vaccines encoding for VP4 and VP7. Efficacy of the vaccines was determined by challenge with 100 ID50 of homotypic rotavirus. Each of the three vaccines was effective in protecting mice against infection after rotavirus challenge as determined by reduction (P < .001) in virus excretion in mice receiving the DNA vaccines. These results demonstrate that DNA vaccination has potential as a new approach for control of rotavirus infections.

Epidemiology of diarrhea among expatriate residents living in a highly endemic environment.

OBJECTIVE: To determine the etiology of diarrhea among expatriate residents living in a developing country and identify risk factors for travelers' diarrhea that are difficult to evaluate in tourist populations. DESIGN: Clinic based case-control study. SETTING: Primary care travel medicine clinic in Kathmandu, Nepal. PARTICIPANTS: A total of 69 expatriate residents with diarrhea, compared with 120 tourists with diarrhea, and 112 asymptomatic resident and tourist controls, selected systematically during a 1-year period. MAIN OUTCOME MEASURES: Risk factors for diarrhea assessed by questionnaire and pathogen prevalence assessed by microbiologic analysis of stool specimens. RESULTS: The dominant risk factors for diarrhea among expatriate residents included younger age (P = .003), shorter duration of stay in Nepal (P < .001), and eating out in restaurants (P = .01). Eating raw vegetables, salads, fresh fruit, or ice served in restaurants was not significantly associated with diarrhea. Longer duration of residence was linearly correlated with protection. Enteric pathogens were identified in 44 (64%) of 69 residents with diarrhea compared with 100 (83%) of 120 tourists with diarrhea, with enterotoxigenic Escherichia coli, Campylobacter, and Shigella predominant for both groups. Pathogens were also found in stools from 32 (37%) of 87 asymptomatic resident controls and 13 (52%) of 25 tourist controls. The attack rate of diarrhea among expatriates was estimated to be 49% (95% confidence interval, 37% to 61%) per month during the first 2 years of residence. The highest-risk months were April through July. CONCLUSIONS: Diarrhea among expatriates in a highly endemic environment is a persistent risk. The extremely high prevalence of enteric pathogens among asymptomatic persons reflects widespread exposure. The most important risk factors for travellers' diarrhea are difficult to modify, including younger age, duration of stay, eating in restaurants, and seasonality. Preventive dietary recommendations may not be fully protective, suggesting that pretravel advice should emphasize empiric treatment in addition to strategies to avoid exposure.

DNA vaccines against rotavirus infections.

Plasmid DNA vaccines encoding for murine rotaviral proteins VP4, VP6, and VP7 were tested in adult BALB/c mice for their ability to induce immune responses and provide protection against rotavirus challenge. Serum antibodies were measured by virus neutralization and by ELISA. Cellular immunity was assessed by measuring cytotoxic T cell (CTL) responses. The vaccines were administered by inoculation into cells of the epidermis with an Accell gene gun (Auragen, Inc., Middleton, WI, USA). Each of the three vaccines elicited rotavirus-specific serum antibodies as measured by ELISA. Virus neutralizing antibodies were detected in mice receiving DNA vaccines encoding for VP4 and VP7, but not in those which received the plasmid encoding for VP6. Vaccines encoding for VP4, VP6, or VP7 generated virus-specific CTL responses in recipient mice. Efficacy of the vaccines was determined by challenge with homotypic rotaviruses. Each of the three vaccines was effective in protecting mice against infection after rotavirus (100 ID50) challenge. Significant reductions (p < 0.0002, analysis of variance) in viral excretion measured over a 9 day period were seen in mice receiving the DNA vaccines compared with mice that received control plasmids.

The changing epidemiology of astrovirus-associated gastroenteritis: a review.

Our understanding of the epidemiology of astrovirus-associated gastroenteritis has changed markedly with each improvement in detection method. In early surveys based on electronmicroscopy (EM), astroviruses appeared to be a rare cause of gastroenteritis, being found in fewer than 1% of children with diarrhea, usually in small outbreaks of disease and primarily during the winter season. The development and use of monoclonal antibodies and enzyme immunoassays (EIA) to detect astroviruses led to reports of a higher prevalence (2.5%-9%) of astrovirus infection among patients hospitalized with diarrhea. Astroviruses appeared second only to rotaviruses as a cause of hospitalization for childhood viral gastroenteritis. Studies based on EIA detection of astroviruses indicate that astroviruses are common causes of diarrhea in children worldwide, and that most children are infected during their first two years of life. The elderly and the immunocompromised represent high-risk groups as well. The observations that newborns monitored prospectively rarely have repeat disease and that the rate of detection decreases with increasing age suggest that immunity to astroviruses, as immunity to rotaviruses, may develop early in life. The cloning and sequencing of astroviruses have led to more sensitive assays to detect the viruses by reverse transcription, polymerase chain reaction (RT-PCR). Application of RT-PCR for detection of astroviruses in children in day-care centers showed a marked increase in the detected prevalence of astrovirus-associated diarrhea, the rate of asymptomatic infection, and the duration of shedding of virus among those infected, when compared with studies that used other methods. As with rotaviruses, neither the mode of transmission nor the reservoir of astrovirus infection has been identified. Both immune and molecular-based assays to detect astrovirus serotypes indicate that serotype 1 is most common worldwide, although the predominant serotypes may vary by region and time. In the absence of obvious strategies to prevent astrovirus-associated diarrhea, vaccines might be considered if further studies establish that the disease burden would render such a vaccine cost-effective.

Monoclonal antibodies for detection of Norwalk virus antigen in stools.

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.

Comparison of three monoclonal antibody-based enzyme immunoassays for detection of herpes simplex virus in clinical specimens.

Three commercial monoclonal antibody-based enzyme immunoassays (Herpchek, IDEIA HSV and SureCell HSV) for detection of herpes simplex virus antigen were compared with isolation of virus in cell cultures. A total of 51 culture positive and 49 culture negative consecutively collected specimens that had been stored at -70 degrees C for a period of up to ten months were used in the study. Herpchek, IDEIA HSV and SureCell HSV assays gave a sensitivity of 88.2%, 82.4% and 47.1% respectively, and a specificity of 95.9%, 93.9% and 83.7% respectively compared to cell culture. A blocking antibody test showed that two culture negative specimens contained herpes simplex virus-specific antigens. If these two specimens were considered to be true positive, Herpchek, IDEIA HSV and SureCell HSV assays had a sensitivity of 88.7%, 83.0% and 47.2%, and a specificity of 100%, 97.9% and 85.1% respectively. The positive predictive value (using the resolved sample results) for Herpchek, IDEIA HSV and SureCell HSV was 100%, 97.8% and 78.1% respectively, and the negative predictive value 88.7%, 83.6% and 58.8% respectively. These results demonstrated that Herpchek and IDEIA HSV are sensitive and highly specific assays. Results could be obtained in less than five hours after receipt of specimens. SureCell HSV gave results in 15 minutes, but both the sensitivity and specificity were too low for this test to be considered as a substitute for culture.

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.

Cloning and characterization of human astrovirus immunoreactive epitopes.

We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.

Etiology of acute diarrhea among United States military personnel deployed to South America and west Africa.

A study of acute diarrhea was conducted from 1985 to 1987 among U.S. military personnel participating in routine shipboard exercises in South America and West Africa and ground troops deployed to coastal Ecuador. An enteropathogen was identified in 146 (51%) of 289 acute cases of diarrhea. Enterotoxigenic Escherichia coli, found in 50 (17%) patients with diarrhea, was the most commonly identified enteropathogen. Viral enteropathogens were also found in a high percentage of acute cases of diarrhea: rotavirus was detected in 11% of the patients and Norwalk virus infection in 10%. Most enteric pathogens were acquired in equal frequencies in South America and West Africa, except for rotavirus infection which was identified more often in West Africa and enteroaggregative E. coli infection which was identified more often in South America. Bacterial enteropathogens were frequently resistant to trimethoprim/sulfamethoxazole, but no resistance to quinolone drugs was observed, indicating that quinolone drugs have become important agents for the treatment of diarrhea in South America and West Africa.

Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes.

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.