Bacillus subtilis strains expressing tetanus toxin fragment C (TTFC) were tested as vaccine candidates against tetanus in adult mice. Mice received three intranasal (IN) exposures to 109 spores or 108 vegetative cells of B. subtilis expressing recombinant TTFC. Immunized mice generated protective systemic and mucosal antibodies and survived challenge with 2× LD100 of tetanus toxin. Isotype analysis of serum antibody indicated a balanced Th1/Th2 response. Lyophilized vaccines stored at 45°C for ≥12 months, remained effective. Immunized conventional and SCID mice remained well, and no histological changes in brain or respiratory tract were detected. Lyophilized/reconstituted B. subtilis tetanus vaccines administered IN to mice appear safe, heat-stable, and protective against lethal tetanus challenge.

ABSTRACT Bacillus subtilis vaccine strains engineered to express either group A bovine or murine rotavirus VP6 were tested in adult mice for their ability to induce immune responses and provide protection against rotavirus challenge. Mice were inoculated intranasally with spores or vegetative cells of the recombinant strains of B. subtilis. To enhance mucosal immunity, whole cholera toxin (CT) or a mutant form (R192G) of Escherichia coli heat-labile toxin (mLT) were included as adjuvants. To evaluate vaccine efficacy, the immunized mice were challenged orally with EDIM EW murine rotavirus and monitored daily for 7 days for virus shedding in feces. Mice immunized with either VP6 spore or VP6 vegetative cell vaccines raised serum anti-VP6 IgG enzyme-linked immunosorbent assay (ELISA) titers, whereas only the VP6 spore vaccines generated fecal anti-VP6 IgA ELISA titers. Mice in groups that were immunized with VP6 spore vaccines plus CT or mLT showed significant reductions in virus shedding, whereas the groups of mice immunized with VP6 vegetative cell vaccines showed no difference in virus shedding compared with mice immunized with control spores or cells. These results demonstrate that intranasal inoculation with B. subtilis spore-based rotavirus vaccines is effective in generating protective immunity against rotavirus challenge in mice.

Human astroviruses have been shown in numerous studies to be an important cause of gastroenteritis in young children worldwide. The present communication addresses their characterization by use of oligonucleotide microarray hybridization. The system developed consists of an RT-PCR using primers of low degeneracy capable of detecting all eight serotypes of human astroviruses. RT-PCR products are then hybridized against a microarray consisting of short oligonucleotide probes 17–18 nucleotides in length. Cy3-labeled ssDNA targets are generated using a Cy3-labeled primer in the RT-PCR. The non-labeled strand is enzymatically digested, and the labeled target is rescued by column purification. This method of generating labeled target uses equimolar concentrations of the amplifying primers and does not compromise assay sensitivity for initial detection of the virus. Hybridization can be performed without the need for additional amplification. Although the amplicon spans a relatively conserved region of the astrovirus genome, the use of short probes enables type distinction despite such limited diversity. Probes differing by as little as a single nucleotide can be used to distinguish isolates. The microarray developed was capable of distinguishing representatives of the eight known serotypes of human astroviruses.

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.

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.

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.

ABSTRACT 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.

A total of 95 clinical samples were cultured for periods of 2 and 7 days in centrifuged A-549 shell vials before and after freezing and thawing of specimens. In addition, centrifuged A-549 shell vials were tested directly for varicella-zoster virus without incubation using a direct fluorescent antibody (DFA) technique. Twenty-seven specimens were positive by at least one method. The sensitivity for DFA on unincubated A-549 shell vials was 85.2%; for unfrozen 2-day cultures, 88.9%; for unfrozen 7-day cultures, 92.6%; for freeze-thaw 2-day cultures, 92.6%; and for freeze-thaw 7-day cultures, 96.3%. Freeze-thawed specimens cultured for 7 days yielded the highest number of positive results with conspicuous cell-to-cell spread as a sign of viral replication.

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.

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.

A-549 and MRC-5 cells were compared for the detection of varicella-zoster virus (VZV) by conventional tube cell culture and by shell vial assay using fresh specimens. VZV was detected in 33 (32.4%) of 102 specimens by one or all of these methods. In conventional tube culture, seven (21.2%) of 33 were positive in MRC-5 cells and 24 (72.7%) of 33 were positive in A-549 cells, a threefold increase in sensitivity. In shell vial assay, 30 (90.9%) of 33 were positive in MRC-5 cells and 31 (93.9%) of 33 were positive in A-549 cells. The samples tested by A-549 shell vial assay had more positives showing plaque formation, The two shell vial procedures gave the highest levels of sensitivity. A-549 cells provided a more sensitive method of detecting VZV in clinical specimens in both conventional tubes and shell vial assays than did MRC-5 cells.

Two commercially available immunofluorescence monoclonal antibody (MAB) reagents (Bartels, Baxter Healthcare, Issaquah, WA; and Symex, Broken Arrow, OK) were evaluated as a means for detecting parainfluenza virus (PIV) both in shell-vial cultures and directly in clinical specimens. Bartels reagents are used in an indirect immunofluorescence assay (IFA) format and exist as MABs reactive with all three PIV sero-types, individually and in a pool. Symex reagents, also available individually and in a trivalent pool, are used in a direct immunofluorescence assay (DFA) format. Among a total of 299 respiratory specimens, 24 yielded PIV. In a shell-vial culture confirmation test, both the individual and pooled monoclonal antibody reagents from both Bartels and Symex detected all 24 PIV isolates. There were three apparent false-positive results with the Bartels pooled IFA reagents. Of the 299 specimens, 160 were also tested directly for the presence of PIV. There were 13 positive specimens among these 160. The Bartels and Symex monoclonal antibody reagents detected similar percentages of positive samples when used for direct detection (that is, 78.6-85.7). No false-positive results were obtained with any of the reagents in the direct-detection format.