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.

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.

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.

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.

Protective CTL-dependent immunity and enhanced immunopathology in mice immunized by particle bombardment with DNA encoding an internal virion protein.

Anti-viral CTL were induced in vitro using a particle bombardment device or "gene-gun" to deliver plasmid DNA encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV). Using this plasmid we were able to study T cell-mediated immunity in the absence of a neutralizing Ab response. Upon a single DNA immunization, a nearly 2 log10 reduction in splenic viral titers was observed 3 days after LCMV infection. After two or three immunizations a greater than 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. C57BL/6 mice immunized with DNA encoding the nucleoprotein gene were also challenged with LCMV intracranially. Upon intracranial challenge, vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. Using limiting dilution analysis it was possible to detect LCMV-specific CTL precursors in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at day 4 after LCMV challenge. Thus DNA vaccines are capable of inducing an anti-viral T cell response that can inhibit viral replication and mediate either protective immunity or immunopathology. Vaccination with DNA may therefore provide a useful alternative to current viral or subunit vaccines once the efficacy of immunization with DNA is optimized.

DNA vaccines: a novel approach to immunization.

Direct DNA inoculations are being developed as a method of subunit vaccination. Plasmid DNAs encoding influenza virus hemagglutinin glycoproteins have been tested for the ability to provide protection against lethal influenza challenges. In immunization trials using inoculations of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens were protected against the lethal challenge. Good protection was achieved by intramuscular, intravenous and intradermal injections. In mice, 95% protection was achieved by gene gun delivery of 250-2500 times less DNA than the saline inoculations. Successful DNA vaccination by multiple routes of inoculation and the high efficiency of gene-gun delivery highlight the potential of this promising new approach to immunization.

Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin.

Immunization of ferrets with a plasmid DNA expressing influenza virus haemagglutinin (pCMV/H1 DNA) provided complete protection from challenge with the homologous A/PR/8/34 (H1N1) influenza virus. Delivery of DNA-coated gold beads by gene gun to the epidermis was much more efficient than intramuscular delivery of DNA in aqueous solution. The antibody response induced by DNA delivered by gene gun was more cross-reactive than DNA delivered in aqueous solution or after natural infection. This novel approach to vaccination against influenza may afford broader protection against antigenic drift than that provided by natural infection.

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine.

Recently, we demonstrated that direct inoculation of a hemagglutinin 7 (H7)-expressing DNA could vaccinate chickens against a lethal H7 influenza virus challenge. These experiments used a defective-retroviral-based vector to express H7 (p188) (Robinson et al., 1993). Here, we report protective immunizations using a non-retroviral-based vector for H7 expression (pCMV/H7). Unlike the previously used retroviral-based vector, this vector cannot be transmitted as an infectious agent (as a consequence of phenotypic mixing with exogenous or endogenous virus proteins). Vaccination was accomplished by inoculating young, immunocompetent chickens by each of three routes (intravenous, intraperitoneal, and intramuscular) with 100 micrograms of cesium chloride-purified pCMV/H7 DNA in saline. After two immunizations, birds were challenged via the nares with a lethal dose of a highly virulent chicken influenza virus of the H7 subtype. The results of five independent vaccine trials demonstrated protective immunizations in approximately 60% of the pCMV/H7 DNA-inoculated chickens. By contrast, only 3% of the chickens inoculated with control DNA survived the lethal challenge.

Latency and reactivation of Marek's disease virus in B lymphocytes transformed by avian leukosis virus.

The physical and biological state of the Marek's disease virus (MDV) genome in avian leukosis virus (ALV)-transformed cells is characterized using cell lines established from ALV tumours co-infected with the SB-1 strain of MDV. The MDV genome within the ALV-transformed cells was found to be methylated at 5' CpG 3' dinucleotides. Less than 2% of the tumour cells expressed MDV antigen and only one virus plaque that was characteristic of an MDV infection was noted when tumour cells were cocultured with fibroblasts permissive for a productive MDV infection. However, when methylation of the MDV genome was prevented by culturing the tumour cell lines in the presence of 5-azacytidine, both MDV antigen expression and viral replication increased. Based on these results, it appears that MDV resides within the ALV-transformed cells in a latent state and that MDV latency might be influenced, to some extent, by methylation of the MDV genome.