Donald L. Lodmell

DNA vaccination of mice against rabies virus: effects of the route of vaccination and the adjuvant monophosphoryl lipid A (MPL).

Adjuvants are known to strongly enhance immune responses generated by traditional vaccines, but less is known about the effects of adjuvants on vaccination with DNA. In this study, we investigated the use of the immunostimulant monophosphoryl lipid A (MPL(R)) as an adjuvant, and analyzed three routes of DNA vaccination to determine if this adjuvant could enhance anti-rabies virus neutralizing antibody responses. Compared with antibody titers elicited with DNA only, antibody titers were enhanced after initial intradermal (i.d.) and gene gun immunizations with the combination of DNA and MPL(R). Antibody was not detected after primary intramuscular (i.m.) immunization unless MPL(R) was included with the DNA. Surprisingly, antibody titers of MPL(R)-treated mice decreased after i.d. or i.m. booster vaccinations, but increased after gene gun booster vaccinations. In contrast to these varied responses, booster immunizations without MPL(R) via the three different routes consistently increased antibody titers. All mice with detectable levels of neutralizing antibody at the time of challenge survived virus infection. There was no difference in the survival rate between groups of mice that received similar vaccinations with MPL(R)/DNA or DNA only. The data suggest that MPL(R) can enhance the neutralizing antibody response when used with the initial injection of DNA. Suppression of neutralizing antibody responses after i.d. or i.m. booster vaccinations that included MPL(R) suggests that the number of vaccinations, and the route of vaccination, should be carefully considered when MPL(R) is used with DNA vaccines.

Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection.

Accell gene gun particle-mediated immunization with DNA encoding the glycoprotein gene of the challenge virus standard strain of rabies virus was evaluated for its ability to elicit protective levels of serum anti-rabies virus neutralizing antibody. Strong primary and booster neutralizing antibody responses were detected in mice following immunization with 2 micrograms of DNA coated on 2.6-micron gold beads. Protective levels of antibody persisted for over 300 days. Mice challenged intraplantarly 315 days post-primary immunization (225 days post-booster vaccination) survived lethal rabies virus challenge. Our data demonstrate a potentially significant role for gene gun-based delivery of DNA in the field of rabies virus vaccination.

Nanogram quantities of plasmid DNA encoding the rabies virus glycoprotein protect mice against lethal rabies virus infection

Vaccination against virus infections has proven to be an effective strategy in the improvement of human health. In this study, we evaluated two plasmid DNA vaccines expressing the glycoprotein (G) gene of the challenge virus standard (CVS) rabies virus for their ability to elicit neutralizing antibody and protect BALB/cByJ mice against lethal rabies virus challenge. A single inoculation of 10 micrograms of plasmid DNA encoding G protected 100% of the intramuscularly (i.m.) vaccinated mice, and 0.1 microgram of DNA protected 83% of the intradermally (i.d.) vaccinated mice. All mice that survived had serum anti-rabies virus neutralizing antibody titers > or = 1:40 prior to virus challenge. The highest antibody titers were detected in mice that had been inoculated i.m. with 10-100 micrograms of DNA in regenerating muscle. The immunostimulant monophosphoryl lipid A enhanced the neutralizing antibody response of i.d.-vaccinated mice. Anti-rabies virus neutralizing antibody elicited by plasmid DNA vaccination cross-neutralized a global spectrum of rabies virus variants. These results indicate that DNA vaccines could be a solution for providing developing countries with an inexpensive vaccine that is simple to prepare, is highly efficacious and has excellent stability.

Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers

Pre-exposure DNA vaccination protects non-human primates against rabies virus. Post-exposure protection of monkeys against rabies virus by DNA vaccination has not been attempted. Presumably, post-exposure experiments have not been undertaken because neutralizing antibody is usually slow to be induced after DNA vaccination. In this study, we initially attempted to accelerate the induction of neutralizing antibody by varying the route and site of DNA vaccination and booster frequency. Gene gun (GG) vaccinations above axillary and inguinal lymph nodes or in ear pinnae generated higher levels of neutralizing antibody than intradermal (ID) needle vaccinations in the pinnae. Concurrent GG booster vaccinations above axillary and inguinal lymph nodes and in ear pinnae, 3 days after primary vaccination, accelerated detectable neutralizing antibody. GG booster vaccinations also resulted in higher neutralizing antibody levels and increased the durability of this response. Post-exposure vaccination with DNA or the human diploid cell vaccine (HDCV), in combination with an one-time treatment with human rabies immune globulin (HRIG), protected 50 and 75% of the monkeys, respectively, as compared to 75% mortality of the controls. These data will be useful for the refinement, development, and implementation of future pre- and post-exposure rabies DNA vaccination studies.

One-time gene gun or intramuscular rabies DNA vaccination of non-human primates: comparison of neutralizing antibody responses and protection against rabies virus 1 year after vaccination

We have previously shown that Macaca fascicularis (Cynomologus) monkeys receiving a primary and either one or two booster rabies DNA vaccinations are protected against rabies virus. In this study, we determined whether monkeys that had been vaccinated only once via gene gun or intramuscularly (i.m.) with different concentrations of DNA would be protected against rabies virus challenge. Neutralizing antibody responses were assayed for 1 year before the monkeys were challenged. Neutralizing antibody was detected at least 50 days earlier in gene gun vaccinated as compared to i.m. vaccinated animals. Prior to viral challenge, all (6/6, 100%) gene gun vaccinated animals, but only 3/6 (50%) i.m. vaccinated animals seroconverted. In general, antibody titers of the gene gun vaccinated animals were higher than the titers of the i.m. vaccinated animals. There was no correlation between the concentration of DNA used for vaccination, the neutralizing antibody responses elicited and protection against viral challenge. Seven days after viral challenge, a rapid and strong anamnestic antibody response was elicited in 100% of the gene gun vaccinated monkeys and in four i.m. vaccinated monkeys. Neutralizing antibody remained undetectable in two i.m. vaccinated monkeys. Overall, 60% (3/5) of the gene gun vaccinated animals and 87% (5/6) of the i.m. vaccinated monkeys survived viral challenge. This study is the first, to our knowledge, to show long-term protection of non-human primates against a human viral pathogen using a DNA vaccination protocol that did not include a booster immunization.

Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines.

Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime-booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase.

Post-exposure DNA vaccination protects mice against rabies virus

Post-exposure anti-rabies vaccination for individuals who have not previously been immunized against rabies includes a cell culture-derived vaccine and a one time injection of rabies immune globulin. Recent studies have shown DNA vaccinations to be highly effective in rabies pre-exposure experiments, but post-exposure protection has not been achieved. This failure is likely due to the slow onset of DNA vaccine induced antibody production. In an attempt to accelerate the onset of the antibody response, we manipulated variables, such as the route of vaccination and booster frequency. Anti-rabies virus antibody was detected 5 days after the initial DNA vaccination. Using this vaccination protocol and a single non-protective dose of anti-rabies immune serum, we questioned whether mice injected 6 h previously with rabies virus would be protected if a DNA vaccine was substituted for the cell culture-derived human diploid cell vaccine (HDCV). The DNA vaccine protected 87% of the mice (P = 0.00005, compared with unvaccinated control mice). Some 75% of mice receiving HDCV were protected (P = 0.00097, compared with unvaccinated control mice). Mice receiving only anti-rabies immune serum were not protected (P > 0.05 compared to unvaccinated control mice). Thus, post-exposure therapy, substituting a DNA vaccine for HDCV, did not compromise protection against rabies virus.

Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes

SummaryRecent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immunofluorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction.