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.

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.

Characterization of Sri Lanka rabies virus isolates using nucleotide sequence analysis of nucleoprotein gene.

Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

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.

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.

DNA immunization in combination with the immunostimulant monophosphoryl lipid a.

The use of the MPL® immunostimulant, a monophosphoryl lipid A preparation derived from the lipopolysaccharide (LPS) of Salmonella minnesota R595, began with the studies of Johnson et al. (1). It was shown that LPS was a potent adjuvant for protein antigens, even if administered at a different site and a different time than the antigen (2,3). Nonetheless, the toxicity of the LPS precluded its usefulness as a practical adjuvant. Studies by Ribi and co-workers (4-6) and others (7) resulted in the attenuation of the toxicity through exposure to mild acid hydrolytic conditions. The resulting acid hydrolysate was shown to be the 4'-monophosphoryl derivative of the lipid A moiety (8). Numerous biological studies confirmed that this 4'-monophosphoryl lipid A derivative was a potent immunostimulant which lacked many toxic properties of the parent LPS. Subsequent studies determined that mild alkaline treatment resulted in removal of one fatty acid from the MPL, resulting in additional attenuation of toxicity without changing the immunostimulating activity (9). These led to the development of the product MPL which is presently undergoing trials as an adjuvant for several human vaccines. The manufacture, chemical composition and structure of MPL has been detailed by Ulrich and Meyers (10). We will describe our techniques for using MPL as an immunostimulant in mice with the aim of enhancing the magnitude and duration of the protective neutralizing antibody response elicited by a DNA vaccine encoding the glycoprotein of the CVS rabies virus.

Rabies DNA vaccines for protection and therapeutic treatment.

Rabies is a successful zoonotic disease that has persisted over time, achieving worldwide distribution in a variety of species. Annually, in developing countries with limited access to high-quality antirabies biologics, approximately 50,000 individuals and millions of animals die of rabies. Many of these countries continue to use vaccines produced in sheep, goat or suckling mouse brain, with ultraviolet light or phenol inactivation of the virus. Although there are several efficacious rabies vaccines derived from cultured cells, such as the human diploid cell vaccine, they are costly to produce and prohibitively expensive for developing countries. DNA vaccines offer a new and powerful approach for the generation of needed vaccines. They are stable, inexpensive to produce, easy to construct and induce a full spectrum of long-lasting humoral and cellular immune responses. This review concerns the present state of rabies DNA vaccines, and addresses the technology that may enhance their therapeutic efficacy.

DNA immunization protects nonhuman primates against rabies virus.

More than 40,000 people die annually from rabies worldwide. Most of these fatalities occur in developing countries, where rabies is endemic, public health resources are inadequate and there is limited access to preventive treatment. Because of the high cost of vaccines derived from cell culture, many countries still use vaccines produced in sheep, goat or suckling mouse brain. The stability and low cost for mass production of DNA vaccines would make them ideal for use in developing countries. To investigate the potential of DNA vaccines for rabies immunization in humans, we vaccinated Macaca fascicularis (Cynomolgus) monkeys with DNA encoding the glycoprotein of the challenge virus standard rabies virus, or with a human diploid cell vaccine (HDCV). The monkeys then were challenged with a non-passaged rabies virus. DNA or HDCV vaccination elicited comparable primary and anamnestic neutralizing antibody responses. All ten vaccinated monkeys (DNA or HDCV) survived a rabies virus challenge, whereas monkeys vaccinated with only the DNA vector developed rabies. Furthermore, serum samples from DNA- or HDCV-vaccinated monkeys neutralized a global spectrum of rabies virus variants in vitro. This study shows that DNA immunization elicits protective immunity in nonhuman primates against lethal challenge with a human viral pathogen of the central nervous system. Our findings indicate that DNA vaccines may have a promising future in human rabies immunization.

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

Recent 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.

Cross-protection of mice against a global spectrum of rabies virus variants.

Rabies, a continuing worldwide problem, kills tens of thousands of people and millions of animals each year. The problem is most severe in developing countries, where cell culture-derived vaccines are unaffordable and the available nervous tissue-derived vaccines are often of questionable immunogenicity and may produce neurological complications. To determine the feasibility of developing a vaccine with worldwide applicability, we investigated whether recombinant vaccinia viruses expressing either the glycoprotein (G), the nucleoprotein (N), or both the G and N (GN) of the challenge virus strain (CVS) of rabies virus would cross-protect mice against 17 rabies virus isolates representing the spectrum of rabies virus variants found worldwide. The results were compared with the commercially available human diploid cell vaccine (HDCV). Among mice injected with any of the 17 viruses, > or = 95% were protected by vaccination with recombinant viruses expressing G or GN, and > or = 85% of the mice were protected by the HDCV. The recombinant virus expressing N was less protective, protecting against only 11 of the 17 viruses. Antibody prepared against the G of the strains used in the vaccines neutralized all 17 viruses, and sera from mice infected with any one virus variant cross-neutralized all of the other viruses. Thus, no antigenic differences that would potentiate vaccine failures were identified. These studies suggest that a single rabies virus strain or its G would protect globally against wild-type rabies viruses.

Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.

Paralysis of street rabies virus-infected mice is dependent on T lymphocytes.

Street rabies virus (SRV)-infected T-lymphocyte-deficient (nude) mice, in contrast to euthymic mice, did not develop hindlimb paralysis prior to death. To document the role of T lymphocytes in rabies virus-associated paralysis, 10(8) spleen cells from normal immunocompetent euthymic mice were transferred to nude mice and the recipient mice were challenged with SRV. One hundred percent of the reconstituted mice developed paralysis and died. Depletion of T cells from the donor spleen suspension prior to transfer abrogated the development of paralysis but did not prevent the deaths of the recipient animals. Mice receiving 10(8) rabies virus-immune spleen cells did not become paralyzed and did not die. Nude mice inoculated with either rabies virus-immune or normal mouse serum prior to and following SRV inoculation did not develop paralysis. Immune serum protected the mice, whereas animals inoculated with normal serum died. Central nervous system inflammatory responses in nude mice immunologically reconstituted with normal spleen cells were characterized by diffuse cellular infiltrates in the parenchyma and extensive perivascular cuffing. Perivascular infiltrates included CD8+ and CD4+ T lymphocytes and Mac-1+ macrophage-microglial cells. Inflammatory cells in the parenchyma were limited to CD8+ lymphocytes and Mac-1+ cells. These observations indicate that paralysis of SRV-infected mice is dependent on T lymphocytes. Whether injury leading to paralysis is mediated by T lymphocytes or by an influence of T lymphocytes on macrophage-microglial cells or other cells remains to be determined.

Role of CD4+ and CD8+ T cells in murine resistance to street rabies virus.

Mice of the SJL/J and BALB/cByJ inbred strains are naturally resistant to street rabies virus (SRV) injected via the intraperitoneal route. To determine the cellular mechanism of resistance, monoclonal antibodies specific for CD4+ or CD8+ subsets of T cells were used to deplete the respective cell population in SRV-infected animals. Elimination of CD4+ T-helper cells abrogated the production of immunoglobulin G (IgG) neutralizing antibodies in response to rabies virus infection and reversed the resistant status of SJL/J and BALB/cByJ mice. In contrast, in vivo depletion of CD8+ cytotoxic T cells had no measurable effect on host resistance to SRV. These results indicate that serum neutralizing antibodies of the IgG class are a primary immunological mechanism of defense against rabies virus infection in this murine model of disease. CD8+ cytotoxic T lymphocytes, which have been shown to transfer protection in other rabies virus systems, appear to have no role in protecting mice against intraperitoneally injected SRV.

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection.

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.

Impaired phagocytosis by the mononuclear phagocytic system in sapphire mink affected with Aleutian disease.

The phagocytic function of the mononuclear phagocytic system (MPS) in normal sapphire mink and in sapphire mink affected with experimental Aleutian disease was compared. Clearance from blood of carbon particles or 125I-labeled microaggregated human serum albumin, and subsequent measurement of radioactivity in phagocytic organs indicated profound MPS blockade in mink affected with advanced Aleutian disease. In contrast, MPS activity in mink in the early stage of the disease was comparable to that of normal mink. It is suggested that the MPS blockade may be responsible for some pathologic changes in Aleutian disease.

Detection of anti-rabies virus cytotoxic T lymphocytes in mice of four distinct H-2 haplotypes using target cells persistently infected with ERA rabies virus.

Cells persistently infected with Evelyn-Rokitnicki-Abelseth (ERA) rabies virus were established. The cells were used as stimulator and target cells to compare H-2 restricted cytotoxic T lymphocyte (CTL) responses specific for rabies virus in A/WySnJ (H-2a), C57BL/6J (H-2b), BALB/cByJ (H-2d), A.SW/SnJ (H-2s) and SJL/J (H-2s) mice. Using a 51chromium release assay, it was determined that an effector/target (E/T) ratio of 5:1 was necessary to demonstrate specific lysis of ERA virus persistently infected mouse neuroblastoma (MNB) (H-2a), EL-4 (H-2b) and P815 (H-2d) cells. Effectors at an E:T ratio of only 0.05:1 specifically lysed an SV-40 transformed SJL/J mouse fibroblast (SSSV) (H-2s) target monolayer. The CTL destruction of the SSSV monolayer was observed visually following Giemsa staining. This is the first instance in which a detailed method for detection of murine anti-rabies virus CTLs has been reported. Furthermore, it is the first time target cells persistently infected with rabies virus were used as stimulator cells to amplify CTLs in vitro and as target cells in the CTL assay. It also is the initial report in which rabies specific CTLs were characterized in H-2d and H-2s rabies virus infected mice.