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.

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow-up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

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.

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 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.

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.

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.

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.

Induction of an invasive phenotype by human parvovirus B19 in normal human synovial fibroblasts.

OBJECTIVE: To investigate the possible role of human parvovirus B19 as an etiologic agent in rheumatoid arthritis (RA), with particular emphasis on its ability to induce invasiveness in human synovial fibroblasts. METHODS: We established an experimental in vitro system in which normal primary human synovial fibroblasts were treated with or without parvovirus B19-containing human sera for 7 days. The fibroblasts were then tested for their ability to degrade reconstituted cartilage matrix using a well-characterized cartilage invasion assay system. RESULTS: Incubation with parvovirus B19-containing serum induced an invasive phenotype in normal human synovial fibroblasts. B19 serum-treated synovial fibroblasts exhibited an increase in invasion of up to 248% compared with the activity of fibroblasts in media alone, in contrast to B19-negative sera-treated synovial fibroblasts, which exhibited no significant change compared with that in media alone. In addition, preincubation of viremic serum with a neutralizing antibody to B19 abrogated the observed effect. CONCLUSION: These results provide direct evidence regarding the ability of parvovirus B19 to induce invasive properties in normal human synovial fibroblasts. Parvovirus B19 has been proposed as an etiologic agent of RA, and our data provide the first biologic link between exposure to B19 and phenotypic changes in normal human synovial fibroblasts.

Metabolic functions of microbial nucleoside diphosphate kinases.

This article summarizes research from our laboratory on two aspects of the biochemistry of nucleoside diphosphate kinase from Escherichia coli--first, its interactions with several T4 bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleoside triphosphate biosynthesis. We identify some of the specific interactions and discuss whether the complex is linked physically or functionally with the T4 DNA replication machinery, or replisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeted deletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lacking this essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specific interactions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, how does T4 multiply after infection of this host? Finally, the ndk disruption strain has highly biased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of 7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutator phenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenic mechanisms? We conclude with brief references to related work in other laboratories.

Persistent infection of rhesus macaques by the rev-independent Nef(-) simian immunodeficiency virus SIVmac239: replication kinetics and genomic stability.

We generated previously a Nef(-), replication-competent clone of SIVmac239 in which the Rev protein and the Rev-responsive element were replaced by the constitutive transport element (CTE) of simian retrovirus type 1 (A. S. von Gegerfelt and B. K. Felber, Virology 232:291-299, 1997). In the present report, we show that this virus was able to infect and replicate in rhesus macaques. The Rev-independent Nef(-) simian immunodeficiency virus induced a persistent humoral immune response in all monkeys, although viral loads were very low. Upon propagation in the monkeys, the genotype remained stable and the virus retained its in vitro growth characteristics. The infected monkeys showed normal hematological values and no signs of disease at more than 18 months post-virus exposure. Therefore, replacement of the essential Rev regulation by the CTE generated a virus variant that retained its replicative capacity both in vitro and in vivo, albeit at low levels.

T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex.

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.

Oral transmission of primate lentiviruses.

Oral transmission of human immunodeficiency virus type 1 (HIV-1) is well documented in children who become infected postnatally through breast milk. In contrast, epidemiologic surveys have yielded conflicting data regarding oral HIV-1 transmission among adults, even though case reports have described seroconversion and the development of AIDS in adults whose only risk was oral-genital contact. To study oral virus transmission in primate models, we exposed rhesus macaques of various ages to cell-free simian immunodeficiency virus (SIV), including uncloned and molecularly cloned viruses. In neonates, viremia and AIDS developed after nontraumatic oral exposure to several SIV strains. Furthermore, chimeric simian human immunodeficiency viruses containing the HIV-1 envelope can also cross intact upper gastrointestinal mucosal surfaces in neonates. In adult macaques, infection and AIDS have resulted from well-controlled, nontraumatic, experimental oral exposure to different strains of SIV. These findings have implications for the risks of HIV-1 transmission during oral-genital contact.