Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques

The authors examined the efficacy of Bacillus anthracis protective antigen (PA) combined with adjuvants as vaccines against an aerosol challenge of virulent anthrax spores in rhesus macaques. Adjuvants tested included i) aluminum hydroxide (Alhydrogel), ii) saponin QS-21 and iii) monophosphoryl lipid A (MPL) in squalene/lecithin/Tween 80 emulsion (SLT). Animals were immunized once with either 50 micrograms of recombinant PA plus adjuvant, or with Anthrax Vaccine Adsorbed (AVA), the licensed human anthrax vaccine. The serological response to PA was measured by enzyme linked immunosorbent assay. Lymphocyte proliferation and serum neutralization of in vitro lethal toxin cytotoxicity were also assayed. In all vaccine groups, anti-PA IgM and IgG titers peaked at 2 weeks and 4-5 weeks postimmunization, respectively. Five weeks postimmunization, animals in all vaccine groups demonstrated PA-specific lymphocyte proliferation and sera that neutralized in vitro cytotoxicity. Six weeks after immunization, the animals were challenged by aerosol with approximately 93 LD50 of virulent anthrax spores. Animals were bled daily for 1 week to monitor bacteremia, and deaths were recorded. Anti-PA ELISA titers in all groups of immunized animals were substantially increased 2 weeks after challenge. One dose of each vaccine provided significant protection (> 90%) against inhalation anthrax in the rhesus macaques.     

Characterization of membrane translocation by anthrax protective antigen

Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.     

Protective efficacy against malaria of a combination sporozoite and erythrocytic stage vaccine

Most malariologists believe that optimal malaria vaccines will induce protective immune responses against different stages of the parasite's life cycle. A multiple antigen peptide (MAP) vaccine based on the Plasmodium yoelii circumsporozoite protein (PyCSP) protects mice against sporozoite challenge by inducing antibodies that prevent sporozoites from invading hepatocytes. A purified recombinant protein vaccine based on the P. yoelii merozoite surface protein-1 (PyMSP-1) protects mice against challenge with infected erythrocytes, presumably by inducing antibodies against the erythrocytic stage of the parasite. We now report studies designed to determine if the PyMSP-1 vaccine protects against challenge with sporozoites, the stage encountered in the field, and if immunization with a combination of the PyCSP and PyMSP-1 vaccines provides additive or synergistic protection against sporozoite challenge. In two experiments, using TiterMax or Ribi R-700 as adjuvant, 3 of 19 mice immunized with the PyMSP-1 vaccine were completely protected against sporozoite challenge. The remaining mice had significantly delayed onset and lower levels of peak parasitemia than did control mice (11.1 +/- 2.8% vs. 36.7 +/- 1.6% in experiment #2, P < 0.01). Immunization with the combination vaccine reduced by approximately 50% the level of antibodies induced to PyCSP and PyMSP-1, as compared to that induced by the individual components. However, in two experiments, there was evidence of additive protection. Six of 19 (31.6%) immunized with the PyCSP vaccine, 3 of 19 (15.8%) immunized with the PyMSP-1 vaccine, and 10 of 19 (52.6%) immunized with the combination were completely protected against sporozoit challenge. This modest increase in protection in the combination group may be a reflection of additive anti-PyCSP and anti-PyMSP-1 immunity, since mice in the combination group had diminished levels of antibodies to each components. These studies indicate that considerable work may be required to optimize the construction, delivery, and assessment of multi-stage malaria vaccines.     

IL-15 augments CD8+ T cell-mediated immunity against Toxoplasma gondii infection in mice

Cytokines of the Th1 profile are important mediators of protective host immunity against Toxoplasma gondii infection in mice. In this study we describe the effect of the recently identified cytokine, IL-15, on prevention of murine infection with T. gondii. Administration of exogenous rIL-15 with soluble Toxoplasma lysate Ag (TLA) provides complete protection against a lethal parasite challenge, whereas treatment with either rIL-15 or TLA alone is not protective. Following immunization with TLA/rIL-15, there is a significant proliferation of splenocytes expressing the CD8+ phenotype in response to TLA. A significant rise in the level of serum IFN gamma was observed in vaccinated mice. Adoptive transfer of CD8+ T cells, but not CD4+ T cells, from TLA/rIL-15-vaccinated mice protects naive mice from a lethal parasite challenge. These CD8+ T cells exhibit enhanced CTL activity against target macrophages infected with T. gondii. Mice that have been immunized are protected against lethal parasite challenge for at least 1 mo postvaccination. These observations demonstrate that TLA when administered with exogenous rIL-15 generates toxoplasmacidal Ag-specific CD8+ T cells. These T cells proliferate upon exposure to parasite Ag, exhibit long term memory CTL against infected target cells, and may be involved in host immune memory to this parasite.     

Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs

The efficacy of an anthrax vaccine licensed for human use, MDPH-PA, was tested in guinea-pigs intramuscularly challenged with 10, 100 or 1000 LD50 of spores from two virulent strains of Bacillus anthracis, Vollum 1B and Ames. As demonstrated in other investigations, immunization with MDPH-PA provided better protection against challenge with the Vollum 1B strain than with the Ames strain, although vaccine efficacy against the Ames strain was better than previously reported. Enzyme-linked immunosorbent assay of serum antibody titres to B. anthracis protective antigen showed that there was no significant correlation between survival and antibody titres.     

Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 microg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 microg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 microg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 microg. The CTLs induced in BALB/c mice immunized twice with 100 microg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Cytokinin regulates the expression of a soybean beta-expansin gene by a post-transcriptional mechanism

Bacillus anthracis, a Gram positive bacterium, is the causative agent of anthrax. This organism is capsulogen and toxinogenic. It secretes two toxins which are composed of three proteins: the protective antigen (PA), the lethal factor (LF) and the edema factor (EF). The lethal toxin (PA + LF) provokes a subite death in animals, the edema toxin (PA + EF) induces edema. The edema and the lethal factors are internalised into the target cells via the protective antigen. EF and LF exert an adenylate cyclase and a metalloprotease activity respectively. The structure-function relationship of these three proteins were defined using in vitro and in vivo approaches.     

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag85-93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env189-196, SSWDFITV), originally described in the B6.CH-2(bm13) mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.     

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.     

Skeletal bone morphogenetic proteins suppress the expression of collagenase-3 by rat osteoblasts

The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming DeltaANR and DeltaSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.     

Anthrax toxin as a molecular tool for stimulation of cytotoxic T lymphocytes: disulfide-linked epitopes, multiple injections, and role of CD4(+) cells

We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated. This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide. Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously. We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines. First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4(+) helper responses are essential for stimulating specific CTL with the PA-LFn system. Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection.     

Tacrolimus therapy for persistent or recurrent acute rejection after lung transplantation

Purified native F1 antigen from Yersinia pestis was used to assess controlled-release vaccine delivery systems in poly(lactide-co-glycolide) (PLG) microparticles and liposomes. Antigen encapsulated in PLG microparticles induced high serum titres when injected i.p. in mice: mucosal IgA was also detected. Mice immunized with F1 in Alhydrogel or PLGs were protected against subcutaneous challenge with Y. pestis. F1 antigen surface-labelled onto liposome vesicles stimulated high serum titres in Balb/c mice and also induced a mucosal response: F1-labelled liposomes protected mice against challenge with up to 1 x 10(5) organisms. These findings indicate that a significant immune response is induced by immunizing with F1 formulated in PLGs and liposomes and that protection was achieved after only one dose.     

Human diploid cell culture rabies vaccine (HDCV) and purified chick embryo cell culture rabies vaccine (PCECV) both confer protective immunity against infection with the silver-haired bat rabies virus strain (SHBRV)

The demonstration of extensive differences in the antigenic makeups of the silver-haired bat rabies virus (SHBRV) and canine rabies virus (COSRV) strains raised concerns as to whether current licensed rabies vaccines are sufficiently protective against SHBRV. NIH mouse protection test results show that both the human diploid cell culture rabies vaccine (HDCV) and the purified chicken embryo cell rabies vaccine (PCECV) protected against lethal infection with SHBRV as well as the canine rabies strain COSRV. However, in this investigation, the potencies of both vaccines in mice were found to be significantly higher for COSRV than for SHBRV. The in vivo protection data are confirmed by in vitro virus neutralizing antibody (VNA) test results which demonstrate that mice immunized with HDCV or PCECV develop significantly higher VNA titres against COSRV than against SHBRV. In contrast, VNA tests of sera from individuals immunized with HDCV or PCECV showed that humans, as opposed to mice, develop significantly higher VNA titres against SHBRV than against COSRV. These data suggest that HDCV and PCECV will protect humans against infection with the silver-haired but rabies virus strain in addition to canine rabies virus strains.     

Effect of dual-subtype vaccine against feline immunodeficiency virus infection

Dual-subtype feline immunodeficiency virus (FIV) vaccine, consisting of inactivated cells infected with subtypes A (Petaluma strain) and D (Shizuoka strain), was developed and tested for its vaccine efficacy against FIV infection in specific pathogen free (SPF) cats. Animals were monitored for proviral DNA by FIV-specific PCR and for FIV-specific antibody profiles by ELISA and virus-neutralization assays. In addition, blood from challenged cats was inoculated into naive SPF cats to confirm the viral status of the vaccinated cats. All cats immunized with Petaluma vaccine alone were protected against homologous Petaluma challenge, but only one of four cats was protected against heterologous Shizuoka challenge. More importantly, all cats immunized with the dual-subtype vaccine were protected against both Petaluma and Shizuoka challenges. These results suggest that a multi-subtype vaccine approach may provide the broad-spectrum immunity necessary for vaccine protection against strains from different subtypes.     

Influence of the type of family predisposition on the age of ulcer disease onset

Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds to the receptor on mammalian cells and facilitates translocation of EF or LF into the cytosol. PA is the primary component of several anthrax vaccines. In this study we expressed and purified PA from Escherichia coli. The purification of PA from E. coli was possible after transporting the protein into the periplasmic space using the outer membrane protein A signal sequence. The purification involved sequential chromatography through hydroxyapatite, DEAE Sepharose CL-4B, followed by Sephadex G-100. The typical yield of purified PA from this procedure was 500 microg/liter. PA expressed and purified from E. coli was similar to the PA purified from Bacillus anthracis in its ability to lyse a macrophage cell line (J774A.1). The present results suggest that a signal sequence is required for the efficient translocation of PA into E. coli periplasmic space.     

The shared tumor-specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection

A number of human tumor antigens have been characterized recently using cytolytic T lymphocytes (CTL) as screening tools. Some of them are encoded by MAGE-type genes, which are silent in normal tissues except in male germ cells, but are activated in a variety of tumors. These tumor-specific shared antigens appear to be promising targets for cancer immunotherapy. However, the choice of these antigens as targets has been questioned because of the lack of direct evidence that in vivo responses against such antigens can lead to tumor rejection. The antigen encoded by the mouse gene P1A represents the only available animal model system for MAGE-type tumor antigens. We show here that mice immunized by injection of L1210 leukemia cells expressing P1A and B7-1 (L1210.P1A.B7-1) are efficiently protected against a challenge with a lethal dose of mastocytoma P815 tumor cells, which express P1A. Mice immunized with L1210 cells expressing B7-1 but not P1A were not protected. Furthermore, we observed that P1A-transgenic mice, which are tolerant to P1A, were not protected after immunization with L1210.P1A.B7-1. These results demonstrate that the immune response to P1A is the major component of the tumor rejection response observed in normal mice, and support the use of tumor-specific shared antigens as targets for the immunotherapy of human cancer.     

Protection against lethal murine coccidioidomycosis by a soluble vaccine from spherules

The formaldehyde-killed, whole-spherule vaccine, which is protective against lethal challenge of laboratory animals with Coccidioides immitis, was fractionated. It yielded a soluble, multicomponent, subcellular fraction termed the 27K vaccine. This vaccine, when it was accompanied by adjuvant, protected mice against lethal intranasal and intravenous challenge with C. immitis.     

Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein

Immunization with irradiated sporozoites protects animals and humans against malaria, and the circumsporozoite protein is a target of this protective immunity. We now report that adjuvant-free intramuscular injection of mice with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein induced higher levels of antibodies and cytotoxic T lymphocytes against the P. yoelii circumsporozoite protein than did immunization with irradiated sporozoites. Mice immunized with this vaccine had an 86% reduction in liver-stage parasite burden after challenge with 5 x 10(5) sporozoites (> 10(5) median infectious doses). Eighteen (68%) of 28 mice that received two or three doses of vaccine were protected against challenge with 10(2) sporozoites, and the protection was dependent on CD8+ T cells. These studies demonstrate the utility of plasmid DNA immunization against a nonviral infection. By obviating the requirement for peptide synthesis, expression and purification of recombinant proteins, and adjuvants, this method of immunization provides an important alternative for rapid identification of protective B- and T-cell epitopes and for construction of vaccines to prevent malaria and other infectious diseases.     

Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.     

Protection of C3H/He mice from experimental Borrelia burgdorferi infection by immunization with a 110-kilodalton fusion protein

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.