Intracellular delivery of a novel multiepitope peptide vaccine by an amphipathic peptide carrier enhances cytotoxic T-cell responses in HLA-A*201 mice.

Cytotoxic T lymphocytes (CTL) are key players in the neutralization of viruses and killing of tumor cells. However, for generating an optimal CTL response by vaccination, the antigen has to be delivered directly into the cytoplasm for presentation by the conventional MHC class I pathway. To mimic the presentation of multiple epitopes by a tumor or virus infected cell, we have designed a multiepitope peptide vaccine incorporating thee CTL epitopes in tandem with double arginine spacers to facilitate efficient cleavage of the individual epitopes. To deliver the multiepitope peptide vaccine into the cytoplasm of mature dendritic cells for presentation by the MHC class I pathway we made use of an amphipathic peptide carrier. Direct injection of a non-covalent complex of the multiepitope peptide vaccine and amphipathic peptide carrier in an aqueous formulation into HLA-A*0201 (HHD) transgenic mice enhanced the cytotoxic T-cell responses by two to sixfold compared with multiepitope peptide vaccination alone. This novel antigen delivery strategy may find general application in the development of more effective vaccines for the treatment of cancer and infectious disease.

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein.

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 microg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.

Protection against Pseudomonas aeruginosa chronic lung infection in mice by genetic immunization against outer membrane protein F (OprF) of P. aeruginosa.

The Pseudomonas aeruginosa major constitutive outer membrane porin protein OprF, which has previously been shown to be a protective antigen, was targeted as a DNA vaccine candidate. The oprF gene was cloned into plasmid vector pVR1020, and the plasmid vaccines were delivered to mice by biolistic (gene gun) intradermal inoculation. Antibody titers in antisera from immunized mice were determined by enzyme-linked immunosorbent assay, and the elicited antibodies were shown to be specifically reactive to OprF by immunoblotting. The immunoglobulin G (IgG) immune response was predominantly of the IgG1 isotype. Sera from DNA vaccine-immunized mice had significantly greater opsonic activity in opsonophagocytic assays than did sera from control mice. Following the initial immunization and two consecutive boosts, each at 2-week intervals, protection was demonstrated in a mouse model of chronic pulmonary infection by P. aeruginosa. Eight days postchallenge, both lungs were removed and examined. A significant reduction in the presence of severe macroscopic lesions, as well as in the number of bacteria present in the lungs, was seen. Based on these findings, genetic immunization with oprF has potential for development as a vaccine to protect humans against infection by P. aeruginosa.

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways.

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.

Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A.

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T(H)1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA(mut) gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.

Pseudomonas aeruginosa LasD processes the inactive LasA precursor to the active protease form.

LasA and LasD are staphylolytic proteinases which are secreted by the opportunistic pathogen Pseudomonas aeruginosa. We have previously described the purification and characterization of both LasA and LasD, a 21-kDa protein which shares many of the enzymatic properties of LasA. In this follow-up study we describe the isolation of the 42-kDa precursor of LasA (proLasA) and demonstrate the ability of the purified LasD proteinase to cleave the inactive proLasA to the 20-kDa active form of the proteinase.

Construction and use of a nontoxigenic strain of Pseudomonas aeruginosa for the production of recombinant exotoxin A.

To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.

Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa.

We have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow-up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat-killed staphylococci. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N-terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross-reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range. In addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave beta-casein.

ToxR (RegA) activates Escherichia coli RNA polymerase to initiate transcription of Pseudomonas aeruginosa toxA.

The Pseudomonas aeruginosa (Pa) structural gene (toxA), which encodes the exotoxin A protein has been shown to be regulated at the transcriptional level by a protein designated ToxR (also known as RegA). We have previously reported that ToxR directly enhances toxA transcription in vitro; however, in the absence of ToxR, Pa RNA polymerase (RNAP) transcribes toxA with low efficiency. In the present study, we have examined the ability of ToxR to initiate toxA transcription using the heterologous Escherichia coli (Ec) RNAP and found that ToxR can function with Ec RNAP to efficiently transcribe toxA both in vitro and in vivo. Antibodies produced against the sigma 70 subunit of Ec RNAP inhibit ToxR-mediated enhancement of toxA transcription, suggesting that the RNAP holoenzyme (E sigma 70) is required for transcriptional activation of toxA. We further demonstrate that ToxR is required for open-complex formation at the toxA promoter. By selectively deleting toxA upstream sequences, we have localized at 214-bp region containing both the toxA promoter and a putative ToxR-binding site sufficient for ToxR-mediated transcription of toxA.

Active site mutations of Pseudomonas aeruginosa exotoxin A. Analysis of the His440 residue.

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins which use NAD+ as the ADP-ribose donor. By analogy to diphtheria and pertussis toxins, the His440 residue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His440 residue was explored through site-directed mutagenesis which resulted in the production of ETA proteins containing Ala, Asn, and Phe substitutions at the 440 position. The His440-substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed severely reduced ADP-ribosylation activity (> 1000-fold). However, NAD glycohydrolase activity remained intact and in the case of ETAH440N actually increased 10-fold. NAD+ binding is not affected by substitutions at the 440 site as indicated by similar Km values for the ETA variants tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensitive monoclonal antibody as well as sensitivity to proteinase digestion. In view of the location of His440 residue within or close to the proposed NAD(+)-binding site, these results suggest that His440 may be a catalytic residue involved in the transfer of the ADP-ribose moiety to the EF-2 substrate.

ToxR (RegA)-mediated in vitro transcription of Pseudomonas aeruginosa toxA.

Exotoxin A (ETA) has been described as a major virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa. The transcription of the ETA structural gene (toxA) has been shown to be positively regulated by the product of the toxR gene (also called regA). However, the mechanism by which ToxR regulates toxA transcription is still under investigation. We have expressed toxR in Escherichia coli under the control of the T7 promoter and purified the wild-type ToxR protein. We have also produced ToxR as a fusion protein consisting of the first 12 amino acids of the T7 capsid protein attached to the N terminus of the intact ToxR protein. In the present study we have developed and used an in vitro transcription assay in order to investigate the mechanism of ToxR-mediated transcriptional regulation of toxA. Under the conditions of this in vitro assay toxA transcription requires the toxR product in addition to P. aeruginosa RNA polymerase (RNAP). Both the native and the T7::ToxR fusion proteins facilitate initiation of toxA transcription in vitro in the presence of Pseudomonas RNAP. Additional studies using (i) specific enzyme-linked immunosorbent assay; (ii) indirect immunoprecipitation; and (iii) gel-filtration chromatography, indicate that ToxR binds to the purified Pseudomonas RNAP and strengthens the possibility that ToxR may be an alternative sigma factor. Furthermore, the ToxR-mediated transcription of toxA is increased approx. threefold in the presence of crude cytoplasmic extracts from P. aeruginosa ToxR+ or ToxR-RegB- strains, indicating that additional factors play a role in the efficient and optimal transcription of toxA.

Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue.

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety of NAD+ onto eucaryotic elongation factor 2 (EF-2). To study the ETA site of interaction with EF-2, an immobilized EF-2 binding assay was developed. This assay demonstrates that ETA, in the presence of NAD+, binds to immobilized EF-2. Additionally, diphtheria toxin was also found to bind to the immobilized EF-2 in the presence of NAD+. Comparative analysis was performed with a mutated form of ETA (CRM 66) in which a histidine residue at position 426 has been replaced with a tyrosine residue. This immunologically cross-reactive, ADP-ribosyl transferase-deficient toxin does not bind to immobilized EF-2, thus explaining its lack of ADPRT activity. ETA bound to immobilized EF-2 cannot bind the monoclonal antibody TC-1 which specifically recognizes the ETA epitope containing His426. Immunoprecipitation of native ETA by mAb TC-1 is only achieved by incubating ETA in the presence of NAD+. Diethyl pyrocarbonate modification of the His426 residue blocks ETA binding to EF-2 and prevents the binding of the TC-1 antibody. Analogs of NAD+ containing a reduced nicotinamide ring or modified adenine moieties cannot substitute for NAD+ in the immobilized binding assay. Collectively, these data support our proposal that the site of ETA interaction with EF-2 includes His426 and that a molecule of NAD+ is required for stable interaction.

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity.

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.

Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments.

With the determination of the three-dimensional structure of elastase and the probable identification of the active site and key residues involved in proteolytic activity, our knowledge of the molecular details of this interesting protease is rapidly increasing. Pseudomonas elastase appears to be remarkably similar to the Bacillus metalloproteinase thermolysin. A further significant development has been the discovery of the lasA gene and the fact that Pseudomonas elastase and alkaline proteinase appear to act in concert with the LasA protein to display the notable elastolytic activity exhibited by isolates of this organism. Biochemical and genetic studies indicate that LasA is a second elastase which may be an important virulence factor that has been overlooked in previous studies.

Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveals elastolytic activity due to alkaline proteinase and the LasA fragment.

The extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to investigate the role of LasB in virulence and LasA processing, a LasB-negative mutant, PAO1E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201-1. The resulting plasmid, pBRMOB-LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB-positive P. aeruginosa strain, PAO1. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PAO1E supernatant fluids yielded no detectable LasB (less than 1 ng ml-1 LasB). The absence of LasB in PAO1E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunoglobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PAO1E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PAO1 and PAO1E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.

Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant.

This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP-ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate.

Purification of the pyocin S2 complex from Pseudomonas aeruginosa PAO1: analysis of DNase activity.

Pyocin S2 purified from mitomycin C-induced lysates of Pseudomonas aeruginosa strain PAO1 has been shown to consist of a complex of two proteins. Further analysis of the purified S2 complex revealed that the 74 kd S2 pyocin demonstrates DNase activity which can be blocked by S2-specific antisera. Chromosomal DNA from pyocin sensitive cells treated with the pyocin S2 complex in vitro did not show any degradation, suggesting that the 10 kd protein inhibits the DNase activity of the S2 protein. These results suggest an alternative mechanism for the toxicity associated with the S2 pyocin.

Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity.

A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P.A. Schad and B.H. Iglewski, J. Bacteriol. 170:2784-2789, 1988). However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.