Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization

Interaction of CD40 with its ligand (CD154) can induce CD40-bearing APCs to express immune stimulatory accessory molecules that facilitate immune recognition. We evaluated whether a plasmid vector encoding CD154 (pCD40L) could influence the immune response to a transgene protein encoded by coinjected plasmid DNA. We found that coinjection of pCD40L in BALB/c mice enhanced the Ab response to beta-galactosidase induced by i.m. or intradermal injection of placZ, a plasmid DNA vector encoding beta-galactosidase. Furthermore, i.m. or intradermal coinjection of pCD40L with placZ enhanced the generation of CTL specific for P815 cells transfected with placZ. This study indicates that pCD40L can serve as a genetic adjuvant capable of augmenting humoral and cellular immune responses to Ags encoded by plasmid DNA expression vectors.     

Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.     

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.     

Effects of mutating specific residues present near the amino terminus of 2'-5'-oligoadenylate synthetase

In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9-2 isozyme of 2'-5'-oligoadenylate synthetase. In vitro expression of deletion mutants showed that residues 1-9 are required for enzyme activity. Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2'-5'-oligoadenylate synthetase. Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity. Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity. The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria. The bacterially expressed protein was also enzymatically inactive. The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally. Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant. These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties.     

Costimulation provided by DNA immunization enhances antitumor immunity

The interaction of the TCR with MHC class I-bound Ag is insufficient for the priming of CTL unless secondary costimulatory signals are provided. To ascertain the minimum elements required to activate an Ag-specific CTL response in vivo, we injected mice intradermally or i.m. with plasmid DNA encoding a MHC class I-restricted peptide Ag (minigene) and different membrane-bound costimulatory ligands. The minigene-encoded epitope only primed a specific CTL response if injected in the vicinity of an ectopically expressed costimulatory ligand. Vector encoding B7-1 was repeatedly more potent at stimulating a cytolytic response than vector encoding B7-2. In contrast the B7-2-encoding plasmid preferentially enhanced Ag-specific Ab responses when injected with either protein or a cDNA expression vector. Gene vaccination with plasmids encoding OVA and B7-1, but not B7-2, prolonged survival in mice challenged with an OVA-transfected tumor. These results show that functional B7-1 transfection can be achieved in vivo and induces the selective induction of CTL. The data suggest that B7-1 plasmids should be coadministered with naked DNA vaccines that aim to induce tumor-specific cellular immunity.     

Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.     

Context-dependent immunogenicity of an S206G-substituted H-2Db-restricted simian virus 40 large T antigen epitope I variant

SV40 large tumor Ag (Tag) contains four H-2b-restricted (I, II/III, IV, and V) CTL epitopes. A hierarchy exists among these CTL epitopes. CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag, while epitope V-specific CTL are not. The mechanisms that define this hierarchy remain unknown. Initial studies have shown that the locations of epitopes I and V within SV40 Tag do not determine the immunological potencies of these epitopes. Like the wild-type Tag, derivatives in which the locations of epitopes I and V were precisely reversed within Tag failed to induce epitope V-specific CTL, but did induce epitope I-specific CTL. The use of an S206G-substituted epitope I variant (GAINNYAQKL) revealed that the S206G variant sequence induced CTL when located within the native epitope I context, but failed to do so when located within the epitope V context of Tag. Mutagenesis of residues adjacent to the S206G-substituted epitope I variant revealed that the identity of the residue flanking the amino terminus of the S206G variant was critical when it resided within the epitope V location, but not within the epitope I location. These results demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity, but that the relative importance of such effects varies in an epitope-dependent manner.     

Expression and purification of a truncated macrophage colony stimulating factor in Kluyveromyces lactis

A truncated human macrophage colony stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was obtained by using polymerase chain reaction. When inserted into plasmid pCXJ1 and psPHO5 and introduced into Kluyveromyces lactis, it directs the the secretory expression of the biologically active dimeric form of M-CSF. Through a four-step purification protocol, i.e. ammonium sulfate salting out, DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified to homogenerity and show its apparent molecular mass at 21KDa on reduced SDS-PAGE, with a specific activity of 1.21 x 10(7) units/mg protein.     

Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.     

Virus-like particles induce MHC class I-restricted T-cell responses. Lessons learned from the hepatitis B small surface antigen

H-2d mice generated a major histocompatibility complex (MHC) class I (Ld)-restricted T-cell response of defined restriction and epitope specificity to the hepatitis B virus small surface antigen (HBsAg). Here, we compare different vaccination techniques that prime in vivo class I-restricted, murine cytotoxic T lymphocyte (CTL) precursors and specific serum antibody responses. CTL were efficiently primed by the injection of low doses of recombinant native HBsAg particles without adjuvants, by the injection of low doses of denatured HBsAg monomers without adjuvants, by infection with recombinant vaccinia virus carrying a HBsAg-encoding gene, or by intramuscular transfer of plasmid DNA encoding HBsAg under appropriate promoter control. The observation that the injection of 100 ng to 1 microgram of native HBsAg "virus-like particles' (VLP) without adjuvants is an exogenous antigen preparation that efficiently primes class I-restricted CTL responses was unexpected. It reveals a novel aspect of the immunogenicity of VLP for T cells.     

Inhibition of cytolytic T lymphocyte activity by oxysterols

The objective of this study was to investigate the effects of oxysterols (OS), namely 5 alpha-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10(-5) M 5 alpha-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h 51Cr release. At a concentration of 5 x 10(-6) M, 5 alpha-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.     

Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo

OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.     

The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.     

DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction

DNA immunization can induce cytotoxic T lymphocytes (CTL), antibodies, and protection against microbial challenge. The underlying mechanisms remain obscure and must be understood to permit rational manipulation and optimization of the technique. We set out to enhance the intracellular degradation of a viral antigen, with the intent of improving antigen entry into, and presentation by, the class I major histocompatibility complex pathway. We achieved this goal by cotranslational ubiquitination of a plasmid-encoded viral antigen, lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP). We show that native NP is very stable in cell culture, while the ubiquitinated product is so rapidly degraded that it is barely detectable. This rapid degradation leads to more efficient sensitization of target cells in an in vitro cytotoxicity assay, consistent with enhanced antigen presentation, and both degradation and target cell recognition are blocked by a proteasome inhibitor. We have used the plasmid for in vivo studies and find that, remarkably, ubiquitination leads to a complete abrogation of antibody responses, presumably because the encoded protein is so rapidly and completely degraded that insufficient antigen remains to interact appropriately with B cells. In contrast, in vivo CTL induction is improved by ubiquitination of NP. That CTL are induced at all by this rapidly degraded protein may shed light on the mechanism by which CTL are induced by DNA immunization; it has been suggested that CTL induction following intramuscular DNA injection results not from antigen presentation by cells taking up and expressing the DNA but rather from uptake of soluble protein by specialized antigen-presenting cells (APC). It appears to us unlikely that the ubiquitinated protein could function in this manner, since it is so rapidly degraded in vitro and fails to induce antibodies in vivo. Finally, the ubiquitinated protein confers markedly enhanced protection against LCMV challenge. Mice immunized with a plasmid encoding NP show approximately 100-fold reductions in virus titers compared to controls, while mice immunized with a plasmid encoding the ubiquitinated NP show reductions in virus load of at least 5 x 10(4)- to 5 x 10(5)-fold. This is by far the most effective DNA vaccine that we have yet designed. Ubiquitination therefore may improve DNA immunization, but caution is warranted, since immunity to many microbes depends on induction of good humoral immunity, and we show here that this may be prevented by ubiquitination of the encoded protein.     

Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli

A synthetic gene encoding 'core' streptavidin (SAV) [amino acid (aa) residues 13-140 of Streptomyces avidinii SAV] has been efficiently expressed in Escherichia coli from the IPTG-inducible lac promoter of plasmid pET3a. In this system, expression levels are nearly tenfold greater for the synthetic gene than for the corresponding native gene. The synthetic gene was constructed from overlapping oligodeoxyribonucleotides whose sequences were optimized to incorporate codons preferred by highly expressed E. coli genes. Biochemical characterizations by gel methods, aa analysis, N-terminal sequencing, and size exclusion chromatography show that the synthetic gene product purified by affinity chromatography possesses the properties expected for core SAV.     

CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge

CD40/CD40 ligand interactions have a central role in the induction of both humoral and cellular immunity. In this study, we examined whether a plasmid expressing CD40 ligand/trimer (CD40LT) could enhance immune responses in vivo. BALB/c mice were injected with plasmid expressing beta-galactosidase DNA with or without CD40LT DNA or IL-12 DNA, and immune responses were assessed. Mice vaccinated with beta-gal DNA plus CD40LT DNA or IL-12 DNA had a striking increase in Ag-specific production of IFN-gamma, cytolytic T cell activity, and IgG2a Ab. The mechanism by which CD40LT DNA enhanced these responses was further assessed by treating vaccinated mice with anti-IL-12 mAb or CTLA-4 Ig (CTLA4Ig). Production of IFN-gamma and CTL activity was abrogated by these treatments, suggesting that CD40LT DNA was mediating its effects on IFN-gamma and CTL activity through induction of IL-12 and enhancement of B7 expression, respectively. Physiologic relevance for the ability of CD40LT DNA to enhance immune responses by the aforementioned pathways was shown in two in vivo models. First, with regard to CTL activity, mice vaccinated with CD40LT DNA did not develop metastatic tumor following challenge with lethal dose of tumor. Moreover, in a mouse model requiring IL-12-dependent production of IFN-gamma, mice vaccinated with soluble Leishmania Ag and CD40LT DNA were able to control infection with Leishmania major. These data suggest that CD40LT DNA could be a useful vaccine adjuvant for diseases requiring cellular and/or humoral immunity.     

Superiority of the ear pinna over muscle tissue as site for DNA vaccination

Three different vaccination sites were compared for efficiency of immunization with naked DNA. Using the bacterial lacZ gene as a model, all three sites of the mouse (skeletal muscle, dermis of abdominal skin or of the ear pinna) could express the gene product beta-gal but varied in expression time with muscle tissue showing the longest expression. Expression time, however, did not correlate with immune response intensity. The ear pinna was by far the most effective and muscle the least effective priming site for specific humoral and cytotoxic T cell-mediated immune responses. Following intra-pinna DNA inoculation, beta-gal expressing cells were detectable around the injection site and in the major draining lymph node. Efficiency of immunization was also dependent on the promoter and expression vector used. The cytomegalus virus promoter driven pCMV beta vector was superior to the Moloney murine leukemia virus LTR driven BAG vector. LacZ DNA immunization was also compared with cell-based vaccination with lacZ-transfected tumor cells, in which case again the pinna was the best site for inducing strong immune responses. Tumor-specific T cell responses could also be well induced in the pinna, leading to cytotoxic T lymphocyte induction and protective antitumor immunity. Thus, the pinna was found to be a privileged site for induction of antitumor responses and for genetic immunization, an important finding of immediate practical and potential future clinical implications.     

Adenovirus-mediated gene transfer of viral interleukin-10 inhibits the immune response to both alloantigen and adenoviral antigen

Although adenoviral vectors are attractive for gene transfer, their effectiveness is limited by host antiviral immune responses. In this study, we determined if host antiallograft and antiviral immunity could be diminished with an adenoviral vector encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10). AdSV40vIL-10, a vIL-10-expressing adenoviral vector with an SV40 promoter, induced significant prolongation of murine cardiac allograft survival to 32.2 +/- 1.7 days compared to 14.2 +/- 1.0 days for controls (p < 0.01). This effect was specific for vIL-10 encoding vector and could be inhibited by anti-vIL-10 monoclonal antibody (mAb). In vivo administration of adenovirus facilitated the generation of adenovirus-specific cytotoxic T lymphocytes (CTL), whereas treatment with AdSV40vIL-10 prevented CTL priming and generation of virus-specific immunity. AdSV40vIL-10 also induced extended expression of a beta-galactosidase reporter from a co-injected LacZ-encoding adenoviral vector. These results demonstrate that adenovirus-mediated gene transfer and expression of vIL-10 prolong allograft survival and inhibit the immune response to adenoviral antigens, thereby improving the persistence of the vector and extending transgene expression. The efficacy of adenoviral vectors can be improved by incorporating immunosuppressive genes into the vector.