Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers.

Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.

DNA shuffling and vaccines.

One challenge of biotechnology is to find ways to optimize enzymes, cytokines, vaccines or transgenes in new contexts that are typically not found in nature. The approach of DNA shuffling is a test-tube process that takes advantage of recombination to generate libraries of chimeric genes, which can then be screened to identify the encoded proteins improved in one or more functions. DNA shuffling of two or more genes that are structurally similar and homologous in function is particularly efficacious in generating large libraries of functionally novel molecules. Other directed evolution methods, such as those involving directed or random mutagenesis, have several limitations compared to the DNA shuffling recombination process. A wide variety of genes have been submitted to DNA shuffling, and significant improvements in various functional parameters have been obtained. Several examples of the application of DNA shuffling to vaccine development, therapeutics and gene therapy are discussed here.

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg.

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.

DNA vaccination with HuD inhibits growth of a neuroblastoma in mice.

Some patients with small cell lung cancer (SCLC) or neuroblastoma develop an immune response against HuD, a human homologue of the Drosophila protein, elav, which is expressed in the nucleus and to a lesser degree the cytoplasm of neurons and tumor cells. This immune response is characterized by antibodies (anti-Hu) that at high titers are associated with a disease called paraneoplastic encephalomyelitis/sensory neuronopathy, in which infiltrates of T cells are found in the tumor and nervous system. Although all SCLCs express HuD, anti-Hu antibodies are identified in only 17% of patients with SCLC, usually at low titers, and are associated with indolent tumor growth. To determine whether the anti-Hu immune response causes indolent tumor growth, we developed an animal model using HuD DNA immunization. We found that a plasmid coding for a secreted form of HuD induced a strong and specific anti-Hu response. Immunized animals were challenged by s.c. implantation of a neuroblastoma cell line that constitutively expresses HuD. When compared with controls, mice immunized with the secreted HuD showed significant tumor growth inhibition (51% reduction volume; P = 0.0012), and 14% of them had complete tumor rejection. Tumors from these animals showed three times more CD3+ lymphocytic infiltrates than those from control mice and had a higher CD8+:CD4+ ratio. None of the animals developed neurological deficits or neuropathological evidence of nervous system pathology. In this mouse model of neuroblastoma, DNA immunization with HuD resulted in tumor growth inhibition but did not induce neurological disease. This model closely mimics the clinical course of more indolent tumor growth seen in patients with the anti-Hu immune response.

MEF-2 and Oct-1 bind to two homologous promoter sequence elements and participate in the expression of a skeletal muscle-specific gene.

The murine adult IIB myosin heavy chain (IIB MyHC) gene is expressed only in certain skeletal muscle fibers. Within the proximal promoter are two A + T-rich motifs, mAT1 and mAT2, which greatly enhance muscle-specific transcription; myogenic cells contain proteins that bind to these sequences. MEF-2 binds to both mAT1 and mAT2; a mutation abolishing its binding to mAT1 greatly diminishes the activity of the promoter. Both mAT motifs also form complexes with a protein requiring a target sequence typical of POU domain proteins, which migrate in electrophoretic mobility shift assays to the same position as a complex containing purified Oct-1 and which are supershifted by an antibody specific to Oct-1; this protein is therefore probably Oct-1. Footprinting experiments demonstrate that mAT1 is preferentially occupied by MEF-2 and mAT2 by Oct-1 and that these two proteins appear to bind cooperatively to their respective sites. Although the two mAT motifs have sequences that are very similar, they nonetheless exhibit distinct behaviors and perform differently in the activation of the promoter. The contribution of the IIB MyHC gene to specification of the myogenic phenotype is thus at least in part regulated by MEF-2 and Oct-1.

The preferential induction of a Th1 immune response by DNA-based immunization is mediated by the immunostimulatory effect of plasmid DNA.

In the present study, we have investigated the T cell response to the HBsAg, normally secreted as multivalent particles, and to beta-galactosidase, a cytoplasmic antigen, delivered as plasmid DNAs. We found that cytokines characteristic of a Th1 phenotype are produced in mice immunized by these plasmid DNAs. Using repeated injections of low doses of purified antigen, we demonstrated that neither prolonged presence of the antigen nor site of immunization resulted in an immune response with characteristics resembling those obtained with DNA-mediated immunization. Analysis of immune responses induced in mice by coinjection of plasmid DNA and beta-galactosidase or HBsAg demonstrated that the coinjected DNA stimulated a Th1 response against the injected antigen. These data therefore strongly suggest that the strong immune response obtained after intramuscular DNA immunization was due to the adjuvant effect of the plasmid DNA which is also responsible for the selective activation of CD4(+) T cells with a Th1 phenotype.

DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice.

Hepatitis B virus (HBV) remains a serious worldwide health problem and the possibility to control it will depend on the availability of safe, effective and affordable vaccines. Recombinant protein or plasma-derived vaccines containing HBV surface antigen (HBsAg) are safe and generally efficacious, however, they are too expensive for widespread use in areas of HBV endemicity and are only partially effective for treatment of HBV chronic carriers. Immunization of mice by injection of HBsAg-expressing plasmid DNA results in rapid induction of strong and long-lasting humoral and cell-mediated immune responses. Here we report optimization of the humoral response with the use of necrotizing agents, co-expression of cytokines or co-stimulatory molecules and formulation of the DNA with cationic liposomes. DNA-based immunization of HBsAg-transgenic mice can also overcome non-response to HBsAg. Thus, DNA vaccines against HBV may be useful for both prophylactic and therapeutic purposes.

The transcriptional activity of a muscle-specific promoter depends critically on the structure of the TATA element and its binding protein.

We have previously characterized the proximal promoter of the mouse IIB myosin heavy chain (MyHC) gene, which is expressed only in fast-contracting glycolytic skeletal muscle fibers. We show here that the substitution into this promoter of a non-canonical TATA sequence from the IgH gene results in inactivity in muscle cells, even though TATA-binding protein (TBP) can bind strongly to this mutated promoter. Chemical foot-printing data show, however, that TBP makes different DNA contacts on this heterologous TATA sequence. The inactivity of such a non-canonical TATA motif in the IIB promoter context appears to be caused by a non-functional conformation of the bound TBP-DNA complex that is incapable of sustaining transcription. The conclusions imply that the precise sequence of the promoter TATA motif needs to be matched with the specific functional class of upstream activator proteins present in a given cell type in order for the gene to be transcriptionally active.

DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.

Comparison of plasmid DNA preparation methods for direct gene transfer and genetic immunization.

Plasmid DNA is widely used for direct gene transfer in animals to study gene therapy, gene regulation, drug delivery and genetic immunization. Here we compare cesium chloride and anion-exchange purified plasmid DNA for direct gene transfer in mouse muscle and show no differences in efficiency of transfection with reporter genes or in humoral response to DNA-based immunization.

DNA vaccines for emerging infectious diseases: what if?

A novel and powerful method for vaccine research, colloquially known as DNA vaccines, involves the deliberate introduction into tissues of a DNA plasmid carrying an antigen-coding gene that transfects cells in vivo and results in an immune response. DNA vaccines have several distinct advantages, which include ease of manipulation, use of a generic technology, simplicity of manufacture, and chemical and biological stability. In addition, DNA vaccines are a great leveler among re-searchers around the world because they provide unprecedented ease of experi-mentation. To facilitate diffusion of information, an Internet site has been established called THE DNA VACCINE WEB (URL:http://www.genweb.com/dnavax/dnavax.html). In this review, a brief survey is undertaken of the experimental models and preclinical work on DNA vaccines to contribute to a greater awareness of the possibilities for emerging infectious diseases.

DNA-mediated immunization to hepatitis B surface antigen: longevity of primary response and effect of boost.

Intramuscular (i.m.) injection of mice with plasmid DNA expression vectors containing all or part of the hepatitis B virus (HBV) gene encoding the envelope proteins induces a strong humoral response to the HBV surface antigen (HBsAg) which is sustained for up to 74 weeks without boost. After a single i.m. injection of 100 micrograms DNA, antibodies to HBsAg (anti-HBs) reach ELISA titers of 4 x 10(4) in C57BL/6 mice and 10(4) in BALB/c mice, or somewhat less in older mice. Although antibody levels induced by a single injection of DNA do not diminish significantly over time, they can be further increased 10-200-fold by boosting with a second injection of DNA or an injection of recombinant HBsAg protein. Prior injection of DNA does not affect the strength or timing of the boosting effect, suggesting that there is no immune response against the vector itself. Boosting with a second injection of DNA is possible even in BALB/c mice, which are known to have a strong cytotoxic T-lymphocyte response against an epitope on the major HBV envelope protein, indicating that possible destruction of newly transfected muscle fibers is not so quick and efficient as to abort the boosting effect. A single injection of DNA results in a stronger and longer lasting humoral response than does a single injection of recombinant protein.

Role of weight-bearing function on expression of myosin isoforms during regeneration of rat soleus muscles.

The expression of myosin isoforms was studied in regenerated rat soleus muscle during either normal or altered postural activity. Regeneration was induced following injury by venom from the Notechis scutatus scutatus snake. Immunohistochemical analysis showed that, in regenerating soleus muscle after 3 wk of hindlimb suspension, nearly all fibers reacted positively with the myosin heavy chain (MHC) antibody associated with fast-twitch muscle fibers (fast MHC). When 3 wk of recovery with normal weight-bearing activity followed hindlimb suspension, the regeneration soleus muscle exhibited a nearly homogeneous staining with the MHC antibody associated with the slow-twitch muscle fibers (slow MHC). These findings were in accordance with quantitative analysis of the electrophoretic separation of the native myosin isoforms. Immunohistochemical data showed that removal of weight bearing in the 21-day old regenerated soleus muscles resulted in an increase in fast MHC expression. Together, the results of the present study clearly demonstrate that the postural load is an important component in the induction of slow MHC in regenerating muscle and that the control of the expression of MHC in muscle comprising a homogeneous population of fibers deriving from satellite cells appears more homogeneous and more complete than in a nondegenerated one.

DNA vaccines, cyberspace and self-help programs.

Occasionally, a major change in vaccine methodology comes along. Such would appear to be the case with the advent of DNA-mediated immunization, colloquially known as DNA vaccines. This represents a radical new way to deliver antigens; it involves the direct introduction of a plasmid DNA encoding an antigenic protein which is then expressed within cells of the organism. This leads to surprisingly strong immune responses, involving both the humoral and cellular arms of the immune system. DNA-mediated immunization to a single antigen can provide protection against infection by a pathogen. Here, a guide is provided comprising twelve steps to help design and carry out DNA-mediated immunization. This approach to immunization will greatly facilitate studies of immunophysiological responses to antigens of pathogenic organisms. An Internet site (URL: http:@www.genweb.com/Dnavax/dnav ax.html) has been created to help promote this potentially revolutionary approach to vaccination in the service of public health.

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures.

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

DNA-mediated immunization in mice induces a potent MHC class I-restricted cytotoxic T lymphocyte response to the hepatitis B envelope protein.

The particulate form of the major envelope or surface (S) protein of hepatitis B virus (HBV) can be taken up by antigen-presenting cells and processed for class I presentation as an exogenous protein. We have used several DNA plasmid vectors expressing the HBV envelope proteins to determine whether these sequences are able to induce cytotoxic T lymphocyte (CTL) responses in BALB/c mice after intramuscular DNA injection. A potent and specific induction was obtained, which can be detected ex vivo using either specific or nonspecific (interleukin-2) stimulation in cell culture, and the DNA-primed CTL responses are stronger than those obtained with protein injection with either stimulation protocol. The CTL response induced by DNA-based immunization is both canonical and highly specific as indicated by the nature of the epitope presented (amino acids 28-39), the class I allele used (Ld), and the T lymphocytes involved (CD8+). The CTL response is initiated between 3 and 6 days after DNA injection. By 6-12 days after a single DNA injection, ex vivo cytolytic activity is nearly maximal, and similar high levels of activity can still be detected 4 months after injection. The possibility is discussed that the unusual mode of delivery of the antigen to the immune system provided by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation as an exogenous protein in addition to activating potentially the classical endogenous pathway.

Use of plasmid DNA for direct gene transfer and immunization.

Direct gene transfer by intramuscular injection of plasmid DNA encoding an antigenic protein may be used for the purpose of immunization. Several factors influence the uptake and expression of plasmid DNA in skeletal muscle, which in turn influence the immune response to the expressed protein. Physical barriers and other factors may impede the diffusion of the DNA within the muscle tissue or its entry into the muscle fibers. Although the efficiency of gene transfer in normal mouse muscle is low (< 100 fibers per injection site), both humoral and cell-mediated immune responses to the hepatitis B surface antigen (HBsAg) are obtained after the expression of a transferred gene, and these are dose dependent. The efficacy of the immune response can be improved by injection of the DNA in or following pretreatment with a hypertonic solution or with the local anesthetic bupivacaine, and even more so by injecting the DNA into regenerating muscle.

DNA-mediated immunization to the hepatitis B surface antigen. Activation and entrainment of the immune response.

The use of plasmid vectors expressing the HBsAg, along with improved protocols for transfection of muscle fibers (Refs. 3-6 and Davis et al., this volume), have provided the reagents and methods with which to investigate the characteristics of the strong immune response given by this antigen after DNA-mediated immunization. Analysis of the fine specificity of the humoral response provides support for the idea that the HBsAg-bearing particles are formed such that the B and T epitopes are presented to the immune system in a way resembling that of the natural viral or subviral particles. As shown here and elsewhere, DNA-mediated immunization with the HBsAg-expressing plasmid vectors induces strong CTL responses as well as a dominant Th1 phenotype among the splenic lymphocytes of immunized mice. The Th1 cytokine profile can be obtained in two different strains of mice and with two types of proteins, HBsAg and beta-galactosidase. One important line of investigation in the future will be to determine the mechanism of this generic Th1 response to DNA-based immunization. Circumstantial evidence, discussed by Pisetsky et al. (this volume), suggests that the chemical nature of DNA may play a role as an adjuvant (see also Ref. 31), and this hypothesis to explain the cytokine profiles observed after DNA-mediated immunization must now be taken seriously. All the questions raised by this novel method of immunization are of interest for the design of future vaccines, even if DNA itself is ultimately not the vaccinating moiety. The question of antigen presentation is particularly intriguing, since the small amounts of protein produced by DNA-mediated immunization (on the order of nanograms) are capable of inducing strong immune responses at the level of B and T cells. Although initially it seemed obvious that endogenous protein synthesis in cells transfected with plasmid DNA would account for the observed induction of CTL activity, this idea must be examined in light of two well established sets of experimental results. First, the primary events in activation of CD8+ (as well as CD4+) T lymphocytes normally require professional APC capable of furnishing co-stimulatory signals to supplement the consequences of interaction of the T-cell receptor with MHC surface molecules. Second, endogenous synthesis and processing is not the only mechanism of class I epitope presentation, and numerous examples are now known whereby particulate exogenous proteins, such as HBsAg, can be taken up and processed in such a way as to allow class I presentation of peptides. Consideration of these two points suggests that a major contribution to the observed CTL induction afforded by DNA-mediated immunization could come from the sustained presence of the antigenic protein in interstitial spaces or in the circulation, coupled with the ability of the exogenous protein to be processed for class I presentation. This could be true for many other proteins in addition to the HBsAg. This hypothesis eliminates the inconvenient notion that muscle fibers (or other nonleukocyte cells) present antigen in a way compatible with primary activation of T cells. However, muscle tissue can be an important reservoir of the antigen because of the potential for prolonged synthesis of the protein; this could therefore explain the immune entrainment observed after DNA-mediated immunization. Muscle fibers or other cells could also serve to present class I epitopes for the purpose of restimulating and thus expanding the pool of activated CD8+ T lymphocytes. These explanations, though certainly plausible, will require experimental investigation. The small numbers of the transfected cells in vivo, as well as the potential mobility of transfected cells other than muscle fibers, may well render such experimentation difficult. DNA-mediated immunization clearly offers opportunities for obtaining novel insights into immunological mechanisms and immunization processes. It is also likely to promote vacc

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.

Modifications of gene expression in myotonic murine skeletal muscle are associated with abnormal expression of myogenic regulatory factors.

The mouse mutants ADR ("arrested development of righting") and the allelic CRP ("cramp") are characterized by a myotonic phenotype resulting from a dysfunction of the skeletal muscle chloride channel which leads to myotonic trains of actions potentials in response to stimuli. Compared to normal mouse muscle, numerous biochemical modifications have been found in the ADR muscle, and changes are observed in the expression of certain isoforms of contractile proteins. We have therefore measured the levels of the mRNA transcripts encoding the myosin heavy chain isoforms (MyHC) in both mutants. Transcripts for the myogenic regulatory factors were also studied since they are known to play a role in the induction of muscle-specific gene transcription, and their own expression is modified by different electrical activity patterns. In both mutants, the mRNA encoding the IIB MyHC was considerably decreased. In contrast, the mRNAs for the IIA, IIX, and beta/slow MyHCs were increased. The mRNA for the neonatal MyHC mRNA was not detectable, and therefore fiber regeneration does not appear to play a role in these phenomena. Among the myogenic regulatory factors, herculin is the most abundant in adult muscle; however, herculin mRNA undergoes a large decrease in myotonic muscle which does not seem to be related to the changing fiber type. The levels of MyoD and myogenin mRNAs are also modified with the former decreasing and the latter increasing. Qualitatively similar changes are seen in the ADR and CRP mutants; however, they are generally less pronounced in CRP. These observations suggest that specific myogenic factors may be linked to the expression of individual MyHC genes and that abnormal expression of some of the factors may be associated with myotonic muscle pathology.