Use Of DNA Vaccines Research Articles

Gene Vaccination for the Induction of Immune Tolerance

DNA vaccination is a strategy of immunization based on the injection of a gene encoding for a target protein with the goal of eliciting a potentially protective immune response in the host. Compared to traditional immunization procedures, DNA vaccination offers several advantages: increased availability of antigenic peptides because of the endogenous and long-term synthesis of the gene product, improved antigen processing and presentation, possibility of antigen structure modeling through molecular engineering, coexpression of immunologically relevant agents, and low cost of vaccine production. Although the choice of the most appropriate vector for gene transfer may still be controversial, the application of DNA vaccination to the treatment of autoimmune diseases in different experimental animal models has demonstrated the great potential of this procedure for therapeutic purposes. DNA vaccination has been successful in protecting mice from the development of organ-specific autoimmunity (experimental allergic encephalomyelitis (EAE), autoimmune diabetes, experimental arthritis, experimental uveitis) as well as systemic autoimmune disease (systemic lupus erythematosus (SLE), antiphospholipid syndrome). The protection appears to be highly influenced by the capacity of DNA vaccination to modulate immune responses affecting the Th1, Th2 and, importantly, the T cell immunoregulatory arms. We review here the experimental evidence and most recent data supporting the use of DNA vaccination in the induction of immune tolerance.

In ovo DNA immunisation followed by a recombinant fowlpox boost is fully protective to challenge with virulent IBDV

The aim of this study was to investigate the potential use of DNA vaccination delivered in ovo for protecting against challenge with infectious bursal disease virus (IBDV). Using a plasmid expressing the β-galactosidase gene, DNA was successfully delivered to the embryo after in ovo injection and localises to the proventriculus and thymus. The coding sequence for the immunogenic IBDV protein, VP2, was cloned into pCI-neo, creating pCI-Vp2. Complete protection against IBDV was obtained by priming in ovo with pCI-Vp2, followed by boosting with the fowlpox recombinant, fpIBD1, also expressing the VP2 gene. This complete protection was not evident with either of the experimental vaccines on their own. An antibody response was not detected after the prime-boost vaccination, even after chicks had been challenged with IBDV, implying that the DNA prime delivered in ovo stimulated a protective cellular immune response.

On DNA vaccines and prolonged expression of immunogens

Persistent hepatitis B virus (HBV) infection represents a major public health concern because of its association with chronic liver disease and the propensity of the disease to progress to cirrhosis and hepatocellular carcinoma. Despite the availability of a prophylactic vaccine effective in a majority of the population, alternative vaccination strategies are being sought to induce protective responses in healthy non-responders and to boost and broaden T cell responses in chronically infected patients, which may lead to a better control of the virus and/or its eventual complete clearance. In this issue of the European Journal of Immunology immunization of BALB/c mice intramuscularly with a DNA vaccine encoding the hepatitis virus B surface antigen (HBsAg) was shown to result in prolonged secretion of HBsAg into the serum and the elicitation of HBsAg-specific antibodies. In fact, the vaccine was so efficient that the antibodies and HBsAg formed circulating immune complexes and induced kidney and liver lesions similar to those observed in chronically infected patients. This commentary discusses these results in terms of the safety of plasmid DNA-vectored genetic vaccines in general, the use of DNA vaccines expressing HBsAg for the treatment of chronic hepatitis and the consequences of prolonged immunogen expression for the development of protective immune responses.

CpG oligodeoxynucleotide augments HSV-2 glycoprotein D DNA vaccine efficacy to generate T helper 1 response and subsequent protection against primary genital herpes infection in mice

The present study was undertaken to evaluate the efficacy of a combined use of DNA vaccine of HSV-2 glycoprotein D (gD DNA) and CpG oligodeoxynucleotide (ODN) in comparison to gD DNA vaccine alone in inducing immunity against genital HSV-2 infection. Intramuscular vaccination of C57Bl/6 mice with gD DNA followed 48 h later by CpG ODN administration conferred a strong immunity against genital herpes infection. This was concomitant with development of a robust specific IgG2c (an indicator of Th1-type response in C57Bl/6 mice) antibody response as well as IFN-γ production by genital lymph node and spleen cells in vitro. Administration of CpG ODN prior to gD DNA immunization, on the other hand, was inferior to immunization with gD DNA alone in providing protection against macroscopic signs of the disease. Consistent with the in vivo protection data, mice immunized with CpG ODN followed by gD DNA vaccine showed decreased specific lymphoproliferative and IFN-γ responses compared to gD DNA vaccinated mice. In conclusion, these results indicate that timely administration of CpG ODN augments the immunity elicited by gD DNA vaccine, resulting in augmented Th1-type immunity against genital herpes infection in mice. These findings emphasize the value of using CpG ODN in a DNA vaccination scheme against genital herpes and merit also further evaluation in genetic vaccination approaches against other sexually transmitted infections.

DNA vaccination for the priming of neutralizing antibodies against non-immunogenic STa enterotoxin from enterotoxigenic Escherichia coli

In order to test the use of DNA vaccination for its capacity to induce antibodies against the non-immunogenic heat-stable enterotoxin STa from Escherichia coli, BALB/c mice were immunized with plasmid DNA encoding hybrid proteins made by the insertion of wild type STa or insertion of the Cys6Ala, Cys17Ala and Cys6Ala–Cys17Ala STa mutants at positions 195 or 216 of the TEM-1 β-lactamase. No STa specific antibodies could be detected after three plasmid injections, but a subsequent boost with native STa peptide was capable of inducing low levels of neutralizing antibodies, as tested in the suckling mouse assay. Highest STa specific responses were found in mice primed with the double mutated STa inserted in position 195. This plasmid induced highest T-cell responses to the TEM-1 protein, indicating that priming of helper T-cell responses to the carrier protein was essential. Mixed IgG1/IgG2a isotypes also reflected this T helper 1 type priming. Moreover, insertion into loop A of the TEM-1 carrier may be more suitable than insertion into loop B, because of reduced competition between carrier and hapten B cell responses.

Single-Epitope DNA Vaccination Prevents Exhaustion and Facilitates a Broad Antiviral CD8+ T Cell Response during Chronic Viral Infection

Induction of a monospecific antiviral CD8+ T cell response may pose a risk to the host due to the narrow T cell response induced. At the individual level, this may result in selection of CD8+ T cell escape variants, particularly during chronic viral infection. Second, prior immunization toward a single dominant epitope may suppress the response to other viral epitopes, and this may lead to increased susceptibility to reinfection with escape variants circulating in the host population. To address these issues, we induced a memory response consisting solely of monospecific, CD8+ T cells by use of DNA vaccines encoding immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV). We analyzed the spectrum of the CD8+ T cell response and the susceptibility to infection in H-2(b) and H-2(d) mice. Priming for a monospecific, CD8+ T cell response did not render mice susceptible to viral variants. Thus, vaccinated mice were protected against chronic infection with LCMV, and no evidence indicating biologically relevant viral escape was obtained. In parallel, a broad and sustained CD8+ T cell response was generated upon infection, and in H-2(d) mice epitope spreading was observed. Even after acute LCMV infection, DNA vaccination did not significantly impair naturally induced immunity. Thus, the response to the other immunogenic epitopes was not dramatically suppressed in DNA-immunized mice undergoing normal immunizing infection, and the majority of mice were protected against rechallenge with escape variants. These findings underscore that a monospecific vaccine may induce efficient protective immunity given the right set of circumstances.

Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine

Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200–1:6400, average titre is 1:4580) and high CTL activity (67.4±8.4% ( E: T=25:1), 69.6±6.7% ( E: T=50:1) and 71.8±6.2% ( E: T=100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6±12.7% ( E: T=25:1), 57.4±11.7% ( E: T=50:1) and 63.0±6.3% ( E: T=100:1)). However, antibody titres were much lower (1:200–1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.

On the use of DNA vaccines for the prophylaxis of mycobacterial diseases.

The principle of genetic vaccination using naked bacterial plasmid DNA is surprisingly simple, yet the first reports on this technology were only made about a decade ago. In their seminal paper of 1990, Wolff et al. reported that injection of naked plasmid DNA encoding the bacterial enzyme β-

Improved Tuberculosis DNA Vaccines by Formulation in Cationic Lipids

Mice were vaccinated with plasmid DNA (pDNA) encoding antigen 85A (Ag85A), Ag85B, or PstS-3 from Mycobacterium tuberculosis either in saline or formulated for intramuscular injections in VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine) (Vaxfectin; Vical, Inc., San Diego, Calif.) or for intranasal instillations in GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine). These two novel cationic and neutral colipid formulations were previously reported to be effective adjuvants for pDNA-induced antibody responses. The levels of Ag85-specific total immunoglobulin G (IgG) and IgG isotypes were all increased 3- to 10-fold by formulation of pDNA in Vaxfectin. The level of production of splenic T-cell-derived Th1-type cytokines (interleukin-2 and gamma interferon) in response to purified Ag85 and to synthetic peptides spanning the entire Ag85A protein was also significantly higher in animals vaccinated with pDNA formulated in Vaxfectin. Cytolytic T-lymphocyte responses generated by pDNA encoding phosphate-binding protein PstS-3 in Vaxfectin were better sustained over time than were those generated by PstS-3 DNA in saline. Intranasal immunization with Ag85A DNA in saline was completely ineffective, whereas administration in GAP-DLRIE:DOPE induced a positive Th1-type cytokine response; however, the extent of the latter response was clearly lower than that obtained following intramuscular immunization with the same DNA dose. Combined intramuscular and intranasal administrations in cationic lipids resulted in stronger immune responses in the spleen and, more importantly, in the lungs as well. Finally, formulation in Vaxfectin increased the protective efficacy of the Ag85B DNA vaccine, as measured by reduced relative light unit counts and CFU counts in the spleen and lungs from mice challenged with bioluminescent M. tuberculosis H37Rv. These results may be of importance for future clinical use of DNA vaccines in humans.

Protective immunity induced by DNA vaccination of channel catfish with early and late transcripts of the channel catfish herpesvirus (IHV-1)

Seven full-length transcripts encoding four early and three late genes of the channel catfish virus (CCV), ictalurid herpesvirus I (IHV-1), have been cloned following rt-PCR amplification and DNA sequencing. Transcripts were selected based on their predicted association with membrane structures, identification as an envelope glycoprotein, or as a viral capsid protein. The transcripts derived from ORF 6, ORF 7, ORF 8a, ORF 10, ORF 51, ORF 53, and ORF 59 were all shown to be complete and unspliced. Each of the seven ORFs was cloned into a vaccine expression vector designed to support high levels of expression of the inserted sequence in catfish tissues. Solutions of DNA containing one each of the seven CCV ORFs, vector alone or PBS were injected intramuscularly into 4–8 cm catfish. Four to 6 weeks after injection each experimental group was challenged with one LD 50 of CCV. Single injections of DNA expression constructs containing ORF 59, encoding the envelope glycoprotein, or ORF 6, encoding a presumptive membrane protein, were found to elicit the strongest resistance to challenge compared to uninjected, PBS injected or vector injected groups. Even more effective was a combination vaccine pair in which both ORF 59 and ORF 6 expression constructs were injected. Other ORFs did not provide consistent protection to challenge above that observed in control fish. Both percent survival and kinetics of cumulative deaths were improved using the combination DNA vaccine encoding ORF 6 and ORF 59. Both ORF 6 and ORF 59 were able to elicit virus neutralizing antibodies capable of an anamnestic response on viral challenge. We believe this evidence provides adequate proof of principle for the use of DNA vaccines in channel catfish and the effectiveness of the resistance to viral infection they elicit.

Flavivirus DNA Vaccines

The use of DNA-based vaccines is a novel and promising immunization approach for the development of flavivirus vaccines. This approach has been attempted in vaccine development for various virus species, including St. Louis encephalitis, Russian spring-summer encephalitis, Central European encephalitis, dengue serotypes 1 and 2, Murray Valley encephalitis, Japanese encephalitis, and West Nile viruses. However, very little is known about the factors affecting its efficacy. Recently, we demonstrated that a single intramuscular immunization of DNA vaccine of Japanese encephalitis and West Nile viruses protected mice and horses from virus challenge. Administration of these recombinant plasmid vectors resulted in endogenous expression and secretion of extracellular virus-like particles that correlated well with the induction of protective immunity. These results provided evidence that the virus-like particles composed of premembrane/membrane and envelope proteins are essential for eliciting immune responses similar to those induced by live, attenuated virus vaccines. The biosynthesis and protein processing of premembrane/membrane and envelope proteins that preserve the native conformation and glycosylation profiles identical to virion proteins could be determined by the effectiveness of the transmembrane signal sequence located at the amino-terminus of premembrane protein. The use of DNA vaccines in multivalent and/or combination vaccines designed to immunize against multiple flaviviruses is also a promising area of development.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

Induction of antigen specific CD4+ T cell responses by invariant chain based DNA vaccines

In this report, the use of DNA vaccination to induce class II restricted antigen specific proliferative responses was studied. To this end, a construct encoding the invariant chain (Ii) was engineered in which the Class II associated invariant chain peptide (CLIP) sequence was replaced by an immunogenic epitope derived form Heat Shock Protein 60, HSP60 178–186. Transfection studies in vitro showed that this construct can be used to efficiently load MHC class II molecules and present epitopes to MHC class II restricted antigen specific T cells. In addition, we showed that intradermal immunisation of Lewis rats with these constructs induced antigen specific T cells in vivo. Therefore, our Ii-gene constructs can be used to immunise for defined CD4+ T cell epitope sequences.

Myelin oligodendrocyte glycoprotein-DNA vaccination induces antibody-mediated autoaggression in experimental autoimmune encephalomyelitis

One strategy to reestablish self tolerance in autoimmune diseases is based on the use of DNA vaccination to induce ectopic expression of the target autoantigen. We assessed the potential of vaccination with a DNA construct encoding the myelin oligodendrocyte glycoprotein (MOG), an important candidate autoantigen in multiple sclerosis, to induce tolerance and protect against experimental autoimmune encephalomyelitis (EAE). Unexpectedly, mice vaccinated with MOG-DNA developed an exacerbated form of EAE when challenged with either MOG or an unrelated encephalitogen, myelin proteolipid protein. We demonstrate that this is due to the inability of DNA vaccination to tolerize the MOG-specific T cell response and to the concomitant induction of a cytopathic MOG-specific autoantibody response, which is pathogenic, enhancing demyelination, inflammation and disease severity. Our data suggest that tolerogenic strategies for autoimmune diseases based on DNA vaccination should be approached with caution, as the outcome is unpredictable.

Recent advances in the use of DNA vaccines for the treatment of diseases of farmed animals

DNA-based vaccination constitutes one of the most recent approaches to vaccine development. This technology is in principle one of the most simple and yet versatile methods of inducing both humoral and cellular immune responses, as well as protection against a variety of infectious agents. However, although immune responses have been induced in a number of larger species, most information on the efficacy of DNA immunization has been generated in mice. In this review the information available to date about the use of DNA vaccines in farmed animals, including cattle, pigs and poultry, is presented. The areas that need specific attention in the future to bring this technology to the market are discussed, including the issues concerning delivery, safety, compatibility of plasmids in multivalent vaccines and the potential of using immune stimulants as part of a DNA vaccine.

The prime-boost strategy: exciting prospects for improved vaccination

Unprecedented levels of cell-mediated immunity can be stimulated by the consecutive use of DNA vaccines and attenuated poxvirus vectors encoding similar heterologous antigens. This may offer a means of preventive vaccination against diseases for which no effective treatment is available.

DNA Vaccination Using the Fragment C of Botulinum Neurotoxin Type A Provided Protective Immunity in Mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igκ leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.

Exacerbation of viral and autoimmune animal models for multiple sclerosis by bacterial DNA.

Theiler's murine encephalomyelitis virus (TMEV) infection and relapsing-remitting experimental allergic encephalomyelitis (R-EAE) have been used to investigate the viral and autoimmune etiology of multiple sclerosis (MS), a possible Th1-type mediated disease. DNA immunization is a novel vaccination strategy in which few harmful effects have been reported. Bacterial DNA and oligodeoxynucleotides, which contain CpG motifs, have been reported to enhance immunostimulation. Our objectives were two-fold: first, to ascertain whether plasmid DNA, pCMV, which is widely used as a vector in DNA immunization studies, could exert immunostimulation in vitro; and second, to test if pCMV injection could modulate animal models for MS in vivo. We demonstrated that this bacterially derived DNA could induce interleukin (IL)-12, interferon (IFN)gamma, (Th1-promoting cytokines), and IL-6 production as well as activate NK cells. Following pCMV injections, SJL/J mice were infected with TMEV or challenged with encephalitogenic myelin proteolipid protein (PLP) peptides. pCMV injection exacerbated TMEV-induced demyelinating disease in a dose-dependent manner. Exacerbation of the disease did not correlate with the number of TMEV-antigen positive cells but did with an increase in anti-TMEV antibody. pCMV injection also enhanced R-EAE with increased IFNgamma and IL-6 responses. These results caution the use of DNA vaccination in MS patients and other possible Th1-mediated diseases.

Potency of an experimental DNA vaccine against Aujeszky's disease in pigs

Intradermal vaccination with plasmid DNA encoding envelope glycoprotein C (gC) of pseudorabies virus (PrV) conferred protection of pigs against Aujeszky's disease when challenged with strain 75V19, but proved to be inadequate for protection against the highly virulent strain NIA-3. To improve the performance of the DNA vaccine, animals were vaccinated intradermally with a combination of plasmids expressing PrV glycoproteins gB, gC, gD, or gE under control of the major immediate-early promotor/enhancer of human cytomegalovirus. 12.5 μg per plasmid were used per immunization of 5-week old piglets which were injected three times at biweekly intervals. Five out of six animals survived a lethal challenge with strain NIA-3 without exhibiting central nervous signs, whereas all the control animals succumbed to the disease. This result shows the increased protection afforded by administration of the plasmid mixture over vaccination with a gC expressing plasmid alone. A comparative trial was performed using commercially available inactivated and modified-live vaccines and a mixture of plasmids expressing gB, gC, and gD. gE was omitted to conform with current eradication strategies based on gE-deleted vaccines. All six animals vaccinated with the live vaccine survived the lethal NIA-3 challenge without showing severe clinical signs. In contrast, five of six animals immunized with the inactivated vaccine died, as did two non-vaccinated controls. In this test, three of six animals vaccinated with the DNA vaccine survived without severe clinical signs, whereas three succumbed to the disease. Comparing weight reduction and virus excretion, the DNA vaccine also ranged between the inactivated and modified-live vaccines. Thus, administration of DNA constructs expressing different PrV glycoproteins was superior to an adjuvanted inactivated vaccine but less effective than an attenuated live vaccine in protection of pigs against PrV infection. Our data suggest a potential use of DNA vaccination in circumstances which do not allow administration of live attenuated vaccines.

New vaccine strategies against enterotoxigenic Escherichia coli: I: DNA vaccines against the CFA/I fimbrial adhesin

Stimulation of the mammalian immune system by administration of plasmid DNA has been shown to be an important approach for vaccine development against several pathogens. In the present study we investigated the use of DNA vaccines to induce immune responses against an enteric bacterial pathogen, enterotoxigenic Escherichia coli (ETEC). Three plasmid vectors encoding colonization factor antigen 1 (CFA/I), an ETEC fimbrial adhesin, were constructed. Eukaryotic cells transfected with each of these plasmids expressed the heterologous antigen in different compartments: bound to the cytoplasmic membrane (pRECFA), accumulated in the cytoplasm (pPolyCFA) or secreted to the outside medium (pBLCFA). BALB/c mice were intramuscularly (i.m.) inoculated with purified plasmid DNA and the systemic, cellular and secreted CFA/I-specific immune responses were analyzed. The results showed that all three DNA vaccine formulations could elicit CFA/I-specific immune responses. Moreover, cellular location of the plasmid-encoded CFA/I seems to have an important role in the induced immune response. Taken together, these results indicate that DNA vaccines also represent a promising approach against enteric bacterial pathogens.