Table of contents :

advantages drawbacks delivery adjuvants anti-infectious diseases vaccines anti-viral DNA vaccines anti-bacterial DNA vaccines anti-protozoal DNA vaccines anti-cancer DNA vaccines anti-chronic degenerative diseases DNA vaccines anti-toxic DNA vaccines

immunocontraceptive DNA vaccines immunotolerant DNA vaccines immunosuppressive anti-cytokine therapeutic vaccines anti-type I hypersensitivity DNA vaccines oral DNA vaccination in utero DNA vaccines for farm animals web resources  companies manufacturing DNA vaccines plasmid DNA purification DNA vaccines conferences institutions and researchers

• advantages^{ref1, ref2, ref3} :
• stable at room temperature
• coupled with delivery vectors :
• combined artificial immunogens mimicking virus particles : the central core of the virus-lile particle (dsRNA) is coated with a layer of the following conjugate : spermidine (arginine, lysine, ..)--polyglucine--enzyme substrate^{ref1, ref2}
• bacterial ghosts are non-denatured bacterial cell envelopes that are formed by the expression of the plasmid-encoded lysis gene E of bacterial phage fX174 : they can be filled with DNA or drugs through the lysis hole and used as vectors^{ref1, ref2}
• replication-defective Bacteria (e.g. Salmonella typhymurium) can be used as vectors for DNA vaccines as they free the carried plasmid after cell lysis.
• coupled with protein adjuvants (DNA fusion vaccine) :
• A and B subunits of cholera toxin (CT) or the heat-labile enterotoxin from E.coli (LT)^ref
• fragment C of tetanus toxin^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}
• plant viral genes coding coat protein (PVCP) (self-aggregating) drives T_h1 dominant responses
• potato virus X coat protein^ref
• transgenic B lymphocyte immunization (TLI) as antigen-presenting cell (APC)-based genetic vaccines : DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional Ig H chain gene under the control of a B-cell-specific promoter. Using PCR, RT-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, EBV-transformed cell lines, and peripheral blood B lymphocytes of the mature naive phenotype (IgM⁺/IgD⁺/CD27^-). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells expressed the gene, processed the protein and presented to MHC-restricted T lymphocytes a peptide expressed in the transgenic product^ref. A single i.v. injection of transgenic B lymphocytes induce robust and long-lasting T-cell immunity reproducibly and specifically even at very low cell doses (approximately 3 x 10²). T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions^ref. These properties reflect on simple characteristics, such as time synchronization and initial localization to secondary lymphoid organs of APCs endowed with protracted synthesis and presentation of antigen to T cells^ref.
• xenogenic DNA immunization is a mean to overcome immunological ignorance or tolerance which normally prevents responses to self antigens found in tumors. Xenogeneic DNA immunization leads to rotection from syngeneic tumor challenge, inhibition of growth of established tumors and autoimmune depigmentation of cat in mice or dogs with canine malignant melanoma^ref
• fusion with genes coding for cell-penetrating peptides (CPP) (e.g. VP22)
• domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion generated antigen-specific T_h1-type cellular immune responses against a non-immunogenic tumor antigen^ref
• ability to induce immunological danger signals at sites of injection :
• the strong immunogenicity has been attributed to the presence in the plasmid backbone (cloned in bacterial cells, usually E.coli) of particular unmethylated CpG motifs that in humans bind to TLR9 activating plasmacytoid dendritic cells (PDCs) eliciting T_h1 polarization. Methylation of the palindromic CpG sequences of bacterial expression vectors abolished their ability to induce an antibody response to the transgene product (a highly immunogenic viral glycoprotein) in mice. The antibody response could be rescued by concomitant injection of oligonucleotides carrying immunostimulatory sequences. The B cell response to 2 plasmid vectors, both expressing the same viral glycoprotein but containing a different content of the highly stimulatory AACGTT motif, was compared. Comparable B cell responses were induced to the 2 constructs given at an optimal vaccine dose while the vector containing additional palindromic sequences resulted in higher antibody titers at a suboptimal dose. These data indicate that deletion of CpG motifs or methylation of such sequences in plasmid DNA can abrogate the immune response to the vector encoded antigen and might thus enhance their usefulness as gene therapy vehicles^ref.
• inherently favor the induction of cytotoxic cellular immunity since Ag can be presented through endogenous processing pathways in the context of class I MHC^ref
• ability to directly infect APCs
• the host cell provides the post-translational modifications to the antigen, allowing its production with the correct native conformation to live infection
• combination therapy :
• recognition of self-antigens is emerging as a theme for the immune recognition of human cancer^{ref1, ref2, ref3, ref4}. Melanoma and prostate cancer provide usegul models to explore immunization against differentiation antigens^{ref1, ref2, ref3, ref4, ref5}. Interaction between CTLA-4 and B7 on APCs results in down-regulation of activated T cells, attenuating the immune response^ref. Blockade of CTLA-4 results in disruption of the binding of CTLA-4 to the costimulatory molecules B71/2 and enhances the response to antigenic tumors and foreign antigens^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}.
• genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA : the typical objective of genetic immunization is the isolation of polyclonal antibody containing sera or antibody-producing B cells, which can be used to generate monoclonal antibody (MAb)-producing cell lines (either by immortalization or by fusion with myeloma cells to form hybridomas). The same vector can be used for both genetic immunization and antigen production^ref
• drawbacks and risks :
• limited to protein antigens (hence excluding carbohydrate antigens) and adjuvants (unless added to DNA mixture)
• interspecific codon usage bias may be an obstacle for effective induction of immune responses by gene vaccines, and should be corrected by codon optimization^ref
• one issue with DNA vaccine formulation is keeping potential adjuvants and the DNA in proximity. DNA delivery results in protein antigen expression on after several hours and can persist for days, a time period where normally the water soluble resiquimod, could be long gone and unable to play a role in local adjuvanticity^ref.
• bupivacaine can modulate immune responses induced by DNA vaccines as they form specific complexes with DNA and this formulation serves to increase transfectant DNA protection as well as local delivery^ref, but stimulated only modest levels of overall antibody production, with relatively low level of IgG_2a
• levamisole exhibits strongest T_h1 stimulatory activity whereas Tween 80 showed weakest T_h1 activity, as determined by IgG_2a production, and saline formulation induced weak T cell proliferation and DTH, in animals inoculated with a DNA construct expressing the FMDV capsule protein VP1. Furthermore, co-inoculation of levamisole increases the production of IFN-g by > 100-fold as compared to that by DNA inoculation formulated in saline^ref
• potential for atypical processing of bacterial and parasite proteins
• development of secondary anti-DNA autoantibodies occurs^ref, but not always^ref and is usually transient
• in vaccination with recombinant plasmid :
• plasmid DNA purification : current good manufacturing practice (cGMP) conditions for obtaining clinical-grade plasmid DNA
• Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all 3 cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner^ref.
• reduced immunogenicity of DNA vaccine plasmids in mixtures : given alone, each of 9 DNA vaccine plasmids encoding candidate malaria vaccine antigens induced significant antibody titers and, in the 4 cases for which appropriate assays were available, IFN-g responses. Significant suppression or complete abrogation of responses were seen when the plasmids were pooled in a 9-plasmid cocktail and injected in a single site. Removal of single genes from the mixture frequently reduced the observed suppression. Boosting with recombinant poxvirus increased the antibody response in animals primed with either a single gene or the mixture, but, even after boosting, responses were higher in animals primed with single plasmids than in those primed with the 9-plasmid mixture. Boosting did not overcome the suppressive effect of mixing for IFN-g responses. Interactions between components in a multiplasmid DNA vaccine may limit the ability to use plasmid pools alone to induce responses against multiple targets simultaneously^ref.
• in vaccination with recombinant viruses :
• presence of virally encoded inhibitors of innate immunity and adaptive immunity
• presence of neutralizing antibodies in pre-exposed or prevaccinated individuals, which inhibit the initial round of virus infection and replication, thereby reducing the ability of the virus to immunize
• individuals who have never previously exposed to the vaccinating virus generate neutralizing antibody after the first vaccination, thereby precluding homologous prime-boost
• the strong viral antigens will dominate, thereby inhibiting responses to the weaker tumour antigen. This concern arises from the ability of immunodominant epitopes in antigens to quench responses to subdominant or cryptic epitopes. However, such a quenching phenomenon is, in fact, rarely observed with recombinant viral vaccines. Heterologous prime-boost would certainly diminish the probability of such a phenomenon, because the recombinant tumour antigen would be the only one being repetitively boosted.
• in vaccination with recombinant bacteria :
• recombinant bacteria having life cycles that involve both phagolysosomal and cytoplasmic stages (e.g. Listeria monocytogenes) engineered to secrete antigens will load the MHC class II processing pathway during the phagolysosomal phase of its life cycle and the MHC class I pathway during the cytoplasmic phase.
• several recombinant bacteria actively induce infected APCs to secrete proinflammatory cytokines such as IL-12
• bacterial vaccines containing plasmids with eukaryotic promoter and enhancer elements that drive the expression of the antigen gene can transfer the plasmid directly into eukaryotic transcriptional compartments within infected APCs and result in potent immunization
• the expression plasmid containing the in vivo-inducible promoter pagC (P(pagC)) from Salmonella typhimurium and HGV-NS3 gene was introduced into the attenuated Salmonella typhimurium SL7207 to investigate the function of P(pagC). The expressed HGV-NS3 protein was detectable by SDS-PAGE and Western blotting in the recombinant bacteria in the presence of low concentration of Mg²⁺ (<50 mmol/L). When the concentration of Mg²⁺ reached to 50 mmol/L, the amount of expressed HGV-NS3 was decreased significantly. The recombinant bacteria were multiplied in LB medium containing 50 mmol/L of Mg²⁺ and used as a DNA vaccine to orally inoculate C57 mice for 3 times. The results of serum antibodies, T lymphocyte proliferative response and cytotoxic T lymphocyte response of immunized mice showed that the oral vaccine could induce strong humoral and cellular immune responses in mice, which indicates that the P(pagC) is a strong in vivo-inducible promoter and can be used in attenuated Salmonella typhimurium to construct an effective oral vaccine^ref
• antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In vivo monitoring of attenuated S. typhimurium uptake is established using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. The ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. Attenuated S. typhimurium oral DNA vaccine using attenuated S. typhimurium carrying the mouse a-fetoprotein (AFP) gene was found to promote protective immunity against both cancer line CT26-murine a-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development^ref
• in vaccination with recombinant yeasts :
• ?
• unlike intramuscular injection, gene gun delivery of DNA drives a strong T_h2 response. In an effort to counter this, the T_h1 cytokines, IL-12 and IL-23, were genetically fused to the hemagglutinin (HA) gene from influenza APR/8/34, and delivered these DNA constructs to Balb/c mice. Gene gun delivery of the HA gene was able to induce antibody production by all vaccinated mice. Linking of IL-12 caused almost complete suppression of immune responses whereas mice vaccinated with IL-23HA showed long-lived IgG₁ antibody levels. Splenocytes from IL-23HA vaccinated mice also tended to produce more IL-5 and IFN-g after restimulation in vitro than splenocytes from HA vaccinated mice. While codelivery of IL-23 did not change the type of immune response it may increase its longevity following vaccination^ref. A comparative study of domestic pig, minipig and mouse in regard to local tolerance and antigen expression of HIV immunotherapeutic using PMED. Pig/minipig is considered a good model for the safety assessment of DNA vaccines due to the similarity to human skin. Local reactions were evaluated at 10min, 4, 24 and 48h. Histology of administration sites revealed epidermal necrosis with associated dermal inflammation at 10min and 4h, and subsequent regeneration with repair at 24 and 48h. The degree and extent of these changes varied according to species. Domestic pig and minipig showed superficial epidermal necrosis and complete repair, while the mouse showed full-thickness epidermal necrosis and partial repair. Expression of HIV antigen was confirmed using immunohistochemistry in all three species at 4, 24 and 48h. The results showed that PMED is an effective system for DNA vaccine delivery as demonstrated by the antigen expression seen as early as 4h^ref
• expensive
• need for injection => risk of accidental parenteral infections from contaminated needles
• need for skilled medical personnel to deliver jabs
• biodistribution : to monitor the distribution and duration of gene expression of a DNA vaccine in living organisms, the naked DNA encoding firefly luciferase (Fluc) has been used as an imaging reporter gene, and in vivo bioluminescent images have been evaluated in a murine model. Bioluminescence was observed at the injection site and at inguinal lymph node from 10h to 24h post-injection when DNA vaccine encoding Fluc (pcDNA3.1-Fluc) was injected into the bilateral posterior flanks in mice. Fluc gene expressions at injection sites and unilateral posterior flank inguinal lymph node were also confirmed by RT-PCR. However, when pcDNA3.1-Fluc was injected into the mid-dorsum bioluminescent signals were observed at the injection site for up to 14 days post-injection, but no bioluminescent signals were detected in inguinal lymph nodes. Concurrent mRNA expressions of Fluc gene at injection sites but not at inguinal lymph nodes were confirmed by RT-PCR. These findings suggest that optical imaging using Fluc could be useful for monitoring the location, intensity and duration of gene expression of naked DNA vaccines in living animals non-invasively and repetitively^ref

• the design of an efficient DNA vaccine, involves choice of a suitable expression vector, ensuring optimal expression by codon optimization, engineering CpG motifs for enhancing immune responses and providing additional sequence signals for efficient translation. DyNAVacS is a web-based tool created for rapid and easy design of DNA vaccines. It follows a step-wise design flow, which guides the user through the various sequential steps in the design of the vaccine. Further, it allows restriction enzyme mapping, design of primers spanning user specified sequences and provides information regarding the vectors currently used for generation of DNA vaccines. The web version uses Apache HTTP server. The interface was written in HTML and utilizes the Common Gateway Interface scripts written in PERL for functionality. DyNAVacS is an integrated tool consisting of user-friendly programs, which require minimal information from the user. The software is available free of cost, as a web based application^ref
• generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for both HIV-1 and other pathogens. Virus-specific CD8⁺ T lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8⁺ T lymphocyte responses may prove critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Epitope modification strategies can alter CD8⁺ T lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing SIV Gag lacking the dominant D^b-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D^b-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes^ref
• Broad therapeutic application of nucleic acid micro- and nanoparticles will require bioprocesses capable of achieving high loads of structurally intact and functionality active DNA. Condensation of pDNA into nanoparticles has been achieved by sedimentation through R4 peptide and partitioning at a hydrophobic interface. > or = 90% coating efficiency onto microparticles is achieved via this combined bioprocess with the pDNA retaining 85-90% intact supercoil after bioprocessing. SEM analyses of the microparticles produced therefrom reveals bound pDNA and R4 peptide nanoparticles. HPLC and chemical analyses afford quantification of the particle-associated pDNA and R4 peptide along with lactose, raffinose, or trehalose carbohydrate stabilizer, surface coatings uniformly applied by spray freeze-drying. Administration of these particles by gene gun demonstrates delivery to the nucleus of expressive nanoparticles and into rodents and pigs pronounced immunogenicity even after bioprocessing and accelerated degradation. These data support the discovery of a robust bioprocess platform for preparing macromolecule bound bioparticles with potential relevance beyond simple preparation of bioactive DNA vaccine^ref.
• 2 different DNA vaccine designs aimed to elicit CD8⁺ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4⁺ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of T_h responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide (CLIP). This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated T_h-dependent priming of CD8⁺ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8⁺ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization^ref
• 2 different DNA vaccine designs aimed to elicit CD8⁺ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4⁺ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of T_h responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide. This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated T_h-dependent priming of CD8⁺ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8⁺ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization^ref

• HSV-1 amplicon vectors efficiently transduce cultured antigen-presenting cells (APC), including both human and murine dendritic cells as well as primary human chronic lymphocytic leukemia (CLL) B cells. Helper-free amplicons have been shown to be especially well-suited for this purpose, since they do not impair the antigen-presenting functions of these target cells. In vivo, amplicon vectors have been used in preclinical studies aimed at the development of therapeutic cancer vaccines, as well as vaccines for Alzheimer's disease, and selected microbial pathogens. Studies in small animal model systems have shown that ex vivo transduction of irradiated tumor cells with amplicon vectors encoding immunomodulatory cytokines such as IL-2 or GM-CSF can elicit protective responses against a tumor challenge. In an experimental model for cancer immunotherapy, direct transduction of preformed tumors with vectors encoding CD40L resulted in slowed tumor growth or tumor eradication. Other studies have examined the ability of amplicons to elicit immune responses against encoded antigens, and have shown that strong cellular immune responses can be generated against amplicon encoded HIV-1 antigens in mice. Thus, amplicon vectors have shown significant promise as vaccine vectors in a range of settings. These promising initial findings highlight the need to perform additional studies, including experiments to evaluate the immunogenicity of amplicon vectors in additional animal models, possibly including nonhuman primates. Overall, amplicon vectors offer compelling advantages when compared to other vaccine-delivery platforms, which include the capacity to incorporate a very large transgene payload and the potential to efficiently transduce mucosal surfaces. It will be important to design future studies to directly test and exploit these features of the amplicon system. The next few years therefore promise to be an exciting and important period in the development of amplicons as vaccine vectors^ref.
• replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. The ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC), was explored. DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-a as well as IL-6 were assessed. Additionally, activation of DC and induction of CTL were determined in vivo. The role of TLR9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-a upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8⁺ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches^ref
• short-interval intradermal DNA delivery, generates robust T-cell responses within 12 d, of a magnitude sufficient to reject established subcutaneous tumors. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time^ref.
• intramuscular needle and syringe, Biojector^® 2000, and Mini-Jecttrade mark was compared in cynomolgus monkeys. The elicited cellular and humoral immune responses were comparable in monkeys immunized using these different delivery techniques, suggesting that the needle-free approaches to vaccine administration do not significantly improve the immunogenicity of the plasmid DNA vaccine used in the study^ref.
• Packaging plasmids containing CpGs into fusogenic liposomes (FL) derived from conventional liposomes and Sendai virus-derived active accessory proteins is an attractive method for enhancing the efficacy of a DNA vaccine. These CpG-enhanced plasmids (possessing 16 CpG repeats) that were packaged into FL, enhanced ovalbumin (OVA)-specific T cell proliferation and cytotoxic T cell activity after immunization. In fact, vaccination with CpG enhanced plasmid-loaded FL induced effective prophylactic effects compared with 13 repeats CpG containing plasmid in a tumor challenge experiment^ref.
• Depletion or dysfunction of CD4 T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4 T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8 T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4 T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis carinii pneumonia, serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs was used to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19 cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens^ref
• Effects of plasmid type and insertion sequence on in vitro and in vivo multiplication and invasiveness of attenuated Salmonella typhimurium were examined following transformation of the bacteria with eukaryotic expression plasmids pcDNA3 and pCI with or without heterologous gene (Newcastle disease virus F gene). Exogenous plasmids had negative impacts on the replication or invasiveness of the attenuated S. typhimurium in LB broth and/or HeLa cell monolayers as well as on its survival in live chicks. The plasmid pCI had more significant effects than pcDNA3. Introduction of heterologous gene into the plasmids not only exhibited additional negative influence on the host strain but also on their own stability therein. All these results suggest that full consideration should be given to the types of plasmids and their stability within the host strain as well as to their effects on replication and invasiveness of attenuated bacteria as the DNA vaccine delivery vector for improved immune protection^ref
• microorganisms : bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo.
• the ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding b-galactosidase (b-Gal) and BALB/c mice. pBRCMVb, b-Gal expression apparatus having a replication origin from low copy pBR322, was constructed. Comparison of the plasmid stability showed that pBRCMVb remained stable in Salmonella even after oral administration, while pUC-based pCMVb tended to be lost quickly. However, titers for b-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVb. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology^ref. Plasmid stability on oral DNA delivery by the attenuated Salmonella enterica serovar Typhimurium vaccine strain BRD509. A DNA vaccine cassette comprising the C fragment of tetanus toxin under control of the cytomegalovirus (CMV) promoter was ligated into plasmid pcDNA3, pUC18, pBBR122, pACYC184, pRSF1010/CAT, pBR322 and pAT153. In vitro and in vivo stability studies revealed that, with the exception of pcDNA3 and pUC18, the plasmids were retained by BRD509. However, pAT153 was the only plasmid to induce a tetanus toxoid-specific antibody response following oral delivery. Plasmid copy number was found to impact on plasmid stability and the induction of antigen-specific humoral responses^ref.
• The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenesis inlA gene can deliver a functional gene into mammalian cells. The potential use of non-invasive L. lactis strains as DNA delivery vehicle was explored. 2 shuttle Escherichia coli-L. lactis plasmids, pLIG:BLG1 and pLIG:BLG2, were constructed containing a eukaryotic expression cassette with the cDNA of bovine b-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis MG1363(pLIG:BLG1) strain was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in human colon carcinoma Caco-2 cells after 3 hours co-incubation with: i) purified pLIG:BLG1, ii) MG1363(pLIG:BLG1), iii) a mix of MG1363(pLIG) and purified pLIG:BLG1 and iv) MG1363. Both BLG cDNA and BLG expression were only detected in Caco-2 cells co-incubated with MG1363(pLIG:BLG1). There was a decrease in BLG cDNA in Caco-2 cells between 24 and 48 h after co-incubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after co-incubation with MG1363(pLIG:BLG1). Lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine^ref.
• E. coli expressing invasin from Yersinia pseudotuberculosis selectively invade nonphagocytic cells in which b₁-integrin is expressed and accessible. Following internalization the E. coli are degraded in the phagosome. Coexpression of listeriolysin O (LLO) mediates release of the content of the bacteria into the cytosol of the invaded cell. In vitro and in vivo experiments demonstrated that gut epithelial cells failed to be invaded by invasive E. coli, due to a basolateral localization of b₁-integrin. By contrast, selective uptake of invasive bacteria from the intestinal lumen into Peyer's patches was observed ex vivo. Once in this structure, invasive E. coli colocalized with dendritic cells and possibly B cells. Oral administration of invasive E. coli coexpressing the model antigen ovalbumin and LLO from Listeria monocytogenes was able to elicit systemic protection against a lethal challenge of B16 tumor cells expressing ovalbumin. These data demonstrate the selectivity of invasin-mediated invasion to the Peyer's patches and indicate the potential of nonpathogenic, invasive E. coli as an oral vaccine with applications in immunotherapy^ref.
• cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the d-alanine-deficient (Ddal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of d-alanine, was transformed with a plasmid encoding GFP under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of d-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP⁺ cells were observed when d-alanine was omitted. Although transfection efficiency initially increased as a result of d-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival^ref
• non-replicating bacterial minicells simultaneously co-delivering the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) and the corresponding DNA vaccine were tested for the ability to generate protective cellular immune responses in mice. It was found that good protection (89%) was achieved after intramuscular administration, moderate protection (31%) was achieved after intranasal administration, and less protection (7%) was achieved following gastric immunization. These results provide a solid foundation on which to pursue the use of bacterial minicells as a non-replicating vaccine delivery platform^ref.
• pDNAVACCultra (source : Nature Technology Corp.) is a family of DNA vaccine vectors, optimized and minimized to comply with FDA guidelines regarding content and elimination of extraneous materials, was constructed. The resulting vectors are much smaller than existing vectors, drive higher levels of target gene expression, facilitate high throughput cloning applications, and allow simultaneous cloning into multiple vectors that feature various intracellular targeting destinations for the protein product. The ability to control expression and trafficking is intended to provide a rapid, rational approach to cancer therapy and emerging infectious diseases^ref
• in vivo active targeting of DNA vaccine to DCs with Man-lipoplex can enhance cytotoxic T lymphocyte (CTL) activity through efficient gene delivery to dendritic cells (DCs) by mannose receptor-mediated endocytosis. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. Mannosylated cationic liposomes (Man-liposomes) were prepared using cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiomannosylethyl)amino]butyl}formamide (Man-C4-Chol) with cationic lipid. The potency of the mannosylated liposome/pCMV-OVA complex (Man-lipoplex) was evaluated by measuring OVA mRNA in CD11c⁺ cells, CTL activity, and the OVA-specific anti-tumor effect after in vivo administration. An in vitro study using DC2.4 cells demonstrated that Man-liposomes could transfect pCMV-OVA more efficiently than cationic liposomes via mannose receptor-mediated endocytosis. In vivo studies revealed that the Man-lipoplex exhibited higher OVA mRNA expression in CD11c⁺ cells in the spleen and peritoneal cavity and provided a stronger OVA-specific CTL response than intraperitoneal (i.p.) administration of the conventional lipoplex and intramuscular (i.m.) administration of naked pCMV-OVA, the standard protocol for DNA vaccination. Pre-immunization with the Man-lipoplex provided much better OVA-specific anti-tumor effect than naked pCMV-OVA via the i.m. route^ref
• Encapsulating DNA within degradable delivery vehicles such as micro- or nanospheres provides an effective way to protect the DNA from the surrounding environment prior to delivery. The ability to target these vehicles directly to the cell type of interest provides a way to enhance the overall efficiency of DNA delivery. One means of highly specific cell targeting is through the addition to the vehicle surface of ligands that bind specifically to receptors on the surface of the targeted cell type. Covalent conjugation of ligands to the surface of degradable delivery vehicles can be difficult, as the most commonly used vehicle formulations use materials selected for their general chemical inertness^ref
• inert poly-l-lysine (PLL) coated polystyrene particles greatly enhance DNA immunogenicity. Intradermal injection of plasmid DNA encoding for chicken egg ovalbumin (OVA) complexed with PLL-coated polystyrene nanoparticles induced high levels of CD8 T cells as well as OVA-specific antibodies in C57BL/6 mice and furthermore inhibited tumour growth after challenge with the OVA expressing EG7 tumour cell line. Importantly, vaccine efficacy depended critically on the size of the particles used as well as on the presence of the PLL linker. PLL-coated polystyrene nanoparticles of 0.05 mm but not 0.02 mm or 1.0 mm in diameter are highly effective for the delivery of DNA vaccines^ref.
• a first intradermal administration of one DNA vaccine dose combined with the slow-release of a second dose conduct to a priming of the bovine herpesvirus 1-specific immune response similar to the one generated by 2 discrete administrations 4 weeks apart. The first experiment demonstrates the efficacy of the slow-release system with well-characterized Alzet^® osmotic pumps, whereas the second experiment extends the same concept with innovative agarose hydrogel implants. These latter implants are cheaper and more convenient than the osmotic pumps or repeated intradermal administrations since they contribute to an efficient priming of the immune response in a single manipulation of the animals^ref
• exploiting the activity of VP22 to enhance the delivery of antigens encoded by DNA vaccine plasmids^ref

• GM-CSF : to assess GM-CSF immune accessory effects in tumor-bearing mice, an animal tumor model was established by inoculating SP2/0 myeloma cells s.c. into the flank of Balb/c mice and 14 days later, injecting either 400 mg recombinant pcDNA3.1/mGM-CSF or a blank plasmid s.c. or i.m. into the tumor four times. The tumor weight, the activities of CTL and NK, the serum levels of IFN-g, IL-2 and lymphocytes infiltrating in tumor tissue were analysed 8 weeks later with MTT, ELISA and pathological section methods. The tumor lump was reduced in mice injected s.c. (0.880 +/- 0.405 g) or i.m. (0.378 +/- 0.411 g) with pcDNA3.1/mGM-CSF compared with control mice injected s.c. (1.548 +/- 0.221g, P < 0.01)or i.m. (1.554 +/- 0.249g, P < 0.001) with a blank vector. Lymphocyte infiltration in tumor tissues was very apparent in mice injected i.m. with pcDNA3.1/mGM-CSF. In contrast, there was no lymphocyte infiltration in tumor tissues of control mice. In addition, the serum concentrations of IFN-g, IL-2 and the activities of CTL and NK cells were significantly increased in mice injected with pcDNA3.1/mGM-CSF compared with a control mice (P < 0.01)^ref. A novel codon-optimized murine GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag^ref
• dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the TRIF or MyD88 were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection^ref.
• IL-18 : to determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce IFN-g expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFN-g response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant^ref.
• trichostatin-A (TSA) is a specific inhibitor for histone deacetylase (HDAC) and a gene expression enhancer in in vitro DNA viral infection, including HSV-1 and CMV. A study sought to determine if increasing antigen expression of DNA vaccines through inhibition of HDAC-induced chromatin silencing using TSA would enhance DNA vaccine efficacy in vivo. A luciferase assay was used to detect effects of TSA on different promoters in vitro. The effects of TSA on DNA vaccination of mice were determined by neutralization assays for antibody production and interleukin staining for detection of specific T cell responses. Mice receiving TSA in combination with a DNA vaccine exhibited higher antibody responses to the vaccine than mice not given TSA. Co-administration of TSA also enhanced specific CD8 T cells response. Drugs such as TSA that reduce initial gene silencing by preventing histone deacetylation can increase immune responses to DNA vaccination^ref
• seeking to combine the immunogenicity of virus-like particles (VLPs) and the ease of production of plasmid DNA, DNA vaccines expressing VLPs consisting of the MLV Gag and modified MLV Env proteins displaying T cell epitopes have been designed. Such DNA vaccines are remarkably efficient immunogens for inducing cellular immune responses. In contrast to similar plasmids harboring a point mutation preventing VLP formation, they induce protection against a lethal viral challenge in mice. Thus, these "plasmo-retroVLPs" represent a promising second-generation DNA vaccine^ref.
• plasmid DNAs encoding cytokines enhance immune responses to vaccination in models of infectious diseases and cancer. DNAs encoding cytokines that affect T cells [IL-2, IL-12, IL-15, IL-18, IL-21, and the chemokine CCL21] and antigen-presenting cells [GM-CSF] were compared in mouse models as adjuvants to enhance CD8⁺ T-cell responses and tumor immunity. A DNA vaccine against a self-antigen, gp100, expressed by melanoma was used in combination with DNA encoding cytokines and cytokines fused to the Fc domain of mouse IgG₁ (Ig). Cytokine DNAs generally increased CD8⁺ T-cell responses against gp100; ligation to Fc domains further enhanced T-cell responses; adjuvant effects were sensitive to timing of DNA injection; (d) the most efficacious individual adjuvants for improving tumor-free survival were IL-12/Ig, IL-15/Ig, IL-21/Ig, GM-CSF/Ig, and CCL21; and (e) combinations of IL-2/Ig + IL-12/Ig, IL-2/Ig + IL-15/Ig, IL-12/Ig + IL-15/Ig, and IL-12/Ig + IL-21/Ig were most active; and (f) increased adjuvanticity of cytokine/Ig fusion DNAs was not related to higher tissue levels or greater stability. These observations support the potential of cytokine DNA adjuvants for immunization against self-antigens expressed by cancer, the importance of timing, and the enhancement of immune responses by Fc domains through mechanisms unrelated to increased half-life^ref
• nanoparticle-based DNA vaccine delivery system : the nanoparticles were engineered by cooling pre-formed warm microemulsions comprised of emulsifying wax as the oil phase and hexadecyltrimethyl ammonium bromide (CTAB) as the surfactant. However, the poor aqueous stability of the nanoparticles and the emulsifying wax in the nanoparticles may severely limit the applications of the nanoparticles. In the present study, we used lecithin, a more biocompatible material, instead of emulsifying wax, to prepared lecithin-based cationic nanoparticles. The 50% growth inhibition concentration (IC₅₀) of the lecithin-based nanoparticles was found to be more than 1000-fold higher than that of the emulsifying wax-based nanoparticles. Moreover, the stability of the lecithin nanoparticles was also significantly increased. The size of the nanoparticles did not significantly change during a 6-month storage period at room temperature. Finally, when plasmid DNA was adsorbed on their surface, the lecithin nanoparticles successfully transfected cells in culture. These lecithin-based nanoparticles may hold great potentials as a DNA (vaccine) delivery system^ref.
• Den123 : a nontoxic self-assembled dendritic spheroidal nanoparticle made of biodegradable monomers) : footpad DNA immunization in Balb/c mice was done three times using the Bet v 1a gene with or without Den123 with 2-week intervals followed by sensitization with rBetv1 (5 mg) in alum twice in a weekly interval. Different doses of pCMV-Betv1 were used (10 mg and 100 mg). The protective role of different formulations was evaluated by measuring the IgG₁, IgG_2a and IgE antibody production, cytokine release of isolated splenocytes and b-hexosaminidase release from the RBL cells. Higher and increasing ratios of IgG_2a/IgG₁ were seen in mice which received plasmids in combination with Den123. Den123 and DNA vaccine synergistically enhanced the Interferon gamma released from splenocytes. In the presence of Den123, IgE inhibition was independent of the dose and type of the injected DNA. All DNA-pre-immunized mice demonstrated low basophil degranulation. It is therefore concluded that administration of the DNA entrapped in Den123 nanoparticles results in sustained release of plasmids, T_h1/T_h2 balanced immune response with promising IgE inhibition. Also higher amounts of DNA contributed to stronger T_h1 response^ref.
• recombinant reovirus type 3 s₁ attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy^ref.
• targeting antigens to FcgRs on DCs has been demonstrated to enhance antigen presentation. CCL21 / secondary lymphoid tissue chemokine (SLC) has been shown to increase immune responses not only by promoting coclustering of T cells and DCs in the lymph nodes and spleen but also by regulating their immunogenic potential for the induction of T cell responses. Using HPV 16 E7 as a model antigen, a chemotactic-antigen plasmid DNA vaccine (pSLC-E7-Fc) was constructed by linking SLC and Fc gene sequences to each end of E7 and evaluated its potency of eliciting specific immune response. Immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte proliferative and CTL responses than control DNA. All the mice receiving pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous inoculation of TC-1 cells, while those given control DNA all developed tumors. These tumor-free mice were also protected against TC-1 rechallenge. Complete tumor regression with long-term survival occurred in 72% of mice given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis model wherein TC-1 cells were intravenously injected, therapeutic vaccination with pSLC-E7-Fc significantly reduced the number of tumor nodules in the lung. In vivo depletion with antibodies against CD4⁺ or CD8⁺ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced immunotherapeutic effect. These data indicate that the DNA vaccine constructed by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is an effective approach to induce potent anti-tumor immune response via both CD4⁺ and CD8⁺ T cells dependent pathways^ref.
• CIA07 : a combination of 0.5-2.0-kb DNA fragments and modified LPS at a ratio of 100:1 exhibited higher immunostimulating activity than each substance alone, and its antitumor activity was significantly higher than that of Bacillus Calmette-Guerin in a mouse bladder cancer model. The use of Escherichia coli DNA or lipopolysaccharide (LPS) as an immunotherapy is often associated with unacceptable toxicity and insufficient therapeutic effects. In this study, we investigated the efficacy of using a combination of bacterial DNA fragments and LPS as an anticancer agent. LPS was isolated from an E. coli strain expressing short-carbohydrate-chain-containing LPS and subjected to alkaline hydrolysis to remove lipid A. The ability to induce TNF-a release in human whole blood cells was significantly lower for the LPS devoid of lipid A than for its parent form. The immunostimulating activity of E. coli DNA fragments of various sizes were tested. Those of 0.2-0.5kb in size exhibited the highest activity in whole blood assays, whereas those of size 0.5-2.0kb exhibited the highest adjuvant activity in mice. An intraperitoneal injection of CIA07 at a dose of 25mg/kg body weight caused no apparent adverse effects in mice and guinea pigs. Taken together, these data demonstrate that CIA07 exhibits potent immunostimulating activity with no apparent toxicity, and therefore warrant the further development of CIA07 as an immunotherapy for cancer treatment^ref.
• in vivo electroporation of a DNA vaccine adjuvanted with plasmids encoding different cytokines was investigated in large animals. Sheep were injected intramuscularly with a DNA vaccine encoding an antigen of Haemonchus contortus (pNPA) and plasmids encoding different cytokines followed by in vivo electroporation. Plasmids (pCI) carrying the genes of different cytokines including ovine IL-4(pCI-IL4), IL-10(pCI-IL10), GM-CSF(pCI-GMCSF), and MCP-1a(pCI-MCP1a), and pCI-IL4+pCI-GMCSF were co-delivered with pNPA. The results showed that co-delivery of pCI-GMCSF or pCI-IL4+pCI-GMCSF significantly enhanced both antibody responses and T cell proliferation responses to the antigen after two DNA immunisations compared to co-delivery of pCI. In contrast, antibody responses of the sheep that received pCI-IL10 were decreased significantly. Other cytokine expressing plasmids did not significantly alter the measured immune responses. Furthermore, co-delivery of pCI-GMCSF increased IgG₂ response more than IgG₁ responses, suggesting a T_h1 bias. However, the increase in IgG₂ over IgG₁ was less apparent when co-delivery of pCI-IL4 with pCI-GMCSF. Interestingly, the co-delivery of pCI-IL4 alone did not increase the IgG₁ titre, suggesting that both pCI-GMCSF and pCI-IL4 are required for optimal IgG₁ production^ref
• geldanamycin (GA), a heat-shock protein (HSP) 90 inhibitor, induces degradation of HSP90 client proteins, which may promote the presentation of degradation peptides with MHC class I on cancer cells. GA may enhance the efficacy of DNA vaccination, and investigated the therapeutic effect of the combination of GA and a DNA vaccine against HSP90 clients p185^neu and Met. The efficacy of various doses of GA combined with an N-terminal neu (N'-neu) DNA vaccine was investigated in a transplanted tumor constitutively overexpressing endogenous p185(neu). Low-dose (2.5 mg) but not high-dose (10 mg) GA enhanced the effect of N'-neu DNA vaccination on the inhibition of murine bladder tumor-2 tumors in syngeneic C3H mice. Anti-p185(neu) antibody titers were similar among all treated groups. Significantly increased infiltrations of CD8⁺ T cells and NK cells were observed at tumor sites. GA sensitized tumor cells to the cytotoxic effects of lymphocytes. Depletion of CD8⁺ T cells eliminated most of the therapeutic efficacy; in contrast, depletion of CD4⁺ T cells enhanced the therapeutic efficacy. A similar enhancing effect was observed for the combination of GA and a DNA vaccine targeting the Met oncogene. Combination of GA and DNA vaccination against GA-targeted proteins^ref
• DNA vaccine encoding a chimeric fusion between FasL and a truncated version of HIV gp120 as a model antigen. The fusion DNA was used as a priming vaccine, along with boosting with recombinant gp120 protein. Priming with fusion protein DNA resulted in a powerful enhancement of immune responses to the protein boost, and, in the presence of aluminum phosphate, to a strong enhancement in T_h2 type responses. Fas ligand delivered in a separate plasmid also had an adjuvant effect, although it was weaker than that delivered by the fusion protein^ref
• dendriplexes, complexes of dendrons and condensed plasmids containing the gene for protective antigen (PA) of Bacillus anthracis, were encapsulated in poly-lactide-co-glycolide (PLGA) particles using the double emulsion method. The 2 dendrons employed are a dendron with three C(18) chains (C(18) dendron) and one with no attached hydrocarbon chains (the C(0) dendron). 3 types of particles were examined, namely PLGA-C(18) dendriplexes, PLGA-C(0) dendriplexes and the control PLGA-naked DNA system. These were characterised by standard biophysical methods such as photon correlation spectroscopy (PCS) and scanning electron microscopy to select the complexes for in vivo testing. 3 intramuscular immunizations were carried out using 14 mg of DNA per dose at weekly intervals in BALB/c mice. Antibodies against rPA were measured using ELISA. Results indicate that the PLGA-C(18) dendriplex particles produced superior levels of anti-PA IgG antibodies in comparison to animals immunized with the PLGA-C(0) dendriplex particles. The level of antibody production was dependent on the number of immunizations, higher antibody levels being measured after two booster vaccinations. However toxin neutralizing antibodies were absent in all treatment groups, and it is likely that the mice lack protection against lethal toxin and anthrax infection. Further studies are needed to optimize the formulation of DNA vaccines and increase the level of anti-lethal toxin antibodies and enhance their functionality^ref

• anti-infectious diseases vaccines^ref :
• anti-prion vaccines :
• DNA immunization of wild-type mice can break immune tolerance against the prion protein, resulting in the induction of PrP-specific antibody and T-cell responses. PrP immunogenicity was increased by fusion to the lysosomal targeting signal from LIMPII (lysosomal integral membrane protein type II). Although mice immunized with a PrP-LIMPII DNA vaccine showed a dramatic delay in the onset of early disease signs after intracerebral challenge, immunization against PrP also had some deleterious effects. These results clearly confirm the feasibility of using active immunization to protect against TSEs and, in the absence of effective treatments, indicate a suitable alternative for combating the spread of these diseases^ref
• anti-viral vaccines
• anti-HIV-1 DNA vaccine (see also protein subunit vaccine and therapeutic vaccines)^ref : the ability of SIVDnef to induce a high frequency virus-specific CD4⁺ T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains^ref
• enzymatically active reverse transcriptase, but not the inactive mutant^ref
• oral or rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain producing TBI protein (artificial protein containing HIV-1 B and T cell epitopes)^ref
• oral and rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain producing gp-160^ref
• rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain could be perspective for delivery plasmid Env DNA and more effective than oral immunization^ref
• multi-epitope T-cell immunogen (TCI), 392 amino acids in length and containing 80 epitopes (both CD8⁺ CTL and CD4⁺ T_h)^ref
• rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain^ref
• i.m. injection of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine-polyglucin conjugate^ref
• a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope with 3 copies of the murine C3d (mC3d₃) added to the carboxyl terminus^ref
• AVX101^® (source : AlphaVax, Inc.) consists of a weakened strain of VEEV, which acts as a vector to carry HIV genetic material into host cells. AVX101 is the first vaccine to be tested that has been designed specifically to target HIV-1 subtype C, which is most prevalent in South Africa, rather than subtype B, which is predominant in the developed world and for which other vaccine trials are underway
• tgAAC09 (developed by Seattle-based Targeted Genetics and the Columbus Children's Research Institute in Ohio) uses rAAV encoding HIV gag and pro genes and a portion of the reverse transcriptase gene. Unlike many vaccines currently in clinical trials, tgAAC09 potentially could be a single-shot vaccine, which would be useful in developing countries. In addition, animal trials have shown that the vaccine candidate can stimulate both an antibody and a cell-mediated response to HIV. The vaccine focuses on HIV subtype C, which is the most common strain of the virus in India, so India has begun its first-ever phase I clinical trial of an HIV vaccine, which is being conducted by the Indian Council of Medical Research and the International AIDS Vaccine Initiative, is testing the vaccine candidate called The Indian trial is part of a multicountry Phase I trail of tgAAC09, which already has started in Europe with German and Belgian researchers testing the vaccine in partnership with IAVI. IAVI beginning in March 2001 provided $3 million over 18 months to the Indian government to fund HIV vaccine development efforts. The trial, which is expected to take about 15 months to complete, will test the vaccine on 30 male and female volunteers ages 18 to 45 who are HIV-negative and do not have any other significant illnesses. The trial will take place at the National AIDS Research Institute in Pune, India. ICMR Director N.K. The candidate vaccine might stimulate an immune system response that could protect volunteers from potential HIV infection. The volunteers have been made aware of the risks involved, they have been counseled and their health status will be monitored all the time. The European trial -- conducted at the Centre Hospitalier Universitaire Saint-Pierre in Brussels, Belgium -- is testing the safety and immune response of the vaccine in 50 male and female volunteers. If the vaccine is shown to be safe, it might advance to larger clinical trials, which could involve thousands of people at high risk of contracting HIV, such as commercial sex workers. Targeted Genetics expects to report data from the European trial in the first half of 2005. Web resources : Henry J. Kaiser Family Foundation : kaisernetwork.org.
• a DNA vaccine consisting of 7 plasmids encoding 9 HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8⁺ T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial^ref.
• current evidence suggests that a strong induced CD8 HIV-1-specific cell mediated immune response may be an important aspect of an HIV vaccine. The response rates and the magnitude of the CTL responses induced by current DNA vaccines in humans need to be improved and cellular immune responses to DNA vaccines can be enhanced in mice by co-delivering DNA plasmids expressing immune modulators. 2 reported to work well in the mouse systems are IL-12 and CD40L. The cDNA for macaque IL-12 and CD40L were cloned into DNA vectors. Groups of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag alone or in combination with either IL-12 or CD40L. CD40L did not appear to enhance the cellular immune response to SIVgag antigen. However, more robust results were observed in animals co-injected with the IL-12 molecular adjuvant. The IL-12 expanded antigen-specific IFN-g⁺ effector cells as well as granzyme B production. The vaccine immune responses contained both a CD8 component as well a CD4 component. The adjuvanted DNA vaccines illustrate that IL-12 enhances a CD8 vaccine immune response, however, different cellular profile^ref. Both 2-trimer and 4-trimer forms of macaque CD40L were active in B cell proliferation assays using macaque and human cells. Using human cells, 4-trimer macaque GITRL costimulated CD4⁺ T cell proliferation, and abrogated the immunosuppressive effects of CD4⁺CD25⁺ regulatory T cells on a MLR. These molecular adjuvants provide new tools for vaccine development in the SIV system and other macaque models^ref
• prime-boost regimens :
• heterologous plasmid DNA prime, live vector boost vaccination regimen appears especially promising in the nonhuman primate/ simian-human immunodeficiency virus (SHIV) challenge model. A series of intramuscular priming immunizations with a plasmid DNA vaccine expressing SIVgag p39, in combination with plasmid expressed rhesus IL-12, could effectively enhance the immunogenicity and postchallenge efficacy of 2 intranasal doses of recombinant vesicular stomatitis virus (rVSV)-based vectors expressing HIV-1 env 89.6P gp160 and SIVmac239 gag p55 in rhesus macaques. In macaques receiving the combination plasmid DNA prime, rVSV boost vaccination regimen, a significantly increased SIVgag- specific cell-mediated and humoral immune responses and significantly lower viral loads postintravenous SHIV89.6P challenge relative to macaques receiving only the rVSV vectored immunizations were observed. In addition, the plasmid DNA prime, rVSV boost vaccination regimen also tended to increase the preservation of peripheral blood CD4⁺ cells and reduce the morbidity and mortality associated with SHIV89.6P infection. An analysis of immune correlates of protection after SHIV89.6P challenge revealed that the prechallenge SHIV-specific IFN-g ELISpot response elicited by vaccination and the ability of the host to mount a virus-specific neutralizing antibody response postchallenge correlated with postchallenge clinical outcome. The correlation between vaccine-elicited cell-mediated immune responses and an improved clinical outcome after SHIV challenge provides strong justification for the continued development of a cytokine-enhanced plasmid DNA prime, rVSV vector boost immunization regimen for the prevention of HIV infection^ref. A mutant full-sized plasmid DNA vaccine regime in macaques was effective against a homologous challenge^{ref1, ref2} : to evaluate the DNA vaccination regime against a heterologous challenge, a novel plasmid named pSHIV-ZF1*IL-2 was constructed. 4 monkeys were intramuscularly and intradermally injected 4 times with the pSHIV-ZF1*IL-2. Vaccinated monkeys were intravenously challenged with a highly pathogenic, heterologous SHIV at 11 weeks post vaccination. All the vaccinated monkeys suppressed the challenge virus rapidly under the detectable level by 16 weeks post challenge. One vaccinated monkey was protected from a loss of CD4⁺ T cells. These results suggest pSHIV-ZF1*IL-2 alone seems partially effective even against a challenge with a heterologous, pathogenic virus^ref.
• gp120/gag DNA priming- gp120 protein boosting : immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the naive animals. Further, one of 6 immunized animals was completely protected whereas all six naive animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L)^ref
• priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. The DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing 1, 3 or 8 primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations^ref
• given the importance of the HIV-specific cell-mediated immune response in the early control and resolution of HIV infection and the observed correlation between pre-challenge vaccine elicited CTL responses and post challenge outcome in SHIV/rhesus macaque experiments, several candidate plasmid DNA (pDNA) vaccine designs capable of eliciting robust and balanced cell-mediated immune responses to multiple HIV-1 derived antigens in mice were identified for further vaccine development. To rationally construct candidate vaccines for immunogenicity testing, the relative immunogenicity of the individual HIV-derived vaccine antigens (env, gag, pol, nef, tat and vif) and the relative strength of various transcriptional control elements (HCMV, SCMV, HSV Lap1) in Balb/c mice were determined. Next, a number of 1-, 2-, 3- and 4-vector pDNA vaccine designs were tested for their ability to elicit HIV-1 antigen-specific CMI responses. For these studies, Balb/c mice were immunized with a fixed total pDNA vaccine dose of 100 mg in combination with 25 mg plasmid-based murine IL-12 and tested for the induction of HIV-1 antigen-specific CMI responses by IFN-g ELISpot analysis. The results of this study indicate that all pDNA vaccine designs were capable of eliciting CMI responses to multiple HIV-1 antigens. As a result of this iterative comparative analysis, we have identified a number of pDNA vaccine candidates capable of eliciting potent, balanced CMI responses to multiple HIV-1 derived antigens. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection^ref
• optimization of codon usage, enhancement of viral promoter function and selection of secretory leader sequences can work synergistically to improve the final antigen expression and immunogenicity of HIV-1 Env DNA vaccines, indicating they work through different mechanisms. The best result came from the approach that optimized all 3 components in a DNA vaccine design. The levels of HIV-1 env-specific RNA transcripts in transiently transfected 293T cells were higher from the codon-optimized gene than the wild type counterpart. This finding suggested other mechanism may also contribute to the increased antigen expression and immunogenicity of codon-optimized DNA vaccines in addition to the improved tRNA usage in mammalian cells for codon-optimized viral genes as previously reported^ref
• a plasmid DNA vaccine containing a fusion gene consisting of an HIV-1 subtype C gag and a modified subtype C pol was compared to a mixture of gag plus pol or gag plus HIV env plasmids. Plasmid DNA was delivered by intramuscular injection followed by electroporation in vivo. 2 vaccinations were sufficient to induce high levels of Gag- and Pol-specific CD4 and CD8 T cells in peripheral blood. The gag-pol fusion plasmid was as immunogenic as the plasmid mixtures. Thus, DNA vaccination by intramuscular electroporation was an effective means for inducing high levels of Gag- and Pol-specific T cells, and a single gag-pol fusion DNA vaccine was sufficient for eliciting immune responses against both antigens^ref
• recombinant DNA vaccine delivered intramuscularly and recombinant Listeria monocytogenes delivered orally induced CD8⁺ and CD4⁺ T cell immune responses in rhesus macaques and this vaccine protocol showed partial protection against an SIV239 challenge. The SIV antigen-specific immune responses at the time of challenge and during the subsequent infection course. The immune status of the animals, as measured by the frequency of antigen-specific IFN-g secreting PBMCs, at the time of challenge correlates more strongly with viral loads at set point than peak viral loads. The correlation between the immune response and viral load was strongest early, as viral set-point was just being established and disintegrates overtime. This study demonstrates the cellular immune response to SIV at the time of challenge of a nonhuman primate is able to impact on viral set-point following SIV239 challenge. Further, this study demonstrates that as virus replicates the T cell immune response to SIV antigens induced by the vaccine is modulated by antigen encountered by immune cells during viral replication^ref
• a potent DNA vaccine against HIV, combining a vector that takes advantage of the segregation and compartmentalization effect of bovine papilloma virus E2 protein with MultiHIV insert, expressing a fusion gene coding for the non-structural and structural proteins was developed and tested for immunogenicity in mice and humans^ref
• the design and preclinical development of a multigene HIV-1 subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of 2 plasmids, pTHr.grttnC and pTHr.gp150CT, which express a polyprotein derived from Gag, reverse transcriptase (RT), Tat and Nef, and a truncated Env, respectively. Genes included in the vaccine were obtained from individuals within 3 months of infection and selection was based on closeness to a South African subtype C consensus sequence. All genes were codon-optimized for increased expression in humans. The genes have been modified for safety, stability and immunogenicity. Tat was inactivated through shuffling of gene fragments, whilst maintaining all potential epitopes; the active site of RT was mutated; 124 aa were removed from the cytoplasmic tail of gp160; and Nef and Gag myristylation sites were inactivated. Following vaccination of BALB/c mice, high levels of cytotoxic T lymphocytes were induced against multiple epitopes and the vaccine stimulated strong CD8⁺ IFN-g responses. In addition, high titres of antibodies to gp120 were induced in guinea pigs. This vaccine is the first component of a prime-boost regimen that is scheduled for clinical trials in humans in the USA and South Africa^ref
• The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. 5 new MHC class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8⁺ T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates^ref and support a subtype C in-frame gagpol fusion gene vaccine^ref
• recombinant vaccinia virus-based vaccine combined with DNA vaccine has produced a protective immune response against HIV infection in non-human primates. A recombinant vaccinia virus (LC16m8 strain) has been used in children without severe side effects. The vaccinia virus expressing an HIV(89.6)env gene (vLC-Env) alone or combined with a DNA vaccine expressing the HIV(89.6)env gene (pCAG-Env) was characterized in BALB/c mice. Vaccination of vLC-Env induced much higher HIV-specific humoral and cell-mediated immune responses than that of pCAG-Env. Priming with pCAG-Env further enhanced vLC-Env induced immune responses, especially cell-mediated immune response. Moreover, efficient expression of Env protein was achieved following infection of bone marrow dendritic cells by vLC-Env in vitro. Administration of vLC-Env-infected dendritic cells to mice generated a high cell-mediated immune response. These results demonstrate that priming with pCAG-Env and boosting with vLC-Env represents a logical candidate for vaccination against HIV infection^ref.
• in-frame fusion of TNF-a DNA to DNA encoding a large fragment of HIV gp120. The studies were performed using a DNA prime, protein boost regime and a heterologous boosting protein. Fusion of TNF-a DNA enhanced T_h1 related immune responses against both the priming and the boosting gp120. In-frame fusion of IFN-g-encoding DNA at the 5' end of the chimeric molecule, to create a tripartite fusion, had no additional effect on immunogenicity^ref
• immunization of macaques with a reverse transcriptase-deleted SHIV(KU2) (DrtSHIV(KU2)) plasmid that contained HIV-1(HXB2) env and SIV gag-nef induced protection against AIDS caused by challenge virus SHIV89.6P with a heterologous env. We further deleted vif and integrase from DrtSHIV(KU2) and substituted the 3'LTR with SV40 poly A sequences, creating D4SHIV(KU2) (M) and a parallel construct containing gag-nef of HIV-1(SF2), D4SHIV(KU2) (H). 6 macaques received two intramuscular injections of the (M) DNA, and another six received three injections of the (H) DNA. 3 of the latter group received 2 post-challenge boosts with (M) DNA vaccine. 7 virus control macaques were inoculated with SHIV89.6P. All twelve immunized macaques were challenged with SHIV89.6P virus, and CMI responses were measured by ELISPOT assays. Virus control animals all developed progressive infection, whereas vaccinated macaques from both groups controlled virus replication, with plasma viral loads dropping to undetectable levels between weeks 6 and 126 p.i. This DNA vaccine was efficacious even though it encoded Env, Gag, and Nef that were genetically distinct from the proteins in the challenge virus. The DNA vaccine induced broad-based protection without using viral proteins to boost the immunity^ref.
• strategies designed to overcome the usual propensity of CTLs to focus recognition on a limited number of dominant epitopes. In studies of rhesus monkeys expressing the Mamu-A*01 MHC class I allele, we show that variously configured multiepitope plasmid DNA vaccine constructs elicit CTL populations that do not evidence skewing of recognition to dominant epitopes. Nevertheless, repeated boosting of these vaccinated monkeys with different live recombinant vaccine vectors uncovers and amplifies the usual CTL epitope dominance hierarchy. Importantly, in vitro peptide stimulation of PBMCs from monkeys that have received only a multiepitope plasmid DNA priming immunization uncovers this dominance hierarchy. Therefore, the dominance hierarchy of the vaccine-elicited epitope-specific CTL populations is inherent in the T lymphocytes of the monkeys after initial exposure to epitope peptides, and the ultimate breadth of epitope recognition cannot be modified thereafter. This finding underscores the enormous challenge associated with increasing the breadth of CTL recognition through vaccination^ref.
• controlled release of GM-CSF protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific CTL and T_h responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific Ab, T_h, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and T_h, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design^ref.
• novel biocompatible core-shell cationic nanoparticles, composed of an inner hard core of poly(methylmethacrylate) (PMMA) and a hydrophilic tentacular shell bearing positively charged groups and poly(ethyleneglycol) chains covalently bound to the core, were prepared by emulsion polymerization and characterized in vitro and in vivo for DNA vaccine applications. The nanoparticles reversibly adsorbed large amounts of DNA, mainly through electrostatic interactions, preserved its functional structure, efficiently delivered it intracellularly, and were not toxic in vitro or in mice. Furthermore, 2 intramuscular (i.m.) immunizations (4 weeks apart) with a very low dose (1mug) of the plasmid pCV-tat delivered by these nanoparticles followed by 1 or 2 protein boosts induced significant antigen-specific humoral and cellular responses and greatly increased T_h1-type T cell responses and CTLs against HIV-1 Tat^ref
• plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8⁺CD127⁺ memory T cell pools specific for a subdominant HLA-A2-restricted Env_121-129 epitope (KLTPLCVTL). Coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8⁺ T cell function that was partially independent of CD4⁺ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines^ref
• to analyze the immunogenicity of HIV-1 DNA vaccine which expressing the chimeric gene gag-gp120 of Chinese prevalent HIV-1 strain and the immunoregulatory activity of IL-6, DNA vaccine plasmid pVAX1-gag-gp120 and eukaryotic expression plasmid pVAX1-IL6 were constructed and the expression in vitro was detected by RT-PCR and Western blotting, the results showed that the gene of interest expressed in the transfected HeLa cells. To explore the immune response in mice coinoculated with HIV-1 DNA vaccine and IL-6 expression plasmid, BALB/c mice were injected i.m. with eukaryotic expression plasmid pVAX1-IL6 and DNA vaccine plasmid pVAX1-gag-gp120. The specific humoral and cellular immunity in mice could be induced by inoculating separately HIV-1 DNA vaccine plasmid or coinoculating with IL-6 expression plasmid, and the specific killing activities of spleen CTL and the level of serum antibodies in the coinoculation group were significantly higher than those in the separate inoculation group. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination^ref
• a multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2^d-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8⁺ CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria^ref
• recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-g ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4⁺, CD8⁺, and CCR⁺ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity^ref
• estimating effective doses of novel HIV vaccines is challenging. Dose-response analyses of DNA and fowlpox virus HIV vaccines showed that 1 mg of DNA vaccine and 5 x 10⁷ pfu of fowlpox virus booster was immunogenic in macaques. However, this dose was poorly immunogenic in humans. When adjusted for body surface area, the human dose studied was equivalent to a poorly immunogenic lower dose in monkeys. These data provide a rationale for guiding dosing in future trials of HIV vaccine technologies^ref
• bicistronic DNA vaccines that encode for HIV env and caspase-3 mutant (casp 3m) that are expressed via the encephalomyocarditis virus internal ribosomal entry site (IRES) or cytomegalovirus (CMV) promoter-dependent translations. While IRES-casp 3m induced weak apoptosis and caused little reduction in antigen expression, CMV-casp 3m elicited strong apoptosis and led to a marked decrease in the antigen expression. Therefore, IRES-casp 3m augmented HIV-specific immune responses, and IRES-casp 3m induced significant protection against the vaccinia-HIV chimeric virus. These results suggest that the appropriate level of apoptosis is important for DNA vaccine development^ref
• Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine. VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. 50 healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle-free intramuscular injection. Humoral responses (measured by ELISA, Western blotting, and neutralization assay) and T cell responses (measured by ELISA and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured. The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4⁺ and CD8⁺ T cell responses occurred after 3 injections and were in response