When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997-1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000-2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines.

VCL-AB01 is a bivalent plasmid DNA (pDNA)-based anthrax vaccine candidate (composed of pDNA encoding detoxified Protective Antigen (PA) and Lethal Factor (LF) formulated with a cationic lipid system) currently in a Phase 1 trial. The immunogenicity and protective efficacy of this investigational vaccine were evaluated in lethal B. anthracis spore challenge mouse and NHP models. DBA/2 mice received VCL-AB01 intramuscularly (100 μg; 15/sex/group) on Days 7 and 14 (accelerated regimen) or Days 0 and 14 (standard regimen). A third group received Anthrax Vaccine Adsorbed (AVA; Bioport; 50 μL subcutaneously) on Days 0 and 14. Only 10 mice/sex of the 15/sex/group were challenged intratracheally on Day 35 (lethal dose; 1.3 X 106 Sterne strain spores). Sera collected on Days 0, 28 (unchallenged group only) and 50 were evaluated for toxin neutralization activity (TNA). By Day 28, VCL-AB01- vaccinated mice had lower TNA [geometric mean titers (GMT) of 76 and 106 ED50 for accelerated and standard regimens, respectively] than AVA-vaccinated mice (GMT=396 ED50). Four of the 10 animals vaccinated with VCL-AB01 on the accelerated regimen and 2/10 vaccinated on the standard regimen had no detectable TNA, yet both challenged cohorts were highly protected (85% and 95%), with comparable survival to the AVA-vaccinated cohort (90%) and demonstrated significantly (p<0.0001; Kruskal-Wallis Test) increased TNA post challenge compared to non-challenged cohorts at Day 50. Cynomolgus macaques (2/sex/group) received either VCL-AB01 (0.6 mg/mL) or 1.0 mL of PBS intramuscularly on Days 0, 14 and 28. Animals received a lethal challenge (AMES strain spores; Mean = 486 LD50) at 10 weeks and monitored for survival. Sera collected on Days 0, 28, 42 and 70 (just prior to challenge) and 3 weeks post- challenge were evaluated for TNA and serum antibodies. At 10 weeks, just prior to challenge, sera samples were negative for TNA. PA- and LF-specific antibodies were variable, ranging from 21-58 μg/mL and from 2-390 μg/mL, respectively. Despite low antibody titers and absence of TNA, 75% of VCL-AB01 vaccinated animals survived challenge. Survival was associated with an exponential rise in titers observed by 3 weeks post challenge (4748–8231 μg/mL PA-specific antibodies; 3045–12948 μg/mL LF-specific antibodies; TNA = 11,296–23,676 ED50). All control animals died 3-8 days post-challenge. Collectively, these results show that surviving animals with low or absent pre-challenge neutralizing antibodies and/or low PA- and LF-specific antibody titers were able to mount a rapid protective immunological response of a very high magnitude to the lethal spore challenge. These results indicate that: 1) vaccination with pDNA elicits a potent B-cell priming response that can be activated to full effector function extremely rapidly in the face of exposure to B. anthracis; 2) serum antibody and TNA titers are not optimal predictors of protection; and 3) other measures predictive of pDNA vaccine efficacy are needed. Presenting author is employee of Vical Incorporated, manufacturer of the investigational vaccine.

In the late 1980s, Vical and collaborators discovered that the injection into tissues of unformulated plasmid encoding various proteins resulted in the uptake of the plasmid by cells and expression of the encoded proteins. After this discovery, a period of technological improvements in plasmid delivery and expression and in pharmaceutical and manufacturing development was quickly followed by a plethora of human clinical trials testing the ability of injected plasmid to provide therapeutic benefits. In this chapter, we summarize in detail the technologies used in the most recent company-sponsored clinical trials and discuss the potential for future improvements in plasmid design, manufacturing, delivery, formulation and administration. A generic path for the clinical development of plasmid-based products is outlined and then exemplified using a case study on the development of a plasmid vaccine from concept to clinical trial.

A short peptide of the nuclear localization signal (NLS) of HIV-1 Tat protein, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, was modified by adding a specified amino acid at the NH2 and COOH termini. These peptides were simply added to DNA before being added with cationic liposomes to improve intracellular trafficking of the plasmid DNA. The Tat peptides were first mixed with reporter plasmid that express either luciferase, LacZ, or GFP, and then mixed with liposomes, to form the trimeric complex of DNA/peptide/liposome (DPL). The DPL complex was treated to various cell lines, and was compared for transfection efficiency with that obtained by the conventional DNA/liposomes complex. When tested for transfection efficiency employing peptides with end modifications, the peptide with COOH-terminal modification showed highest transfection efficiency. When the DPL complex was formed with various cationic liposomes, DOSPA/DOPE (LipofectamineTM) exhibited better transfection efficiency than other liposomes tested. Meanwhile plasmid DNA mixed only with the Tat NLS peptide showed significantly lower transfection when compared to that obtained with the DPL complex. When an optimal ratio of each DPL component was used with the DOSPA/DOPE liposomes, transfection efficiency was shown to be 100 to 600 fold better in reporter gene expression than that obtained with the DNA/liposomes (without peptide addition) complex. This study shows that the trimeric DPL complex can be simply formed and employed for enhanced expression of a transgene in many different cells with a strong potential for practical applications.

Top of pageAbstract We are testing a number of expression plasmids (pDNA) encoding SARS-CoV antigens to support potential development of a pDNA vaccine against this emerging pathogen. Our constructs are all derived from SARS-CoV Urbani and include the complete coding sequence for the nucleocapsid antigen, the soluble (extracellular) portion of the spike antigen, and two subdomains of the spike antigen termed S1 and S2. The codons used in these constructs were optimized for expression in human cells. These pDNAs were assayed for expression both in vitro and in vivo. Western blots of in vitro transfected cell lysates and culture supernatants were probed with a rabbit polyclonal antibody specific to either spike or nucleocapsid peptides. These assays indicate that the spike antigen is efficiently released from cells in culture whereas the nucleocapsid antigen remains predominately intracellular. All four pDNAs were formulated in either cationic lipid or poloxamer and injected into mouse (50 mg) or rabbit (1 mg) muscle tissue (i.e. 3X @ two week intervals). Sera from pDNA-vaccinated animals will be tested for virus neutralization in vitro. The use of one or more of these pDNA vaccines will be further evaluated for their immunogenicity and safety in animal models.

DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipid-formulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits. Eight or nine animals in each group were challenged with 100x LD50 of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA or PA plus LF pDNA vaccines were protected. In addition, 5 of 9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA plus LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers postchallenge, suggesting little or no spore germination. In contrast, titer increases were seen in AVA animals, and in surviving animals vaccinated with LF pDNA alone. Preclinical evaluation of this cationic lipid-formulated bivalent PA and LF vaccine is complete, and the vaccine has received U.S. Food and Drug Administration Investigational New Drug allowance.

B-cell lymphomas express tumor-specific immunoglobulin, the variable regions of which [idiotype (Id)] can serve as a target for active immunotherapy. Promising results have been obtained in clinical studies of Id vaccination using Id proteins.However, Id protein is laborious and time-consuming to produce. DNA vaccination is an attractive alternative for delivering Id vaccines, because Id DNA can be rapidly isolated by PCR techniques. DNA coding for lymphoma Id can provide protective immunity in murine models. In the present study, we performed a Phase I/II clinical trial to study the safety and immunogenicity of naked DNA Id vaccines in 12 patients with follicular B-cell lymphoma. The DNA encoded a chimeric immunoglobulin molecule containing variable heavy and light chain immunoglobulin sequences derived from each patient's tumor, linked to the IgG2a and kappa mouse immunoglobulin (MsIg) heavy- and light-chain constant regions chains, respectively. Patients in remission after chemotherapy received three monthly i.m. injections of the DNA in three dose escalation cohorts of four patients each (200, 600, and 1800 micro g). After vaccination, 7 of 12 patients mounted either humoral (n = 4) or T-cell-proliferative (n = 4) responses to the MsIg component of the vaccine. In one patient, a T-cell response specific to autologous Id was also measured. Anti-Id antibodies were not detectable in any patient. A second series of vaccinations was then administered using a needle-free injection device (Biojector) to deliver 1800 micro g both i.m. and intradermally (i.d.); 9 of 12 patients had humoral (n = 6) and/or T-cell (n = 4) responses to MsIg. Six of 12 patients exhibited humoral and/or T-cell anti-Id responses; yet, these were cross-reactive with Id proteins from other patient's tumors. Subsequently, a third series of vaccinations was carried out using 500 micro g of human granulocyte-macrophage colony-stimulating factor DNA mixed with 1800 micro g of Id DNA. The proportion of patients responding to MsIg remained essentially unchanged (8 of 12), although humoral or T-cell responses were boosted in some cases. Throughout the study, no significant side effects or toxicities were observed. Despite the modest level of antitumor immune responses in this study, DNA vaccine technology retains potential advantages in developing anti-Id immunotherapies. Additional studies are warranted to optimize vaccine dose, routes of administration, vector designs, and prime-boost strategies. These results will help guide the design of such future DNA vaccine trials.

Tumor vaccines are a promising alternative to chemotherapy for the treatment of metastatic cancer. To be effective and safe, a therapeutic cancer vaccine should specifically target antigens expressed only on metastatic tumor cells. A vaccine directed against the unique surface immunoglobulin or idiotype expressed on non-Hodgkin's B-cell lymphoma fulfills these criteria, as both primary and metastatic tumor cells express tumor specific immunoglobulins. Using the murine 38C13 B-cell lymphoma tumor as a model system, a plasmid DNA vaccine was designed to express a bicistronic mRNA encoding both the light and heavy tumor immunoglobulin (idiotype) proteins expressed on the surface of the 38C13 tumor. To increase the immunogenicity of the plasmid DNA vaccine, each of the murine variable domains (light and heavy) were fused to their respective human immunoglobulin constant domains. In addition, a eukaryotic expression cassette was constructed to effect both high-level expression of the mouse/human chimeric immunoglobulin, and to elicit a protective immune response in vivo. Unique Sfi I restriction sites were used for the rapid cloning of any tumor specific immunoglobulin idiotype domains and a series of plasmid constructs were made to test changes to the J domain and/or the human C domain to insert the Sfi I restriction sites. Such changes were found to have significant effects on both expression and immunogenicity. Vaccination of mice with prototype idiotype vaccines was found to generate a protective immune response to the 38C13 tumor. This study indicates that a novel bicistronic plasmid DNA-based vaccine can be used to develop a tumor specific vaccine against B-cell lymphoma.

MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding five proteins from Plasmodium falciparum and one plasmid DNA encoding human GM-CSF. To evaluate the safety of MuStDO 5, a series of pre-clinical studies were conducted in mice and rabbits. In pharmacology studies in mice, GM-CSF could not be detected in the serum following either intramuscular or a combined intramuscular/intradermal administration of the vaccine, but was readily detected in the muscle following intramuscular administration. In a tissue distribution study in mice, MuStDO 5 plasmid DNA was detected by PCR initially in highly vascularized tissues, while at later time-points the plasmid DNA was detected primarily at the site(s) of injection. In GLP safety studies in mice and rabbits, repeated intramuscular/intradermal administration of the MuStDO 5 vaccine was found to be safe and well tolerated without any evidence of autoimmune pathology.

Erythropoietin (EPO) cDNA was cloned from kidney total RNA of a NZW rabbit. The cDNA comprises a 588-bp open reading frame encoding a 195 amino acid protein with distinguishable regions of high of homology to other mammalian EPOs. Intramuscular injection of mice with a rabbit EPO expression plasmid resulted in a significant hematocrit increase. A rabbit genomic DNA fragment was also cloned using the rabbit EPO cDNA. This 4312-bp genomic DNA fragment contains sequences homologous to the mouse EPO promoter and hypoxia-responsive enhancer. In addition, the genomic DNA also presents a high degree of conservation to other regions involved in hypoxia response. Sequence divergence in the 3' UTR may indicate differences in regulation of mRNA stability or response to low oxygen tension.

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-γ responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8 + and CD4 + T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8 + -dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-γ responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 microg to 1 microg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-microg doses of pPyCSP plus 1 microg pGMCSF with mice primed by 1-microg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-gamma-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-gamma spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.

DNA-based vaccines are considered to be potentially revolutionary due to their ease of production, low cost, long shelf life, lack of requirement for a cold chain and ability to induce good T-cell responses. Twenty healthy adult volunteers were enrolled in a Phase I safety and tolerability clinical study of a DNA vaccine encoding a malaria antigen. Volunteers received 3 intramuscular injections of one of four different dosages (20, 100, 500 and 2500 microg) of the Plasmodium falciparum circumsporozoite protein (PfCSP) plasmid DNA at monthly intervals and were followed for up to twelve months. Local reactogenicity and systemic symptoms were few and mild. There were no severe or serious adverse events, clinically significant biochemical or hematologic changes, or detectable anti-dsDNA antibodies. Despite induction of excellent CTL responses, intramuscular DNA vaccination via needle injection failed to induce detectable antigen-specific antibodies in any of the volunteers.

Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials to test their safety and efficacy in healthy human volunteers. A safety concern unique to this new class of vaccines is the potential risk of deleterious integration into host cell genomic DNA following direct intramuscular injection. To address this issue experimentally, a preclinical safety study was conducted in mice to determine the structural nature of plasmid DNA sequences persisting in total muscle DNA at both 30 and 60 days following a single intramuscular injection of a plasmid expressing the Plasmodium falciparum circumsporozoite protein. In a protocol described for the first time, total DNA was extracted from muscle tissue and was subsequently linearized with a restriction endonuclease to enable agarose gel size fractionation of all extrachromosomal plasmid DNAs from high molecular weight mouse genomic DNA. Using PCR assays to quantitate plasmid-specific sequences, it was found that the amount of plasmid DNA persisting in muscle tissue varied but averaged about 10 fg per microgram of genomic DNA (in the range of 1500 copies per 150,000 genomes). In two of four separate experimental injections of mouse muscle, PCR assays of genomic DNA fractions indicated that agarose gel purification removed plasmid DNA down to a level of < or =3 copies per 150,000 mouse genomes. In the two other experimental samples, 3-30 copies of plasmid DNA remained associated with purified genomic DNA. The time following injection (i.e., 30 or 60 days) was not a factor in the number of copies of plasmid associating with genomic DNA and it was not possible to conclude if such sequences were covalently linked to genomic DNA or simply adventitiously associated with the genomic DNA. However, if an assumption is made that the highest level plasmid DNA found associated with genomic DNA (i.e., 30 copies) represented covalently integrated plasmid inserts and that each insert resulted in a mutational event, the calculated rate of mutation would be 3000 times less than the spontaneous mutation rate for mammalian genomes. This level of integration, if it should occur, was not considered to pose a significant safety concern.