Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stage Plasmodium falciparum DNA vaccine combined with escalating dose human GM-CSF DNA.

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.

Plasmid vaccines and therapeutics: from design to applications.

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.

Immunogenicity of a plasmid DNA vaccine encoding chimeric idiotype in patients with B-cell lymphoma.

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.

The development of a bicistronic plasmid DNA vaccine for B-cell lymphoma.

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.