Terry J. Higgins

First Human Trial of a DNA-Based Vaccine for Treatment of Human Immunodeficiency Virus Type 1 Infection: Safety and Host Response

A DNA-based vaccine containing human immunodeficiency virus type 1 (HIV-1) env and rev genes was tested for safety and host immune response in 15 asymptomatic HIV-infected patients who were not using antiviral drugs and who had CD4+ lymphocyte counts of > or = 500 per microliter of blood. Successive groups received three doses of vaccine (30, 100, or 300 microg) at 10-week intervals in a dose-escalation trial. Vaccine administration induced no local or systemic reactions, and no laboratory abnormalities were detected. Specifically, no patient developed anti-DNA antibody or muscle enzyme elevations. No consistent change occurred in CD4 or CD8 lymphocyte counts or in plasma HIV concentration. Antibody against gp120 increased in individual patients in the 100- and 300-/microg groups. Some increases were noted in cytotoxic T lymphocyte activity against gp160-bearing targets and in lymphocyte proliferative activity. The safety and potential immunogenicity of an HIV-directed DNA-based vaccine was demonstrated, a finding that should encourage further studies.

Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine.

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.

Vaccination of Seronegative Volunteers with a Human Immunodeficiency Virus Type 1 envlrev DNA Vaccine Induces Antigen-Specific Proliferation and Lymphocyte Production of β-Chemokines

There is a pressing need to test novel vaccine concepts in an effort to develop an effective vaccine for human immunodeficiency virus (HIV) type 1. A phase I clinical study was done to test the immunogenicity of an HIV env/rev DNA vaccine, which was administered intramuscularly to HIV-1-seronegative persons. Subjects received 3 doses of vaccine at a single concentration (100 or 300 microgram) at 0, 4, 8, and 24 weeks. In at least 1 of multiple assays, the 6 subjects who received the 300-microgram dose had DNA vaccine-induced antigen-specific lymphocyte proliferative responses and antigen-specific production of both interferon-gamma and beta-chemokine. Furthermore, 4 of 5 subjects in the 300 microgram-dose group responded to both the rev and env components of the vaccine. The responses did not persist within inoculated individuals and scored in different individuals at different times in the trial. This study supports that HIV-1 DNA vaccine antigens can stimulate multiple immune responses in vaccine-naive individuals, and it warrants additional studies designed to enhance DNA vaccine immunogenicity.

Enhancement of protective humoral (Th2) and cell-mediated (Th1) immune responses against herpes simplex virus-2 through co-delivery of granulocyte-macrophage colony-stimulating factor expression cassettes.

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) could in theory attract antigen‐presenting cells in muscle following intramuscular DNA immunization, resulting in enhanced antigen‐specific immune responses. Thus, such adjuvants could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of GM‐CSF cDNA as a vaccine adjuvant for herpes simplex virus (HSV)‐2 in a mouse challenge model. GM‐CSF cDNA co‐injection enhanced levels of specific IgG, IgE and IgA against HSV‐2 gD protein significantly higher than gD plasmid vaccination alone. Moreover, GM‐CSF co‐injection induced a dramatic increase in IgG1 levels, as compared to IgG2a levels, suggesting a Th2 bias in the response. T helper cell proliferation and secretion of cytokines (IL‐2 and IFN‐γ) were significantly increased by GM‐CSF cDNA co‐injection. When challenged with a lethal dose of HSV‐2, GM‐CSF co‐injection increased survival rates to 90 %, an improvement as compared to gD vaccination alone (60 – 63 %). Furthermore, GM‐CSF cDNA co‐injection reduced herpetic lesions and resulted in a faster recovery from lesions. These data indicate that GM‐CSF cDNA enhances both humoral and cellular immune responses and enhances vaccine efficacy, resulting in reduced HSV‐2‐derived morbidity as well as mortality.

DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans

Humoral and cellular immune responses have been produced by intramuscular vaccination with DNA plasmids expressing HIV-1 genes, suggesting possible immunotherapeutic and prophylactic value for these constructs. Vaccination with these constructs has decreased HIV-1 viral load in HIV-1-infected chimpanzees. In addition, naive (i.e. non-HIV-1-infected) chimpanzees were protected against a heterologous challenge with HIV-1. Ongoing phase I clinical trials show that therapeutic vaccinations indeed boost anti-HIV-1 immune responses in humans. A therapeutic phase I trial on humans with these constructs induced a good safety profile and also demonstrated an immunological potentiation. These findings indicate that further studies with these constructs in humans are warranted.

T-cell responses induced in normal volunteers immunized with a DNA-based vaccine containing HIV-1 env and rev.

Objective: An effective HIV-1 vaccine will likely need to induce strong cell-mediated immunity in humans. Therefore, we examined the ability of a DNA HIV-1 vaccine to induce a T-cell response in HIV-1 seronegative humans. Design: Individuals were enrolled in a phase I clinical trial of safety and immune responses to an env/rev-containing plasmid at doses of 100, 300 or 1000 μg. Peripheral blood mononuclear cells (PBMC) samples were analyzed by standard lymphocyte proliferation, cytotoxic T lymphocyte (CTL) and ELISPOT techniques. Results: PBMCs from subjects immunized with doses as low as 300 μg proliferated in vitro to env (four of six) or rev (three of six) proteins. Importantly, when the dose of vaccine was increased to 1000 μg of DNA, lymphocytes secreted IFN-gamma in an ELISPOT assay following in vitro stimulation with env (three of six) or rev (four of six) proteins. Conclusion: We observed HIV-1 DNA plasmid vaccines induce CD4 T-helper cell responses in humans. We observed a discrepancy in the CD4 versus CD8 response suggesting the importance of analyzing both compartments in clinical evaluation. Furthermore, this report demonstrates the high level of immunogenicity of rev and its importance as a component of a prophylactic vaccine for HIV-1.

Phase I Study of a Herpes Simplex Virus Type 2 (HSV-2) DNA Vaccine Administered to Healthy, HSV-2-Seronegative Adults by a Needle-Free Injection System

ABSTRACT We conducted a double-blind, vehicle-controlled, dose escalation safety and immunogenicity trial of a candidate herpes simplex virus type 2 (HSV-2) surface glycoprotein D2 (gD2) DNA vaccine administered by use of a needle-free device. Sixty-two healthy adults were randomized using a 4:1 vaccine-to-placebo ratio. Half of the participants were HSV-1 seronegative, and all were HSV-2 seronegative. Vaccine doses included 100 μg, 300 μg, 1,000 μg or 3,000 μg of a plasmid expressing the gD2 protein. Subjects received vaccine at 0, 4, 8, and 24 weeks. Some subjects received an additional 1,000-μg boost at 52 weeks. We found that the vaccine was safe and well tolerated, with most adverse events being local site reactions. No dose-limiting toxicities were observed. gD2-specific cytotoxic T-lymphocyte and lymphoproliferation responses were detected 2 weeks after the third vaccine injection in one of four HSV-1-seronegative, HSV-2-seronegative participants who received 3,000 μg of vaccine. A DNA-based vaccination strategy against HSV-2 appears to be safe and may generate a vaccine-specific cellular immune response, but high vaccine doses are likely needed to elicit an immune response in most vaccinees.

Plasmid DNA-expressed secreted and nonsecreted forms of herpes simplex virus glycoprotein D2 induce different types of immune responses

Herpes simplex viruses (HSVs) are significant pathogens and major targets of vaccine development. Several attempts have been made to develop prophylactic and therapeutic vaccines for HSV types 1 and 2. Although these vaccines elicit strong humoral responses, the overall impact on pathology has been disappointing. An effective vaccine for HSV must induce both humoral and cellular immune responses. DNA vaccines are ideal candidates for HSV vaccines because they induce both types of immune responses. This study showed that the type of immune response generated by immunization with DNA vaccines is modulated by expression of various forms of an antigen, each with a different cellular localization. Expression of cell-associated forms of HSV-2 glycoprotein D (gD) induces primarily a Th1 response, whereas expression of secreted gD results in a Th2 response. Immunization with plasmids expressing different forms of the antigen may increase the efficacy of a vaccine.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Herpes Simplex Virus DNA Vaccine Efficacy: Effect of Glycoprotein D Plasmid Constructs

The impact of vaccination with plasmid DNA encoding full-length glycoprotein D (gD) from herpes simplex virus (HSV) type 2 (gD2), secreted gD2, or cytosolic gD2 was evaluated in mice and guinea pigs. Immunization with plasmids encoding full-length gD2 or secreted gD2 produced high antibody levels, whereas immunization with DNA encoding cytosolic gD2 resulted in significantly lower antibody titers in both species (P<.001). Vaccination with DNA encoding full-length or secreted gD2 significantly reduced acute disease in mice and guinea pigs (both P<.001) and subsequent recurrent disease in guinea pigs (P<.05). In guinea pigs, immunization with DNA encoding cytosolic gD2 did not protect from acute or recurrent disease, whereas in mice it did protect, but not as well as DNA encoding full-length or secreted gD2. None of the vaccines resulted in improved virus clearance from the inoculation site, and none significantly reduced recurrent disease when used as a therapeutic vaccine in HSV-2-infected guinea pigs.