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.

Antiviral therapy prolongs suppression of viral replication and allows for significant immune reconstitution but has not been effective in eradicating reservoirs of virus, which produce resurgent viremia when highly active antiretroviral therapy (HAART) is discontinued. Immune-based therapy may provide an additional antiviral effect. We vaccinated stable HIV-positive patients on HAART with an HIV plasmid vaccine to determine safety, immunogenicity, and therapeutic potential. Volunteers received a combination of two HIV DNA plasmid constructs, which drive expression of env/rev and gag/pol genes. The vaccine was well tolerated with no toxicity. CD4 and CD8 lymphocyte counts did not change significantly among volunteers. CD8 MHC class I-restricted responses to HIV antigens were assayed. Eight of 13 vaccinees responded after vaccination with detectable ELISpot result. Importantly, we observed a difference in viral detection events in vaccinated compared to control patients. Three out of the five placebo recipients had "viral blips" (transient elevations of HIV RNA) during follow-up (10/49 assays) while these were only present in one of 13 vaccinees on one occasion (1/130 assays; p<0.04). The decrease in the frequency of transient viremia and failure suggests that DNA immunization with CD8-generating vaccines in HAART-controlled HIV-positive subjects may have therapeutic potential.

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.

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.

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.

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.

A DNA vaccine encoding glycoprotein D (gD) of herpes simplex virus type 2 (pHSV-gD2) was injected via parenteral and mucosal routes to determine the optimal route of delivery for immune stimulation. Generation of distal mucosal immunity following parenteral vaccination was also evaluated. While all routes of DNA vaccine administration resulted in systemic cellular and humoral responses, the intra-muscular (i.m.) and intra-dermal (i.d.) routes of delivery produced the highest responses. Furthermore, i.m. and i.d. routes produced mucosal humoral responses that were comparable to those obtained via mucosal routes. Specific pHSV-gD2 PCR signals were detected in the Peyer's patches (PP) within hours following vaccination and antigen specific IgA was detected in secretions and supernatants from gut fragment cultures. Furthermore, antigen specific CD4(+) cells were found in PP. Collectively these results suggest that the DNA vaccine stimulated a response in the PP, a major inductive site for mucosal responses.

The immunogenicity and efficacy of a herpes simplex virus type 2 glycoprotein D (gD2) DNA vaccine formulated with bupivacaine was evaluated using murine and guinea pig models of genital herpes. Animals received three doses of 100 microg of gD2 plasmid or control plasmid intramuscularly prior to intravaginal challenge with HSV-2. Immunization induced HSV ELISA and neutralizing antibody in serum and ELISA antibody in the vaginal secretions of all animals evaluated. Following intravaginal HSV-2 challenge, vaginal viral replication was reduced in both models with peak reductions of greater than 99%. Immunization also decreased the number of animals developing any clinical disease (p < 0.001) and the severity of the acute disease (total lesion score 6.4 versus 0.6 in guinea pigs, p < 0.001). Further recurrent lesion days were reduced from 14.5 to 4.9 days in immunized guinea pigs (p < 0.001). DNA immunization with gD2 + bupivacaine was effective in reducing clinical disease and viral replication in both guinea pigs and mice.

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.

A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts ≥500/μl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 μg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-μg dose group also developed increased MIP-1α levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 μg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4(+)-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.

The advent of new technology and the unmet needs of old and new epidemics of infectious diseases have stimulated a new era of vaccinology. One of the most novel approaches employs plasmid DNA engineered to express one or more genes of the pathogen in mammalian cells. Plasmids may also express cytokine or costimulatory molecules to 'direct' the immune response and/or express altered forms of the antigen to direct it to a specific intracellular compartment or a specific extracellular receptor. The quality of immune responses generated by DNA vaccines in animals has previously only been equaled by live attenuated viral vaccines. The immune stimulating activity of DNA vaccines, combined with their versatility, suggests vast potential for these vaccines.

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.

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-gamma) 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.

Using fyn PTK as a template, a series of phosphopeptides 1–11 spanning in length from 1–14 amino acids was prepared. Kinetic evaluation of 1–11 suggest that CD45 does not have a strong preference for its N- or C-terminal amino acids and that extended phosphopeptides are not required for efficient substrate turnover.

Using a high throughput colorimetric bioassay three aporphine alkaloids, annonaine, nornuciferine and roemerine, were isolated and identified as the CD45 protein tyrosine phosphatase inhibitor metabolites of Rollinia ulei. The nor-aporphine alkaloids, anonaine (1) and nornuciferine (3), exhibited more potent inhibitory activity than that of the N-methylated alkaloid roemerin (2).