Richard B. Ciccarelli

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.

In vivo protective anti‐HIV immune responses in non‐human primates through DNA immunization

Abstract: An effective immune response involves the specific recognition of and elimination of an infectious organism at multiple levels. In this context DNA immunization can present functional antigenic proteins to the host for recognition by all arms of the immune system, yet provides the opportunity to delete any genes of the infectious organism which code for antigens or pieces of antigens that may have deleterious effects. Our group has developed the use of nucleic acid immunization as a possible method of vaccination against Human immunodeficiency virus type 1 (HIV‐1) [1,2,3,10,11,12]. Sera from non‐human primates immunized with DNA vectors that express the envelope proteins from HIV‐1 contain antibodies specific to the HIV‐1 envelope. These sera also neutralize HIV‐1 infection in vitro and inhibit cell to cell infection in tissue culture. Analysis of cellular responses is equally encouraging. T cell proliferation as well as cytotoxic T cell lysis of relevant env expressing target cells were observed. In addition, evidence that DNA vaccines are capable of inducing a protective response against live virus was demonstrated using a chimeric SIV/HIV (SHIV) challenge in vaccinated cynomologous macaques. We found that nucleic acid vaccination induced protection from challenge in one out of four immunized cynomolgus macaques and viral load was lower in the vaccinated group of animals versus the control group of animals. These data encouraged us to analyze this vaccination technique in chimpanzees, the most closely related animal species to man. We observed the induction of both cellular and humoral immune responses with a DNA vaccine in chimpanzees. These studies demonstrate the utility of this technology to induce relevant immune responses in primates which may ultimately lead to effective vaccines.

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 90u2009%, an improvement as compared to gD vaccination alone (60u2009–u200963u2009%). 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.

Safety and immune responses to a DNA-based human immunodeficiency virus (HIV) type I env/rev vaccine in HIV-infected recipients: follow-up data.

To the Editor—We recently reported the safety and immunologic response to a DNA vaccine containing env/rev in a group of untreated healthy human immunodeficiency virus (HIV)– infected subjects with CD4 lymphocyte counts 1500 cells/mL [1]. No significant adverse experiences occurred, with some evidence for antibody responses and occasional cell-mediated immune responses. We now report an additional cohort immunized with a 300-mg dose by jet injection (JI) or by needle (N), follow-up safety data over 12.5 years for the whole cohort, and results of boosting with 1 mg of vaccine. The safety profile for the vaccine continues to be excellent. No participant experienced a significant adverse response requiring interruption or withdrawal from study. Anti-DNA antibody was detected in 1 subject with a borderline antinuclear antibody level before vaccination, and 2 others had transient chemistry abnormalities immediately before boosting, with resolution 2 weeks later. Local mild-to-moderate reactions were noted with 9 of 17 JI injections, compared with 4 of 54 with N injections. We compared the immune responses to primary vaccine series (300 mg 3 3) administered by JI versus N, measured at entry, week 21 (1 week after 3d dose), and week 36 (4 months after 3d dose). Responses were defined as in our original paper [1]. No subject in either 300-mg group had a >20% increase or decrease in CD4 lymphocyte count, compared with the value at entry. No subject had a >0.5 log change in viral titer in plasma, compared with the value at entry. For lymphoproliferative response, we defined response as stimulation index (SI) >4 above baseline value. Four subjects in the JI group were studied: 1/4 responded to gp120 and rev antigens; 1/4 responded only to rev. Three patients in the N group were studied: 3/3 responded to gp120; none responded to rev. We defined an antibody response as a geometric mean titer >23 the baseline value. After JI, 0/5 responded, whereas 3/5 immunized by N responded. For immune responses to 1 mg boosting, we compared values at time of boost (>6 months after primary immunization series) with those 2 and 4 weeks later. Five of the 12 subjects available for boosting had begun antiretroviral therapy. No subject had a >20% increase or decrease in CD4 lymphocyte count, compared with the value at boost. For HIV quantitation in plasma, no subject had a >0.5 log change in viral titer, compared with titer at boost. When stimulated with gp160, none of 12 subjects showed a rise in SI (range, 0.8–5.1 before boost and 0.8–4.5 after boost); SIs increased in 3 of 12 subjects in response to rev (range, 0.5–5 before and 0.7–6.1 after). The responders were not receiving antiretroviral therapy. Three of 10 assessable subjects had a >2-fold geometric mean titer rise, compared with levels at boost; none had been receiving antiretroviral therapy. There were no clear differences in CD4 cell count, viral load, or immunologic responses between patients immunized by JI and those immunized by N, although more lymphoproliferative activity (LPA) and antibody increases were seen in the N-injected cohort. Boosting of all cohorts (30 mg-, 100 mg-, and both 300-mg groups) with 1 mg of the construct resulted in occasional LPA and antibody responses (without regard to whether they had initiated antiretroviral therapy) and no significant changes in CD4 cell counts or viral loads. Adverse experiences were minor, although more local tenderness was seen after JI. No subject withdrew because of toxicity. Safety monitoring for 12.5 years did not reveal any adverse events that were interpreted as related to the vaccine. The study began in 1994, before the dynamic daily turnover in CD4 lymphocyte counts and viral load was recognized [2, 3]. It is now theorized that viral replication and CD4 turnover must be suppressed to elicit a potentially protective immune response to HIV vaccine [4, 5]. We are currently conducting such a trial, using env/revand gag/pol-containing constructs in patients whose viral production is maximally suppressed with highly active antiretroviral therapy [6].

HIV-1 DNA vaccines and chemokines

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. The technology, although it dates back to the 1950s, has had an insurgence of interest within the past few years following concurrent research papers. The basic technology is being applied broadly to viral, bacterial and parasitic infections. It has also been demonstrated that genes delivered via plasmid expression vectors result in expression of functional proteins in the inoculated host. Further, injection of plasmids encoding cytokine, chemokine or co-stimulatory molecules, also referred to as immunomodulatory plasmids can lead to the further expansion of this technology to include directed immunology. We have been developing DNA technology specifically with a focus as a vaccine against HIV-1 infection. We report that such vaccines can stimulate immune responses in a variety of relevant animal systems including humoral and cellular responses as well as the production of beta-chemokines. We describe that the beta-chemokines can both modulate the immune response induced by DNA vaccines and be modulated by the DNA vaccines in the murine and chimpanzee models as well as in humans.

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.

A Gag-Pol/Env-Rev SIV239 DNA vaccine improves CD4 counts, and reduce viral loads after pathogenic intrarectal SIVmac251 challenge in Rhesus Macaques

DNA vaccines are an important vaccine approach for many infectious diseases including human immunodeficiency virus (HIV). Recently, there have been exciting results reported for plasmid vaccination in pathogenic SHIV model systems. In these studies, plasmid vaccines supplemented by IL-2 Ig cytokine gene adjuvants or boosted by recombinant MVA vectors expressing relevant SIV and HIV antigens prevented CD4(+) T-cell loss and lowered viral loads following pathogenic challenge. However, similar results have not been reported in a direct pathogenic macaque challenge model. Here we report on a study of the ability of a multiplasmid SIV DNA vaccine in a pathogenic SIV251 rhesus mucosal challenge study. We observed that pGag/Pol+pEnv/Rev plasmid vaccines could not prevent SIV infection; however, vaccinated animals exhibited significant improvement in control of viral challenge compared to control animals. Furthermore, vaccinated animals exhibited protection against CD4(+) T-cell loss.

Systemic and Mucosal Immunity Is Elicited after Both Intramuscular and Intravaginal Delivery of Human Immunodeficiency Virus Type 1 DNA Plasmid Vaccines to Pregnant Chimpanzees

DNA vaccines encoding human immunodeficiency virus type 1 (HIV-1) env/rev and gag/pol were delivered intravaginally (IVAG) and intramuscularly (IM) to 2 pregnant chimpanzees. Vaccination was well tolerated and each chimpanzee developed antibodies (up to 1 year later) to both vaccines. Placental transfer of anti-Env and anti-Gag IgG was demonstrated in both maternal/infant pairs. Specific IgG was also demonstrated in saliva, vaginal, and rectal washes after IVAG immunization. Predominantly anti-HIV-1 IgA was detected in the milk of both mothers after both IM and IVAG immunization. Cellular responses included Gag-specific proliferation of lymphocytes and cytotoxic T lymphocytes against both antigens. These data suggest a strategy for induction of mucosal and systemic responses after both IM and IVAG delivery of DNA vaccines in a primate model and could ultimately be useful in lowering maternal-to-fetal transmission of HIV-1, perinatally and through breastfeeding.

Nucleic acid immunization of chimpanzees as a prophylactic/immunotherapeutic vaccination model for HIV-1: prelude to a clinical trial.

Vaccine development strategies have often utilized recombinant envelope glycoproteins which usually generate strong humoral immune responses but which do not generate strong cytotoxic T lymphocytes (CTL). A recent novel experimental vaccination approach involves the technology known as nucleic acid immunization in which DNA plasmids expressing a gene of interest is injected intramuscularly in experimental animals. These expressed proteins then are presented to the immune system with the subsequent development of strong antibody and cellular (particularly CTL) immune responses. These types of immune responses have been elicited in rodents as well as nonhuman primates including chimpanzees. Results from studies on nucleic acid immunization of HIV-1 infected chimpanzees with envelope glycoprotein expressing constructs indicated that this method was able to decrease substantially HIV-1 viral load in these chimpanzees. These data are useful for the development and implementation of human phase I clinical trials with HIV constructs expressing various genes from the HIV-1 genome.