Mark L. Bagarazzi

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.

Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial

BACKGROUND Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. METHODS Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). FINDINGS Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49·5%) of 107 VGX-3100 recipients and 11 (30·6%) of 36 placebo recipients had histopathological regression (percentage point difference 19·0 [95% CI 1·4-36·6]; p=0·034). In the modified intention-to-treat analysis 55 (48·2%) of 114 VGX-3100 recipients and 12 (30·0%) of 40 placebo recipients had histopathological regression (percentage point difference 18·2 [95% CI 1·3-34·4]; p=0·034). Injection-site reactions occurred in most patients, but only erythema was significantly more common in the VGX-3100 group (98/125, 78·4%) than in the placebo group (24/42, 57·1%; percentage point difference 21·3 [95% CI 5·3-37·8]; p=0·007). INTERPRETATION VGX-3100 is the first therapeutic vaccine to show efficacy against CIN2/3 associated with HPV-16 and HPV-18. VGX-3100 could present a non-surgical therapeutic option for CIN2/3, changing the treatment outlook for this common disease. FUNDING Inovio Pharmaceuticals.

Immunotherapy Against HPV16/18 Generates Potent TH1 and Cytotoxic Cellular Immune Responses

CD8+ T cells with cytolytic activity are induced after therapeutic human papillomavirus vaccination in humans. Shocking HPV into Submission Human papillomavirus (HPV) infection is frequently asymptomatic but can lead to the development of cervical cancer in infected women. Current vaccines against HPV are quite effective at preventing infection; however, there is no vaccine to help those already infected. Now, Bagarazzi et al. report that a therapeutic vaccine for HPV can induce an immune response in a phase 1 study. VGX-3100 is a candidate vaccine for the high-risk HPV serotypes 16 and 18. Here, 18 women previously treated for cervical neoplasia—a precursor to cervical cancer—were given the DNA vaccine VGX-3100 by electroporation—where a small localized electric pulse accompanies the injection—in a range of doses. Previous attempts at DNA vaccination have proved less than successful in clinical trials; however, preclinical studies suggest that electroporation may greatly enhance the efficacy of the vaccine. The authors show that the electroporation-delivered VGX-3100 induces a robust HPV-specific immune response in previously infected individuals and that the vaccine is safe and immunogenic. Although efficacy remains to be tested in a larger trial, the enhanced immune response elicited by VGX-3100 may attack HPV-infected cells, potentially inducing cancer regression in individuals already infected with HPV. Despite the development of highly effective prophylactic vaccines against human papillomavirus (HPV) serotypes 16 and 18, prevention of cervical dysplasia and cancer in women infected with high-risk HPV serotypes remains an unmet medical need. We report encouraging phase 1 safety, tolerability, and immunogenicity results for a therapeutic HPV16/18 candidate vaccine, VGX-3100, delivered by in vivo electroporation (EP). Eighteen women previously treated for cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) received a three-dose (intramuscular) regimen of highly engineered plasmid DNA encoding HPV16 and HPV18 E6/E7 antigens followed by EP in a dose escalation study (0.3, 1, and 3 mg per plasmid). Immunization was well tolerated with reports of mild injection site reactions and no study-related serious or grade 3 and 4 adverse events. No dose-limiting toxicity was noted, and pain was assessed by visual analog scale, with average scores decreasing from 6.2/10 to 1.4 within 10 min. Average peak interferon-γ enzyme-linked immunospot magnitudes were highest in the 3 mg cohort in comparison to the 0.3 and 1 mg cohorts, suggesting a trend toward a dose effect. Flow cytometric analysis revealed the induction of HPV-specific CD8+ T cells that efficiently loaded granzyme B and perforin and exhibited full cytolytic functionality in all cohorts. These data indicate that VGX-3100 is capable of driving robust immune responses to antigens from high-risk HPV serotypes and could contribute to elimination of HPV-infected cells and subsequent regression of the dysplastic process.

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.

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.

Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes

DNA immunization has been investigated as a potential immunization strategy against infectious diseases and cancer. To enhance a DNA vaccines ability to induce CTL response in vivo, we co-administered CD80 and CD86 expression cassettes along with HIV-1 immunogens. This manipulation resulted in a dramatic increase in MHC class I-restricted and CD8+ T-cell-dependent CTL responses in both mice and chimpanzees. This strategy of engineering vaccine producing cells to be more efficient T-cell activators could be an important tool for optimizing antigen-specific T-cell-mediated immune responses in the pursuit of more rationally designed vaccines and immune therapies.

Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids

ObjectiveTo assess HIV-1 DNA vaccination and co-immunization with interleukin (IL)-12 and IL-10 as immunotherapy in the HIV-1 infected chimpanzee model system. MethodsFour chimpanzees that were infected with HIV-1-IIIB for longer than 4 years and remained symptom free were immunized with HIV-1 plasmid vaccines. Two chimpanzees were immunized with DNA plasmids that encoded env/ rev, gag/ pol along with a plasmid that encoded both chains of human IL-12. A third animal was immunized with HIV-1 DNA vaccine constructs and co-immunized with an IL-10 expressing plasmid. Finally a control animal received the HIV-1 DNA vaccine constructs alone. ResultsThere was no evidence of systemic toxicity associated with the administration of the DNA vaccines or the cytokine-expressing plasmids. We observed that the IL-12/HIV-1 DNA vaccinated animals had enhanced proliferative responses to multiple HIV-1 antigens at multiple time points. The animal that was co-immunized with HIV-1 and IL-10 did not have any changes in the proliferative responses. Finally, the control chimpanzee demonstrated moderate increases in the proliferative responses to HIV-1 antigens. The animal that received HIV-1 vaccines alone and the animals co-immunized with IL-12 all had declines in viral load over the course of the study, however, the decrease in viral loads were transient in all animals. ConclusionImmunization of HIV-1 infected chimpanzees with DNA based vaccines containing the env, gag and pol genes can transiently boost the env specific proliferative responses. Co-administration of IL-12 expressing plasmids further leads to transient boosting of the proliferative response to the core protein, p24 as well. However, at these doses the impact on viral load is minimal.

DNA Priming-Protein Boosting Enhances Both Antigen-Specific Antibody and Th1-Type Cellular Immune Responses in a Murine Herpes Simplex Virus-2 gD Vaccine Model

It has previously been reported that herpes simplex virus (HSV)-2 gD DNA vaccine preferentially induces T-helper (Th) 1-type cellular immune responses, whereas the literature supports the view that subunit vaccines tend to induce potent antibody responses, supporting a Th2 bias. Here, using an HSV gD vaccine model, we investigated whether priming and boosting with a DNA or protein vaccine could induce both potent antibody and Th1-type cellular immune responses. When animals were primed with DNA and boosted with protein, both antibody and Th-cell proliferative responses were significantly enhanced. Furthermore, production of Th1-type cytokines (interleukin-2, interferon-gamma) was enhanced by DNA priming-protein boosting. In contrast, protein priming-DNA boosting produced antibody levels similar to those following protein-protein vaccination but failed to further enhance Th-cell proliferative responses or cytokine production. DNA priming-protein boosting resulted in an increased IgG2a isotype (a Th1 indicator) profile, similar to that induced by DNA-DNA vaccination, whereas protein priming-DNA boosting caused an increased IgG1 isotype (a Th2 indicator) profile similar to that seen after protein-protein vaccination. This result indicates that preferential induction of IgG1 or IgG2a isotype is determined by the type of priming vaccine used. Thus, this study suggests that HSV DNA priming-protein boosting could elicit both potent Th1-type cellular immune responses and antibody responses, both of which likely are important for protection against HSV infection.

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.

Development of a multicomponent candidate vaccine for HIV-1.

Nucleic acid or DNA immunization represents a novel approach to both vaccine and immune therapeutic development. DNA vaccination induces antigen-specific cellular and humoral immune responses through the delivery of non-replicating transcription units which drive the synthesis of specific foreign proteins within the inoculated host. We have previously reported on the potential use of DNA immunization as a novel vaccine strategy for HIV-1. We found that both antigen-specific cellular and humoral immune responses could be induced in vivo with various DNA vaccine constructs against different antigenic targets within HIV-1. In order to enhance the DNA vaccines ability to elicit cell-mediated immune responses, we co-delivered plasmids encoding costimulatory molecule B7 and interleukin-12 genes with DNA vaccine for HIV-1. We observed a dramatic increase in both antigen-specific T helper cell proliferation and CTL response. Eventual development of successful vaccines for HIV-1 would likely involve targeting multiple antigenic components of the virus to direct and empower the immune system to protect the host from viral infection. We present here the utility of multicomponent DNA immunization to elicit specific humoral and cell-mediated immune responses against different antigenic targets of HIV-1 as well as the ability of this immunization strategy to achieve significant enhancements of antigen-specific cellular immune responses.