A randomized controlled trial of treatment with intermittent negative pressure for intermittent claudication.

OBJECTIVE: We investigated the effects of lower extremity intermittent negative pressure (INP) treatment for 1 hour two times daily for 12 weeks on the walking distance of patients with intermittent claudication (IC). METHODS: Patients with IC were randomized to treatment with -40 mm Hg INP (treatment group) or -10 mm Hg INP (sham control group). Pain-free walking distance (PWD) and maximal walking distance (MWD) on a treadmill, resting and postexercise ankle-brachial index, resting and postischemic blood flow (plethysmography), and quality of life (EQ-5D-5L and Vascuqol-6) were measured at baseline and after 12 weeks of treatment. RESULTS: A total of 72 patients were randomized, and 63 had data available for the intention-to-treat analyses. The between-group comparisons showed a significant change in the PWD, favoring the treatment group over the sham control group (estimated treatment effect, 50 m; 95% confidence interval [CI], 11-89; P = .014). The PWD had increased by 68 m (P < .001) in the treatment group and 18 m (P = .064) in the sham control group. No significant difference was found in the change in the MWD between the two groups (estimated treatment effect, 42 m; 95% CI, -14 to 97; P = .139). The MWD had increased by 62 m (P = .006) in the treatment group and 20 m (P = .265) in the sham control group. For patients with a baseline PWD of <200 m (n = 56), significant changes had occurred in both PWD and MWD between the two groups, favoring the treatment group (estimated treatment effect, 42 m; 95% CI, 2-83; P = .042; and estimated treatment effect, 62 m; 95% CI, 5-118; P = .032; respectively). Both overall and for the group of patients with a PWD <200 m, no significant differences were found in the changes in the resting and postexercise ankle-brachial index, resting and postischemic blood flow, or quality of life parameters between the two groups. CONCLUSIONS: Treatment with -40 mm Hg INP increased the PWD compared with sham treatment in patients with IC. For the patients with a baseline PWD of <200 m, an increase was found in both PWD and MWD compared with sham treatment.

Effects of intermittent negative pressure treatment on circulating vascular biomarkers in patients with intermittent claudication.

The aim of this study was to investigate the effects of lower extremity intermittent negative pressure (INP) treatment for 1 hour twice daily for 12 weeks, on circulating vascular biomarkers in patients with intermittent claudication. Patients were randomized to treatment with -40 mmHg INP (treatment group), or -10 mmHg INP (sham control group). Venous blood samples were collected at baseline and after 12 weeks, and concentrations of vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, von Willebrand factor (vWF), l-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) were analyzed. A larger proportion of the patients in the treatment group (25/31) had a reduction in vWF levels after 12 weeks, compared to the sham control group (17/30) (p = 0.043). Within the treatment group there was a significant mean (SEM) reduction in the concentration of vWF of -11% (4) (p = 0.019), whereas there was no significant change in the levels of vWF in the sham control group (1% (6); p = 0.85). There were no significant differences in the change of any of the biomarker levels between the groups after 12 weeks of treatment. In conclusion, there were no differences in the change of the circulating levels of the measured biomarkers between the treatment group and the sham control group after 12 weeks of INP treatment. However, the observed changes in vWF might indicate a beneficial effect of INP treatment on endothelial activation and endothelial injury. Clinicaltrials.gov Identifier: NCT03640676.

Lower Extremity Intermittent Negative Pressure for Intermittent Claudication. Follow-Up after 24 Weeks of Treatment.

BACKGROUND: Treatment with lower extremity intermittent negative pressure (INP) of -40 mm Hg for one hour twice daily for 12 weeks, increases walking capacity in patients with intermittent claudication (IC). However, the effects of INP treatment beyond 12 weeks have not been elucidated. The aim of the present study was to investigate the clinical effects of INP treatment after 24 weeks in patients with IC. METHODS: This was a follow-up study after a randomized sham-controlled trial, where patients randomized to the active treatment group were offered to continue treatment for 12 additional weeks (24 weeks in total). Treatment with -40 mm Hg INP was applied in a pressure chamber sealed around the lower leg, and the patients were instructed to treat themselves at home one hour in the morning and one hour in the evening. Pain free walking distance (PWD), maximal walking distance (MWD), resting ankle-brachial index (ABI) and post exercise ABI were measured at baseline, after 12 and 24 weeks. RESULTS: Ten out of 32 patients (31%) from the active treatment group in the initial trial were included in this follow-up study. At baseline, PWD was (mean ±SD) 151 ± 91 m and MWD was 362 ±159 m. There was a significant increase in both PWD and MWD after 24 weeks of treatment, compared to baseline (ANOVA; P= 0.006 and P= 0.012, respectively). Post hoc tests revealed that PWD increased significantly from baseline to 12 weeks (mean 81 m; 95% CI [6, 156]; P = 0.032), and that MWD increased significantly from 12 to 24 weeks (mean 145 m; 95% CI [22, 268]; P = 0.018). There were no significant changes in resting ABI or post exercise ABI during the 24-week treatment period (ANOVA; P= 0.157 and P= 0.450, respectively). CONCLUSION: Both PWD and MWD improved after treatment with - 40 mm Hg INP for one hour twice daily for 24 weeks, compared to baseline. The main improvement in PWD occurred during the first 12 weeks of treatment, whereas the main improvement in MWD occurred between 12 and 24 weeks of treatment.

Intermittent mild negative pressure applied to the lower limb in patients with spinal cord injury and chronic lower limb ulcers: a crossover pilot study.

STUDY DESIGN: Randomized, assessor-blinded crossover pilot study. OBJECTIVES: To explore the use of an intermittent negative pressure (INP) device for home use in addition to standard wound care (SWC) for patients with spinal cord injury (SCI) and chronic leg and foot ulcers before conducting a superiority trial. SETTING: Patient homes and outpatient clinic. METHODS: A 16-week crossover trial on 9 SCI patients (median age: 57 years, interquartile range [IQR] 52-66), with leg ulcers for 52 of weeks (IQR: 12-82) duration. At baseline, patients were allocated to treatment with INP + SWC or SWC alone. After 8 weeks, the ulcers were evaluated. To assess protocol adherence, the patients were then crossed over to the other group and were evaluated again after another 8 weeks. Lower limb INP treatment consisted of an airtight pressure chamber connected to an INP generator (alternating 10 s -40mmHg/7 s atmospheric pressure) used 2 h/day at home. Ulcer healing was assessed using a photographic wound assessment tool (PWAT) and by measuring changes in wound surface area (WSA). RESULTS: Seven of nine recruited patients adhered to a median of 90% (IQR: 80-96) of the prescribed 8-week INP-protocol, and completed the study without side effects. PWAT improvement was observed in 4/4 patients for INP + SWC vs. 2/5 patients for SWC alone (P = 0.13). WSA improved in 3/4 patients allocated to INP + SWC vs. 3/5 patients in SWC alone (P = 0.72). CONCLUSIONS: INP can be used as a home-based treatment for patients with SCI, and its efficacy should be tested in an adequately sized, preferably multicenter randomized trial.

Intermittent negative pressure applied to the lower limb increases foot macrocirculatory and microcirculatory blood flow pulsatility in people with spinal cord injury.

STUDY DESIGN: Experimental prestudy and poststudy. OBJECTIVES: Examine the acute effects of intermittent negative pressure (INP) applied to the lower limb on foot circulation in people with spinal cord injuries (SCIs). SETTING: Vascular laboratory, Oslo University Hospital. METHODS: Twenty-four people with SCI (median age 59 years, range 29-74) were exposed to lower leg INP (-40 mm Hg) using an air-tight pressure chamber connected to an INP generator. The contralateral leg was placed outside the pressure chamber. We continuously measured arterial blood flow velocity (ultrasound Doppler), skin blood flow (laser Doppler), skin temperature of the dorsum of the foot, heart rate (ECG) and systemic blood pressure (Finometer) during 5-min baseline (atmospheric pressure), followed by 10-min INP (alternating 10 s -40 mm Hg and 7 s atmospheric pressure), and 5-min post-INP (atmospheric pressure). Skin blood flow was measured on the foot placed outside the pressure chamber. A mixed effects regression model was applied to estimate the effect of INP on blood flow. To quantify flow fluctuations, we calculated cumulative up-and-down changes in arterial blood flow velocity per minute. RESULTS: Flow fluctuations increased during INP compared to baseline [32.3 cm/s/min (95% CI 26.9 to 37.7) vs. 15.2 cm/s/min (95% CI 9.8 to 20.6), P < 0.001]. Peak blood flow velocity and skin blood flow was reached 2-3 s after the onset of negative pressure and increased 33% (95% CI 16 to 46, P < 0.001) and 11% (95% CI -4.1 to 60, P = 0.14) above baseline, respectively. CONCLUSIONS: INP induced increased foot arterial blood flow fluctuations compared to baseline. SPONSORSHIPS: The Norwegian Research Council provided funding to Otivio (grant: 241589).

Therapeutic DNA vaccination using in vivo electroporation followed by standard of care therapy in patients with genotype 1 chronic hepatitis C.

Clearance of infections caused by the hepatitis C virus (HCV) correlates with HCV-specific T cell function. We therefore evaluated therapeutic vaccination in 12 patients with chronic HCV infection. Eight patients also underwent a subsequent standard-of-care (SOC) therapy with pegylated interferon (IFN) and ribavirin. The phase I/IIa clinical trial was performed in treatment naive HCV genotype 1 patients, receiving four monthly vaccinations in the deltoid muscles with 167, 500, or 1,500 µg codon-optimized HCV nonstructural (NS) 3/4A-expressing DNA vaccine delivered by in vivo electroporation (EP). Enrollment was done with 2 weeks interval between patients for safety reasons. Treatment was safe and well tolerated. The vaccinations significantly improved IFN-γ-producing responses to HCV NS3 during the first 6 weeks of therapy. Five patients experienced 2-10 weeks 0.6-2.4 log10 reduction in serum HCV RNA. Six out of eight patients starting SOC therapy within 1-30 months after the last vaccine dose were cured. This first-in-man therapeutic HCV DNA vaccine study with the vaccine delivered by in vivo EP shows transient effects in patients with chronic HCV genotype 1 infection. The interesting result noted after SOC therapy suggests that therapeutic vaccination can be explored in a combination with SOC treatment.

DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time.

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA(27-35). We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2(+) patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2(-) control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4(+) and PSMA(27)-specific CD8(+) T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4(+) T-cell response and PSMA(27)-specific CD8(+) T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4(+) and CD8(+) vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA(27) vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.

Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation.

In vivo electroporation (EP) has been shown to augment the immunogenicity of plasmid DNA vaccines, but its mechanism of action has not been fully characterized. In this study, we show that in vivo EP augmented cellular and humoral immune responses to a human immunodeficiency virus type 1 Env DNA vaccine in mice and allowed a 10-fold reduction in vaccine dose. This enhancement was durable for over 6 months, and re-exposure to antigen resulted in anamnestic effector and central memory CD8(+) T-lymphocyte responses. Interestingly, in vivo EP also recruited large mixed cellular inflammatory infiltrates to the site of inoculation. These infiltrates contained 45-fold-increased numbers of macrophages and 77-fold-increased numbers of dendritic cells as well as 2- to 6-fold-increased numbers of B and T lymphocytes compared to infiltrates following DNA vaccination alone. These data suggest that recruiting inflammatory cells, including antigen-presenting cells (APCs), to the site of antigen production substantially improves the immunogenicity of DNA vaccines. Combining in vivo EP with plasmid chemokine adjuvants that similarly recruited APCs to the injection site, however, did not result in synergy.

The acute effects of different levels of intermittent negative pressure on peripheral circulation in patients with peripheral artery disease.

Intermittent negative pressure (INP) applied to the lower leg induces acute increase in arterial and skin blood flow. The aim of this study was to identify the optimal level of INP to increase blood flow in patients with lower extremity peripheral artery disease (PAD). We investigated the acute effects of different levels of INP in 16 subjects (7 women and 9 men, mean (SD) age 71(8) years) diagnosed with PAD. During application of INP in a pressure chamber sealed below the knee, arterial blood flow was continuously recorded in the dorsalis pedis artery or tibialis posterior artery (ultrasound Doppler), and skin blood flow was continuously recorded at the pulp of the first toe (laser Doppler). Different pressure levels (0, -10, -20, -40, and -60 mmHg) were tested in randomized order. Maximal arterial blood flow relative to baseline (median [25th, 75th percentiles]) was: 0 mmHg; 1.08 (1.02, 1.13), -10 mmHg; 1.11 (1.07, 1.17), -20 mmHg; 1.18 (1.11, 1.32), -40 mmHg; 1.39 (1.27, 1.91) and -60 mmHg; 1.48 (1.37, 1.78). Maximal laser Doppler flux (LDF) relative to baseline was: 0 mmHg; 1.06 (1.02, 1.12), -10 mmHg; 1.08 (1.05, 1.16) -20 mmHg; 1.12 (1.06, 1.27), -40 mmHg; 1.24 (1.14, 1.50) and -60 mmHg; 1.35 (1.10, 1.70). There were significantly higher maximal arterial blood flow and maximal LDF at -40 mmHg compared with -10 mmHg (P = 0.001 and P = 0.025, respectively). There were no significant differences in maximal arterial blood flow and maximal LDF between 0 and -10 mmHg (both P = 1.0), or between -40 and -60 mmHg (both P = 1.0). INP of -40 mmHg was the lowest negative pressure level that increased blood flow.

Taking electroporation-based delivery of DNA vaccination into humans: a generic clinical protocol.

We are presently aware of two early-phase DNA vaccine clinical trials in humans using electroporation-enhanced vaccine delivery. Moreover, two phase I immunogenetherapy studies are in progress and several tolerability studies have been performed on healthy volunteers. We have used knowledge from these studies to compose a template for clinical protocols involving electroporation-mediated gene delivery. In this template the emphasis will be on aspects related to electroporation. In addition, we will discuss general topics concerning electroporation-augmented DNA vaccination in human subjects.

The acute effects of lower limb intermittent negative pressure on foot macro- and microcirculation in patients with peripheral arterial disease.

BACKGROUND: Intermittent negative pressure (INP) applied to the lower leg and foot increases foot perfusion in healthy volunteers. The aim of the present study was to describe the effects of INP to the lower leg and foot on foot macro- and microcirculation in patients with lower extremity peripheral arterial disease (PAD). METHODS: In this experimental study, we analyzed foot circulation during INP in 20 patients [median (range): 75 (63-84yrs)] with PAD. One leg was placed inside an air-tight vacuum chamber connected to an INP-generator. During application of INP (alternating 10s of -40mmHg/7s of atmospheric pressure), we continuously recorded blood flow velocity in a distal foot artery (ultrasound Doppler), skin blood flow on the pulp of the first toes (laser Doppler), heart rate (ECG), and systemic blood pressure (Finometer). After a 5-min baseline sequence (no pressure), a 10-min INP sequence was applied, followed by 5-min post-INP (no pressure). To compare and quantify blood flow fluctuations between sequences, we calculated cumulative up-and-down fluctuations in arterial blood flow velocity per minute. RESULTS: Onset of INP induced an increase in arterial flow velocity and skin blood flow. Peak blood flow velocity was reached 3s after the onset of negative pressure, and increased 46% [(95% CI 36-57), P<0.001] above baseline. Peak skin blood flow was reached 2s after the onset of negative pressure, and increased 89% (95% CI 48-130), P<0.001) above baseline. Cumulative fluctuations per minute were significantly higher during INP-sequences compared to baseline [21 (95% CI 12-30)cm/s/min to 41 (95% CI 32-51)cm/s/min, P<0.001]. Mean INP blood flow velocity increased significantly ~12% above mean baseline blood flow velocity [(6.7 (95% CI 5.2-8.3)cm/s to 7.5 (95% CI 5.9-9.1)cm/s, P = 0.03)]. CONCLUSION: INP increases foot macro- and microcirculatory flow pulsatility in patients with PAD. Additionally, application of INP resulted in increased mean arterial blood flow velocity.

[DNA--the future in vaccine technology?]. / DNA--fremtidens vaksine?

DNA vaccines represent a new and promising technology that uses DNA to encode the antigen(s) of interest, instead of inoculating with attenuated or inactivated microbes or isolated antigens. Antigen is produced within the transfected cells minaicking a real life viral infection. This vaccine modality has been shown to elicit strong cellular immune responses and is promising for treating diseases where traditional vaccine approaches have failed. In spite of promising results in small animal models, DNA vaccines have so far proven less potent in human clinical trials. In this review we provide a general overview on the mechanisms of action for DNA vaccines, discuss potential benefits of traditional vaccine approaches and review current strategies for improving the immunogenicity of DNA vaccines to enable the successful transfer of the technology can be successfully transferred from mice to men.

Application of intermittent negative pressure on the lower extremity and its effect on macro- and microcirculation in the foot of healthy volunteers.

Intermittent negative pressure (INP) applied to the lower leg and foot may increase peripheral circulation. However, it is not clear how different patterns of INP affect macro- and microcirculation in the foot. The aim of this study was therefore to determine the effect of different patterns of negative pressure on foot perfusion in healthy volunteers. We hypothesized that short periods with INP would elicit an increase in foot perfusion compared to no negative pressure. In 23 healthy volunteers, we continuously recorded blood flow velocity in a distal foot artery, skin blood flow, heart rate, and blood pressure during application of different patterns of negative pressure (-40 mmHg) to the lower leg. Each participant had their right leg inside an airtight chamber connected to an INP generator. After a baseline period at atmospheric pressure, we applied four different 120 sec sequences with either constant negative pressure or different INP patterns, in a randomized order. The results showed corresponding fluctuations in blood flow velocity and skin blood flow throughout the INP sequences. Blood flow velocity reached a maximum at 4 sec after the onset of negative pressure (average 44% increase above baseline, P < 0.001). Skin blood flow and skin temperature increased during all INP sequences (P < 0.001). During constant negative pressure, average blood flow velocity, skin blood flow, and skin temperature decreased (P < 0.001). In conclusion, we observed increased foot perfusion in healthy volunteers after the application of INP on the lower limb.

The effects of intermittent negative pressure on the lower extremities' peripheral circulation and wound healing in four patients with lower limb ischemia and hard-to-heal leg ulcers: a case report.

Peripheral circulation is severely compromised in the advanced stages of peripheral arterial disease. Recently, it was shown that the application of -40 mmHg intermittent negative pressure (INP) to the lower leg and foot enhances macro- and microcirculation in healthy volunteers. In this case report, we describe the effects of INP treatment on four patients with lower limb ischemia and hard-to-heal leg and foot ulcers. We hypothesized that INP therapy may have beneficial hemodynamic and clinical effects in the patients. Four patients (age range: 61-79 years) with hard-to-heal leg and foot ulcers (6-24 months) and ankle-brachial pressure indices of ≤0.60 on the affected side were included. They were treated with an 8-week intervention period of -40 mmHg INP (10 sec negative pressure and 7 sec atmospheric pressure) on the lower limbs. A custom-made vacuum chamber was used to apply INP to the affected lower leg and foot for 2 h per day. After 8 weeks of INP therapy, one ulcer healed completely, while the other three ulcers were almost completely healed. These cases suggest that INP may facilitate wound healing. The theoretical foundation is that INP assists wound healing by improving blood flow to the small blood vessels in the affected limb, increasing the flow of oxygen and nutrients to the cells.

Synergistic effect of low dose cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation.

OBJECTIVE: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. METHODS: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4 x 20 ms impulses at 200 V/cm) 24 h prior to the transplantation. Group A (n=5) received CsA (2.5 mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5 microg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5 mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. RESULTS: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233+/-123 mmHg, vs 44+/-8 mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II-IIIA). hIL-10 serum levels on day 5 were 14+/-7 pg/ml in group B vs. 0 in group A. CONCLUSIONS: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantation.

Green fluorescent protein modified to bind DNA initiates production of anti-DNA antibodies when expressed in vivo.

Studies have clearly demonstrated that DNA itself is not or scarcely immunogenic in experimental animals. We have previously demonstrated that linking human polyomavirus large T-antigen to DNA rendered DNA immunogenic irrespective of the source or the structure of DNA. As an alternative to this artificial system, in vivo expression of the DNA binding protein large T-antigen of human polyomaviruses also resulted in the production of anti-DNA antibodies. This observation demonstrates that the large T-antigen concept is operational in vivo and supports the idea that complex formation between a non-self DNA-binding protein and DNA renders DNA immunogenic in analogy to a hapten-carrier model. To further investigate this model, the DNA binding domain (DBD) of a self-protein (glucocorticoid receptor) was linked to a non-DNA binding non-self protein, the green fluorescent protein (GFP). Immunization of mice with an expression plasmid for this fusion protein resulted in the production of anti-DNA antibodies, while mice inoculated with either a plasmid encoding the GFP or a plasmid encoding the DBD of the glucocorticoid receptor failed to produce anti-DNA antibodies. These results demonstrate that DNA may become immunogenic through in vivo association with any non-self DNA binding protein. Considering these data in context of results obtained with the polyomavirus large T-antigen, one may conclude that viral DNA-binding proteins may affect the regulation of immune tolerance to DNA and nucleosomes in vivo.

DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer.

We are evaluating the use of electroporation (EP) to deliver a novel DNA vaccine, p.DOM-PSMA(27). This vaccine encodes a domain (DOM) of fragment C of tetanus toxin to induce CD4(+) T cell help, fused to a tumor-derived epitope from prostate-specific membrane antigen (PSMA) for use in HLA-A2(+) patients with recurrent prostate cancer. We report on safety and tolerability and on antibody response to DOM as a first indication of the effect of EP in patients. In this open label phase I/II, two-arm, dose escalation trial DNA was delivered either by intramuscular injection or by intramuscular injection followed by EP (DNA+EP), with five patients per dose level. Three vaccinations were given at 0, 4, and 8 weeks,with booster doses at 24 and 48 weeks; here we allowed crossover between study arms if supported by the safety and immunological data. In the 20 patients in the first two dose cohorts we observed that beyond brief and acceptable pain at the injection site, EP did not appear to add toxicity to the vaccination. We evaluated humoral responses to DOM. Low anti-DOM IgG antibody responses were observed after intramuscular injection of DNA without EP (at week 12: mean 1.7- vs. 24.5-fold increase over baseline with DNA+EP). These could be boosted by delivery of DNA+EP at later time points. Delivery of DNA+EP at all five vaccinations yielded the highest levels of anti-DOM antibody. Responses persisted to 18 months of follow-up. These data establish EP as a potent method for stimulating humoral responses induced by DNA vaccination in humans.

In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells.

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.

Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques.

Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log(10) increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

A novel electroporation device for gene delivery in large animals and humans.

Intramuscular injection of plasmid DNA followed by electrical stimulation (electroporation) is an efficient method for achieving therapeutic levels of encoded proteins or eliciting efficient immune responses in smaller animals such as mice and rats. Electroporation in larger animals and humans poses new technical challenges, the main difficulty being to maintain efficacy while limiting invasiveness and pain. Here we present data using a new device for combined injection and electroporation in large animals and humans. The device injects DNA through two needles during insertion into the muscle and thus distributes the injection volume along the needles which also serve as electrodes. Since the electrical field is strongest close to the needle-electrode, a near perfect match between the DNA and the electric field is achieved. We show that using moderate amounts of DNA: (1) muscle tissue is transfected along the entire length of the needle path, (2) the efficacy is higher compared to when the DNA is injected between the electrodes, (3) level of protein expression can be tightly controlled by the number of treatments, and (4) efficient immunization is achieved.