Neuroprotection with the cannabigerol quinone derivative VCE-003.2 and its analogs CBGA-Q and CBGA-Q-Salt in Parkinson's disease using 6-hydroxydopamine-lesioned mice.

The quinone derivative of the non-psychotropic cannabinoid cannabigerol (CBG), so-called VCE-003.2, has been recently investigated for its neuroprotective properties in inflammatory models of Parkinson's disease (PD) in mice. Such potential derives from its activity at the peroxisome proliferator-activated receptor-γ (PPAR-γ). In the present study, we investigated the neuroprotective properties of VCE-003.2 against the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA), in comparison with two new CBG-related derivatives, the cannabigerolic acid quinone (CBGA-Q) and its sodium salt CBGA-Q-Salt, which, similarly to VCE-003.2, were found to be active at the PPAR-γ receptor, but not at the cannabinoid CB1 and CB2 receptors. First, we investigated their cytoprotective properties in vitro by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-003.2 at a concentration of 20 µM, which was not reversed by the blockade of PPAR-γ receptors with GW9662, supporting its activity at an alternative site (non-sensitive to classic antagonists) in this receptor. We also found CBGA-Q and CBGA-Q-Salt being cytoprotective in this cell assay, but their effects were completely eliminated by GW9662, thus indicating that they are active at the canonical site in the PPAR-γ receptor. Then, we moved to in vivo testing using mice unilaterally lesioned with 6-OHDA. Our data confirmed that VCE-003.2 administered orally (20 mg/kg) preserved tyrosine hydroxylase (TH)-positive nigral neurons against 6-OHDA-induced damage, whereas it completely attenuated the astroglial (GFAP) and microglial (CD68) reactivity found in the substantia nigra of lesioned mice. Such neuroprotective effects caused an important recovery in the motor deficiencies displayed by 6-OHDA-lesioned mice in the pole test and the cylinder rearing test. We also investigated CBGA-Q, given orally (20 mg/kg) or intraperitoneally (10 mg/kg, i.p.), having similar benefits compared to VCE-003.2 against the loss of TH-positive nigral neurons, glial reactivity and motor defects caused by 6-OHDA. Lastly, the sodium salt of CBGA-Q, given orally (40 mg/kg) to 6-OHDA-lesioned mice, also showed benefits at behavioral and histopathological levels, but to a lower extent compared to the other two compounds. In contrast, when given i.p., CBGA-Q-Salt (10 mg/kg) was poorly active. We also analyzed the concentrations of dopamine and its metabolite DOPAC in the striatum of 6-OHDA-lesioned mice after the treatment with the different compounds, but recovery in the contents of both dopamine and DOPAC was only found after the treatment with VCE-003.2. In summary, our data confirmed the neuroprotective potential of VCE-003.2 in 6-OHDA-lesioned mice, which adds to its previous activity found in an inflammatory model of PD (LPS-lesioned mice). Additional phytocannabinoid derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection in 6-OHDA-lesioned mice, but their effects were lower compared to VCE-003.2, in particular in the case of CBGA-Q-Salt. In vitro studies confirmed the relevance of PPAR-γ receptors for these effects.

Cannabidiol and Other Cannabinoids in Demyelinating Diseases.

A growing body of preclinical evidence indicates that certain cannabinoids, including cannabidiol (CBD) and synthetic derivatives, may play a role in the myelinating processes and are promising small molecules to be developed as drug candidates for management of demyelinating diseases such as multiple sclerosis (MS), stroke and traumatic brain injury (TBI), which are three of the most prevalent demyelinating disorders. Thanks to the properties described for CBD and its interesting profile in humans, both the phytocannabinoid and derivatives could be considered as potential candidates for clinical use. In this review we will summarize current advances in the use of CBD and other cannabinoids as future potential treatments. While new research is accelerating the process for the generation of novel drug candidates and identification of druggable targets, the collaboration of key players such as basic researchers, clinicians and pharmaceutical companies is required to bring novel therapies to the patients.

Neuroprotection with the Cannabidiol Quinone Derivative VCE-004.8 (EHP-101) against 6-Hydroxydopamine in Cell and Murine Models of Parkinson's Disease.

The 3-hydroxyquinone derivative of the non-psychotrophic phytocannabinoid cannabigerol, so-called VCE-003.2, and some other derivatives have been recently investigated for neuroprotective properties in experimental models of Parkinson's disease (PD) in mice. The pharmacological effects in those models were related to the activity on the peroxisome proliferator-activated receptor-γ (PPAR-γ) and possibly other pathways. In the present study, we investigated VCE-004.8 (formulated as EHP-101 for oral administration), the 3-hydroxyquinone derivative of cannabidiol (CBD), with agonist activity at the cannabinoid receptor type-2 (CB2) receptor in addition to its activity at the PPAR-γ receptor. Studies were conducted in both in vivo (lesioned-mice) and in vitro (SH-SY5Y cells) models using the classic parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). Our data confirmed that the treatment with VCE-004.8 partially reduced the loss of tyrosine hydroxylase (TH)-positive neurons measured in the substantia nigra of 6-OHDA-lesioned mice, in parallel with an almost complete reversal of the astroglial (GFAP) and microglial (CD68) reactivity occurring in this structure. Such neuroprotective effects attenuated the motor deficiencies shown by 6-OHDA-lesioned mice in the cylinder rearing test, but not in the pole test. Next, we explored the mechanism involved in the beneficial effect of VCE-004.8 in vivo, by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-004.8 at a concentration of 10 µM, which was completely reversed by the addition of antagonists, T0070907 and SR144528, aimed at blocking PPAR-γ and CB2 receptors, respectively. The treatment with T0070907 alone only caused a partial reversal, whereas SR144528 alone had no effect, indicating a major contribution of PPAR-γ receptors in the cytoprotective effect of VCE-004.8 at 10 µM. In summary, our data confirmed the neuroprotective potential of VCE-004.8 in 6-OHDA-lesioned mice, and in vitro studies confirmed a greater relevance for PPAR-γ receptors rather than CB2 receptors in these effects.

Pulmonary artery pseudoaneurysm after radiofrequency ablation: report of two cases.

We report two cases of pulmonary arterial pseudoaneurysms (PAs) following percutaneous radiofrequency ablation (PRFA). The first patient was a 74-year-old Caucasian man who was treated for a secondary location of an advanced melanoma. A computed tomography scan at 72 h after the procedure, performed for basithoracic pain, hyperthermia and haemoptysis, revealed a 17-mm PA within the ablative zone. A lobectomy was performed. The second patient was an 80-year-old white man followed up for a right apical lung adenocarcinoma. Massive haemoptysis occurred 24 h after PRFA; emergent contrast-enhanced CT and pulmonary arteriography revealed a pulmonary artery PA (20 mm diameter), which was embolised with coils. The initial clinical course was satisfactory; however, 15 days after the procedure, the patient unfortunately presented a new massive haemoptysis and died a few hours later. The long ablation duration and the multiple repositioning of the electrodes might have been risk factors for this rare and potentially lethal complication.

Vaxfectin adjuvant improves antibody responses of juvenile rhesus macaques to a DNA vaccine encoding the measles virus hemagglutinin and fusion proteins.

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.m. or 100 µg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 µg of DNA plus Vaxfectin than after 100 µg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection.

The herpes simplex type 2 (HSV-2) envelope glycoprotein (gD2) was evaluated as a potential antigen candidate for a plasmid DNA (pDNA)-based HSV-2 vaccine. The pDNA was formulated with Vaxfectin, a cationic lipid-based adjuvant, and tested in a murine HSV-2 lethal challenge model. gD2 was expressed as full-length (FL) and secreted (S) gD2 forms. A 0.1 µg pDNA dose was tested to distinguish treatment conditions for survival and a 100 µg pDNA dose was tested to distinguish treatment conditions for reduction in vaginal and latent HSV-2 copies. Vaxfectin-formulated gD2 pDNA significantly increased serum IgG titres and survival for both FL gD2 and S gD2 compared with gD2 pDNA alone. Mice immunized with FL gD2 formulated with Vaxfectin showed reduction in vaginal and dorsal root ganglia (DRG) HSV-2 copies. The stringency of this protection was further evaluated by testing Vaxfectin-formulated FL gD2 pDNA at a high 500 LD(50) inoculum. At this high viral challenge, the 0.1 µg dose of FL gD2 Vaxfectin-formulated pDNA yielded 80 % survival compared with no survival for FL gD2 pDNA alone. Vaxfectin-formulated FL gD2 pDNA, administered at a 100 µg pDNA dose, significantly reduced HSV-2 DNA copy number, compared with FL gD2 DNA alone. In addition, 40 % of mice vaccinated with adjuvanted FL pDNA had no detectable HSV-2 viral genomes in the DRG, whereas all mice vaccinated with gD2 pDNA alone were positive for HSV-2 viral genomes. These results show the potential contribution of Vaxfectin-gD2 pDNA to a future multivalent HSV-2 vaccine.

Evaluation of a plasmid DNA-based anthrax vaccine in rabbits, nonhuman primates and healthy adults.

VCL-AB01, a cationic lipid-formulated plasmid DNA (pDNA)-based vaccine that contains genes encoding genetically detoxified Bacillus anthracis protective antigen (PA) and lethal factor (LF), was assessed in a Phase 1, dose-escalating clinical trial in healthy adults for safety and immunogenicity, and in nonhuman primates for immunogenicity and efficacy against challenge with a lethal dose of B. anthracis spores. Healthy 18-45 year old subjects were randomly assigned to receive either the investigational vaccine containing 0.2 mg, 0.6 mg, or 2 mg of total pDNA per dose, or saline placebo, administered at 0, 1 and 2 months. The 0.2 mg and 0.6 mg dose levels were generally well tolerated; however, dose-limiting reactogenicity was observed among subjects given the first 2 mg dose and the remaining two injections in the 2 mg group were reduced to 0.6 mg. Dose-related increases in seroconversion frequencies were observed. Overall, 10%, 33.3% and 80% of subjects in the 0.2, 0.6 and 2 mg groups, respectively, developed antibodies to PA and/or LF as measured by ELISA; however, antibodies with toxin neutralizing activity (TNA) were detected in only one subject. In monkeys that received a 0.6 mg dose three times at 2 week intervals, low levels of antibodies were detected by ELISA but not by the TNA assay in all animals just prior to challenge. Despite the absence of TNA, 75% animals survived the lethal challenge. In summary, VCL-AB01 was generally well tolerated in humans at a dose that provided immunity in monkeys despite the lack of robust TNA titers in either species.

Physical characterization and in vivo evaluation of poloxamer-based DNA vaccine formulations.

BACKGROUND: Plasmid DNA (pDNA) vaccines have generated significant interest for the prevention or treatment of infectious diseases. Broader applications may benefit from the identification of safe and potent vaccine adjuvants. This report describes the development of a novel polymer-based formulation to enhance the immunogenicity of pDNA-based vaccines. METHODS: Plasmid DNA was formulated with a nonionic block copolymer, poloxamer CRL1005, and the cationic surfactant benzalkonium chloride (BAK) to produce a thermodynamically stable, self-assembling system. The influence of parameters such as polymer concentration and BAK composition on the immune responses was evaluated in mice vaccinated with pDNA encoding influenza nucleoprotein. RESULTS: At concentrations of 7.5 mg/ml CRL1005, 0.3 mM BAK and 5 mg/ml pDNA, CRL1005/BAK/pDNA particles had a mean diameter of 261 +/- 0.2 nm and a surface charge of - 11.6 +/- 0.9 mV. The negative surface charge and atomic force microscopy images suggested that pDNA binds to BAK adsorbed to the surface of poloxamer particles. The CRL1005/BAK/pDNA formulation significantly enhanced antigen-specific cellular and humoral immune responses, and increased transgene levels in muscle and serum. The complexity of the formulation was reduced by replacing the commercial BAK, which is a mixture of four alkyl chains, with a C14 BAK homolog. The substitution yielded an analytically preferable formulation with equivalent physical characteristics and immunogenicity. CONCLUSIONS: The results suggest that the CRL1005/BAK/pDNA formulation may enhance immunogenicity by improving the delivery of pDNA-based vaccines. This formulation is currently being evaluated for the prevention of CMV-associated disease in a phase 2 clinical trial.

A TaqMan reverse transcription polymerase chain reaction (RT-PCR) in vitro potency assay for plasmid-based vaccine products.

A TaqMan-based reverse transcription polymerase chain reaction (RT-PCR) assay has been developed as an in vitro potency assay to measure the most immediate biological activity of plasmid DNA (pDNA)-based products. The assay measures transgene-specific messenger RNA (mRNA) from cultured cells transfected with VCL-CB01, a bivalent pDNA-based human cytomegalovirus (CMV) vaccine. The forward and reverse primers have been designed to make the RT-PCR reaction selective for plasmid-derived mRNA and to allow discrimination of expression levels of individual plasmids in a multivalent pDNA vaccine. The relative potency of a vaccine lot is assessed by transfecting reference and test samples into cultured cells in parallel and analyzing total RNA from the cells by RT-PCR. Statistical analysis of dose response data from reference material supports a parallel-line model for calculating relative potency. Preliminary data demonstrate the ability of this assay to distinguish product potencies at 50, 75, 150, and 200% of the reference material. In addition, forced degradation of pDNA demonstrates that a decrease in relative potency as measured by the RT-PCR assay in vitro correlates well with a decrease in CMV DNA vaccine-mediated humoral immune responses in mice injected with the same material.

IL-2 plasmid electroporation: from preclinical studies to phase I clinical trial.

Electroporation (EP)-assisted intralesional delivery of Interleukin-2 (IL-2) plasmid (pDNA) has the potential to increase the local concentration of the expressed cytokine for an extended time in the injected tumors while minimizing its systemic concentration, in comparison with systemic delivery of the recombinant cytokine. Nonclinical Investigational New Drug application-enabling studies were performed in mice to evaluate the effect of intratumoral administration of murine IL-2 pDNA on local expression and systemic distribution of IL-2 transgene as well as the inhibition of established tumor growth. The safety of repeated administrations of a human IL-2 pDNA product candidate with EP was evaluated in rats. Following the nonclinical safety and efficacy studies, a human IL-2 pDNA product candidate intralesionally administered with EP to metastatic melanoma patients is currently being investigated in a phase I clinical trial.

Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza A challenge.

The feasibility of a linear expression cassette (LEC)-based influenza A DNA vaccine was demonstrated in mice, using a lethal dose (LD90) of a mouse-adapted A/Hong Kong/8/68 (H3N2) influenza strain. LECs expressing hemagglutinin (HA) from either the homotypic H3N2 or the heterotypic H1N1 (A/Puerto Rico/8/34) influenza virus were produced by polymerase chain reaction and either phosphodiester- or phosphorothioate-modified oligonucleotide primers. Survival subsequent to lethal viral challenge was used as a primary end point; weight loss was the secondary end point. Survival and weight loss data showed that protection can be achieved in mice with 50 microg of phosphate-buffered saline-formulated LEC DNA or 2 microg of Vaxfectin-formulated LEC DNA. Survival correlated with neutralizing antibody titers (hemagglutination inhibition, HAI); titers obtained after vaccination with LEC were equivalent to those obtained with HA (H3N2) plasmid DNA control. Vaccination with heterotypic H1 HA-LEC DNA provided no protection against viral challenge.

Nuclear gene delivery: the Trojan horse approach.

The nuclear envelope represents a formidable barrier to the transfer of plasmids to the cell nucleus, particularly in nondividing cells. The probability of intact plasmids arriving in the nucleus by a passive process is extremely low. There is substantial evidence in the literature that describes the transport of macromolecules, including plasmids, to the nucleus as a very inefficient process, and so far attempts to affect the active transport through the nuclear pores have achieved limited success. Several approaches have been attempted to improve nuclear transport of plasmids, including the condensation of plasmids to unimolecular complexes of minimal hydrodynamic diameter to favour passive transport through the nuclear pore complex, and the incorporation of nuclear localisation signals in the plasmid or in the delivery system to enhance the active transport of plasmids through the nuclear pores.

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

II. Cationic lipid-formulated plasmid DNA-based Bacillus anthracis vaccine: evaluation of plasmid DNA persistence and integration potential.

Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.

Gene medicines: the end of the beginning?

First-generation gene medicines and genetic vaccines represent a promising new class of therapeutics that have the potential to prevent, correct, or modulate genetic or acquired diseases. The rational design of synthetic gene delivery and expression systems continues to be essential to enable the precise temporal and spatial control of transgene expression in vivo. With the tantalizing efficacy results and outstanding safety profile observed with nonviral, plasmid-based product candidates in early clinical trials, a multidisciplinary approach remains critical to further improve the effectiveness, reduce the manufacturing costs, and maintain the safety of gene therapeutics and vaccines for their successful development. This commentary provides an historical perspective on somatic gene therapy and briefly addresses the rate-limiting steps in effective gene transfer and expression. The importance of understanding plasmid pharmacokinetics after administration by conventional routes in animal models and in humans is emphasized. Pharmaceutical scientists have a pivotal role to play in deciphering the key biological parameters to effective gene transfer and designing gene delivery systems that will enable plasmid-based products to become an integral part of the future medical armamentarium.

Development of anthrax DNA vaccines.

Over 120 years ago, Pasteur and Greenfield developed an in vitro procedure for producing a live-attenuated Bacillus anthracis bacterial culture capable of protecting livestock from anthrax disease. Since then, anthrax has become one of the best characterized bacterial pathogens with regard to mechanism of toxicity and vaccine development. Most developments have used live-attenuated strains, bacterial supernatants or protein subunit approaches. Recently, novel plasmid DNA (pDNA) approaches to a safe and effective anthrax vaccine have been proposed. This review summarizes the history of anthrax, the need for new vaccines and recent developments in pDNA-based vaccines, leading to the initiation of a human phase I clinical trial in a significantly shorter timeframe than in traditional vaccine development.

Clinical Development of a Cytomegalovirus DNA Vaccine: From Product Concept to Pivotal Phase 3 Trial.

2013 marks a milestone year for plasmid DNA vaccine development as a first-in-class cytomegalovirus (CMV) DNA vaccine enters pivotal phase 3 testing. This vaccine consists of two plasmids expressing CMV antigens glycoprotein B (gB) and phosphoprotein 65 (pp65) formulated with a CRL1005 poloxamer and benzalkonium chloride (BAK) delivery system designed to enhance plasmid expression. The vaccine's planned initial indication under investigation is for prevention of CMV reactivation in CMV-seropositive (CMV⁺) recipients of an allogeneic hematopoietic stem cell transplant (HCT). A randomized, double-blind placebo-controlled phase 2 proof-of-concept study provided initial evidence of the safety of this product in CMV⁺ HCT recipients who underwent immune ablation conditioning regimens. This study revealed a significant reduction in viral load endpoints and increased frequencies of pp65-specific interferon-γ-producing T cells in vaccine recipients compared to placebo recipients. The results of this endpoint-defining trial provided the basis for defining the primary and secondary endpoints of a global phase 3 trial in HCT recipients. A case study is presented here describing the development history of this vaccine from product concept to initiation of the phase 3 trial.

Preclinical evaluation of Vaxfectin-adjuvanted Vero cell-derived seasonal split and pandemic whole virus influenza vaccines.

Increasing the potency and supply of seasonal and pandemic influenza vaccines remains an important unmet medical need which may be effectively accomplished with adjuvanted egg- or cell culture-derived vaccines. Vaxfectin, a cationic lipid-based adjuvant with a favorable safety profile in phase 1 plasmid DNA vaccines trials, was tested in combination with seasonal split, trivalent and pandemic whole virus, monovalent influenza vaccines produced in Vero cell cultures. Comparison of hemagglutination inhibition (HI) antibody titers in Vaxfectin-adjuvanted to nonadjuvanted vaccinated mice and guinea pigs revealed 3- to 20-fold increases in antibody titers against each of the trivalent influenza virus vaccine strains and 2- to 8-fold increases in antibody titers against the monovalent H5N1 influenza virus vaccine strain. With the vaccine doses tested, comparable antibody responses were induced with formulations that were freshly prepared or refrigerated at conventional 2-8°C storage conditions for up to 6 mo. Comparison of T-cell frequencies measured by interferon-gamma ELISPOT assay between groups revealed increases of between 2- to 10-fold for each of the adjuvanted trivalent strains and up to 22-fold higher with monovalent H5N1 strain. Both trivalent and monovalent vaccines were easy to formulate with Vaxfectin by simple mixing. These preclinical data support further testing of Vaxfectin-adjuvanted Vero cell culture vaccines toward clinical studies designed to assess safety and immunogenicity of these vaccines in humans.

Preclinical evaluation of the immunogenicity and safety of plasmid DNA-based prophylactic vaccines for human cytomegalovirus.

Human cytomegalovirus (CMV) establishes a lifelong persistent infection characterized by periods of latency and sporadic viral replication and is a major infectious cause of birth defects following congenital infection. Currently, no licensed vaccine is available that would prevent CMV infection. In an effort to develop a prophylactic CMV vaccine, the effects of different formulations, immunization routes and delivery devices on the immunogenicity of plasmid DNA (pDNA)-based vaccines were evaluated in rabbits and mice. Compared with PBS- and poloxamer-based formulations, significantly higher antibody responses were obtained with pDNA formulated with Vaxfectin (®) , a cationic lipid-based adjuvant. With low vaccine doses, the intradermal (ID) route resulted in higher antibody responses than obtained when the same dose was administered intramuscularly (IM). Since the IM route allowed injection of larger volumes and higher doses than could be administered at a single ID site, better antibody responses were obtained using the IM route. The needle-free injection system Biojector (®) 2000 and electroporation devices enhanced antibody responses only marginally compared with responses obtained with Vaxfectin (®) -formulated pDNA injected IM with a needle. A single-vial Vaxfectin (®) formulation was developed in a dosage form ready for use after thawing at room temperature. Finally, in a GLP-compliant repeat-dose toxicology study conducted in rabbits, single-vial Vaxfectin (®) -formulated vaccines, containing pDNA and Vaxfectin (®) up to 4.5 mg and 2 mg/injection, respectively, showed a favorable safety profile and were judged as well-tolerated. The results support further development of a Vaxfectin (®) -formulated pDNA vaccine to target congenital CMV infection.

A novel therapeutic cytomegalovirus DNA vaccine in allogeneic haemopoietic stem-cell transplantation: a randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Cytomegalovirus reactivation occurs within 6 months in 60-70% of cytomegalovirus-seropositive patients after allogeneic haemopoietic stem-cell transplantation (HSCT), mainly due to immunosuppression associated with the procedure. Pre-emptive antiviral therapy reduces incidence of cytomegalovirus disease but can be toxic. To reduce the potential for disease and subsequent need for such antiviral drugs, we aimed to assess safety and efficacy of a cytomegalovirus therapeutic DNA vaccine compared with placebo. METHODS: In this exploratory double-blind, placebo-controlled, parallel group, phase 2 trial, up to 80 donor-recipient pairs and 80 unpaired recipients undergoing allogeneic HSCT were planned for enrolment at 16 transplant centres in the USA. Eligible recipients were cytomegalovirus-seropositive, 18-65 years old, without high-risk primary disease, T-cell depletion, previous vaccination for cytomegalovirus, or autoimmune diseases. We randomly allocated participants in both parallel groups in a 1:1 ratio to receive a cytomegalovirus therapeutic DNA vaccine (TransVax; Vical, San Diego, CA, USA) or placebo before conditioning and at 1, 3, and 6 months after transplantation. The vaccine contains plasmids encoding cytomegalovirus glycoprotein B and phosphoprotein 65 formulated with poloxamer CRL1005 and benzalkonium chloride. Randomisation was done by sequential allocation based on Pocock and Simon's method, and stratified by site, donor-recipient HLA matching status, and donor's cytomegalovirus serostatus. The primary outcome was the occurrence rate of clinically significant viraemia resulting in initiation of cytomegalovirus-specific antiviral therapy in the per-protocol assessable population. We assessed rates of adverse events in all participants who received at least one dose of vaccine or placebo. This study is registered with ClinicalTrials.gov, number NCT00285259. FINDINGS: We randomly allocated 108 participants (94 HSCT recipients and 14 paired donors) between June 29, 2006, and Dec 11, 2009. Enrolment of the paired arm was halted in February 2008 for logistical reasons. Safety was assessed in all participants; the efficacy population was restricted to 74 unpaired recipients. Groups were balanced for demographic and clinical variables. 19 (48%) of 40 vaccine recipients required cytomegalovirus-specific antiviral therapy, compared with 21 (62%) of 34 controls (p=0·145). However, during follow-up vaccine significantly reduced the occurrence and recurrence of cytomegalovirus viraemia and improved the time-to-event for viraemia episodes compared with placebo. The vaccine was well-tolerated; only one participant discontinued after an allergic reaction. Incidence of common adverse events after HSCT (eg, graft-versus-host disease or secondary infections) did not differ between groups. INTERPRETATION: We show proof of concept for an immunotherapeutic cytomegalovirus vaccine (TransVax) for clinically significant viraemia in the HSCT setting. The reported safety and efficacy outcomes support further development in a phase 3 trial, notwithstanding a lack of significant reduction in the use of cytomegalovirus-specific antiviral therapy compared with placebo in this phase 2 trial. FUNDING: Vical and US National Institute of Allergy and Infectious Diseases.