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.

Vaxfectin(®) is a lipid-based adjuvant initially developed for use with plasmid DNA (pDNA) vaccines. Here we present detailed nonclinical assessments performed prior to Vaxfectin(®)'s first-in-man use, as an adjuvant in the H5N1 influenza vaccine VCL-IPT1. Following IM delivery to rabbits, VCL-IPT1 pDNA localized primarily to injection sites, where levels steadily declined over the 2 months examined. Risk of pDNA integration into genomic DNA was negligible. Toxicology studies in rabbits revealed mild inflammatory/immune responses at injection sites characteristic of IM vaccine delivery; Vaxfectin(®) directly contributed to these responses. These data support clinical development of H5N1 pDNA vaccines, and also present an encouraging profile for further development of Vaxfectin(®) as an adjuvant for vaccines in general.

Plasmid DNA (pDNA) vaccines represent an alternative to conventional inactivated influenza vaccines that are likely to experience supply constraints during a pandemic. Several Vaxfectin-formulated pDNA vaccines were tested in mice and ferrets for efficacy against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) influenza virus strain; the vaccines encoded influenza A virus hemagglutinin (HA), and/or nucleoprotein (NP), and M2 protein. Complete protection from death and disease was achieved in mice and ferrets with 2 doses of a Vaxfectin-formulated vaccine containing H5 HA, NP, and M2 plasmids and in ferrets with only 1 dose. A Vaxfectin-formulated vaccine containing NP and M2 pDNA provided significant protection against death in mice and provided some benefit in ferrets (i.e., 17% survival, delayed time to illness and death, and significant reduction in viral load compared with that in negative control animals). These experiments support the clinical testing of pDNA vaccine candidates that may ultimately increase global vaccine supply options during pandemics.

Experiments were conducted with a cationic lipid-formulated pDNA vaccine (VCL-AB01) to evaluate the models used to determine biodistribution, persistence and the potential for integration (into genomic DNA) of plasmid DNA-based vaccines. Mice were injected with a high-dose volume of 50 μL unilaterally containing ~2.42 × 1011 plasmid copy numbers (PCN) or a low-dose volume of 20 μL bilaterally (~9.67 × 1010 PCN). Rabbits were injected bilaterally with a 0.5 mL (~1.37 × 1011 PCN) volume. Injection site muscle tissue was harvested two days, one month, and two months postinjection for the low-dose murine and rabbit models and two days and two months postinjection for the high-dose murine model. Total DNA was extracted and analyzed by real-time quantitative PCR for sequences specific to the injected pDNA. The geometric mean PCN/μg of total DNA from the high and low dose models were compared to determine if injection volume impacts clearance and/or persistence. Results from these studies showed that PCN clearance over two months was similar in mice injected with 20 μL and rabbits injected with 0.5 mL, but PCN clearance was slower in mice injected with similar PCN in 50 μL (1.33 x 1013 PCN) compared to 20 μL (5.3 x 1012 PCN),. Persistence at two months in the rabbit and low-dose murine models was comparable, with geometric mean of 5.22 × 103 PCN/μg of total DNA for the low-dose volume murine model and 2.81 × 103/μg DNA for the rabbit model. Interanimal variability in persistence was not impacted by dose volume.

VCL-AB01 is a bivalent plasmid DNA (pDNA)-based anthrax vaccine candidate (composed of pDNA encoding detoxified Protective Antigen (PA) and Lethal Factor (LF) formulated with a cationic lipid system) currently in a Phase 1 trial. The immunogenicity and protective efficacy of this investigational vaccine were evaluated in lethal B. anthracis spore challenge mouse and NHP models. DBA/2 mice received VCL-AB01 intramuscularly (100 μg; 15/sex/group) on Days 7 and 14 (accelerated regimen) or Days 0 and 14 (standard regimen). A third group received Anthrax Vaccine Adsorbed (AVA; Bioport; 50 μL subcutaneously) on Days 0 and 14. Only 10 mice/sex of the 15/sex/group were challenged intratracheally on Day 35 (lethal dose; 1.3 X 106 Sterne strain spores). Sera collected on Days 0, 28 (unchallenged group only) and 50 were evaluated for toxin neutralization activity (TNA). By Day 28, VCL-AB01- vaccinated mice had lower TNA [geometric mean titers (GMT) of 76 and 106 ED50 for accelerated and standard regimens, respectively] than AVA-vaccinated mice (GMT=396 ED50). Four of the 10 animals vaccinated with VCL-AB01 on the accelerated regimen and 2/10 vaccinated on the standard regimen had no detectable TNA, yet both challenged cohorts were highly protected (85% and 95%), with comparable survival to the AVA-vaccinated cohort (90%) and demonstrated significantly (p<0.0001; Kruskal-Wallis Test) increased TNA post challenge compared to non-challenged cohorts at Day 50. Cynomolgus macaques (2/sex/group) received either VCL-AB01 (0.6 mg/mL) or 1.0 mL of PBS intramuscularly on Days 0, 14 and 28. Animals received a lethal challenge (AMES strain spores; Mean = 486 LD50) at 10 weeks and monitored for survival. Sera collected on Days 0, 28, 42 and 70 (just prior to challenge) and 3 weeks post- challenge were evaluated for TNA and serum antibodies. At 10 weeks, just prior to challenge, sera samples were negative for TNA. PA- and LF-specific antibodies were variable, ranging from 21-58 μg/mL and from 2-390 μg/mL, respectively. Despite low antibody titers and absence of TNA, 75% of VCL-AB01 vaccinated animals survived challenge. Survival was associated with an exponential rise in titers observed by 3 weeks post challenge (4748–8231 μg/mL PA-specific antibodies; 3045–12948 μg/mL LF-specific antibodies; TNA = 11,296–23,676 ED50). All control animals died 3-8 days post-challenge. Collectively, these results show that surviving animals with low or absent pre-challenge neutralizing antibodies and/or low PA- and LF-specific antibody titers were able to mount a rapid protective immunological response of a very high magnitude to the lethal spore challenge. These results indicate that: 1) vaccination with pDNA elicits a potent B-cell priming response that can be activated to full effector function extremely rapidly in the face of exposure to B. anthracis; 2) serum antibody and TNA titers are not optimal predictors of protection; and 3) other measures predictive of pDNA vaccine efficacy are needed. Presenting author is employee of Vical Incorporated, manufacturer of the investigational vaccine.

Cytolytic T-lymphocyte-mediated killing is thought to be an important effector mechanism in controlling viral infections. Recently, we reported that intramuscular injection of plasmid DNA containing the nucleoprotein (NP) gene of the influenza virus resulted in generating nucleoprotein-specific cytolytic T cells and antibodies. Gene-injected mice were subsequently protected from a lethal challenge with live influenza virus. Here we show that a single intramuscular injection of a small dose of nucleoprotein plasmid DNA generates nucleoprotein-specific cellular and humoral immune responses that last 1 year. The cellular response is associated with the CD8* subpopulation of T cells. Thus, plasmid DNA injections can be used to induce longlasting immune responses against the viral gene product without an exposure to live virus itself.