Adrian Vilalta

Use of Vaxfectin Adjuvant with DNA Vaccine Encoding the Measles Virus Hemagglutinin and Fusion Proteins Protects Juvenile and Infant Rhesus Macaques against Measles Virus

ABSTRACT A measles virus vaccine for infants under 6 months of age would help control measles. DNA vaccines hold promise, but none has provided full protection from challenge. Codon-optimized plasmid DNAs encoding the measles virus hemagglutinin and fusion glycoproteins were formulated with the cationic lipid-based adjuvant Vaxfectin. In mice, antibody and gamma interferon (IFN-γ) production were increased by two- to threefold. In macaques, juveniles vaccinated at 0 and 28 days with 500 μg of DNA intradermally or with 1 mg intramuscularly developed sustained neutralizing antibody and H- and F-specific IFN-γ responses. Infant monkeys developed sustained neutralizing antibody and T cells secreting IFN-γ and interleukin-4. Twelve to 15 months after vaccination, vaccinated monkeys were protected from an intratracheal challenge: viremia was undetectable by cocultivation and rashes did not appear, while two naïve monkeys developed viremia and rashes. The use of Vaxfectin-formulated DNA is a promising approach to the development of a measles vaccine for young infants.

Herpes simplex virus type 2 tegument proteins contain subdominant T-cell epitopes detectable in BALB/c mice after DNA immunization and infection

Cytotoxic T cells are important in controlling herpes simplex virus type 2 (HSV-2) reactivation and peripheral lesion resolution. Humans latently infected with HSV-2 have cytotoxic T cells directed against epitopes present in tegument proteins. Studies in mice of immunity to HSV have commonly focused on immunodominant responses in HSV envelope glycoproteins. These antigens have not proved to be an effective prophylactic vaccine target for most of the human population. The murine immune response against HSV tegument proteins has not been explored. We analysed cellular responses in BALB/c mice directed against the tegument proteins encoded by UL46, UL47 and UL49 and against the envelope glycoprotein gD after DNA vaccination or HSV-2 infection. After DNA vaccination, the splenocyte T-cell response to overlapping peptides from UL46 and UL47 was more than 500 gamma interferon spot-forming units per 10(6) responder cells. Peptide truncation studies, responder cell fractionation and major histocompatibility complex binding studies identified several CD8(+) and CD4(+) epitopes. Cellular responses to tegument protein epitopes were also detected after HSV-2 infection. Tegument proteins are rational candidates for further HSV-2 vaccine research.

Sustained protective rabies neutralizing antibody titers after administration of cationic lipid-formulated pDNA vaccine

Published data indicate that formulation of pDNA with cationic lipids could greatly enhance the response to a pDNA vaccine in larger mammals. The present work tested the influence of several pDNA:cationic lipid formulations on rabies neutralizing titers. Plasmid expressing Rabies G protein (CVS strain) was evaluated in vivo for ability to elicit neutralizing titers. pDNA:DMRIE-DOPE formulated at two DNA:cationic lipid molar ratios was compared in mice to a Vaxfectin™-pDNA formulation. Mouse data indicate that Vaxfectin™ is more effective than DMRIE-DOPE in eliciting neutralizing titers. In addition, the ratio of pDNA to DMRIE-DOPE can also affect neutralizing titers. Our data show that sustained neutralizing titers (120 days) can be obtained after a single administration of DMRIE-DOPE-formulated pDNA in rabbits.

Vaxfectin®, a cationic lipid-based adjuvant for protein-based influenza vaccines

Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV formulated with Vaxfectin, a cationic lipid-based adjuvant. Increasing doses of Vaxfectin resulted in increased hemagglutination-inhibition or anti-TIV ELISA antibody titers, with up to a 200-fold increase obtained with 900 microg of Vaxfectin. A >or=10-fold dose-sparing effect was demonstrated with Vaxfectin formulations. Vaxfectin preferentially increased IgG2 titers compared to IgG1 titers, resulting in a balanced IgG isotype distribution. Lower doses of Vaxfectin (30 microg) did not enhance antibody responses, but increased the number of IFN-gamma secreting T-cells by up to 18-fold. The data demonstrate that Vaxfectin enhances Th1 responses with protein-based seasonal influenza vaccine, and suggest that cellular or humoral immune responses may be preferentially induced by modifying the Vaxfectin:antigen ratio in the vaccine formulation.

Comparison of Rabbit and Mouse Models for Persistence Analysis of Plasmid-Based Vaccines

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.

Analysis of biomarkers after intramuscular injection of Vaxfectin®-formulated hCMV gB plasmid DNA

Cationic lipids have been used as delivery systems to enhance the performance of vaccines and immunotherapeutics. However, little is known about the effect of administration of cationic lipid-formulated vaccines on gene expression. This study used DNA microarrays (39,000 transcripts) to characterize early changes in gene expression patterns in mouse muscle 1 and 2 days after intramuscular (i.m.) injection of a hCMV gB plasmid DNA (pDNA) vaccine formulated with the cationic lipid system Vaxfectin; gene expression profiles were compared to those obtained after i.m. injection of pDNA in PBS. Analysis of the DNA microarray data indicated that approximately 1% of the represented transcripts were modulated at least 2-fold compared to the PBS samples at both time points. Functional analysis of the modulated genes revealed that transcripts involved in antigen processing and presentation, apoptosis and the Toll-like receptor pathway were significantly enriched. In addition, confirmation of local and systemic modulation of subsets of biomarkers was achieved using Real-Time PCR and Cytometric Bead Assays. Time course and magnitude of cellular infiltration (F4/80+ and CD11b+ cells) to the injection site was changed in response to formulation of hCMVgB pDNA with Vaxfectin. Since the expression level of the pDNA-encoded transgene in the muscle was not affected by formulation Vaxfectin mechanism of action is expected to rely primarily on modulation of immune pathways and not on an increase in transfection of the antigen-encoding pDNA. Taken together, these data help explain the Vaxfectin-dependent robust enhancement of immune responses.