William F. Swain

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine.

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-naïve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.

Phase 1 safety and immune response studies of a DNA vaccine encoding hepatitis B surface antigen delivered by a gene delivery device.

This study was designed to determine the safety and immunogenicity in volunteers of a DNA vaccine consisting of a plasmid encoding hepatitis B surface antigen delivered by the PowderJect XR1 gene delivery system into human skin. Seven healthy adult volunteers received two immunizations at one of three forces of delivery on day 0 and 56. The vaccine was well tolerated. One of six seronegative volunteers developed high titers of persistent HBsAb after a single immunization. In retrospect, this volunteer may have had previous exposure to hepatitis B. Our study suggests that the hepatitis B DNA vaccine given by this gene delivery system may induce a booster response, but the vaccine at the extremely low DNA dose used (0.25 microg) did not induce primary immune responses.

Soybean genetic engineering - commercial production of transgenic plants

Abstract Until recently, the improvement of soybean ( Glycine max ) by genetic engineering has been limited due to the lack of an efficient transformation system for introducing foreign genes into regenerable tissues. We report the production of transgenic plants using an electric discharge to inject DNA-coated gold particles into soybean meristems; the transformed phenotype segregated in a mendelian fashion and the introduced genes were stable for up to six generations. The technique has been developed into a commercially viable process for introducing any foreign gene into any variety of soybean.

Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene

Equine influenza virus infection remains one of the most important infectious diseases of the horse, yet current vaccines offer only limited protection. The equine immune response to natural influenza virus infection results in long-term protective immunity, and is characterized by mucosal IgA and serum IgGa and IgGb antibody responses. DNA vaccination offers a radical alternative to conventional vaccines, with the potential to generate the same protective immune responses seen following viral infection. Antigen-specific antibody isotype responses in serum and mucosal secretions were studied in ponies following particle-mediated delivery of hemagglutinin (HA)-DNA vaccination on three occasions at approximately 63-day intervals. One group of four ponies were vaccinated at skin and mucosal sites and the another group were vaccinated at skin sites only. All ponies were subjected to a challenge infection 30 days after the third vaccination. Skin and mucosal vaccination provided complete protection from clinical signs of infection, while skin vaccination provided partial protection; DNA vaccination provided partial protection from viral shedding. DNA vaccination generated only IgGa and IgGb antibody responses, which occurred with a higher frequency in the skin and mucosa vaccinated ponies. No mucosal IgA response was generated prior to challenge infection and IgA responses were only detected in those ponies which shed virus postchallenge. These results demonstrate that HA-DNA vaccination induces IgG(a) and IgG(b) antibody responses which are associated with protection in the absence of mucosal IgA responses. In addition, additional DNA vaccinations of mucosal sites increased protection and the frequency of seroconversion in ponies.

Transgenic cotton resistant to herbicide bialaphos

Resistance to bialaphos, a non-selective herbicide, was intro duced into cotton through genetic engineering. A gene encoding phosphinothric in acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, β-glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta®) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commerically important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets

Immunogenicity and efficacy of baculovirus-expressed and DNA-based equine influenza virus hemagglutinin vaccines in mice

Two fundamentally different approaches to vaccination of BALB/c mice with the hemagglutinin (HA) of A/Equine/Kentucky/1/81 (H3N8) (Eq/KY) were evaluated, that is, administration of HA protein vs administration of HA-encoding DNA. Each vaccine was tested for its immunogenicity and ability to provide protection from homologous virus challenge. HA protein was synthesized in vitro by infection of Sf21 insect cells with a recombinant baculovirus. Intranasal administration of this vaccine induced virus-specific antibodies, as measured by enzyme-linked immunosorbent assay (ELISA), but did not induce virus neutralizing (VN) antibodies. This route of administration provided partial protection from virus challenge, but interestingly, this protection was completely abrogated, rather than enhanced, by co-administration of 10 micrograms of cholera holotoxin. As a second approach, mice were directly vaccinated in vivo by Accell gene gun delivery of plasmid DNA encoding the Eq/KY HA gene. This approach induced VN antibodies as well as virus-specific ELISA antibodies. When two doses of DNA vaccine were administered 3 weeks apart, mice were not protected from challenge, although they cleared the infection more rapidly than control mice. However, when the second DNA vaccination was delayed until 9 weeks after the first, 9 out of 10 vaccinated mice were completely protected. These results indicate that the time between initial and booster DNA vaccinations may be an important variable in determining DNA vaccination efficacy.

Mucosal co-administration of cholera toxin and influenza virus hemagglutinin-DNA in ponies generates a local IgA response

We have previously demonstrated that equine influenza virus hemagglutinin (HA) DNA vaccination protects ponies from challenge infection, and induces protective IgGa and IgGb responses. However, this approach does not induce a nasal IgA response. The objective of this study was to examine the value of cholera toxin (CT) administration as an adjuvant for intranasal HA DNA vaccination, and to measure protection 3 months after DNA vaccination. After an immunogenic dose of CT was determined, ponies were immunized on two occasions by intranasal administration of HA DNA and cholera toxin, or HA DNA alone. Ponies in both groups received two additional HA DNA particle mediated vaccinations at skin and mucosal sites. Antibody responses, and protection from challenge infection 3 months after the last vaccination were studied and compared to an influenza virus naive control group. Ponies in both vaccination groups produced virus-specific IgG antibodies in serum following vaccination and showed clinical protection from challenge infection 3 months after the last vaccination. Co-administration of CT plus HA DNA vaccination induced a nasal IgA response. In addition, analysis of antibody titers in nasal secretions indicated local production of nasal IgGb, which was amplified by CT administration.

Methods for Particle-Mediated Gene Transfer into Skin

During the past 5 yr, particle-mediated delivery techniques have been developed as a physical means for gene transfer into various eukaryotic systems, including plants, insects, fish, and mammals (1-7). For mammalian somatic tissues, this technology, popularly known as the gene gun method, has been shown effective in transfection of skin, liver, pancreas, muscle, spleen, and other organs in vivo (3,4); brain, mammary, and leukocyte pnmary cultures or explants ex vivo (2,5-7); and a wide range of different mammalian cell lines in vitro (3,6,7).

Preparations for Particle-Mediated Gene Transfer Using the Accell® Gene Gun

Particle-mediated delivery involves coating materials onto the surface of dense sub-cellular sized (0.5-5 mm) particles and accelerating the particles to sufficient velocity to penetrate target cells. The technique was invented by Sanford and Wolf at Cornell University (1) to transfer DNA into intact plant cells (2), and was further developed into an effective process for producing genetically engineered crop plants by several groups (reviewed in 3). Subsequent work has shown that this method is generally applicable for transferring materials including DNA, RNA, proteins, peptides and pharmacological compounds into a wide variety of tissue and cell types in vivo, ex vivo, or in vitro (reviewed in 4).