Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies.

Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies each. Two vaccination groups were DNA vaccinated on four different occasions with combinations of plasmids encoding the gB, gC, and gD glycoproteins or plasmids encoding the immediate early (IE) and early proteins (UL5) of EHV-1, using the PowderJect XR research device. Total dose of DNA/plasmid/vaccination were 25 microg. A third group comprised unvaccinated control ponies. All ponies were challenge infected with EHV-1 6 weeks after the last vaccination, and protection from clinical disease, viral shedding, and viremia was determined. Virus neutralizing antibodies and isotype specific antibody responses against whole EHV-1 did not increase in either vaccination group in response to vaccination. However, glycoprotein gene vaccinated ponies showed gD and gC specific antibody responses. Vaccination did not affect EHV-1 specific lymphoproliferative or CTL responses. Following challenge infection with EHV-1, ponies in all three groups showed clinical signs of disease. EHV-1 specific CTLs, proliferative responses, and antibody responses increased significantly in all three groups following challenge infection. In summary, particle-mediated EHV-1 DNA vaccination induced limited immune responses and protection. Future vaccination strategies must focus on generating stronger CTL responses.

Regional antibody and cellular immune responses to equine influenza virus infection, and particle mediated DNA vaccination.

We have previously demonstrated that hemagglutinin (HA) gene vaccination and influenza virus infection generate protective antibody responses in equids. However, these antibody responses differ substantially in that particle mediated DNA vaccination does not induce an immunoglobulin A (IgA) response. A study was performed to investigate the regional immunoregulatory mechanisms associated with these different immune responses. Ponies were either vaccinated with equine HA DNA vaccines at skin and mucosal sites, infected with influenza virus or left untreated and influenza-specific antibody responses and protection from challenge infection was studied. In a subset of ponies, lymphocytes from peripheral blood (PBLs), nasopharyngeal mucosal tissue, or lymph nodes (LNLs) were collected for measurement of influenza virus-specific lymphoproliferative responses, local antibody production and IL-2, IL-4 and IFN-gamma mRNA production by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). DNA vaccination and influenza virus infection induced humoral immunoglobulin Ga (IgGa) and immunoglobulin Gb (IgGb) production and lymphoproliferative responses that were positively correlated with IFN-gamma mRNA production. However, there were marked differences in immune response in that only influenza infection induced an IgA response, and the regional distribution of lymphoproliferation, IFN-gamma and antibody responses. Responses to DNA vaccination occurred in PBLs and in lymph nodes draining DNA vaccination sites, while influenza virus infection induced responses in PBLs and hilar LNLs. In summary, common features of immune responses to either influenza virus infection or DNA vaccination were virus-specific IgGa, IgGb and IFN-gamma responses, which are associated with protection from infection, even when the regional distribution of these immune responses varied depending on the site of immune encounter.

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.

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.

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).

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.

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.

In vivo gene transfer to skin and wound by microseeding.

BACKGROUND: Gene transfer to skin has many potential applications but lacks a safe, practical delivery method. This report presents a new technique, microseeding, for in vivo gene transfer to skin and wounds and for DNA-mediated vaccination. The plasmid DNA solution was delivered directly to the target cells of the skin by a set of oscillating solid microneedles driven by a modified tattooing device. MATERIALS AND METHODS: Skin and partial-thickness excisional wounds in pigs were microseeded with either hEGF expression plasmid or beta-galactosidase expression plasmid. Human EGF was also delivered by single injection or particle bombardment. hEGF expression in wound fluid and in target tissue was determined by ELISA with anti-hEGF-specific antibodies. Additionally, weanling pigs were microseeded with a hemagglutinin of swine influenza virus expression plasmid and production of anti-HA-specific antibodies was determined by blocking ELISA. RESULTS: hEGF expression in microseeded partial thickness wounds (5664 pg/site) and skin sites (969 pg/site) peaked 2 days after transfection being four- to seven-fold higher than gene transfer by a single intradermal injection and two- to three-fold higher than particle-mediated gene transfer. The beta-galactosidase-expressing cells were detected in dermis and epidermis. Pigs microseeded with HA expression plasmid were protected from infection by the Swine influenza virus. CONCLUSIONS: These results demonstrate that microseeding is a simple and effective method for in vivo gene transfer to skin and wounds and is more efficient than single injection and particle-mediated gene transfer.

Immunization of pigs with a particle-mediated DNA vaccine to influenza A virus protects against challenge with homologous virus.

Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.

Coadministration of DNA encoding interleukin-6 and hemagglutinin confers protection from influenza virus challenge in mice.

This study was conducted to investigate whether Accell gene gun coadministration of DNA encoding human interleukin-6 (IL-6) would enhance protective immune responses in mice to an equine influenza A virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone exhibited accelerated clearance of homologous challenge virus but were not protected from infection. In contrast, mice that received both HA and IL-6 DNA had no detectable virus in their lungs after challenge. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.

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.

Manipulation of immune responses via particle-mediated polynucleotide vaccines.

Polynucleotide vaccines are a new approach to immunization that promises qualitative advances in vaccine technology. These vaccines mimic infection in that they result in expression of pathogen gene products in situ, which can elicit both cell-mediated immune responses and humoral responses. This approach has been applied primarily to vaccines against viral diseases, but may be significant for vaccines directed toward bacterial pathogens. Auragen has developed a generally applicable gene transfer technology and, for vaccine applications, has focused on particle-mediated gene transfer to epidermis. Results demonstrate that Accell polynucleotide vaccines induce immune responses toward human immunodefficiency virus (HIV) antigens, influenza A virus antigens, and hepatitis B virus (HBV) antigens in rodent,s swine and primates. Cellular immune responses toward these antigens have been demonstrated in rodents. In a swine influenza a challenge model Accell vaccination provides protection equivalent to that of a commercial killed-whole-virus vaccine. Vaccination of mice by this method toward a Chlamydia pneumoniae major outer-membrane protein elicits a species-specific antibody response.

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).

Particle-mediated gene transfer with transforming growth factor-beta1 cDNAs enhances wound repair in rat skin.

Based on preliminary but variable results with direct DNA transfer into wounds, we evaluated in vivo gene transfer by particle-mediated DNA delivery to rat skin to determine whether overexpression of TGF-beta1 at the site of skin incisions would result in a significant improvement in repair. Optimization of the method with viral promoter-luciferase reporter constructs indicated that expression of luciferase activity persisted up to 5 d and was promoter, pressure, and site dependent (ventral > dorsal). Using cytomegalovirus (CMV)-driven human alpha1-antitrypsin, transgene expression was immunolocalized within keratinocytes of the stratum granulosum at 24 h. We measured tensile strength of skin incisions at 11-21 d in both normal and diabetic rats transfected with TGF-beta1 expression vectors at surgery. Native murine TGF-beta1 under an SV40 promoter produced positive effects, while wound strengthening was more pronounced in diabetic animals using a CMV-driven construct. Transfection of rat skin with constitutively active, mutant porcine TGF-beta1 under the control of the CMV and Moloney murine leukemia virus promoters significantly increased tensile strength up to 80% for 14-21 d after surgery. Transfection 24 h before surgery was more effective. Particle-mediated gene delivery can be used to deliver viral promoter-cytokine expression constructs into rat skin in a safe, efficient, and reproducible fashion. The extent of wound repair, as evidenced by enhanced tensile strength, can be markedly improved in tissues transfected with TGF-beta1 expression constructs.

Particle-mediated nucleic acid immunization.

Nucleic acid immunization involves the direct in vivo administration of antigen-encoding plasmid DNA molecules that results in the de novo production of correctly folded microbial antigens at the site of DNA delivery. While this process can lead to the development of neutralizing antibody responses recognizing authentic protein conformations, in vivo antigen production also results in epitope presentation via the MHC class I antigen processing pathway, leading to the elicitation of cytotoxic cellular immune responses. Recent efforts in the authors' laboratories have focused on use of the Accell gene delivery system (gene gun) to achieve the direct, intracellular delivery of small quantities of DNA into cells of the epidermis. The gene gun approach to nucleic acid vaccination capitalizes on the synergistic combination of an effective DNA delivery system and a target tissue that serves as a major immunological inductive site. Experimental gene gun-based nucleic acid vaccines can achieve potent humoral and cytotoxic cellular immune responses in rodent models following immunization with as little as 16 ng of DNA. Equally strong responses have also been elicited in larger animals, such as pigs and monkeys, following epidermal immunization with as little as 2 to 4 micrograms of DNA.

Gene transfer.

The principles and techniques of gene transfer are presented. Common concepts are defined, and the frequently used transfer vectors are described herein. Several gene transfer applications are discussed briefly. Areas of particular interest to the plastic surgeon such as gene transfer to skin and wounds are included.

In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair.

This report details the transfer of a human epidermal growth factor (hEGF) expression plasmid to porcine partial-thickness wound keratinocytes by particle-mediated DNA transfer (Accell). After gene transfer an external sealed fluid-filled wound chamber was used to protect the wound, provide containment of the exogenous DNA and expressed peptide, and permit sampling of the wound fluid. Analysis of wound fluid for hEGF and total protein, an indicator of reformation of the epithelial barrier, showed that wounds bombarded with the hEGF plasmid exhibited a 190-fold increase in EGF concentration and healed 20% (2.1 days) earlier than the controls. EGF concentrations in wound fluid persisted over the entire 10-day monitored period, decreasing from 200 pg/ml to 25 pg/ml over the first 5 days. Polymerase chain reaction results showed that plasmid DNA was present in the wound for at least 30 days. These findings demonstrate the possible utility of in vivo gene transfer to enhance epidermal repair.

Cotransformation frequencies of foreign genes in soybean cell cultures.

Through the use of electroporation and a soybean (Glycine max L.) protoplast system, we generated stably transformed cell lines expressing a number of foreign genes (neomycin phosphotransferase,ß-glucuronidase, chloramphenicol acetyl transferase, and phosphinothricin acetyl transferase). Selected and unselected marker genes were cointroduced either linked on a single plasmid or as separate plasmids. Calli expressing multiple genes were recovered, and Cotransformation frequencies were established for both cases. Our results show a 50% cotransformation frequency in the case of linked genes. In situations in which two genes are introduced on independent plasmids, cotransformation frequencies are 18%-27%. Similar rates of cotransformation were observed among various marker pairs.

Inheritance and expression of foreign genes in transgenic soybean plants.

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R(1)) and second (R(2)) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for beta-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for beta-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R(1) generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R(2) generation. R(1) plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R(2) generation, demonstrating recovery of both homozygous and heterozygous R(1) plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.