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.

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).

Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor.

Class-switched, affinity-matured murine monoclonal antibody (MAb)-producing cell lines were generated against the Flt-3 receptor in less than 4 weeks following polynucleotide immunizations, used in conjunction with repetitive immunizations, multiple sites (RIMMS). Plasmid DNA encoding Flt-3/Fc was coated onto gold particles, which were subsequently propelled into the epidermis of mice using biolistic particle bombardment using the Accell gene gun. Pools of immune peripheral lymph node cells were somatically fused 13 days after the onset of delivery of DNA encoding the target antigen. To determine if early responses could be augmented, DNA-encoding murine GM-CSF was delivered 3 days prior to the Flt-3/Fc DNA immunizations. The data presented demonstrates the successful identification and characterization of class-switched, affinity-matured MAbs that bind to the Flt-3 receptor. When compared to conventional methodologies or intramuscular targeted DNA-based immunization for the generation of MAbs, use of the gene gun in conjunction with RIMMS allows for a more rapid production of affinity-matured MAb-producing cell lines.

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.

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.

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.

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.