Project Leader:

Dr. Sylvia van den Hurk

Background: Carefully selected functional sequences of DNA have great potential as vaccines. The DNA must encode important protective antigens from the pathogen of interest. When introduced into the cells of the animal to be protected, the DNA is transcribed and the resulting foreign protein is recognized by the immune system. As a result, antibodies are formed and a cellular immune response is stimulated.

The objectives described in this project include optimization of DNA vaccination by improving the technology for plasmid delivery, by: a) targeting the expressed antigens to antigen presenting cells; b) by combining several plasmids or genes into one DNA vaccine; and c) by heterologous DNA prime-protein boost regimens. Since there is evidence that DNA vaccines may overcome the difficulties encountered in inducing protective immunity in neonates, the DNA vaccines were also tested in newborn animals.

The use of plasmids to introduce the desired DNA into the cell offers many advantages. Plasmids can be produced relatively quickly and easily, their quality can be precisely controlled, the vaccine DNA is not integrated into the DNA of the vaccinated animal, and there is no immune response to the plasmid itself. Further benefits include longevity of the immune response, the potential for neonatal immunization, a broad stimulation of the immune system (including both cellular and humoral responses) and possibly immunization against several pathogens with a single vaccine.

Experiments on mice provide very useful information about the basic form that the vaccine should take and about methods of modulating the immune response. It has been much easier to induce immune responses in mice than in most other species, especially when intramuscular injection is the route by which the vaccine is given. Yet it is important to use the target domestic animal to answer questions about dose, specific immune responses and method of delivery.

We have now identified viral genes that encode the proteins that induce protective immunity against a number of important respiratory and enteric infections in cattle. They include bovine herpesvirus-1 (BHV-1), bovine parainfluenzavirus-3, bovine respiratory syncytial virus, bovine viral diarrhea virus, Manheimia haemolytica and Haemophilus somnus. Ultimately we intend to generate multivalent DNA vaccines that consist of a mixture of plasmids, plasmids expressing multiple genes, or a combination of these.

Objectives: The long-term objective of this project is to develop nucleic acid immunization as a technology for use in food-producing animals. The specific objectives are to:

1. Develop improved methods for plasmid delivery.
2. Induce both humoral and cytotoxic lymphocyte responses by DNA immunization.
3. Develop combination and chimeric DNA vaccines.
4. Target plasmid expressed antigen to antigen-presenting cells.
5. Induce immunity in neonates in the presence of maternal antibodies.
6. Improve duration of immunity induced by DNA vaccines.

Progress:
1. Develop improved methods for plasmid delivery. We evaluated a gene gun for DNA immunization of cattle and found that it is an appropriate device for delivery of small amounts of plasmid on gold particles into different vaccination sites, so this technology allows us to reduce the amount of plasmid needed for vaccination. In addition, we used a needle-free device, called Biojector® to deliver plasmid DNA in a non-invasive manner. Indeed, the protein (antigen) amounts expressed after Biojector®-mediated delivery, as well as the immune responses induced, were significantly higher than those achieved by delivery with needle and syringe. Thus, these studies provided two non-invasive effective delivery methods for DNA vaccines.

2. Induce both humoral and CTL responses by DNA immunization. For a vaccine to be effective we need to induce both antibody and cell-mediated immunity (CMI). Glycoproteins B (gB) and D (gD) have been identified as major protective glycoproteins against BHV-1. We already knew that gD induces strong neutralizing antibody responses, and we expected that gB would induce strong CMI and confirmed that a gB DNA vaccine indeed induces antigen-specific cytotoxicity in mice and cattle. Once we confirmed this, we wanted to more precisely define the parts, or epitopes, of the gB molecule that most readily give rise to the CTL response. The results showed that potentially useful CTL epitopes lay between amino acids 328 – 342 of BHV-1 gB, so we could use shorter segments of gB to use induce this response. Furthermore, the immunostimulatory properties of cytokine IL-12 have led to the idea that IL-12 may be used as a vaccine adjuvant. We found that IL-12 enhanced the CTL response when mice were immunized intra-muscularly or in the dermis with the gene gun, but not intra-dermally.

3. Develop combination and chimeric DNA vaccines. A truncated secreted form of BHV-1 gD (tgD) and the amino-terminal portion of gB (gBb) were used to test various methods of delivering multiple genes in one vaccine. We found that a dicistronic plasmid system with the genes expressed as separate proteins can be used to express two individual antigens successfully. We attempted to improve expression using a plasmid encoding a chimeric protein consisting of tgD fused to gBb. Mice immunized with a chimeric tgD-gBb plasmid had significant gB- and gD- specific antibody and T cell responses when compared with control mice and mice immunized with separate plasmids encoding tgD and gBb in a single formulation. These results suggest that immunization with plasmids encoding chimeric antigens may represent a promising approach in the field of DNA vaccination. We are now using this approach to vaccinate simultaneously against type 1 and type 2 BVDV.

4. Target plasmid-expressed antigen to antigen-presenting cells. The antigen produced by the DNA vaccine must be taken up by antigen-presenting cells (APCs) for an effective immune response to develop. To improve the chance for antigen presentation, we planned to target expressed antigen to APCs and possibly up-regulate these cells, by creating chimeric proteins between the antigen and a ligand for an APC, i.e. CD154. Our results demonstrated that using CD154 to target plasmid-expressed antigen significantly enhanced immune responses induced by a DNA vaccine. The objective of the next study was to determine whether this DNA vaccine encoding bovine CD154 linked to tgD induced improved tgD-specific immune responses in cattle. Although the expression of an antigen as a chimeric protein with CD154 qualitatively altered immune responses in cattle, there was no difference in the level of protection.

5. Induction of immunity in neonates in the presence of maternal antibodies. Conventional vaccines do not usually work in very young animals because their immune system is still immature and the passive immunity they have acquired from their mothers may neutralize the vaccine. To test whether immune responses can be induced in the presence of maternal antibodies by DNA immunization or by immunization with proteins formulated with oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs, we created plasmids containing both a gene encoding an antigen and multiple CpG motifs. We found that a regimen of priming with the plasmids and boosting with tgD formulated with CpG ODN induced the strongest immune responses. Therefore, DNA immunization or immunization with protein formulated with CpG ODN is an effective strategy to vaccinate neonates that possess high levels of maternal antibodies.

6. Assess duration of immunity induced by DNA vaccines. To evaluate whether DNA immunization or immunization with protein formulated with CpG ODN would induce long-term memory responses, we compared the ability of each vaccine preparation to prime the immune system. Regardless of the vaccine used for priming, vaccinated lambs developed significantly increased antibody responses. However, lambs immunized with tgD and CpG ODN developed significantly higher titers after two weeks, and had significantly higher titers than all other groups after boosting, suggesting that this group had the best memory response. We further evaluated the biological activity of the tgD-specific antibodies and found that the virus neutralization titres were highest in the tgD/CpG group, which suggests that these vaccination regimens may lead to protective immunity. Investigating cellular immune responses, we found that after secondary immunization, the tgD/CpG group developed the highest number of proliferating lymphocytes, confirming the vaccination with tgD and CpG ODN induces the best memory responses.

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