Patterson: CD8 Broadening Strategies
The major goal of the project is to develop an HIV CD8+ T cell vaccine targeted to dendritic cells in the skin that will induce a broad CTL response, and to test this hypothesis in a repeated low dose NHP challenge model.
An effective HIV-1 T-cell vaccine would be capable of stimulating a broad, persistent cytotoxic T-cell response supported by helper T-cells at the three major barriers to HIV infection: the genital and rectal mucosa, the regional lymph nodes, and blood. Researchers at the Patterson-led consortium aim to develop a candidate HIV-1 vaccine that will target dendritic cells in the skin as the main antigen presenting cells and stimulate a broad response to HIV-1 gag.
The research consortium has several projects that aim to accomplish this goal. The researchers are developing a system to deliver a vaccine through the skin using micro-needle sugar patches containing viral vectors coding for HIV vaccine genes. The sugars quickly dissolve releasing the viral vectors to directly contact skin dendritic cells, which act as antigen-presenting cells that stimulate the immune system to produce T-cell responses. The researchers are also exploring how to enhance the diversity of T-cell responses by vaccination that will result in a broad HIV CD8 response on HIV infection. One hypothesis for the lack of breadth of T-cell responses on vaccination is competition for space on the surface of the antigen-presenting cells. The researchers are trying to overcome this problem by dividing the HIV-1 vaccine genes into a series of mini-genes and incorporating them into separate vectors. This strategy will reduce the number of HIV-1 epitopes expressed by any single presenting cell and could thus broaden the response.
Another strategy under study at the Patterson consortium is to construct adenovirus vectors coding for fusion proteins between ubiquitin and HIV-1 gag and thus increase the number of epitopes on the presenting cell surface.
- Develop and assess the immunogenicity of a rAd5 vaccine delivered to target Langerhans cells and other skin dendritic cells and stimulate mucosal and systemic CD8 responses.
- Define the optimal vaccination regimen (route, depth and device) to deliver rAd vaccine to dendritic cells in the skin that will induce systemic and mucosal immunity.
- To test rAd5 constructs coding for gag vaccine genes that have been modified to generate a large pool of CD8 memory cells recognizing multiple CTL epitopes.
- Based on the results of objects 1-3 design a comparable cognate Ad SIV vaccine and evaluate immunogenicity in Cynomolgus macaques followed by assessment of efficacy by repeated low dose intrarectal challenge with SIV.
In the original proposal the Patterson-led consortium aimed to develop a T-cell vaccine that will target skin dendritic cells (DC) via a patch consisting of an array of sugar-based micro-needles. Adenovirus vectors carrying HIV vaccine genes are embedded in the sugar micro-needles, which, upon application to the skin, quickly dissolve releasing the vaccine in close proximity to DC.
Adenovirus vectors embedded in dry sugar are stable, can transduce cells, and can induce expression of encoded transgenes following storage for over a month at up to 40°C. Demonstration of heat stability suggests that storage of the vaccine product in a dry sugar preparation makes the product suitable for use in third world counties where refrigeration facilities may not be available.
In vivo small animal immunogenicity studies have been performed with patches containing dried adenovirus vector containing a transgene coding for a model antigen, ovalbumin. Results have shown induced CD8+ T cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization.
To increase the level of immune response, the researchers aim to increase the level of expression of MHC peptide complexes, thought to be a factor in determining dominance. For this purpose the researchers have constructed adenovirus vectors coding for fusion proteins between ubiquitin and gag. These fusion vectors have been tested on human DCs and shown more effective targeting of the recombinant antigen to the proteasome than unmodified antigen. However despite increased targeting of the proteasome, there was no improvement or even a reduction in the magnitude and breadth of gag-specific CD8 T cell responses induced when compared with unmodified gag genes. Several possibilities have been explored to explain these findings. The possibility that the ubiquitin fusion proteins are preferentially directed to the MHC class I pathway with a consequent reduction in presentation by MHC class II was investigated, but CD4 responses were found to be induced by the vaccine. An explanation for the reduced immunogenicity was provided by studies showing that human DCs transfected with ubiquitin gag fusion proteins have a reduced ability to up-regulate co-stimulatory molecules, a requirement for effective T cell stimulation. By comparison fusion of another protein with ubiquitin, Melanin A, did not interfere with the ability of DC to up regulate co-stimulatory molecules suggesting that the deleterious effect of fusion was associated with gag. In addition ubiquitin gag fusion proteins were found to cause increased secretion of the immunosuppressive cytokine IL-10 in transfected cells. It still remains possible that fusion of ubiquitin to other HIV proteins may enhance immunogenicity.
A further goal is to overcome competition on the DC surface by responding T cells recognizing different epitopes, since competition may restrict the number of epitopes recognized. The researchers have constructed a series of 10 adenovirus vectors containing gag mini-genes, each coding for 4-5 epitopes, that have a ubiquitin fusion at the 5’ end and an HA tag at the 3’ end. In preparation for testing their vaccine approach in Cynomolgus macaques comparable recombinant Adenoviruses have been made containing SIV gag mini genes fused to ubiquitin. Studies in the murine model in which animals receive a single vaccination have shown that vaccination with SIV gag mini genes induces a breadth of response similar to that obtained with full length gag. In the human primary in vitro system comparison of full length ubiquitinated gag and ubiquitinated mini genes consistently showed that stimulation with the mini genes increased the number of epitopes recognized.
The vaccine was tested in groups of 8 MHC comparable Cynomolgus macaques and in 16 unvaccinated controls. Four vaccines were tested; full length unmodified gag, full length ubiquitinated gag, 7 ubiquitinated gag mini genes and 7 non-ubiquitinated gag mini genes. For each vaccine three intradermal DNA primes were given followed by an intradermal rAd5 boost. The mini genes were given at 7 separate sites on the back of the animal to avoid antigenic competition. The vaccinated animals responded by mounting a strong antigen-specific CD4 response, of particular interest was that most of the responding CD4 cells expressed the integrin molecules α4 and β7 that mediate migration to mucosal tissues, the site of infection. A weaker antigen-specific CD8 was induced and these cells did not show strong expression of α4 and β7. The animals were challenged intrarectally at 10 weekly intervals with a low dose with SIVMac 251. Encouragingly, vaccination mediated protection as indicated by a higher number of exposures being required for infection and somewhat surprisingly the highest protection was provided by the unmodified full length gag protein. The researchers speculated that this positive result might be due to the skin delivery route of the vaccine and thus animals were challenged with virus after intramuscular vaccination. However, this route of vaccination also provided protection from challenge. Further immunological analyses are being performed to elucidate the mechanism of protection.