Patterson Vaccine Discovery Consortium

OVERVIEW:

An effective HIV-1 T-cell vaccine would be capable of stimulating a broad, persistent cytotoxic T-cell response supported by helper T-cells and B-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 VDC aims to develop a candidate HIV-1 vaccine that will evade host anti-vector immunity and stimulate a broad response to clade C of HIV-1.

The VDC has several projects that aim to accomplish this goal. The researchers are developing a system to deliver a vaccine through the skin using microneedle sugar patches in which 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. They are also investigating whether coating vectors with polymers can reduce non-specific immune responses that might detract from vaccine efficacy.

The researchers are also exploring how to enhance the diversity of T-cell responses in response to natural infection or vaccination. One hypothesis for the lack of T-cell responses 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 VDC 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.

RESEARCH OBJECTIVES:

1. Develop and assess the immunogenicity of a combined adjuvant and rAd5 vaccine engineered to evade anti vector immunity target Langerhans cells and stimulate mucosal and systemic CD8 responses.

2. Define the optimal vaccination regimen (route, depth and device) of microneedle sugar patches to deliver rAd vaccine to dendritic cells in the skin that will induce systemic and mucosal immunity.

3. Generate a large pool of CD8 memory cells recognizing multiple CTL epitopes.

4. 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 SIV vaginal challenge.

PROGRESS:

The Patterson-led VDC is developing 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 generation of ovalbumin-specific CD8 T cell responses in Peyer’s Patches as well as blood and spleen. To gain a better insight into the effectiveness with which the sugar needle patches deliver the vaccine to the skin preliminary studies have been performed in pigs which have a skin structure and properties similar to that of human skin. These studies point to changes that will need to be made in the needle design to ensure optimal skin vaccine delivery.

Polymer coating of the vector may prevent widespread systemic distribution of the vector, resulting in reduction of excessive non-specific inflammation particularly in the liver. The researchers have successfully attached the TLR2 ligand, PAM2Cys, to the polymer coated vector. Immunogenicity studies have shown that the response to the polymer coated vector was equivalent to those induced by the naked vector.

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 to very efficiently target the recombinant antigen to the proteasome.

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. Transfection experiments confirm that these constructs express ubiquitin- and HA-tagged gag protein fragments and construction of corresponding adenovirus vaccine vectors is ongoing. In preparation for testing their vaccine approach in Cynomolgus macaques comparable Adenovirus vectors have been made containing SIV gag mini genes.