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Patterson Progress Report Abstracts continued

 
Submitted January 15, 2012

This project aims to develop a CD8 T cell vaccine that will induce responses against multiple epitopes and overcome the problem of virus immune escape. The vaccine will be delivered to the skin, a tissue rich in dendritic cells and essential for the initiation of a T cell response, by sugar micro-needles patches containing the embedded vaccine. We have shown that the adenovirus vaccine vector in a dried sugar formulation maintains viability at high temperatures for several months and demonstrated in the mouse that the sugar patch delivery system generates good immune responses. More recently we have shown in the mouse that skin vaccination with patches induces antigen-specific CD8 responses in the genital tract that can kill target cells. We have tested two strategies to stimulate a broad cytotoxic CD8 T cell response. In the first strategy HIV gag genes have been fused withubiquitin to more efficiently target the proteasome and increase MHC peptide complexes on the cell surface. Vaccine vectors containing these fusions have been tested and do very effectively enhance proteasomal targeting, however, although some modest increase in response to certain epitopes has been seen in vaccinated mice overall we have observed reduced immunogenicity. The second strategy designed to broaden the response is to fragment the vaccine gene and clone segments into separate vectors. The aim is to express all the potential epitopes but reduce the number of different epitopes expressed on individual dendritic cells and overcome competition between T cells recognising different epitopes, a factor thought to limit the breadth of the response. To test this hypothesis 7 SIV gag gene segments spanning the whole of gag have been cloned into Ad5 and tested in a human in vitro priming system and encouragingly, compared to the full length gag, the mini gene vectors induced a marked increase in the number of epitopes recognised. A DNA prime Ad5 boost approach has been adopted to test the new vaccines in Mauritian Cynomolgus macaques (MCM). Animals have been vaccinated intradermally with either full length unmodified gag, full length gag fused to ubiquitin or the 7 mini gene fragments each fused to ubiquitin. The limited number of MHC haplotypes expressed by these animals has enabled us to have groups of control and vaccinated animals with a similar range of MHC genetic backgrounds which will enable data to be more clearly interpreted. These animals have been challenged at 10 weekly intervals intrarectally with low doses of SIVmac 251. Vaccination induced good CD4 and CD8 responses with about 50% of CD4 cells expressing mucosal homing markers. The vaccinated groups showed statistically significant (p=0.015) resistance to challenge compared to controls. This encouraging result is the first report of vaccination with gag employing a replication defective vector that has conferred resistance to infection. Since fusion with ubiquitin reduces immunogeniticy we vaccinated MCM with mini gene fragments that are not fused to ubiquitin. These animals will be challenged with SIVmac 251 intrarectally in January 2012.

Thus of a number of strategies that were tested two, skin vaccination and gene fragmentation, may facilitate future HIV vaccine development.

 
Submitted March 1, 2011

This project aims to develop a CD8 T cell vaccine that will induce responses against multiple epitopes and overcome the problem of virus immune escape. The vaccine will be delivered to the skin, a tissue rich in dendritic cells and essential for the initiation of a T cell response,by sugar micro-needles patches containing the embedded vaccine. We have shown that the adenovirus vaccine vector in a dried sugar formulation maintains viability at high temperatures for several months and demonstrated in the mouse that the sugar patch delivery system generates good immune responses. We have tested two strategies to stimulate a broad cytotoxic CD8 T cell response. In the first strategy HIV gag genes have been fused with ubiquitin to more efficiently target the proteasome and increase MHC peptide complexes on the cell surface. Vaccine vectors containing these fusions have been tested and do very effectively enhance proteasomal targeting, however, although some modest increase in response to certain epitopes has been seen in vaccinated mice overall we have not observed an improvement in immunogenicity. The second strategy designed to broaden the response is to fragment the vaccine gene and clone segments into separate vectors. The aim is to express all the potential epitopes but reduce the number of different epitopes expressed on individual dendritic cells and overcome competition between T cells recognising different epitopes, a factor thought to limit the breadth of the response. In preparation for a challenge study in Mauritian Cynomolgus macaques (MCM) to test this hypothesis 7 SIV gag gene segments spanning the whole of gag have been cloned into Ad5 and tested in a human in vitro priming system. Encouragingly, compared to the full length gag, the mini gene vectors induced a marked increase in the number of epitopes recognised when tested in a human in vitro priming assay. A DNA prime Ad5 boost approach has been adopted to test the mini gene vaccination hypothesis in MCM. Animals are now being vaccinated (January –May 2011) and will subsequently challenged intra rectally with SIV. The limited number of MHC haplotypes expressed by these animals has enabled us to have groups of control and vaccinated animals with a similar range of MHC genetic backgrounds which will enable data to be more clearly interpreted. We have also tested a new microbial adjuvant in mouse and initial experiments suggest that it induces a more poly-functional immune response. Finally, work designed to explain the STEP trial outcome suggests that vaccination of individuals immune to adenovirus serotype 5 caused activation and expansion of memory CD4 T cells expressing mucosal homing molecules thus increasing the number of susceptible targets at the site of HIV transmission.

 
Submitted July 26, 2010

As the project has developed, and with lessons learnt, some of the aims detailed in the original proposal have altered in line with new data . As the consortium enters our Year 5, the major goal of the project is still focused on developing a HIV CD8+ T cell vaccine targeted to dendritic cells in the skin that will induce a broad CTL response, and to test our hypothesis in the Cynomologus macaque repeated low dose challenge model.

In the original proposal we aimed to develop a micro-needle patch composed of dried sugars and our adenovirus vaccine vectors, which would deliver the “vaccine” to the skin dendritic cells. In vivo murine experiments have identified the cells targeted by this delivery system and shown that it can successfully induce a CD8 T cell response. The mechanics of delivery have been investigated initially in the murine model and thereafter in the porcine model as a direct comparator for human skin. Our data indicate that delivery of the “vaccine” is affected by the shape of the micro-needle, with pyramid shaped needles being more effective at penetrating the skin than cone shaped needles.

However, further research and development of the micro-needle patch system is still required, and thus delivery of the “vaccine” in the Cynomolgus macaque challenge study will be done using Becton Dickinson Technologies intradermal delivery system Microcone™. This device is available to the consortium following successful collaborative negotiations. The Microcone™ represents BD Technologies R&D model of the commercially developed Micro Injection System™ licensed by sanofi pasteur for use with their influenza vaccine.

To address the aim of improving the magnitude and breadth of the CD8 T cell response, we have employed two strategies:

  • Firstly the gag gene has been fused with ubiquitin to increase proteasome processing. Initially we made fusion constructs fused to 4 ubiquitin repeats but as these proved to be unstable mono ubiquitin gag gene constructs were made. Proteasomal targeting of gag was significantly enhanced in these constructs and subsequently tested in in vivo murine immunogenicity experiments and in a human in vitro priming system
  • Secondly a series of Adenovirus vectors have been constructed each containing a different fragment of the gag gene with the aim of avoiding competition on the surface of the antigen presenting cell between different clones of T cells that are specific for different gag epitopes. HIV CN54 gag has been divided into 10 fragments, with each fragment predicted to express 4-5 epitopes, and SIVmac251 gag into 7 fragments and vectors produced

Fusion protein constructs tested in vitro in mouse and ex vivo in human dendritic cells showed more effective targeting of gag to the proteasome than than unmodified gag. However, on vaccination of mice or in the human in vitro priming system there was no improvement in the magnitude of gag-specific CD8 T cell responses induced when compared with unmodified gag genes. There are several possibilities to explain these results and we are currently working on strategies to overcome this problem.

In parallel we have generated a series of 7 vectors each containing a separate segment of SIVmac251 gag fused to 1xUb as well as unmodified and 1xUb full length SIVmac251 gag constructs. As the SIV challenge study is imminent we have given priority to immunogenicity testing of these constructs. Thus far full length 1xUb gag and several 1xUb micro-fragments have shown immunogenicity in vivo in mice and we expect soon to have accumulated sufficient data to confirm whether fragmentation broadens the response. In the human in vitro priming system we have preliminary data suggestive of response broadening, more experiments are in progress aiming to confirm this.

Submitted January 13, 2010 (Interim Report)

This project aims to develop a polymer-coated adenovirus vector vaccine to evade adenoviral neutralizing antibody and to target skin dendritic cells. Delivery to the skin is by sugar micro-needles patches containing the embedded vaccine. The HIV vaccine genes have been modified to stimulate a broad cytotoxic CD8 T cell response. We have now shown that the adenovirus vector in a dried sugar formulation maintains viability at high temperatures for several months. Vector construction is well advanced with production and testing of adenovirus vectors containing full length gag genes alone or fused to mono or tetra ubiquitin sequences. As anticipated, fusion gene products show enhanced proteasomal targeting. In addition, 10 adenovirus vectors designed to broaden the immune response containing ubiquitin fusion mini genes spanning the whole gag have been made and shown expected expression in vitro. Equivalent constructs containing 7 SIV gag mini genes for Cynomolgus macaque challenge studies are completed. In mouse studies vaccination with the micro-needle patches has been optimised to target skin DC and induce immune responses. Polymer coating the virus and retargeting with the TLR 2 ligand, PAM2Cys, reduces distribution to other tissues and thus increases the safety profile of the vaccine. In addition, induction of Gag T cell responses by vaccination with a coated adenovirus vector has been demonstrated and preliminary experiments suggest generation of central memory cells. Co-vaccination with a new microbial adjuvant suggests in initial experiments a more poly-functional immune response. Finally, work designed to explain the STEP trial outcome suggests that vaccination of individuals immune to adenovirus serotype 5 caused activation and expansion of memory CD4 T cells expressing mucosal homing molecules thus increasing the number of susceptible targets at the site of HIV transmission.

 
Submitted July 31, 2009

This project aims to develop a polymer-coated adenovirus vector vaccine to evade adenoviral neutralizing antibody and to target skin dendritic cells (DC). Delivery to the skin is by sugar micro-needles patches containing the embedded vaccine. The HIV vaccine genes have been modified to stimulate a broad cytotoxic CD8 T cell response. We have now shown that the adenovirus vector in a dried sugar formulation maintains viability at high temperatures for several months. Vector construction is well advanced with production of Ad5 vectors containing full length gag genes alone or fused to mono or tetra ubiquitin sequences; fusion gene products show enhanced proteasomal targeting. In addition, 10 Ad5 vectors designed to broaden the immune response containing ubiquitin fusion mini genes spanning the whole have gag have been made and shown expected expression in vitro. Equivalent constructs containing 7 SIV gag mini genes for cynomolgus challenge studies are near completion. In mouse studies vaccination with the micro-needle patches has been optimised to target skin DC and induce immune responses. Polymer coating the virus and retargeting with the TLR 2 ligand, PAM2Cys, reduces distribution to other tissues and thus increases the safety profile of the vaccine. In addition, induction of gag T cell responses by vaccination with coated Ad vector has been demonstrated and preliminary experiments suggest generation of central memory cells. Co-vaccination with a new microbial adjuvant suggest in initial experiments a more poly-functional immune response. Finally, work designed to explain the STEP trial outcome suggest that vaccination of individuals immune to Ad5 caused activation and expansion of memory CD4 T cells expressing mucosal homing molecules thus increasing the number of susceptible targets at the site of HIV transmission. 

 
Submitted January 5, 2009 (Interim Report)

Our consortium is developing a T-cell vaccine that will target skin dendritic cells (DC) via a patch consisting of an array of 25-40 sugar-based micro-needles. Adenovirus vectors carrying HIV vaccine genes are embedded in the sugar micro-needles which, on application to the skin, quickly dissolve releasing the vaccine in close proximity to DC. Since our 2008 annual report we have shown that adenovirus vectors embedded in dry sugar are stable, can transduce cells and 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 has potential benefits in developing a 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. We are currently developing a system in which our adenovirus vector is coated with a polymer permitting cellular ligands to covalently attach and mediate DC entry; these ligands can also act as adjuvants in the system to induce DC maturation. A further potential advantage is that 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. We 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. These initial “proof of concept” experiments are encouraging for us to test further ligands to achieve higher responses. Results also indicated that vaccination with naked vector led to a broad delivery of vector as indicated by transfection of resident lymph node DC whereas those animals vaccinated with polymer-coated vector transfection was limited to local tissue DC. Our aim is to develop a vaccine that induces a broad array of cytotoxic lymphocytes (CTL) that recognise dominant and sub-dominant epitopes. Two approaches are being pursued. Firstly we aim to increase the level of expression of MHC peptide complexes, thought to be a factor in determining dominance. For this purpose we 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. Secondly, we aim to overcome competition on the DC surface by responding T cells recognising different epitopes, since competition may restrict the number of epitopes recognized. We have constructed a series of 10 shuttle plasmids 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.

 
Submitted August 1, 2008

This project aims to develop a polymer-coated adenovirus vector vaccine to evade adenoviral neutralizing antibody and to retarget the ‘shielded’ vector to skin dendritic cells (DC). The vaccine will be delivered to the skin by sugar micro needles containing the embedded vector which is formulated into a patch comprising 25-30 needles of up to 1500μm in length. The vector in which HIV vaccine genes are engineered is modified to stimulate a broad cytotoxic CD8 T cell response. The T cell vaccine will be given together with a vector coding for HIV-like particles to stimulate antibody and CD4 T cell responses.

We had previously identified sugar formulations for microneedle manufacture that would maintain up to 80% of vector viability after drying. We have tested an additional sugar known to give improved needle physical properties and found that it effectively conserves vector viability.

Animal studies with polymer shielded retargeted virus, previously shown to infect mouse dendritic cells in the presence of neutralising antibodies, have now been performed. Vaccination of mice with the re-targeted shielded vector has been found to induce an immune response equivalent to the level of that attained with naked viral vector and future work will aim to refine and further enhance the immunogenicity of the insert encoded by the modified vector. Preliminary experiments in mice with pre-existing anti-vector neutralising antibodies have demonstrated that polymer ‘shielded’ vector can induce an immune response in Ad5 sero-positive mice and shows promise for future development. We have also discovered that ‘shielded’ vector primarily transduces dendritic cells at the site of vaccination whereas naked vector is disseminated and transduces lymph node resident dendritic cells and stromal cells. Thus the shielded vector should have a better safety profile.

Experiments in mice with microneedles containing FITC-dextran have shown that the FITC-dextran can be effectively delivered to skin and lymph node dendritic cells by this route of delivery. Preliminary experiments with microneedles containing vector encoding ovalbumin as a model antigen have demonstrated induction of systemic antigen-specific CD8 T cells.

Steady progress has been made in vector construction. Vectors are being made with 10 gene fragments of HIV gag fused to ubiquitin to enhance and broaden the HIV T cell response. Plasmid constructs have been shown to express each gene fragment and of the fragments have been cloned into shuttle vectors, the penultimate manipulation before vector production. Ad5 vectors containing gag or gag fused to one or four ubiquitin genes have been made and studies in cell lines indicate that gag fused to four ubiquitin most efficiently targets the proteasome and thus may be most effective in inducing CD8 responses.

With the ongoing debate regarding the unforeseen outcome of the STEP vaccine trial we have investigated possible reasons why vaccination with adenovirus serotype 5 vectors may increase the incidence of HIV infection in individuals with high pre-existing neutralising antibodies to the virus. Our work suggests that vaccination of individuals with immunity to Ad5 activates Ad-specific CD4 memory cells that have homing molecules that mediate migration to mucosal surfaces where HIV transmission occurs and thus increase the potential number of HIV target cells.

To summarise, our main achievements in this reporting period are:

  • Improved formulation for microneedle patches
  • Successful vaccination of mice with polymer shielded virus
  • Demonstration in mice that cutaneous application of microneedle patches can deliver Ad5 vector encoded antigens to dendritic cells and induce systemic CD8 T cell responses
  • Progress in making Ad vector mini gene constructs
  • Construction of vectors containing ubiquitin-gag fusion genes and demonstration that fusion protein gag is more efficiently targeted to the proteasome.
  • Analysis of the reasons for increased incidence of HIV infection I the STEP HIV vaccine trial.
 
Submitted January 15, 2008 (Interim Report)

This project aims to develop a polymer-coated adenovirus vector vaccine to evade adenoviral neutralizing antibody and to retarget the ‘shielded’ vector to skin dendritic cells (DC). The vaccine will be delivered to the skin by sugar micro needles containing the embedded vector which is formulated into a patch comprising 25-30 needles up to 1500μm in length. The vector in which HIV vaccine genes are engineered is modified to stimulate a broad cytotoxic CD8 T cell response. The T cell vaccine will be given together with a vector coding for HIV-like particles to stimulate antibody and CD4 T cell responses.

Initial studies for delivery of the vector have analyzed the sugar mixture that gives optimal preservation of infectivity whilst maintaining the strength and rigidity of the needles. We find that up to 80% of infectivity can be achieved in vector preparations dried in high sugar concentrations. Vector preserved in a high sugar concentration will be embedded in the core of the needle and an outer coating of sugar at a lower concentration will be added to give the needle the necessary penetration properties.

The polymer shield will protect the adenovirus from neutralizing antibodies but requires a cellular ligand attached to the coating to mediate uptake by dendritic cells. In studies using mouse DC we found that a toll-2 receptor (TLR2) ligand, Pam2Cys, attached to the shield efficiently mediates uptake by DC and induces an activated state in infected DC that is a requirement for effective stimulation of an immune response. Using OT1 TCR transgenic CD8+T cells specific for ovalbumin (aa 256-264) as a model readout system, we observe an increase in both proliferation and differentiation (interferon production and granzyme expression) induced by DC transduced by a Pam2Cys modified Ad5 Ova vector compared with the unmodified Ad5 Ova vector. We are currently assessing the immunogenicity of Pam2Cys-pc-rAd5 vectors in vivo.

The HIV vaccine gene has been fused with the ubiquitin gene and the fusion protein shown to be more efficiently directed to the proteasome, the first step in forming cell surface MHC-antigenic complexes that will stimulate expansion of antiviral T cells. In addition to testing these gag modified constructs in vivo in mice they will be tested in an in vitro human priming system in which DC are pulsed with vaccine vector, cultured with autologous lymphocytes and after several rounds of stimulation the breadth and magnitude of the response measured. The human in vitro priming system has been successfully established using Ad vector containing an unmodified gag gene.

To summarise, our main achievements in this reporting period are:

  • Construction of engineered gag genes and micro-genes, including ubiquitin-linked and HA-tagged fusions.
  • Establishment of a vector production system for adenovirus serotype 11 vectors, and generation of a range of adenovirus serotype 5 and 11 vectors expressing reporter and antigen genes.
  • Comparative evaluation and optimization of formulations for desiccation and dry storage of adenoviral vaccine vectors.
  • Demonstration that a TLR2 agonist retargets polymer coated Ad vectors to murine dendritic cells.
 
Submitted August 1, 2007

This project aims to develop a polymer coated adenovirus vector vaccine to evade adenoviral neutralizing antibody and to retarget the ‘shielded’ vector to skin dendritic cells (DC) The vaccine will be delivered to the skin by sugar micro needles containing the embedded vector which is formulated into a patch comprising 25-30 needles up to 1500μm in length. The vector in which HIV vaccine genes are engineered is modified to stimulate a broad cytotoxic T cell response. The T cell vaccine will be given together with vector coding for HIV-like particles to stimulate antibody and CD4 T cell responses.  Initial studies for delivery of the vector have analyzed the sugar mixture that gives optimal preservation of infectivity whilst maintaining the strength and rigidity of the needles. This will be achieved by embedding the vector in the core of the needle in a high concentration of sugar and adding an outer coating of sugar at a lower concentration to give the needle the necessary penetration properties. The polymer shield will protect the adenovirus from neutralizing antibodies but requires a cellular ligand attached to the shield to mediate uptake by dendritic cells. In studies using mouse DC we found that a toll-2 ligand attached to the shield efficiently mediates uptake by DC and further induces and activated state in infected DC that is a required for effective stimulation of an immune response. The HIV vaccine gene has been fused with the ubiquitin gene and the fusion protein shown to be more efficiently directed to the proteasome, the first step in forming cell surfaces MHC-antigenic complexes that will stimulate expansion of antiviral T cells.

 
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