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Pantaleo Progress Report Abstracts continued

 
Submitted January 31, 2011 (Interim Report)

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors, the strategies implemented in the Vaccine Discovery Core include: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses, b) the generation of replication competent (rc-) poxvirus vectors, and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. During the first half of year 5, the Discovery Core has focused its efforts on establishing industrial standard QA/QC procedures for the generation of clinical grade pre-seeds of the selected NYVAC candidates for GMP production. In parallel, generation of NYVAC constructs combining the replication competent strategy (NYVAC-KC) and multiple gene deletion strategy are well underway. These new constructs could potentially serve as a third generation improved NYVAC candidates if they meet the criteria as defined in the PTVDC selection algorithm.

The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. In the first half of year 5, we have started an immunogenicity study in non-human primates comparing the priming ability of replication deficient NYVAC vs replication competent NYVAC-KC for protein boost with or without DNA. Data is expected by Q2 2011.

 
Submitted September 13, 2010

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors, the strategies implemented in the Vaccine Discovery Core include: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses, b) the generation of replication competent (rc-) poxvirus vectors, and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. During year 4, the Discovery Core has completed the generation of multiple replication competent NYVAC constructs expressing the optimized immunogens, as well as constructs combining the replication competent strategy (NYVAC-KC) and multiple gene deletion strategy targeting at TLR antagonists and combined chemokine and interferons pathways. Further, new approaches on immunogen design based on the program for automated computation of evolutionary conservation and population conservation are under development


The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. In year 4, data from both innate and adaptive immunity filters have identified the lead candidate to be moved forward to NHP and clinical studies. The 2nd NHP study further confirmed that scarification is most likely the best immunization route for replication competent NYVAC and that there is no or little boosting effect when giving multiple immunizations of NYVAC. In addition, initial data have been obtained on the effect of Resiquimod adjuvant on recall T-cell responses. The data indicated that the addition of Resiquimod improved HIV-specific CD8 T-cell responses induced by poxvirus vectors.


For the Clinical Core, initial immunogenicity analysis using IFN-g ELISpot assay has shown better response in terms of 1) number of responders, 2) breadth and 3) magnitude in the 3xDNA arm compared to the 2xDNA arm.

 
Submitted February 5, 2010 (Interim Report)

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors, the strategies implemented in the Vaccine Discovery Core include: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses, b) the generation of replication competent (rc-) poxvirus vectors, and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. During the first half of year 4, the Discovery Core has completed the generation of multiple replication competent NYVAC constructs expressing the optimized immunogens. In parallel, generation of NYVAC constructs combining the replication competent strategy (NYVAC-KC) and multiple gene deletion strategy are well under way. DNA and unmodified NYVAC expressing SIVmac239 GagPolNef and gp140 are also generated. Further, new approaches on immunogen design based on the program for automated computation of evolutionary conservation and population conservation is under development

The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. In the first half of year 4, data from both innate and adaptive immunity filters has identified the lead candidate to be moved forward to NHP and clinical studies. The 2nd NHP study further confirmed that scarification is most likely the best immunization route for replication competent NYVAC and that there is no or little boosting effect when giving multiple immunizations of NYVAC. In addition, initial data has been obtained on the effect of Resiquimod adjuvant on recall T-cell responses.

The data indicated that the addition of Resiquimod improved HIV-specific CD8 T-cell responses induced by poxvirus vectors.

For the Clinical Core, initial immunogenicity analysis using IFN-g ELISpot assay has shown better response in terms of 1) number of responders, 2) breadth and 3) magnitude in the 3xDNA arm compared
to the 2xDNA arm.

 
Submitted September 15, 2009

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors, the strategies implemented in the Vaccine Discovery Core include: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses, b) the generation of replication competent (rc-) poxvirus vectors, and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. During the 3rd year of the program, the Discovery Core has generated 3 combined NYVAC mutants and 3 multi-gene deletion mutants. Based on the large set of in vitro immunological data, we have identified the lead rc-NYVAC vector and are moving rapidly with integrating this strategy with that of gene deletion as well as implementing modifications in the immunogens. Further, pathogenicity studies in new born mice have also demonstrated a satisfactory safety profile of the lead replication competent NYVAC vector.

The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. In year 3, we have refined selection algorithm based on the combination of innate and adaptive immunity filters/assays with transcriptional analysis and pre-clinical evaluation in the NHPs model. Improvements in all innate and adaptive immunity filters and in the expression of the transgene by the newly generated NYVAC mutants compared to unmodified NYVAC is required for a Go decision for moving into further evaluation in the NHPs model. Further we have completed a pilot NHP study with the lead rc-NYVAC. The data from the study demonstrated improved immunogenicity with rc-NYVAC in comparison to unmodified NYVAC and also provided the initial indication of the best immunization route for rc-NYVAC.

For the Clinical Core, all volunteers in the EV03/ANRSVAC20 trial have completed the immunization and the initial immunogenicity results are expected by Q3 2009.

 
Submitted February 1, 2009 (Interim Report)

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors, the strategies implemented in the Vaccine Discovery Core include: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses, b) the generation of replication competent poxvirus vectors, and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. During the 1st half of the 3rd year, several multi-gene deletion NYVAC mutants, replication competent NYVAC mutants and combined (replication competent plus deletion) NYVAC mutants have been successfully generated. In vitro immunogenicity studies are underway to compare and select the “best-in-class” to be moved forward. Furthermore, significant advances have been made with the immunogen design and development of trimeric Env immunogen encoded within NYVAC and DNA is well underway. A quantitative neutralizing antibody assay for the detection of poxvirus antibodies is in advanced phase of development.

Based on the validated pre-clinical filters, ie: a) maturation assays of monocyte- derived DC, b) cytokine secretion from THP-1 macrophage cell line, c) T-cell proliferation assays using CFSE following stimulation with parental and modified NYVAC or MVA and d) expression gene profile by microarray; the Immunology Core has been systematically testing the mutants generated by the Discovery Core using the various immunological filters/assays. The combined use of these assays has allowed the identification of a number of promising mutants and provided guidance on the generation of new mutants combining the different strategies. Furthermore, a pilot immunogenicity study in non-human primates (NHP) has started and will provide us with an indication on the best immunization route as well as the immunogenicity of rc-NYVAC in NHP.

For the Clinical Core, immunizations are completed for the phase II study EV03/ANRSVAC20 and the initial safety and immunogenicity result is expected by Q1 2009.

 
Submitted September 1, 2008

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors, the strategies implemented in the Vaccine Discovery Core include: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses, b) the generation of replication competent poxvirus vectors, and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. During the 2nd year of the program, the Discovery Core has accomplished the standardization of the standard operating procedures (SOPs) and the release criteria for the preparation of the virus stocks, crucial for the comparison of the biological effects of the different NYVAC and MVA mutants. Multiple NYVAC and MVA single and double gene deletion mutants have been generated. Go/No-go decisions for the further development of multiple deletion mutants are taking place based on the characterization performed within the Immunology Core. For the generation of replication competent virus, four viruses with replication competence have been successfully generated. Pathogeneicity and in vitro immunogenicity studies are underway to compare and select the “best-in-class” to be moved forward. Furthermore, significant advances have been made with the immunogen design and prioritization to move the best immunogen forward is underway.

The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. The Immunology Core has accomplished the validation of the following assays/filters: a) maturation assays of monocyte- derived DC, b) cytokine secretion from THP-1 macrophage cell line, c) T-cell proliferation assays using CFSE following stimulation with parental and modified NYVAC or MVA and d) expression gene profile microarray. The combined use of these assays has allowed the characterization of the biologic effects of NYVAC and MVA parental viruses and of gene deletion NYVAC & MVA mutants. In addition, significant advances have been made with the generation of the polyallelic peptide and testings are ongoing within the Immunology Core.

For the Clinical Core, the phase II study EV03/ANRSVAC20 is fully enrolled and the initial safety and immunogenicity result is expected by Q1 2009.

 
Submitted January 31, 2008 (Interim Report)

The overall goal of PTVDC is to design and bring forward “best-in-class” poxvirus-based (NYVAC and MVA) HIV vaccine candidates with an enhanced ability to induce HIV-specific T cell responses.

The strategies implemented in the Vaccine Discovery Core includes: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses; b) the generation of replication competent poxvirus vectors; and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. For the last reporting period, the Discovery Core has accomplished the standardization of the SOPs and the release criteria for the preparation of the virus stocks which is crucial for the comparison of the biological effects of the different NYVAC and MVA mutants. With regard to the generation of gene deletion mutants, two additional single gene deletion mutants have been generated. Go/No-go decisions for the further development of double-deletion mutants are taking place based on the characterization performed within the Immunology Core. For the generation of replication competent virus, the genes restoring replication competence have been successfully inserted into NYVAC-C, and safety data generated in new-born mice show that the replication competent NYVAC has a pathogenicity identical to the non-replication competent MVA. Furthermore, significant advances have been made with the immunogen design and prioritization to move the best immunogen forward is underway.

The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. The Immunology have accomplished the validation of the following assays/filters: a) maturation assays of monocyte-derived DC, b) cytokine secretion from THP-1 macrophage cell line, c) T-cell proliferation assays using CFSE following stimulation with parental and modified NYVAC or MVA and d) expression gene profile microarray. The combined use of these assays has allowed the characterization of the biologic effects of NYVAC and MVA parental viruses and of single gene deletion NYVAC mutants. In addition, significant advances have been made with the generation of the polyallelic peptide and is ready to be tested within the Immunology Core.

 
Submitted September 1, 2007

In order to increase the immunogenicity by at least 10 fold of the poxvirus vectors (NYVAC, MVA and ALVAC), the strategies implemented in the Vaccine Discovery Core included: a) the deletion of virus-specific genes encoding proteins that have been implicated in the evasion of innate immune responses; b) the generation of replication competent poxvirus vectors; and c) the generation of a combined (multiple gene deletion mutant plus replication competent) poxvirus vector. To date, six out of the ten planned NYVA-C-C/MVA-C mutants with single deletions have been generated and the genes restoring replication competence have been inserted into NYVAC-C and the final construct is expected to be ready in Q3 2007. Evaluation of the optimized expression of gag, pol, nef and env genes and different optimization algorithms are being developed. Vaccinia-specific CD4 and CD8 T-cells have been quantified and a large number of vaccine-specific epitopes have been identified. Preliminary evidence for the adjuvant effect of MegaCD40L on T-cell responses has been obtained. The Immunology Core has the role of developing and applying pre-clinical filters to guide the selection of the newly developed vectors. The experimental methods for the isolation of different subsets of DC’s and of direct and indirect presentation are being developed. Strategies to quantify the total number of antigen-specific CD4 and CD8 T-cells have been developed and are in the process of validation. Protocols for the gene array using the Illumina platform have been standardized, validated and ready to determine the gene expression profile of vaccine induced T-cell responses.

 
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