Koup Progress Report Abstracts continued
Efforts in Year 5 and the no cost extension (NCE) included the provision of cellular assay services to clinical and NHP trials, conclusion of ICS standardization activities, and optimization of molecular technologies for interrogating CD8+ and CD4+ immune responses. Specifically, we provided ELISpot, ICS and gene arrays to the DCVAX001 clinical trial and coordinated CSF services to this trial of Dr Steinman’s unique dendritic cell targeting vaccine. ELISpot and ICS assays were performed for an NHP trial conducted by the Pantaleo VDC, and over 500 gene arrays were performed by VGTI in support of the Picker (Parks VDC) NHP CMV/LAV trial.
We qualified 7 and 10 color ICS panels for clinical use, and an 8 color panel for NHP use, and successfully transferred each assay from its source lab to another core laboratory. Championed by Drs Roederer and DeRosa, we successfully concluded a head-to head comparison of the HVTN and VIMC ICS assays which demonstrated remarkable comparability. Through additional development efforts at the FHCRC and VRC labs, we now offer a variety of ICS panels to VDCs, enabling the selection of an assay more closely matched to the parameters of interest—CD4+, CD8+, memory markers--in an HIV vaccine trial.
Our molecular immune correlates initiative has now optimized the Fluidigm assay, applied this innovative technology to specimens from the HIV/CMV cohort and the RV144 specimens and determined molecular profiles of antigen specific CD8+ and CD4+ T cells in those sample sets.
Other important accomplishments during this reporting period include:
- Completion of two years of performance monitoring of the standardized IFNγ ELISpot assay across our clinical core labs and harmonized use of the BIDMC ELISpot across NHP labs;
- Transferred and characterized PBMC samples of African origin to the Duke Cell Repository and developed an online specimen request system to make these and other well characterized PBMC specimens available to CAVD members;
- Development of a gene array database from an integrative bioinformatics meta-analysis of immune response profiles from HIV infected subjects, including elite controllers, chronic viremic and chronic treated aviremic subjects, and additional datasets with different viruses (CMV, Yellow Fever); and
- Updated of our assay utilization algorithm, which includes several second tier assays to probe vaccine-induced memory B-cell responses (B-cell ELISpot), and more in-depth analysis of both CD4+ and CD8+ T cells
During the NCE the CTC-VIMC focused on restructuring the consortium for its second 5 year performance period. Going forward, increased emphasis is placed on the core services we provide to the VDCs, with less emphasis on discovery research. Our renamed Clinical Trials Testing Core (CTTC), now under Dr Nicole Frahm’s leadership, and the NHP Core maintain their regular activities of supporting clinical and NHP trials. The CTTC also initiated work on optimizing a B cell ELISpot. Our Molecular Immune Correlates Discovery (MICD) initiative morphed into a MIC Core poised to provide molecular profiling (gene array and Fluidigm) for CAVD clinical and NHP trials. Our Central Cell Repository (formerly the Proficiency Testing Core) continued its mission of providing high quality, characterized PBMC for use in assay development, qualification and comparisons, and for proficiency testing. The Central Reagent Core continues to purchase and distribute peptides and standard reagents to the VIMC laboratories and other CAVD groups. Additionally, plans were finalized for activities of a small scientific discovery program that will assess new approaches to T cell immune monitoring through discovery research.
The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) provides standardized T cell assays to CAVD members through its network of four GCLP-compliant clinical laboratories and three nonhuman primate (NHP) laboratories. The comparability of the IFN-g ELISpot and a seven color ICS
assays performed in our clinical laboratories is assured through a program of assay performance monitoring.
The CTC-VIMC also provides advanced T cell research services and assays, such as epitope mapping and gene arrays, through its discovery team laboratories. CAVD members can access a variety of peptides, including SIVmac239 and E543, and the PTE Global and Clade A pools through our Reagent Core. Our PBMC repository at Duke contains fully characterized specimens from both HIV seropositive
and negative donors, including a growing subset of specimens from Africa, which can be accessed to support assay development and validation projects.
In its fourth year of operation, CTC-VIMC services have been utilized to: support immune monitoring of two clinical trials; assist in the re-analysis of data from HVTN trials; evaluate NHP studies of new vaccine candidates and adjuvants; and facilitate the development of an ADCC assay by our antibody counterpart, the CA-VIMC.
Our scientists have also maintained a robust discovery effort, collaborating to further characterize antigen-specific T cell responses. Our Molecular Immune Correlates Discovery (MICD) team has refined tetramer sorting techniques for CD4 and CD8 cells and is now systematically applying the methodology to heterologous cohorts of human donors and NHP specimens (e.g., HIV+, HIV-/CMV+, and vaccinees.)
Once sorted, clinical and NHP specimens are successively interrogated by TCR sequencing, gene array and qPCR, and advanced single cell technologies (e.g., Fluidigm and CyTOF) to identify new targets for immune correlate analysis and markers for assays. Current efforts have advanced our knowledge of T cell phenotype, function and the correlates of immune protection. CTC-VIMC now stands able to provide state-of-the-art technology for assessing T cell reactivity to the CAVD community.
Year 4 activities for the CTC-VIMC include:
- Supporting the Ho, EuroVac, DC VAC001 and Shattock clinical trials and the Picker (Parks IAVI VDC) NHP trial;
- Qualifying new ICS assays for clinical (7 color) and NHP (8 color) laboratories;
- Quarterly performance monitoring of the standardized IFNγ ELISpot assay in our 4 clinical core labs and harmonizing use of the BIDMC ELISpot across 3 NHP labs; and
- Ramping up the new Molecular Immune Correlates Discovery (MICD) initiative.
Thus, our year 4 agenda balances T cell immunological CAVD infrastructure refinement with an innovative discovery initiative.
A new MICD workgroup comprised of members representing our PBMC Repository (Proficiency Testing Core), NHP Scientific Core and two of our discovery teams (Immune Correlates and the Innate and Mucosal Immunity), will work collaboratively to bring new technology to bear on efforts to further characterize antigen-specific T cell responses. This MICD group will use epitope mapping, tetramer staining and TCR clonotyping to evaluate the breadth and clonality of immune responses in cohorts of clinical and NHP specimens. Goals for this year include characterizing responses in CMV+, HIV- and HIV+ (acute and LTNP) human subjects (including B*57 and A*022 responders) using PBMC and gut biopsies. NHP specimens will include MamuA01 animals that have received DNA/Ad vaccine and others that have received the CMV vaccine. These same specimens will then undergo microarray analysis to determine gene specific responses, which will then be validated through the use of the Fluidigm technology. Use of Fluidigm single cell technology will provide additional information on dynamic range and the heterogeneity of expression.
Efforts in the first half of year 4 have focused on refining the tetramer sorting and staining techniques, epitope mapping repository specimens, and establishing the availability of other specimens for analysis. Currently, we are finalizing a 3 hour tetramer staining and sorting procedure capable of distinguishing CD4 responses, as well as the originally planned CD8 response. Once finalized, cell sorting will begin, followed by clonotyping, microarrays and the Fluidigm analyses. Planning for expansion of this project in year 5, we continue to build alliances with a variety of vaccine development groups to enable testing a broad variety of vaccine responses.
Begun in 2006, the Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) is comprised of 19 of the world’s leading T cell immunologists who work in concert to provide optimized immune testing in support of AIDS vaccine research. The CTC-VIMC is part of the Collaboration for AIDS Vaccine Discovery (CAVD), public private partnership funded by the BMGF with 19 research consortia dedicated to the development of a vaccine for AIDS. Our mission is to facilitate AIDS vaccine discovery by standardizing T cell assays for non-human primate (NHP) vaccine research and clinical trials of vaccine candidates.
Now completing its third year of performance, the CTC-VIMC has standardized the ELISpot assay across all of its central laboratories, and is completing the testing of a new and improved intracellular cytokine staining assay. These two assays will form the foundation for T cell immunologic testing of new vaccines developed within the CAVD. The CTC-VIMC is completing the monitoring of 2 ongoing CAVD clinical trials, and is making plans to evaluate T cell immunogenicity in 2 upcoming trials. The CTC-VIMC has also developed a suite of advanced T cell assays that can be applied to past and future clinical trials. The CTC-VIMC is also embarking on a broad effort to identify molecular immunologic correlates of protective immunity. This effort will apply a range of molecular immunologic techniques to evaluate T cell responses in HIV pathogenesis, HIV/SIV vaccines, and SIV infection, and will allow for the identification of new immune targets of protective immunity to HIV.
As recommended by our Scientific Advisory Board (SAB), the Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) has implemented a new discovery agenda. In this post-Step era, our SAB advised rebalancing our research agenda, increasing effort on the evaluation of alternative T cell assays, with reciprocal reductions of proficiency testing efforts for standardized T cell assays. To this end, the VIMC has initiated studies to compare second tier T cell assays. These studies complement our clinical and NHP core activities and the ongoing research conducted by our discovery teams.
The new assay comparison studies include contrasting the CFSE assay developed by Dr Rafick Sékaly’s group against the Cultured ELISpot developed by Sir Andrew McMichael’s lab, and the Viral Inhibition Assay developed by the IAVI Core lab against the multiparameter flow assay with perforin from Dr Giuseppe Pantaleo’s team. Plans are on target to initiate pilot studies in January 2009. These results will inform finalization of the study protocols for the larger comparisons to be conducted later in the spring.
Another comparison study being planned involves an evaluation of the cryomedia used for the preservation of PBMCs. Undertaken in conjunction with the HSC, our Proficiency Testing Core will split samples from new leukaphresis volunteers at our Duke PBMC Repository and comparatively evaluate the performance of the new HSC cryomedia with that currently used at Duke.
Directed by Dr Mario Roederer from our Immune Correlates Discovery Team, our VIMC has developed new 7 and 10 color panels for multi-parameter flow cytometry for clinical use; similar panels for NHP labs are also progressing. To assure broad utility, the new clinical panels rely on commercially available reagents to identify, quantify and characterize antigen-specific T cells. The 7 color panel includes a viability marker; CD3, CD4, and CD8 (to identify T cell lineages); and IFN-γ, IL-2, and TNF-α (to identify the antigen-specific T cells and their response profiles). The 10 color panel adds CD45RA, CD28, and CCR7 (to identify the particular subset of T cells that is responding). The development, optimization and planned qualification of the new clinical assays have been VIMC-wide activities, involving representatives from all core groups and most discovery teams.
Our Core groups continue to achieve their respective objectives. Both the Certification Validation and Testing Core and the NHP have now standardized ELISpot assays. An evaluation of the IAVI and HVTN ELISpot assays yielded comparable results, enabling use of either assay in upcoming clinical trials, and providing insight into the comparability of results from previously conducted trials that used either assay. Directed by Dr Norm Letvin, the NHP Core, now expanded to 5 NHP laboratories, has harmonized an ELISpot assay across all labs and plans proficiency testing for the spring. Both clinical and NHP PBMC repositories continue to accrue specimens. Efforts are underway to increase the number of human HIV+ and African donors; NHP specimens include monkeys vaccinated in a prime/boost regimen. The VIMC Reagent Core has received Ad5 and HIV peptides to match the VRC inserts and anticipates receipt of SIV E543 and 239 peptides in 2009. These reagents will be made available to the CAVD members through the VIMC CSF website.
The goals of the Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) are (1) to create a consortium of immune testing laboratories within the top HIV vaccine pre-clinical and clinical trials networks and (2) to bring together leading scientists to identify new targets and assays for use in T cell immune monitoring, and to then optimize and standardize the best assays for use in comparing different vaccine platforms.
The scientific core groups and discovery teams that comprise the CTC-VIMC enjoyed significant progress in achieving their objectives during the second year of performance. Specific achievements include:
- establishing the equivalence of two different validated ELISpot assays;
- beginning the development of a multicolor ICS assay using commercially available reagents;
- collaborating with multiple non-human primate (NHP) laboratories to coordinate ELISpot and ICS testing;
- establishing human and NHP PMBC repositories for assay development and proficiency testing;
- determining the utility of PTE peptides for cross-trial comparisons;
- identifying new T-cell functions that correlate with protection from disease and progression;
- optimizing a cultured ELISpot assay;
- assessing the importance of Th17 cells in the mucosal response to gut flora;
- establishing genomic profiles relevant to several vaccine platforms and trials; and
- establishing a collaboration to study the genomics of exposed-uninfected cohorts.
The second year of performance culminated with an annual Scientific Advisory Board and Investigator’s Meeting in Washington, DC where the group charted a revised scope of work for Year 3, directing attention to comparative evaluations of alternative T cell assays, further expansion of the PBMC repositories, and additional collaborations with other CAVD consortia evaluating vaccine candidates and technological advances in the preservation of specimens.*
*The recommendations from the SAB meeting have already been distributed to our Project Officers, Drs Nina Russell and Jose Esparza. A copy of these recommendations can be supplied upon request.
The CTC-VIMC has made significant progress toward establishing standardized ELISpot and multi-color flow cytometry panels during the first six months of this second year of performance. A pilot study comparing the IAVI and HVTN ELISpot assays, directed by our CTV Core (J. Gilmour), has revealed strong concordance between the two assays. Once confirmed in a larger study, this concordance will enable the consortium to deploy either assay in our CTV clinical Core labs with minimal technology transfer and assay cross-validation effort. Further, this demonstration of the consistency in results between the two assays should facilitate comparative interpretation of past vaccine trials conducted across these two HIV clinical trial networks.
A secondary gain afforded by the pilot study is the opportunity it provided for CTC-VIMC collaborators to refine their understanding of the demands of collaborative research. Our VIMC has now established a framework for developing, approving and initiating study plans across consortium collaborators--processes that will facilitate future collaborations, including our new initiative to develop multi-color flow cytometry panels. This ICS effort, spearheaded by the Immune Correlates Discovery Team (M. Roederer and G. Pantaleo), has focused on developing assays that utilize commercially available reagents, flow cytometers with standardized configurations and QC procedures, and concentrates on markers for CD3, CD4 and CD8 T cells, TNFα, IFNγ and IL2, and a marker that can distinguish naïve from memory T cells. Plans are to complete disaster check comparisons of the new 7 color assay across several VIMC labs by March 2008. These simultaneous comparisons with existing ICS assays will assure that the new ICS assay merits further development.
The CTC-VIMC’s other core functions have also progressed. The Proficiency Testing Core’s (PTC) (J Cox) PBMC Repository (T Denny) is now fully equipped, has successfully completed a GCLP audit and has leukopheresed 30 volunteers--half of the target sample. The protocol for including 10 HIV+ subjects in the PBMC bank is completing IRB approval. Another member of the PTC (C Gray) will expand the repository with the addition of PBMCs from HIV+ and HIV- African volunteers. The Reagent Core is finalizing orders for peptides that match the VRC HIV vaccine inserts and evaluating the need for SIV MAC 239 peptides. An electronic order form has been launched on the CAVD CSF web-portal along with descriptive information on the VIMC’s assays. We are optimistic that these materials will increase the visibility of VIMC services available to CAVD members.
Our NHP Core continues efforts to develop its NHP PBMC Repository and anticipates the availability of PBMCs that can be shared with other CAVD members. Work progresses on standardizing an ELISpot assay across the BIDMC, VRC and BPRC labs, while ICS panel development proceeds in parallel with the clinical effort described above. New collaborations with the IAVI and OHSU NHP laboratories enrich the efforts to standardize T cell assays and extend pre-clinical testing capacity.
The efforts of CTC-VIMC Discovery Teams have also advanced in the last six months. Optimization studies conducted by the Long Term T Cell Memory DT have focused on the cultured IFNg ELISpot (10 day expansion of peptide stimulated PBMC assayed by ELISpot). T cell responses specific to common Flu, EBV and CMV CD8 epitopes and to the immunodominant antigen of M. tuberculosis, Antigen 85A were defined in buffy coats using ex vivo IFN-g ELISpot. Mapping of these responses were performed using individual peptides in parallel with peptide pools in three different matrix formats. Peptide-specific T cell responses mapped in buffy coats were also detected reproducibly, by cultured ELISpot. IL-2 titrations (2 manufacturers), culture periods, human AB versus fetal bovine serum, cell concentration and the effect of additional cytokines have also been tested.
The Clade-Specific T Cell DT has designed the Env PTE-C patch peptide set, which complement the global Env PTE peptides to cover deep into PTEs with frequencies greater than 5% in subtype C circulating strains. This new PTE peptide set will enable more accurate assessment of the vaccine-elicited responses for subtype C strains. Design and synthesis of global PTE peptides spanning the HIV-1 Tat, Vif, Vpr, Vpu, and Rev gene products are complete; these peptides currently await QC and testing in proficiency studies.
The Innate and Mucosal Immunity DT has used a multipronged approach to identify effector cells and molecules of innate and mucosal immunity. The Douek group at the VRC used multiparametric flow cytometry to characterize T cells subsets and their functions in different mucosal compartments (gut and broncho-alveolar lavages), the Rosenthal group assessed the induction of innate immunity effector molecules (defensins, SLIPs) in mucosal compartments using primary ovarian cells, and the Sekaly’s group used gene array technology and multiprametric flow cytometry to identify gene signatures in mucosal compartments and in effector molecules of innate immunity associated with potent activation of these cells and antiviral or immunomodulatory function. Bioinformatic analysis will allow the development of focused arrays that can be used in clinical trials to assess the innate and mucosal responses induced by vaccines.
The goals of the Comprehensive T Cell Vaccine Immune Monitoring Consortium are (1) to create a consortium of immune testing laboratories within the top HIV vaccine pre-clinical and clinical trials networks and (2) to bring together leading scientists to identify new targets and assays for use in T cell immune monitoring, and to then optimize and standardize the best assays for use in comparing different vaccine platforms. There are three objectives:
- Establish an international network of four Central and seven Satellite laboratories for T cell immune testing by building Good Clinical Laboratory Practice (GCLP) infrastructure and capacity through coordination, standardization, certification, and Quality Assurance (QA). As part of this effort, Discovery Teams will develop new assays and technologies for monitoring T cell immune responses to HIV vaccines that will be incorporated into the laboratory assay repertoire.
- Establish one Central and up to four Satellite laboratories for pre-clinical evaluation of T cell immune responses to candidate vaccines in non-human primates. As part of this effort, and in coordination with the Discovery Teams, advances in T cell immune monitoring will be incorporated into the assays performed on non-human primate samples.
- Provide administrative and scientific structure that will facilitate the evaluation of new assays developed by the discovery teams leading to rapid validation, reagent standardization, training, and distribution to central laboratories.
The end product of this effort will be a system that allows for the intelligent comparison of T cell immunogenicity of pre-clinical and clinical HIV vaccine platforms.