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von Briesen Progress Report Abstracts continued

 
Submitted January 31, 2011

The goal of the "Global HIV Vaccine Research Cryorepository (GHRC)" project is to establish a central cryorepository for the advanced and standardized banking of all types of specimens necessary for the development of new vaccine candidates and to develop improved cryoprocedures and cryoequipment. Elements of this technology are miniaturized cell containers, which allow the partial withdrawal of sample material without thawing the remaining samples and which can be sent worldwide in a frozen state. Cryodevices for well-defined freezing and thawing processes are completing the concept. The encoded double data storage represents another element of the cryobank technology and takes place for one in a central database and second attached to the frozen samples. This principle and its technical solution provide nearly complete mismatch-proof which is essential for any sample storage. The deep-freeze technology is completed by a suitable databank and cryobank control architecture that ensures optimal logistical workflows.

In the reporting period, the HIV Specimen Cryorepository (HSC) has continued to improve and deploy standardized workflows, cryoprocedures and the newly developed equipment. The mass-production of HIV-1 Env-pseudoviruses is ongoing and reagents are constantly distributed to the CA-VIMC core and regional labs. Within the “Study 0002”, which has been completed in November 2010, 48,721ml of 67 different pseudoviruses had been produced. In this context, a validation of the automated pseudovirus production system took place last November and excellent progress was attested. Furthermore, the collaboration with the CAVIMC is currently extended to the production of Infectious Molecular Clones (IMC) by HSC. As the CAVD community is aware of our of bioreagent production, we received different requests for the production of pseudoviruses and antibodies at the annual CAVD meeting in Seattle. Regarding cryoprocedures, HSC developed a new cryomedia generation based on synthetic additives, chemically fully defined, endotoxin-free and xeno- and protein free in 2010. The media show high PBMC recovery and viability and they are currently tested in the labs of different GHRC partners.

As to the cryotechnology, the repository and the new developments become generally known. In July 2010, the minister of health in the Cape Region of South Africa, Theuns Botha, visited the HSC to get acquainted with our repository and new technologies. His visit resulted in the shipment of a mobile BSL-3 lab to Cape Town in November 2010. Likewise a visit of the German Federal Minister of Health, Philipp Roesler, is scheduled for 8 March 2011. Also, in cooperation with the WHO, the organization of seminars to introduce the new cryoequipment in Brazil and China has started recently.

Finally, the activities to largely increase the biobank at FhG-IBMT have started in autumn 2010. The extension will be funded by the European Union and the Saarland Government with 13 Mio Euro and also includes a scale-up of the BSL-3 facility.

 
Submitted June 30, 2010

The goal of the GHRC project (Global HIV Vaccine Research Cryorepository ) is to establish a state-of-the-art specimen repository where samples and reagents relevant for HIV vaccine development are stored under controlled low-temperature conditions and to provide high quality specimens, tools and procedures, having a direct benefit to the CAVD community. Within the first three years (project phase 1), the HSC has successfully developed these processes, cryoequipment and bioreagents. The past year has been used to employ, improve and distribute the new cryotechnology platform, to develop new cryomedia and to establish new cooperations in the field of R&D as well as for technology transfer.

Special attention has been given to link the HSC with the CHAVI laboratories and repositories. The cooperation has been discussed in six face-to-face meetings at HSC in Sulzbach, Duke University and NICD in Johannesburg. The meetings at Duke University have also been used to foster the cooperation with the CAVIMC. As a first part of the cryotechnology transfer, a “Port Reader Station” has been transferred to David Montefiori’s lab. It is used to read out our smart cryovials automatically and to import the sample data directly into the lab management system. Thus, one part of the sample-data-sample loop has been successfully integrated.

The research in cryomedia and improved cryoprocedures led to the development of two further xeno-free cryomedia without any animal or human components (e.g., serum) and three labs ordered the media in 2010 to evaluate them for their research purposes. Furthermore, a crossconsortia lab study to evaluate the media for PBMC cryopreservation started in November 2009 with the CA-VIMC lab at the University of Birmingham (Christina Ochsenbauer). The pseudovirus production is continuing successfully and since September 2009, several test stocks have been produced with the automated cell culturing system. The efforts to certify the system under GCLP have been strengthened with the help of external experts and the goal is to get the system fully optimized and validated in November 2010. Finally, with 44 material shipments since August 2009, the envisioned HSC service function has been strongly used by the CAVD.

Summing up, all activites could be executed successfully, and promising cooperations have been initiated, both of which was again emphasized by our External Advisory Board. For the coming year, we would like to introduce our scalable biobanking expertise into other areas (e.g., Gates Foundation programs as Malaria, TB, etc.) and to initiate the discussion about future possibilities to support the CAVD program.

 
Submitted March 1, 2010 (Interim Report)

The goal of the "GHRC - Global HIV Vaccine Research Cryorepository" project is to establish a central cryorepository for the advanced and standardized banking of all types of specimens necessary for the development of new vaccine candidates. Elements of this technology are miniaturized cell containers, which allow the partial withdrawal of sample material without thawing the remaining samples and which can be sent worldwide in a frozen state. Cryodevices for well-defined freezing and thawing processes as well as an operational cryobank is completing the entire concept. The encoded double data storage forms another element of the cryobank technology and takes place for one in a central database and second attached to the frozen samples. This principle and its technical solution provide nearly complete mismatch-proof which is essential for therapeutical sample storage. The deep-freeze technology is completed by suitable databank and cryobank control architecture that ensures optimal logistical workflows.

Over the past three years, the HIV Specimen Cryorepository (HSC) project has successfully developed, delivered and executed processes, prototypes, and fully operational hard- & software solutions supporting the Collaboration for AIDS Vaccine Discovery (CAVD).

In the current project phase, our goal is to implement these developments and solutions within
other consortia. In this context, Tom Denny (Duke University) visited the HSC in autumn 2009 to get to know the GHRC technology and equipment. In addition, a meeting with Bart Haynes (Duke University) took place during the CAVD Annual Meeting 2009 in order to discuss the cooperation possibilities of the CHAVI and CAVD sample repositories. Both partners stressed their interest in a collaboration regarding sample and data exchange and harmonization.

Thus, a site visit of a GHRC delegation at Duke University is planned for March 2010 to discuss and continue with these issues. Since sample tracking and IT issues play a crucial role, this
topic will be reviewed during a “CHAVI and CAVD relational database organization, interactions and data mining methods“ meeting in May 2010.

With respect to the cryobiology, three consortia ordered the new GHRC serum free cryomedia in 2010 to test them in their research.

As to the pseudovirus production, first stocks have been produced by the automated production system Cellerity. The focus lies now on the optimization and validation of the processes according to GCLP.

 
Submitted September 30, 2009

The goal of this project is to establish a state-of-the-art specimen repository (Global HIV
Vaccine Research Cryorepository - GHRC) where samples and reagents relevant for HIV vaccine development can be stored under controlled low-temperature conditions, for longterm
preservation and for sharing among collaborating laboratories. After comprising the entire laboratory-cryo-chain in the second project year, the third year has been used to test and optimize the operability of the new cryotechnology platform with the goal to approve the operations. Likewise, the research activities and existing cooperations have been fostered. Serum free cryomedia and improved cryoprocedures, which were developed within the first years, were further improved and tested in a multi-center study (“PBMC Cryo Project”) in collaboration with the Duke University. We found that the serum free GHRC-CryoMedia are appropriate to substitute the common used FCS containing cryomedia. Regarding the cryoequipment development, the volume production of several designs and prototypes of cryosubstrates, which have been engineered in the first years, has started. The second generation of access tower and workbench prototypes has been further investigated and optimized. Six platforms of the third generation were installed at HSC for the storage and handling of the GHRC samples. Simultaneously, the cryoelectronic tank infrastructure has evolved into the equipment of the access towers with 24 electronic rack systems. The specimen preparation workflow management system ChameleonLab has been further developed and adopted to the latest SOPs and the new cryodevices have been integrated into the workflow management system. Concerning the sample collection, 1058 types of sample materials are actually stored in the cryobank and can be ordered via the online inventory www.ghrc.info. Within the cooperation between CA-VIMC and HSC to produce standardized pseudoviruses, 22,210 vials of more than 60 different pseudovirus strains (Clade A, B, C, A/E, B/C) have been produced under GCLP conditions. Repeated shipments were sent to 10 different CA-VIMC core and regional laboratories. According to the Global Access Strategy and in the spirit of the Global Vaccine Enterprise, all
services and developments are so far freely available to CAVD consortia.

 
Submitted February 1, 2009 (Interim Report)

The goal of the "GHRC - Global HIV Vaccine Research Cryorepository" project is to establish a central cryorepository for the advanced and standardized banking of all types of specimens necessary for the development of new vaccine candidates. Elements of this technology are miniaturized cell containers, which allow the partial withdrawal of sample material without thawing the remaining samples and which can be sent worldwide in a frozen state. Devices for well-defined freezing and thawing as well as a cryobank concept go along with that. The encoded double data storage forms another element of the cryobank technology and takes place for one in a central database and second attached to the frozen samples,. This principle and its technical solution provide nearly complete confusion-proofness which is essential for therapeutical sample storage. The deep-freeze technology is completed by suitable databank and cryobank control architecture that ensures optimal logistical workflows. Furthermore, the access to clinical specimens derived from "early infections", i.e., from seroconverters, is a second goal of the consortium. Therefore, the collaboration with Primary Sites in developing countries is considered crucial.

After installation of the GHRC project in the first year, the entire laboratory-cryo-chain could be closed in the second year. Equipment and procedures, which have been developed in this period, have been implemented and are in use now. All milestones which are due from month 25 to month 30 could be met as outlined in the proposal. The progress compared to these milestones is summarized below:

Several prototypes of new cryosubstrates have been produced, tested and redesigned. A final version of the tubes has been developed and will be commercially available in spring 2009. The second generation of industrially made cryoequipment prototypes has been developed and evaluated in 2008. As the devices are ready for general use, the South African Primary Site at Stellenbosch University in Cape Town has been fully equipped with an ASKION Access Tower and Freezer Handling Box, a storage tank and the laboratory management system ChameleonLab in October 2008. Furthermore, a fully automated handling solution for the cryosubstrates has been developed in an external project. The data management work in the third project year concentrates on the interface between the Central Cryobank Information System (EurocryoDB), and the other ICT related components of the HIV Cryo-Technology Platform (i.e., cryosubstrate with flash memory, lab workflow system ChameleonLab, smart tank, etc.). In this context, different tools and interfaces were released and we continued the efforts to link our data with other relevant HIV information sources and external services such as the ATLAS system of the VISC. Within the large scale pseudotype virus production, 15 different pseudoviruses have been manufactured and the Neutralization Serotype Discovery Program conducted by the CA-VIMC has started. To automate the pseudovirus production, a complete cell culture and transfection automation system has been installed and GCLP conformity is being implemented. Finally, SOPs for optimized cryopreservation have been sent to the Primary Sites and other CAVD labs for evaluation and standardization of procedures.

 
Submitted September 1, 2008

The goal of this project is to establish a state-of-the-art specimen repository (Global HIV Vaccine Research Cryorepository - GHRC) where samples and reagents relevant for HIV vaccine development can be stored under controlled low-temperature conditions, for longterm preservation and for sharing among collaborating laboratories.

During the second year, the entire laboratory-cryo-chain could be comprised as the following conditions for the project have been set. Due in the cooperation between CA-VIMC and HSC to produce standardized pseudoviruses, Good Clinical Laboratory Practice (GCLP) had been introduced at the laboratory and the approval to work with GMO was obtained in year two. With regard to scientific issues, different combinations of serum free cryomedia and improved cryoprocedures were tested and interim SOPs for freezing and thawing procedures were developed within the first year. These media and SOPs were further developed in the second year. Two cryomedia batches with different  formulas have been manufactured by the company PAA Laboratories GmbH according to certified standards (GMP). The media are currently tested within the labs of our consortium partners and will then be tested within other CAVD labs.

Different designs and prototypes of cryosubstrates have been developed in the first year. The injection molding production started in the second year and we are cooperating with the newly founded company Perma Cryo Technology (PCT) to produce the substrates industrially. Regarding the cryoequipment development, we were able to win the company ASKION to enter into this development during the first project year. The first company-made prototypes of the cryoequipment had been delivered in December 2007 and the second generation of prototypes had been delivered in July 2008. The new version of EurocryoDB was evaluated and released. Simultaneously, we completed the first version of the GHRC Web Portal which offers a specimen catalog to search and order HIV specimens of the GHRC cryorepository. Concerning the sample collection, 419 types of samples are actually stored in the cryobank. These can be ordered via the online inventory www.ghrc.info. Furthermore, we reacted to needs emerging within the CAVD consortium by offering three newly defined activities, which had not been planned at the outset of the project:

  • The production of monoclonal antibodies
  • Centralized production of GCLP conform HIV-1 Env-pseudovirus stocks
  • PBMC cryo project for standardized processing and cryopreservation between HSC, CA-VIMC and CTC-VIMC has been initiated in July 2008.

According to the Global Access Strategy and in the spirit of the Global Vaccine Enterprise, all services and developments are so far freely available to CAVD consortia.

 
Submitted January 31, 2008 (Interim Report)

The goal of the "GHRC - Global HIV Vaccine Research Cryorepository" project is to establish a central cryorepository for the advanced and standardized banking of all types of specimens necessary for the development of new vaccine candidates. Elements of this technology are miniaturized cell containers, which allow the partial withdrawal of sample material without thawing the remaining samples and which can be sent worldwide in a frozen state. Devices for well-defined freezing and thawing as well as a cryobank concept go along with that. The encoded double data storage, which takes place both for one in a central database and two attached to the frozen samples, forms another element of the cryobank technology. This principle and its technical solution provides nearly complete confusion-proof which is essential for therapeutical sample storage. The deep-freeze technology is completed by suitable databank- and cryobank- control architecture that ensures optimal logistical workflows. Furthermore, the access to clinical specimens derived from "early infections", i.e., from seroconverters, is a second goal of the consortium. Therefore, the collaboration with Primary Sites in developing countries is considered crucial. The first year has been used to install the GHRC project, i.e., to expand the cryofacility, to handle infectious material under BSL-3 condition, to develop equipment design concepts and to define processes. These processes and developments now result in first prototypes and specifications. All milestones which are due from month 13 to month 18 could be met as outlined in the proposal. The progress made thus far compared to these milestones is displayed in the summary below:

The installation of the P3-laboratory and the integration of the HIV cryobank into the FhG-IBMT cryobank was completed in the summer of 2007. Several prototypes of new cryosubstrates have been produced, tested and redesigned. The latest version is actually under evaluation. With respect to cryoequipment, the first companymade prototypes of the freezer handling box and access tower system for cryotanks have been produced and are actually under evaluation. The key element for the electronic cryotank infrastructure is a new rack system for long-term cryostorage. As to the information system, the GHRC’s ICT infrastructure consists of the Central Cryobank Information and Specimen Administration System eurocryoDB, the Specimen Preparation Workflow Management System ChameleonLab for the HIV cryo laboratory and the GHRC Web portal. An extended version of eurocryoDB was released in October 2007. In a next step the online registration of HIV specimens will be implemented. The HSC, as central service facility, has proved to be well suited for the pseudotype virus stock production and has started this process. As the pseudotype production for large scale vaccine trials needs automation, a complete cell culture and transfection automation system is envisioned for this purpose.

 
Submitted September 1, 2007

The goal of this project is to establish a state-of-the-art specimen repository (Global HIV Vaccine Research Cryorepository - GHRC) where samples and reagents relevant for HIV vaccine development can be stored in controlled low-temperature conditions, for long-term preservation and for sharing among collaborating laboratories. The following activities happened during the first project year:

The installation of a P3-laboratory could be completed within the first project year. After a planning phase of 4 months, the construction started in the end of January 2007 with an extraordinary tight work schedule. All operations could be managed without any delay within only 6 ½ months. On August 17, the laboratory has been approved by local authorities according to German law to handle infectious agents of category 3.

With regard to scientific issues, different cryomedia were tested and interim SOPs for freezing and thawing procedures of PBMCs with increased survival rate and SOPs for ELIspot assays with fresh or frozen PBMCs are available. Different designs and prototypes of cryosubstrates have been developed and prototypes of the HIV cryosubstrates are available. An advanced web based information, management and automation system for innovative HIV cryobanking has been developed, which supports in particular the HIV Cryotechnology Platform with its advanced cryocomponents (i.e. cryosubstrate with flash memory, smart tanks, etc). As to sample collection, we have just initiated the first sample shipments of other CAVD consortia. Regarding the Primary Sites, five sites could be selected and evaluated. The South African site has already obtained the ethical approval for this study by the local authorities and has started to collect samples for CAVD. Furthermore, contacts to the German HIV Network of Competence and the German seroconverters study could be established. Newly defined activities are going to be implemented, such as centralized monoclonal antibody production and pseudovirus stock production for neutralization assays. Finally, all coordination &management activities and milestones, as set-up of consortium agreement and establishment of External Advisory Board, could be met within the first project year.

 
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