An automated HIV-1 Env-pseudotyped virus production for global HIV vaccine trials.
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2012-01
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Abstract
Background
Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed.Methodology/principal findings
The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO(2) incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity.Conclusions
An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.Type
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Schultz, Anke, Stefanie Koch, Martina Fuss, Angela S Mazzotta, Marcella Sarzotti-Kelsoe, Daniel A Ozaki, David C Montefiori, Hagen von Briesen, et al. (2012). An automated HIV-1 Env-pseudotyped virus production for global HIV vaccine trials. PloS one, 7(12). p. e51715. 10.1371/journal.pone.0051715 Retrieved from https://hdl.handle.net/10161/33635.
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Scholars@Duke
Marcella Sarzotti-Kelsoe
Ongoing Applied Activities
•I direct a Global Quality Assurance Program, which I developed and pioneered here at Duke University, to oversee compliance with Good Clinical Laboratory Practice Guidelines in three HIV vaccine trial networks (CHAVI, CAVD, Duke HVTN, EQAPOL, Duke VTEU) involving domestic and international laboratory sites.
•I also direct a Global Proficiency Testing Program for laboratories testing for neutralizing antibody function in individuals infected with HIV or vaccinated against HIV. The Program was launched in 2009.
•I provide assistance and oversight for endpoint assay standardization, qualification and validation, as well as for the QSU of the GMP facility at DHVI, which will manufacture HIV vaccine products for first-in-man Phase I trials.
Past Basic Research
•Development of T cell responses in neonates.
•Neonatal T cell receptor Vβ repertoire diversity in the peripheral T cell pool.
•The role of heat shock protein, as a natural adjuvant, at eliciting innate and adaptive immune responses.
•Development of the T cell receptor repertoire in naïve, immunodeficient infants, given bone marrow or thymic transplantation.
•Thymic output, T cell diversity and T cell function in long-term human SCID chimeras.
•Telomere length in T cells from SCID chimeras.
David Charles Montefiori
Dr. Montefiori is Professor and Director of the Laboratory for HIV and COVID-19 Vaccine Research & Development in the Department of Surgery, Division of Surgical Sciences at Duke University Medical Center. His major research interests are viral immunology and HIV and COVID-19 vaccine development, with a special emphasis on neutralizing antibodies.
Multiple aspects of HIV-1 neutralizing antibodies are studied in his laboratory, including mechanisms of neutralization and escape, epitope diversity among the different genetic subtypes and geographic distributions of the virus, neutralizing epitopes, requirements to elicit protective neutralizing antibodies by vaccination, optimal combinations of neutralizing antibodies for immunoprophylaxis, and novel vaccine designs for HIV-1. Dr. Montefiori also directs large vaccine immune monitoring programs funded by the NIH and the Bill & Melinda Gates Foundation that operate in compliance with Good Clinical Laboratory Practices and has served as a national and international resource for standardized assessments of neutralizing antibody responses in preclinical and clinical trials of candidate HIV vaccines since 1988.
At the onset of the COVID-19 pandemic he turned his attention to SARS-CoV-2, with a special interest in emerging variants and how they might impact transmission, vaccines and immunotherapeutics. His rapid response to emerging SARS-CoV-2 variants of concern provided some of the earliest evidence of the potential risk the variants pose to vaccines. In May 2020, his laboratory was recruited by the US Government to lead the national neutralizing antibody laboratory program for COVID-19 vaccines.
His laboratory utilizes FDA approved validated assay criteria to facilitate regulatory approvals of COVID-19 vaccines. He has published over 750 original research papers that have helped shape the scientific rationale for antibody-based vaccines.
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