Abstract
BackgroundInfections 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 FindingsThe 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 CO2 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.ConclusionsAn 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.
Highlights
The Human Immunodeficiency Virus (HIV) continues to threaten human health
The automated production was aimed to be conducted under Good Clinical Laboratory Practice (GCLP)-conditions due to the intended use for HIV vaccine trials
The robotic manipulator arm (RoMa) handles the automation friendly cell culture flasks (RoboFlasksH) within the system mediating the transport from the transfer station to the Flask Flipper and vice versa (Figure 2B)
Summary
The Human Immunodeficiency Virus (HIV) continues to threaten human health. In the year 2010, around 34 million people were living with HIV as reported by the world health organization (UNAIDS World AIDS Day Report, 2011; www. unaids.org). The MRKAd5 vaccine of the STEP and Phambili phase IIb trials consisted of an attenuated adenovirus 5 with inserted gag, pol and nef genes. Both trials were stopped in September 2007 due to enhancement of HIV infections rather than control [6]. The phase III study in Thailand (RV144) used a combination of a recombinant canarypox vector vaccine (ALVAC) and booster injections with recombinant glycoprotein 120 subunit vaccine (AIDSVAX) It induced, with 31.2% efficacy, partial protection [7]. The low level of efficacy observed provided a way to assess whether neutralizing antibodies play an important role in vaccine-induced protection [8,9]. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed
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