An Automated HIV-1 Env-Pseudotyped Virus Production for Global HIV Vaccine Trials

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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.

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Most cited references 25

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Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120.

C. A. Derdeyn, J M Decker, J Sfakianos … (2000)

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC(50)) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log(10) higher than the mean IC(50) for CXCR4 (X4) isolates (P = 0. 0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC(50) for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log(10) higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC(50) for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log(10) lower than the IC(50) for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.

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Magnitude and Breadth of the Neutralizing Antibody Response in the RV144 and Vax003 HIV-1 Vaccine Efficacy Trials

David C. Montefiori, Chitraporn Karnasuta, Ying Huang … (2012)

Background. A recombinant canarypox vector expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pro, and membrane-linked gp120 (vCP1521), combined with a bivalent gp120 protein boost (AIDSVAX B/E), provided modest protection against HIV-1 infection in a community-based population in Thailand (RV144 trial). No protection was observed in Thai injection drug users who received AIDSVAX B/E alone (Vax003 trial). We compared the neutralizing antibody response in these 2 trials. Methods. Neutralization was assessed with tier 1 and tier 2 strains of virus in TZM-bl and A3R5 cells. Results. Neutralization of several tier 1 viruses was detected in both RV144 and Vax003. Peak titers were higher in Vax003 and waned rapidly in both trials. The response in RV144 was targeted in part to V3 of gp120.vCP1521 priming plus 2 boosts with gp120 protein was superior to 2 gp120 protein inoculations alone, confirming a priming effect for vCP1521. Sporadic weak neutralization of tier 2 viruses was detected only in Vax003 and A3R5 cells. Conclusion. The results suggest either that weak neutralizing antibody responses can be partially protective against HIV-1 in low-risk heterosexual populations or that the modest efficacy seen in RV144 was mediated by other immune responses, either alone or in combination with neutralizing antibodies.

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Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa.

Susan Allen, J. Suarez Gonzalez, Victoria R Polonis … (2006)

A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.

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Author and article information

Contributors

Jean-Pierre Vartanian: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2012

Publication date (Electronic): 27 December 2012

Volume: 7

Issue: 12

Electronic Location Identifier: e51715

Affiliations

[1 ]Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany

[2 ]Department of Virology, Saarland University, Homburg, Germany

[3 ]Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America

[5 ]ICREA Infection Biology Laboratory, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain

Institut Pasteur, France

Author notes

* E-mail: andreas.meyerhans@ 123456upf.edu

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: SK AS MS-K DAO. Performed the experiments: SK AS MF ASM. Analyzed the data: SK AS MS-K DAO. Contributed reagents/materials/analysis tools: DM. Wrote the paper: AM AS. Conceptional outline and participating by adding key points to the manuscript: AM MS-K DM HVB HZ.

Article

Publisher ID: PONE-D-12-32221

DOI: 10.1371/journal.pone.0051715

PMC ID: 3531445

PubMed ID: 23300558

SO-VID: 66c6dc4c-a8ee-4a6e-82ee-edf3e218b60a

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 16 October 2012

Date accepted : 5 November 2012

Page count

Pages: 10

Funding

This article derived from a collaboration of the Fraunhofer Institute for Biomedical Engineering, the “Global HIV Vaccine Research Cryorepository Consortium” (principal investigator: HVB) and the “Comprehensive Antibody Vaccine Immune Monitoring Consortium” (principal investigator: DM) within the “Collaboration for AIDS Vaccine Discovery”, funded by the Bill and Melinda Gates Foundation (#OPP38580_01). Automation of cell culture and HIV pseudovirus production was established in exclusive cooperation with TECAN funded as a strategic investment by the Fraunhofer Society, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

An Automated HIV-1 Env-Pseudotyped Virus Production for Global HIV Vaccine Trials

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Abstract

Background

Methodology/Principal Findings

Conclusions

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Most cited references 25

Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120.

Magnitude and Breadth of the Neutralizing Antibody Response in the RV144 and Vax003 HIV-1 Vaccine Efficacy Trials

Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa.

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