A novel method for the titration of recombinant virus stocks by ELISPOT assay

Cheong, Siew Chiat; Clément, Nathalie; Velu, Thierry; Brandenburger, Anne-Nicole

doi:10.1016/s0166-0934(03)00061-2

Cited by 6 publications

(4 citation statements)

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“…The sera were classified for neutralizing activity as described in Section 3.6. Cheong et al (2003) used an ELISPOT approach to titer stocks of a recombinant virus expressing human IL-2 by plating infected cells onto anti-IL-2 Ab coated ELI-SPOT plates. In a novel application for a neutralization assay for the SARS coronavirus, an ELISPOT analyzer was used to count whole cells actually growing on ELISPOT plate membranes and infected with a pseudovirus, which also encoded a Lac Z gene that made cells blue upon staining with X-gal (Han et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Novel microneutralization assay for HCMV using automated data collection and analysis

Abai

Smith

Wloch

2007

Journal of Immunological Methods

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In addition to being sensitive and specific, an assay for the assessment of neutralizing antibody activity from clinical trial samples must be amenable to automation for use in high-volume screening.To that effect, we developed a 96-well microplate assay for the measurement of HCMV-neutralizing activity in human sera using the HCMV-permissive human cell line HEL-299 and the laboratory strain of HCMV AD169. The degree to which neutralizing antibodies diminish HCMV infection of cells in the assay is determined by quantifying the nuclei of infected cells based on expression of the 72 kDa IE1 viral protein. Nuclear IE1 is visualized using a highly sensitive immunoperoxidase staining and the stained nuclei are counted using an automated ELISPOT analyzer. The use of Half Area 96-well microplates, with wells in which the surface area of the well bottom is half the area of a standard 96-well microplate plate, improves signal detection compared with standard microplates and economizes on the usage of indicator cells, virus, and reagents. The staining process was also streamlined by using a microplate washer and data analysis was simplified and accelerated by employing a software program that automatically plots neutralization curves and determines NT 50 values using 4-PL curve fitting.The optimized assay is not only fast and convenient, but also specific, sensitive, precise and reproducible and thus has the characteristics necessary for use in measuring HCMV neutralizing activity in the sera of vaccine trial subjects such as the recipients of Vical's HCMV pDNA vaccine candidates.

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Section: Discussionmentioning

confidence: 99%

Novel microneutralization assay for HCMV using automated data collection and analysis

Abai

Smith

Wloch

2007

Journal of Immunological Methods

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“…Vector titres were determined by an ELISPOT assay and are expressed as transducing units (t.u.) (Cheong et al, 2003). A slight increase in vector production was observed (two-to threefold) at day 1 after transfection in hypoxia (6 % O 2 ), but titres remained lower than those produced in standard conditions (day 3, 20 % O 2 ).…”

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confidence: 89%

Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

Servais

Caillet-Fauquet

Draps

et al. 2006

Journal of General Virology

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Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1a) that interacts with a 'hypoxia-responsive element' (HRE), the consensus sequence of which ( A / G CGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.The parvovirus Minute virus of mice, MVM(p), replicates preferentially in oncogenic-transformed cells, where it completes a lytic life cycle (Rommelaere & Cornelis, 1991). This property is exploited in gene-therapy vectors derived from autonomous parvoviruses (Russell et al., 1992;Cornelis et al., 2004). The MVM(p) genome is composed of two transcription units, encoding two sets of viral proteins, NS (non-structural) and VP (capsid), under the control of the P4 and P38 promoters, respectively (Cotmore et al., 1983). The non-structural protein NS1 plays an important role in viral DNA replication (Nüesch et al., 1998), is toxic in transformed cells (Caillet-Fauquet et al., 1990) and is required for transactivation of the P38 promoter, which is otherwise only expressed at a very low basal level (Rhode, 1985).The mechanisms underlying the preferential expression of MVM(p) in transformed cells are only poorly understood, but it is at least in part linked to the upregulation of the P4 promoter in these cells compared with normal cells. We therefore wanted to determine whether P4 activity could be further enhanced and whether this could improve vector production. We have explored the possibility of producing MVM vectors under low oxygen tension (hypoxia). Gene expression and production of the human erythrovirus B19 virus are increased under these conditions (Caillet-Fauquet et al., 2004;Pillet et al., 2004). Regulation of promoter activity in hypoxia occurs through the interaction of the transcriptional regulator hypoxia-inducible factor (HIF-1) with a 'hypoxia-responsive element' (HRE) (Semenza, 2001). The MVM(p) promoter contains four putative HREs (consensus sequence A / G CGTG), two in each of the sense and antisense orientations.The presence of the HRE in the promoter region is necessary, but not sufficient, for promoter regulation. Other cellular elements are probably...

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“…This method is based on the expression of the transgene and not DNA amplification, as is the case for in situ hybridisation. It can therefore not be excluded that these vectors do have a slight defect in DNA amplification that cannot be detected by Southern blotting but that prevents the accumulation of sufficient amounts of vector DNA to be detected by in situ hybridisation [31]. The titres of these stocks are equivalent to those of first-generation vectors and 70% of them are free of RCV compared with 0% for first-generation stocks produced under the same conditions.…”

Section: Downstream Of the Transgene A) Defective Particlesmentioning

confidence: 99%

Autonomous parvovirus vectors: preventing the generation of wild-type or replication-competent virus

Brandenburger

Velu

2004

J. Gene Med.

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The preferential expression of autonomous parvoviruses in tumour cells and their oncolytic activity has attracted attention to the potential use of these viruses as vectors for cancer gene therapy. Moreover, they are non-pathogenic in adult animals and they seem to be associated with low or no immunogenicity. Other interesting features are their episomal replication and high stability. Vectors derived from the autonomous parvoviruses MVM(p) or H1 express proteins that can directly or indirectly interfere with tumour development. They retain cis- and trans-acting sequences required for viral DNA amplification; the transgene replaces part of the capsid coding genes. Their development has been hampered by low titres and contamination with replication-competent virus (RCV) that is generated through homologous recombination with helper plasmids. Several approaches have been used to avoid recombination between vectors and helpers. In most instances, reducing the homology up- or downstream of the transgene in either the vector or the helper did not significantly affect RCV production. However, completely eliminating homology downstream of the transgene, splitting VP genes on different helpers or pseudotyping vectors resulted in the production of RCV-free stocks. Although VP-containing particles could sometimes be identified in these stocks by in situ hybridisation, they did not amplify and are therefore not true RCV. The integration of capsid-coding sequences into packaging cells also reduced contamination by RCV and allowed for the amplification of vectors through serial infections. Great progress has been made recently towards the generation of truly RCV-free stocks of vectors derived from autonomous parvoviruses H1 and MVMp. Combining these new vectors with a new packaging cell line should greatly facilitate their development.

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A novel method for the titration of recombinant virus stocks by ELISPOT assay

Cited by 6 publications

References 10 publications

Novel microneutralization assay for HCMV using automated data collection and analysis

Novel microneutralization assay for HCMV using automated data collection and analysis

Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

Autonomous parvovirus vectors: preventing the generation of wild-type or replication-competent virus

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