Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Gardner, Thomas J.; Bolovan-Fritts, Cynthia; Teng, Melissa W.; Redmann, Veronika; Kraus, Thomas; Sperling, Rhoda; Moran, Thomas M.; Britt, William J.; Weinberger, Leor S.; Tortorella, Domenico

doi:10.1128/cvi.00644-12

Cited by 20 publications

(35 citation statements)

References 64 publications

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“…We examined protein expression of immediate early genes by immunoblot (Figure 1). Consistent with published data (Gardner et al, 2013; Teng et al, 2012), both IE1 and IE2-YFP proteins were detected as early as 6 hours post-infection (hpi) and through 24hpi (Figure 1a, lanes 2-4 and 6-8). Mock-infected cells (Figure 1A, lanes, 1 and 5) validated the specific detection of viral gene products and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) immunoblot confirmed equivalent protein loading (Figure 1A, lanes 9-12).…”

Section: Resultssupporting

confidence: 92%

“…A CMV strain of AD169 engineered to express a viral chimeric protein consisting of the IE2 protein product and yellow fluorescent protein (YFP) (AD169 IE2-YFP ) was employed to visualize and quantify virus-infected nuclei. Previous studies indicated that the AD169 IE2-YFP demonstrated similar growth kinetics to the parental AD169 strain (Gardner et al, 2013). Our initial experiment examined mock-infected and AD169 IE2-YFP -infected human lung fibroblasts (MRC5) (multiplicity of infection (MOI): 3).…”

Section: Resultsmentioning

confidence: 83%

“…CMV AD169 IE2-YFP and TB40/E FLAG-YFP were propagated as previously described (Gardner et al, 2013). Infectious virus yield was assayed on human fibroblasts by median tissue culture infective dose (TCID 50 ).…”

Section: Methodsmentioning

confidence: 99%

“…Following a one-hour inoculation cells were washed with media and then fresh media without drug was added back to the wells. At 18 hpi, the plates were analyzed with an Acumen e X3 cytometer for the number of IE2-YFP positive cells/well based on IE2-YFP fluorescent intensity/well (Gardner et al, 2013). Fluorescent emission above 2 standard deviations of background was registered as positive signal.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, assessment of IE gene expression can serve as a rapid proxy for the measurement of CMV infection. We and others have utilized CMV virus strains encoding fluorescent chimeric proteins to measure IE expression in infected cells (Gardner et al, 2013; Straschewski et al, 2010). …”

Section: Introductionmentioning

confidence: 99%

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Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

et al. 2015

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Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169IE2-YFP) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z′ scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

et al. 2015

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Direct enhancement of viral neutralising antibody potency by the complement system: a largely forgotten phenomenon

Mellors,

Carroll

2024

Cell. Mol. Life Sci.

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Neutralisation assays are commonly used to assess vaccine-induced and naturally acquired immune responses; identify correlates of protection; and inform important decisions on the screening, development, and use of therapeutic antibodies. Neutralisation assays are useful tools that provide the gold standard for measuring the potency of neutralising antibodies, but they are not without limitations. Common methods such as the heat-inactivation of plasma samples prior to neutralisation assays, or the use of anticoagulants such as EDTA for blood collection, can inactivate the complement system. Even in non-heat-inactivated samples, the levels of complement activity can vary between samples. This can significantly impact the conclusions regarding neutralising antibody potency. Restoration of the complement system in these samples can be achieved using an exogenous source of plasma with preserved complement activity or with purified complement proteins. This can significantly enhance the neutralisation titres for some antibodies depending on characteristics such as antibody isotype and the epitope they bind, enable neutralisation with otherwise non-neutralising antibodies, and demonstrate a better relationship between in vitro and in vivo findings. In this review, we discuss the evidence for complement-mediated enhancement of antibody neutralisation against a range of viruses, explore the potential mechanisms which underpin this enhancement, highlight current gaps in the literature, and provide a brief summary of considerations for adopting this approach in future research applications.

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Valspodar limits human cytomegalovirus infection and dissemination

et al. 2021

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Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Cited by 20 publications

References 64 publications

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

Direct enhancement of viral neutralising antibody potency by the complement system: a largely forgotten phenomenon

Valspodar limits human cytomegalovirus infection and dissemination

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