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Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System

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Abstract

Purpose

In order to improve the detection limit of existing HIV diagnostic assays, we explored the use of a temperature-responsive magnetic nanoparticle reagent system in conjunction with cyanovirin-N for HIV recognition to rapidly and efficiently concentrate viral particles from larger sample volumes, ~ 1 ml.

Methods

Cyanovirin-N (CVN) mutant, Q62C, was expressed, biotinylated, and then complexed with a thermally responsive polymer-streptavidin conjugate. Confirmation of protein expression/activity was performed using matrix assisted laser desorption/ionization (MALDI) and a TZM-bl HIV inhibition assay. Biotinylated CVN mutant recognition with gp120 was characterized using surface plasmon resonance (SPR). Virus separation and enrichment using a thermoresponsive magnetic nanoparticle reagent system were measured using RT-PCR.

Results

Biotinylated Q62C exhibited a KD of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1 × 105 virus copies in 100 μl, using pNIPAAm-Q62C within 30 minutes. Additionally, the same reagent system achieved nearly 9-fold enrichment by processing a 10-times larger sample of 1000 μl (Fig. 3).

Conclusion

This work demonstrated a temperature-responsive reagent system that provides enrichment of HIV using antiviral lectin CVN for recognition, which is potentially amenable for use in point-of-care settings.

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Scheme 1
Fig. 1
Fig. 2
Fig. 3

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Abbreviations

CVN:

Cyanovirin-N

HIV:

Human immunodeficiency virus

LCST:

Lower critical solution temperature

LFA:

Lateral flow assay

MALDI:

Matrix-assisted laser desorption ionization

mNP:

Magnetic nanoparticles

pNIPAAm:

Poly(N-isopropylacrylamide)

SA:

Streptavidin

SPR:

Surface plasmon resonance

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Reggie Gausman and Jose Ortega for technical assistance with the RT-PCR work. Funding support was provided by the University of Washington, the University of Washington CFAR Clinical Research and Retrovirology Core (P30-AI-027757) and the ACTG Laboratory Center (UM1-AI-106701), NIH GM100558 and CA174581, and the National Science Foundation Graduate Research Fellowship Program. BJN is an employee of Nexgenia, a company that is commercializing stimuli-responsive reagents for life science applications.

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Authors and Affiliations

  1. Department of Bioengineering, University of Washington, Box 355061, Seattle, Washington, 98195, USA

    Joseph C. Phan, Selvi Srinivasan, Kim A. Woodrow & James J. Lai

  2. Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington, 98104, USA

    Robert W. Coombs

  3. Nexgenia, Inc., 4000 Mason Rd., Fluke Hall, Suite 312-1, Seattle, Washington, 98195, USA

    Barrett J. Nehilla

Authors
  1. Joseph C. Phan
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Correspondence to Kim A. Woodrow or James J. Lai.

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Phan, J.C., Nehilla, B.J., Srinivasan, S. et al. Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System. Pharm Res 33, 2411–2420 (2016). https://doi.org/10.1007/s11095-016-1980-7

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  • DOI: https://doi.org/10.1007/s11095-016-1980-7

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