Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

The work presented here was supported by the Canadian Institutes of Health Research (CIHR) (grants DCB-120266, PPP-133377 and HBF-348967 to A.G.).

1. Cells and Transfections
<ol>
	<li>Culture HEK 293T cells in Dulbecco&#39;s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Prepare a 2 x 105 cells/ml suspension in the cell culture medium. Add 500, 100 and 1,000 &#181;l of the cell suspension to each well of 24-well, 96-well and 12-well plates, for viral production, cell viability and immune activation assays, respectively (Figure 2A).</li>
	<li>Gently swirl the plates and incuba...

<ol>
	<li>Hoxie, J. A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+and+gene+therapies+for+HIV.">Novel cell and gene therapies for HIV.</a> Cold Spring Harb Perspect Med. 2, a007179 (2012).</li><li>Bobbin, M. L., Burnett, J. C., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+approaches+for+treatment+of+HIV-1+infection.">RNA interference approaches for treatment of HIV-1 infection.</a> Genome Med. 7, 50 (2015).</li><li>Allers, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+cure+of+HIV+infection+by+CCR5Delta32/Delta32+stem+cell+transplantation.">Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation.</a> Blood. 117, 2791-2799 (2011).</li><li>DiGiusto, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+hematopoietic+stem+cell+based+gene+therapy+for+HIV-1+infection:+considerations+for+proof+of+concept+studies+and+translation+to+standard+medical+practice.">Development of hematopoietic stem cell based gene therapy for HIV-1 infection: considerations for proof of concept studies and translation to standard medical practice.</a> Viruses. 5, 2898-2919 (2013).</li><li>Burke, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Cellular+Resistance+to+HIV-1+Infection+In+Vivo+Using+a+Dual+Therapeutic+Lentiviral+Vector.">Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.</a> Mol Ther Nucleic Acids. 4, e236 (2015).</li><li>DiGiusto, D. 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J., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+durable+treatment+of+HIV-1+infection+using+RNA+interference.">Toward a durable treatment of HIV-1 infection using RNA interference.</a> Prog Mol Biol Transl Sci. 102, 141-163 (2011).</li><li>Blazquez, L., Fortes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+interference+(U1i)+for+Antiviral+Approaches.">U1 interference (U1i) for Antiviral Approaches.</a> Adv Exp Med Biol. 848, 51-69 (2015).</li><li>McIntyre, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=96+shRNAs+designed+for+maximal+coverage+of+HIV-1+variants.">96 shRNAs designed for maximal coverage of HIV-1 variants.</a> Retrovirology. 6, 55 (2009).</li><li>Sajic, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+modified+U1+snRNAs+to+inhibit+HIV-1+replication.">Use of modified U1 snRNAs to inhibit HIV-1 replication.</a> Nucleic Acids Res. 35, 247-255 (2007).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE-directed+discovery+of+potent+shRNA+inhibitors+of+HIV-1.">SHAPE-directed discovery of potent shRNA inhibitors of HIV-1.</a> Mol Ther. 20, 820-828 (2012).</li><li>Scarborough, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Conserved+Target+Site+in+HIV-1+Gag+RNA+is+Accessible+to+Inhibition+by+Both+an+HDV+Ribozyme+and+a+Short+Hairpin+RNA.">A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA.</a> Mol Ther Nucleic Acids. 3, e178 (2014).</li><li>Lain&#233;, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+cleavage+of+HIV-1+RNA+by+new+SOFA-HDV+ribozymes+and+their+potential+to+inhibit+viral+replication.">In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication.</a> RNA Biol. 8, 343-353 (2011).</li><li>Scarborough, R. J., L&#233;vesque, M. V., Perreault, J. P., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Evaluation+of+Clinically+Relevant+SOFA-HDV+Ribozymes+Targeting+HIV+RNA.">Design and Evaluation of Clinically Relevant SOFA-HDV Ribozymes Targeting HIV RNA.</a> Methods Mol Biol. 1103, 31-43 (2014).</li><li>Scarborough, R. J., Adams, K. L., Daher, A., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+Inhibition+of+HIV-1+Production+by+Short+Hairpin+RNAs+and+Small+Interfering+RNAs+Targeting+a+Highly+Conserved+Site+in+HIV-1+Gag+RNA+Is+Optimized+by+Evaluating+Alternative+Length+Formats.">Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats.</a> Antimicrob Agents Chemother. 59, 5297-5305 (2015).</li><li>Sarzotti-Kelsoe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+validation+of+the+TZM-bl+assay+for+standardized+assessments+of+neutralizing+antibodies+against+HIV-1.">Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1.</a> J Immunol Methods. 409, 131-146 (2014).</li><li>Campos, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+lasting+control+of+viral+rebound+with+a+new+drug+ABX464+targeting+Rev+-+mediated+viral+RNA+biogenesis.">Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis.</a> Retrovirology. 12, 1-15 (2015).</li><li>Zhu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR/Cas9+system+inactivates+latent+HIV-1+proviral+DNA.">The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA.</a> Retrovirology. 12, 22 (2015).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J Virol. 76, 1004-1014 (2002).</li><li>Shan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+PCR+assay+for+quantification+of+HIV-1+RNA.">A novel PCR assay for quantification of HIV-1 RNA.</a> J Virol. 87, 6521-6525 (2013).</li><li>Clerzius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PKR+activator,+PACT,+becomes+a+PKR+inhibitor+during+HIV-1+replication.">The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication.</a> Retrovirology. 10, 96 (2013).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Methods Enzymol. 182, 50-68 (1990).</li><li>Battisti, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+activity+between+the+trans-activation+response+RNA-binding+protein,+TRBP2,+and+cyclin+T1+on+HIV+type+1+expression+and+viral+production+in+murine+cells.">Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.</a> AIDS Res Hum Retroviruses. 19, 767-778 (2003).</li><li>Bannwarth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+regulation+of+TRBP1+promoter+by+NF-Y+transcription+factor+in+lymphocytes+and+astrocytes.">Cell-specific regulation of TRBP1 promoter by NF-Y transcription factor in lymphocytes and astrocytes.</a> J Mol Biol. 355, 898-910 (2006).</li><li>Clerzius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAR1+interacts+with+PKR+during+human+immunodeficiency+virus+infection+of+lymphocytes+and+contributes+to+viral+replication.">ADAR1 interacts with PKR during human immunodeficiency virus infection of lymphocytes and contributes to viral replication.</a> J Virol. 83, 10119-10128 (2009).</li><li>Burugu, S., Daher, A., Meurs, E. F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT.">HIV-1 translation and its regulation by cellular factors PKR and PACT.</a> Virus Res. 193, 65-77 (2014).</li><li>ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+HIV-1+with+RNA+interference:+a+multiple+shRNA+approach.">Silencing of HIV-1 with RNA interference: a multiple shRNA approach.</a> Mol Ther. 14, 883-892 (2006).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1.">Optimal design and validation of antiviral siRNA for targeting HIV-1.</a> Retrovirology. 4, 80 (2007).</li><li>Mandal, D., Feng, Z., Stoltzfus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs.">Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.</a> J Virol. 84, 12790-12800 (2010).</li><li>Chang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA(Lys3)-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters.">Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.</a> Mol Ther. 6, 481-489 (2002).</li><li>Chang, L. J., Liu, X., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1.">Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.</a> Gene Ther. 12, 1133-1144 (2005).</li><li>Daniels, S. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP.">U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.</a> Nucleic Acids Res. 39, e71 (2011).</li><li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds.">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li><li>Riss, T. L., et al., Sittampalam, G. 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The HIV-1 production assay described was performed using HEK293T cells (Figure 2) and is similar to assays used to screen HIV-1 RNA for effective ribozyme13, shRNA10,29, siRNA30, and U1i RNA11,31 target sites. Using different methods to quantify HIV-1 production, most studies have measured viral production 48 hr after co-transfection of an HIV-1 expression plasmid with candidate RNAs. Following the production of HIV-1, immature virions undergo proteolytic cleav...

A limitation of current HIV-1 treatments is that they must be chronically administered to prevent disease progression. Transplant of HIV-1 resistant T lymphocyte, or hematopoietic stem cells, has the potential to provide long term control of HIV-1 replication in the absence of drug therapy1,2 and may also be an effective approach to attain an HIV-1 cure3. One way to render cells resistant to HIV-1 replication is to insert one or more genes coding for anti-HIV-1 RNAs or peptides into an infected individual&#39;s cells during an autologous transplant4. Several candidate anti-HIV-1 genes have been designed with some entering clinical trials in combinations of two5 or three6, to prevent the development of HIV-1 resistance to any single gene.
Anti-HIV-1 RNAs are among the top candidates for combination gene therapy due to their low potential to elicit immune responses and because they are transcribed from very short gene sequences. Some anti-HIV-1 RNAs have been designed to target viral entry and integration. However, most anti-HIV-1 RNAs target post-integration steps in the viral life cycle (Figure 1). Post-integration inhibitors include decoy RNAs, targeting the HIV-1 regulatory proteins Tat or Rev1, and antisense-based RNAs, targeting different sites in HIV-1 RNA, such as ribozymes7, shRNAs8 and U1i RNAs9. Methods that have been used to compare the efficacy of anti-HIV-1 RNAs include monitoring viral replication in cells transduced with genes coding for candidate RNAs and measuring viral production in cells transiently transfected with plasmids expressing candidate RNAs and an HIV-1 expression plasmid10-13. We have previously used an HIV-1 production assay to screen HIV-1 RNA for new ribozyme target sites13-15. These methods have since been refined to optimize the format of an RNA interference molecule expressed from plasmid DNA as an shRNA or delivered as a synthetic siRNA16. The assay measures the production of mature viruses from human embryonic kidney (HEK) 293T cells, and can be used to compare the effects of inhibitors that target post-integration steps in the HIV-1 replication cycle (Figure 1). For inhibitors that target pre-integration steps, alternative assays such as a TZM-bl cell infectivity assay17 are needed to evaluate antiviral efficacy.
Major safety concerns for the delivery of anti-HIV-1 RNAs in the clinic include potential off-target effects on human RNAs or proteins, and activation of innate immune sensors. To evaluate the toxicity of anti-HIV-1 siRNAs, we have used a cell viability assay in different cell lines16. We also measured activation of the double stranded RNA immune sensors, RNA activated protein kinase R (PKR) and Toll like receptor 3 (TLR3), as well as expression of the interferon stimulated gene, ADAR1 p150. These assays can be used to confirm that the efficacy of anti-HIV-1 RNAs is not due to indirect effects on cell viability or immune sensor activation. They are also useful in excluding candidate RNAs with potential toxicities from further development.
In the following protocols, procedures to identify new therapeutic RNAs and optimize the format of existing ones are described. The methods are useful for screening RNA based post-integration inhibitors of HIV-1 replication and could be adapted to screen other post-integration inhibitors, such as small molecules targeting Rev mediated export of viral RNA18 or CRISPR/Cas systems designed to target integrated HIV-1 DNA19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Gibco</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 96 well, 24 well, 6 well.</td>
			<td>Corning</td>
			<td>353075, 353047, 353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes Axygen</td>
			<td>Corning</td>
			<td>311-08-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Low molecular weight Poly I:C</td>
			<td>InvivoGen</td>
			<td>3182-29-6</td>
			<td></td>
		</tr>
		<tr>
			<td>DharmaFECT-1</td>
			<td>Dharmacon</td>
			<td>T-2001-01</td>
			<td>transfection reagent for synthetic RNAs</td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Mirus</td>
			<td>MIR 2300</td>
			<td>transfection reagent for RNA expression plasmids</td>
		</tr>
		<tr>
			<td>Nonidet P40 (NP-40)</td>
			<td>USB</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr>
			<td>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</td>
			<td>Perkin Elmer</td>
			<td>1450-522</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate scintillation counter</td>
			<td>Perkin Elmer</td>
			<td>1450-024</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M-2128</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>Bio-rad</td>
			<td>1706930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis buffer tablets</td>
			<td>Roche</td>
			<td>4693159001, 4906837001</td>
			<td>protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Bio-rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel running chamber</td>
			<td>Hoefer</td>
			<td>SE600</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer cell</td>
			<td>Bio-rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein ladder EZ-Run</td>
			<td>Thermo Fisher</td>
			<td>BP3603-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-rad</td>
			<td>162-0094</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody stripping solution</td>
			<td>Millipore</td>
			<td>2504</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL - Pierce</td>
			<td>Thermo Fisher&#160;</td>
			<td>PI32106</td>
			<td></td>
		</tr>
		<tr>
			<td>ADAR1 antibody</td>
			<td></td>
			<td></td>
			<td>from Dr. B.L. Bass</td>
		</tr>
		<tr>
			<td>phospho-T446-PKR antibody</td>
			<td>Abcam</td>
			<td>ab32036</td>
			<td></td>
		</tr>
		<tr>
			<td>phospho-S396-IRF3 antibody</td>
			<td>Cell Signaling</td>
			<td>4947</td>
			<td></td>
		</tr>
		<tr>
			<td>PKR antibody</td>
			<td></td>
			<td></td>
			<td>from Dr. A. Hovanessian</td>
		</tr>
		<tr>
			<td>IRF3 antibody</td>
			<td>Cell Signaling</td>
			<td>11904</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin antibody</td>
			<td>Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-labeled goat anti-rabbit</td>
			<td>KPL</td>
			<td>474-1506</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-labeled goat anti-mouse</td>
			<td>KPL</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>6226-79-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

A general schematic of the procedures is shown in Figure 2 with an example transfection plan for three test RNAs and a control RNA provided in Figure 2B. For viral production and cell viability assays, the read-out for each test construct is normalized to a negative control. Replicates are transfected in sets, so that each test RNA is normalized to its adjacent negative control. This is done to avoid inaccurate data related to the time between complexing ...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

The overall goal of this procedure is to compare the efficacy and toxicity of new RNAs targeting HIV-1 production. This method can help identify new RNA molecules for using HIV-1 gene or drug therapy, such as short hairpin or small interfering RNAs, ribozymes, and RNA decoys. The main advantage of this technique is that several new RNA designs can be screened simultaneously with a quick turnaround time.Although this method was designed to evaluate new RNA drug or gene therapy for HIV, it can also be applied to other systems, such as small molecules or CRISPR tasks. We first had the idea for this method when we were setting up experiments to screen a large number of ribozymes for anti-HIV effects. To begin, prepare a suspension of human embryonic kidney 293T cells at two times 10 to the fifth cells per milliliter in DMEM with 10%FPS and 1%penicillin streptomycin.Add 500, 100 and 1, 000 microliters of the cell suspension to each of the wells of 24, 96, and 12-well plates for viral production, cell viability, and immune activation assays, respectively. Gently swirl the plates and incubate them overnight at 37 degrees Celsius with 5%carbon dioxide to 50%to 70%confluency. For the viral production assay, prepare a 10 nanogram per microliter dilution of an HIV-1 expression plasmid and add 10 microliters to each assay tube.Next, prepare five micromolar dilutions of test RNAs and a negative control RNA. Then add 2.5 microliters or 10 microliters of each test RNA and negative control dilution to the corresponding tubes for 25 nanomolar or 100 nanomolar final concentrations. For the cell viability assay, prepare five micromolar dilutions of test RNAs and a 10 milligram per milliliter dilution of the positive control RNA, low molecular weight poly(I:C)Add two microliters of the test RNA or the positive control RNA dilutions to the corresponding tubes for 100 nanomolar or 200 micrograms per milliliter final concentrations, respectively.For the immune activation assay, add 20 microliters of test RNA or positive control RNA dilutions, as prepared for the cell viability assay, to the corresponding tubes for 100 nanomolar or 200 micrograms per milliliter final concentrations, respectively. Then add 50, 25, or 75 microliters of DMEM to each transfection tube for viral production, cell viability, and immune activation assays, respectively. For viral production assays, bring cells and prepared transfection tubes to a biosafety level three, or BSL-3, laboratory before the next step.Add two microliters of the transfection reagent sequentially to the transfection tubes, and incubate 15 to 20 minutes to allow complexes to form. Add the entire transfection mixture from each microtube drop-wise to the corresponding positions in the cell culture plates. Gently swirl, and incubate the plates for 48 hours at 37 degrees Celsius and 5%carbon dioxide.To carry out the viral production assay, after removing the 24-well cell culture plates from the incubator, transfer them to the BSL-3 cell culture hood. Gently swirl the plates and then transfer 150 microliters of the supernatant from each well to a corresponding well in a 96-well flat-bottom plate to be used to quantify HIV-1 production. Transfer five microliters of supernatant to the corresponding wells in a 96-well plate containing 25 microliters of a viral disruption cocktail.After incubating the mixture for five minutes at room temperature, transfer the plate to a radioactivity work station. Next, prepare a radioactive cocktail, and add 25 microliters to each well of viral supernatant and disruption cocktail. Incubate the plates at 37 degrees Celsius for two hours.Spot five microliters of the reaction mixture onto corresponding squares in a glass fiber/DEAE filter mat paper, and allow the spots to dry for 10 minutes. Use 2x SSC buffer to wash the papers five times for five minutes each, followed by two one-minute washes with 95%ethanol. Allow the papers to dry before sealing them in sample bags.Clip sample bags containing the filter mat paper into a cassette, and insert the cassette into a microplate scintillation counter. Then start the counter. For any viral quantification method, divide the values obtained for each test RNA by the adjacent negative control and multiply this value by 100 to get the percent inhibition of HIV-1 production for each replicate of the test RNAs.To carry out a cell viability assay, after removing the 96-well plates from the incubator, add 20 microliters of five milligrams per milliliter MTT and DPBS to each well. Then incubate the plates for three hours at 37 degrees Celsius. Add 150 microliters of acidified isopropanol with detergent to each well, and incubate the plates at room temperature for two hours.Then use a microplate spectrophotometer to determine the absorbance at 570 nanometers. Calculate the relative MTT metabolism for each positive control and test RNA by dividing the value obtained for each sample by its adjacent transfection control. For the immune activation assay, after removing the 12-well plates from the incubator, aspirate the culture medium and use DPBS to gently wash the cells twice.Then add 70 microliters of cold lysis buffer containing protease and phosphotase inhibitors to each well, and incubate the plates on ice for 10 minutes. Transfer cell lysates to microtubes, and fast freeze them by immersing the tubes in liquid nitrogen. After allowing the samples to thaw, freeze-thaw them two more times, for a total of three freeze-thaw cycles.Next, centrifuge the lysates at 15, 700 times g and 4 degrees Celsius for 15 minutes to pellet the cell debris. Then, following electrophoresis and transfer to a membrane, reveal protein bands by incubating the membrane in Ponceau S for one minute. Then wash it with double-distilled water.Use the bands and protein ladder as a guide to cut the membrane at 80 and 55 kilodaltons. Now, use TBS with 0.05%TWEEN 20 or TBST to wash out the Ponceau S staining. Add TBST with 5%non-fat milk to completely cover the membranes, and incubate them at room temperature with agitation for one hour.Transfer the membranes into TBST with 3%BSA and antibodies diluted 1:1, 000 for ADAR, phosphor-PKR, and phosphor-IRF3 for the top, middle, and bottom pieces of the membrane, respectively. Incubate at 4 degrees Celsius with agitation overnight. The following morning, use TBST to wash the membranes five times for five minutes each, before transferring to TBST with 5%non-fat milk and 1:5, 000 peroxidase labeled goat anti-rabbit secondary antibodies for one hour.After visualizing the protein bands on films, use a stripping solution to wash the middle and bottom pieces of the membrane for 10 minutes, before incubating the middle piece of the membrane in PKR antibodies and the bottom piece in IRF3 antibodies. As a positive control, probe the bottom membrane with antibodies to Actin according to the text protocol. A general schematic of the assays is shown here with an example transfection plan for three test RNAs and a control RNA.For viral production and cell viability assays, the readout for each test construct is normalized to a negative control. Using the HIV-1 production assay in cells transfected with a plasmid expressing HIV-1 strain NL4-3, the percent of RT activity in cells co-transfected with one of the siRNAs shown here, compared to cells transfected with a long Dicer substrate nonsense siRNA, was calculated to identify the optimal length of RNA interference molecules targeting a conserved site in HIV-1 RNA. Using the most effective siRNAs identified, the percent metabolism of MTT was determined for transfected cells.Along double-stranded RNA, poly(I:C)reduced cell viability only at the highest dose. No significant reduction in cell viability was observed for siRNAs targeting the 1498 site in HIV-1 RNA, regardless of their length. As demonstrated here by Western blot, the same set of RNAs was evaluated in the immune activation assay.Under conditions where poly(I:C)activated the expression of ADAR1-p150 and induced phosphorilation of PKR and IRF3, no significant effect on any inactivation markers could be observed for any of the test RNAs. Once mastered, this technique can be done in seven days, if it's performed properly. While attempting this procedure, it is important to use high quality preparations of both RNA and DNA for transfection.Furthermore, to include the proper negative and positive controls. Following this procedure, other methods like HIV infection of transduced cells, can be used to evaluate the long-term efficacy and toxicity of potential anti-HIV candidates. After its development, this technique allowed us to identify an accessible and conserved target site in HIV RNA and optimize the formats of ribozymes, shRNAs and siRNAs targeting it.After watching this video, you should have a good understanding of how to quickly screen new RNA candidates targeting HIV production for efficacy using the viral production assay, and potential toxicity using the immune activation and cell viability assays. Don't forget that working with HIV and radiolabeled nucleotides can be extremely hazardous. Precautions such as using certified biosafety and radioactivity laboratories should always be taken while performing this procedure.

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...