The authors wish to acknowledge the patients who participated in the many studies of HIV-1.
J.M.C. was a Research Professor of the American Cancer Society with support from the F.M. Kirby Foundation.

<ol>
	<li>Amara, R. R., Villinger, F., Altman, J. D., Lydy, S. L., O'Neil, S. P., Staprans, S. I., Montefiori, D. C., Xu, Y., Herndon, J. G., Wyatt, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+a+mucosal+challenge+and+prevention+of+AIDS+by+a+multiprotein+DNA/MVA+vaccine.">Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.</a> Vaccine. 20, 1949-1955 (2002).</li><li>Dinoso, J., Kim, S., Blankson, J., Siliciano, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Dynamics+of+Elite+Suppressors+in+HIV-1+Infection.">Viral Dynamics of Elite Suppressors in HIV-1 Infection.</a> Conference on Retroviruses and Opportunistic Infections. , (2008).</li><li>Dinoso, J. B., Kim, S. Y., Wiegand, A. M., Palmer, S. E., Gange, S. J., Cranmer, L., O'Shea, A., Callender, M., Spivak, A., Brennan, T., Kearney, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+intensification+does+not+reduce+residual+HIV-1+viremia+in+patients+on+highly+active+antiretroviral+therapy.">Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 9403-9408 (2009).</li><li>Doria-Rose, N. A., Klein, R. M., Manion, M. M., O'Dell, S., Phogat, A., Chakrabarti, B., Hallahan, C. W., Migueles, S. A., Wrammert, J., Ahmed, R., Nason, M., Wyatt, R. T., Mascola, J. R., Connors, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+and+phenotype+of+human+immunodeficiency+virus+envelope-specific+B+cells+from+patients+with+broadly+cross-neutralizing+antibodies.">Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies.</a> J. Virol. 83, 188-199 (2009).</li><li>Dornadula, G., Zhang, H., Uitert, B. V. a. n., Stern, J., Livornese, L., Ingerman, M. J., Witek, J., Kedanis, R. J., Natkin, J., DeSimone, J., Pomerantz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+HIV-1+RNA+in+blood+plasma+of+patients+taking+suppressive+highly+active+antiretroviral+therapy.">Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy.</a> JAMA. 282, 1627-1632 (1999).</li><li>Gandhi, R. T., Zheng, L., Bosch, R. J., Chan, E. S., Margolis, D. M., Read, S., Kallungal, B., Palmer, S., Medvik, K., Lederman, M. M., Alatrakchi, N., Jacobson, J. M., Wiegand, A., Kearney, M., Coffin, J. M., Mellors, J. W., Eron, J. 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G. <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+persistent+low-level+viremia+in+individuals+who+control+human+immunodeficiency+virus+in+the+absence+of+antiretroviral+therapy.">Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy.</a> J. Virol. 83, 329-335 (2009).</li><li>Havlir, D. V., Bassett, R., Levitan, D., Gilbert, P., Tebas, P., Collier, A. C., Hirsch, M. S., Ignacio, C., Condra, J., Gunthard, H. F., Richman, D. D., Wong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+predictive+value+of+intermittent+viremia+with+combination+hiv+therapy.">Prevalence and predictive value of intermittent viremia with combination hiv therapy.</a> JAMA. 286, 171-179 (2001).</li><li>Jordan, M. R., Kearney, M., Palmer, S., Shao, W., Maldarelli, F., Coakley, E. P., Chappey, C., Wanke, C., Coffin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+standard+PCR/cloning+to+single+genome+sequencing+for+analysis+of+HIV-1+populations.">Comparison of standard PCR/cloning to single genome sequencing for analysis of HIV-1 populations.</a> J Virol Methods. 168, 114-120 (2010).</li><li>Kaufmann, D. E., Kavanagh, D. G., Pereyra, F., Zaunders, J. J., Mackey, E. W., Miura, T., Palmer, S., Brockman, M., Rathod, A., Piechocka-Trocha, A., Baker, B., Zhu, B., Gall, S. L. e., Waring, M. T., Ahern, R., Moss, K., Kelleher, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+CTLA-4+by+HIV-specific+CD4++T+cells+correlates+with+disease+progression+and+defines+a+reversible+immune+dysfunction.">Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction.</a> Nat Immunol. 8, 1246-1254 (2007).</li><li>Kearney, M., Maldarelli, F., Shao, W., Margolick, J. B., Daar, E. S., Mellors, J. W., Rao, V., Coffin, J. M., Palmer, S. <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+Population+Genetics+and+Adaptation+in+Newly+Infected+Individuals.">HIV-1 Population Genetics and Adaptation in Newly Infected Individuals.</a> J. Virol. 83, 2715-2727 (2008).</li><li>Kearney, M., Palmer, S., Maldarelli, F., Shao, W., Polis, M. A., Mican, J., Rock-Kress, D., Margolick, J. B., Coffin, J. M., Mellors, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+polymorphism+at+drug+resistance+sites+in+HIV-1+protease+and+reverse+transcriptase.">Frequent polymorphism at drug resistance sites in HIV-1 protease and reverse transcriptase.</a> AIDS. 22, 497-501 (2008).</li><li>Kearney, M., Spindler, J., Shao, W., Maldarelli, F., Palmer, S., Hu, S. L., Lifson, J. D., Kewal Ramani, V. N., Mellors, J. W., Coffin, J. M., Ambrose, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+simian+immunodeficiency+virus+encoding+HIV-1+reverse+transcriptase+persists+in+macaques+despite+antiretroviral+therapy.">Genetic diversity of simian immunodeficiency virus encoding HIV-1 reverse transcriptase persists in macaques despite antiretroviral therapy.</a> Journal of Virology. 85, 1067-1076 (2011).</li><li>Keele, B. F., Giorgi, E. E., Salazar-Gonzalez, J. F., Decker, J. M., Pham, K. T., Salazar, M. G., Sun, C., Grayson, T., Wang, S., Li, H., Wei, X., Jiang, C., Kirchherr, J. L., Gao, F., Anderson, J. A., Ping, L. H., Swanstrom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+transmitted+and+early+founder+virus+envelopes+in+primary+HIV-1+infection.">Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection.</a> Proc. Natl. Acad. Sci. U.S.A. , 105-7552 (2008).</li><li>Liu, S. L., Rodrigo, A. G., Shankarappa, R. G., Learn, H., Hsu, L., Davidov, O., Zhao, L. P., Mullins, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+quasispecies+and+resampling.">HIV quasispecies and resampling.</a> Science. 273, 415-416 (1996).</li><li>Mahalanabis, M., Jayaraman, P., Miura, T., Pereyra, F., Chester, E. M., Richardson, B., Walker, B., Haigwood, N. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple,+linked+human+immunodeficiency+virus+type+1+drug+resistance+mutations+in+treatment-experienced+patients+are+missed+by+standard+genotype+analysis.">Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis.</a> J. Clin. Microbiol. 43, 406-413 (2005).</li><li>Palmer, S., Wiegand, A. P., Maldarelli, F., Bazmi, H., Mican, J. M., Polis, M., Dewar, R. L., Planta, A., Liu, S., Metcalf, J. A., Mellors, J. W., Coffin, J. 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No conflicts of interest declared.

HIV-1 infected individuals on combination antiretroviral treatment (cART) or who naturally control the infection have very low viral loads, typically around 1 copy per ml of plasma4, 11, 12, 17, 18. Viral loads in patients with natural control, often fluctuate around an individual set point1, 2, 17. The sensitivity of the assays described herein is highly dependent on the input volume of plasma. Generally, we have been working with 7 ml of plasma, but positive results can be obtained from smaller am...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Random hexamers (500 ug/ml)</td>
			<td>Promega</td>
			<td>C118A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Taqman buffer A</td>
			<td>Applied Biosystems</td>
			<td>4304441</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNAsin (40 U/ul)</td>
			<td>Promega</td>
			<td>N211B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RT enzyme (200 U/ul) </td>
			<td>Strategene</td>
			<td>600107-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amplitaq Gold DNA polymerase</td>
			<td>Applied Biosystems</td>
			<td>4311814</td>
			<td>6-pack</td>
		</tr>
		<tr>
			<td>Amplitaq 10XGold PCR buffer II</td>
			<td>Applied Biosystems</td>
			<td>4311814</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>Magnesiumcloride 25 mM</td>
			<td>Applied Biosystems</td>
			<td>4311814</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>1M Tris-HCl buffer, pH 8.0, 5 mM</td>
			<td>Invitrogen</td>
			<td>AM9855G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III reverse transcriptase enzyme, 200 U/ul </td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III 10X buffer</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>DTT 100 mM</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>Platinum Taq DNA polymerase High Fidelity 5 U/ul</td>
			<td>Invitrogen</td>
			<td>11304-102</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X High Fidelity PCR Buffer</td>
			<td>Invitrogen</td>
			<td>11304-102</td>
			<td>comes with the enzyme</td>
		</tr>
		<tr>
			<td>Proteinase-K 20 mg/ml</td>
			<td>Ambion</td>
			<td>2546</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Guanidinium Thiocyanate solution 6M</td>
			<td>FlukaBiochemica</td>
			<td>50983</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycogen 20 mg/ml</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>19516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Sigma-Aldrich</td>
			<td>E702-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Molecular grade water </td>
			<td>Gibco/Invitrogen</td>
			<td>10977-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dNTPs (25 mmol each) </td>
			<td>Bioline</td>
			<td>BIO-39025</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<td>Name of Euipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Easy Seal Centrifuge Tubes (16x73mm)</td>
			<td>Seaton Scientific</td>
			<td>6041</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>White Delrin crowns </td>
			<td>Seaton Scientific</td>
			<td>4020</td>
			<td>Caps for the Easy Seal Tubes</td>
		</tr>
		<tr>
			<td>Tube Rack for Optiseal Bell-Top Tubes, 8.9 ml</td>
			<td>Beckman</td>
			<td>361642</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hex driver &frac12; inc opening</td>
			<td>Seaton Scientific</td>
			<td>4013</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>cap removal tupe</td>
			<td>Seaton Scientific</td>
			<td>4014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ml serological pipettes</td>
			<td>Costar</td>
			<td>4051</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipetboy</td>
			<td>any</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml Transfer pipettes </td>
			<td>ISC Bioexpress</td>
			<td>P-5005-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5.8 ml Dispossable transfer pipettes, fine tip</td>
			<td>VWR</td>
			<td>14670-329</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml centrifuge tubes</td>
			<td>Falcon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml centrifuge tubes</td>
			<td>any</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris buffer Saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>T5030-100TAB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge and rotor </td>
			<td>Sorval</td>
			<td>90SE and T-1270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-well PCR plates</td>
			<td>Biorad</td>
			<td>HSS-9601</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml Reagent reservoir </td>
			<td>Costar</td>
			<td>4870</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microamp optical 96-well reaction plate</td>
			<td>Applied Biosystems</td>
			<td>N801-0560</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optical plate covers </td>
			<td>Applied Biosystems</td>
			<td>4333183</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Compression Pad</td>
			<td>Applied Biosystems</td>
			<td>4312639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microseal A film</td>
			<td>Biorad</td>
			<td>MSA-5001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 ml centrifuge tubes </td>
			<td>Any</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1% agarose gels (E-gel, invitrogen)</td>
			<td>Invitrogen</td>
			<td>G-7008-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E-base (Invitrogen)</td>
			<td>Invitrogen</td>
			<td>EB-M03</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
EDTA Collection tubes any brand

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.

Watch this Scientific Journal Video about Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays at JoVE.com

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

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 ...

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Immunology and Infection

31.3K Views.  NCI-Frederick. 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.

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

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 ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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. ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.