HIV-1 DNA LOAD ANALYSIS IN PERIPHERAL BLOOD LYMPHOCYTES AND MONOCYTES FROM NAÏVE AND

HAART-TREATED INDIVIDUALS

HUZAIFA UMAR20142894

Dept. 0f BiogineeringCyprus International University

OUTLINES Introduction

HIV

Immune Cells

Highly Active Antiretroviral Therapy (HAART)

Objective

Results

Discussion

Conclusion

Critique

INTRODUCTION The human immunodeficiency virus (HIV) is a lentivirus (a subgroup of retrovirus) that causes the acquired

immunodeficiency syndrome (AIDS) and replicates in a host cell through the process of reverse transcription

(Weiss,1993).

Its transmitted as single stranded RNA. And upon entry to the target cells, it uses its own reverse transcriptase

enzyme to produce DNA from its RNA genome. This new DNA is then incorporated into the host's genome by an

integrase enzyme (Smith and Daniel, 2006).

Types of HIVHIV-1HIV-2

Weiss RA. (May 1993). "How does HIV cause AIDS?". Science 260 (5112): 1273–9. Bibcode:1993Sci...260.1273W. doi:10.1126/science.8493571. PMID 8493571Smith JA, Daniel R. (2006). "Following the path of the virus: the exploitation of host DNA repair mechanisms by retroviruses". ACS Chem Biol. 1 (4): 217–26)

INTRODUCTION CON… The immune system is a system within an organism that protects the organism against disease caused by

pathogens (virus to parasitic worm) and disorders in the system can autoimmune diseases "Inflammatory

Cells and Cancer" (Lisa and Zena, 2010).

HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells),

macrophages, and dendritic cells (Berlier et al., 2005).

Lisa M. Coussens and Zena Werb (2010). Journal of Experimental Medicine, March 19, 2001, vol. 193, no. 6, pages F23– 26, Retrieved Aug 13, 2010)Berlier W, Bourlet T, Lawrence P, Hamzeh H, Lambert C, Genin C et al. (2005). "Selective sequestration of X4 isolates by human genital epithelial cells:

Implication for virus tropism selection process during sexual transmission of HIV". J Med Virol. 77 (4): 465–74)

Objective

The main objective of the study is to evaluate HIV-1

DNA load in PBLs and Monocytes from both long-term

HAART-treated and antiretroviral naïve HIV-1 infected

patients.

Thirty-four HIV-1 treated by a combination of two different nucleoside reverse transcriptase

inhibitor (NRTI) and one non-nucleoside reverse inhibitor (NNRTI) for 24-36 months.

And 34 HIV-1 Naïve patients were enrolled in this study. All patient were recorded 30-72

months before the analysis.

Whole blood sample were collected from HIV-1 positive patients by venipuncture in EDTA

containing tube for each patients.

Materials and MethodPatients and Sample Collection

www.celebritydiagnosis.com

ISOLATION OF PBLS AND MONOCYTES Pheripheral blood mononuclear cells (PBMCs) were separated by Ficoll gradient (Ficoll-

Hystopaque, Pharmacia, Uppsala, Sweden).

CD14+ monocytes was isolated by CD14+ Miltenyi Purification kit (Miltenyi, Auburn, CA).

Both PBLs and CD14+ monocytes were analysed using Flow cytometry and all samples

showed a purity percentage >96% of CD11b/CD14+ cells (96.2-99.5%).

Flow cytometry analysis of PBLs by FIT-anti-CD15 and PE-anti-CD14 monoclonal antibody

(Becton-Dickinson, Palo Alto, CA) displayed cell contaminant <1% with a PBL purity

percentage >99%

CD4+ CELL COUNT AND RNA VIRAL LOAD DETERMINATION

CD4+ lymphocyte count was determine by flow cytometry (Perfetto, 1999).

And plasma RNA viral load by Quantiplex HIV-1 3.0 assay branched DNA kit

(Siemens, East Walpole, MA) respectively.

The lower HIV-1 RNA detection limit was determined at 50 copies/ml.

DNA was extracted from PBLs and monocytes by DNA easy kit. The content of

each sample was determine by spectrophotometer analysis at 260/270 and stored at -

80oC.

Perfetto SP, McCrary G. Immunophenotyping and assessment of cell function by three-color flow cytometry of peripheral blood lymphocytes. In: Michael N, Kim JH, editors. HIV protocols. Totowa NJ: Humana Press; 1999. p. 397e407.

QUANTITATIVE DETERMINATION OF HIV-1 DNA VIRAL LOAD

The quantitative determination of HIV-1 DNA load was performed by SYBR green real time PCR analysis.

DNA detection was carried out in 20 ul PCR mixture volume containing Taq DNA polymerase, 200nM

HIV-1 gag SK431/SK462 primers & 300ng of DNA extracted from PBLs /Monocyte (Gibellin et el.,

2004).

HIV-1 gag gene amplification was carried out as follows: Initial activation of DNA polymerase at 95oC for

15 min; 45 circle in four steps: 94 oC for 10 s, 60 oC for 30 s, 72 oC for 45 s and 78 oC.

Amplification, data acquisition and single analysis of flourescence (78 oC) were carried out by light cycler

instrument (Roche, Mannheim, Germany) using light Cycler 5.3.2 software (Roche).

Gibellini D, Vitone F, Gori E, La Placa M, Re MC. Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by SYBR green real time RT-PCR technique in HIV-1 seropositive patients. J Virol Methods 2004;115:183e9.

The data was analyzed by the Shapiro-Wilk normality test.

The Correlation was evaluated using Kendall’s rank correlation test. And Wilcoxon’s

was employed for mean rank analysis.

The data were expressed as mean + standard deviation (SD).

Samples with <50 HIV-1 DNA copies/106 cells and/or with <50 plasma RNA viral

copies/ml were both considered 49.

Statistical Analysis

RESULTSTable 1. HIV positive patients

HAART-treated (NRTI & 1 NNRTI)

Antiretroviral naïve

Number of patients 34 (27 men and 7 women) 34 (27 men and 7 women)

Age 47+ 9.5 ( range 27 – 69) 42+ 9 (range 23 – 66)

Months of treatment 24 – 36 -----

CD4+ T-cell count (cell/ul) 584+ 297 (range 197 - 1120) 24 + 10 (range 11- 914)

% CD4+ T cell 29 + 12 (range 7-52) 24 + 10 (range 1 – 45)

RNA viral load (copies/ml) <50 28528 + 45746 (range 652 - 272000

The values were expressed as mean + standard deviationNRTIs : Nucleoside Reverse Transcriptase Inhibitors (eg. Zidovudine, lamuvidine, stavurdin)NNRTIs : Non-Nucleoside Reverse Transcriptase Inhibitors (eg. Navirapine) All patients were first recorded as infected by current serological screening methods 30 – 72 months before the

analysis.

CONT.Table 2. HIV-1 DNA load in HAART and antiretroviral naïve individuals

HAART-treated HIV-1 DNA load

Antiretroviral naïve HIV-1 DNA load

PBLs (copies/106 cells) 632 + 698 (range 78 – 3052) 2063 + 2144 (range 126 - 9736

Monocytes (copies/106 cells) 61 + 29 (range 49 – 187) 273 + 329 (range 54 – 1749)

PBLs/monocytes ratio 10 + 11 (range 1.6 – 22.2) 9 + 6.5 (range 2.3 – 11)

The values were expressed as mean + standard deviation HIV-1 DNA load detected in PBLs from both patients, was significantly higher in naïve patients than in HAART treated individual (P > 0.001; Wilcoxon rank sum test)The monocyte HIV-1 burden was significantly higher in naïve patients than in HAART treated individual (P > 0.001; Wilcoxon rank sum test) Analysis of HAART treated or naïve HIV DNA PBLs/monocytes ratio reveal no significance correlation.

DISCUSSION This study demonstrated that, the PBLs for HAART-treated individuals after 34 months

of effective treatment showed significant decrease in the total HIV-1 DNA load when

compare with PBLs from naïve patients. And the data obtained are in agreement with

several previous papers (Ibanez et al., 1999; Ngo-Giang-Huong et al., 2001; De Rossi et

al., 2002; Sarmati et al., 2005)

HIV-1 DNA viral load in HAART and naïve partients was compared with CD4 cell

counts, indicating a strong association between viral load and immunological

parameters (McDermont et al.,1991; Lafeuillade et al., 2001; Riva et al., 2003).

CONCLUSION This study indicate that effective long-term HAART significantly decreases the HIV-

1 DNA load both in PBLs and monocytes in HAART treated patient compared to

naïve individuals.

Although RNA viral load and CD4+ cell count represent the main parameters to

monitor HIV-1 infection, but this study also suggest that HIV-1 DNA load

quantitative analysis in PBLs and monocytes may be considered an important

approach to study the HIV-1 reservoir and the effectiveness of HAART therapy in

HIV-1 seropositive patients.

CRITIQUE

The patients (samples) suppose to be confined in the same place and fed

with the same food because food variation may affect the result.

Standard dilution and samples suppose to be run in triplicate to achieve

reproducible result instead of duplicate.

The extraction procedure suppose to be stated in the paper because it is

the most sensitive stage in DNA analysis.

Other kits such as COBAS AmpliPrep/COBAS Kits, Roche Amplicor

HIV-1 Monitor Test kits suppose to be employed to verify the results.

Thanks for Listening

?

Observation