Detection of Diverse Variants of HIV-1 proviral DNA load using TaqMan real-time PCR in isolate CD4+ cells.

ABSTRACT

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4+ T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we developed a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The study involves the detection of HIV proviral DNA from blood sample of treated patients and their current status of Plasma RNA load and CD4+ count for correlation.



INTRODUCTION

Individuals infected with HIV-1 are generally followed by monitoring the plasma viral RNA loads and CD4+ cell counts, which are markers of viral replication and immune function, respectively (Mellors et al., 1996; Pantaleo et al., 1993). The plasma viral RNA level predicts minimally the rate of subsequent CD4+ cell decline (Rodríguez et al., 2006) and is used commonly as a marker of the outcome of highly active antiretroviral therapy (HAART). Although the plasma viral RNA level in the majority of patients receiving HAART declines below the detection limit within a few months after the start of therapy (Gallant et al.,2006), proviral DNA is detected persistently in peripheral blood mononuclear cells (PBMCs) or lymphoid tissue (Chun et al., 1997; Sonza et al., 2001). Several studies have suggested that measuring the proviral DNA level may be important for evaluating the longterm effectiveness of HAART (Burgard et al., 2000; De Milito et al., 2003; Pellegrin et al., 2003), while this is sometimes conflicting (Soriano-Sarabia et al., 2007).

The HIV-1 proviral DNA load could be an alternative viral marker, as it is known that proviral DNA persists in infected cells, even after prolonged successful HAART as evidenced by undetectable plasma RNA viral load. A decline in DNA might indicate a long-term impact of the HAART on the reservoirs and the long-term effectiveness of the treatment. But data regarding the decline in DNA are sometimes conflicting. Some authors noted decreased levels after one year of antiretroviral triple combination therapy and others reported stable HIV-1 DNA levels over several years in ART naive patients.

The quantification of HIV type 1 proviral DNA in peripheral blood mononuclear cells (PBMC) have previously been reported, and many of them are based either on the principle of conventional PCR requiring post PCR analysis or on real-time PCR on total PBMC, using specific probe detection.



Real-Time PCR Method

Real-time PCR is becoming increasingly popular as a method for the quantitative detection of DNA and RNA viruses. Besides its research applications, an accurate determination of the viral load, whether cell associated or free in biological fluids, can provide critical information in the clinical setting for the diagnosis and staging of acute and chronic viral infections, as well as for guiding treatment interventions and assessing their efficacy. In addition, real-time PCR has been applied for screening purposes to measure the level of contaminating viruses in blood donations or plasma pools for the production of blood derivatives.

HIV Diversity

The circulating strains of human immunodeficiency virus type 1 (HIV-1) have been classified into 3 major phylogenetic groups, termed M, N, and O, all of which cause HIV/AIDS in infected individuals. GroupM (for “major”) comprises the great majority of HIV-1 infection worldwide and has been further subdivided into 12 distinct lineages, termed subtypes (AD, FH, and J), and circulating recombinant forms (AB, AE, AG, and AGI). Group O (for “outlier”) comprises many fewer isolates that are geographically restricted to west central Africa. Finally, group N (for “non-M/non-O”) was discovered recently and is the least widespread of all HIV-1 lineages. Viruses belonging to this group have thus far only been identified in a handful of individuals, all of whom were residents of Cameroon. Sequence variation among members of these various lineages is extensive, with nucleotide sequence variation in the env gene with a range of 18% among the different group M subtypes and circulating recombinant forms (CRFs) to nearly 42% among members of all 3 groups. Given this extent of diversity, it is not surprising that the great majority of nucleic acidbased diagnostic tests originally designed for group M, subtype B viruses have reduced sensitivities in detecting viruses from other groups and subtypes.


Methods

Assay design

Quantification of the HIV-1 proviral DNA is increasingly used in the clinical follow-up of HIV-1infected individuals. However, due to the high degree of genetic variability of HIV-1, the identification of a PCR amplicon that guarantees the same efficiency of amplification with all the genetic subtypes of the main (M) HIV-1 group represents a major challenge to the design of a universal assay. We have developed an HIV-1 group M-specific quantitative realtime PCR assay that measures the HIV-1 proviral DNA load with a similar degree of sensitivity and accuracy regardless of the viral genetic subtype. The success of this protocol for the quantification of HIV-1 proviral DNA relies on the optimized design of primers and probe, which ensures the correct quantification of virtually all circulating group-M HIV-1 strains. Moreover, this assay is so robust that it can quantify DNA derived from a crude lysate with the same degree of accuracy as with purified DNA. As a starting point, we selected a region spanning parts of the HIV-1 LTR, which is highly conserved among all circulating group-M HIV-1 subtypes. The selected region is completely conserved in about 96% of the HIV-1 sequences present in the NCBI database. A realtime PCR assay was developed by designing two specific primers, and a probe within the selected LTR region.

We evaluated the accuracy and sensitivity of the HIV-1 of different genetic subtypes, some of which were carrying mutations in either the primers or the probe sequences. The modified assay was able to accurately measure proviral DNA from all the primary HIV-1 isolates tested with similar efficiency regardless of genetic subtype as well as from clinical samples derived from HIV-1-infected patients. The assay involves quantification in PBMC of HIV-1 seropositive patients. As 95 to 99% of infected cells are CD4+ cells, we developed a relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. Here, we have developed a multiplex real-time PCR for quantification of HIV-1 LTR DNA and the CCR5 gene in CD4+ cells using TaqMan probes. Sequence-specific hybridisation probes provide the most specific real-time analysis of amplified target sequences.


Cd4+ cell isolation

CD4+ cells were isolated from 10 ml of patient EDTA whole blood samples by an immunomagnetic method using anti-CD4 coated magnetic beads (Dynabeads® CD4 Positive Isolation Kit) according to the manufacturer's protocol and were stored at -80°C until use. The complete process takes approximately 3 hours and the purity of the CD4+ cell preparation was about 99% as estimated by Becton Dickinson Fascan Flow Cytometer technology.

DNA purification

DNA was extracted from purified patient CD4+ cells diluted in 200 µl PBS, using the Sigma Aldrich, GeneElute, (Cat. no. NA2020), according to the manufacturer's protocol. Particular attention was paid on DNAse and RNAse free materials. Depending on the number of samples, the whole DNA purification process required approximately 1 hour.


HIV-1 DNA real time PCR assay

A series of dilutions of HIV-1 DNA standard corresponding to 106 to 100 was included in each experiment in order to generate an external standard curve. The PCR mixture (total volume 20 μl) in nuclease free water contained MyTaq™ HS DNA Polymerase, final concentrations of 3mM MgCl2 and 0.5μM of each primer and 5μl of purified DNA or negative control. All samples were analysed in duplicate. The amplification protocol for HIV-1 on the CFX96™ (IVD) BIORAD was as follows: a 5 min denaturation step at 95°C for polymerase activation, of 45 cycles consisting of 15 seconds at 92°C, 1 minute at 65°C. The fluorescence was measured at the end of each elongation step. A fragment from the CCR5 gene was amplified in parallel with the HIV-1 LTR gene to quantify the total number of investigated cellular purified DNA of CD4+ cells.

Sample collection

The study based on the selected patient those who have undergone treatment with the Anti-HIV treatment of HOOIMM plus. Here, we have reported the status of Qualitative proviral DNA, Quantitative HIV-1 RNA RT-PCR, Western blot Assay, qualitative antibody assays for all the five treated HIV patients. Nevertheless, CD4 cells enumeration at present.

Technical resources for HIV-1 status.
  • Identification of serostatus: HIV COMB approved by USFDA
  • Detection of HIV-1/2 antibody and band profiles: Genelab HIV Blot 2.2 approved by USFDA
  • HIV-1 (RT-PCR) Quantitative assay: Real time Cobas Taqman approved by USFDA
Results:

It has been found that after the treatment, all the four patients were HIV asymptomatic seropositive, since their report showing positive with HIV COMB (Followed the Manufacture's protocol) (Table 1.1). Plasma viral load were detectable for all treated patient. The table 1.2: show different western blot band profile in the serum (Detection Genelab HIV Blot 2.2 USFDA) confirming the HIV Antibodies and HIV type. The blood samples were reported HIV-1 type for all individual. Table 1.1, Figure1, 1.2 and Table 1.3: shows proviral DNA not detected for all five treated patient.

Table 1.1: Patient characteristics

Table.1.2: Showing western blot band profiling of the 5 treated patients.

HIV INVIVO HUMAN CLINICAL STUDIES

In the Invivo human clinical studies, this drug were provided as a treatment to the HIV patients through HAKH Medical Foundation and the improvements in the patients in terms of physical as well as pathological were recorded in proper, controlled condition through standard procedure and all the blood test pertaining to HIV were undertaken through Molecular Diagnostic Services in association with Reliance Life Science and the reports & blood samples were maintained for more than 3-5 yrs for repeatability.

The clinical observation of this drug revealed the following improvements in the patients:
  • CD4 count doubled in the patient and CD8 count declined to 30-50%.
  • 7-8 folds decrease in HIV viral load counts within 5-6 weeks of the treatment.
  • Viral load count decreased to less than detectable limit (>100) within 5-7 months of the treatment.
  • 4-6 pounds increment in weight within 2-4 months.
  • Relief from HIV symptoms.
  • Prophylactic against opportunistic infections.
  1. Phenomenal change in DNA-PCR Antigen test Patients those continued the treatment for 18-20 months after attaining the viral load to less than detectable limit and adhering to instruction strictly had recorded HIV DNA-PCR Antigen Qualitative test ’Not Detected’ and the test interpreted ‘the blood sample to be negative for the presence of HIV”. And so the medication were completely stopped for almost one year and again undergone for HIV DNA-PCR Antigen test (Qualitative) and recorded ‘Not Detected’. The same test was repeated for 2nd, 3rd & 4th year after completely stopping the medications and the patients recorded ‘Not Detected’ for the test. The method for the confirming the presence of HIV antigen is HIV DNA-PCR Antigen test (Qualitative) test by Nested PCR Technique.
  2. A major breakthrough on HIV research through Antigen Vs Antibody Test. HIV antibodies remain in the body for a long time to come although HIV antigen is completely evaded from the body.
Methods : 6ml serum sample is to be drawn from the HIV patients who were earlier confirmed HIV positive through Western blot test and later undergone complete treatment with Unani medicine HOO-IMM PLUS. The sample is to be cautiously handled as per the NABL guidelines and to be sent to a renowned and reliable PCR Molecular diagnostic lab. 3 ml of the sample is to be allocated for Western Blot Antibody test and the other 3 ml for HIV DNA-PCR Antigen test.

Results : Our study showed that the above mentioned tests showed that the blood sample is positive for Western blot Antibody test & Negative for HIV DNA-PCR Antigen test. This directly interprets that at present the person is HIV negative though he still carries HIV antibodies authenticating his earlier infection with HIV. Conclusion: It could be concluded that our body immune system creates cell mediated immunity responses towards HIV antigen to produce antibodies that may persist for a long time, even after the antigen is completely eliminated from the body. The other best example is Mycobacterium Tuberculosis antibodies. The human body can respond to antigen in many different ways.

These fall into two major categories:
  • Antibody-mediated immunity. Antibodies, dissolved in blood, lymph, and other body fluids bind the antigen and trigger a response to it. (This form of immunity is also called humoral immunity).
  • Cell-mediated immunity (CMI). T cells (lymphocytes) bind to the surface of other cells, display the antigen and trigger a response and may remain in the body for a long time. The response may involve a) other lymphocytes and b) a ny of the other white blood cells (leukocytes)
HIV AIDS treatment is not complicated, knowing what drugs you are taking, how they work against HIV, and why it is so important to take them as prescribed (on time, with or without food, etc.) can really help in the fight against HIV. HIV AIDS treatment is safer, if your treatment consists of toxic free, side-effect free medications. HIV AIDS treatment becomes more comfortable, if your treatment doesn’t ask for switching on to new drug combination because of drug resistance. HIV AIDS treatment is not complicated, knowing what drugs you are taking, how they work against HIV, and why it is so important to take them as prescribed (on time, with or without food, etc.) can really help in the fight against HIV. HIV AIDS treatment is safer, if your treatment consists of toxic free, side-effect free medications. HIV AIDS treatment becomes more comfortable, if your treatment doesn’t ask for switching on to new drug combination because of drug resistance.