Monitoring processed, mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen
© Baird et al; licensee BioMed Central Ltd. 2005
Received: 28 February 2005
Accepted: 12 April 2005
Published: 12 April 2005
Protease inhibitors (PIs) block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.
Assembly and transport of the 55 kDa gag (p55gag) and 160 gag-pol (p160gag-pol) proteins to the inner plasma membrane is essential for the packaging of the viral genomic RNA, host tRNALys,3 primer, as well as for interactions with HIV-1 envelope glycoproteins . Budding and virus release initiates the processing of the gag and gag-pol precursor proteins. This processing step likely requires the dimerization of two gag-pol precursors (at least in the region of protease) that permits a low-efficiency cleavage of the precursor proteins and release of fully active protease (PR) homodimers . These enzymes then complete protein maturation to produce an infectious virus particle. Thus, protease inhibitors (PI) appear to be most active at blocking HIV-1 replication following budding of the immature virus particle [4, 6]. In contrast other antiretroviral drugs (ARV) such as nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI), block reverse transcription during intracellular HIV-1 replication .
To date, the best method to monitor inhibition of HIV-1 replication is to evaluate virus concentrations in the plasma . Several commercial, FDA-approved assay kits (HIV-1 Quantiplex (bDNA) assay, AMPLICOR HIV-1 MONITOR assay, NucliSens HIV-1 assay) involve measuring virus levels via reverse transcription-PCR amplification of genomic HIV-1 RNA . It is important to recognize however, that these assays cannot monitor the pharmacodynamic properties of many antiretroviral agents immediately following treatment initiation. Protease inhibitors block HIV-1 protease processing following virus release from cells in contrast to NNRTIs or NRTIs that inhibit during an intracellular replication step, i.e. reverse transcription. The half life of plasma virus is estimated to be approximately 6 hrs [13, 18]. but the half life of activated CD4+ cells infected with and producing HIV even in the presence of PIs is approximately 1.2 days [13, 18, 19] during phase I decay An assay measuring levels of viral RNA does not distinguish between the immature virus (processing blocked by PIs) and infectious virus, both of which encapsidate HIV-1 genomic RNA. The estimated time required for protease inhibitors to clear the majority of free virus particles from the circulation and activated cells (not latently infected or quiescent cells) is approximately 4 weeks. Thus, a viral RNA assay performed on plasma does not provide a complete assessment of PI activity for at least 1–4 weeks.
In this study, we tested the ability of three different assays to measure the quantity of both infectious virions and defective/immature virus particles in the plasma of HIV-infected patients who started treatment with a PI-containing regimen. The performance of three assays was validated in vitro utilizing HIV-1 infected cell lines in the presence or absence of PIs and other ARVs. These tests were followed by in vivo analyses using plasma samples from patients receiving a PI-based treatment regimen. The following provides a brief summary of the first two assays that could detect the effects of PI activity in vitro but failed in vivo.
The first assay involved measurement of infectious virus potential. We serially diluted cell-free culture supernatants from chronically HIV-infected U87.CD4.CCR5 cells treated with PIs. This diluted and undiluted plasma was then added to uninfected peripheral blood mononuclear cells (PBMC). Although this assay could be used to measure infectious potential of high titer viruses in tissue culture, only plasma containing extremely high viral loads (> 104 viral RNA copies/ml) could support any HIV-1 infection of PHA/IL-2 treated PBMC regardless of the patients treatment status (data not shown). Concentrating the virus by ultracentrifugation did little to increase infectious titer of virus from plasma.
The second assay involved PCR amplification of strong-stop viral DNA found in cell-free virus. Previous reports have shown that viral DNA is found HIV-1 particles [9, 17] but that steric hindrance or the lack of dNTP substrates limit reverse transcription and presence of viral DNA to 1:1000 to 1:10,000 virions [1, 2]. We have shown that a defective protease abolishes the synthesis of any HIV-1 DNA in virus particles [1, 2]. HIV-1 strong-stop DNA was not detected by PCR amplification in virus produced from the chronically infected cells in the presence of PIs (data not shown). However, viral loads of >10,000 RNA copies/ml were required in patients to even detect the presence of HIV-1 DNA in plasma, which is consistent with previous findings. Thus, this assay was not effective for those patients starting PI therapy with lower viral loads (<103–4 RNA copies/ml).
In contrast to the assays described above, an anti-p24 Western blot was successful in measuring both in vitro and in vivo PI effects and was the simplest in design and application. To initially test this assay we infected U87.CD4.CXCR4 cells with a wild type HXB2 virus or the protease inhibitor resistant virus, RF (containing PR mutations V82F and I84V) . Following established infection and stable virus production over three days (as measured by RT activity in the culture supernatant), cultures were treated with 0.2 and 20 nM lopinavir (LPV) or 2 and 200 nM nevirapine (NVP). The higher concentration of each drug was approximately 100-fold greater than the reported IC50 values (i.e. lower concentrations of each drug) [11, 14, 15]. Cell free culture supernatant (1 ml) was then harvested at 0, 4, 8, 24, and 72 h post drug addition. Virus was pelleted from the supernatant by ultracentrifugation (35,000 g for 1 h) and then resuspended in 50 μl of sodium-dodecyl sulfate (SDS) lysis buffer (1% SDS, 10% glycerol, 10% β-mercaptoethanol, 0.04 M Tris pH 6.8); of which 10 μl were heated to 95°C, separated on Tris-HCl-12.5% polyacrylamide precast gels (Bio-Rad), and transferred onto polyvinylidene difluoride membranes (Immobilon P; Millipore)by electroblotting (BioRad). Membranes were incubated with blocking reagent (5% milk-0.05% Tween in phosphate-bufferedsaline) for 1 h at room temperature then hybridized with a mouse anti-p24 monoclonal antibody (diluted 1:1,000; Fitzgerald Industries International, Inc.) overnight at 4°C. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG1 antiserum (diluted 1:40,000; Pierce) for 3 hours. Immune complexes were visualized with the ECL system (Amersham) according to the manufacturer's instructions and films were analyzed using BioRad Quantity One software.
Results and discussion
To test the utility of this simple Western blot assay to monitor initial PI treatment, nine patients were enrolled into the A5036s Substudy of the ACTG Clinical Trial Substudy A5014 . All of the patients in A5014 were ARV treatment naïve and were randomized to receive either LPV+ the non-nucleoside RT inhibitor NVP or NVP + three nucleoside RT inhibitors: lamivudine (3TC) + stavudine (D4T) + abacavir (ABV). All participants and investigators in this study were blinded to the treatment arms. Ten ml of blood was drawn into Acid Citrate Dextrose (ACD) tubes prior to the first drug administration, then 4, 8, 12, 24, 72 hours, three days and four weeks post drug administration. Two aliquots of 3.5 ml of plasma were shipped on dry ice to CWRU and then stored at -70°C prior to analyses.
In addition to the Western blot analyses, the sub-study also called for a measure of infectious potential by HIV-1 found in plasma. For these tests we exposed HIV-negative peripheral blood mononuclear cells to plasma samples obtained prior to and immediately following treatment with the PI- or non PI-containing HAART regimen. Only plasma samples of one patient (of 9) resulted in productive infection of PHA/IL-2 treated PBMC cultures suggesting that this not a sensitive assay. Unfortunately, no assessment of PI activity could be evaluated using this infectious assay since this patient was treated with NVP and the three NRTIs. It is unlikely that viral levels in plasma is the sole factor contributing to the ability of plasma virus to infect PBMC cultures since all patients in this substudy had viral RNA loads approximately 104 copies/ml at initiation of treatment. The level of virus production or success of PBMC infections did not increase if the virus was concentrated from plasma by ultracentrifugation. This concentration step would also remove any residual drug in plasma that might affect infectivity of virus in plasma after the initial treatment.
We predicted that the p55:p24 ratio would increase during first three days of PI treatment with the possible dips in this ratio between PI dosages (every 12 h). Preliminary data with patients starting a PI-containing treatment regimen suggest a PI-mediated inhibition of p55 processing within 8–12 h of treatment (data not shown). However, these studies were performed with patients starting RIT+SAQ or IND-containing treatment regimens and not with patients treated with LPV. As indicated by the results of tissue culture infection experiments shown in Fig. 1, the p55:p24 ratio should remain stable in plasma samples obtained from patients receiving non-PI containing HAART regimens (i.e. NVP+3TC+D4T+ABV) since neither NRTI nor NNRTI inhibit processing of the gag or gag-pol precursors.
Although only one example of these analyses is shown (Fig. 2B, panel I and II), the Western blot results of plasmas from each of four patients treated with the NVP+3TC+D4T+ABV combination showed a constant ratio of p55:p24 following treatment. In patients randomized to receive the LPV+NVP regimen, the ratio of p55:p24 increased at 72 h following the initial dosing (Fig. 2B). This increase in the p55:p24 ratio was maintained after 4 weeks of PI treatment. Previous findings revealed that HAART resulted in a drop in RNA and plasma infectivity in one day , however, the efficacy of ARV treatment can be affected by factors such as drug concentrations, compliance, potency, and selection of ARV resistant quasispecies. Unfortunately, two of the patients randomized to receive the LPV+NVP combination dropped out of the 5036 sub-study prior to the 72 h sample collection, i.e. the time that is likely required to detect a LPV block on viral protein maturation. In one patient, the p24 band on the Western blot was below the limit of detection in all plasma samples. There was an apparent delay in LPV activity following treatment in vivo as compared to treatment in tissue culture (Figs. 1 and 2). A longer time was likely required to attain inhibitory concentrations in blood or other tissues whereas the effect of LPV on newly produced virus particles was immediate in tissue culture.
In summary HIV protease inhibitors block the processing of p55gag and p160gag-pol precursor proteins during virus budding or following virus release. However, the protease inhibitor does not impede incorporation of genomic HIV-1 RNA into the virus particle. Thus, following PI treatment, viral load assays based on detection of viral RNA measure both noninfectious, immature virus particles and virions found in plasma. We have developed a method to measure the anti-HIV activity of a protease inhibitor using a simple approach. In three patients treated with LPV+NVP, the ratio of unprocessed p55:processed p24 increased at 72 hours and over the next four weeks of treatment. In contrast, the ratio of HIV-1 p55:p24 did not change over the four weeks of study in patients treated with an NNRTI-containing regimen (NVP+3TC+D4T+ABV). This pilot study, though limited in patient number, has provided evidence that an HIV-1 p24 Western blot can be used to immediately measure the antiviral activity of protease inhibitors. Preliminary in vitro data also suggests that inability of PIs to block PI-resistant HIV-1 in patients could be assessed within 3 days of treatment. Based on these findings we are now testing the utility of this assay in highly PI experienced patients to predict the success of a new PI-containing treatment regimen within 3 days of starting this therapy. In addition, this study indicates that western blot is an excellent tool for the evaluation of the activity of protease inhibitors in vitro, and may be useful in evaluating new drugs putatively active against isolates resistant to current agents, or to evaluate the activity of different combinations of protease inhibitors using a more insightful measure than viral infectivity.
Research for this study was performed at Case Western Reserve University (E.J.A.) and was supported by funds from Social and Scientific Systems Inc. and the NIH/NIAID AIDS Clinical Trial Group. We thank the AACTG5014 and AACTG 5036 s team for their coordination of samples collection and support. Additional support was provided to EJA from the National Institute of Allergy and Infectious Diseases, NIH (AI49170). All virus work was performed in the Biosafety Level 2 and 3 facilities of the CWRU Center for AIDS Research (AI36219).
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