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. 1999 May 17;189(10):1545-54.
doi: 10.1084/jem.189.10.1545.

Constant mean viral copy number per infected cell in tissues regardless of high, low, or undetectable plasma HIV RNA

R D Hockett  1 , J M Kilby, C A Derdeyn, M S Saag, M Sillers, K Squires, S Chiz, M A Nowak, G M Shaw, R P Bucy
Affiliations

Constant mean viral copy number per infected cell in tissues regardless of high, low, or undetectable plasma HIV RNA

R D Hockett et al. J Exp Med. .

Abstract

Quantitative analysis of the relationship between virus expression and disease outcome has been critical for understanding HIV-1 pathogenesis. Yet the amount of viral RNA contained within an HIV-expressing cell and the relationship between the number of virus-producing cells and plasma virus load has not been established or reflected in models of viral dynamics. We report here a novel strategy for the coordinated analysis of virus expression in lymph node specimens. The results obtained for patients with a broad range of plasma viral loads before and after antiretroviral therapy reveal a constant mean viral (v)RNA copy number (3.6 log10 copies) per infected cell, regardless of plasma virus load or treatment status. In addition, there was a significant but nonlinear direct correlation between the frequency of vRNA+ lymph node cells and plasma vRNA. As predicted from this relationship, residual cells expressing this same mean copy number are detectable (frequency <2/10(6) cells) in tissues of treated patients who have plasma vRNA levels below the current detectable threshold (<50 copies/ml). These data suggest that fully replication-active cells are responsible for sustaining viremia after initiation of potent antiretroviral therapy and that plasma virus titers correlate, albeit in a nonlinear fashion, with the number of virus-expressing cells in lymphoid tissue.

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Figures

Figure 3
Figure 3
Representative photomicrographs of ISH for HIV RNA. (A) Suspension of normal PBMC infected with HIV-1 in vitro. (B) HIV RNA+ cell identified in lymph node frozen tissue section. (C) Lymph node section of patient HADE showing HIV RNA in germinal center of lymph node scored as 1+ and two individual positive single cells. (D) Lymph node section of patient TRRA showing HIV RNA in germinal center of lymph node scored as 3+ and two individual positive single cells.
Figure 1
Figure 1
Plasma samples from HIV-infected patients were obtained and immediately separated into 1-ml aliquots, which were analyzed by each of two methods with blinded results. In addition, a standard preparation of HIV RNA was obtained from the National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Program and analyzed by the UAB QC-RT-PCR procedure. Roche Monitor® assay was performed as per manufacturer's instructions. Specimens with viral loads >5 × 105/ml were analyzed after appropriate dilution. The UAB QC-RT-PCR procedure was performed as described in Materials and Methods.
Figure 2
Figure 2
Analysis of HIV RNA+ cells by LDA and single-cell UAB QC-RT-PCR. (A) LDA for the frequency of HIV vRNA–expressing cells on five untreated patients described in Table I. After dilution of lymph node cells in 96-well microtiter plates, each well was harvested and QC-RT-PCR performed. For the graph in A, wells were scored positive or negative for HIV vRNA only. The number of lymph node cells diluted per well is plotted along the x-axis, and the percent negative wells by QC-RT-PCR is plotted along the y-axis. The frequency of HIV vRNA–expressing cells per 106 lymph node cells was calculated by Poisson statistics. (B) Frequency histograms for four of the LDAs graphed in A for quantification of copies per well. The range of copies per well for each bar on the x-axis represents 0.3 log10 multiples of copies of HIV vRNA. Only positive wells (>1,000 copies) were used for the calculation of mean HIV vRNA copies per well. See Table I for summary of other patient data and calculations of HIV vRNA quantity in different pools.
Figure 2
Figure 2
Analysis of HIV RNA+ cells by LDA and single-cell UAB QC-RT-PCR. (A) LDA for the frequency of HIV vRNA–expressing cells on five untreated patients described in Table I. After dilution of lymph node cells in 96-well microtiter plates, each well was harvested and QC-RT-PCR performed. For the graph in A, wells were scored positive or negative for HIV vRNA only. The number of lymph node cells diluted per well is plotted along the x-axis, and the percent negative wells by QC-RT-PCR is plotted along the y-axis. The frequency of HIV vRNA–expressing cells per 106 lymph node cells was calculated by Poisson statistics. (B) Frequency histograms for four of the LDAs graphed in A for quantification of copies per well. The range of copies per well for each bar on the x-axis represents 0.3 log10 multiples of copies of HIV vRNA. Only positive wells (>1,000 copies) were used for the calculation of mean HIV vRNA copies per well. See Table I for summary of other patient data and calculations of HIV vRNA quantity in different pools.
Figure 4
Figure 4
Relationship of plasma viral load to HIV vRNA–expressing cells in lymph node tissue. Each point represents a separate biopsy specimen paired with the plasma viral load at time of biopsy. HIV vRNA+ cells were measured by ISH, and plasma viral loads were determined by the Roche Monitor® ultra-sensitive assay. The lines connecting points with the same symbols represent the same patient before and after HAART, as shown in Table I. Overall regression line (using the log-transformed data) of these data: slope = 1.6; r =  0.95 (P < 10−8).
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