Abstract

The follicular dendritic cell network (FDC) in lymphoid tissues (LTs) is the major site of human immunodeficiency virus (HIV) storage in presymptomatic and late stages of disease. However, little is known about the rate of virus accumulation during the acute and early stages. In situ hybridization and quantitative image analysis were used to determine the amount of virus bound to the FDC network during the first year of infection. The FDC pool was already >7.0 log10 copies of HIV RNA/g LT in the first year, and 2 patients biopsied within 2–4 days of symptom onset had 7.3 and 8.2 log10 copies of HIV RNA/g LT, respectively. There was no correlation between duration of infection and accumulation of HIV into the FDC network. These data suggest that a large pool of infectious virus is established soon after infection and that initiation of antiretroviral therapy when symptoms of primary HIV infection are recognized is unlikely to prevent substantial accumulation of virus in the FDC network.

In the presymptomatic and late stages of human immunodeficiency virus (HIV)-1 infection, large quantities of virus are stored in lymphoid tissue (LT) by follicular dendritic cells (FDCs) [1–3]. In the late stages of the infection (clinical AIDS), the total body pool of FDC-associated virus has been estimated to be between 1010 and 1011 virions, equivalent to >1013 copies of the major viral core protein P24 [4]. There are at least 3 potentially important implications of this accumulation of virus for immune function, virus persistence, and the success of antiretroviral therapy. First, HIV antigens dominate the FDC network, where B and T cells interact to generate immune responses, and may interfere with the response to other antigens. Second, the high concentration of virus in the FDC pool found in patients with long-standing infection is associated with involution and collapse of follicles, further compromising the host's ability to mount and maintain a functional immune response to antigens [1, 5, 6]. Third, the FDC pool also represents a potential source of virus [1, 2, 4, 7] to perpetuate infection because of the ability of FDCs to reactivate infectious virus complexed with neutralizing antibody [8] or (alternatively) release infectious virus attached to FDCs through an independent interaction of antibody with antigen [9]. A >2500-fold reduction in this pool has been documented after initiation of highly active antiretroviral therapy (HAART), but even after 6–12 months of therapy, the pool of stored virus in some patients is still >106 virions [10, 11]. How persistence of virus and viral antigen stored in the FDC pool may eventually affect the success of HAART in fully reconstituting the immune system remains to be determined. Previous studies have suggested that accumulation of virions into the FDC pool may occur slowly [12]. If true, this would provide a rationale for early identification of patients (during the seroconversion illness) and intervention to minimize damage to the network of FDCs and diminish the residue of persistent infection that HAART and immune responses must contain. Using in situ hybridization (ISH) and quantitative image analysis (QIA), we set out to examine the rate at which virus accumulates into the FDC compartment to determine what (if any) potential exists for early therapy to eradicate the infection.

Materials and Methods

Subject identification and enrollment of patients

Patients were recruited from ongoing investigations of primary and early HIV-1 infection at 4 sites: the University of Minnesota (UMN; n = 2), the University of Colorado Health Sciences Center (UCHSC; n = 2), the University of Washington (UW; n = 8), and the University of California, San Diego (UCSD; n = 9). Of the 21 patients reported here, 15 experienced symptoms of an acute retroviral syndrome (median duration, 7 days; range, 4–45 days), and thus the date of seroconversion was based on the first day of symptom onset. The remaining 6 did not report any symptoms, and the date of seroconversion was assigned as the midpoint between the last negative and first positive HIV antibody test.

At entry, subjects completed standardized interviews to enable collection of clinical and epidemiologic information, and baseline laboratory samples were obtained. Subjects who agreed to lymph node biopsy had tissue removed by means of standard surgical techniques. Two of the biopsies were axillary, and 19 were inguinal. Specimens that were preserved appropriately for in situ hybridization and were obtained from individuals in the first year of infection were shipped to the UMN for analysis.

HIV-1 serology, T-cell subset analysis, and plasma HIV-1 RNA

HIV-1 antibody testing was done by EIA and Western blot techniques according to the manufacturers' directions. T-cell subset analysis was done by using flow cytometry. At UW, HIV-1 RNA in plasma was initially determined by use of the first-generation bDNA assay (Chiron, Emeryville, CA), which has a lower limit of detection of 10,000 copies of HIV-1 RNA/mL plasma. Plasma samples that were near or below the limit of detection of the bDNA assay (<12,000 copies/mL of HIV-1 RNA) were subsequently retested by use of the Amplicor method (Roche, Somerville, NJ), which has a lower limit of detection of 400 copies of HIV-1 RNA/mL plasma. At UCSD, UCHSC, and UMN, HIV-1 RNA in plasma was determined by using the Amplicor method.

ISH and QIA

The number of HIV-1 RNA copies in virions associated on FDCs was determined by combining ISH, as described elsewhere [4]. In brief, 10 sections that were 8 μm thick were cut from different regions of the node, adhered to siliconized glass slides, and deparaffmized. After treatments to facilitate diffusion of probe, the sections were hybridized to a collection of antisense 35S-labeled riboprobes complementary to ∼90% of full-length HIV genomic RNA sequences. After hybridizing and washing, the sections were coated with nuclear track emulsion, exposed for 24 h, developed, and stained (figure 1). Probe bound to viral RNA in virions associated with FDCs and their processes generates a diffuse hybridization signal of silver grains scattered over germinal centers. The number of silver grains is proportional to the number of viral RNA copies, and, consequently, the latter can be estimated from the number of silver grains, the specific activity of the probe, the exposure time, and the efficiency of 0.5 grains/dpm for 35S. This method of estimating copy number has been validated and is reproducible within ±15% [4]. Video images of autoradiographs, illuminated with epipolarized light, were captured, and silver grains were automatically enumerated with the Metamorph software program (v. 2.5; Image 1, West Chester, PA). The number of HIV RNA copies was expressed as log10/g LT by estimating the weight of the individual sections from the product of the area (A), thickness (T), and density (D) of the section (A × T × D).

Figure 1

This figure illustrates the large quantity of human immunodeficiency virus (HIV) RNA (8.2 log10 copies HIV RNA/g lymphoid tissue [LT]) trapped onto the follicular dendritic cell (FDC) network in axillary LT obtained from an individual exposed to HIV through anal intercourse 2 days after onset of symptoms associated with acute HIV infection. After hybridization to 35S-labeled HIV-specific riboprobes, probe bound to viral RNA in virions complexed with the FDC network generates a diffuse signal of silver grains in a germinal center which have a yellow color when illuminated by an epipolarized light source. There are also many productively infected cells in the paracortical region. The hybridization signal generated by high intracellular concentrations of HIV-1 RNA is concentrated over infected cells (arrow).

Figure 1

This figure illustrates the large quantity of human immunodeficiency virus (HIV) RNA (8.2 log10 copies HIV RNA/g lymphoid tissue [LT]) trapped onto the follicular dendritic cell (FDC) network in axillary LT obtained from an individual exposed to HIV through anal intercourse 2 days after onset of symptoms associated with acute HIV infection. After hybridization to 35S-labeled HIV-specific riboprobes, probe bound to viral RNA in virions complexed with the FDC network generates a diffuse signal of silver grains in a germinal center which have a yellow color when illuminated by an epipolarized light source. There are also many productively infected cells in the paracortical region. The hybridization signal generated by high intracellular concentrations of HIV-1 RNA is concentrated over infected cells (arrow).

Results

We obtained lymphoid tissue from 20 men and 1 woman a median of 133 days (range, 2–294 days) after onset of symptoms associated with primary HIV infection. Biopsies were performed on 3 of the patients within 10 days of onset of symptoms, or about the third week of infection, based on a median incubation period of 12–14 days from exposure to symptoms [13]. These patients were considered to be in the acute stages of HIV infection, because they had not as yet formed antibodies to HIV-1 at the time of biopsy. The other biopsies spanned the periods 15–90 days (4 biopsies), 91–180 days (7 biopsies), and 180–294 days (7 biopsies) after onset of symptoms. We subsequently refer to these intervals as early-stage HIV-1 infection.

Contrary to our initial hypothesis, we found that virus could accumulate to very high levels in the FDC pool as early as 2–4 days after onset of symptoms (table 1, figure 1) and that there was no correlation between the amount of virus in this pool and duration of illness (r2 = −.18, P = .4; figure 2). In the 2 axillary biopsies from patients who had been exposed rectally and who had symptoms associated with primary HIV infection sufficiently severe to see a physician or require brief hospital-ization, the FDC pool of virus was >7 log10 copies of HIV-1 RNA/g. In the axillary lymph node from the patient at day 2 of symptoms, the FDC pool of virus was equivalent to that measured in the presymptomatic and late stages of disease. The estimated total body FDC pool of virus in this 70-kg individual was 8.2 log10 copies of HIV-1 RNA/g, corresponding to 1.1 × 1011 copies of viral RNA, or 6 × 1010 virions (2 copies of viral RNA/viral particle). There was no difference between the mean size of the FDC pool of virus in the first 120 days and that after 120 days (the point at which plasma HIV-1 RNA becomes predictive of the rate of progression to AIDS [13], table 1). In addition, there was no significant difference in the mean size of the FDC pool of virus between patients sampled in the intervals of 0–14, 15–90, 91–180, and 181–300 days from symptom onset (7.2, 6.8, 7.5, and 6.8 log10 copies HIV-1 RNA/g, respectively; Kruskall Wallis test, P = .5).

Table 1

Production and storage of human immunodeficiency virus (HIV)-1 in lymphoid tissues in first year of infection.

Table 1

Production and storage of human immunodeficiency virus (HIV)-1 in lymphoid tissues in first year of infection.

Figure 2

The size of the pool of human immunodeficiency virus (HIV)-1 RNA associated with follicular dendritic cells (FDCs) is not related to duration of infection. The log10 transformed data from table 1 on size of FDC pool of virus have been plotted against days after onset of symptoms associated with acute HIV infection. There is no statistically significant correlation between size of pool and duration of infection (r2 = −.18; P = .4).

Figure 2

The size of the pool of human immunodeficiency virus (HIV)-1 RNA associated with follicular dendritic cells (FDCs) is not related to duration of infection. The log10 transformed data from table 1 on size of FDC pool of virus have been plotted against days after onset of symptoms associated with acute HIV infection. There is no statistically significant correlation between size of pool and duration of infection (r2 = −.18; P = .4).

Discussion

The FDC pool of virus is established by the time symptoms associated with primary HIV infection are recognized, based on these data. We observed 7–8 log10 copies of HIV-1 RNA/g of LT sampled within a few days of symptom onset, similar to levels associated with late-stage disease. The fact that this tissue was both axillary and from patients with rectal exposure illustrates the speed at which systemic dissemination occurs after mucosal transmission. This finding was a surprise to us, because it has been reported that accumulation of virus into this pool is gradual [12]. These discrepant results may represent a sampling error, because the number of patients reported by Pan-taleo et al. and by us is relatively small. However, the finding of several patients with significant deposition of virus into the FDC pool prior to detection of antibodies suggests that, whatever the mechanism, virus deposition into this compartment can occur rapidly. We observed significant intersubject variability in the FDC pool of virus, and FDC levels of virus did not correlate with time from onset of symptoms of HIV se-roconversion (table 1). This variability demonstrates the very complex relationship formed between the evolving host immune response and rate of viral replication in the earliest stages of infection. This has also been documented in the pool of peripheral virus [13]. Although not documented in this report, the early association of virus and FDCs may initiate the pathologic changes in follicles that lead to the changes seen in later stages of infection. Collectively, these findings on the early accumulation of virus into the FDC pool make it unlikely that antiretroviral therapy initiated as soon as symptoms are recognized will necessarily prevent deposition of large enough quantities of virus in the FDC pool or the FDC network. However, early treatment might limit injury and reduce the FDC pool of virus to a level at which pathologic changes are reversed and virus-specific CD4T cells are preserved, enabling infected individuals to mount and maintain a more efficient immune response to HIV-1 infection [14]. We think that these potential benefits to the immune system, rather than virus loads in the LT reservoir per se, offer a persuasive rationale for early therapy and that further research into those questions is warranted.

Acknowledgments

We thank Aldan Harken, M.D., Melodie Bahan, Timothy Leonard, Gerald Sedgewick, John Erbe, and Steven Wietgrefe for preparation of the manuscript, figures, and statistical analysis.

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Institutional review board-approved informed consent was obtained from all individuals prior to lymph node biopsy.
Financial support: NIH grant support (AI28246, AI30731, AI01338, AI38858, AI 43638, AI27670, AI36214, AI29164, and AI43752 and ACTG grant 2-U01-A127664-06, AI451536-01).