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Journal of Virology, June 2008, p. 5548-5561, Vol. 82, No. 11
0022-538X/08/$08.00+0     doi:10.1128/JVI.00124-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the Follicular Dendritic Cell Reservoir of Human Immunodeficiency Virus Type 1 ^,

Brandon F. Keele,^1, Loubna Tazi,² Suzanne Gartner,³ Yiling Liu,³ Trever B. Burgon,^1, Jacob D. Estes,^1,¶ Tyler C. Thacker,^1,|| Keith A. Crandall,² Justin C. McArthur,³ and Gregory F. Burton^1*

Department of Chemistry and Biochemistry,¹ Department of Biology, Brigham Young University, Provo, Utah 84602,² Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21287³

Received 17 January 2008/ Accepted 24 March 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Throughout the natural course of human immunodeficiency virus (HIV) infection, follicular dendritic cells (FDCs) trap and retain large quantities of particle-associated HIV RNA in the follicles of secondary lymphoid tissue. We have previously found that murine FDCs in vivo could maintain trapped virus particles in an infectious state for at least 9 months. Here we sought to determine whether human FDCs serve as an HIV reservoir, based on the criteria that virus therein must be replication competent, genetically diverse, and archival in nature. We tested our hypothesis using postmortem cells and tissues obtained from three HIV-infected subjects and antemortem blood samples obtained from one of these subjects. Replication competence was determined using coculture, while genetic diversity and the archival nature of virus were established using phylogenetic and population genetics methods. We found that FDC-trapped virus was replication competent and demonstrated greater genetic diversity than that of virus found in most other tissues and cells. Antiretrovirus-resistant variants that were not present elsewhere were also detected on FDCs. Furthermore, genetic similarity was observed between FDC-trapped HIV and viral species recovered from peripheral blood mononuclear cells obtained 21 and 22 months antemortem, but was not present in samples obtained 4 and 18 months prior to the patient's death, indicating that FDCs can archive HIV. These data indicate that FDCs represent a significant reservoir of infectious and diverse HIV, thereby providing a mechanism for viral persistence for months to years.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A major obstacle in the treatment of human immunodeficiency virus (HIV) disease is the establishment of viral reservoirs that provide a continuing source of infectious virus (3, 6, 14, 25, 39, 40, 49). HIV reservoirs not only maintain viral infectivity but also harbor drug-resistant and immune-escape quasispecies that may contribute to ongoing viral evolution under drug- or immune-selective pressures. Much attention has been focused on the latently infected CD4⁺ T-cell reservoir (7, 15, 62) and, to a lesser extent, the macrophage reservoir (9, 32, 51); however, little information exists regarding follicular dendritic cells (FDCs) as an HIV reservoir (16, 29).

HIV reservoirs have been characterized as cells or tissues that restrict virus replication and preserve replication-competent HIV for long periods of time (35). Reservoirs will contain infectious virus, having a greater stability (i.e., a longer half-life) than virus in cells where active viral expression is ongoing (3). Because of this stability, virus accumulates over time, thus creating an "archive" possessing higher overall genetic diversity than that of the non-reservoir-associated virus (5, 34, 35). Because viral reservoirs present major obstacles to the success of antiretroviral (ARV) therapy, we sought to characterize the FDC HIV reservoir.

FDCs are localized in the follicles of all secondary lymphoid tissues, where they help establish and maintain potent, long-term immunoglobulin G (IgG) and IgE humoral immunity (22, 26, 31). FDCs trap and retain antigens on the surfaces of their dendritic processes, including intact HIV virions. These trapped antigens and virus particles exist in the form of immune complexes (4, 24, 52). FDC-trapped antigens and virus particles have a half-life of approximately 2 months and are maintained in their native nondegraded state (52, 59). Because HIV is held on the surface of the FDC, it does not actively replicate or further evolve, despite retaining the ability to infect nearby trafficking cells (52). Importantly, although FDCs themselves remain uninfected, they reside immediately adjacent to activated T and B cells in the germinal center and the long dendritic processes of FDCs interact directly with these adjacent lymphocytes.

During primary HIV infection, massive quantities of virus are trapped on FDCs (1, 2, 45, 58). In fact, FDCs likely represent the largest repository of HIV, with a viral burden estimated to contain 1.5 x 10⁸ copies of viral RNA per gram of lymphoid tissue (19, 20). FDC-associated virus remains throughout the course of disease until the ultimate demise of the FDCs. Furthermore, throughout the natural course of infection, active viral replication in T cells can be observed surrounding germinal centers where the FDC resides. In a nonpermissive murine model, FDC-trapped virus possessed an in vivo half-life of 8 weeks and infectious virus could be rescued from FDCs for at least 9 months after a single injection of virus (52). Importantly, the maintenance of infectivity of FDC-trapped HIV did not require either infection of the FDC or replication of the virus. Studies using human FDCs in vitro indicate that HIV is preserved in a replication-competent state for about 1 month (52, 53). In contrast to the case for other dendritic cells, FDCs do not express CD209 (dendritic cell-specific, ICAM-3-grabbing nonintegrin [DC-SIGN]) for the maintenance of infectivity and trapped virus particles are not internalized (36). FDC maintenance of viral infectivity requires Fc and/or complement receptors on the FDC and the presence of a specific antibody that reacts with the virion (24, 53, 54).

Although FDCs have been suggested as a possible HIV reservoir because of their ability to retain virus in a replication-competent form for many months in a murine model (52), direct testing of FDCs isolated from infected human subjects has not been performed. Here we tested the hypothesis that FDCs are a reservoir of HIV in humans. We found that FDC-trapped virus was replication competent and possessed greater genetic diversity than that of virus found in most other cells and tissues tested, suggesting the accumulation of genetic variants over time. Consistent with the archival nature of virus reservoirs, HIV on FDCs had genotypes similar to those of viral variants detected from peripheral blood mononuclear cells (PBMCs) obtained at 21 and 22 months, but not 4 and 18 months, prior to the patient's death. Thus, we report here that FDCs do indeed fulfill the criteria of an HIV reservoir in humans and have the ability to contribute to persisting disease. A greater understanding of the nature of the FDC reservoir should provide the foundation needed to devise more successful strategies to target this important source of infectious HIV.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient population. Lymph nodes (LN) and spleen sections were obtained at autopsy from three HIV-infected subjects (JHU559, -614, and -621) at Johns Hopkins Hospital, Baltimore, MD (Table 1). The LN (and in one instance, i.e., for subject JHU614, splenic fragments) from each patient were collected and used for the isolation and culture of specific cell types. In some instances (i.e., for subjects JHU559 and -614) mid-frontal gyrus, deep white matter, meninges, and bone marrow (for subject JHU614 only) were also obtained for direct viral DNA isolation. This research has complied with all relevant federal guidelines and institutional policies.

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TABLE 1. HIV-infected subjects involved in this study

cell_isolation_from_secondary_lymphoid_tissues.">FDC and CD4⁺ T-cell isolation from secondary lymphoid tissues. FDC and CD4⁺ T-cell populations were isolated from LN or splenic fragments. Briefly, tissue was cut into small cubes (2 mm³) and digested in RPMI 1640 (HyClone, Logan, UT) containing DNase I (150 IU; Sigma, St. Louis, MO), collagenase D (20 mg; Roche Applied Science, Indianapolis, IN), and gentamicin (50 µg/ml; Invitrogen, Carlsbad, CA). Tissues were then incubated at 37°C for 45 min, with periodic mixing. The freed cells were removed and transferred to medium supplemented with antibiotics and heat-inactivated fetal bovine serum (FBS) (33%, vol/vol) (HyClone, Logan, UT). The remaining undigested tissue sections were again incubated in fresh medium containing the above enzyme cocktail, and newly released cells were collected as before. After the second digestion, any remaining tissue was gently passed in and out of a sterile pipette to further disassociate any remaining cells. All collected cells were pooled, washed in fresh medium to remove enzymes, and resuspended in fresh medium.

FDCs were isolated using positive selection. Cells were incubated on ice (with gentle agitation) with the primary antibody HJ2 (mouse IgM monoclonal antibody that binds human FDCs [kindly provided by Moon Nahm, University of Alabama at Birmingham]), after which they were washed in fresh medium and labeled for 30 min with a secondary antibody (rat anti-mouse IgM) conjugated to magnetic beads (Dynabeads; Invitrogen, Carlsbad, CA). Cells were isolated by magnet separation and washed thoroughly to remove residual contaminating cells. In some instances, FDCs were isolated via fluorescence-activated cell sorting using a FACSVantage SE system with FACSDiva software (BD Biosciences, San Jose, CA) with HJ2 as the primary antibody, followed by a goat F(ab')₂ anti-murine IgM-fluorescein isothiocyanate-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). In all cases, the preparations of FDCs were subjected to 3,000 rad of irradiation before use to block the proliferation of any possible contaminating lymphocytes that could otherwise be activated to support viral replication. Even though we failed to detect the presence of HIV DNA in our irradiated FDC preparations, the possibility that a rare contaminating HIV-infected T cell was present cannot be unambiguously ruled out. The lymphoid tissue cells remaining after FDC enrichment were subjected to the depletion of B cells, macrophages, NK cells, and CD8⁺ T cells, followed by collection of the remaining CD4⁺ T cells by using negative selection (Miltenyi Biotec, Auburn, CA). The isolated CD4⁺ T cells were not mitogen activated or otherwise stimulated before use. Our enrichment procedures typically yielded cell purities of 75 to 95%. To ensure that FDC-trapped HIV was replication competent, whenever insufficient tissue was available for both coculture and direct virus sequencing (i.e., in the cases of the JHU559 and JHU621 samples), we prioritized the use of isolated FDCs for coculture and virus rescue. In the case of subject JHU614, we obtained sufficient tissue to isolate FDCs for both virus rescue coculture and direct virus sequencing without culture.

cell_isolation_from_the_peripheral_blood_of_an_HIV-infected_subject.">CD4⁺ T-cell isolation from the peripheral blood of an HIV-infected subject. Antemortem peripheral blood samples were collected from HIV-infected subject JHU614. These cells were subjected to density gradient separation using Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ) according to the manufacturer's directions. The cells were then subjected to DNA lysis as described below.

cell_isolation_from_the_peripheral_blood_of_healthy_donors.">CD4⁺ T-cell isolation from the peripheral blood of healthy donors. CD4⁺ T cells were obtained from healthy donors after informed consent. These cells were subjected to density gradient separation as described above, after which they were depleted of macrophages, NK cells, B lymphocytes, and CD8 T cells and the remaining CD4⁺ T cells were collected using a CD4 negative selection kit as described above. These cells were activated for 3 days using phytohemagglutinin (5 µg/ml), after which the cells were washed to remove the mitogen and then resuspended and maintained throughout culture in complete RPMI (containing RPMI, interleukin-2 [20 U/ml; Roche Applied Science, Indianapolis, IN], 20% fetal bovine serum, gentamicin [50 µg/ml], nonessential amino acids, HEPES [10 ml/500 ml RPMI], and glutamine).

cells_from_HIV-positive_(HIV⁺)_subjects.">Detection of infectious HIV from FDCs and CD4⁺ T cells from HIV-positive (HIV⁺) subjects. Rescue bioassays were performed to determine the infectious nature of virus on FDCs or in CD4⁺ T cells from HIV⁺ subject JHU559. Immunoselected FDCs or identical numbers of CD4⁺ T cells were obtained from the same LN collected at autopsy. These cells were cultured without in vitro activation with allogeneic, mitogen-activated CD4⁺ T cells obtained from the peripheral blood of a healthy donor. The cocultures were maintained for 18 days, during which culture fluid was removed and the production of p24 was measured using an antigen capture enzyme-linked immunosorbent assay (Beckman Coulter, Fullerton, CA) to detect productive infection. The cells from these cocultures were also harvested, and their DNA was isolated and subjected to nested PCR amplification as described below. Furthermore, immunoselected FDCs and CD4⁺ T cells (both isolated from the same LN or piece of splenic tissue) were obtained from HIV⁺ subjects JHU621 and JHU614 and cocultured for at least 6 days in the same manner as that described above, after which the cellular DNA was also isolated and subjected to nested PCR amplification. The HIV amplicons obtained from these cells were subjected to DNA sequencing and phylogenetic analysis as described below.

Proviral DNA and viral RNA isolation. Cells were lysed using 50 µl DNA lysis buffer (0.01% sodium dodecyl sulfate, 0.001% Triton X-100, 0.42 µg/ml proteinase K in TE [10 mM Tris and 1 mM EDTA]). Cell lysis and protein digestion were performed at 56°C overnight, after which the DNA was aliquoted and stored at –80°C. RNA was isolated by adding 160 µl of chloroform-800 µl RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) and the solution was vortexed and then centrifuged at 13,000 x g for 15 min at 4°C. Equal volumes of the aqueous phase from each sample were transferred to a fresh tube containing an equal volume of isopropanol. This tube was vortexed and incubated at –20°C for 30 min and then centrifuged at 4°C for 30 min at 13,000 x g. The RNA pellet was washed once in 75% ethanol (1.0 ml), air dried, and resuspended in an equal volume of RNase-free water. Pretreatment of RNA with DNase I (Invitrogen, Carlsbad, CA) was performed to exclude DNA contamination. Equal volumes of RNA were then reverse transcribed using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Equal volumes of cDNA were PCR amplified under the same conditions as those used for the isolated DNA.

PCR amplification of HIV env and pol genes. Using nested-set PCR technology, we examined the entire env gene and/or a portion of the pol gene containing all of the protease (Pro) and the first 248 amino acids of the reverse transcriptase (RT) genes. Genomic DNA or cDNA was used at empirically determined optimal concentrations for the first round of PCR amplification. Multiple first-round amplifications (three or more) were pooled to guard against selection bias. For the second-round PCR, 1 or 2 µl of the pooled first-round reaction mixture was added as the template. All reactions were performed in 50-µl volumes and included 50 µM deoxynucleoside triphosphates, 0.40 µM of each primer, 3 mM MgCl₂, PCR buffer II, and 2.5 U Expand HiFi Long-Taq polymerase (Roche Applied Science, Indianapolis, IN), having an error rate of 6.1 x 10^–6. The PCR cycling conditions for both rounds of amplification consisted of an initial denaturation at 94°C for 2 min, followed by 10 cycles of 94°C for 11 s, 55°C for 31 s, and 68°C for 4 min. This denaturation was followed by an additional 24 cycles, with 20 s added to the extension of each cycle, followed by a final extension at 72°C for 12 min. All PCRs were performed with precautionary procedures to safeguard against contamination. All reagents were aliquoted in a DNA-free hood, and all amplicons were physically separated from nonamplified DNA. Replicate control PCRs were performed without template to demonstrate no carryover contamination. The outer primers for the complete env gene were 160out3 (5'-GTTCTGCCAATCAGGGAAGTAGCCTTGTGTGTGG-3') and 160out5 (5'-CCTATGGCAGGAAGAAGCGGAGACAGCGACGAA-3') and the inner primers were nJL69 (5'-TAATAGAAAGAGCAGAAGACAGT-3') and JL71 (5'-TTTTGACCACTTGCCACCCAT-3'), which produced an approximately 2.6-kb amplicon (HXB2, nucleotides 6225 to 8795) (30). For the examination of HIV protease-RT sequences, an approximately 1-kb fragment (HXB2, nucleotides 2253 to 3278) was PCR amplified. The outer primers were Polout5 (5'-CAGAGCCAACAGCCCCACAAGAG-3') and Polout3 (5'-TTGCCCAATTCAATTTTCCCACTAACTT-3'). The inner primers were Polin5 (5'-CCTCTCAGAAGCAGGAGCCGATAGA-3') and Polin3 (5'-TCAATTTTCCCACTAACTTCTGTATGTCATT-3').

Cloning and sequencing HIV genes. PCR-amplified products were T/A cloned into the vector pCR3.1 (Invitrogen, Carlsbad, CA) by directly ligating the vector with fresh PCR product at 15°C overnight. STBL2 bacteria (Invitrogen, Carlsbad, CA) were then transformed via heat shock (25 s at 42°C) and plated on LB plates with 100 µg/ml ampicillin. Individual colonies were selected and screened for correctly sized inserts via a modified toothpick boiling method. Briefly, bacteria were incubated in a 96-well, V-bottom plate at 30°C overnight. Cells were then centrifuged and lysed, and genomic DNA and proteins were precipitated and removed by centrifugation. The plasmid DNA in the supernatant was then subjected to electrophoresis, and plasmids with inserts were identified by relative size. Insert-positive clones were then cultured in 5 ml LB broth at 30°C, with shaking at 225 rpm. Clean, sequence-ready, plasmid DNA was purified using the QIAprep spin column (Qiagen, Valencia, CA). Clones for each gene were randomly selected and sequenced using the BigDye (version 3.1) terminator method (Applied Biosystems, Foster City, CA), with a battery of previously developed primers. After the sequencing reaction, the samples were purified using the DyeEx terminator removal kit (Qiagen, Valencia, CA). Sequences were then analyzed on an ABI-3730XL DNA analyzer at Brigham Young University's core facility. Both strands of each viral insert were sequenced, with all genes having at least two overlapping independent sequences per site. All contigs were trimmed, and a consensus sequence was made with Sequencher (Gene Codes, Ann Arbor, MI).

Phylogenetic analysis of HIV from FDCs and other cells/sites. We aligned sequences by using the default settings in ClustalX 1.81 (61). The sequences were then manually adjusted and checked for insertions and deletions (indels) to preserve codon structure using MacClade 4 (Sinauer Associates, Sunderland, MA). Phylogenies were estimated using maximum likelihood (13), with nodal support assessed via bootstrapping (1,000 pseudoreplicates) (12) as implemented in PHYML (18). We also estimated phylogenies by using Bayesian methods (23) coupled with the Markov Chain Monte Carlo inference as implemented in MrBayes, version 3.1.2 (48). Model selection for these analyses followed the procedure outlined previously by Posada and Buckley (41), as implemented in ModelTest, version 3.6 (43). For the techniques using Bayesian methods (23) coupled with the Markov chain Monte Carlo method, two independent analyses were run, with each consisting of four chains. Each Markov chain started from a random tree and ran for 2.0 x 10⁷ cycles, with sampling every 1,000th generation. In order to confirm that our Bayesian analyses converged and mixed well, we monitored the fluctuating value of likelihood and compared means and variances of all likelihood parameters and likelihood scores from the independent runs using the program Tracer, version 1.4 (http://beast.bio.ed.ac.uk/tracer). These phylogenetic analyses were performed on the supercomputing cluster of the College of Life Sciences at Brigham Young University.

The genetic diversity of FDC-trapped HIV and virus obtained from other tissues and cells was determined using a phylogenetic approach to estimate nucleotide diversity implemented in LAMARC, version 2.0.2 (27). This program provided a maximum likelihood estimate of theta ( = 2N_eµ), where N_e is the inbreeding effective population size and µ is the per site mutation rate.

Genotype network reconstruction and genetic diversity estimates. To compare FDC and PBMC RT genetic sequences in subject JHU614 within a 95% parsimony limit, a haplotype network was constructed using the program TCS, version 1.21 (8). The networks are constructed so that colored squares (putative outgroups) and circles represent actual cloned sequences, the sizes of which are proportional to the number of clones with the same sequence. Each open circle represents a putative quasispecies in the evolutionary pathway. Solid lines connecting a network suggest that two quasispecies can be connected with at least a 95% degree of confidence. Broken lines in a network represent a more tenuous connection.

Analyses of sequence data for recombination. It is well known that HIV-1 undergoes recombination (46, 47, 57) and that this can potentially impact estimates of both population genetic parameters (50) and phylogeny estimation (44). The impact of recombination on phylogeny estimation seems to be more severe when the recombination event involves roughly equal divisions across the gene (44). Otherwise, the tree estimated is the history of the majority of the gene sequence after the recombination event. To test the robustness of our phylogenetic conclusions to recombination, we used a network approach (TCS) that allows for recombination (42) and has an associated test for it (10). While there is no doubt that recombination is occurring, there are no identifiable recombinants in these data by using this approach. The sequences were very closely related (there was typically only a single nucleotide difference among sequence variants), and those few that were more distantly related were not connected to disparate places in the network (a telltale sign of recombination). Thus, our analyses indicate that recombination does not significantly impact our conclusions.

Nucleotide sequence accession numbers. GenBank accession numbers for the sequences determined in this study are EF440689 to EF441210, EU009131 to EU009140, and EU330488 to EU330524. Each sequence is identified by using the subject name, the tissue and cell type, and the clone designation (e.g., JHU559_CFDC_3D6). When a sequence was derived from a coculture, we added "cc" immediately before the clone identification (e.g., JHU559_CFDC_cc3D9).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
FDCs retain infectious HIV. Because the most essential aspect of an HIV reservoir is the harboring of infectious virus, we first sought to determine whether FDC-trapped virus was replication competent. We reasoned that if FDC-trapped HIV was infectious, we could transfer the infection to susceptible CD4 T cells and detect viral protein production and/or viral DNA. We therefore immunoselected FDCs from a postmortem LN obtained from HIV-infected subject JHU559 and used these as the only source of input virus in a rescue bioassay (Table 2). The FDCs used in each culture well (i.e., 10,000 cells) contained 62 pg of HIV p24 per milliliter of cell culture fluid. When this input virus on FDCs was cultured with activated allogeneic CD4⁺ T-cell targets, we detected the production of significant quantities of HIV p24 (59,000 and 65,000 pg/ml at 9 and 18 days of culture, respectively). To confirm that FDC-trapped HIV was infectious, we performed additional cocultures with FDCs obtained postmortem from two additional subjects, JHU614 and JHU621 (Table 2). To detect the transmission of infection in these assays, we harvested the target cells from the cocultures and examined them for the presence of HIV DNA (Table 2). While HIV DNA could not be amplified from FDCs themselves (which bear only HIV RNA), it was amplified from all of the FDC cocultures, confirming its infectious nature.

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TABLE 2. FDC-trapped HIV is replication competenta

FDC-trapped HIV is genetically diverse. We first analyzed the pol gene from virus obtained by coculture of FDCs and T cells obtained from subjects JHU559 (Fig. 1), JHU621 (Fig. 2), and JHU614 (Fig. 3). In samples from subjects JHU559 and JHU621, FDC HIV and virus from T cells formed distinct clades in the phylogenetic tree, demonstrating the compartmentalization of virus populations. In the third subject (JHU614), however, virus from the FDCs and T cells was substantially more intermixed. We also noted a relationship between viruses in spatially separated secondary lymphoid tissues; FDC viruses from the spleen clustered not only with splenic T cells but also with T-cell-derived viruses from the axillary LN (i.e., SFDC_cc3F6 and SFDC_cc3H8 were related to ST_ccC3C9, ST_ccC3C9b, ST_ccC3E9, A1T_ccC11, A1T_ccB1E5, and A1T_ccT1C3). In all but one of these instances (i.e., ST_ccC3E9 was identical to SFDC_cc3F6), the T-cell-derived HIV differed from the FDC virus by a single, nonsynonymous nucleotide change in the RT gene, resulting in the following amino acid alterations: T128A for ST_ccC3C9 and ST_C3C9b, E203G for A1T_ccC11, and E224G for A1T_ccT1C3, where the first amino acid listed at a given position was present in FDCs and the second was present in the T cells. No known drug resistance was associated with these changes.

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FIG. 1. Phylogenetic analysis of HIV Pro-RT obtained from FDC and CD4 T-cell cocultures from subject JHU559. FDCs and CD4 T cells (2 x 104 cells per well) were immunopurified and cocultured with mitogen-activated, allogeneic T-cell blasts. Following 9 days of culture, cells were harvested, and their DNA was collected. HIV pol was amplified by nested PCR, cloned, and sequenced as described in the text. Phylogenetic analysis was performed using both maximum likelihood (ML) and Bayesian methods, which generated identical topologies. Statistical analysis is provided for both analyses and is displayed as bootstrap values in the numerator and posterior probability (by using the Bayesian Markov Monte Carlo method) in the denominator. Statistical support of 50% or greater is shown. Each sequence was identified by the cell type from which the viral gene was cloned, followed by "cc" (to indicate that it was derived from coculture) and the clone designation (e.g., FDC_cc458). The corresponding sequence deposited with GenBank is prefaced by the identifier for the infected subject (e.g., JHU559_FDC_cc458). BMCMC, Bayesian methods coupled with the Markov chain Monte Carlo method.

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FIG. 2. Phylogenetic analysis of HIV Pro-RT obtained from FDC and CD4 T-cell cocultures using cells obtained from subject JHU621 (as described for Fig. 1). Phylogenetic analysis was performed using both maximum likelihood (ML) and Bayesian methods, which generated identical topologies. Statistical analysis is provided for both analyses and is displayed as bootstrap values in the numerator and posterior probability (by using the Bayesian Markov Monte Carlo method) in the denominator. Each sequence was identified by the cell type from which the viral gene was cloned, followed by "cc" (to indicate that it was derived from coculture) and the clone designation (e.g., FDC_cc42B). The corresponding sequence deposited with GenBank is prefaced by the identifier for the infected subject (e.g., JHU621_FDC_cc42B). BMCMC, Bayesian methods coupled with the Markov chain Monte Carlo method.

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FIG. 3. Phylogenetic analysis of HIV RT obtained from FDC and CD4 T-cell cocultures from subject JHU614 (as described for Fig. 1). Phylogenetic analysis was performed using both maximum likelihood (ML) and Bayesian methods, which generated identical topologies. Statistical analysis is provided for both analyses and is displayed as bootstrap values in the numerator and posterior probability (by using the Bayesian Markov Monte Carlo method) in the denominator. Each sequence was identified by the tissue and cell type from which the viral gene was cloned, followed by "cc" (to indicate that it was derived from coculture), and the clone designation (e.g., CFDC_cc3B8). The corresponding sequence deposited with GenBank is prefaced by the identifier for the infected subject (e.g., JHU614_CFDC_cc3B8). Tissue identifiers are A (axillary LN), C (cervical LN), and S (spleen). BMCMC, Bayesian methods coupled with the Markov chain Monte Carlo method.

We also assessed the genetic relatedness of gp160 sequences from subject JHU559. In this analysis, we examined both FDC and T-cell viral sequences derived from coculture and directly cloned sequences obtained from uncultured HIV DNA from the bone marrow, the brain (the meninges and mid-frontal gyrus), and from a second LN where immunoselection and culture of cells were not performed prior to sequencing (Fig. 4). FDC-associated virus formed a monophyletic group (a distinct evolutionary lineage containing all the descendants from a common ancestor). Furthermore, virus on FDCs was genetically heterogeneous, as evidenced by the presence of multiple branches within the monophyletic group and the long branch lengths of the FDC-derived sequences. In contrast to HIV obtained from LN FDCs, virus from the unfractionated LN was not monophyletic but clustered with a number of different sites, including the brain and the bone marrow; however, virus from this tissue still contained substantial genetic diversity, as indicated by the relatively long branch lengths compared to those for virus from other tissues.

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FIG. 4. Phylogenetic analysis of uncultured (bone marrow, brain [meninges and mid-frontal gyrus], and an unfractionated LN) and cocultured (FDCs and T cells) HIV gp160 sequences derived from multiple sites in subject JHU559. Phylogenetic analysis was performed using both maximum likelihood (ML) and Bayesian methods, which generated identical topologies. Statistical analysis is provided for both analyses and is displayed as bootstrap values in the numerator and posterior probability (by using the Bayesian Markov Monte Carlo method) in the denominator. Each sequence was identified by the tissue and cell type from which the viral gene was cloned and the clone designation (e.g., BM_C5). The corresponding sequence deposited with GenBank is prefaced by the identifier for the infected subject (e.g., JHU559_BM_C5). When FDC and T-cell-derived sequences were obtained from coculture, "cc" prefaced the clone designation (e.g., FDC_cc34). Cocultured FDCs and T cells were obtained from the same LN used for Fig. 1. Tissue sites are bone marrow (BM), meninges (MEN), mid-frontal gyrus (MFG), and unfractionated lymph node (LN). BMCMC, Bayesian methods coupled with the Markov chain Monte Carlo method.

We next examined RT sequences of uncultured and cocultured virus from multiple sites in subject JHU614, who was treated only with RT inhibitors (Fig. 5). FDC-trapped virus was found distributed throughout the phylogenetic tree. However, we also noted examples of completely segregated virus that could be found in both the FDC and T-cell compartments, regardless of whether the samples originated from uncultured or cocultured material.

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FIG. 5. Phylogenetic analysis of uncultured and cocultured HIV RT sequences obtained from multiple sites in subject JHU614 (as described for Fig. 1). Phylogenetic analysis was performed using a Bayesian method. Statistical analysis is displayed as the posterior probability (by using the Bayesian Markov Monte Carlo method). For sequence identification of the displayed data, see Fig. S1 in the supplemental material; the identification utilizes the nomenclature explained for Fig. 1 to 4.

Because HIV in a presumed reservoir is predicted to have a high degree of genetic diversity due to the accumulation of multiple quasispecies over time, we measured the genetic diversity of FDC-trapped HIV and compared diversity levels with those of virus from other cell and tissue types. We selected subjects JHU559 and JHU614 for study because we could compare the diversity of sequences obtained from multiple cells and tissues. Genetic diversity was estimated by theta ( = 2N_eµ); N_e is the effective population size, and µ is the mutation rate per site per generation (Table 3). Theta estimates for subject JHU559 indicated that the FDC-derived sequences were more diverse (i.e., higher theta value) than all other sequences examined, with the exception of those derived from the unfractionated LN. An analysis of JHU614 sequences indicated that LN FDCs possessed quasispecies having a greater diversity than virus from all other tissue-derived cells. We also noted a difference in the overall diversity between FDC sequences derived from the LNs and the spleen, where the LN FDC quasispecies demonstrated a threefold increase over the splenic quasispecies. The effect of coculture on the genetic diversity of FDC-trapped HIV appeared to be minimal, as evidenced by the similar theta estimates from sequences derived from either cultured or uncultured samples.

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TABLE 3. Theta estimates of viral diversitya

FDC-trapped HIV contains archived viral variants. Because long-term reservoirs contain archival virus, we compared JHU614 viral sequences trapped on FDCs obtained postmortem with those from antemortem PBMCs to obtain evidence of quasispecies that were extant at earlier times. We reasoned that if FDC-trapped HIV included archived variants, we would observe FDC isolates having genotypes identical or very closely related to those present in the PBMC samples obtained many months prior to the patient's death. We therefore constructed a genotype network comprised of FDC and PBMC-derived viral RT sequences from JHU614 (Fig. 6). This network shows the number of nucleotide changes existing between different genotypes (comprised of various numbers of identical sequences). It was readily apparent that the FDC genotypes were distributed throughout the network of PBMC-derived sequences, consistent with the presence of multiple quasispecies extant at different time intervals during the course of disease (Fig. 6). FDC samples also contained genotypes with few (e.g., H39 and H40) as well as multiple nucleotide changes (e.g., H91 and H92) separating the genotypes, consistent with virus trapping throughout the disease course. Most importantly, the FDC genotypes were closely related to those of many previously collected PBMC samples. Notably, the H26 genotype consisted of six identical sequences: five derived from FDCs and one from a PBMC sample collected 22 months prior to the patient's death. Furthermore, eight PBMC genotypes, H27, H30, H37, H47, H29, H36, H46, and H48, differed from the H26 genotype (largely derived from FDCs) by only a single nucleotide and the PBMC sequences were all obtained from samples collected more than 20 months prior to the patient's death (Fig. 6). Five of the single-nucleotide changes for the above genotypes (i.e., V75A for H46, M184R for H47, D17G for H48, T139A for H29, and V241E for H36) were nonsynonymous, but none conferred drug resistance by itself. We also noted a difference in the overall number of nucleotide changes present in samples obtained over 20 months prior to the patient's death compared to samples obtained 4 and 18 months before death (Fig. 6). In general, the genotypes derived from the oldest two PBMC samples showed fewer nucleotide changes than those obtained closer to death, as evidenced by the numerous small circles connecting the genotypes in Fig. 6, where each small circle represents a putative ancestor with a single nucleotide alteration.

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FIG. 6. Genotype network analysis of HIV RT sequences obtained from postmortem FDCs and antemortem PBMCs from JHU614. Each solid line represents one mutational step and connects two quasispecies with at least a 95% confidence level. Small circles between genotype connections indicate a requirement for an additional mutational step per circle encountered between the genotypes. Broken lines indicate a more tenuous connection, requiring 10 or more mutational steps connecting genotypes. The sizes of the solid squares and circles are proportional to the genotype frequency, with the smallest size representing only one sequence. In the key, numbers following PBMC indicate the month and last two digits of the year in which the sample was collected.

We also examined drug resistance mutations from FDC- and PBMC-derived virus to determine whether the same resistant quasispecies were represented (Table 4). The K103N mutation (nonnucleoside RT inhibitor [NNRTI] resistance) was found in 47 of 85 (55%) FDC and 14 of 70 (20%) PBMC-derived sequences. Of the latter 14 PBMC resistance quasispecies, all were found in samples obtained over 20 months prior to the patient's death. We observed five additional drug resistance mutations on the FDCs: K103S, V108I, Y188C, Y188H, and T215Y. All of these mutations confer resistance to NNRTIs except T215Y, which provides resistance to azidothymidine. Lastly, we discovered a resistance mutation, Y181C (NNRTI resistance), which could be detected only in a single PBMC clone obtained 18 months prior to the patient's death. Thus, all but one of the seven drug resistance genotypes found in subject JHU614 were present on the FDCs. Collectively, these data indicated the existence of archival virus on FDCs, thereby satisfying this criterion of a reservoir.

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TABLE 4. ARV resistance mutations from post- and antemortem samples from JHU614a

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Viral reservoirs present major obstacles to the successful treatment of HIV infection (3, 6). In the present study, we examined human FDCs isolated from secondary lymphoid tissues obtained postmortem from infected subjects and found that FDC-trapped HIV was indeed a reservoir. This virus was infectious, genetically diverse, and included archived isolates with drug resistance mutations that were not seen in other cells or tissue sites. Furthermore, in contrast to other HIV reservoirs, where each infected cell harbors on average one virus, a single FDC may trap and retain multiple, genetically diverse, replication-competent virus particles. Thus, FDC-trapped HIV has the potential to contribute to virus transmission, persistence, and diversification.

The replication-competent nature of FDC-trapped HIV was determined in two different ways: a sensitive virus rescue bioassay and cloning of virus quasispecies from uninfected T-cell targets that were cocultured with virus-bearing FDCs. When the production of p24 was assessed in a coculture of FDCs obtained from subject JHU559, we noted the transmission of a significant spreading infection, as evidenced by increasing p24 production over the period of culture. In fact, less than 100 pg of HIV p24 present on the input FDCs led to the production of over 65 ng of p24 in just 18 days, a more than 650-fold increase. The establishment of a productive infection indicates that FDCs trapped intact virus particles as opposed to just viral proteins. It is also important to note that the FDCs themselves did not require an activation step to transmit infection, as would be the case with a latent T-cell reservoir. The amount of p24 generated in our cocultures in this study is consistent with our previous observations examining FDC transfer of virus, where a few picograms of HIV on FDCs produced 12 ng p24/ml after culture (52). These data from human FDCs are also consistent with previous observations based on using xenogeneic cocultures of virus-bearing murine FDCs and human CD4 T-cell targets, where we found that 40 pg of p24 trapped on murine FDCs in vivo resulted in the production of nanogram levels of p24 in vitro (52). Thus, these data in both human and murine systems confirm that FDC-trapped HIV is readily transmissible.

Virus trapping and retention on FDCs are mediated primarily by specific antibodies and/or complement proteins coupled with immune complex receptors on FDCs (21, 24, 53). The antibodies involved in virus trapping would likely consist of both neutralizing and nonneutralizing forms, although the antibodies involved may have differing affinities, which in turn could affect the stability of the virus over time. We postulate that the FDC network of HIV may actually favor the maintenance of infectious virus particles. This preference could occur as a consequence of trapping virus with bound antibody, which from in vitro studies maintains infectivity, a part of which is mediated by inhibiting gp120 shedding (53). A number of other features of FDCs may also protect virus from degradation, including the presence of high levels of thiol groups on FDC surfaces that create a reducing microenvironment (60), the envelopment of virions in the FDCs' dendritic processes that are known to sequester conventional protein antigens from the surrounding cells (55, 56), and the presence of complement regulatory proteins, such as CD55 (decay accelerating factor) (28).

Genetic analysis of FDC-trapped HIV indicated the presence of a diverse repertoire of viral quasispecies. We found instances where FDC-trapped virus was genetically distinct from virus present in other cells; however, we also detected examples where FDC HIV was found associated with virus from other cells and tissues. Our interpretation is that some of this "virus mixing" between FDCs and T-cell sequences reflects the mobile nature of T cells as they traffic throughout the secondary lymphoid tissues. The presence of similar virus quasispecies on FDCs and T cells could arise in different ways. The T cells could acquire virus from FDCs as they entered the germinal center microenvironment, or alternatively, they could enter the lymphoid tissue and produce virus that became trapped on FDCs. A third alternative could occur where a previously infected, virus-expressing cell releases progeny virus that is in turn transported into secondary lymphoid tissues, where it both infects susceptible lymphocytes and becomes trapped on FDCs. These alternatives are not mutually exclusive, and we envision that within this dynamic tissue compartment, all three scenarios could occur simultaneously.

We do not yet understand whether virus trapped on FDCs at different times during the disease course has different association/dissociation kinetics. However, if FDCs trap HIV throughout the disease course, we expect multiple virus quasispecies to accumulate, thereby increasing the overall diversity of HIV in this site. When we analyzed the genetic diversity (i.e., theta) of virus from multiple cells and tissues in two independent infected subjects, we found that viral sequences trapped on FDCs demonstrated greater diversity than did those on most other cells and tissues. Because theta estimates are derived from a linear function, one can directly analyze the values between different sites to compare the overall differences in diversity from one site to another. In general, FDC-associated virus was about twofold more diverse than sequences obtained from other tissue sites, regardless of whether env or pol genes were compared. Exceptions to this observation were the unfractionated LN cells obtained from JHU559 and the PBMC sample from JHU614 collected 18 months prior to the patient's death. In the latter instance, the sample shows diversity similar to that of virus obtained from the LN FDCs. It was also apparent that PBMC samples segregated into two populations having theta values that differ by fivefold (i.e., 0.02 for samples collected 21 and 22 months prior to the patient's death or 0.1 in samples collected at 4 or 18 months before death). This segregation based on diversity is further supported by the genotype network analysis of virus from PBMCs, where the two older samples show a low number of nucleotide changes between genotypes and the more recent ones demonstrate a greater number of changes.

We also noted a difference in sequence diversity of virus samples from FDCs in the LN versus those from the spleen, with a threefold decrease in diversity noted in the splenic samples. The different theta values in the two types of lymphoid tissue were paralleled in JHU614 samples by the distinct genetic differences observed between HIV on FDCs in the LN and those in the spleen. At this time, we do not know whether these observations represent a fundamental difference in virus localization in different types of secondary lymphoid tissues or are simply limited to a single specific patient. In the present study, we were unable to address this question because of the lack of splenic tissue samples from more than one infected subject. Differences in virus localization in different secondary lymphoid tissues support the concept that at least in some subjects, similar tissues may harbor unique virus isolates, each of which could contribute to persisting infection. Further studies with additional blood and tissue samples will undoubtedly help resolve a number of these questions.

HIV reservoirs possess archived virus that is acquired throughout the course of disease. We looked for evidence of archival virus by examining drug resistance mutations from FDCs and comparing them to resistance mutations present in PBMC samples collected several months prior to the patient's death. We found mutations on FDCs that were present in our earliest samples of blood but that then disappeared in blood samples obtained closer to death. We reason that as subject JHU614 discontinued and then reinstituted ARV therapy, there were periods of time when drug concentrations would be low enough to allow the virus to mutate, while at the same time be present in a high enough concentration to provide selective pressure for the establishment of resistance mutations. We presume that in the absence of drug treatment, these mutations possessed no selective advantage and were lost from the circulating virus pool but remained trapped on FDCs. It should be noted that the FDC-derived sequences with this mutation were detected in both uncultured and cultured FDCs, indicating the infectious nature of these virus variants. Another strong argument for the archival nature of FDC HIV is the genotypic analysis of virus derived from FDC and PBMC samples. These genotypes indicate a very close relationship (i.e., one or two nucleotide changes) and, in one case, an identical one between the FDC and a majority of the PBMC genotypes collected at 21 and 22 months antemortem. Taken together, these data strongly support the archival nature of FDC-trapped HIV.

Since the first identification of HIV on FDCs, it has been difficult to directly measure virus in this compartment (11, 37). To help understand this important site, investigators have implemented mathematical modeling to estimate viral dynamics within lymphoid tissue. These dynamics include the original estimates of viral reservoirs, with a biphasic decay rate during antiretroviral treatment (38). These models accurately estimated the rapid reduction of the lymphoid tissue compartment, but could not directly estimate virus trapped on FDCs (33). A more recent mathematical model, which directly estimates on/off rates for virus trapped on FDCs, concludes that these cells can be reservoirs of infectious virus, thus offering additional support to the data reported here (17). Furthermore, this model predicts that a patient with a rapid dissociation of virus on FDCs may have a better clinical outcome with successful therapy. Those subjects with slow dissociation kinetics of FDC virus may have worsening clinical prognoses with more frequent drug failures. As we gain greater understanding of the contributions of FDC-trapped HIV in pathogenesis, we should be able to directly test the above estimates.

Here we report the genetic characterization of HIV trapped on human FDCs. This study illustrates the infectious and diverse nature of virus in this reservoir. Moreover, the presence of archival forms coupled with the ability of FDCs to preserve virus infectivity over time suggests the continued presence of diverse virus that could reignite and perpetuate infection throughout the disease course. A better understanding of this large and diverse repository of infectious virus may be critical in the goal of controlling HIV and understanding how to defeat the virus in this reservoir.

 
This research complied with all relevant federal and institutional policies. The authors acknowledge that they have no financial or other relationships that would pose a conflict of interest relating to the research presented in this study.

This work was supported by grants from the National Institutes of Health, National Institute of Allergy and Infectious Disease (AI39963 and AI57007), National Institutes of Health, National Institute of General Medicine (GM66276), and the American Foundation for AIDS Research (106365-33-RGRL).

 
* Corresponding author. Mailing address: Rm. C211A BNSN, Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602. Phone: (801) 422-4917. Fax: (801) 422-0153. E-mail: greg_burton{at}byu.edu

Published ahead of print on 2 April 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294-0024.

Present address: Department Microbiology and Immunology, Stanford University, Stanford, CA 94305.

^¶ Present address: AIDS Vaccine Program, NCI-Frederick, Fort Detrick Campus, Frederick, MD 21702.

^|| Present address: Tuberculosis Research Unit, USDA Agricultural Research Service, Ames, IA 50201.

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Journal of Virology, June 2008, p. 5548-5561, Vol. 82, No. 11
0022-538X/08/$08.00+0     doi:10.1128/JVI.00124-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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