Viral reservoirs present major obstacles to the successful treatment of HIV infection (3
). 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
). 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
), 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
). 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.