Tissue is the issue: how altruistic people with HIV are changing the HIV tissue reservoir landscape

So far, extensive investigations in humans have been unable to characterize the large, complex HIV reservoirs that must be eradicated to achieve a cure. This is because most human studies have only sampled blood – with some limited sampling of cerebrospinal fluid, genital secretions and the gut [1]. However, HIV has been detected in all tissues and anatomic compartments, including (but not limited to) brain, lymph nodes, fat and spleen [2]. These tissues are largely inaccessible in living individuals – even through the most sophisticated techniques. Additionally, large amounts of tissues and cells are needed to fully characterize the burden and anatomic location of HIV-infected cells, beyond what is typically feasible in routine or even experimental biopsy procedures. Postmortem tissues from autopsies can be of value for this analysis, but the time from death to tissue preservation is often too long for adequate preservation of viable cells or HIV nucleic acids. Clinical data about the donor is also often not known, such as whether a tissue donor continued antiretroviral therapy (ART) prior to death or not, which would complicate the interpretation of the presence of HIV RNA or proteins in tissues.

To begin to address this, nonhuman primates, such as chimpanzees and rhesus macaques have been used to provide a useful animal model for interrogating HIV reservoirs and a wide variety of potential therapeutics [3]. However, primate models have limitations and they differ from humans in rates of disease progression, reservoir dynamics, and immune responses, among others [4]. An example of this is the Merck Ad5 trivalent HIV-1 vaccine trial (STEP trial), which found no protection from HIV infection or slowing of disease progression despite initial results in various macaque studies that had seemed promising [5,6]. The trial actually had to be stopped early when an independent safety review discovered high levels of pre-existing Ad5 immunity in some participants who may have actually increased their risk of HIV acquisition [5]. This is just one of many examples where animal models insufficiently reproduce human reality and why human-centered studies are still of utmost importance.

Why tissues matter

Describing and quantifying HIV persistence in all anatomic compartments is important because the blood compartment (mostly naive T cells) is likely not representative of how HIV behaves in tissues, where most T cells are differentiated effector memory (the main HIV targets) [7,8] and where HIV also infects other target cells (e.g., macrophages) [9,10].

There is ongoing controversy over how the HIV reservoirs in various tissues correlate with each other and how these discrete compartments influence HIV reservoir dynamics. In fact, despite ART, immune activation and dysfunction persist in the setting of HIV, and likely impact the size and distribution of the tissue HIV reservoirs throughout the body [11]. The vast majority of HIV-infected cells that persist during ART reside in the gut or in follicles within secondary lymphoid tissues. There is also controversy as to whether replication-competent HIV persists in myeloid-derived cells of the central nervous system, which see far lower concentrations of antiretroviral drugs than plasma [12].

For these reasons, it is increasingly important to interrogate the immune cells resident in a tissue as these tissue-resident immune cells are poorly understood – particularly in humans and especially in the setting of an immune system perturbed by HIV. Understanding these better may provide the key to deciphering the sequelae of chronic HIV infection such as endothelial dysfunction, cardiomyopathy, encephalitis, nephropathy, etc. [13]. Local inflammation in the tissues is perturbed, which is likely reflected by aberrant immune cell subpopulations [14–16]. With a greater understanding of these populations and the way in which they are perturbed, we will have a better chance not only of eliminating these reservoirs, but potentially reversing the deranged inflammation at these sites.

Understanding the dynamics of cells in these reservoirs is also important, as clonal expansion of HIV-infected cells enables self-renewal of the HIV reservoir, and thus likely contributes to HIV persistence during ART [7]. Genetic analyses of HIV proviruses and integration sites in persons with HIV (PWH) who are ART-suppressed have provided evidence that clonal expansion of HIV-infected CD4⁺ T cells is a key mechanism of viral persistence [17]. Expansion of HIV-infected cells can be driven by antigen stimulation, but it remains unclear to what extent this process maintains the HIV reservoir, especially in nonblood tissues [7]. For example, latently infected cells in the gut may be particularly vulnerable to antigen-induced expansion given their proximity and ongoing contact with environmental antigens, including resident viruses, bacteria and fungi [18]. Conversely, HIV reservoirs in the brain likely have limited antigen exposure due to the stricter filtering imposed by the blood–brain barrier [19]. The reservoirs in all other tissues are likely somewhere between these two extremes due to varied exposure to antigenic stimulation and the diapedesis of immune cells.

The role of altruistic PWH

Researchers have found that some individuals with terminal cancer are willing to participate in research at the end of life, even if there is no chance of altering the course of their underlying illness [20]. There are, naturally, many people who are altruistic and who would take part in a research study that may not benefit them. A sense that the research may benefit their friends, family or mankind seems to inspire them. Knowing that one may be near the end of life may make this sense of altruism all-the-more acute and cause one to feel that participation in scientific research could serve as a final, meaningful contribution to society-at-large. It is also possible that well-informed, altruistic and appropriately consented human volunteers who happen to be at the end of their lives could improve the way therapeutics move from the laboratory to the clinic by altering the balance between risk and benefit that is used to determine what research should proceed [21].

The ‘Last Gift’ is an end-of-life HIV research study conducted at the University of California San Diego, CA, USA, that has the goal of understanding the distribution of HIV throughout the human body [21,22]. The study involves collecting biological samples and detailed clinical data from participants before death and then performing an autopsy soon after death.

The desire to perform rapid autopsies developed due to a need to preserve the integrity of nucleic acids, proteins and cellular physiology for further study. For over a decade, rapid (also previously called ‘warm’) autopsies have been carried out in cancer [20,23–25] and neurodegenerative disease [26] research. In these previous studies, extraordinary steps were taken to rapidly process select tissues of interest (usually within 2–6 h of death) in order to preserve nucleic acid sequences [27]. We sought to replicate these efforts in a systematic way with a cohort of PWH to further HIV research and lay the groundwork for developing a cure [28].

In a recent paper, our group described the results after obtaining tissues from six individuals undergoing long-term ART as part of the Last Gift cohort [22]. Samples were obtained from 28 different tissues. Sequences were analyzed to determine phylogenetic and genetic relationships, and bioinformatic approaches were used to track how infected cells dispersed and migrated across tissues. HIV DNA was detected in most tissues and all HIV DNA populations within individuals were highly related, but evidence of compartmentalization within tissues could be detected. In general, HIV DNA populations were genetically diverse, but our group also identified populations of proviruses in different tissues that were identical in sequence; in the setting of a genetically diverse population repeated recovery of identical sequences suggests that these proviruses are members of a clone [22,29]. These putative clonal members were detected in a number of tissues and provide useful insights into how infected cells are distributed in individuals and will shed additional insight on in vivo viral dissemination.

Conclusion & future perspective

The search for a cure for HIV could benefit from individuals who are terminally ill and voluntarily participate in biomedical research. For example, autopsy studies permit sampling from multiple organs at the time of death, allowing us to understand how the virus distributes throughout the body and how these tissue reservoirs correlate with each other and the blood. These volunteers also allow us to better understand ethical issues associated with this type of research. Because the numbers of participants in rapid autopsy cohorts like ours is thus far small, there is a great deal of heterogeneity among the participants. We understand that this heterogeneity is a challenge, but we like to consider it also an asset. In fact, the field of HIV is filled with examples of unique individuals driving research advancements (e.g., Berlin patient) and collecting as much detail as possible about each participant allows a bit of ‘personalized science’ that could prove useful for other diseases. For instance, our cohort has already had two participants with amyotrophic lateral sclerosis and their tissues may be useful for understanding amyotrophic lateral sclerosis in addition to the contribution they have made to HIV research. Similarly, the cohort contains several patients with epithelial neoplasms (e.g., oral and rectal cancers) and these tumor microenvironments can be examined as a group to discern their peculiar impact on the immune system and HIV reservoir – a lesson that could have an impact on thousands of PWH who are treated for similar neoplasms every year [30].

In order to overcome these limitations, rapid autopsy programs need to expand and include a wider swath of the population. It will not be easy, and an impressive degree of coordination and extraordinary teamwork is required for a successful rapid autopsy program. However, as programs like this gain acceptance, it will be important to broaden outreach to communities that might not otherwise seek out posthumous tissue donation [31]. Cultural beliefs and perceptions of donation may influence who participates in body donation at the end of life. Nevertheless, a global meta-analysis found that all cultures and demographic studies exhibited positive perceptions of donation and expressed a sense of altruism and camaraderie with the broader community as a driver for this altruistic attitude toward research [32]. Perhaps the topic of posthumous tissue donation is not as delicate or controversial as we perceive. The research community would greatly benefit from further research into the impact of cultural, logistical and system-wide barriers to donation and rapid tissue processing. We would never have made the dramatic scientific progress we have made to date without a close partnership between PWH and the research community. These examples teach us that those partnerships can endure and be fulfilling even as people approach the end of life. The gift that is given by participants who consent to rapid autopsy is a terminal kindness, the gift of a lifetime, the Last Gift.