INTRODUCTION

Anatomic sites exposed to the environment are colonized with bacteria, eukaryotes, and viruses, cumulatively referred to as the microbiome. The microbiome is diverse, and the composition widely dependent upon the unique environmental factors present across the body. A millennium of coevolution with the host has led to a predominantly symbiotic relationship, with several microbiome-produced metabolites benefitting the host [1,2▪]. Alterations to the composition of the gastrointestinal (GI) tract microbiome have been associated with several disease states [3] and are considered important contributors to progressive infection in people with HIV (PWH) and Simian Immunodeficiency Virus (SIV)-infected Asian macaque nonhuman primates (NHPs). Here, we review recent work on the role of the microbiome composition on HIV and SIV acquisition and disease progression (Fig. 1). 

Box 1:

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FIGURE 1:

The primate microbiome is a diverse community of bacterial, eukaryotic, and viral constituents residing in the gastrointestinal lumen (as shown) and other external interfaces. Antigenic and metabolic stimulation by the microbiome may prime vaccine outcomes, alter susceptibility to lentiviral acquisition, modulate lentiviral disease progression, and fluctuate during postexposure prophylaxes. These interventions and disease states may also alter homeostasis of the healthy microbiome. A detailed understanding of the mechanisms underpinning these dynamic relationships will inform novel, improved therapeutic interventions. 1. Corley MJ, Sacdalan C, Pang APS, et al. Abrupt and altered cell-type specific DNA methylation profiles in blood during acute HIV infection persists despite prompt initiation of ART. PLoS Pathog 2021;17:e1009785.

Although experiments involving animal models contribute significantly to HIV research, questions remain regarding the usefulness of these models to microbiome-related disease outcomes. Major advantages of animal models involve longitudinal potential and the ability to control for variables such as diet, antibiotic use, and behaviorally-associated risk factors – parameters that influence the composition of the GI tract microbiome irrespective of infection. Animal models also provide the ability to analyze anatomic sites that can only rarely be obtained from humans. On the other hand, there exists species variability in the composition of the microbiome in these models as compared to human participants [4] and these models are incapable of recapitulating the complex variables that contribute to microbiome individuality across diverse human populations. These limitations suggest that animal models should complement biomedical research, providing mechanistic insights and a framework against which therapeutic interventions can be tested prior to clinical implementation.

Approaches for measurement of the microbiome generally fall into three broad categories – those that involve nucleic acid analysis, those that involve mass spectroscopic and/or proteomic analysis, and those that involve culture analysis [5] – each methodology having its own strength. Nucleic acid analysis is, by far, the most common modality. Sequencing the DNA of bacterial 16S rRNA is robust, bioinformatic pipelines for analysis are well accepted, and bacterial taxa within the microbiota are well annotated. However, 16S analysis identifies only the bacterial component of the microbiome and does not directly provide details related to the functionality. As compared to 16S analysis, metagenomic analysis of microbiome-derived DNA or RNA adds significant advantage in that it is unbiased, broadens bacterial assignment, captures nonbacterial taxa, and permits more robust functional inference. To best understand the metabolic activity of the microbiome, however, metabolomic and proteomic analyses are also required [6].

Metabolites produced by the microbiome disseminate systemically, calibrating host immunity beyond antigenic interface. One of the most commonly studied microbiome-derived metabolites is trimethylamine N-oxide (TMAO), which contributes to cardiovascular disease through currently unknown mechanisms [7,8]. Too, tryptophan catabolites have been associated with HIV disease and may be related to tryptophan metabolizing bacterial taxa [9], and short chain fatty acid (SCFA) production by bacteria represents a significant source of energy for the host [10]. A mechanistic determination of the contribution of nonhost metabolites to health and disease represents an important but underexplored area of research.

MICROBIOME-ASSOCIATED DETERMINANTS OF HIV ACQUISITION

Understanding how the microbiome contributes to the risk of sexually transmitted HIV infections is an essential next step in ending the HIV epidemic. Indeed, increased risk of HIV acquisition in heterosexual women is strongly associated with the presence of a Lactobacillus nondominant vaginal microbiome [11], and in men who have sex with men (MSM) with increased intestinal Prevotella:Bacteroides[12]. Although initial reports describing these findings were cross-sectional, recent retrospective analyses and models have strengthened and refined these associations. As described by Fulcher et al.[13▪▪], seroconversion in at-risk MSM was found to associate with unique microbiome-associated features. Compared to similarly at-risk individuals that did not seroconvert over the study period, individuals that seroconverted had decreased frequencies of several Bacteroides species and increased serum inflammatory cytokines and bioactive lipids preceding HIV acquisition. Similarly, Sui et al.[14] observed unexpected variation in cohort susceptibility to low-dose intrarectal SIV in the rhesus macaque model. Here, susceptibility to SIV acquisition was associated with frequencies of rectal CCR5+Ki67+ CD4+ T-cells and in turn, fecal Prevotella:Bacteroides ratios.

To experimentally determine whether a dysbiotic microbiome might contribute to increased susceptibility, we recently utilized the antibiotic vancomycin to induce dysbiosis in rhesus macaques [15▪▪]. As predicted, we observed that pronounced dysbiosis was associated with an increase in susceptibility to low-dose intrarectal SIV infection. Surprisingly, however, increased susceptibility was not associated with direct measures of the microbiome but rather, with measures of perturbed antimicrobial immunity– namely, reduced rectal TH17 and TH22 frequencies and increased rectal PLA2G2A, GZMB, LTF, DUOX2, and SUCNR1 transcripts. Collectively, these findings suggest that microbiome-responsive features in host immunity – more so than specific taxa – tune host susceptibility to rectal HIV infection. It remains unclear whether perturbations of the preinfection microbiome alone might influence disease progression beyond sexual acquisition. The outcome of experimental studies assessing these relationships will be important for the assessing the long-term potential of preexposure prophylaxes or repeated antibiotic exposure prior to sexual acquisition.

VACCINATING FROM WITHIN: COMMENSALS AND THE PRE-EXISTING REPERTOIRE

Although vaccine development has long harnessed adjuvants to stimulate pathogen-associated molecular pattern receptors [16], only recently have findings described the role of individuals’ microbiomes in vaccine outcomes. The administration of broad-spectrum antibiotics to individuals prior to seasonal influenza vaccination impairs the generation of antibody responses in subjects with low preexisting antibody titers in addition to enhancing inflammatory signatures [17]. These perturbations have the potential both to negate protective immunity and to alter the efficacy of functional-derived immunity. Lessons learned from studying the role of the microbiome in HIV vaccine development may lead to the identification of novel modalities and strategies.

Eliciting an improved antibody response to HIV envelope is considered the most probable target for generating protective immunity due to the early emergence of anti-Env antibodies after HIV infection. Previous work has described epitope mimicry between gp41 and commensal bacterial antigens [18,19]. A recent publication by Mayer-Blackwell et al.[20▪] reports that from a global survey of 1470 HIV Vaccine Trials Network (HVTN) participants, 58% of prevaccinated, unexposed participants had detectable anti-gp41 IgG titers – with large geographic variation – whereas anti-gp120 immunoglobulin G (IgG) was seen only in 1.5% of participants. Using prevaccination fecal samples, these authors were able to correlate several taxa of the order Eubacteriales with preexisting antigp41 IgG levels. Importantly, these authors noted a strong correlation between the magnitude of pre versus postvaccination antigp41 IgG levels, and linear epitope mapping further revealed a focusing of the gp41 response, suggestive of a recall response [18].

As naturally occurring antigp120 envelope responses expand too late in infection to effectively clear HIV [21], understanding whether and how preexisting antigp120 antibodies are generated will guide vaccine-elicited expansion. In infant rhesus macaques immunized with varied gp120 protein-based formulations, Jiang et al.[22] observed that that age was the biggest driver of antigp120 IgG plasma levels variation in their study. To determine whether the developing microbiome might prime gp120 immunity, the authors surveyed the fecal microbiome and observed that vaccination induced specific shifts in Sutterella and Rodentibacter species, which further correlated with antigp120 IgG plasma levels across modalities. From metagenomic inference analyses, the authors identified that SCFA and bile acid (BA) gene abundance increased in tandem with Suterella frequencies and correlated with antigp120 IgG, suggesting that targeting SCFA and BA pathways may improve gp120 vaccine efficacy.

Antibodies against the conserved CD4-induced (CD4i) epitope work cooperatively with CD4 binding-site antibodies to potently inhibit HIV infection in vitro[23,24]. It remains unclear whether the brief conformational exposure of CD4i is sufficient to account for estimated anti-CD4i antibody titers in vivo. To ascertain whether the antigenic identity of CD4i antibodies may be commensally derived, Biswas et al.[25] passively immunized mice with CD4i antibodies 4E9C and 916B2, and isolated antiidiotypic antibodies (aID mAbs). Using these aID to a random 12-mer phage library, these authors identified consensus peptide sequences by homology. Of aID found to target microbial-associated sequences, the authors found epitope homology with Nocardioidaceae and Vibrio vulnificus sequences. Using matching, synthesized peptides, these authors demonstrated that a single peptide recognized by Vibrio vulnificus reacted to previously identified CD4i mAbs 4E9C, 17b, and 1F4F and to several of their identified aID. Epitopes encoded by this and other cross-reactive bacterium may be useful as a mimetic to prime CD4i in vaccine strategies [24] or in the purposeful design of oral probiotics [26,27] to boost vaccine efficacy.

ANTIRETROVIRALS… ANTIVIRAL OR ANTIBACTERIAL?

Potential roles for antiretrovirals (ARVs) in modulating the intestinal microbiome in PWH have been widely described [28]. Unlike with HIV infection - where the date of acquisition is often unknown – the introduction of ARVs is a chronologically definable event and samples from stage-matched individuals show commonalities in their microbiome and microbial-translocation associated markers [29,30]. Too, the introduction of ARVs in SIV-infected rhesus macaque models induces immediate effects on the intestinal microbiome [31,32]. However, changes in the microbiome are neither uniformly observed nor categorically similar after lentiviral infection, suggesting that further research is warranted.

Defining an ‘ARV-associated microbiome’ is complicated by the use of varying ARV regimens across individuals [28]. As characterized by Pinto-Cardoso et al.[33], a cross-sectional examination of the fecal microbiomes of ARV-treated patients reveals that although individuals on long-term ART collectively have distinct diversity measures as compared to uninfected individuals, there remain modest but appreciable variations in measures of the microbiome and translocation markers between patients on differing regimens. Too, Hanttu et al.[34] reported increased alpha diversity in individuals switching from an efavirenz- to a protease inhibitor-based regimen.

Some of the confounding effects of cocktail regimens can be resolved by testing the effects of ARVs – individually or in combination – on bacterial taxa in vitro. A methodical survey of over 1000 marketed drugs against 40 gut bacterial strains revealed that 27% of nonantibiotic drugs affected the growth of at least one taxon in vitro[35]. To assess the effects of HIV ARVs more specifically on commensal taxa, Rubio-Garcia et al. cultured 16 ARVs with gut and vaginal human commensal bacteria in vitro and showed that five have antibacterial activity [36], expanding on previous reports [37]. Additionally, Wallace et al. explored the possibility that the prolonged use of ARVs may contribute to antiviral resistance in susceptible bacterial taxa [38▪]. In addition to developing antiviral resistance against the primary ARV, several taxa concerningly developed cross-resistance to other antivirals or, antibacterial resistance. It remains to be seen whether similar findings are observed in patients receiving long term ARVs as either post or preexposure prophylaxes.

Despite observed sensitivities of individual bacterial taxa to antivirals in vitro, we previously demonstrated that short courses of ARVs in healthy rhesus macaques in vivo led only to modest perturbations in the intestinal microbiome and intestinal immunity [39]. However, the administration of ARVs led to a generalizable instability of the microbiome, a measure which has conceptually been observed in other studies assessing the microbiome:ARV interface in vivo[40]. It remains unclear whether ARV-associated bacterial perturbations stem from lentiviral-associated mucosal pathologies, direct antibacterial activity, or targeting of nonbacterial components of the microbiome, as further discussed below.

HARNESSING THE POWER OF THE MICROBIOME

Therapeutics designed to improve the intestinal microbiome have gained favor as a way to reduce disease progression or to boost immune reconstitution after ARV therapy. As covered more extensively in this issue, wholesale efforts to manipulate the microbiome in HIV and SIV infection include probiotic regimens [41–45] and fecal microbial translocation [46–49]. We and others have considered that supplementation of nutrients and metabolites to stimulate or mimic commensal metabolic output may more naturally improve microbiome function, by allowing the host and microbiome to reciprocally recalibrate themselves [50–52]. In murine models, metabolite supplementation can significantly improve intestinal immune function in models of intestinal disease [53,54]. To improve immune function in immunological non-responders (INRs), Lu et al.[55] provided INR study participants with whole-protein enteral nutrition formula supplementation. With just 3 months of daily treatment, supplemented participants exhibited improved circulating T-cell counts, and reduced microbial translocation and intestinal E. coli. In a more targeted approach, we recently provided oral sodium butyrate to SIV-infected, ARV-treated macaques and assessed measures of immune reconstitution [56]. No differences were observed in measures of disease progression, the intestinal microbiome, or butyrate-responsive transcriptional pathways as compared to controls, however, suggesting that sodium-butyrate administration is insufficient to improve intestinal immune parameters in chronically SIV-infected macaques. Additional nutritional studies are needed to shed light on the metabolic regimens that best improve immune reconstitution.

NONBACTERIAL COMPONENTS OF THE MICROBIOME

Metagenomic sequencing, in theory, allows for the identification of all members of the microbiome and recent data are beginning to explore how eukaryotic and viral constituents contribute to health and disease. Indeed, nonbacterial components of the microbiome can induce significant immunological responses. For example, the recently identified Tritrichomoas muscalis has been identified as a eukaryotic member of the murine microbiome in some animal facilities and alone induces significant innate and adaptive immune responses in vivo[57]. Moreover, human infection with cytomegalovirus is sufficient to drive expansion of large frequencies of CD4+ and CD8+ T-cells in vivo[58] and the presence of viral STDs such as HPV increase susceptibility to sexually-acquired HIV acquisition [59].

Early analysis of the nonbacterial components of the GI microbiome in PWH and SIV-infected NHPs demonstrated an expansion of the virome and suggested that the expanded virome could contribute to GI tract pathology [60,61]. Subsequent analysis found that expansion of the virome was also present in plasma of PWH [62]. In PWH, the degree of virome expansion (particularly Anelloviridae, Flaviviridae, and Adenoviridae) and bacterial dysbiosis correlate with measures of immunosuppression in participants [63–66], suggesting that lack of immune responses against particular viruses may be responsible. The composition of the virome, particularly bacteriophages, is also likely influenced by administration of ARVs in PWH [67▪]. Whether and how the expanded virome contributes to disease and inflammation in PWH is an active area of research, and interventional studies aimed at reducing particular viruses may lead to reduced inflammation.

The eukaryotic component of the microbiome too is gaining attention, particularly characterization of opportunistic yeast and fungi. The complexity of the eukaryotic microbiome has been recently explored in several anatomic sites of PWH, with alterations apparent both in the GI tract and oral cavity [68–70]. Indeed, several taxa associated with fungal opportunistic infections are routinely expanded in PWH – including Candida albicans and Pneumocystis jirovecii – and correlate with the degree to which the PWH are immunocompromised. The mechanisms underlying these expansions are currently unclear [68–70]. Little exploration of the mycobiome in longitudinally obtained samples from SIV-infected Asian macaques has been performed and further work is merited.

CONCLUSION

The anatomy and functionality of the GI tract is important for nearly all facets of health and is a focus of considerable research effort in PWH and animal models of HIV. There are considerable immunological perturbations within the GI tract of both PWH and SIV-infected Asian NHPs which are associated with focal areas of damage to the epithelial barrier and accumulating studies have explored how the composition of the luminal microbiome may also contribute [71,72]. The degree to which the microbiome is ‘druggable’ is an active area of research and how individual microbiome-altering therapeutics may benefit PWH and other individuals with diseases where the microbiome may contribute will continue to benefit from placebo-controlled interventional trials in large cohorts of genetically diverse study participants.

Acknowledgements

We would like to thank the NIH Medical Arts Branch for their assistance in generating the figure in this manuscript.

Financial support and sponsorship

Funding for this study was provided in part by the Division of Intramural Research/NIAID/NIH. The content of this publication does not necessarily reflect the views or policies of DHHS, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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