Study Participant Characteristics
To investigate the effects of IRIS on HIV populations, we identified three groups of HIV infected study participants for detailed investigations: (i) individuals with active TB with clinical signs and symptoms and microbiologic confirmation at cART initiation, who were diagnosed with TB-IRIS after starting cART, according to protocol specified criteria based on AIDS Clinical Trials Group Criteria (updated January 10, 2009) (20-22) (TBI; N=9). (ii) Individuals enrolled with active TB who did not develop IRIS (TBNI; N=9) and; (iii) individuals enrolled without TB who did not develop IRIS (NTBNI; N=10). The demographic characteristics of these groups were comparable (Table S1); the median age of study participants (54% female) was 38 years, with no significant differences in age among study groups (P > 0.05; unpaired t-tests). All study participants had chronic HIV infection and the majority (18 of 28 participants) were infected with subtype B, C, or CRF02_AG. Study participants had numerous comorbid complications, including Kaposi sarcoma, chronic hepatitis B, Cryptosporidium, herpes simplex virus, and recent Pneumocystis pneumonia (Table S1). Study participants with TB-IRIS experienced symptoms of IRIS within a median of 11 days (IQR 11, 14 days) of starting cART. They continued cART and most underwent additional corticosteroid therapy for variable periods until IRIS symptoms were resolved [median of 113 days (IQR 71, 336)]. Eight of nine individuals with TBI received corticosteroids, and 4 were on immunosuppressive therapy at the time of sampling shortly after cART initiation.
CD4+ T cell increases after cART initiation
PWH typically experience an increase in CD4+ T cell numbers within the first 6 months of initiating cART (23), with slightly reduced rates of increase in TB coinfected people (23, 24). Prior to cART initiation, all study participants had CD4+ T cell counts <100 cells/mm3, indicating advanced disease (25). There was no difference in baseline CD4+ T cell levels in the blood among study groups (Table S1, average of 45 cells/mm3; 6.5% of total lymphocytes; p > 0.05). Within the first 5 weeks of cART, study participants with IRIS experienced a transient increase in CD4+ T cell levels (total cells and percent of total lymphocytes), followed by reductions in these cell numbers (Figure S1). Overall, there were no significant differences in the rate of increase in CD4+ T cells in the blood during the first 6 months of cART, consistent with recent observations (26), and total CD4+ T cell counts increased in the blood by an average of +3 cells per week during the first 26 weeks of cART (p > 0.05; linear mixed-effects model). For the next 1-2 years of cART, CD4+ T cell levels continued to rise by an average of +1 cell per week.
Increased levels of soluble inflammatory and coagulation markers with IRIS
Substantial immune activation is a hallmark of IRIS (25, 27, 28). We evaluated the plasma levels of 20 pro-inflammatory biomarkers prior to and following cART initiation in our cohort. Study participants with TBI had higher levels of D-dimer, MPO, IL-27, IFNγ, IL-6, TNF-α, IP-10, hyaluronic acid, and soluble (s)CD14, both prior to cART initiation (p < 0.05; Kruskal-Wallis tests; Table S2) and during IRIS (p < 0.05; Kruskal-Wallis tests; Table S3), compared to those who did not develop IRIS. No significant elevations were detected in pre-cART levels of CRP, IL-2, IL-10, IL-8, soluble CD163 or hepcidin (Table S2). These data demonstrate that individuals with TB-IRIS had substantial systemic inflammation, as previously reported (26).
Higher pre-therapy plasma HIV RNA levels in people who develop IRIS
To investigate the effects of IRIS on HIV populations, we first quantified pre-therapy HIV RNA levels in plasma prior to cART. Study participants who developed IRIS after cART initiation had higher pre-therapy plasma HIV RNA levels compared to those who did not (Log10 median [Range] copies RNA/mL plasma: TBI 5.9 [4.9 - 6.6]; TBNI 5.1 [4 - 5.5]; NTBNI 4.7 [4.3-6.4]) (Figure 1a). Several cytokines (e.g. TNF-α and IL-2) were also elevated in TBI participants (Tables S2 and S3), which have been correlated with increased HIV RNA production in vitro (29-32). When we analyzed cytokine levels and HIV viremia prior to cART initiation, we found strong correlations between pre-therapy plasma HIV RNA levels and a number of soluble biomarkers, most of which were significantly elevated at baseline in the TBI group: TNF-α, IL-2, IL-6, IL-10, CXCL-10, IL-27 and D-dimer (R = 0.4 - 0.8; p < 0.05; Spearman correlations) (Figure S2).
After initiating cART, HIV RNA levels declined in plasma with typical multiphase decay kinetics in all study participants (33-36). Within 49 weeks of starting cART, all participants achieved plasma HIV RNA levels below the lower limit of detection (LOD) of commercially available assays (<50 copies/mL) and remained below this LOD for the duration of the study period (Figure S3). After long-term cART, quantification of HIV viremia with sensitive single copy assay (37) (Table S4) revealed detectable levels in 80% of all participants, with no significant differences in levels of viremia among the study groups (p = 0.52; Kruskal-Wallis tests). Therefore, although study participants with IRIS had higher pre-therapy viremia than those without IRIS, cART sufficiently inhibited viral replication below commercial LOD in all study participants within the first year of therapy.
The size of HIV infected cell populations is comparable with or without IRIS
We investigated the effects of TB-IRIS on the size and composition of HIV infected cell populations by quantifying the abundance of HIV infected cells in peripheral blood by measuring levels of cell-associated (CA) HIV DNA levels in the peripheral blood lymphocytes (38, 39). For this analysis, pre-cART HIV infected cells were available for a subset of study participants. Since CD4+ cell populations may shift significantly after cART initiation, especially during IRIS events (12, 40), we quantified the levels of HIV infected cells as the concentration of HIV infected cells in PBMC (copies of CA HIV DNA per mL blood) (Figure S4a), as well as in CD4 cell populations (copies of CA HIV DNA per million CD4+ T cells) (Figure S4b).
Prior to cART initiation, there were no significant differences in the proportion of HIV infected cells among study groups (CA HIV DNA in TBI versus TBNI and NTBNI groups: p > 0.05; Kruskal-Wallis tests). Comparable levels of HIV infected cells in the absence of cART indicate that the higher pre-therapy in HIV RNA levels in the TBI group could not be attributed to larger proportions of HIV infected cells.
After cART initiation, and as expected (29, 38, 41, 42), CA HIV DNA levels (37, 39) decreased in all evaluable study participants (Figure S5) as infected cells were eliminated. CA HIV DNA levels in the TBI study group remained comparable to the TBNI and NTBNI study groups after short-term cART and with prolonged cART (TBI vs TBNI and NTBNI; p > 0.05, linear mixed-effects model). These data indicate that IRIS events neither expanded nor contracted the overall levels of HIV infected cells.
No increases in cell-associated HIV RNA during or following IRIS
Next, we investigated the effects of IRIS on CA HIV RNA production (37) in vivo, and quantified HIV RNA levels of total PBMC populations, normalized to the levels of HIV DNA present in the cells from which they were derived. Pre-therapy HIV RNA production (CA HIV RNA:DNA ratios) in cell derived material was substantial in all study participants tested regardless of study group (Figure 1b), and although pre-therapy plasma viremia was higher in those with TBI, there were no significant differences in pre-therapy CA HIV RNA production between individuals with TBI and TBNI. CA HIV RNA:DNA ratios decreased significantly after cART initiation in all study participants and there were no significant differences in HIV RNA production among study groups, during IRIS after short-term cART (Figure 1c), or after long-term cART (Figure 1d) (p>0.05; Kruskal-Wallis tests). Analysis of this limited dataset demonstrated that, despite clinical and laboratory evidence of vigorous broad increases in pro-inflammatory mediators, those with IRIS did not have elevated levels of CA HIV RNA in PBMC compared to individuals without IRIS.
TB-specific HIV infected CD4 cells are present, expand and persist after long-term cART
TB-antigen (TB-Ag) driven polyclonal expansion of CD4+ T cells is an important mechanism implicated in IRIS pathogenesis (14, 43) and is reported to originate from cells that were present prior to cART initiation (18). We utilized a series of functional assays, where we stimulated CD4+ T cells derived from patient PBMC with TB-Ag (purified TB protein derivative; PPD) to determine if after long-term cART, these TB-Ag responsive CD4+ T cells included HIV infected cells, and if such cells were capable of HIV production in response to TB-Ag. We adapted the ultrasensitive HIV-Flow (44) approach to detect HIV infected CD4+ T cells obtained after prolonged cART, which produced virus in response to PPD exposure. PBMCs were available from 15 study participants (5 participants per study group) after long-term cART. We enriched for CD4+ T cells via magnetic-bead depletion of CD8+, CD56+ and CD19+ cells. CD4+ T cells were cultured with IL-2 and PPD or antiCD3/antiCD28 beads (Dynabeads®) or a control with no activation agent. Growth media contained a nucleoside analogue and integrase inhibitor (200nM lamivudine and raltegravir respectively) to block new cycles of HIV infection during the stimulation. After 36 hours, cells were stained for production of p24 using a dual antibody staining strategy (28B7/KC57) (44).
In the absence of stimulation, 3 study participants with TB and 2 without TB had spontaneous HIV p24 production (Figure 1e). In the presence of PPD, the majority of the TB co-infected group (8/10 participants) exhibited HIV p24 production in 0.5-3.5 cells/million CD4+ T cells in response to PPD, which was greater than the TB-Ag response in the NTB group (p < 0.05). These results indicate that a proportion of HIV infected cells were TB-specific, activated by TB-Ag, and can produce virus. TB-specific HIV infected cells were present in individuals with and without IRIS and there were no statistically significant differences in concentrations of TB-specific HIV infected cells between those with and without IRIS (Median [95% CI Upper; Lower] = 1.2 [1.2; 1.9] versus 0.5 (1.0; 2.4) respectively; p > 0.1 Kruskal-Wallis).
We further quantified the HIV populations in cells that were responsive to TB in 2 study participants with TBI (P6 and P7). PBMC obtained from P6 during IRIS (week 12 of cART), and from both individuals after long-term cART (week 96 and week 144 from P6 and P7 respectively) were incubated with PPD for 4 hours, sorted into CD4+ IFNg+/- subsets, and then further sorted on CD45RO+/-. HIV single copy assays were used to investigate levels of CA HIV DNA in CD4+ CD45RO+ IFNg+ cells and substantial levels of HIV DNA were present in cells from both individuals; 8,300 copies HIV DNA per million TB-Ag responsive cells were present during IRIS in P6; and 6,300 and 8,700 copies HIV DNA per million TB-Ag responsive cells after long-term cART in P6 and P7 respectively). These results demonstrated that TB-Ag responsive CD4+ T cells obtained during IRIS and after long-term cART contained substantial numbers of HIV infected cells and that overall, TB-specific HIV infected cells make up a proportion of CD4+ T cell populations that were undergoing reconstitution during IRIS. These cells persist and remain responsive to TB-Ag after prolonged cART, long after TB resolution.
TB-IRIS is associated with more diverse HIV and HIV infected cell populations
The persistence of TB-specific HIV infected CD4+ T cells in co-infected people suggests that TB and IRIS have long-term effects on the structure of HIV infected populations. To investigate their effects on the composition and structure of HIV populations, we obtained single genome sequences from plasma derived HIV RNA and PBMC derived HIV DNA prior to and after the initiation of cART in 25 of the study participants. We used phylogenetic and population genetics approaches to define the structure of these populations, and we determined if HIV populations shifted after the introduction of cART. As our studies identified that TB-specific HIV infected cells were present throughout cART, we also analyzed the HIV populations present in the cells and media from the ex vivo TB-Ag stimulations of CD4+ T cells after long-term cART from P6 and P7.
We first determined the genetic diversity of HIV populations by measuring average pairwise distance (APD) of single genome sequences (Table S5). No significant differences in the genetic diversity of plasma HIV RNA were detected in any of the study groups prior to initiating cART (Figure 2a; p > 0.05 for all comparisons). Several of the participants in the TBI had relatively high genetic diversity in plasma HIV RNA (APD >1.9%), which is higher than reported in a number of previous studies (45-48). The source of this increased genetic diversity is uncertain and further analysis revealed no correlation between plasma HIV RNA levels and APD (p > 0.05; Spearman correlation), however there was a strong positive correlation between IL-2 levels and APD of pre-therapy plasma HIV RNA populations (Figure 2b) (R = 0.73, p = 0.03; Spearman correlation), supporting the possibility of antigen driven proliferation and expansion of the cellular viral reservoir in those with TBI, which may be driving the genetic diversity of HIV populations.
Although the diversity of pre-therapy plasma HIV RNA populations was comparable among all study groups, there were minor differences in PBMC derived HIV DNA populations. Pre-therapy APD of PBMC HIV DNA populations in participants with TBI was modestly higher than corresponding levels in NTBNI groups (Figure 2c, Table S5: APD 1.8% versus 1.3% respectively; p = 0.04; Kruskal-Wallis tests). Study participants with TBI also had more diverse HIV DNA populations during IRIS compared to the diversity of HIV DNA populations after short-term cART in non-IRIS groups (Figure 2d, APD 1.8% versus 1.2% respectively; p = 0.03). After prolonged cART, however, there were no significant differences in genetic diversity of post-IRIS HIV DNA populations compared to those who had no prior IRIS event or pre-existing TB (APD 1.9% versus 1.3% respectively; p > 0.05; Figure 2e). A temporal trend analysis of genetic diversity in study participants who developed symptoms of IRIS revealed a greater overall diversity in HIV DNA populations on cART compared to those who did not experience IRIS (Figure 2f; p = 0.01; linear mixed-effects), demonstrating a greater overall genetic diversity in TBI study participants.
Evidence of potential clonal expansions of HIV infected cells during IRIS
To further investigate the structure of HIV populations we performed detailed phylogenetic analysis of single genome sequences. In general, sequences from TBI, TBNI and NTBNI groups were highly diverse; however, in each group there were examples of identical sequences, suggesting they were potential clones. Additional clonal prediction scores (49) (Figure S6) confirmed these identical sequences had high probability of being members of cell clones. Phylogenetic analysis of single genome HIV sequences derived from CA HIV DNA populations revealed that portions of these populations comprised of identical sequences, which may represent clonally expanded HIV infected cell populations. Potential HIV clones (49) were comparable among study groups; median (range) frequencies of HIV clones in all study groups combined was 5% (0 - 38) prior to cART initiation, 13% (0 - 43) shortly after cART initiation and 18.5% (0 - 59) after long-term cART (p > 0.05 among all study groups; Kruskal-Wallis tests). We did observe an increase in the frequency of identical HIV DNA sequences in participants with TBI after short-term cART (pre-therapy median [range] = 0% [0 - 19] versus short-term cART 21% [0 - 43]; p = 0.03; One-way Anova test) (Figure 3a), but not in those who did not experience IRIS (Figure 3b and 3c).
After prolonged cART, identical sequences remained detectable in all study groups, with no further increases in the TBI group after IRIS. We did not observe a relationship between the proportion of identical HIV DNA sequences in the TBI group with continuous cART (p = 0.9; Poisson regression analyses), but in the absence of IRIS, there was an increase in the proportion of identical sequences detected on cART (p = 0.0003 in the TBNI group and p = 0.02 in the NTBNI group).
This phylogenetic analysis of HIV populations suggested fundamental differences in the structure of CD4 populations of the TBI group. To characterize the structure of the entire CD4 population, we determined the distribution T cell receptor rearrangements using high throughput sequencing, from which we investigated the diversity of the populations using Shannon entropy analyses. Figure 3d illustrated the relatively low Shannon index of the TBI group, indicating a highly diverse population, while the Shannon index for TBNI was significantly higher (p = 0.04; Kruskal-Wallis test). These findings indicate that in the absence of previous IRIS, CD4+ T cell diversity in HIV/TB co-infected individuals is less diverse after long-term cART, CD4+ T cell expansions associated with IRIS include HIV infected cells and imply a more polyclonal population of expanded HIV infected cells with IRIS.
Long-term shifts in HIV populations after IRIS
Overall, phylogenetic analyses revealed that pre-therapy plasma and PBMC derived HIV populations were highly related; single genome sequences derived from plasma and PBMC appeared to be intermingled, with plasma and PBMC-derived sequences sharing large branches on the phylogenetic tree, and few distinct branches having bootstrap supports >70 (Figure S7). To rigorously investigate whether the HIV populations in PBMC and plasma were distinct, we performed detailed analyses for population subdivision and migration.
Population subdivision analysis (46) revealed pre-therapy HIV populations in plasma RNA and PBMC DNA as well-mixed, and derived from the same viral population (“panmictic populations”; Table S6). The presence of identical sequences may bias panmixia analyses, therefore we repeated these analyses with individual groups of identical sequences collapsed to single lineages; these repeat analyses confirmed that the pre-therapy populations were panmictic. An independent analyses for population shifts using migration analysis (Slatkin Maddison (50)) also determined that pre-therapy sequences in plasma and PBMC were intermingled, with no evidence of compartmentalization or non-random migration of variants (Table S7). Taken together, these data demonstrate that HIV populations in plasma and PBMC prior to cART initiation are highly diverse and well mixed.
To investigate the fate of these pre-therapy viral populations after cART initiation, we repeated these population genetics analyses using single genome sequences obtained from PBMC after short-term cART. After initiating cART, HIV populations continued to be genetically diverse. However, detailed fine structure analysis using population subdivision analyses revealed that population shifts took place in some participants. As illustrated in Figure 4, HIV DNA populations from 1/9 TBI participants (Figure 4a) and 3/16 without IRIS (Figure 4b and 4c) were shifted and were no longer panmictic with pre-therapy plasma HIV RNA populations after short-term cART. These data suggest that shifts in HIV-infected cell populations occur in some individuals early after cART initiation, but the presence of these shifts was not more frequent with IRIS.
We also analyzed PBMC DNA derived HIV populations after prolonged cART to determine if IRIS affected long-term persistence of HIV-infected cell populations. As shown in Figure 4, HIV populations after long-term cART remained highly genetically diverse, however, population shifts were significantly more common after long-term cART in individuals who experienced IRIS after starting therapy (6/9 participants with TBI) compared to those who did not experience IRIS (2/16 NI participants; p = 0.03; Fisher-Exact test). In addition, all study participants with TBI had evidence of compartmentalization of HIV DNA populations (Slatkin-Maddison, probability of compartmentalization p = 0.002) while only 3/16 individuals without IRIS had evidence of compartmentalization. The differences in the frequency of compartmentalization in individuals with prior IRIS events was highly significant (p = 0.0001, Fisher’s exact test). Taken together, these data indicate that TB-IRIS events fundamentally shift the long-term composition of HIV populations persisting during cART.
One study participant in the TBI group, P13, discontinued cART due to nonadherence and experienced a rebound in viral replication (186,000 copies HIV RNA/mL plasma) after 76 weeks of suppression of viral replication with cART. We analyzed the genetic characteristics of the viremia to determine if the rebound virus was distinct from pretherapy virus, as predicted by the panmixia and compartmentalization analyses. As shown in Figure S8, the predominant virus producing lineage was not clustered with the majority of proviral lineages detected during IRIS or after 64 weeks of cART. Panmixia analyses revealed the rebounded plasma viral population was distinct from pretherapy (Table S8), demonstrating that the HIV reservoir was distinct from pretherapy viremia. Of note, the rebound viremia was also distinct from the HIV DNA population at week 64 (Table S8), suggesting rebound viremia emerged from a highly diverse HIV DNA population.
These analyses indicate profound shifts in HIV populations after IRIS; the lineages of HIV infected cells persisting after IRIS, do not reflect the viral populations present in plasma at the time cART was introduced. Furthermore, these data suggest that the majority of infected cells that were producing HIV virions at cART initiation did not persist in the PBMC compartment after TB-IRIS. Furthermore, rebound viremia in P13 did not originate from the full spectrum of viral lineages replicating prior to cART initiation, but rather from a limited proportion of the proviral lineages persisting on cART. This finding suggests that in this individual, defective proviral lineages dominated the HIV infected cell populations that persisted on cART and not virus producing lineage largely responsible for rebound viremia.
TB antigen-driven expansion of HIV infected cells as a mechanism of HIV persistence
We showed that during IRIS and after long-term cART, some TB-Ag responsive cells from those with IRIS harbor HIV proviruses. Next, we investigated the possibility of clonal expansion of HIV infected cells of this phenotype as a possible mechanism of HIV persistence in these individuals, and if these cells were capable of producing HIV virions after long-term cART. The cellular yield from the ex vivo stimulation studies was insufficient to do detailed integration site analyses, therefore populations were analyzed using single genome sequencing and phylogenetic analyses. During IRIS, we obtained 20 sequences from CD4+CD45RO+IFNg+ cells derived from P6. After long-term cART we obtained 36 sequences from P6 and 26 sequences from P7 from cells of the same phenotype. Several variants in both study participants were detected multiple times and first detected during IRIS.
In P6, there were 3 viral variants detected in HIV DNA populations during IRIS, with evidence of potential persistence via clonal expansion in response to TB antigen (Figure 5a). Two of these variants (Figure 5a, Variants 1 and 3) were first detected in CD4+CD45RO+IFNg+ T cell populations responsive to TB antigen during IRIS. Identical sequences were detected after 96 weeks of cART in this patient in HIV DNA populations from the same TB responsive cell phenotype, suggesting that these cells were present during IRIS and persisted for prolonged periods after IRIS subsided and TB infection resolved. Variant 3 was also detected in the HIV RNA derived from the supernatant of the cell culture after PPD stimulation, indicating that the cell of origin activated by TB-antigen stimulation was capable of producing HIV virions.
In P7, there were 2 viral variants detected during IRIS in PBMC HIV DNA populations with identical sequences detected after 144 weeks of cART in CD4+CD45RO+IFNg+ cells (Figure 5b). Variant 1 was detected in PBMC obtained prior to cART initiation, demonstrating potential persistence of these variants from pre-therapy lineages. HIV DNA populations within TB-Ag responsive cells were panmictic with pre-therapy HIV RNA populations.
These findings demonstrate that TB-specific cells can harbor HIV proviruses and produce HIV virions within the context of coinfection and active TB disease prior to cART initiation. We found that HIV infected TB-specific CD4+ T cells with memory phenotypes can produce virions. Furthermore, both TB co-infection and IRIS can shape the HIV infected cell populations that persist on cART, potentially through mechanisms like clonal expansion. Finally, these proviruses can produce virions, inducible by TB antigens after years of cART, highlighting their clinical relevance and importance in considerations for viral remission strategies.