Introduction
HIV pre-exposure prophylaxis (PrEP) has proven effective in specific populations with oral tenofovir disoproxil fumarate (TDF)/emitricitabine (FTC), tenofovir (TFV) alafenamide (TAF)/FTC, vaginal tenofovir (TFV) gel, vaginal dapivirine ring (DPV) and injectable long-acting cabotegravir [1–9]. As with contraception, having multiple PrEP choices likely increases uptake, persistence and adherence of PrEP regimens with product choices influenced by many variables beyond efficacy [10–13]. Biological and pharmacological variables, which vary between men and women, also influence HIV acquisition and prevention, but are infrequently compared directly.
HPTN 069/ACTG 5305 was a prospective safety and tolerability trial evaluating maraviroc (MVC) alone or in combination with FTC or TDF in comparison to TDF/FTC, the only PrEP regimen proven effective at that time. Nonoverlapping side effect profiles and coverage of community-acquired TFV or FTC resistance were considered potential benefits of MVC-containing regimens. Perhaps, the most significant challenge to PrEP product development is the inability to evaluate concentration-response in Phase 2 prior to Phase 3 efficacy trials. Substitutes have included site of HIV acquisition assessments of mucosal tissue antiretroviral pharmacokinetics, pharmacodynamic concentration-response evaluations using ex-vivo HIV challenge of mucosal tissue as a potential indicator of clinical HIV protection, and immunological changes that might affect HIV infectivity. Sex-based differences in colorectal tissue, an important route of HIV acquisition in MSM, transgender women (TGW) and cisgender women (CGW), are not well studied.
We conducted a mucosal tissue substudy within HPTN 069/ACTG 5305 that compared CGW with MSM with respect to pharmacokinetics, pharmacodynamic and mucosal immunology. This substudy represents one of the largest studies comparing colorectal and cervical tissue and a unique opportunity to compare these variables in CGW and MSM. We report important differences in colorectal tissue susceptibility to HIV infection between MSM and CGW both before and during antiretroviral dosing.
Materials and methods
Study design and population
We report the mucosal tissue pharmacokinetics, pharmacodynamic and flow cytometry results of CGW (first reported here) in comparison to previously reported MSM results [14]. This tissue substudy was nested within HPTN 069/ACTG 5305 study (NCT01505114), a Phase 2 prospective, randomized, controlled, double-blind trial of the safety and tolerability of three candidate PrEP regimens, MVC 300 mg, MVC 300 mg/FTC 200 mg (MVC/FTC) and MVC 300 mg/TDF 300 mg, compared with a control group who received TDF 300 mg/FTC 200 mg [15,16]. Substudy participants followed the same study protocol as well as collection of plasma and peripheral blood mononuclear cells (PBMCs), cervicovaginal fluid (CVF) and rectal fluid at Weeks 24, 48 and 49 for pharmacokinetics, and cervical and colorectal biopsies for pharmacokinetics, ex-vivo HIV infectivity (pharmacodynamic) and colorectal tissue flow cytometry at Baseline, Week 24, 48 and 49. All study sites were in the US.
Sample processing
Plasma, PBMC, rectal fluid and colorectal biopsy collection and processing was previously reported [14]. Participants self-collected CVF with a Dacron swab; two exocervical biopsies were collected using Tischler forceps (reported previously) [17]. Blood plasma, PBMC lysates, CVF, rectal fluid and biopsy homogenates were immediately frozen, stored and later shipped to JHU for drug concentration analysis. Frozen explant supernatants (batch shipped) and flow cytometry samples (overnight shipped) went to University of the Pittsburgh for analysis.
Pharmacology
Drug concentration analysis used ultra-performance liquid chromatograph tandem mass spectrometry (UPLC-MS/MS) [18–20]. CVF, rectal fluid and tissue results were reported as mg drug per mg of sample; lower limit of quantitation (LLOQ) listed for these matrices is based on median sample weights (Table 1).
Table 1 -
Drug concentration summary pooling Week 24 and Week 48 when study drugs were prescribed, comparing cisgender women to MSM and transgender women by biological matrix analysed and drug analyte.
|
Analyte |
Units |
LLOQa
|
Number |
Drug concentration median (IQR) |
Below LLOQ (%) |
| Matrix |
|
|
|
Pooled |
CGW |
MSM |
Pooled |
CGW |
MSM |
Pooled |
CGW |
MSM |
| Plasma |
MVC |
ng/ml |
0.5 |
125 |
44 |
81 |
15.6 (6.8, 31.4) |
22.9 (10.0, 38.1)∗
|
13.3 (4.8, 19.9) |
8.8 |
9.1 |
|
|
TFV |
ng/ml |
0.31 |
81 |
34 |
47 |
57.2 (36.0, 112.0) |
53.8 (29.3, 98.3) |
67.6 (36.0, 114.0) |
8.6 |
14.7 |
4.3 |
|
FTC |
ng/ml |
0.31 |
106 |
44 |
62 |
133.5 (49.0, 346.3) |
105.5 (13.4, 333.5) |
166.5 (51.3, 376.0 |
11.3 |
18.2 |
6.5 |
| PBMC |
TFV-DP |
fmol/106 cells |
0.135 |
82 |
36 |
46 |
54.5 (34.2, 93.4) |
64.0 (24.9, 117.8) |
52.3 (36.2, 82.0) |
8.5 |
13.9 |
4.3 |
|
FTC-TP |
pmol/106 cells |
5.405 |
106 |
44 |
62 |
5.7 (2.5, 8.7) |
5.4 (0.3, 9.3) |
5.7 (3.2, 8.5) |
11.3 |
18.2 |
6.5 |
| Rectal fluid |
MVC |
ng/mg |
0.021 |
94 |
13 |
81 |
2.9 (0.4, 15.7) |
5.7 (0.1, 27.9) |
2.8 (0.5, 14.4) |
11.7 |
15.4 |
11.1 |
| Colorectal tissue |
MVC |
ng/mg |
0.010 |
93 |
13 |
80 |
0.7 (0.2, 1.4) |
0.5 (0.1, 0.9) |
0.7 (0.3, 1.5) |
10.8 |
15.4 |
10.0 |
|
TFV |
ng/mg |
0.003 |
54 |
8 |
46 |
1.4 (0.8, 2.1) |
1.5 (0.4, 2.0) |
1.4 (0.8, 2.1) |
5.6 |
0.0 |
6.5 |
|
FTC |
ng/mg |
0.013 |
72 |
10 |
62 |
0.7 (0.3, 1.1) |
0.3 (0.1, 0.5)∗
|
0.7 (0.4, 1.1) |
9.7 |
20.0 |
8.1 |
|
TFV-DP |
fmol/mg |
2.618 |
54 |
8 |
46 |
32.3 (19.1, 85.5) |
318.4 (89.4, 526.9)∗
|
26.1 (17.5, 52.7) |
7.4 |
0.0 |
8.7 |
| Cervical tissue |
MVC |
ng/mg |
0.014 |
42 |
42 |
– |
– |
0.03 (BLQ, 0.11) |
– |
– |
42.9 |
– |
|
TFV |
ng/mg |
0.003 |
32 |
32 |
– |
– |
BLQ (BLQ, BLQ) |
– |
– |
90.6 |
– |
|
FTC |
ng/mg |
0.017 |
39 |
39 |
– |
– |
BLQ (BLQ, 0.3) |
– |
– |
53.8 |
– |
|
TFV-DP |
ng/mg |
3.401 |
32 |
32 |
– |
– |
BLQ (BLQ, BLQ) |
– |
– |
84.4 |
– |
| Cervicovaginal fluid |
MVC |
ng/mg |
0.001 |
43 |
43 |
– |
– |
0.08 (0.01, 0.22) |
– |
– |
9.3 |
– |
|
TFV |
ng/mg |
0.011 |
33 |
33 |
– |
– |
0.37 (0.07, 1.02) |
– |
– |
9.1 |
– |
|
FTC |
ng/mg |
0.044 |
40 |
40 |
– |
– |
1.9 (0.24, 4.76) |
– |
– |
17.5 |
– |
Participants are included regardless of adherence category. Total number exceeds tissue substudy study population due to pooling Week 24 and Week 48 visits.FTC, emtricitabine; FTC-TP, FTC triphosphate; IQR, interquartile range; LLOQ, lower limit of assay quantitation; MVC, maraviroc; TFV, tenofovir; TFV-DP, TFV diphosphate.
aOther than plasma, LLOQ listed in table is based upon median sample mass or cell count and assay drug amount per sample LLOQ (PBMC TFV-DP 2.5 fmol/sample and FTC-TP 100 pmol/sample; rectal fluid MVC 0.5 ng/Merocel sponge; cervical and colorectal tissue MVC 0.05 ng/sample, TFV 0.05 ng/sample, FTC 0.25 ng/sample, and TFV-DP 50 fmol/sample; cervicovaginal fluid MVC 0.05 ng/Dacron swab, TFV 0.625 ng/Dacron swab and FTC 2.5 ng/Dacron swab).
∗P < 0.005 based on all PK eligible participants, regardless of adherence.
Ex-vivo HIV challenge
Cervical and colorectal biopsies were placed in tissue culture media, challenged with HIV for 2 h, and supernatant harvested over 10–14 days [17,21]. Differences in cervical and colorectal methods, respectively, include biopsy number (one vs. four), HIV challenge dose (5 x 104 vs. 1 x 104 TCID50), supernatant sampling (4, 7, 10 vs. 4, 7, 10, 14), analysis volume (0.7 vs. 0.5 ml). Unit of analysis was median biopsy cumulative log10 p24 antigen concentration. LLOQ for p24 was 12.5 pg/ml.
Cell phenotype
The isolation of colorectal biopsy mononuclear cells and flow cytometry analysis for CD3+, CD4+, CD8+ and CD45+ [surface (FCS) and intracellular markers (FCI)] and CD38+, CD69+, HLA-DR+, CCR5+, CXCR4+, Ki67+ (FCS only) was described previously [14,22].
Data analysis
Nonparametric descriptive statistics, comparisons tests for differences among regimens or study visits (Kruskall--Wallis or paired Friedman tests) or between sex at birth, regimens or visits (Wilcoxon ranked sum tests), and correlation (Spearman test) were used.
We defined daily ‘adherence’ using HPTN 066 thresholds for TFV and FTC; ‘percentage adherence’ is the percentage of participants meeting this definition [20]. The MVC adherence threshold, 4.6 ng/ml, was determined by the lowest concomitant MVC concentration in TFV and FTC adherent participants on combination regimens. The MVC threshold demonstrated 93% sensitivity and 78% specificity identifying daily adherence defined by TFV and FTC thresholds.
Drug concentration vs. p24 response modelling used the sum of molar tissue drug concentrations to account for combination drug regimens. Modelling explored two, three and four-parameter Imax models [E0 baseline p24 without drug, Imax maximum p24 change on drug, IC50 molar drug concentration at half-maximal effect and slope term (Hill coefficient)], weighting schemes for heteroscedasticity, ± biopsy weight adjustment, ± MVC concentration correction (x0.18) to account for pharmacokinetic (no incubation) vs. pharmacodynamic (2-h incubation) processing differences [23] and ± imputation of baseline and/or BLQ values. Goodness-of-fit was assessed using the correlation matrix, coefficient of variation, and Schwartz and Akaike information criterion (Phoenix WinNonlin v.8; Certara, Cary, North Carolina, USA).
Results
Enrollment and participant characteristics
The substudy included 37 CGW, whose pharmacokinetic, pharmacodynamic and cell phenotype results are first reported here, and the previously reported cohort of 54 MSM (Fig. 1) [14]. The analysis set of 91 participants, excluded six enrolled substudy participants without postdose pharmacokinetic, pharmacodynamic or flow cytometry sampling. Paired comparisons of baseline and active drug visits included 84 participants and excluded seven CGW without baseline biopsies.
;)
Fig. 1: Study enrolment and outcome availability summary for the parent HPTN 069/ACTG A5305 study and the mucosal tissue substudy that enrolled 91 participants from within the larger parent study.
CGW height was lower than in the MSM group (P < 0.001), but age, weight and creatinine clearance were similar (Supplemental Table 1, https://links.lww.com/QAD/C102). Pooling sex groups, there were no differences in these variables across study arms. Nineteen CGW (51%) reported use of hormonal contraceptives, including depot medroxyprogesterone acetate (N = 3) and other progestins (N = 16), of whom six were also on oestrogens.
Adherence
Across regimens, 79% of CGW met the protocol definition of adherence, 12% lower than the 90% adherent in MSM (P = 0.045). Overall adherence changed little from Week 24 (87%) to Week 48 (85%).
Pharmacokinetics
For on drug periods (Week 24 and Week 48), plasma, PBMC, rectal fluid, cervicovaginal fluid and colorectal tissue concentrations fell below the LLOQ in 0–20% of samples depending on sex and matrix-analyte pair, without CGW-MSM differences (Table 1). Sex differences were observed in plasma MVC (1.7-times higher in CGW), colorectal tissue FTC (2.3-times higher in MSM) and colorectal tissue TFV-DP (12.2 times higher in CGW, all P < 0.005). When excluding nonadherent participants, the only additional pharmacokinetic difference was a 59% higher PBMC TFV-DP in MSM (P = 0.044). The differences in plasma MVC (1.6-times higher in CGW, P < 0.001), colorectal tissue FTC (2.0-times higher in MSM, P = 0.016) and colorectal tissue TFV-DP (13.8-times higher in CGW, P < 0.001) remained statistically significant and of similar magnitude. We observed no pharmacokinetic differences among the four regimens when pooling study week across all participants (Table 1).
In contrast to all other matrices, cervical tissue drug concentrations fell below the LLOQ in 43–91% of samples. Paired median colorectal to cervical tissue concentration ratios, using the analyte-matrix LLOQ where necessary for BLQ values, indicate differences of 12 times (MVC only), 276 times (TFV), three times (FTC) and 93 times (TFV-DP), though with only 8–11 observations per drug analyte.
Tissue cell HIV infectivity
Biopsy weight differences were observed among regimens for cervical tissue at Week 24 (P = 0.05), colorectal tissue at Weeks 48 (P = 0.048) and 49 (P = 0.028), and between MSM and CGW (each week P ≤ 0.002). Therefore, explant p24 results are biopsy weight-adjusted. However, the weight-adjusted and nonweight-adjusted explant p24 antigen values were highly correlated for both cervical (r = 0.963) and colorectal tissue (r = 0.983). In addition, there were trivial differences in goodness-of-fit assessments in PK-PD modelling whether or not using biopsy weight-adjusted p24.
For the 29 CGW with pre and postdose cervical biopsies, cervical explant p24 expression was not different across study visits when pooling drug regimens (Fig. 2). When comparing on drug visits to baseline, the only difference was a one log10 p24 reduction in the MVC/FTC arm at Week 24 (P = 0.03). We found greater p24 suppression in FTC containing arms compared to others (P ≤ 0.05). The frequency of greater than one log10 reductions from baseline was (Week 24 and 48 range): MVC only 0%, MVC/FTC 0–33%, MVC/TDF 0–25% and TDF/FTC 38–44% (Fig. 3). Concomitant use of progestin-containing birth control was associated with lower cervical explant p24 at baseline, median log10 (IQR) 1.76 (0.96, 2.29) pg/ml per mg, when compared with CGW without progestin use, 2.65 (1.97, 3.31) pg/ml per mg (P = 0.009). There was no consistent relationship (seen at both Week 24 and 48) with hormonal contraceptive use and p24 results for either cervical or colorectal tissue.
Fig. 2: Explant p24 antigen concentration (cumulative biopsy weight-adjusted) change from baseline by tissue type, sex group and antiviral drug regimen (median and interquartile range).
Fig. 3: Antiretroviral concentration vs. p24 antigen response plots of 120 cervical tissue (left) and 243 rectal tissue (right) homogenates.
In cervical tissue concentration-response modelling, the data best fit a two-parameter sigmoid Imax model with mean (95% confidence interval) E0 2.1 (1.8–2.3) log10 p24 antigen (pg/ml per mg) and IC50 1.20 (0.09–2.31) pmol/mg. Although the coefficient of variation for these estimates is acceptable (6 and 46%, respectively) and the parameter estimate statistically significant, the IC50 estimate should be viewed tentatively, as only 7–40% of cervical tissue pharmacokinetic results were above the LLOQ and only 20% of those values were observed above the estimated IC50.
For the 11 CGW with colorectal explant p24 results (Fig. 2), biopsy weight-adjusted colorectal tissue explant p24 expression was not different at any visit (Week 24, 48 or 49) compared with Baseline. The MSM reductions at Week 24, 48 and 49, were 1.8, 1.5 and 0.7 log10, respectively (all P < 0.001) (Fig. 2). When compared with MSM, CGW p24 was higher at every visit, including predrug baseline: Baseline 0.31 log10 difference (P = 0.046), Week 24 1.01 log10 difference (P = 0.015), Week 48 1.19 log10 difference (P = 0.016) and Week 49 0.61 log10 difference (P = 0.011).
Combining all participant data, statistically significant p24 reductions relative to baseline were observed at all visits (P < 0.001). For on drug periods (Weeks 24 and 48), there were differences among study drug regimens (both P < 0.001), with reductions compared with baseline seen in FTC- and TDF-containing regimens at Week 24 and 48 (all P < 0.001) and FTC-containing regimens at Week 49 (P < 0.033). The frequency of reductions from baseline greater than one log10 on drug for each regimen was (range of Week 24 and 48 visits): MVC only 18–23%, MVC/FTC 73–80%, MVC/TDF 64–82% and TDF/FTC 79–80% (Fig. 3).
In colorectal concentration-response modelling, the data best fit a three-parameter Imax model with mean (95% confidence interval) E0 2.7 (2.5–2.9) pg/ml/mg, IC50 0.55 (0.22–0.87) pmol/mg tissue and Imax 2.2 (1.9–2.4) pg/ml/mg. The coefficients of variation for the three parameters were 6, 30 and 4%, respectively. In contrast to cervical tissue explant modelling, 64% of colorectal tissue drug concentrations (including Week 49) were above the LLOQ and 81% of these were greater than the estimated IC50 (Fig. 3). Direct comparisons between cervical tissue and colorectal tissue p24 results should not be made due to methodological differences. (Note: the best colorectal and cervical model fits included uniform weighting, MVC dilution correction and LLOQ/10 imputation for baseline drug concentrations. IC50 estimates were sensitive to imputation of drug values below LLOQ, so, these were excluded from modelling.)
Colorectal tissue cell phenotype
When comparing flow cytometry results in CGW (11 participants with 39 observations) to MSM (54 participants with 214 observations) including all regimens at each study visit, the only difference at Baseline was 4% higher CD38+/CD8+ FCS (activated suppressor cells) in CGW compared with MSM (P = 0.029). With only two or three CGW per regimen, we did not test regimen-specific differences between sexes. On study drug, the only consistent sex-based difference (same direction of statistically significant change) was CD69+/CD8+ FCS (tissue resident memory suppressor cells) at Week 24, 48 and 49 (all CGW to MSM ratios ≤7%, all P < 0.046). CD69+/CD8+ cells were also increased 3% at Baseline in women using progestins for contraception (P = 0.03).
Pooling colorectal tissue results (both sexes), differences among study weeks were observed only for CD3+ FCI (all regimens and MVC-containing regimens, P < 0.01), CCR5+/CD8+ FCS (MVC-containing regimens, P = 0.001), CCR5+/CXCR4+/CD8+ FCS (all regimens and MVC-containing regimens, P ≤ 0.01) and CD69+/CD4+ FCS (all regimens and MVC-containing regimens P < 0.001). In general, CD3+, CCR5+/CD8+ and CCR5+/CXCR4+/CD8+ (all FCS) rose at one or more of weeks 24, 48 and 49 compared with baseline in at least one drug regimen. Compared with earlier visits, CD69+/CD4+ FCS fell at Week 49, though only in MVC containing regimens. Nearly all the individual participant changes in these few surface markers were between a two-fold increase or 50% decrease (Fig. 4).
Fig. 4: Colorectal tissue flow cytometry changes over time relative to a reference visit (baseline or postdrug) expressed as a ratio on the y-axis are displayed by several layers of groupings along the x-axis, starting with surface marker subset, study arm, study week (numerator of ratio) and sex (MSM beside CGW).
Discussion
This tissue substudy substantially extends pharmacokinetics, ex-vivo HIV infectivity and tissue flow cytometry observations in CGW on oral antiretroviral candidate PrEP regimens, enabling direct comparison to HIV seronegative MSM in the same study. Our key finding is increased HIV infectivity of colorectal tissue biopsies of CGW compared with MSM following ex-vivo HIV challenge. CGW values were two-fold higher at baseline (0.31 log10) and increased on antiretrovirals. Four earlier PrEP studies including both sexes were too small to compare HIV infectivity differences; we were only able to make comparisons by including more women and pooling all regimens [23–26]. The impact and mechanism of this sex difference remains to be understood but underscores the need for critical inclusion of populations at risk of HIV very early in PrEP development.
The baseline HIV infectivity difference cannot be explained by any immunologic measure we assessed as the only consistent CGW vs. MSM difference was a very modest seven percentage higher CD69/CD8 FCS, a difference also seen in MTN-007 [27]. Our immunological findings are limited, however, without cytokines or immunohistochemistry to provide absolute cell subset numbers and anatomic co-localization of cell subsets [28].
HIV infectivity differences cannot be fully explained by pharmacologic differences because p24 differences were seen before study drug dosing, higher FTC concentrations in MSM colorectal tissue are too small to explain the difference given the PK/PD modelling and higher colorectal tissue TFV-DP would be expected to confer reduced HIV infectivity in CGW, making this finding incongruous with the HIV infectivity observation. The CGW colorectal TFV-DP concentrations, however, fall within the previously reported range, 206 and 1329 fmol/mg, for daily oral TDF dosing; this leaves MSM colorectal tissue TFV-DP results as the anomaly, more consistent with single dose results [20,24,29,30].
We suggest hormonal differences as the source of the HIV infectivity differences, but only as an explanation by exclusion. Exogenous progestin in our study, however, was associated with reduced, not increased HIV infectivity at baseline. Two other clinical studies designed to compare the impact of a specific progestin, depot medroxyprogesterone acetate (DMPA), reported either no difference or an increase in active drug concentrations in cervical tissue in the presence of DMPA and no impact on cervical tissue HIV infectivity. However, neither study assessed the DMPA impact on colorectal tissue pharmacology or HIV infectivity [31,32]. We had too few CGW using DMPA to assess this.
CGW have a four-fold greater risk of HIV acquisition through unprotected receptive anal intercourse (URAI) compared to MSM in one meta-analysis, though this should be viewed tentatively, as it is derived from one retrospective observational study in heterosexual couples compared to three prospective studies in MSM [33–37]. No prospective clinical studies directly compare URAI risk in CGW and MSM, which would provide the strongest evidence comparing URAI HIV transmission risk in CGW and MSM to corroborate the meta-analysis and our ex vivo HIV infectivity findings. Obtaining such definitive evidence is doubtful given few seroconversion endpoint studies including both CGW and MSM/TGW, challenges capturing reliable CGW anal sex data, and confounding of much higher frequency of receptive vaginal sex compared to anal sex in CGW.
Some have argued the need for higher antiretroviral drug concentrations for oral TDF/FTC PrEP in CGW compared to MSM to achieve the same level of protection as in MSM [38,39]. These studies attributed HIV acquisition differences to TDF/FTC pharmacokinetic differences in cervicovaginal compared with colorectal tissue -- abundantly evident in our present study -- as systemic active drug concentrations (PBMC TFV-DP and FTC-TP) do not differ between CGW and MSM [29,40]. Our findings suggest there may also be physiologic, possibly hormonal differences in colorectal HIV infectivity that may be relevant in TGW on gender-affirming hormonal therapy (GAHT). Reductions in TFV and FTC analytes have been reported inconsistently in TGW on GAHT [29,40–42].
Our finding of no HIV infectivity suppression in cervical or colorectal tissue explants for the MVC only arm concurs with several studies of MVC by both oral, rectal and vaginal dosing routes, using a variety of methods [19,23,43,44]. This finding may also further explain findings from our main study results wherein five out of 406 MSM acquired HIV; four out of these five seroconverters were randomized to the maraviroc only arm [16]. In several reports, including ours, combination of MVC with either TDF or FTC (oral) or with dapivirine (vaginal ring) reported significant HIV suppression indicating the assay performed as expected to indicate HIV suppression in comparison to MVC alone, suggesting consistency in findings [19,45]. However, several groups have reported that MVC is not fairly tested by explant HIV challenge due to substantial loss of MVC during incubation in culture media. This loss is diminished with TFV-DP and FTC-TP, as they are trapped within cells [23,44,46]. We are not aware of similar testing of DPV in vitro, but it is highly lipophilic and probably more resistant to loss during the HIV incubation step. Taking account of this loss of MVC from tissue, we improved our concentration-response model fitting. These MVC findings highlight the critical importance of understanding the impact of analytical conditions on both physiology and pharmacology before application in clinical studies.
Even excluding the MVC only arm, the three combination drug regimens also failed to consistently suppress HIV replication in our cervical explants. Again, interesting to consider this in light of the main study finding where none of 188 women study participants acquired HIV [15]. Others also reported no HIV suppression with a single dose MVC/TDF oral dose using similar HIV challenge methods [43]. Significant HIV suppression has been reported with a single MVC/TDF oral dose, though using double the MVC/TDF dose, a higher HIV challenge titer, and RNA, not p24, as primary readout [45]. Extending the comparison to vaginal PrEP products, single agent formulations indicate substantial reductions of 1.1–1.5 log10 (TFV film and gel) to 1.0–1.7 log10 reduction (DPV film and gel) [19,47–50].
The 1.9 log10 p24 suppression in the combination arms was higher than all the previous studies, among them < 1 log10 suppression with oral TDF/FTC in all MSM study, MTN-017 [51]. This was due to baseline p24 higher and p24 LLOQ lower than previous reports, raising caution when comparing explant results across studies. The only other oral study of TDF/FTC with colorectal explant testing in women, RMP-02/MTN-006, did not show any p24 reduction, but performed biopsies only 30 min after a single dose [24]. Prior to this report, colorectal tissue p24 suppression with rectal dosing exceeded that seen with oral TDF/FTC dosing; e.g. reduction of 1.1 log10 p24 and 1.9 log10 with a rectal optimized TFV gel and hypo-osmolar TFV douche, respectively, both with p24 baseline and p24 LLOQ values comparable to MTN-017 [26,51,52].
Our greatest limitation is the small number of CGW on each regimen with colorectal biopsies, preventing regimen-specific CGW-MSM comparisons, especially for pharmacokinetic endpoints. Still, we report the largest HIV prevention study of CGW colorectal explant p24 analysis. The inability to assess MVC's antiviral effect in the explant challenge model further limited assessment of HIV infectivity to only three arms. The practical limitation of very few cervical biopsies captured when compared with colorectal biopsies reduces pharmacologic assay sensitivity in cervical tissue, thus, making it difficult to quantitatively understand how much lower drug concentrations were in cervical tissue compared to colorectal tissue. Assays that are more sensitive are now available, which could add precision to the colorectal-cervical pharmacokinetic comparisons.
In summary, we identified significantly higher HIV infectivity of colorectal tissue in CGW when compared with MSM, seen before, during, and after study drug dosing, consistent with at least one meta-analysis of clinical studies. At best, these CGW vs. MSM differences are only partly attributable to immunologic and pharmacologic measures we assessed. More work is needed to understand the mechanism of this difference and to understand any impact on PrEP dosing recommendations for CGW who have URAI. Our results also reinforce the need for earlier comparative studies of HIV risk and PrEP interventions in all people at risk of HIV acquisition.
Acknowledgements
The authors would like to recognize the contributions of study participants, other members of the HPTN 069/ ACTG A5305 study team, FHI 360, the pharmaceutical sponsors who provided study drugs, Gilead Sciences Inc. and ViiV Healthcare, and the study staff at the participating sites in the tissue substudy: Johns Hopkins University, Baltimore, Maryland (UM1-AI-069465); University of California, Los Angeles, California (UM1-AI-069424); and the University of Pittsburgh, Pittsburgh, Pennsylvania (UM1-AI-069494, UL1-RR-024153, UL1-TR-000005).
The HIV Prevention Trials Network is funded by the National Institute of Allergy and Infectious Diseases (UM1AI068619, UM1AI068613, UM1AI1068617), the AIDS Clinical Trials Group (ACTG; UM1AI068636) and the Microbicide Trials Network (MTN; UM1AI068633 and UM1AI106707), with co-funding from the National Institute of Mental Health, and the National Institute on Drug Abuse, all components of the U.S. National Institutes of Health. Dr Sekabira received support from the HPTN International Scholars Program. Gilead Sciences and ViiV Healthcare provided study drugs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflicts of interest
I.M.G. is an employee of Orion Biotechnology, Ontario, Canada.
R.J.L. has served on advisory boards for Gilead Sciences and Merck, Inc, and received honoraria from Roche and Janssen.
K.H.M. has served on scientific advisory boards for Gilead Sciences and Merck, Inc. and has received unrestricted research grants from Gilead Sciences, Merck, Inc, and Janssen.
K.R.A. has received an educational grant from Gilead Sciences (2018) and served on an advisory board for Gilead Sciences (2020).
Y.C.M. has received research funding from Hologic, Quanterix, Becton-Dickinson, and Ceres. She also received funding support to Johns Hopkins University from miDiagnostics.
C.W.H. has received funding for research from Gilead, Merck, ViiV/GSK, and served on advisory boards for Gilead Sciences, Merck, ViiV/GSK and Population Council.
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