Ensuring access to preferred contraceptive methods for women and couples is essential to securing the well-being and autonomy of women.1,2 This aspect is particularly relevant to HIV infection, where family planning reduces the risk of unintended pregnancies among women living with HIV. Although the strategies to prevent mother-to-child HIV transmission have been extremely successful, there is still a limited risk of intrauterine and breast-milk HIV transmission and also a risk of children being orphaned due to premature maternal death that could be prevented by adequate contraception. Among the different methods of contraception, condoms are highly recommended because they have the advantage of providing a physical barrier against sexually transmitted infections, including HIV. However, the effectiveness of condoms as contraceptives is <80%, whereas hormonal contraceptives (HC) have an effectiveness of 94% and 91% for injectable and oral preparations, respectively.1 In addition, compared with condoms, the use of HC is more readily controlled by women, conferring them a degree of choice and autonomy.
In the context of HIV infection, the use of HC has been hotly debated. There is evidence that the use of HC may increase the risk of HIV transmission and acquisition and may accelerate progression of HIV infection in women who are not on antiretroviral therapy.3–12 In addition, compared with HIV-infected women using non-hormonal birth control methods, HIV-infected women on HC had higher HIV loads in genital secretions.13 Pregnancy, a state of heightened female hormone secretion, has also been associated with modest increases both in the risk of HIV acquisition and in HIV loads in the genital tract.14,15 The results of these studies might have been influenced by socioeconomic and behavioral characteristics of the study population that are difficult to separate from those directly generated by the use of HC on the risk of HIV acquisition, transmission, or progression. However, studies of HC in animal models of simian immunodeficiency virus or simian-HIV acquisition also found an increased risk of infection in animals treated with supraphysiologic doses of female hormones.16,17 It should be noted that the effect of hormonal contraceptives on transmission of HIV or other primate lentiviruses and on disease progression remains controversial, with multiple studies failing to demonstrate any associations.4,5,18–23
Human and animal studies sought to elucidate the mechanism(s) that mediate the potential increased risk of HIV infection and disease progression associated with the use of HC. These ultimately found 2 important, non-mutually exclusive potential mechanisms: (1) changes in the female reproductive tract24–28 and (2) attenuation of cell-mediated immunity (CMI).16,17,29–33
Among the HC, depot medoxyprogesterone acetate (DMPA) is one of the most widely used worldwide, including areas of high prevalence of HIV infection such as sub-Saharan Africa and Southeast Asia.34 In the US, women enrolled in the Women's Interagency HIV Study reported roughly equal use of DMPA and oral HC.35 DMPA is administered intramuscularly every 3 months, ensuring better compliance than oral HC, which typically require daily administration. Furthermore, DMPA maintains effectiveness when coadministered with efavirenz, which typically decreases the effectiveness of progestin implants.36 However, DMPA is also commonly used to promote female genital tract infection with simian-HIV and simian immunodeficiency virus in non-human primates taking advantage of its local effects on the female reproductive tract.16 It is also purported to depress CMI through its dual action on progestin and glucocorticoid receptors, which are widely expressed by lymphocytes and other mononuclear cells.37–41
In this study, we investigated the CMI in participants of the AIDS Clinical Trials Group (ACTG) study A5283.42 The goal of the immunologic component of the study was to compare functional CMI against HIV and varicella-zoster virus (VZV), T-cell activation (Tact) and regulation, and inflammatory biomarkers before and after DMPA administration and to examine potential associations of immunologic and inflammatory characteristics with medroxyprogesterone acetate (MPA) plasma concentrations.
SUBJECTS AND METHODS
The study was a 12-week, multicenter, open-label, nonrandomized trial in which a single dose of 150 mg DMPA was administered intramuscularly to nonpregnant, premenopausal HIV-1-infected women ≥15-year old, who did not use DMPA for at least 6 months before enrollment and were on a stable combination antiretroviral regimen (cART) containing lopinavir with ritonavir boost (LPV/r) administered twice daily for at least 12 weeks before enrollment. All subjects had plasma HIV-1 RNA ≤400 copies per milliliter within 30 days of study entry and were required to continue on cART for the 12 weeks of the study. Blood samples for MPA and progesterone concentrations, plasma HIV RNA levels, CD4+ cell counts and immunologic and inflammatory measurements were collected at study entry (week 0, pre-DMPA administration), 4 weeks (putative peak of MPA) and 12 weeks (putative trough of MPA) after DMPA administration.
Processing of Samples for Immunologic Assays
Peripheral blood mononuclear cells (PBMCs) were cryopreserved at the clinical site laboratories following a standardized protocol (http://www.hanc.info/labs/Pages/SOPs.aspx). All laboratories were in good standing with the Immunology Cryopreservation Quality Assurance program.43 Cryopreserved PBMCs were shipped within 7 days of collection to a central repository where cells were stored in liquid N2 until shipment in liquid N2 dewars to the testing laboratory at the University of Colorado Anschutz Medical Center. This procedure ensured optimal viability and functionality of the PBMCs.44 Plasma was separated and cryopreserved in 1 mL aliquots by the processing laboratories and batch shipped on dry ice. All specimens from each subject were tested in the same run to avoid confounders potentially introduced by interassay variability.
Dual-color fluorospot kits for IFNγ and IL2 (Mabtech, Cincinnati, OH) were used as per the manufacturer's instructions. PBMCs were thawed and rested over night. Cells with viability ≥70% before and after resting were added at 250,000 PBMCs/well in 100 μL of RPMI 1640 with glutamine (Gibco, Langley, CA) containing 10% human AB serum (Nabi, Los Angeles, CA) and 1% antibiotics (Gibco, Langley, CA) and stimulated in duplicate wells with HIV-inactivated virions and control (gift of Dr. Jeff Lifson45; 6 μg/mL), VZV-inactivated cell lysate, and mock-infected control prepared as previously described46 at a preoptimized concentration, phytohemagglutinin A (PHA; Sigma, St. Louis, MO; 0.01 μg/mL) or anti-CD3 and anti-CD28 mAb (CD3/CD28; Mabtech; 0.1 μg/mL). After 36 hours at 37°C in a 5% CO2-humidified atmosphere, plates were washed; bound IFNγ was detected with 7-B6-1-FS FITC and bound IL-2 with 11-Biotin. Spots were revealed using a mixture of anti-FITC-Green fluorochrome (IFNγ) and SA-Red fluorochrome (IL-2) and analyzed with an Immunospot II plate reader (CTL, Shaker Heights, OH). Results were reported as mean spot-forming cells (SFC)/105 PBMCs in antigen- or mitogen-stimulated wells after subtraction of the mean SFC in control wells.
Lymphocyte Proliferation Assay
The lymphocyte proliferation assay (LPA) was performed on freshly thawed PBMCs with a viability ≥70% as previously described.47 Stimulants consisted of HIV antigen and control (1.5 μg/mL), VZV antigen and control (0.1 μg/mL), CD3/CD28 (0.1 μg/mL), and PHA (2.5 μg/mL). Results are presented as stimulation indices calculated by dividing the median counts per minute (cpm) in the antigen-stimulated wells, by the median cpm in the control wells.
Flow Cytometry Assays
T-cell subsets were enumerated in freshly thawed cryopreserved PBMCs. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD3-AF488 (Biolegend, San Diego, CA; clone HIT3a), anti CD4-APC/Cy7 (Biolegend; RPA-T4), anti-CD25-PE/Cy7 (Biolegend; BC96), anti-HLA-DR-PerCP/Cy5.5 (Biolegend; L243), anti-CD39-APC (Biolegend; A1), and anti-CD38-PECy7 (Biolegend; HIT2). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, San Jose, CA), and stained with anti-IL10-APC (R & D Systems; 127107), anti-FOXP3-PE (Biolegend; 206D), anti-TGFβ-PerCP/Cy5.5 (Biolegend; TW4-2F8), and anti-IL35-PE (eBioscience, San Diego, CA; ebic6) and analyzed with Guava easyCyte 8HT and FlowJo (Treestar, Ashland, OR). Subsets were expressed as percentages of the parent CD4+ and CD8+ T-cell populations.
IL6, IL8, IL10, IFNγ, and TNFα plasma levels were measured by multiplex bead array and TGFβ by ELISA as previously described.48 The bead array assays used the MILLIPLEX MAP High Sensitivity Human Cytokine Magnetic Bead Panel kit (Millipore, Billerica, MA; HSCYTMAG-60SK) on the Bio-Rad Bio-Plex 200 instrument following the manufacturers' instructions. The lower level of detection (LLOD) was 0.08–1.01 pg/mL and the dynamic range 13–2000 pg/mL. Data were analyzed using Bio-Plex manager 5.0 software (Bio Rad, Hercules, CA) and concentrations were interpolated on the manufacturer's standard curve using PRISM software (Graphpad, La Jolla, CA). The LLOD for the TGFβ ELISA was 31 pg/mL and the dynamic range 31–4000 pg/mL. The optical density (OD) was measured with a Multiscan FC ELISA reader (Thermo Fisher, Waltham, MA) using a 450-nm filter. Test TGFβ concentrations were calculated by interpolating the test ODs on the standard curve built with quantitative controls provided by the manufacturer.
All analyses were conducted on subjects with assay results available for immunologic and inflammatory end points using nonparametric statistical approaches, unless otherwise noted. The effects of hormonal contraceptive MPA on those immunologic markers were evaluated based on intrasubject changes using nonparametric Wilcoxon signed-rank test. Spearman's correlation test was used to assess associations between immunologic biomarkers and associations of immunologic markers against MPA pharmacokinetic parameters. All analyses were conducted using SAS (SAS Institute Inc., Cary, MA).
Characteristics of the Study Population
The study used blood samples from 24 HIV-infected women with a median age of 32 years (Table 1). As per inclusion criteria, all participants had HIV plasma RNA ≤400 copies per milliliter on LPV/r-containing cART. CD4+ cell numbers varied from 326 to 1367 cells per microliter (median = 622 cells/μL) at study entry.
Functional Cell-mediated Immunity of HIV-Infected Women Before and After a Single Dose of DMPA
At entry, women had robust HIV-, VZV-, PHA- and CD3/CD28-stimulated LPA responses (Fig. 1), including median [interquartile ranges (IQRs)] LPA stimulation indices for HIV of 51 (5, 74), for VZV 22 (3, 67), for CD3/CD28 65 (11, 135), and for PHA 105 (33, 166). Using a threshold ≥3 for HIV and VZV and ≥5 for CD3/CD28 and PHA to define positive results, 78% had positive qualitative results for HIV, 77% for VZV, 86% for CD3/CD28 and 90% for PHA. There were no significant quantitative or qualitative changes in the LPA responses after DMPA administration (Fig. 1).
IFNγ fluorospot results at entry showed median (IQR) SFC/105 PBMCs of 83 (4, 152) for HIV, 3 (1, 12) for VZV, 133 (47, 233) for CD3/CD28, and 133 (34, 221) for PHA (Fig. 2A). At 4 and 12 weeks after DMPA administration, HIV, CD3/CD28, and PHA IFNγ SFC did not appreciably change compared with baseline (Fig. 2A). VZV IFNγ SFC did not change from baseline to week 4, but increased at week 12 by a median of 2 SFC/105 PBMC (fold-rise = 1.4, P = 0.007). IL2 SFC were highly correlated with those of IFNγ and showed similar changes or lack thereof over time (Fig. 2B).
CD4+ and CD8+ T-cell Subsets of HIV-Infected Women Before and After a Single Dose of DMPA
Activated T cells were identified by the expression of CD25 and by dual expression of CD38 and HLADR (Table 2). There were small changes in the distribution of activated T-cell subsets in the course of the study including a median decrease of 0.5% in CD4+CD25+% at week 12 that reached statistical significance (from a median of 3.42%–2.84%, P = 0.03); and lesser decreases at week 12 of 0.05% in CD8+CD25+% (from 0.39% to 0.34%; P = 0.08) and 0.7% in CD8+CD38+HLADR+% (from 4.47% to 3.79%, P = 0.06).
Regulatory T-cell subsets were characterized by expression of FOXP3, dual expression of CD25 and FOXP3, CD39, IL10, IL35, and TGFβ (Table 2). At week 4 after DMPA administration, there was a small but statistically significant increase of 0.06% in CD8+FOXP3+% (from 0.85% to 0.91%, P = 0.02). At week 12, there was a statistically significant decrease of 0.5% in CD4+IL35+% (from 1.86% to 1.35%, P = 0.02) and a trend increase of 1.5% in CD4+CD39+% (from 10.1% to 11.6%, P = 0.08).
Soluble Biomarkers of HIV-Infected Women Before and After a Single Dose of DMPA
Pregnancy, which is a state of heightened female hormone secretion, is also characterized by increased production of inflammatory and regulatory cytokines.49 Pro-inflammatory (IL6, IL8, IFNγ, and TNFα) and regulatory cytokines (IL10 and TGFβ) were measured before and after DMPA administration. There were no significant changes over time in plasma concentrations of IL6, IL8, IL10, IFNγ, or TNFα. TGFβ had a small but significant decrease at week 12 (P = 0.04).
Relationship Between MPA Plasma Concentrations and Immunologic Parameters in HIV-Infected Women
Correlation analyses were performed to determine the relationship of MPA Cmax and area under the concentration curve (AUC) with changes in functional immune responses (ratios of post-DMPA over baseline results), T-cell subsets (differences in percentages from baseline to post-DMPA) and/or plasma biomarkers (ratios of post-DMPA over baseline results). High MPA Cmax were associated with increased HIV IFNγ fluorospot fold-rises at week 12 over baseline (r = 0.64, P = 0.003; Fig. 3), CD3/CD28 IFNγ at week 4 (r = 0.51, P = 0.04; Fig. 3), CD4+IL35+% at weeks 4 and 12 (r ≥ 0.51, P ≤ 0.02; Fig. 3), and CD4+TGFβ+% at week 4 (r = 0.44, P = 0.04; Fig. 3). In addition, high MPA AUC was associated with increases in CD4+IL35+% at week 4 and 12 compared with baseline (r = 0.47, P = 0.03 for both; Fig. 3) and with a decrease in the plasma concentration of IL10 (r = −0.46, P = 0.03; Fig. 3).
This exploratory analysis of the effect of DMPA on CMI of HIV-infected women on effective cART failed to show any attenuation of their CMI after a single dose of DMPA. This result was unexpected based on the accumulated evidence that suggested a downregulatory and anti-inflammatory effect of progestins, in general, and of MPA, in particular, on CMI.16,17,29,33,37–40,50–54 In contrast to these studies, we observed an increase in VZV-specific IL2 and IFNγ fluorospot responses after DMPA administration compared with baseline. In addition, we found positive associations of the MPA Cmax with the fold-increase of HIV-specific IFNγ responses from baseline to week 12 and of CD3/CD28-stimulated IFNγ responses from baseline to week 4. A mechanism to explain the association of DMPA administration with the CMI increases that we observed is unclear. Based on previous in-vitro data, it is unlikely that the mechanism involved a direct effect of MPA on the cells of the immune system. However, an indirect effect mediated by the decrease of estrogen secretion in DMPA recipients55 is a potential explanation because of the strong immune-regulatory effect of estrogen.56–66
It is important to note that our study evaluated women on effective cART, a characteristic shared by other studies that failed to detect any detrimental effects of DMPA on HIV disease progression or transmission studies.18,21,67 By contrast, studies that documented detrimental effects of DMPA were generally conducted in the absence of cART.7,8 A stimulatory direct effect of MPA and of other steroidal hormones on HIV replication has been described.68 The use of cART might attenuate the direct effect of MPA on HIV replication, thus contributing to the difference in findings between DMPA studies conducted in the presence or absence of cART. If the deleterious effect of MPA on the CMI of HIV-infected women is indeed mediated by the enhancement of HIV replication, the current trend toward increased cART utilization may improve the safety of DMPA administration to HIV-infected women.
Tact markers decreased after DMPA administration, an effect that was predicted by in-vitro studies showing that MPA decreased dendritic cell and monocyte activation, and production of pro-inflammatory cytokines.30,31,33,39,40 However, we were not able to demonstrate a decrease in proinflammatory cytokine plasma levels. A recent study showed increased Tact in HIV-uninfected women receiving DMPA compared with women using oral or no hormonal contraceptives.69 The divergence may be related to differences in study populations, HIV-infected vs uninfected, and longitudinal vs cross-sectional designs. The longitudinal study design allowed us to use subjects as their own controls, thus avoiding potential biases introduced by differences in exposures to other factors that may promote Tact.
Based on previous studies,53,54 we expected an increase in circulating T-cell regulation (Treg) subsets after DMPA administration. In fact, we observed a correlation between high MPA Cmax and AUC with an increase in CD4+IL35+% and in CD4+TGFβ+% after DMPA administration compared with baseline, which was consistent with a stimulatory effect of MPA on Treg. However, the changes in the proportion of Treg were heterogeneous among study participants, and at the group level, there was no consistent increase in the proportion of Treg over time on DMPA. This suggests that the stimulatory effect of MPA on Treg subsets may be offset by the DMPA-mediated decrease in estrogen, which is also a Treg inducer, perhaps even more potent than MPA.64,65
Our study had limitations and virtues. Because this was an exploratory analysis, we did not adjust for multiple comparisons. This increased our ability to detect changes and generate hypotheses but might have also allowed the introduction of spurious associations. Although the number of participants in this study was limited, their ages, which varied from 15 to 47 years, overlap with the age range of 15–44 years considered by the CDC as representative for the use of contraceptive methods in the US and with previously published demographics of DMPA in HIV-infected users.70,71 The limited number of participants in this analysis was counterbalanced by the longitudinal design using intrasubject comparison approaches, which minimized the introduction of DMPA-unrelated variables. Furthermore, longitudinal samples of each participant were assayed jointly to minimize the effect of interassay variability.
Although our findings need to be confirmed, these preliminary results are encouraging because they support the notion that the effectiveness of DMPA as a contraceptive agent in the context of cART overpowers its potential attenuation of immune defenses in HIV-infected women. Our results also show that the effect of in-vivo administration of hormones have consequences that may not be anticipated from their effect in vitro, probably because of feedback mechanisms that are intact in vivo and change the overall hormonal homeostasis and cannot be readily reproduced in vitro. More studies are needed to evaluate the cumulative effect of multiple doses of DMPA on the immune system of HIV-infected women and its effect on HIV transmission.
The authors are grateful for the patients' commitment and participation in this study. We thank Dr. Karin Klingman for her overall contributions to the A5283 protocol and for critical review of this manuscript. The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the views of the Office of the Global AIDS Coordinator, the National Institutes of Health, or the US Department of State. The following individuals assisted in conducting A5283: Becky Straub, RN, MPH, and Miriam Chicurel-Bayard, RN, BSN—UNC Global CTU: Chapel Hill CRS (Site 3201); Rachel K. Scott, MD, MPH, and Patricia Tanjutco, MD—MedStar WA Hospital Center (Site 5023); Jenny Baer, RN, and Jennifer Forrester, MD—Cincinnati CRS (Site 2401); Mariam Aziz, MD, and Maureen McNichols, RN—Rush University Medical Center/Ruth M. Rothstein CORE Center (Site5083); Mary Adams, RN, and Christine Hurley, RN—University of Rochester CRS (Site 1101); Sheila Dunaway, MD, and Eric Helgeson, RN—University of Washington ACTG CRS (Site1401); Donna McGregor, NP—Northwestern University CRS(Site 2701); Steven Zeichner, MD, and Connie Trexler, RN—Children's National (Site5015); Rodrigo Diaz-Velasco, MD, and Elvia Perez-Hernandez, MPH—San Juan Hospital (Site 5031); Sharon Nachman, MD, Denise Ferraro, FNP, and Erin Infanzon—SUNY Stony Brook NICHD CRS (Site 5040); and Patricia Riley and Sheila Bradford—Tulane University New Orleans NICHD CRS (Site 5095).
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