Objective
To evaluate whether cervicovaginal secretions inhibit HIV-1 infectivity in an in vitro model, and estimate concentration of immune mediators.
Study Design
We enrolled midtrimester pregnant and regularly menstruating (nonpregnant) women. Cervicovaginal lavage was collected at 2 visits and incubated with HIV-1 and TZM-bl cells. Infectivity was compared with positive controls. Concentrations of immune mediators were compared between groups.
Results
At enrollment, cervicovaginal lavage inhibited IIIB virus 88.2% and 82.4%, and BaL virus 72.8% and 77.9%, among pregnant (n = 13) and nonpregnant women (n = 9), respectively. At second visit, cervicovaginal lavage inhibited IIIB 89.7% and 82.5%, and BaL 77.4% and 69.9% among pregnant (n = 15) and nonpregnant women (n = 8), respectively (all P ≤ .04). Adjusting for body mass index, race, and protein content of cervicovaginal lavage, antimicrobials were suppressed but cytokines and chemokines were not markedly different in pregnancy.
Conclusion
Cervicovaginal secretions significantly suppress HIV-1 infectivity in this model. Concentrations of certain immune mediators are altered in pregnancy.
Women account for half of all people living with HIV-1 globally and for 60% of those in sub-Saharan Africa. This figure represents a significant overall increase in the number of adult women infected with HIV globally since 2001. The vast majority of incident HIV worldwide is caused by heterosexual intercourse. The lower genital tract is one of the most susceptible areas in the body for HIV acquisition. During vaginal intercourse, women are twice as likely to contract HIV from their male partner as men are from a female partner. As more women of reproductive age become HIV-infected, they may become pregnant and are at risk of transmitting HIV to their fetus. As a result of infection in women, there are now nearly 2 million children living with HIV, the vast majority of these perinatally infected.
A large, rigorous study following over 10,000 women done in Rakai, Uganda, found that women were at significantly increased risk of HIV acquisition during pregnancy. Data from this community cohort with longitudinal data were analyzed for the incidence rate of HIV during pregnancy, and compared with the incidence rate during periods of nonpregnancy. The incidence rate was 2.3 per 100 person years in pregnancy as compared with 1.1 per 100 person years in nonpregnant women. This difference in incidence rates resulted in an incident rate ratio of HIV acquisition in pregnancy of 2.16 (95% confidence interval, 1.39–3.37) after adjusting for age, marital status, education, number of sex partners, genital ulcer disease, and condom use. The data are not entirely consistent, however, as several smaller studies have not demonstrated such an association.
The biologic reasons for a potentially increased risk of HIV acquisition during pregnancy have not been elucidated. It has been suggested that mucosal immunity in the genital tract is compromised during pregnancy. Concentrations or expression of certain antimicrobial peptides, cytokines, and chemokines have been shown to be altered under certain conditions in pregnancy, such as bacterial vaginosis, trichomoniasis, or premature rupture of membranes.
A number of studies have examined the use of a TZM-bl indicator assay as an in vitro surrogate of HIV-1 infectivity. This infectivity assay is the World Health Organization (WHO) preferred infectivity assay and is commonly used in HIV vaccine research. It is considered to be more standardized than traditional peripheral blood mononuclear cell (PBMC) infectivity assays. The assay has been studied to measure the impact of genital tract secretions on prevention of HIV infectivity but its performance testing cervicovaginal lavage (CVL) from pregnant women has not been explored. Our purpose in this study was 3-fold. First, we sought to assess whether CVL fluid would suppress HIV-1 infection of target cells differentially in pregnant and nonpregnant women, second, to evaluate whether protective immune mediator concentrations were altered in pregnancy, and third, determine whether cytokines, chemokines and anti-HIV molecules results vary when expressed per unit volume vs per unit protein.
Materials and Methods
We enrolled HIV-negative pregnant and nonpregnant women between the ages of 18 and 35 presenting for care at our tertiary care institution. Pregnant women were offered enrollment if they were between 14 and 26 weeks’ gestation as determined by best obstetric estimate. Nonpregnant women were offered enrollment if they had regular menses for the previous 3 months. Exclusion criteria were pregestational diabetes mellitus, chronic hypertension requiring medications, antibiotic use within 2 weeks of specimen collection, use of hormonal contraception, current or planned cerclage, planned termination of pregnancy, known fetal anomalies, or symptomatic vaginal discharge requiring doctor visit within 2 weeks of enrollment.
All participants signed written, informed consent. The study was approved by the Women and Infant’s Hospital Institutional Review Board on Oct. 6, 2008, protocol number 08-0115. At enrollment, baseline data were collected, including demographic information, basic medical and obstetric risks, and vaginal practices. All women underwent CVL collection performed in a standard manner. The 10 mL normal saline was instilled into the vaginal cavity with the stream directed toward the external os of the cervix. The fluid was allowed to pool in the posterior fornix, and then aspirated. At second study visits, CVL was collected in the same manner.
Pregnant women were in the third trimester at the time of follow-up. Nonpregnant women were enrolled during the proliferative phase of the menstrual cycle and follow-up was performed when they were periovulatory. On the same day as collection, CVL was centrifuged at 1500 g for 10 minutes and the supernatant was frozen at −80°C until used in the TZM-bl assay.
The HIV-1 strains used, IIIB (×4), a virus that infects via the CXCR4 coreceptor and BaL (R5), which infects via the CCR5 coreceptor, thought to be a more common viral coreceptor for sexual transmission were kindly provided by Dr P. Gupta (University of Pittsburgh, Pittsburgh, PA). Virus stocks were propagated in PHA-stimulated human PBMC and stored frozen at −80°C. Details on this assay have been previously described.
The light intensity of each well was measured using a luminometer and expressed as relative light units (RLU). Uninfected cells and cells incubated with CVL only were used to determine background luminescence. HIV-1 incubated in media alone before adding it to the TZM-bl cells was used as positive control. TZM-bl cells were incubated with secretions alone and media alone were used a negative controls and determination of background values. Viability of TZM-bl cells on treatment with CVL was quantified using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison,WI) according to manufacturer’s instructions.
The RLU were expressed as median values, percent inhibition as compared with virus-only positive control set at 100%, and after adjustment for background luminescence. Comparisons were made between pregnant and nonpregnant groups by Wilcoxon rank sum test, and each group was tested against the positive control. The van Elteren test was used to compare medians adjusting for body mass index (BMI) and race. Statistical significance was considered P < .05.
Antimicrobial peptides, cytokines, and chemokines that have been previously shown to have an impact on HIV infectivity were measured in the CVL of pregnant and nonpregnant women. Secretory leukocyte protease inhibitor (SLPI), macrophage inflammatory protein-3-alpha (MIP)-3α, and elafin were measured using enzyme linked immunosorbent assay (ELISA) kits from R & D Systems (Minneapolis, MN) according to manufacturer’s instructions. Standards for each ELISA were resuspended in phosphate buffered saline (PBS). Samples were diluted in 1× PBS. Antimicrobials were quantified based on standard curves obtained using an ELISA reader (Dynex, Chantilly, VA). Human beta defensin (HBD) 2 was assayed using ELISA test kit from PeproTech (Rocky Hill, NJ) according to manufacturer’s instructions. CVL were assayed for 14 different chemokines and cytokines (BioRad, Hercules, CA), using a multiplex bead assay (Luminex Corp., Austin, TX) as previously described. Total protein concentration in each CVL sample was determined using the BCA Protein Assay kit from Fisher Scientific (Hampton, NH), according to manufacturer’s instructions. Concentration of each molecule was expressed as picograms/milliliter as well as per 10 μg of protein and compared between pregnant and nonpregnant women. The van Elteren test was used to compare concentrations between groups after controlling for BMI and race.
Results
A total of 31 subjects were enrolled in the study, 20 pregnant and 11 nonpregnant. Thus, we obtained 13 usable samples from women pregnant in the second trimester, 15 in the third trimester, and 17 samples from nonpregnant women at various stages of the menstrual cycle. In the latter group, sample numbers were too low to allow cycle dependent stratification. Pregnant women were slightly older, but of similar race, with the majority being white. Nearly all participants had completed high school. Pregnant women were more likely to be married ( Table 1 ). Using the TZM-bl assay, we investigated whether anti-HIV activity in CVL is similar in pregnancy as we have shown in nonpregnant women. As seen in Tables 2 and 3 , when compared with nonpregnant samples, CVL from pregnant subjects collected at enrollment and second visits markedly suppressed infectivity of both X4 and R5 viral strains. These studies indicated further that inhibitory capacity was slightly lower, but not dramatically so, in the presence of IIIB ( Figure , A and C) and BaL ( Figure , B and D) virus in CVL collected at enrollment or at return visit. Within the limits of the sample size, no difference was detected in infectivity between visits for a given viral strain, or for viral inhibition between pregnant and nonpregnant women (not shown).
| Characteristic | Pregnant (n = 13) | Nonpregnant (n = 9) |
|---|---|---|
| Age, y | 24 (18–33) | 21 (18–35) |
| Gestational age, wk | 23.5 (15–26) | Not applicable |
| Race | ||
| Black/African-American | 2 (11.1) | 2 (18.2) |
| White | 12 (66.7) | 7 (63.6) |
| Other/more than 1 race | 4 (22.2) | 2 (18.2) |
| Insurance | ||
| Private | 6 (30.0) | 6 (54.6) |
| Medicaid | 8 (40.0) | 0 |
| Other | 4 (20.0) | 0 |
| Uninsured | 2 (10.0) | 5 (45.5) |
| Marital status | ||
| Single | 10 (50.0) | 9 (81.8) |
| Married | 10 (50.0) | 2 (18.2) |
| Education level | ||
| < High school | 2 (10.0) | 0 |
| High school/equivalent | 8 (40.0) | 1 (9.1) |
| Some college | 8 (40.0) | 8 (72.7) |
| College graduate | 2 (10.0) | 2 (18.2) |
| Employment | ||
| Unemployed | 8 (40.0) | 2 (18.2) |
| Employed full-time | 6 (30.0) | 4 (36.4) |
| Employed part-time | 5 (25.0) | 5 (45.5) |
| Other | 1 (5.0) | 0 |
| Income level | ||
| <$10,000 | 2 (11.1) | 4 (36.4) |
| $10,000-24,999 | 8 (44.4) | 1 (9.1) |
| $25,000-49,999 | 4 (22.2) | 3 (27.3) |
| ≥$50,000 | 4 (22.2) | 3 (27.3) |
| Body mass index, kg/m 2 | 25.9 (20.1–40.5) | 24.4 (20.6–39.8) |
| Variable | Pregnant (n = 13) Median (range) | P value a | Nonpregnant (n = 9) Median (range) | P value a |
|---|---|---|---|---|
| IIIb % inhibition | 88.2% (21.3–99.1) | .0002 | 82.4% (−10.4 to 93.9) | .02 |
| BaL % inhibition | 72.8% (−1.7 to 95.7) | .0005 | 77.9% (−7.2 to 91.3) | .04 |
a P value for comparison to no inhibition (positive control) by signed rank test.
| Variable | Pregnant (n = 15) Median (Range) | P value | Nonpregnant (n = 8) Median (range) | P value |
|---|---|---|---|---|
| IIIb % inhibition | 89.7% (−19.1 to 98.3) | .0002 | 82.5% (−1.8 to 91.1) | .02 |
| BaL % inhibition | 77.4% (−11.1 to 97.5) | .004 | 69.9% (−10.3 to 83.1) | .04 |
To understand whether immune parameters in CVL change with pregnancy, an analysis was carried out in which CVL from nonpregnant and pregnant women were analyzed for antimicrobial, cytokine, and chemokine levels. As seen in Tables 4 and 5 , data are expressed both as pg/mL ( shaded boxes ) and pg/10 μg protein ( clear boxes ). Data are presented in both ways owing to our finding of an elevation in protein concentration that occurs in CVL during pregnancy. Overall protein concentration was significantly higher among pregnant women, median 139.8 μg/mL (range, 48.3–514.2) in CVL compared with 22.6 μg/mL (range, 1.0–1139.5) ( P = .03) in CVL from nonpregnant women after controlling for race and BMI.
| Variable | Pregnant (n = 13) Median (range) | Nonpregnant (n = 9) Median (range) | P value a | P value b |
|---|---|---|---|---|
| Gestational age, wks | 23.5 (15–26) | |||
| RANTES | ||||
| pg/mL | 6.0 (4.2–92.2) | 5.1 (4.2–17.9) | .03 c | .05 |
| pg/10μg protein | 0.8 (0.2–2.8) | 1.9 (0.2–50.6) | .1 | .1 |
| Eotaxin | ||||
| pg/mL | 28.4 (0–136.1) | 18.0 (0–92.3) | .2 | .1 |
| pg/10μg protein | 2.5 (0–6.7) | 8.1 (0–66.7) | .06 | .2 |
| Fractalkine | ||||
| pg/mL | 86.7 (44.2–1475.7) | 44.2 (44.2–351.1) | .1 | .09 |
| pg/10μg protein | 8.5 (1.6–37.4) | 29.7 (1.8–440.2) | .1 | .09 |
| G-CSF | ||||
| pg/mL | 12.1 (3.5–2795.6) | 6.7 (3.5–24447.5) | .1 | .1 |
| pg/10μg protein | 1.7 (0.5–73.9) | 7.2 (0.7–214.5) | .2 | .6 |
| IL-1α | ||||
| pg/mL | 56.2 (9.9–1812.2) | 6.5 (2.7–1838.5) | .02 c | .03 c |
| pg/10μg protein | 4.0 (0.8–35.2) | 4.4 (1.9–26.9) | .9 | .7 |
| IL-1RA | ||||
| pg/mL | 24588.8 (1991.5–84614.6) | 776.4 (0.1–57124.8) | .03 c | .03 c |
| pg/10μg protein | 1283.0 (289.3–5908.1) | 405.1 (0.3–2133.9) | .06 | .2 |
| IL-6 | ||||
| pg/mL | 6.3 (4.5–103.9) | 4.9 (4.0–751.6) | .2 | .2 |
| pg/10μg protein | 0.9 (0.3–3.5) | 4.4 (0.8–39.8) | .01 c | .03 c |
| IL-8 | ||||
| pg/mL | 243.4 (0–4443.1) | 32.9 (0–36052.4) | .3 | .3 |
| pg/10μg protein | 13.0 (0–143.5) | 6.3 (0–316.4) | .7 | .9 |
| IP-10 | ||||
| pg/mL | 165.0 (0–933.5) | 0 (0–2970.4) | .2 | .1 |
| pg/10μg protein | 10.1 (0–132.3) | 0 (0–27.0) | .1 | .1 |
| MCP-1 | ||||
| pg/mL | 18.2 (4.2–94.6) | 8.5 (4.2–890.5) | .8 | .5 |
| pg/10μg protein | 0.9 (0.6–2.6) | 7.4 (2.0–48.2) | .002 c | .001 c |
| MIP-1α | ||||
| pg/mL | 30.2 (13.4–108.2) | 19.6 (2.6–455.0) | .3 | .2 |
| pg/10μg protein | 2.2 (0.9–8.3) | 7.8 (1.2–176.0) | .04 c | .09 |
| MIP-1α | ||||
| pg/mL | 20.4 (0–585.3) | 7.3 (0–660.8) | .3 | .1 |
| pg/10μg protein | 1.9 (0–11.4) | 2.4 (0–72.7) | .6 | .6 |
| TNF-α | ||||
| pg/mL | 0 (0–145.8) | 0 (0–13.0) | .3 | .1 |
| pg/10μg protein | 0 (0–2.8) | 0 (0–0.1) | .2 | .1 |
| GM-CSF | ||||
| pg/mL | 0.2 (0–91.8) | 0 (0–6.1) | .2 | .3 |
| pg/10μg protein | 0.1 (0–1.8) | 0 (0–1.2) | .1 | .1 |
| Elafin | ||||
| pg/mL | 32310 (7510–79160) | 35930 (12270–89800) | .7 | .5 |
| pg/10μg protein | 3749.1 (146.1–7503.0) | 25718.6 (162.5–181990.0) | .02 c | .03 c |
| SLPI | ||||
| pg/mL | 58426 (0–224000) | 18304 (3404–230000) | .4 | .4 |
| pg/10μg protein | 2790.8 (0–21060.9) | 9500.4 (1842.9–77655.4) | .06 | .07 |
| HBD2 | ||||
| pg/mL | 710 (100–8220) | 1110 (30–9650) | .6 | .3 |
| pg/10μg protein | 35.1 (8.8–1164.9) | 211.1 (39.3–4265.4) | .03 c | .03 c |
| MIP-3α | ||||
| pg/mL | 24 (4–660) | 40 (4–3420) | > .99 | .8 |
| pg/10μg protein | 1.4 (0.1–24.5) | 22.5 (0.8–42.7) | .01 c | .04 c |
a P value by Wilcoxon rank sum test for difference between groups;
b P value by van Elteren test for difference between groups, adjusting for overweight BMI and white race;
| Variable | Pregnant (n = 15) Median (range) | Nonpregnant (n = 8) Median (range) | P value a | P value b |
|---|---|---|---|---|
| Gestational age, wk | 34 (28–38) | — | — | — |
| RANTES | ||||
| pg/mL | 6.0 (4.2–47.2) | 5.1 (4.2–28.3) | .2 | .1 |
| pg/10μg protein | 0.9 (0.1–2.1) | 3.1 (1.3–42.3) | .004 c | .01 c |
| Eotaxin | ||||
| pg/mL | 20.9 (0–133.6) | 17.1 (0–48.9) | .4 | .3 |
| pg/10μg protein | 2.9 (0–12.6) | 6.0 (0–41.1) | .04 c | .1 |
| Fractalkine | ||||
| pg/mL | 44.2 (0–625.2) | 44.2 (0–360.4) | .8 | .2 |
| pg/10μg protein | 9.0 (0–22.5) | 37.1 (0–451.4) | .02 c | .1 |
| G-CSF | ||||
| pg/mL | 8.5 (0–14202.9) | 6.7 (3.5–54.2) | .1 | .3 |
| pg/10μg protein | 1.2 (0–205.3) | 4.5 (1.1–67.9) | .08 | .5 |
| IL-1α | ||||
| pg/mL | 49.1 (5.9–2193.1) | 6.9 (2.7–41.3) | .01 c | .01 c |
| pg/10μg protein | 2.7 (0.7–31.7) | 4.3 (0.7–33.8) | .7 | .3 |
| IL-1RA | ||||
| pg/mL | 40898.3 (408.4–85612.1) | 442.7 (0–14440.9) | .002 c | .01 c |
| pg/10μg protein | 3012.9 (161.5–7003.0) | 272 (0–5929.3) | .01 c | .01 c |
| IL-6 | ||||
| pg/mL | 4.5 (2.9–70.8) | 4.2 (3.5–29.9) | .5 | .3 |
| pg/10μg protein | 0.8 (0.1–2.2) | 3.3 (1.2–40.8) | .006 c | .04 c |
| IL-8 | ||||
| pg/mL | 118.0 (0–4100.3) | 25.8 (0–778.1) | .1 | .4 |
| pg/10μg protein | 11.4 (0–281.9) | 13.7 (0–104.3) | .8 | .7 |
| IP-10 | ||||
| pg/mL | 165.0 (0–2141.8) | 0 (0–144.3) | .06 | .01 c |
| pg/10μg protein | 8.0 (0–82.9) | 0 (0–19.4) | .1 | .09 |
| MCP-1 | ||||
| pg/mL | 7.6 (2.8–103.0) | 6.5 (4.2–20.9) | .7 | .7 |
| pg/10μg protein | 0.8 (0.1–2.7) | 3.3 (1.3–42.4) | .006 c | .008 c |
| MIP-1α | ||||
| pg/mL | 30.2 (0–103.5) | 21.1 (0–66.6) | .2 | .2 |
| pg/10μg protein | 3.9 (0–17.3) | 8.7 (0–77.3) | .09 | .3 |
| MIP-1α | ||||
| pg/mL | 13.6 (0–267.8) | 10.5 (0–103.3) | .4 | .09 |
| pg/10μg protein | 2.4 (0.1–7.7) | 7.7 (0–13.8) | .2 | .6 |
| TNF-α | ||||
| pg/mL | 0 (0–59.9) | 0 (0–23.2) | > .99 | .2 |
| pg/10μg protein | 0 (0–1.6) | 0 (0–3.1) | .8 | .8 |
| GM-CSF | ||||
| pg/mL | 1.2 (0–27.6) | 0 (0–5.6) | .2 | .04 c |
| pg/10μg protein | 0.2 (0–1.0) | 0 (0–0.8) | .5 | .06 |
| Elafin | ||||
| pg/mL | 29390 (12270–81700) | 28405 (18240–54900) | .5 | .6 |
| pg/10μg protein | 3025.2 (186.9–12345.1) | 15375 (4184.5–186122.4) | .02 c | .04 c |
| SLPI | ||||
| pg/mL | 31616 (0–68672) | 22759 (6586–98452) | > .99 | .5 |
| pg/10μg protein | 2551.5 (0–21898.6) | 11764.9 (2069.4–146571.4) | .02 c | .1 |
| HBD2 | ||||
| pg/mL | 900 (10–8740) | 640 (80–5140) | .8 | .8 |
| pg/10μg protein | 36.8 (3.5–1239.4) | 451 (69.5–2110.4) | .03 c | .004 c |
| MIP-3α | ||||
| pg/mL | 4 (4–104) | 16 (4–5140) | .1 | .2 |
| pg/10μg protein | 1.6 (0.1–15.3) | 16.3 (1.1–2110.4) | .01 c | .04 c |
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