Methodological Issues in Sampling the Local Immune System of the Female Genital Tract in the Context of HIV Prevention Trials

Abstract

Citation Jespers V, Francis SC, van de Wijgert J, Crucitti T. Methodological issues in sampling the local immune system of the female genital tract in the context of HIV prevention trials. Am J Reprod Immunol 2011; 65: 368–376 The spread of HIV continues unabated in the most vulnerable populations of the world. HIV prevention methods, such as a vaginal microbicide, a mucosal vaccine, pre‐exposure prophylaxis or a vaccine, are urgently needed in the fight against new infections. We must make a commitment to supporting innovative research and product design, so that one or more of these products provide a halt to the spread of HIV. Above all, these products should be proven to be safe and not negatively disturb the local immune system in a way that facilitates or enhances heterosexual transmission of HIV. HIV specific and non specific cellular and humoral local vaginal immunity must be assessed in clinical trials when testing prevention products for safety or efficacy. A proven, well‐documented and standardized sampling strategy will provide high quality data to be able to assess both safety and local immune responses. In this paper, we will discuss methods for vaginal immunology sampling in the context of clinical trials.

Characteristics of HIV target CD4 T cells collected using different sampling methods from the genital tract of HIV seronegative women

“…Consistent with the distinct FGT compartments sampled by CVL and CB (cervicovaginal versus endocervical), we observed no association in CD4 T cell yields between the two methods ( Fig 1B ). In addition, the cellular yields from the CB samples in our study were comparable or higher than those described in other studies where CVLs were not collected prior to CB [12, 13], indicating that CVL followed by CB represents a feasible sampling strategy for assessing T cell immunobiology of the genital mucosa. In a comparison with CB samples collected without preceding FS from 6 women over 12 visits, we noted that CB yields obtained were not significantly reduced by a prior FS ( S1 Fig ).…”

“…Previous studies have used minimally-invasive methods, such as CVL [26], endocervical flocked swabs [13, 27], cytobrushes [7], and menstrual cup [28], for cellular characterization of the FGT, while others have used more invasive cervical or endometrial biopsies [7, 29] in order to attain sufficient cellular yields, but few studies exist that compare multiple minimally-invasive methods. In this study, we sought to longitudinally characterize and compare T cell yields and phenotypes using 3 minimally-invasive FGT sampling methods.…”

“…Sexual, reproductive, medication, and genitourinary symptom histories were collected at each study visit. Cervicovaginal specimens were collected by the same clinician for all participants during speculum pelvic examination in the following distal to proximal order to reduce any potential effect of one collection method on the subsequent[13]: 1) CVL with 10ml phosphate buffered saline (PBS) directed toward the cervical os i.e., the opening in the center of the ectocervix, and vaginal walls x 1 minute lavage and placed in 5mL human AB serum (Atlanta Biologicals), 2) two sequential endocervical FS (COPAN Innovation, Italy) placed in 20mL complete media (RPMI -1640, penicillin-streptomycin, L-glutamine (Gibco) with 10% heat inactivated fetal bovine serum [FBS]), 3) two sequential endocervical CB collected in 20mL complete media each. Specimens were collected and immediately transported on ice for real-time processing and immune phenotyping.…”

Differences in the concentration and correlation of cervical immune markers among HPV positive and negative perimenopausal women

“…The ratio of immune marker concentration to total protein for each specimen was created to minimize differences in collection across individuals and then this ratio was log-transformed to reduce the effect of outliers and normalize the distribution of values. This normalization procedure has been used in other settings involved in the measurement of local immune markers related to other sexually transmitted diseases such as HIV-1 [14,15], and minimizes the observed correlation of immune marker concentration and specimen weight (data not shown). Therefore, immune marker concentrations are expressed as [ng of immune marker]/[mg of total protein].…”

Comparison of normalization methods for measuring immune markers in cervical secretion specimens

“…Different sampling methods have been employed to measure immunologic markers including cervical or cervicovaginal swabs and direct collection of cervical secretions using absorbent sponges such as Weck-cell, sno-strips, or opthalamic sponges (Jespers et al, 2011). Sponges generally have higher concentrations of measured markers compared to other diluted specimen types such as cervicovaginal lavage, but are subject to a high degree of inter-individual variability in the amount of specimen collected (Quesnel et al, 1997; Snowhite et al, 2002).…”