Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C.

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at -20 degrees C. DBS were created by spotting 50-microl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at -20 degrees C or at 37 degrees C with approximately 85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at -20 degrees C, and 398 stored at 37 degrees C. A chi-square test showed fewer positive reactions for DBS stored at 37 degrees C (55%) than for those stored at -20 degrees C (78%) (P < 0.0001). Samples stored at -20 degrees C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37 degrees C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37 degrees C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.

Upregulation of human cytomegalovirus by HIV type 1 in human lymphoid tissue ex vivo.

HIV-1 copathogens are believed to play a critical role in progression to AIDS. Human cytomegalovirus (HCMV) has a high prevalence in the general population and is a common copathogen in HIV-1-infected individuals. Important events in copathogen interactions with HIV-1 take place in lymphoid tissue where critical events in HIV-1 disease occur. Here, we used an experimental system of human lymphoid tissue ex vivo to investigate interactions of HCMV with HIV-1. We inoculated ex vivo blocks of human lymphoid tissue with a recombinant strain of HCMV, expressing the green fluorescent protein, and HIV-1 and monitored viral replication and the phenotype of productively infected cells. HCMV readily replicated in tissue blocks as revealed by the release of HCMV viral DNA and an increasing number of viral-positive cells. Immunophenotyping of HCMV-infected cells showed a preferential infection of activated lymphocytes. The number of these cells significantly increased in HIV-1-coinfected tissues. Accordingly, HCMV replication was enhanced 2- to-3 fold. This upregulation occurred in tissues infected with either CXCR4- or CCR5-utilizing HIV-1. Thus, HIV-1 creates new targets for HCMV, which may explain the strong association of HCMV with HIV-1 infection in vivo. Ex vivo-infected human lymphoid tissue constitutes a model to study the mechanisms of HCMV tissue pathogenesis and its interactions with HIV-1 and this model may provide new targets for anti-HIV-1 therapy.

Systemic and mucosal differences in HIV burden, immune, and therapeutic responses.

BACKGROUND: Mucosal tissues represent major targets for HIV transmission but differ in susceptibility and reservoir function by unknown mechanisms. METHODS: In a cross-sectional study, HIV RNA and infectious virus were compared between oral and genital compartments and blood in HIV-infected women, in association with clinical parameters, copathogens, and putative innate and adaptive HIV inhibitors. RESULTS: HIV RNA was detectable in 24.5% of women from all 3 compartments, whereas 45% had RNA in only 1 or 2 sites. By comparison, infectious HIV, present in blood of the majority, was rare in mucosal sites. Innate mediators, secretory leukocyte protease inhibitor and thrombospondin, were highest in mucosae. Highly active antiretroviral therapy was associated with an 80% decreased probability of shedding. Multivariate logistic regression models revealed that mucosal HIV RNA was associated with higher plasma RNA, infectious virus, and total mucosal IgA, but not IgG. There was a 37-fold increased probability of detecting RNA in both genital and oral specimens (P = 0.008; P = 0.02, respectively) among women in highest versus lowest IgA tertiles. CONCLUSIONS: Mucosal sites exhibit distinct characteristics of infectious HIV, viral shedding, and responses to therapy, dependent upon both systemic and local factors. Of the putative innate and adaptive mucosal defense factors examined, only IgA was associated with HIV RNA shedding. However, rather than being protective, there was a striking increase in probability of detectable HIV RNA shedding in women with highest total IgA.

In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories.

The first product to be clinically evaluated as a microbicide contained the nonionic surfactant nonoxynol-9 (nonylphenoxypolyethoxyethanol; N-9). Many laboratories have used N-9 as a control compound for microbicide assays. However, no published comparisons of the results among laboratories or attempts to establish standardized protocols for preclinical testing of microbicides have been performed. In this study, we compared results from 127 N-9 toxicity and 72 efficacy assays that were generated in five different laboratories over the last six years and were performed with 14 different cell lines or tissues. Intra-assay reproducibility was measured at two-, three-, and fivefold differences using standard deviations. Interassay reproducibility was assessed using general linear models, and interaction between variables was studied using step-wise regression. The intra-assay reproducibility within the same N-9 concentration, cell type, assay duration, and laboratory was consistent at the twofold level of standard deviations. For interassay reproducibility, cell line, duration of assay, and N-9 concentration were all significant sources of variability (P < 0.01). Half-maximal toxicity concentrations for N-9 were similar between laboratories for assays of similar exposure durations, but these similarities decreased with lower test concentrations of N-9. Results for both long (>24 h) and short (<2 h) exposures of cells to N-9 showed variability, while assays with 4 to 8 h of N-9 exposure gave results that were not significantly different. This is the first analysis to compare preclinical N-9 toxicity levels that were obtained by different laboratories using various protocols. This comparative work can be used to develop standardized microbicide testing protocols that will help advance potential microbicides to clinical trials.

In vitro and in vivo: the story of nonoxynol 9.

There is an urgent need to expand the range of interventions to prevent HIV transmission and acquisition, especially those that can be controlled by women. Microbicides, defined as antimicrobial products that can be applied topically for the prevention of HIV and other sexually transmitted infections, may offer one of the most promising preventive interventions, because they could be inexpensive, readily available, and widely acceptable. The first microbial product to be clinically evaluated contained Nonoxynol-9 (nonylpenoxypolyethoxyethanol [N-9]), a nonionic surfactant, as the active agent. This article presents a review of the in vitro, ex vivo, and animal model data on the safety of N-9 and a critical analysis of their predictive power based on the results of multiple safety and efficacy trials.

HIV-inducing factor in cervicovaginal secretions is associated with bacterial vaginosis in HIV-1-infected women.

OBJECTIVE: Certain cervicovaginal lavage (CVL) fluid samples obtained from HIV-1-infected and uninfected women stimulate in vitro HIV-1 replication. This activity, HIV-inducing factor (HIF), changes when CVL fluid is heated. We sought to confirm a previous observation that HIF was associated with bacterial vaginosis (BV). METHODS: HIF was measured in unheated and heated CVL fluid obtained from HIV-1-infected women and compared with the presence of BV by Nugent scores, other genital tract conditions, and cervicovaginal HIV-1 shedding. RESULTS: Among the 295 women studied, 54% of CVL samples had HIF activity and 21% showed heat-stable HIF activity. In adjusted logistic regression, heat-stable HIF was associated with BV (odds ratio [OR]=51.7, 95% confidence interval [CI]: 5.0, 530.7) and with intermediate flora (OR=43.3, 95% CI: 3.6, 521.1); heat-labile HIF was not associated with BV. Neither heat-stable nor heat-labile HIF was associated with other cervicovaginal conditions nor, after controlling for plasma viral load, with genital tract HIV-1 shedding. CONCLUSION: We confirmed the association of HIF with BV and attribute it to the heat-stable component. Heat-stable activity is also associated, although less strongly, with intermediate vaginal flora. We propose that heat-stable HIF is a result of products of BV-associated bacteria.

CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue by selective induction of CC-chemokines.

In infected individuals, human immunodeficiency virus type 1 (HIV-1) exist as a "swarm" of quasi species compartmentalized in tissues where individual viral variants may interact locally. We have used human lymphoid tissue, where the critical events of HIV disease occur, to study local interactions in model HIV-1 binary swarms ex vivo. We infected tissue blocks with binary mixtures consisting either of CCR5-dependent and CXCR4-dependent variants or of 2 dual-tropic HIV-1 variants, of which one is skewed to utilization of CXCR4 and the other of CCR5. HIV-1 variants that use CXCR4 suppress replication of CCR5-dependent HIV-1 variants, whereas CCR5-dependent HIV-1 variants do not affect replication of CXCR4-dependent HIV-1. CC-chemokines that inhibit replication of CCR5-dependent HIV-1 variants were up-regulated by CXCR4-dependent HIV-1, thus possibly contributing to this suppression. Tissue-specific chemokine/cytokine network modulations triggered by individual HIV-1 variants may be an important mechanism of local interactions among HIV-1 quasi species in infected tissue.

Recommendations for the nonclinical development of topical microbicides for prevention of HIV transmission: an update.

The development of methods to prevent HIV infection is critical to curbing the rising epidemic. Topical microbicides represent a potential new strategy for reduction of HIV transmission. The purpose of this article is to update and expand upon the nonclinical recommendations of a previously published document on the development of microbicides prepared by the International Working Group on Microbicides. The nonclinical studies discussed here represent general concepts and regulatory considerations that are pertinent to the development of topical microbicides for prevention or reduction of HIV transmission. Essential early steps in product development include the determination of antiviral activity, cytotoxicity, mechanism of action, pathways to resistance, and cross-resistance to approved drugs. Other parameters to consider include activity against vaginal microflora and pathogens that cause sexually transmitted diseases. Before and during clinical trials, nonclinical data on toxicology and pharmacokinetics should be obtained. Finally, product quality issues, including microbicide formulation characteristics, interaction with other products, and stability, should be addressed.

HIV type 1 and cytomegalovirus coinfection in the female genital tract.

The relationship between human immunodeficiency virus (HIV) type 1 and human cytomegalovirus (CMV) was studied in blood, saliva, and cervicovaginal lavage (CVL) specimens from 33 HIV-1-infected women. An association between HIV-1 RNA and CMV DNA was found in the CVL specimens, which also were tested for cytokine levels. Women with detectable CMV DNA in CVL specimens were more likely to have higher interleukin (IL)-1 beta and IL-8 levels than were women with undetectable CMV DNA in CVL specimens. More than 1 strain of CMV was detected in specimens from 2 patients. These results suggest mechanisms by which CMV coinfection could affect HIV-1 disease progression.

Women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA loads.

Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1-positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120-specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load.

Multicenter evaluation of use of dried blood and plasma spot specimens in quantitative assays for human immunodeficiency virus RNA: measurement, precision, and RNA stability.

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at -70 degrees C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.