Drug synergy of tenofovir and nanoparticle-based antiretrovirals for HIV prophylaxis.

BACKGROUND: The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV). METHODOLOGY/PRINCIPAL FINDINGS: Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d ∼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC50) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI50 = 0.07) at a 1∶50 ratio of IC50 values and additive effects (CI50 = 1.05) at a 1∶1 ratio of IC50 values. TFV combined with NP-SQV at a 1∶1 ratio of IC50 values also showed strong synergy (CI50 = 0.07). CONCLUSIONS: ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.

Drug-eluting fibers for HIV-1 inhibition and contraception.

Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract.

Cross-reactive T cell responses in HIV CRF01_AE and B'-infected intravenous drug users: implications for superinfection and vaccines.

Abstract We previously observed limited cross-reactive T cell responses in two HIV-1-superinfected injection drug users (IDUs) before superinfection [Ramos A, et al.: J Virol 2002;76(15):7444-7452]. To elucidate the role of such responses in superinfection we examined cross-reactive T cell responses in IDUs infected with a single HIV-1 subtype. In this study, IFN-gamma ELISPOT assays were performed using recombinant vaccinia constructs and peripheral blood mononuclear cells (PBMCs) from 43 IDUs singly infected with CRF01_AE or B' from the same cohort as the superinfected IDUs. PBMCs were from time points corresponding to pre- (early) or post- (late) superinfection in the superinfected IDUs. We observed that most singly infected IDUs had cross-reactivity in samples from early (84% of CRF01_AE and 78% of B'-infected IDUs) and late (96% of CRF_01AE and 77% of B'-infected IDUs) time points. Frequent homologous reactivity at early (67% of CRF-01AE and 100% of B') and late (84% of CRF01_AE-infected and 100% of B'-infected IDUs) time points was also observed. Cross-reactive responses were predominantly to Pol and were broader and higher in CRF01_AE than in B'-infected IDUs (medians of 825 vs. 90 and 585 vs. 60 spot-forming units/10(6) PBMCs at early and late time points, respectively). Our results show that cross-reactive responses were more prevalent with greater height and breadth in singly infected IDUs than previously observed in corresponding collection time points of superinfected IDU. Thus, low or absent cross-reactivity may have contributed to the previously observed superinfections. These data are relevant for understanding superinfection and improving vaccine design.

Increase in sexual risk behavior and prevalence of Chlamydia trachomatis among adolescents in Northern Thailand.

BACKGROUND: Monitoring changes in adolescent sexual risk behaviors and sexually transmitted infections is critical for evaluating the effectiveness of human immunodeficiency virus and other prevention programs, but population-based data on adolescents in Thailand are limited. We report findings from 2 cross-sectional surveys conducted in 1999 and 2002 among 15-to 21-year-old vocational students. METHODS: In 1999 and 2002, 1725 and 966 students, respectively, were interviewed using computer-assisted self-interview methods. Urine samples were collected and tested for Chlamydia trachomatis and Neisseria gonorrhoeae by polymerase chain reaction. RESULTS: From 1999 to 2002 C. trachomatis prevalence increased from 3.2% to 7.5% (P <0.001) in women and from 2.5% to 6.0% (P <0.001) in men. There was an increase in the reported mean lifetime number of steady sexual partners among both men (3.4-4.7, P = 0.01) and women (2.5-3.3, P <0.001), and in the mean lifetime number of casual partners among men (1.1-2.1, P <0.001) and women (0.3-1.1, P = 0.04). Reported consistent condom use decreased significantly among women with casual partners (43%-19%, P = 0.03) but not among men (25%-31%, P = 0.31). CONCLUSIONS: Our study identified important increases in the prevalence of chlamydial infection and in sexual risk behaviors among Thai adolescents over a 3-year period. These findings are consistent with other studies suggesting profound social changes are changing norms of adolescent sexual behavior in Thailand, and highlight the need for adolescent sexual health services and prevention programming.

A randomized, placebo-controlled trial to assess the safety and acceptability of use of carraguard vaginal gel by heterosexual couples in Thailand.

OBJECTIVES: To determine the safety and acceptability of use of Carraguard, a carrageenan-derived candidate microbicide gel, during sexual intercourse in women and men. STUDY DESIGN: We conducted a 6-month randomized, placebo-controlled trial among sexually active, couples at relatively lower risk for HIV infection in northern Thailand. METHODS: Women inserted 1 applicator of study gel vaginally every time the couple had sex. Safety was assessed by symptom report and genital examination of both partners and by changes in vaginal flora. Acceptability was assessed by participant interview. RESULTS: Overall, 55 couples were randomized, 28 to Carraguard use and 27 to the methyl-cellulose placebo gel group. Retention and study gel use were similarly high in both study groups; use of gel without condoms was reported in more than 95% of vaginal sex acts. The 2 study groups were similar in the proportions of women and men with symptoms or with genital findings without epithelial disruption, of men with findings with epithelial disruption, and of women with abnormal genital flora, whereas more women in the placebo group had findings with epithelial disruption. Women and men in both groups reported that the gel and applicator were acceptable. CONCLUSIONS: Carraguard can safely be used an average of 2 to 3 times per week during sex and is acceptable to Thai women and men.

Infection with hepatitis C virus among HIV-infected pregnant women in Thailand.

OBJECTIVE: The purpose of this study was to describe the epidemiology of coinfection with hepatitis C virus (HCV) and HIV among a cohort of pregnant Thai women. METHODS: Samples from 1771 pregnant women enrolled in three vertical transmission of HIV studies in Bangkok, Thailand, were tested for HCV. RESULTS: Among HIV-infected pregnant women, HCV seroprevelance was 3.8% and the active HCV infection rate was 3.0%. Among HIV-uninfected pregnant women, 0.3% were HCV-infected. Intravenous drug use by the woman was the factor most strongly associated with HCV seropositivity. Among 48 infants tested for HCV who were born to HIV/HCV coinfected women, two infants were HCV infected for an HCV transmission rate of 4.2% (95% 0.51-14.25%). CONCLUSIONS: HCV seroprevalence and perinatal transmission rates were low among this Thai cohort of HIV-infected pregnant women.

Human papillomavirus (HPV) detection among human immunodeficiency virus-infected pregnant Thai women: implications for future HPV immunization.

BACKGROUND: Human immunodeficiency virus (HIV)-infected women are at increased risk for developing cervical cancer and for infection with human papillomavirus (HPV). Prophylactic vaccines targeting HPV types 16 and 18 are being evaluated for efficacy among young women. GOAL: The goal was to assess the prevalence of HPV among HIV-infected pregnant women in Bangkok and to evaluate the need for prophylactic HPV vaccines studies in this population. STUDY DESIGN: The study population consisted of 256 HIV-infected pregnant women who participated in a mother-to-child HIV transmission trial. Stored cervicovaginal lavage samples were tested for the presence of HPV DNA by polymerase chain reaction with PGMY09/11 primers and reverse line-blot hybridization for determination of anogenital HPV types. RESULTS: HPV prevalence was 35.5% (91/256); high-risk HPV prevalence was 23.4% (60/256). HPV type 16 or 18 was present in 8.2% (21/256). Almost half of all infections were multiple. Furthermore, overall HPV detection was associated with abnormal cervical cytology (P<0.001) and higher HIV-plasma viral load (P=0.007). CONCLUSIONS: Only one-quarter of HIV-infected pregnant women in Bangkok had high-risk HPV types; less than 10% had HPV types 16 or 18. As the HPV prevalence is expected to increase during HIV disease, prophylactic vaccines targeting HPV types 16 and 18 should be studied among HIV-infected women not yet infected with these HPV types and not previously exposed.

Evaluation of a new single-parameter volumetric flow cytometer (CyFlow(green)) for enumeration of absolute CD4+ T lymphocytes in human immunodeficiency virus type 1-infected Thai patients.

Use of the standard dual-platform flow cytometric method for determination of CD4(+) T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4(+) T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlow(green) (Partec), a single-parameter SP volumetric FCM. The performance of CyFlow(green) was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4(+) and CD8(+) T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlow(green) were compared with those obtained with the bead-based three-color TruCOUNT system (R(2)=0.96; mean bias, -69.1 cells/microl; 95% confidence interval [CI], -225.7 to+87.5 cells/microl) and the FACSCount system (R(2)=0.97; mean bias, -40.0 cells/microl; 95% CI, -165.1 to+85.1 cells/microl). The correlation of the CD4(+) T-lymphocyte counts obtained by the two bead-based systems was high (R(2)=0.98). Interestingly, CyFlow(green) yielded CD4(+) T-lymphocyte counts that were 21.8 and 7.2 cells/microl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4(+) T-lymphocyte counts were <250 CD4(+) T-lymphocyte counts/microl range or 17.3 and 5.8 cells/microl less, respectively, when CD4(+) T-lymphocyte counts were <200 cells/microl. The single-parameter CyFlow(green) volumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

Frequency of HIV-1 dual subtype infections, including intersubtype superinfections, among injection drug users in Bangkok, Thailand.

OBJECTIVES: To estimate the frequency and incidence of dual HIV-1 subtype infections, including superinfections, among recent seroconvertors from a cohort of injection drug users (IDUs). METHODS: A total of 1209 HIV-negative IDUs were followed in a prospective cohort study at 15 methadone clinics in Bangkok, Thailand. After 2308 person-years (PY) of follow-up, 133 seroconverted to HIV-1, of which approximately 20% were subtype B and 80% were CRF01_AE (formerly called subtype E). Specimens from 126 individuals were available at time of first seropositive test and specimens from 80 of these 126 individuals were also available more than 12 months later. For each infected participant, we calculated the amount of time to superinfection, loss to follow-up, or to the closest visit more than 12 months after the time of initial seropositivity. RESULTS: Of all 126 seroconverters seen at the time of the first seropositive test result, there was no apparent case of concurrent dual subtype infection detected despite 2301 PY of observation. Overall, the incidence of superinfection was 2.2 per 100 PY [95% confidence interval (CI), 0.3-7.8]. The 1-year incidence of CRF01_AE superinfection following subtype B primary infection was 3.9 per 100 PY (95% CI, 0.1-21.9) and the incidence of subtype B superinfection following CRF01_AE primary infection was 1.5 per 100 PY (95% CI, 0.04-8.3). CONCLUSIONS: Determination of the frequency and incidence of dual HIV-1 subtype infection demonstrates that HIV-1 superinfection is not uncommon in a population with high HIV-1 incidence with more than one circulating strain.

CCR5 promoter human haplogroups associated with HIV-1 disease progression in Thai injection drug users.

BACKGROUND: An evolutionary-based analysis of the CC chemokine receptor 5 gene (CCR5) promoter region has identified nine stable human haplogroups, within which certain haplogroups appear to influence HIV-1 disease progression differentially among Caucasians and African-Americans. OBJECTIVE: To assess the influence of CCR5 haplogroups on HIV-1 disease progression in a Thai population. DESIGN: Haplogroup analysis of HIV-1-seropositive injection drug users (IDU) participating in a prospective cohort study in Bangkok. All were documented seroconverters with a median follow-up time of 3.5 years (range, 0.2-7.0). METHODS: From a cohort of 130 IDU, 106 (81.5%) were genotyped for the CCR2b-64I, CCR5-delta32 and seven CCR5 promoter alleles constituting the CCR5 haplogroups. Survival curves and adjusted Cox proportional hazards models were used to assess the effect of haplogroups on the time from HIV-1 infection until CD4 count < 200 x 10(6) cells/l. RESULTS: The most common CCR5 haplogroups were HHC (61.8%), followed by HHE (15.6%) and HHF*2 (14.6%). HHE was associated with an accelerated CD4 count decline to < 200 x 10(6) cells/l (adjusted relative hazard, 1.88; 95% confidence interval, 1.05-3.36; P = 0.02). CONCLUSIONS: This is the first evidence that the CCR5 haplogroup E speeds the decline of the CD4 cell count and may lead to accelerated disease progression among HIV-infected Thais. These new observations highlight the need for additional studies involving populations in Asia.

Frequent human leukocyte antigen class I alleles are associated with higher viral load among HIV type 1 seroconverters in Thailand.

The loss of viral control by the host may be due to the evolution of viruses with mutations that limit presentation by human leukocyte antigen (HLA) to cytotoxic T cells. The authors hypothesized that the consequence of such evolution might be that persons with common HLA class I alleles would be less able to control viremia, on average, than would those with rare alleles. HLA class I typing was completed for 128 injection drug users who seroconverted in a prospective cohort study in Bangkok, Thailand. Logistic regression was used to model viral load (greater than or equal to the median) at 9 and 12 months after seroconversion with an HLA score that profiled the relative prevalence of each individual's alleles. At 12 months after seroconversion, injection drug users with the most common HLA alleles (highest quartile HLA score) had an almost 4-fold increased risk for higher viral load (> or = 32,055 copies/mL) than injection drug users with less common HLA alleles (adjusted odds ratio, 3.92; 95% confidence interval, 1.3-11.8). These findings support the importance of frequency-dependent effects of host genes on HIV type 1 evolution in different populations and suggest that HLA-driven viral evolution critically influences control of viremia in early HIV type 1 infection.

Early markers of HIV-1 disease progression in a prospective cohort of seroconverters in Bangkok, Thailand: implications for vaccine trials.

BACKGROUND: Some candidate HIV-1 vaccines may not prevent HIV-1 infection but may alter the course of disease. Surrogate endpoints based on early laboratory makers in HIV-1-infected persons who are antiretroviral therapy (ART)-naive will be useful for evaluating vaccine efficacy in slowing disease progression (VEp). We examined pretreatment HIV-1 viral loads and CD4 cell counts in recent HIV-1 seroconverters to inform selection of these endpoints. METHODS: We studied 130 newly HIV-1-infected injection drug users identified from a prospective cohort of initially uninfected persons in Bangkok during 1995 through 1998. We analyzed trends in HIV-1 viral loads and CD4 cell counts as well as progression to the surrogate endpoint, defined as 2 consecutive CD4 cell counts of fewer than 350 cells/mm, during 24 months after the first HIV-1 seropositive (FP) visit. RESULTS: Median HIV-1 RNA copies/mL with interquartile ranges were 43,693 (14,320-94,767) at the FP visit, 46,924 (16,273-104,314) at 6 months, 28,446 (11,292-54,325) at 12 months, and 18,080 (8713-54,059) at 18 months. HIV-1 viral loads at the FP visit and at 18 months were positively correlated (r = 0.53, P < 0.0001). Of 130 participants, 12% reached the surrogate endpoint by 6 months, 16% by 12 months, and 27% by 18 months. In Cox regression analyses, HIV-1 viral loads of more than 50,000 copies/mL at the FP visit (hazard ratio [HR] = 2.3, 95% confidence interval [CI]: 1.1-4.8) and first CD4 cell count of 500 or fewer cells/mm (HR = 7.6, 95% CI: 3.2-17.6) were independently associated with faster progression to the surrogate endpoint. CONCLUSIONS: Participants with high HIV-1 RNA levels and low CD4 cell counts close to the time of seroconversion were more likely to experience early immunologic progression. Approximately one quarter of seroconverters reached the surrogate immunologic endpoint within 18 months of their FP visit and before starting ART, suggesting the utility of this endpoint for analyses of VEp in some ongoing and planned HIV-1 vaccine efficacy trials.

Rapid whole-blood finger-stick test for HIV antibody: performance and acceptability among women in northern Thailand.

Although use of rapid HIV antibody tests of finger-stick blood specimens could expand voluntary counseling and testing in areas where fear of venipuncture and delays in learning test results are barriers, there is little information on performance and acceptability of these tests in Asia. We used the Hema. Strip HIV-1/2 test (Saliva Diagnostic Systems, Vancouver, WA) in a prospective cohort study of HIV seroincidence among women in northern Thailand from 1998 to 1999. Nurses obtained whole-blood specimens by finger-stick testing and provided test results and counseling at each visit. Acceptability of the rapid test was assessed at the first 6-month follow-up visit. HIV-1 seroprevalence among the 804 women screened at enrollment was 3.1%. Positive rapid test results from 25 women were confirmed by enzyme immunoassay and Western blot analysis using serum obtained by venipuncture. Of the 741 women who returned for follow-up, 56% preferred specimen collection by finger-stick testing to venipuncture, 80% preferred immediate rather than delayed test results, 79% preferred the rapid test method to typical testing methods, and 97% were satisfied with the test method used. Results from this study demonstrate the utility and acceptability of the rapid finger-stick test for HIV antibody among women in northern Thailand.

Risk factors for sexually transmitted diseases in northern Thai adolescents: an audio-computer-assisted self-interview with noninvasive specimen collection.

BACKGROUND: Previous studies of sexual behavior and sexually transmitted diseases (STDs) in Thai adolescents may have been limited by participation bias and underreporting of stigmatized behaviors. GOAL: The goal was to increase knowledge about risk behaviors and STDs among youths in Thailand. STUDY DESIGN: Students aged 15 to 21 years completed an audio-computer-assisted self-interview. Oral fluid was tested for HIV antibodies and urine was tested for Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids with polymerase chain reaction. RESULTS: Of 1736 invited students, 1725 (99.4%) agreed to participate. Overall, C trachomatis infection was detected in 49 (2.8%), and there were five cases (0.3%) each of infection with N gonorrhoeae and HIV. Among those who reported sexual intercourse, the prevalence of chlamydial infection was 3.7% among men and 6.1% among women. Logistic regression analysis showed age-adjusted factors associated with chlamydial infection among men to be parents' occupation in agriculture, having sold sex, having a sex partner who had been pregnant, and the number of casual sex partners during lifetime. Among women, age-adjusted factors were parents' occupation in agriculture, number of casual partners during lifetime, having an older sex partner, and perception of higher HIV infection risk. CONCLUSION: These adolescents had high rates of unprotected intercourse and are at risk for STDs. Prevention programs should emphasize use of effective contraceptive methods, including condom use; reducing the number of sex partners (stressing the risk a partner of older age may pose to female adolescents); and reducing engagement in commercial sex.