Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Co-Expression and Combined Prognostic Value of CSPG4 and PDL1 in TP53-Aberrant Triple-Negative Breast Cancer.

Background: In triple-negative breast cancer (TNBC), PDL1/PD1-directed immunotherapy is effective in less than 20% of patients. In our preliminary study, we have found CSPG4 to be highly expressed together with PDL1 in TNBCs, particularly those harboring TP53 aberrations. However, the clinical implications of co-expressed CSPG4 and PDL1 in TNBCs remain elusive. Methods: A total of 85 advanced TNBC patients treated in the Hunan Cancer Hospital between January 2017 and August 2019 were recruited. The expressions of CSPG4 and PDL1 in TNBC tissues were investigated using immunohistochemistry (IHC). The RNA-seq dataset from the TCGA-BRCA project was further used to analyze the mRNA expression of CSPG4 and PDL1 in TP53-aberrant TNBCs. Cox proportional hazards model and Kaplan-Meier curves with Logrank test was used to analyze the effects of CSPG4 and PDL1 on survival. TNBC cell lines were further used to investigate the molecular mechanism that were involved. Results: TP53 aberrations occurred in more than 50% of metastatic TNBCs and were related to higher tumor mutation burden (TMB). In TCGA-BRCA RNA-seq dataset analysis, both CSPG4 and PDL1 levels were high in TNBCs, especially in TP53-aberrant TNBCs. IHC assay showed nearly 60% of advanced TNBCs to be CSPG4-positive and about 25% to be both CSPG4-positive and PDL1-positive. The levels of CSPG4 and PDL1 were high in TNBC cell lines as revealed by flow cytometry and immunoblotting compared with non-TNBC cells. Univariate Cox regression analysis indicated that CSPG4 positivity was a significant risk factor for progression-free survival in metastatic TNBCs, with a hazard ratio (HR) of 2.26 (P = 0.05). KM curves with Logrank test also identified high level of CSPG4 as a significant risk factor for overall survival in advanced breast cancers in TCGA-BRCA samples (P = 0.02). The immunoblotting assays showed that EMT-related pathways were involved in CSPG4-mediated invasion. Conclusions: CSPG4 expression level is associated with PDL1 positivity in TP53-aberrant TNBC cells. Patients with CSPG4 expression have poor treatment response and poor overall survival. Co-expressed CSPG4 and PDL1 may have an important prognostic value and provide new therapeutic targets in TNBC patients. CSPG4 might mediate tumor invasion and PDL1 overexpression through EMT-related pathway.

Infection with multiple HIV-1 founder variants is associated with lower viral replicative capacity, faster CD4+ T cell decline and increased immune activation during acute infection.

HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants' samples was sequenced close to transmission (median 21 days post infection, IQR 18-41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF.

Factors associated with vaccination completion and retention among HIV negative female sex workers enrolled in a simulated vaccine efficacy trial in Kampala, Uganda.

BACKGROUND: Female sex workers (FSWs) at substantial risk of HIV are potentially a suitable group for HIV prevention trials including vaccine trials. Few HIV vaccine preparatory studies have been conducted among FSWs in Sub-Saharan Africa (SSA); data are therefore limited on acceptability of vaccine trial procedures. We determined vaccination completion and one-year retention among FSWs in Kampala, Uganda. METHODS: We conducted a prospective study that simulated a vaccine efficacy trial among HIV negative FSWs (18-49 years). Hepatitis B vaccine (Engerix B) was used to mimic an HIV vaccine product. Volunteers received 1 ml intramuscular injection at 0, 1 and 6 months, and made additional visits (3 days post-vaccination and months 3, 9 and 12). They were censored at that visit if diagnosed as HIV positive or pregnant. We collected socio-demographic, behavioral and clinical data at baseline, 6 and 12 months and fitted Poisson regression models with robust standard error to find factors associated with vaccination completion and retention. RESULTS: We enrolled 290 volunteers (median age 27 years) of whom 230 reached a study end-point as follows: 7 became HIV infected, 11 became pregnant and 212 completed both the vaccination schedule and 12-month visit giving a retention of 77.9% (212/272). Vaccination completion was 82.4%. Non-retention at 1 year was more likely among those reporting symptoms of genital ulcer disease (GUD) in the past 3 months (IRR 1.90; 95% CI 1.09-3.32) and those < 35 years; (IRR 6.59; 95% CI 2.11-20.57). Non-completion of the vaccination schedule was associated with being < 35 years (IRR 13.10; 95% CI 1.89-90.92, reporting GUD symptoms (IRR 3.02; 95% CI 1.71-5.33) and reporting consistent condom use with new sexual partners (IRR 2.57; 95% CI 1.10-6.07). CONCLUSIONS: FSWs are at substantial risk of HIV infection and yet willing to participate in HIV vaccine and prevention research; young FSWs should be empowered, and those reporting GUD symptoms need close follow up to improve participation in future HIV vaccine trials.

Chondroitin sulfate proteoglycan 4 enhanced melanoma motility and growth requires a cysteine in the core protein transmembrane domain.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell surface proteoglycan that enhances malignant potential in melanoma and several other tumor types. CSPG4 functions as a transmembrane scaffold in melanoma cells to activate oncogenic signaling pathways such as focal adhesion kinase (FAK) and extracellular signal regulated kinases 1,2, that control motility, invasion and anchorage independent growth. Here, we demonstrate that CSPG4 promotes directional motility and anchorage independent growth of melanoma cells by organizing and positioning a signaling complex containing activated FAK to lipid rafts within the plasma membrane of migrating cells. This FAK-containing signal transduction platform, which consists of syntenin-1, active Src and caveolin-1 requires the cytoplasmic domain of CSPG4 for assembly. Enhanced directional motility promoted by this complex also requires a CSPG4 transmembrane cysteine residue C2230. Substituting C2230 with alanine (CSPG4) still permits assembly of the signaling complex, however Src remains in an inactive state. CSPG4 also fails to promote anchorage independent growth and activation of extracellular signal regulated kinases 1,2. Therapies that target the transmembrane domain of CSPG4 could be a novel strategy for limiting progression by disrupting its function as a compartmentalized motogenic and growth-promoting oncogenic signaling node.

HIV incidence and predictors of inconsistent condom use among adult men enrolled into an HIV vaccine preparedness study, Rustenburg, South Africa.

INTRODUCTION: Understanding HIV incidence and risk behaviour among populations being considered for HIV vaccine studies is necessary for the appropriate design of trials. METHODS: Between May 2012 and June 2015, we recruited men aged 18-49 years from urban and peri-urban areas of Rustenburg, a mining town in the North West Province, South Africa. Men who reported HIV-risk behaviour were followed for nine to 12 months to determine HIV incidence and factors associated with condom use. RESULTS: A total of 400 HIV uninfected men were enrolled; 366 (91.5%) had at least one follow-up visit and were included in the analysis; 47.6% were under 25 years of age. HIV incidence was 1.9 per 100 person-years (95% CI: 0.79-4.56). Among heterosexual men (N = 339), 80.8% reported having vaginal intercourse with multiple partners in the past three months, among whom 74.1% reported inconsistent condom use. Sixty-eight percent reported vaginal intercourse with new female partners, of whom 40.6% reported inconsistent condom use. Over half (55.6%) of men who had sex with men (N = 27) reported anal intercourse with multiple male partners in the past three months, of whom 68.2% reported using condoms inconsistently. Men who had more than two female partners in the last three months (n = 121) were more likely to use condoms inconsistently (aOR 4.31, 95% CI: 1.34-13.8); in contrast, those with more than one new female sex partner (aOR 0.13, 94% CI 0.04-0.44), and whose sexual debut was after 19 years of age (aOR 0.39, 95% CI: 0.15-1.01) were less likely to use condoms inconsistently. CONCLUSION: HIV incidence was low and similar to other studies of heterosexual men in South Africa. To identify men at high risk for HIV for enrolment in prevention trials, future researchers may need to focus on those who report early sexual debut and who report having multiple sexual partners. Men in newer relationships appear to use condoms more frequently.

Point-of-care HIV RNA testing and immediate antiretroviral therapy initiation in young adults seeking out-patient care in Kenya.

: We evaluated outcomes of an HIV-1-testing intervention using rapid HIV tests followed by point-of-care Xpert Qual testing for HIV-1 RNA. Of 706 young urgent-care seeking participants evaluated, 24 (3.4%) had chronic HIV (antibody-positive), 3 (0.4%) acute HIV-1 (Qual-positive, antibody-negative), and 3 (0.4%) early HIV-1 infection (Qual-positive, antibody-discordant). Overall, 21 (70.0%) diagnosed patients started antiretroviral therapy after a median of 4 days (range 0-71). HIV-1 RNA testing led to an increase in confirmed diagnoses by 25%.

Reported willingness to participate in a hypothetical HIV vaccine trial and its translation to actual participation among healthy adults-Experience from Kenya.

OBJECTIVE: To evaluate initial reported willingness to participate in a hypothetical HIV vaccine clinical trial and actual participation of volunteers in a longitudinal observational study. METHODS: We recruited HIV negative male and female volunteers aged 18-45 years into a longitudinal observational study at KAVI-ICR Kangemi in Kenya, to serve as a pool from which to draw participants into a phase I HIV vaccine clinical trial. A structured questionnaire was used to collect information regarding willingness to join a HIV vaccine clinical trial in the future. Study follow-up visits were every 6 months. RESULTS: A total of 105 participants were screened and 100 (M46:F54) were enrolled into the observational study. Ninety- four per cent of those enrolled expressed willingness to participate in a future HIV vaccine trial. Altruism and desire to learn the body's response to the vaccine were the most motivating factors at 40% and 25% respectively. At the onset of a 40-person phase I HIV vaccine trial, 86 observational study participants who had previously expressed willingness to participate were contacted but only 26 (30%) came for information. All 26 consented to participate and after screening for eligibility, 24 were eligible. Of the 24, 15 were enrolled. These numbers were not adequate; hence the vaccine trial employed other recruitment methods to meet the deficit. CONCLUSION: Observational "pools" of cohorts may not provide adequate number of participants into vaccine clinical trials even if they report willingness; therefore supplementary recruitment methods such as direct community recruitment, passive approach, and snowballing need to be in place.

In vitro elucidation of the role of pericellular matrix in metastatic extravasation and invasion of breast carcinoma cells.

Numerous studies have demonstrated the importance of altered hyaluronan metabolism to malignant progression of multiple tumor types, including breast carcinomas. Increased hyaluronan (HA) metabolism in the stroma of primary tumors promotes activation of oncogenic signaling pathways that impact tumor initiation, growth, and invasion. Carcinoma cell synthesis and assembly of HA-rich pericellular matrices induces a stromal-independent phenotype, which is associated with cancer progression. Although the pro-tumorigenic role of stromal HA is well established, a novel but unexplored hypothesis is that carcinoma cell-associated HA pericellular matrices promote metastasis of circulating tumor cells. Here, we report the development of an in vitro assay that employs microfluidic techniques to directly measure the importance of an HA-rich pericellular matrix in the entry of carcinoma cells into ectopic sites. This model provides the capability to visualize specific steps in metastasis, which is difficult using animal models. The results show that the presence of a HA-rich pericellular matrix correlates to the invasive and metastatic potential of breast carcinoma cells. Furthermore, enzymatic removal or pharmacologic inhibition of HA synthesis significantly inhibits carcinoma cell extravasation and invasion in this model system. These results implicate pericellular HA-rich carcinoma cell associated pericellular matrices in colonization of ectopic sites by circulating tumor cells and support specific targeting of this matrix to limit metastasis in patients.

Prediction of extended high viremia among newly HIV-1-infected persons in sub-Saharan Africa.

OBJECTIVE: Prompt identification of newly HIV-infected persons, particularly those who are most at risk of extended high viremia (EHV), allows important clinical and transmission prevention benefits. We sought to determine whether EHV could be predicted during early HIV infection (EHI) from clinical, demographic, and laboratory indicators in a large HIV-1 incidence study in Africa. DESIGN: Adults acquiring HIV-1 infection were enrolled in an EHI study assessing acute retroviral syndrome (ARS) symptoms and viral dynamics. METHODS: Estimated date of infection (EDI) was based on a positive plasma viral load or p24 antigen test prior to seroconversion, or the mid-point between negative and positive serological tests. EHV was defined as mean untreated viral load ≥5 log10 copies/ml 130-330 days post-EDI. We used logistic regression to develop risk score algorithms for predicting EHV based on sex, age, number of ARS symptoms, and CD4 and viral load at diagnosis. RESULTS: Models based on the full set of five predictors had excellent performance both in the full population (c-statistic = 0.80) and when confined to persons with each of three HIV-1 subtypes (c-statistic = 0.80-0.83 within subtypes A, C, and D). Reduced models containing only 2-4 predictors performed similarly. In a risk score algorithm based on the final full-population model, predictor scores were one for male sex and enrollment CD4<350 cells/mm3, and two for having enrollment viral load >4.9 log10 copies/ml. With a risk score cut-point of two, this algorithm was 85% sensitive (95% CI: 76%-91%) and 61% specific (55%-68%) in predicting EHV. CONCLUSIONS: Simple risk score algorithms can reliably identify persons with EHI in sub-Saharan Africa who are likely to sustain high viral loads if treatment is delayed. These algorithms may be useful for prioritizing intensified efforts around care linkage and retention, treatment initiation, adherence support, and partner services to optimize clinical and prevention outcomes.

Computational analysis of antibody dynamics identifies recent HIV-1 infection.

Accurate HIV-1 incidence estimation is critical to the success of HIV-1 prevention strategies. Current assays are limited by high false recent rates (FRRs) in certain populations and a short mean duration of recent infection (MDRI). Dynamic early HIV-1 antibody response kinetics were harnessed to identify biomarkers for improved incidence assays. We conducted retrospective analyses on circulating antibodies from known recent and longstanding infections and evaluated binding and avidity measurements of Env and non-Env antigens and multiple antibody forms (i.e., IgG, IgA, IgG3, IgG4, dIgA, and IgM) in a diverse panel of 164 HIV-1-infected participants (clades A, B, C). Discriminant function analysis identified an optimal set of measurements that were subsequently evaluated in a 324-specimen blinded biomarker validation panel. These biomarkers included clade C gp140 IgG3, transmitted/founder clade C gp140 IgG4 avidity, clade B gp140 IgG4 avidity, and gp41 immunodominant region IgG avidity. MDRI was estimated at 215 day or alternatively, 267 days. FRRs in untreated and treated subjects were 5.0% and 3.6%, respectively. Thus, computational analysis of dynamic HIV-1 antibody isotype and antigen interactions during infection enabled design of a promising HIV-1 recency assay for improved cross-sectional incidence estimation.

Differences in acute retroviral syndrome by HIV-1 subtype in a multicentre cohort study in Africa.

OBJECTIVE: Symptoms of acute retroviral syndrome (ARS) may be used to identify patients with acute HIV-1 infection who seek care. ARS symptoms in African adults differ by region. We assessed whether reporting of ARS was associated with HIV-1 subtype in a multicentre African cohort study representing countries with predominant HIV-1 subtypes A, C, and D. METHODS: ARS symptoms were assessed in adults enrolling within 6 weeks after the estimated date of infection in an acute and early HIV-1 infection cohort study. HIV-1 subtype was determined by POL genotyping. We used log-binomial regression to compare ARS symptom prevalence among those with subtype A vs. C or D, adjusting for sex, time since enrolment, and enrolment viral load. RESULTS: Among 183 volunteers ascertained within 6 weeks after estimated date of infection, 77 (42.0%) had subtype A, 83 (45.4%) subtype C, and 23 (12.6%) subtype D infection. Individuals with subtype A were 1.40 (95% confidence interval: 1.17, 1.68) times as likely as individuals with subtypes C or D to report any ARS symptoms; each individual symptom other than rash was also more prevalent in subtype A than in subtype C or D, with prevalence ratios ranging from 1.94 (1.40, 2.70) for headache to 4.92 (2.24, 10.78) for lymphadenopathy. CONCLUSION: Individuals with subtype A were significantly more likely than individuals with subtypes C or D to report any ARS symptoms. HIV-1 subtypes may help explain differences in ARS that have been observed across regions in Africa, and may impact the yield of symptom-based screening strategies for acute HIV infection detection.

Infection Staging and Incidence Surveillance Applications of High Dynamic Range Diagnostic Immuno-Assay Platforms.

BACKGROUND: Custom HIV staging assays, including the Sedia HIV-1 Limiting Antigen (LAg) Avidity EIA and avidity modifications of the Ortho VITROS anti-HIV-1+2 and Abbott ARCHITECT HIV Ag/Ab Combo assays, are used to identify "recent" infections in clinical settings and for cross-sectional HIV incidence estimation. However, the high dynamic range of chemiluminescent platforms allows differentiating recent and long-standing infection on signal intensity, and this raises the prospect of using unmodified diagnostic assays for infection timing and surveillance applications. METHODS: We tested a panel of 2500 well-characterized specimens with estimable duration of HIV infection with the 3 assays and the unmodified ARCHITECT. Regression models were used to estimate mean durations of recent infection (MDRIs), context-specific false-recent rates (FRRs) and correlation between diagnostic signal intensity and LAg measurements. Hypothetical epidemiological scenarios were constructed to evaluate utility in surveillance applications. RESULTS: Over a range of MDRIs (reflecting recency discrimination thresholds), a diluted ARCHITECT-based RITA produced lower FRRs than the VITROS platform (FRR ≈ 0.5% and 1.5%, respectively at MDRI ≈ 200 days), and the unmodified diagnostic ARCHITECT produces incidence estimates with comparable precision to LAg (relative SE ≈ 17.5% and 15%, respectively at MDRI ≈ 200 days). ARCHITECT S/CO measurements were highly correlated with LAg optical density measurements (r = 0.80), and values below 200 are strongly predictive of LAg recency and duration of infection less than 1 year. CONCLUSIONS: Low quantitative measurements from the unmodified ARCHITECT obviate the need for additional recency testing, and its use is feasible in clinical staging and incidence surveillance applications.

Biphasic Dependence of Glioma Survival and Cell Migration on CD44 Expression Level.

While several studies link the cell-surface marker CD44 to cancer progression, conflicting results show both positive and negative correlations with increased CD44 levels. Here, we demonstrate that the survival outcomes of genetically induced glioma-bearing mice and of high-grade human glioma patients are biphasically correlated with CD44 level, with the poorest outcomes occurring at intermediate levels. Furthermore, the high-CD44-expressing mesenchymal subtype exhibited a positive trend of survival with increased CD44 level. Mouse cell migration rates in ex vivo brain slice cultures were also biphasically associated with CD44 level, with maximal migration corresponding to minimal survival. Cell simulations suggest that cell-substrate adhesiveness is sufficient to explain this biphasic migration. More generally, these results highlight the potential importance of non-monotonic relationships between survival and biomarkers associated with cancer progression.

Dynamics and Correlates of CD8 T-Cell Counts in Africans with Primary Human Immunodeficiency Virus Type 1 Infection.

In individuals with HIV-1 infection, depletion of CD4+ T cells is often accompanied by a malfunction of CD8+ T cells that are persistently activated and/or exhausted. While the dynamics and correlates of CD4 counts have been well documented, the same does not apply to CD8 counts. Here, we examined the CD8 counts in a cohort of 497 Africans with primary HIV-1 infection evaluated in monthly to quarterly follow-up visits for up to 3 years in the absence of antiretroviral therapy. Statistical models revealed that (i) CD8 counts were relatively steady in the 3- to 36-month period of infection and similar between men and women; (ii) neither geography nor heterogeneity in the HIV-1 set-point viral load could account for the roughly 10-fold range of CD8 counts in the cohort (P > 0.25 in all tests); and (iii) factors independently associated with relatively high CD8 counts included demographics (age ≤ 40 years, adjusted P = 0.010) and several human leukocyte antigen class I (HLA-I) alleles, including HLA-A*03:01 (P = 0.013), B*15:10 (P = 0.007), and B*58:02 (P < 0.001). Multiple sensitivity analyses provided supporting evidence for these novel relationships. Overall, these findings suggest that factors associated with the CD8 count have little overlap with those previously reported for other HIV-1-related outcome measures, including viral load, CD4 count, and CD4/CD8 ratio. IMPORTANCE: Longitudinal data from 497 HIV-1 seroconverters allowed us to systematically evaluate the dynamics and correlates of CD8+ T-cell counts during untreated primary HIV-1 infection in eastern and southern Africans. Our findings suggest that individuals with certain HLA-I alleles, including A*03 (exclusively A*03:01), persistently maintain relatively high CD8 counts following HIV-1 infection, a finding which may offer an intriguing explanation for the recently reported, negative association of A*03 with HIV-1-specific, broadly neutralizing antibody responses. In future studies, attention to HLA-I genotyping data may benefit in-depth understanding of both cellular and humoral immunity, as well as the intrinsic balances of these types of immunity, especially in settings where there is emerging evidence of antagonism between the two arms of adaptive immunity.

Performance of the Bio-Rad Geenius HIV1/2 Supplemental Assay in Detecting "Recent" HIV Infection and Calculating Population Incidence.

OBJECTIVE: HIV seroconversion biomarkers are being used in cross-sectional studies for HIV incidence estimation. Bio-Rad Geenius HIV-1/2 Supplemental Assay is an immunochromatographic single-use assay that measures antibodies (Ab) against multiple HIV-1/2 antigens. The objective of this study was to determine whether the Geenius assay could additionally be used for recency estimation. DESIGN: This assay was developed for HIV-1/2 confirmation; however, quantitative data acquired give information on increasing concentration and diversity of antibody responses over time during seroconversion. A quantitative threshold of recent HIV infection was proposed to determine "recent" or "nonrecent" HIV infection; performance using this cutoff was evaluated. METHODS: We tested 2500 highly characterized specimens from research subjects in the United States, Brazil, and Africa with well-defined durations of HIV infection. Regression and frequency estimation were used to estimate assay properties relevant to HIV incidence measurement: mean duration of recent infection (MDRI), false-recent rate, and assay reproducibility and robustness. RESULTS: Using the manufacturer's proposed cutoff index of 1.5 to identify "recent" infection, the assay has an estimated false-recent rate of 4.1% (95% CI: 2.2 to 7.0) and MDRI of 179 days (155 to 201) in specimens from treatment-naive subjects, presenting performance challenges similar to other incidence assays. Lower index cutoffs associated with lower MDRI gave a lower rate of false-recent results. CONCLUSIONS: These data suggest that with additional interpretive analysis of the band intensities using an algorithm and cutoff, the Geenius HIV-1/2 Supplemental Assay can be used to identify recent HIV infection in addition to confirming the presence of HIV-1 and HIV-2 antibodies.

Viral load criteria and threshold optimization to improve HIV incidence assay characteristics.

OBJECTIVE: Assays for classifying HIV infections as 'recent' or 'nonrecent' for incidence surveillance fail to simultaneously achieve large mean durations of 'recent' infection (MDRIs) and low 'false-recent' rates (FRRs), particularly in virally suppressed persons. The potential for optimizing recent infection testing algorithms (RITAs), by introducing viral load criteria and tuning thresholds used to dichotomize quantitative measures, is explored. DESIGN: The Consortium for the Evaluation and Performance of HIV Incidence Assays characterized over 2000 possible RITAs constructed from seven assays (Limiting Antigen, BED, Less-sensitive Vitros, Vitros Avidity, BioRad Avidity, Architect Avidity, and Geenius) applied to 2500 diverse specimens. METHODS: MDRIs were estimated using regression, and FRRs as observed 'recent' proportions, in various specimen sets. Context-specific FRRs were estimated for hypothetical scenarios. FRRs were made directly comparable by constructing RITAs with the same MDRI through the tuning of thresholds. RITA utility was summarized by the precision of incidence estimation. RESULTS: All assays produce high FRRs among treated patients and elite controllers (10-80%). Viral load testing reduces FRRs, but diminishes MDRIs. Context-specific FRRs vary substantially by scenario - BioRad Avidity and Limiting Antigen provided the lowest FRRs and highest incidence precision in scenarios considered. CONCLUSION: The introduction of a low viral load threshold provides crucial improvements in RITAs. However, it does not eliminate nonzero FRRs, and MDRIs must be consistently estimated. The tuning of thresholds is essential for comparing and optimizing the use of assays. The translation of directly measured FRRs into context-specific FRRs critically affects their magnitudes and our understanding of the utility of assays.

Comparison of HIV incidence estimated in clinical trial and observational cohort settings in a high risk fishing population in Uganda: Implications for sample size estimates.

BACKGROUND: Clinical trial participants may differ from the source population due to the demands of trial participation and self-selection, inadvertent selection of a lower-risk group, or both. We investigated the HIV risk status of volunteers in a Simulated Vaccine Efficacy Trial (SiVET) nested within a prospective observational cohort of fisher folks in South Western Uganda. METHODS: Volunteers aged 18-49 years, at high risk for HIV from fishing communities in Masaka district were recruited into an observational cohort and followed quarterly. High risk was defined as a self-report, of at least one of the following in the past three months; sexually transmitted infections, unprotected sex with >1 partner or a new sexual partner, use of recreational drugs, weekly alcohol use, and/or frequent travel. Volunteers who had at least three months of follow-up in the observational cohort were consecutively enrolled in SiVET, administered Hepatitis B vaccine at months (0, 1, 6) and followed-up three days post vaccinations to mimic a vaccine trial schedule. HIV incidence over the next 12 months was compared between SiVET and the observational cohort studies. RESULTS: Between January 2012 and February 2013, 575 individuals were enrolled in the observational cohort, of whom 282 were enrolled in SiVET between July 2012 and February 2013. Despite similar pattern of reported risk behaviour in both studies, HIV incidence was higher in observational cohort, 11.4 cases/100 PYO [95% CI: 7.4-17.7] compared to 3.8 [95% CI: 2.0-7.0] in SiVET (p<0.01). SiVET volunteers tended to be men, having some education and longer-term residents, all factors that are also associated with lower HIV risk. CONCLUSION: We observed a lower HIV incidence in SiVET than in the observational cohort. The two populations differed significantly in demographics but not in reported risk. HIV incidence estimates from observational cohorts must be used with caution to estimate the trial study size.

CD4:CD8 lymphocyte ratio as a quantitative measure of immunologic health in HIV-1 infection: findings from an African cohort with prospective data.

In individuals with human immunodeficiency virus type 1 (HIV-1) infection, CD4:CD8 lymphocyte ratio is often recognized as a quantitative outcome that reflects the critical role of both CD4(+) and CD8(+) T-cells in HIV-1 pathogenesis or disease progression. Our work aimed to first establish the dynamics and clinical relevance of CD4:CD8 ratio in a cohort of native Africans and then to examine its association with viral and host factors, including: (i) length of infection, (ii) demographics, (iii) HIV-1 viral load (VL), (iv) change in CD4(+) T-lymphocyte count (CD4 slope), (v) HIV-1 subtype, and (vi) host genetics, especially human leukocyte antigen (HLA) variants. Data from 499 HIV-1 seroconverters with frequent (monthly to quarterly) follow-up revealed that CD4:CD8 ratio was stable in the first 3 years of infection, with a modest correlation with VL and CD4 slope. A relatively normal CD4:CD8 ratio (>1.0) in early infection was associated with a substantial delay in disease progression to severe immunodeficiency (<350 CD4 cells/µl), regardless of other correlates of HIV-1 pathogenesis (adjusted hazards ratio (HR) = 0.43, 95% confidence interval (CI) = 0.29-0.63, P < 0.0001). Low VL (<10,000 copies/ml) and HLA-A*74:01 were the main predictors of CD4:CD8 ratio >1.0, but HLA variants (e.g., HLA-B*57 and HLA-B*81) previously associated with VL and/or CD4 trajectories in eastern and southern Africans had no obvious impact on CD4:CD8 ratio. Collectively, these findings suggest that CD4:CD8 ratio is a robust measure of immunologic health with both clinical and epidemiological implications.