skip to main content

Title: A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples

Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an early virological biomarker of HIV-1 infection. Here, To improve p24 detection in patients missed by immunological tests that dominate the diagnostics market, we show a click chemistry amplified nanopore (CAN) assay for ultrasensitive quantitative detection. This strategy achieves a 20.8 fM (0.5 pg/ml) limit of detection for HIV-1 p24 antigen in human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation of the CAN assay in a pilot cohort shows p24 quantification at ultra-low concentration range and correlation with CD4 count and viral load. We believe that this strategy can improve the utility of p24 antigen in detecting early infection and monitoring HIV progression and treatment efficacy, and also can be readily modified to detect other infectious diseases.

; ; ; ; ; ; ; ; ; ; ; ;
Award ID(s):
Publication Date:
Journal Name:
Nature Communications
Nature Publishing Group
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir.

  2. Drug-resistant HIV-1 has caused a growing concern in clinic and public health. Although combination antiretroviral therapy can contribute massively to the suppression of viral loads in patients with HIV-1, it cannot lead to viral eradication. Continuing viral replication during sub-optimal therapy (due to poor adherence or other reasons) may lead to the accumulation of drug resistance mutations, resulting in an increased risk of disease progression. Many studies also suggest that events occurring during the early stage of HIV-1 infection (i.e., the first few hours to days following HIV exposure) may determine whether the infection can be successfully established. However, the numbers of infected cells and viruses during the early stage are extremely low and stochasticity may play a critical role in dictating the fate of infection. In this paper, we use stochastic models to investigate viral infection and the emergence of drug resistance of HIV-1. The stochastic model is formulated by a continuous-time Markov chain (CTMC), which is derived based on an ordinary differential equation model proposed by Kitayimbwa et al. that includes both forward and backward mutations. An analytic estimate of the probability of the clearance of HIV infection of the CTMC model near the infection-free equilibrium is obtainedmore »by a multitype branching process approximation. The analytical predictions are validated by numerical simulations. Unlike the deterministic dynamics where the basic reproduction number $ \mathcal{R}_0 $ serves as a sharp threshold parameter (i.e., the disease dies out if $ \mathcal{R}_0 < 1 $ and persists if $ \mathcal{R}_0 > 1 $), the stochastic models indicate that there is always a positive probability for HIV infection to be eradicated in patients. In the presence of antiretroviral therapy, our results show that the chance of clearance of the infection tends to increase although drug resistance is likely to emerge.

    « less
  3. Dai, Tianhong ; Wu, Mei X. ; Popp, Jürgen (Ed.)
    The SARS-CoV-2 pandemic has revealed the need for rapid and inexpensive diagnostic testing to enable population-based screening for active infection. Neither standard diagnostic testing, the detection and measurement of viral RNA (via polymerase chain reaction), or serological testing (via enzyme-linked immunosorbent assay) has the capability to definitively determine active infection. The former due to a lack of ability to distinguish between replicable and inert viral RNA, and the latter due to varying immune responses (ranging from latent to a complete lack of immune response altogether). Despite many companies producing rapid point-of-care (POC) tests, none will address the global scale of testing needed and few help to combat the ever growing issue of testing resource scarcity. Here we discuss our efforts towards the development of a highly manufacturable, microfluidic device that instantly indicates active viral infection status from ~ 20 μL of nasal mucus or phlegm and requires no external power. The device features a biotin functionalized silicon nanomembrane within an acrylic body containing channels and ports for sample introduction and analysis. Virus capture and target confirmation are done using affinity-based capture and size-based occlusion respectively. Modularity of the device is proven with bead and vaccinia virus capture as we workmore »towards testing with both pure SARS-CoV-2 virus and human samples. With success on all fronts, we could achieve an inexpensive POC diagnostic which can determine an individual’s infection status, aiding containment efforts in the current and future pandemics. In addition to direct viral detection, our method can be used as a rapid POC sample preparation tool that limits the application of PCR reagents to those samples which already display viral size and antigen-based positivity through our device.« less
  4. Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 10 5 copies/μl) and exhibited a limit of detection (0.38 copies/μl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.
  5. Abstract

    The transmission dynamics of HIV are closely tied to the duration and overlap of sexual partnerships. We develop an autonomous population model that can account for the possibilities of an infection from either a casual sexual partner or a long-term partner who was either infected at the start of the partnership or has been newly infected since the onset of the partnership. The impact of the long-term partnerships on the rate of infection is captured by calculating the expected values of the rate of infection from these extended contacts. The model includes three stages of infectiousness: acute, chronic, and virally suppressed. We calculate HIV incidence and the fraction of new infections attributed to casual contacts and long-term partnerships allowing for variability in condom usage, the effect of achieving and maintaining viral suppression, and early intervention by beginning HAART during the acute phase of infection. We present our results using data on MSM HIV transmission from the CDC in the U.S. While the acute stage is the most infectious, the majority of the new infections will be transmitted by long-term partners in the chronic stage when condom use is infrequent as is common in long-term relationships. Time series analysis ofmore »the solution, as well as parameter sensitivity analysis, are used to determine effective intervention strategies.

    « less