A mAb for the detection of the antiretroviral drug emtricitabine : AIDS

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A mAb for the detection of the antiretroviral drug emtricitabine

Youngpairoj, Ae S.a; Vanderford, Thomas H.a; Reed, Matthew S.b; Granade, Timothy C.a; Pau, Chou-Ponga; Pohl, Janb; Switzer, William M.a; Heneine, Walida

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AIDS 36(13):p 1890-1893, November 1, 2022. | DOI: 10.1097/QAD.0000000000003357
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Abstract

Antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) are the cornerstones of HIV prevention and elimination strategies and are very effective when taken consistently [1]. Antiretroviral regimens containing the nucleoside reverse transcriptase inhibitor emtricitabine (FTC) are widely used in first-line therapy of HIV-infected persons. Daily PrEP with the combination of FTC and tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) is also recommended for HIV prevention in populations at risk for infection [2]. Inadequate adherence to daily ART and PrEP has been shown to reduce effectiveness and public health benefit [3–7]. Pharmacologic monitoring of adherence to these regimens provides an objective measurement of compliance and has been shown to predict the efficacy of PrEP [4,8]. Current methods to measure drugs rely on complex analytical tests using liquid chromatography and tandem mass spectrometry that are costly, require skilled personnel, and are not implementable at point-of-care (POC) testing sites [9–11]. Furthermore, due to limitations in self-reporting and pill-counting methods of adherence monitoring [12,13], a direct method for measuring FTC levels provides an objective marker that could have broad implications on the ability of care providers to monitor and counsel adherence to ART and PrEP [14]. Thus, there is a need to develop a simple, scalable, and inexpensive assay to detect antiretroviral drugs for adherence monitoring in people on PrEP and ART. Antibody-based tests, such as lateral flow diagnostic strips, allow for the development of POC assays that can provide real-time feedback to patients to improve adherence [14]. Several groups have developed immunodiagnostic tests for tenofovir detection demonstrating the validity of this approach on small antiretroviral drugs [15–17]. However, tenofovir concentrations vary substantially in persons treated with either TDF or TAF requiring different assay modifications and varied reference cutoffs [16,18,19]. Because FTC dosage is the same in ART and PrEP regimens, we posit that an antibody-based FTC assay will provide a universally simple tool for adherence monitoring of ART and PrEP. Thus, we developed an FTC-specific antibody to facilitate the development of these critical tools.

Three BALB/C mice were immunized with FTC conjugated to an extremely immunogenic carrier molecule, the Limulus polyphemus hemocyanin II. After 5 months, serologic responses to FTC were assayed to determine the magnitude and specificity of the responses for FTC. Twenty-nine hybridomas were generated from the mouse with the best anti-FTC responses, expanded for 3 weeks, and screened for reactivity and specificity to FTC. Twelve antibodies were chosen for further characterization.

Due to FTC's small size (247.2 Da) relative to antibody-binding surfaces, quantitation by enzyme immunoassay (EIA) using a single antibody can only be performed in a competitive format where known concentrations of free FTC would compete with an immobilized FTC-substrate for antibody binding. Only one antibody (5D2) exhibited specificity for FTC (Fig. 1a) in this assay. The dynamic linear range of the purified antibody for FTC detection was from 100 μg/ml to 3 μg/ml (Fig. 1b). The limit of detection for this assay was estimated by comparing the number of wells at 1.56 and 3.125 μg/ml that had greater than 0.1 difference in absorbance from the paired no FTC condition. All 10 of the 3.125 μg/ml wells were distinguishable from no FTC (Fig. 1c) while only seven of 10 of the 1.56 μg/ml wells were distinguishable from no FTC (Fig. 1d). Thus, the limit of detection of our FTC EIA is estimated to be 3 μg/ml.

F1
Fig. 1:
Competitive enzyme immunoassay characterization of anti-FTC antibodies.

Because FTC is a nucleoside analog, the mAb 5D2 was tested for cross-reactivity to nucleosides and FTC structural analogs and anabolites. A competitive EIA was used to evaluate the reactivity of 5D2 to naturally occurring structural analogs consisting of nitrogenous nucleic acid bases, ribonucleosides, and deoxyribonucleosides. 5D2 did not bind to any of the 10 ribonucleosides or deoxyribonucleosides tested, nor did it bind to FTC-triphosphate, the pharmacologically active anabolite of FTC (Fig. 1e). Assessment of two structurally similar antiretroviral drugs, lamivudine (3TC) and abacavir, along with the pharmacological intermediate, FTC-diphosphate, further demonstrated the high specificity of 5D2 binding to FTC (Fig. 1f). FTC is an analog of deoxycytidine with two significant differences: first, a sulfur substitution for the 3′ carbon, a motif that it shares with 3TC, and second, a fluorine addition to the pyrimidine ring which is unique to FTC. Given that 5D2 does not recognize cytidine, deoxycytidine, nor 3TC, we hypothesize that the fluoride-modified pyrimidine ring is the primary antigenic motif recognized by 5D2. Indeed, fluorine substitutions, which are widely used in biomedical drug development [20], have been shown to increase antibody affinity when employed in cocaine-hapten and anti-cancer sialyl-Tn vaccine studies [21,22]. However, despite strong affinity for the native FTC structure, this antibody does not bind to the di-phosphate or tri-phosphate anabolites. It is possible that the phosphate groups could first, mask the antigenic contribution of the 5′ hydroxyl; second, sterically hinder 5D2 from binding to the putative fluoro-pyrimidine antigen; or third, modify the fluoro-pyrimidine motif's chemical properties (e.g., hydrophobicity, electrochemical charge, etc.). Further studies are needed to determine the exact immunological contacts between 5D2 and FTC.

Taken together, the present data support the utility of 5D2 in antibody-based assays developed for clinical adherence monitoring, including POC test formats that allow immediate and low-cost detection in clinical settings. Because daily dosing is known to concentrate FTC in the urine to high levels, ranging between 10 and 100 μg/ml [23], our data point to the feasibility of 5D2 use in POC lateral flow assays for urine testing which has additional feasibility and acceptability advantages [24]. For ART, a simple urine test for FTC may allow for enhanced adherence counseling and might better inform the need for viral load and drug resistance testing. In sum, despite the challenges of raising antibodies to a small nucleoside analog, we were able to generate a highly specific antibody to FTC. The availability of 5D2 will enable the development of low-cost antibody-based adherence tests for FTC to improve the effectiveness of PrEP and ART.

Acknowledgements

Author contributions: Conceptualization: C.-P.C., T.C.G., J.P., W.M.S., and W.H.; methodology: A.S.Y., M.S.R., T.C.G., C.-P.P., J.P., W.M.S., and W.H.; validation: A.S.Y., M.S.R., T.C.G., and C.-P.P.; formal analysis: A.S.Y., T.H.V., M.S.R., T.C.G., and C.-P.P.; investigation: A.S.Y., M.S.R., T.C.G., and C.-P.P.; resources: M.S.R., J.P.; data curation: A.S.Y., T.H.V.; writing – original draft preparation: A.S.Y., T.H.V., and W.H.; writing – review and editing: A.S.Y., T.H.V., M.S.R., T.C.G., C.-P.P., J.P., W.M.S., W.H.; visualization: T.H.V.; supervision: J.P., W.M.S., and W.H.; project administration: J.P., W.M.S., and W.H.; funding acquisition: W.H. All authors have read and agreed to the published version of the article.

The findings and conclusions of this article are those of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention (CDC).

Conflicts of interest

A.S.Y, T.C.G., C-P.P., J.P., W.M.S., and W.H. are named in US Government patents (US20210253738) and patent applications on ‘Monoclonal antibody for the detection of the antiretroviral drug emtricitabine (FTC, 2′,3′-DIDEOXY-5-FLUORO-3′-THIACYTIDINE)’.

References

1. Saag MS, Benson CA, Gandhi RT, Hoy JF, Landovitz RJ, Mugavero MJ, et al. Antiretroviral drugs for treatment and prevention of HIV infection in adults: 2018 recommendations of the International Antiviral Society-USA Panel. JAMA 2018; 320:379–396.
2. World Health Organization. Updated recommendations on first-line and second-line antiretroviral regimens and postexposure prophylaxis and recommendations on early infant diagnosis of HIV. Geneva: World Health Organization; 2018.
3. Bangsberg DR, Hecht FM, Charlebois ED, Zolopa AR, Holodniy M, Sheiner L, et al. Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population. AIDS 2000; 14:357–366.
4. Dimitrov DT, Masse BR, Donnell D. PrEP adherence patterns strongly affect individual HIV risk and observed efficacy in randomized clinical trials. J Acquir Immune Defic Syndr 2016; 72:444–451.
5. Montaner JS, Reiss P, Cooper D, Vella S, Harris M, Conway B, et al. A randomized, double-blind trial comparing combinations of nevirapine, didanosine, and zidovudine for HIV-infected patients: the INCAS Trial. Italy, The Netherlands, Canada and Australia Study. JAMA 1998; 279:930–937.
6. Paterson DL, Swindells S, Mohr J, Brester M, Vergis EN, Squier C, et al. Adherence to protease inhibitor therapy and outcomes in patients with HIV infection. Ann Intern Med 2000; 133:21–30.
7. Viswanathan S, Justice AC, Alexander GC, Brown TT, Gandhi NR, McNicholl IR, et al. Adherence and HIV RNA suppression in the current era of highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2015; 69:493–498.
8. Kimulwo MJ, Okendo J, Aman RA, Ogutu BR, Kokwaro GO, Ochieng DJ, et al. Plasma nevirapine concentrations predict virological and adherence failure in Kenyan HIV-1 infected patients with extensive antiretroviral treatment exposure. PLoS One 2017; 12:e0172960.
9. Gandhi M, Glidden DV, Liu A, Anderson PL, Horng H, Defechereux P, et al. Strong correlation between concentrations of tenofovir (TFV) emtricitabine (FTC) in hair and TFV diphosphate and FTC triphosphate in dried blood spots in the iPrEx open label extension: implications for preexposure prophylaxis adherence monitoring. J Infect Dis 2015; 212:1402–1406.
10. Landovitz RJ, Beymer M, Kofron R, Amico KR, Psaros C, Bushman L, et al. Plasma tenofovir levels to support adherence to TDF/FTC preexposure prophylaxis for HIV prevention in MSM in Los Angeles, California. J Acquir Immune Defic Syndr 2017; 76:501–511.
11. Marrazzo JM, Ramjee G, Richardson BA, Gomez K, Mgodi N, Nair G, et al. Tenofovir-based preexposure prophylaxis for HIV infection among African women. N Engl J Med 2015; 372:509–518.
12. Haberer JE, Kiwanuka J, Nansera D, Ragland K, Mellins C, Bangsberg DR. Multiple measures reveal antiretroviral adherence successes and challenges in HIV-infected Ugandan children. PLoS One 2012; 7:e36737.
13. Kagee A, Nel A. Assessing the association between self-report items for HIV pill adherence and biological measures. AIDS Care 2012; 24:1448–1452.
14. Drain PK, Bardon AR, Simoni JM, Cressey TR, Anderson P, Sevenler D, et al. Point-of-care and near real-time testing for antiretroviral adherence monitoring to HIV treatment and prevention. Curr HIV AIDS Rep 2020; 17:487–498.
15. Pratt GW, Fan A, Melakeberhan B, Klapperich CM. A competitive lateral flow assay for the detection of tenofovir. Anal Chim Acta 2018; 1017:34–40.
16. Gandhi M, Bacchetti P, Spinelli MA, Okochi H, Baeten JM, Siriprakaisil O, et al. Brief report: Validation of a urine tenofovir immunoassay for adherence monitoring to PrEP and ART and establishing the cutoff for a point-of-care test. J Acquir Immune Defic Syndr 2019; 81:72–77.
17. Gandhi M, Wang G, King R, Rodrigues WC, Vincent M, Glidden DV, et al. Development and validation of the first point-of-care assay to objectively monitor adherence to HIV treatment and prevention in real-time in routine settings. AIDS 2020; 34:255–260.
18. Johnson KA, Niu X, Glidden DV, Castillo-Mancilla JR, Yager J, MaWhinney S, et al. Lower urine tenofovir concentrations among individuals taking tenofovir alafenamide versus tenofovir disoproxil fumarate: implications for point-of-care testing. Open Forum Infect Dis 2021; 8:ofab200.
19. Okochi H, Louie A, Phung N, Zhang K, Tallerico RM, Kuncze K, et al. Tenofovir and emtricitabine concentrations in hair are comparable between individuals on tenofovir disoproxil fumarate versus tenofovir alafenamide-based ART. Drug Test Anal 2021; 13:1354–1370.
20. Muller K, Faeh C, Diederich F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007; 317:1881–1886.
21. Song C, Zheng XJ, Liu CC, Zhou Y, Ye XS. A cancer vaccine based on fluorine-modified sialyl-Tn induces robust immune responses in a murine model. Oncotarget 2017; 8:47330–47343.
22. Cai X, Tsuchikama K, Janda KD. Modulating cocaine vaccine potency through hapten fluorination. J Am Chem Soc 2013; 135:2971–2974.
23. Haaland RE, Martin A, Livermont T, Fountain J, Dinh C, Holder A, et al. Brief report: Urine emtricitabine and tenofovir concentrations provide markers of recent antiretroviral drug exposure among HIV-negative men who have sex with men. J Acquir Immune Defic Syndr 2019; 82:252–256.
24. Marrazzo JM, Scholes D. Acceptability of urine-based screening for Chlamydia trachomatis in asymptomatic young men: a systematic review. Sex Transm Dis 2008; 35: (11 Suppl): S28–S33.
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