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Developmentofaptamerbased HIV-1 entryinhibitorprophylactic drugs Grace London CSIR, Biosciences Emerging Health Technologies Platform Pretoria, South Africa 2 nd International Conference and Exhibition on Pathology
Properties of Aptamers Aptamers are antibodies nucleic acids with properties of Generated by simple in vitro process called SELEX High affinity and specificity Small in size and fold in 3 D structure (e.g RNA aptamers) Joubert et al., 21 Resistant to nucleases and chemically stable Low toxicity and non-immunogenic
Applications of aptamers Marro et al., 25 Rotherham et al., 212 Rusconi et al., 24 Green et al., 21 Appl. Microbiol. Biotechnol., 25, Nov 11, 1-8
Anti-gp12 aptamers as HIV-1 entry inhibitors Apt 1 Apt 2 Apt 1 bind gp12 trimer Apt 2 bind recombinant gp12 Apt = aptamer Khati et al., 23; Cohen et al., 28
HIV-1 entry and inhibitors HIV HIV gp41 gp12 CCR5 CXCR4 gp12- CD4 receptor binding CD4 gp12 Co-receptor binding gp41 Mediated Fusion HIV Host Cell Pierson and Doms., Current Top Microbiol Immunol (23) 281 pp 1-27
Outline of the study 1. Evaluate efficacy of anti-gp12 aptamer subtype C 2. Test toxicity 3. Map aptatope s HIV-1 on 4. Tes t gp12 synergy with other entry inhibitors agains t HIV- 1
Aptamers inhibit entry of HIV-1 subtype C Env pseudoviruses Aptamer IC 5 Env clone Stages of disease Geographic location Apt 1 Apt 2 CAP45.2..G3 Acute/early S.Africa 2.6.3 ZM233M.PB6 Acute/early Zambia 4.9.1 ZM249M.PL1 Acute/early Zambia >5.6 ZM53M.PB12 Acute/early Zambia 2.1.8 ZM19F.PB4 Acute/early Zambia 14.1 >5 ZM197M.PB7 Acute/early Zambia 19.2.4 CAP21.2.E8 Acute/early S.Africa 2.2.1 ZM135M.PL1a Acute/early Zambia 2.8 NT ZM214M.PL5 Acute/early Zambia 17.6.2 DU172.17 Acute/early S.Africa 12.1.6 DU156.12 Acute/early S.Africa 3.3.6 DU422.1 Acute/early S.Africa >5.3 CAP8.2..F6 Acute/early S.Africa 1.2 1 CAP61.2..F1 Acute/early S.Africa.3.4 CAP63.2..A9J Acute/early S.Africa 1.7.1 CAP84.2..32J Acute/early S.Africa 29.1 >5 CAP85.2..9J Acute/early S.Africa 16.1.1 CAP239.2..G3J Acute/early S.Africa 7.1.2 RP1.12* Acute/early S.Africa 4.7.6 RP4.3 Acute/early S.Africa >5.5 COT6.15 Chronic S.Africa.9 >5 COT9.6 Chronic S.Africa 6.1 >5 DU151.2 Acute/early S.Africa >5 >5 DU123.6 Acute/early S.Africa >5 >5 Conc Acute/early S.Africa 5.1 CAP288.2..5 Acute/early S.Africa 9.5.3 CAP26.2..E8 Acute/early S.Africa 3.8.7 CAP244.2..D3 Acute/early S.Africa 2.3.4 RP6.6 Acute/early S.Africa 3.7.4 CAP88.2..B6J Acute/early S.Africa 1.6 1 IN8362.25 Acute/early India 18.2 NT IN1395.211 Acute/early India.2 NT 5 nm = No inhibition NT = Not titred * viruses using CXCR4 coreceptor Apt 1 = inhibited 84 % viruses Mean IC 5 6.6 ± 8.1 nm Apt 2 = inhibited 79 % viruses Mean IC 5.4 ±.3 nm % viruses neutralized 84% 79% Mean IC 5 (nm) 6.6 ± 8.1.4 ±.3
Aptamers inhibit entry of PBMC HIV-1 subtype C Virus control Apt 2 Aptamer[nM] Aptamer][nM] Mean IC 8 in PMBC = 8 ± 11.8 nm Aptamer[nM] Aptamer [nm]
Aptamers subtype inhibit entry of HIV-1 C in Macrophages Du422 IC 8 = 6 Du42 nm IC2 8 = 6 nm COT9 IC 8 = 29 COT nm 7.5 2 IC 8 9 = 29 nm UCLA1 (nm) 7.5 2 Virus 5. Du422 COT *** p Control < IC NS 8 = 6 nm IC 8 9= 29 nmvir UCLA1.1 * u p s 1 (nm) <C o. n t 5 rol 5. IC 8 7.5 2.5 NS: not NS 2 significant UCLA1 Virus control (nm) 1 *** p <.1 Virus Apt 2Control )l * p < 2.. m.5 5. 5 *** p <.1 3.6 11 33 1 CoNnStr 3.6 11 33 1 NS: not * p <.5 Control significant Apt UCLA1 (nm) ol Aptamer [nm] NS: not significant amer [nm]. 2.5 1 UCLA1 (nm) 3.6 11 Contr 3.6 11 33ADA 1 SW 33 14 1 ol Control P24 (ngpm24l)(ngml) P24 (ngml) p24 (ng/ml) p24 (ng/ml) IUCC8LA=12( UCIC L8A1 = (n 3 M ) n6mn) SW11 M nmada3. IC 8 4 = 26 U n C M LA1 IC 6 UCLA1 (nm) (nm) 8 = 3 3 2 nm 3 4 IC ADA 8 SW14 = 26 nm 2 IC IC 8 8 = 3 nm 3. 4 3.6 33 1 Control 11 33 1 p24 (ng/ml)p24 (ng/ml) 1 3 3 4 2 1 2 3 2 3.6 11 33 1 3.6 11 33 1 ntrol 1 Co Control UCLA1 (nm) 1 UCLA1 2 (nm) 1 1 3.6 11 33 1 3.6 11 33 1 Contr ol Contro l AptaUmCeLrA[n1M(n]M) er[nm] p24 (ng/ml) 3.6 11 33 1 UCLA1 (nm) p24 (ng/ml) p24 (ng/ml) p24 (ng/ p24 (ng/ml) Control AptamU CLA1 (nm) 3.6 11 33 1 UCLA1 (nm) Control Control Mean IC 8 in MDM = 23 ± 1.4 nm
Aptamers exhibit no cytotoxicity TZM-bl MTS based-assay 12 12 PBMC MTS based-assay 1 1 % Cell Viability 8 8 6 6 4 4 % Cell Viability 2 2
Aptamers interact with conserved residues on gp12 Aptamers bind to amino acids within the coreceptor (CoRbs) CCR5 binding site Mufhandu, H et al., J. Virol.(212,)86(9), pp. 4989
Synergy of aptamers with inhibitors HIV-1 entry Du156 T2 ZM249 T2 1 1 U A C p L t A 2 1 + T b 2 1 % inhibition 75 75 1 5 Du156 T 2 5 1 7525 25 75 ZM249 T 2 2 UACpLtA21 TT22 UCLA1+T2 UCLA1 T2 %%inihnibhiit % bioint I i n o h n i 5 5.1.1 1 1 1.1.1 1 1 1 25.1 1 Du172 b12.1 1 1 1 1 75 75 µm Lo 2 g 5 Synergism :CI=.3.9 Antagonism:CI=>1.1 SW7 b12.1 1 1 1 UCLA1+b12 UCLA1 b12 1 1 5 5 UCLA1+b12 Apt 2 + b12 UCLA1 Apt 2 75 75 T2 25 25 b12 5 5.1.1 1 1 1.1 25 25 bition Du172 b12 µm (Log)( SW7 b12.1 1 1 1.1.1 1 1 1.1.1 1 1 1 µm (Log)
Summary agains t Antigp12 subtype C isolates( A s concentration efficacious in RN aptamer are nanomolar) HIV- 1 They interact with conserved residues on gp12, delay virus resistance Not toxic in different cell types Synergy with other entry inhibitors, combination therapy with other drugs Anti-gp12 aptamers can be developed as entry inhibitor drugs
Acknowledgements Collaborators NICD (Lynn Morris) UKZN (Alexander Pym) UCT & GSH (B. Mayosi) University of Oxford, UK (William James) Funding The Scripps Research Institute, USA (Dennis Burton) Reagents Los Alamos National Lab, USA (Basil I Swanson) IAVI NIH AIDS Reagents
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