Research Publications/Outputs

Permanent URI for this community

http://hdl.handle.net/10204/11481

Browse

Now showing 1 - 6 of 6

Comparison of the antiviral activity of the microbicide candidate griffithsin and its tandemers derivatives against different modes of HIV-1 transmission
(2020-05) Alexander, Kabamba; Malatji, Kanyane; Mulaudzi, Takalani
Tandemers 2MG, 2MG3, 3MG and 4MG are derivatives of the potent anti-HIV-1 microbicide candidate griffithsin (GRFT). We compared these compounds anti-HIV-1 activity to GRFT using the viruses CAP206.08 and CAAN5342.A2 that have decreased sensitivity to this lectin. The 2MG and 2MG3 tandemers had similar activity to GRFT against cell-free and cell-associated viruses, while 3MG and 4MG were significantly more potent. Furthermore, the restoration of the 234N or 295N glycan in these viruses, known to increase sensitivity to GRFT, also increased sensitivity to 2MG and 2MG3, and not to 3MG and 4MG. In addition, GRFT resistant viruses generated in-vitro were equally resistant to 2MG and 2MG3 while they had considerably low resistance to 3MG and 4MG. Lastly, all five compounds showed increased inhibitory activity in seminal and vaginal simulants although the effect was more pronounced in the former. These data support further studies of tandemers as potential microbicides.
Development of a multiplex HIV/TB diagnostic assay based on the microarray technology
(2023-09) Malatji, Kanyane; Singh, Advaita A; Thobakgale, C; Alexandre, Kabamba
Currently there are diagnostic tests available for human immunodeficiency virus (HIV) and tuberculosis (TB); however, they are still diagnosed separately, which can delay treatment in cases of co-infection. Here we report on a multiplex microarray technology for the detection of HIV and TB antibodies using p24 as well as TB CFP10, ESAT6 and pstS1 antigens on epoxy-silane slides. To test this technology for antigen-antibody interactions, immobilized antigens were exposed to human sera spiked with physiological concentrations of primary antibodies, followed by secondary antibodies conjugated to a fluorescent reporter. HIV and TB antibodies were captured with no cross-reactivity observed. The sensitivity of the slides was compared to that of high-binding plates. We found that the slides were more sensitive, with the detection limit being 0.000954 µg/mL compared to 4.637 µg/mL for the plates. Furthermore, stability studies revealed that the immobilized antigens could be stored dry for at least 90 days and remained stable across all pH and temperatures assessed, with pH 7.4 and 25 °C being optimal. The data collectively suggested that the HIV/TB multiplex detection technology we developed has the potential for use to diagnose HIV and TB co-infection, and thus can be developed further for the purpose.
In vitro study on efficacy of PHELA, an African traditional drug against SARS-CoV-2
(2022-06) Matsabisa, MG; Alexandre, Kabamba B; Ibeji, CU; Tripathy, S; Erukainure, OL; Malatji, Kanyane; Chauke, Sipho H; Okole, Blessed N; Chabalal, HB
In 2019, coronavirus has made the third apparition in the form of SARS-CoV-2, a novel strain of coronavirus that is extremely pathogenic and it uses the same receptor as SARS-CoV, the angiotensin converting enzyme 2 (ACE2). However, more than 182 vaccine candidates have been announced; and 12 vaccines have been approved for use, although, even vaccinated individuals are still vulnerable to infection. In this study, we investigated PHELA, recognized as an herbal combination of four exotic African medicinal plants namely; Clerodendrum glabrum E. Mey. Lamiaceae, Gladiolus dalenii van Geel, Rotheca myricoides (Hochst.) Steane & Mabb, and Senna occidentalis (L.) Link; as a candidate therapy for COVID-19. In vitro testing found that PHELA inhibited> 90% of SARS-CoV-2 and SARS CoV infection at concentration levels of 0.005 mg/ml to 0.03 mg/ml and close to 100% of MERS-CoV infection at 0.1 mg/ml to 0.6 mg/ml. The in vitro average IC50 of PHELA on SARS-COV-2, SARS-CoV and MERS-COV were ~ 0.01 mg/ml. Secondly in silico docking studies of compounds identifed in PHELA showed very strong binding energy interactions with the SARS-COV-2 proteins. Compound 5 showed the highest afnity for SARS-COV-2 protein compared to other compounds with the binding energy of - 6.8 kcal mol-1. Our data showed that PHELA has potential and could be developed as a COVID-19 therapeutic.
New transmission-selective antimalarial agents through hit-to-lead optimization of 2-([1,1'-Biphenyl]-4-carboxamido)benzoic acid derivatives
(2022-09) Reader, J; Opperman, DFL; Van der Watt, ME; Theron, Anjo; Leshabane, M; Da Rocha, S; Turner, J; Garrabrant, K; Piña, I; Mills, C
Malaria elimination requires multipronged approaches, including the application of antimalarial drugs able to block human-to-mosquito transmission of malaria parasites. The transmissible gametocytes of Plasmodium falciparum seem to be highly sensitive towards epidrugs, particularly those targeting demethylation of histone post-translational marks. Here, we report exploration of compounds from a chemical library generated during hit-to-lead optimization of inhibitors of the human histone lysine demethylase, KDM4B. Derivatives of 2-([1,1'-biphenyl]-4-carboxamido) benzoic acid, around either the amide or a sulfonamide linker backbone (2-(arylcarboxamido)benzoic acid, 2-carboxamide (arylsulfonamido)benzoic acid and N-(2-(1H-tetrazol-5-yl)phenyl)-arylcarboxamide), showed potent activity towards late-stage gametocytes (stage IV/V) of P. falciparum, with the most potent compound reaching single digit nanomolar activity. Structure-activity relationship trends were evident and frontrunner compounds also displayed microsomal stability and favourable solubility profiles. Simplified synthetic routes support further derivatization of these compounds for further development of these series as malaria transmission-blocking agents.
Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications
(2020-04) Singh, Advaita Acarya; Pooe, O; Kwezi, Lusisizwe; Lotter-Stark, T; Stoychev, Stoyan H; Alexandra, Kabamba B; Gerber, Isak B; Bhiman, JN; Vorster, J; Pauly, M; Zeitlin, L; Whaley, K; Mach, L; Steinkellner, H; Morris, L; Tsekoa, Tsepo L; Chikwamba, Rachel K
Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.
Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
(2022-08) Singh, Advaita A; Pillay, Priyen; Naicker, Previn; Alexandre, Kabamba B; Malatji, Kanyane; Mach, L; Steinkellner, H; Vorster, J; Chikwamba, Rachel K; Tsekoa, Tsepo L
The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant XTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and XTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.

Browse

Browsing Research Publications/Outputs by browse.metadata.impactarea "Array Print Compan Diagnostics"

Results Per Page

Sort Options