Molecular dynamic simulations to investigate the structural impact of known drug resistance mutations on HIV-1C integrase-dolutegravir binding

Resistance associated mutations (RAMs) threaten the long-term success of combination antiretroviral therapy (cART) outcomes for HIV-1 treatment. HIV-1 Integrase (IN) strand transfer inhibitors (INSTIs) have proven to be a viable option for highly specific HIV-1 therapy. The INSTI, Dolutegravir is recommended by the World Health Organization for use as first-line cART. This study aims to understand how RAMs affect the stability of IN, as well as the binding of the drug Dolutegravir to the catalytic pocket of the protein. A homology model of HIV-1 subtype C IN was successfully constructed and validated. The site directed mutator webserver was used to predict destabilizing and/or stabilizing effects of known RAMs while FoldX confirmed any changes in protein energy upon introduction of mutation. Also, interaction analysis was performed between neighbouring residues. Three mutations known to be associated with Raltegravir, Elvitegravir and Dolutegravir resistance were selected; E92Q, G140S and Y143R, for molecular dynamics simulations. The structural quality assessment indicated high reliability of the HIV-1C IN tetrameric structure, with more than 90% confidence in modelled regions. Change in free energy for the three mutants indicated different effects, while simulation analysis showed G140S to have the largest affect on protein stability and flexibility

Drug resistance mutations against protease, reverse transcriptase and integrase inhibitors in people living with HIV-1 receiving boosted protease inhibitors in South Africa

The South African national combination antiretroviral therapy (cART) roll-out program started in 2006, with over 4.4 million people accessing treatment since it was first introduced. HIV-1 drug resistance can hamper the success of cART. This study determined the patterns of HIV-1 drug-resistance associated mutations (RAMs) in People Living with HIV-1 (PLHIV-1). Receiving first (for children below 3 years of age) and second-line (for adults) cART regimens in South Africa. During 2017 and 2018, 110 patients plasma samples were selected, 96 samples including those of 17 children and infants were successfully analyzed. All patients were receiving a boosted protease inhibitor (bPI) as part of their cART regimen. The viral sequences were analyzed for RAMs through genotypic resistance testing. We performed genotypic resistance testing (GRT) for Protease inhibitors (PIs), Reverse transcriptase inhibitors (RTIs) and Integrase strand transfer inhibitors (InSTIs). Viral sequences were subtyped using REGAv3 and COMET. Based on the PR/RT sequences, HIV-1 subtypes were classified as 95 (99%) HIV-1 subtype C (HIV-1C) while one sample as 02_AG

Structural comparison of diverse HIV-1 subtypes using molecular modelling and docking analyses of integrase inhibitors

CITATION: Isaacs, Darren et al. 2020. Structural comparison of diverse HIV-1 subtypes using molecular modelling and docking analyses of integrase inhibitors. Viruses, 12(9):936, doi:10.3390/v12090936.The original publication is available at: https://www.mdpi.comENGLISH ABSTRACT: The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance.Publisher's versio

Fas mediated(CD95L) periferal T-cell Apotosis marker in monitoring HIV-1 disease progression in adults in Yaoundé, Cameroon

CITATION: Ikomey, G. M. et al. 2016. Fas mediated(CD95L) periferal T-cell Apotosis marker in monitoring HIV-1 disease progression in adults in Yaoundé, Cameroon. International Journal of Immunology, 4(1): 1-5, doi:10.11648/j.iji.20160401.11.The original publication is available at http://www.sciencepublishinggroup.com/j/ijisFas (CD95) / FasL are hallmarks of apoptosis involvement in pathogenesis of HIV. We assess changes in soluble Fas /FasL, CD4 % and HIV-1 viral load in patients prior to the initiation of antiretroviral therapy (ART) and 6 months thereafter. A prospective longitudinal study on sixty consented HIV-1 positive adults. sFas and sFasL levels were measured by ELISA. CD4 cell counts and HIV-1 viralloads were measured using standard methods. Samples were analysed according to the manufacturers’ guidelines.There was a significant positive correlation between HIV-1 viral load and FasL at six months (M6) on treatment [r = +0.49, (0.03)]. There were no correlation between sFas/FasL and CD4 cell counts [ r = -33 (0.16), -31 (0.17) - 23 (0.03) respectively]. The significant correlation between sFasL and HIV-1 viral load at six months of ART suggests that sFasL could be a signal biomarker for HIV-1 disease progression. We have shown in this study that high levels of sFasL depict high HIV-1 viral loads and advance state of the HIV disease. These biomarker should be investigated further in other settings.http://www.sciencepublishinggroup.com/home/indexhttp://article.sciencepublishinggroup.com/html/10.11648.j.iji.20160401.11.htmlPublisher's versio

Peripheral blood lymphocyte proviral DNA predicts neurocognitive impairment in clade C HIV

CITATION: Ruhanya, V. et al. 2020. Peripheral blood lymphocyte proviral DNA predicts neurocognitive impairment in clade C HIV. Journal of NeuroVirology, 26:920–928, doi:10.1007/s13365-020-00882-9.The original publication is available at https://link.springer.comIt is not known if proviral DNA in the periphery corresponds to cognitive status in clade C as it does in clade B and recombinant forms. A cross-sectional study was conducted on participants investigated for HIV-associated neurocognitive impairment in South Africa. HIV-1 proviral DNA was quantified using a PCR assay targeting a highly conserved HIV-1 LTR-gag region. Fifty-four (36.7%) participants were cognitively impaired and 93 (63.3%) were not impaired. Forty-three (79.6%) of the cognitively impaired participants were female and 11 (20.4%) were male. There was no significant age difference between cognitively impaired and unimpaired participants (p = 0.42). HIV-1 DNA in cognitively impaired PLWH was significantly higher than in cognitively normal individuals (p = .016). Considering impaired participants, lymphocyte HIV-1 DNA was significantly higher in males than females (p = 0.02). There was a modest positive correlation between lymphocyte HIV-1 DNA and global deficit scores (GDS) r = 0.176; p = 0.03). The two measures of viral load, lymphocyte HIV-1 DNA copies/million and plasma RNA copies/ml, were positively correlated (r = 0.39; p < .001). After adjusting for other covariates, age, sex, treatment status, and the interactions between impairment and treatment, the multivariate regression showed association between proviral load and neurocognitive impairment; omega effect size was 0.04, p value = 0.010. The burden of HIV-1 peripheral blood lymphocyte proviral DNA corresponds to neurocognitive impairment among individuals infected with clade C disease. Therefore, therapeutic strategies to reduce the HIV-1 proviral DNA reservoir in lymphocytes may improve neurocognitive outcomes in PLWH.Poliomyelitis Research Foundation (PRF)National Research Foundation (NRF)South African Medical Research Council (SAMRC)Publisher's versio

Structural comparison of diverse HIV-1 subtypes using molecular modelling and docking analyses of integrase inhibitors

The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance

Chronic obstructive pulmonary disease (COPD) : neutrophils, macrophages and lymphocytes in patients with anterior tuberculosis compared to tobacco related COPD

CITATION: Guiedem, E., et al. 2018. Chronic obstructive pulmonary disease (COPD): neutrophils, macrophages and lymphocytes in patients with anterior tuberculosis compared to tobacco related COPD. BMC Research Notes, 11:192, doi:10.1186/s13104-018-3309-6.The original publication is available at https://bmcresnotes.biomedcentral.comObjective: The inflammatory profile of chronic obstructive pulmonary disease (COPD) related to tobacco is known in certain studies while that of the post tuberculosis form is not yet known. This study aimed to evaluate the levels of neutrophils, macrophages and lymphocytes cells in sputum of COPD patients with history of smoking or anterior tuberculosis. Enumeration of cells in samples was analyzed using standard microscopy. Results: We enrolled 92 participants, 46 (50%) were COPD subjects comprising 22 (47.83%) smokers and 24 (52.17%) with anterior tuberculosis while 46 (50%) healthy persons constituted the control group. The levels of neutrophils, lymphocytes and monocytes were statistically higher in COPD patients compared to the control group with p-values of 0.0001 respectively. Neutrophils levels were higher in COPD patients with history of tobacco than in COPD patients with anterior tuberculosis with a mean rate of 4.72 × 106/ml and 2.48 × 106/ml respectively (p = 0.04). The monocytes and lymphocytes levels were not statistically different between the two sub-groups of COPD patients with p-value of 0.052 and 0.91 respectively. Neutrophils are the only inflammatory cells that were significantly higher in COPD patients with history of smoking as compared to COPD patients with anterior tuberculosis.https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-018-3309-6Publisher's versio

Kinetics of CD4+ T-cell recovery amongst HIV load suppressed patients on first-line antiretroviral therapy in Yaoundé, Cameroon

HIV infected patients on Antiretroviral Therapy (ART) are exposed to various immunological disorders. Immune reconstitution is one of the most challenging problem linked to morbidity and mortality in HIV patients. This study aimed at evaluating the kinetics of CD4+ T-cell recovery amongst HIV load suppressed patients on first-line ART in Yaoundé, Cameroon. This was a retrospective cohort study performed at the care and treatment units of the Yaoundé University Teaching Hospital and Essos Hospital Center, with viral suppressed patients initiated on ART between March and July 2015. Data were collected using a standard form and analyzed using R.3.6.2 software. A p&lt;0.05 was considered statistically significant for a 95%CI. Of the 499 viral suppressed participants, 32% (n=160) were male and 68% (n=339) female; 33% and 40% had severe and moderate immunodepression at baseline, respectively; 9% and 28% remain respectively on the same immunological state. CD4+ T-cell count increased by 73%, 49% and 29% for patients that started treatment, with CD4+ &lt;150 cells/ml, 150&lt;CD4+&lt;350 cells/ml and 350&lt;CD4+&lt;500 cells/ml, respectively and 14%, 34% and 40% reached a target of 500 cells/ml or more after 4 years of treatment. Elder patients and males were likely to have CD4+ T-cells less than 350 Cells/ml. Approximately 35% of patient started treatment with CD4+ T-cells &lt;350 Cells/ml. CD4+ T-cells increased significantly during 4 years of treatment but, just 29% in average achieved CD4+ ≥ 500 cells/ml. CD4 T-cells recovery represent and important challenge in the immunological monitoring of long-term HIV infected patients on ART

HIV-1 Resistenz-Analysen von nicht-therapierten Patienten aus Südafrika und Tansania und Charakterisierung eines neuen HIV-1 Subtyp C proviralen molekularen Klons

The acquired immunodeficiency syndrome (AIDS) is currently the most infectious disease worldwide. It is caused by the human immunodeficiency virus (HIV). At the moment there are ~33.3 million people infected with HIV. Sub-Saharan Africa, with ~22.5 million people infected accounts for 68% of the global burden. In most African countries antiretroviral therapy (ART) is administered in limited-resource settings with standardised first- and second-line ART regimens. During this study I analysed the therapy-naïve population of Cape Town, South Africa and Mwanza, Tanzania for any resistance associated mutations (RAMs) against protease inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors. My results indicate that HIV-1 subtype C accounts for ~95% of all circulating strains in Cape Town, South Africa. I could show that ~3.6% of the patient derived viruses had RAMs, despite patients being therapy-naïve. In Mwanza, Tanzania the HIV drug resistance (HIVDR) prevalence in the therapy-naïve population was 14.8% and significantly higher in the older population, >25 years. Therefore, the current WHO transmitted HIVDR (tHIVDR) survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Based on the prevalence rates of tHIVDR in the study populations it is recommended that all HIV-1 positive individuals undergo a genotyping resistance test before starting ART. I also characterized vif sequences from HIV-1 infected patients from Cape Town, South Africa as the Vif protein has been shown to counteract the antiretroviral activity of the cellular APOBEC3G/F cytidine deaminases. There is no selective pressure on the HIV-1 Vif protein from current ART regimens and vif sequences was used as an evolutionary control. As the majority of phenotypic resistance assays are still based on HIV-1 subtype B, I wanted to design an infectious HIV-1 subtype C proviral molecular clone that can be used for in vitro assays based on circulating strains in South Africa. Therefore, I characterized an early primary HIV-1 subtype C isolate from Cape Town, South Africa and created a new infectious subtype C proviral molecular clone (pZAC). The new pZAC virus has a significantly higher transient viral titer after transfection and replication rate than the previously published HIV-1 subtype C virus from Botswana. The optimized proviral molecular clone, pZAC could be used in future cell culture and phenotypic HIV resistance assays regarding HIV-1 subtype C.Das erworbene Immundefektsyndrom (“acquired immunodeficiency syndrome”, AIDS), verursacht durch das Humane Immundefizienzvirus (HIV), ist derzeit die häufigste Infektionskrankheit weltweit. Zirka 33,3 Millionen Menschen sind gegenwärtig mit HIV infiziert, wobei hiervon etwa 22,5 Millionen Infizierte (68%) in den Ländern südlich der Sahara leben. In den meisten dieser Länder ist die antiretrovirale Therapie (ART) in nur zwei standardisierten Medikamentenkombinationen verfügbar. In dieser Arbeit wurden nichttherapierte Patienten aus Kapstadt (Südafrika) und Mwanza (Tansania) auf resistenzassoziierte Mutationen (RAMs) gegen Protease Inhibitoren, nukleosidische- und nichtnukleosidische Reverse Transkriptase Inhibitoren analysiert. Meine Ergebnisse zeigten, dass in 3,6 % der Patienten RAMs gefunden wurden, obwohl diese nicht vortherapiert waren. In der Patientengruppe aus Tansania wurden sogar in 14,8 % der Patientenviren RAMs gefunden. Dieses Patientenkollektiv war signifikant älter als 25 Jahre und damit außerhalb der von der WHO beobachteten Altersgruppe. Meine Studie legt nahe, dass die WHO-Kriterien zur Überwachung der Übertragung von resistenten HIVs die Weitergabe von resistenten Viren unterschätzt, da Patienten über 25 Jahre ausgeschlossen werden. Weiterhin wurden vif Sequenzen von HIV-1 infizierten Patienten aus Kapstadt charakterisiert, da bereits gezeigt wurde, dass das HIV Vif Protein die antiretrovirale Aktivität der Cytidin Deaminase APOBEC3G/F antagonisieren kann. Da jedoch keine Medikamenten induzierte Selektion auf diesen Sequenzen liegt, wurden diese zur Analyse der viralen Evolution verwendet. Phenotypische Resistenzanalysen basieren gegenwärtig meist auf dem HIV Subtyp B, jedoch sind die meisten Infizierten in Südafrika und sogar weltweit mit Subtyp C infiziert. Deshalb war es ein Ziel dieser Arbeit einen proviralen HIV Subtyp C Plasmid zu entwickeln. Dazu wurde das Virus aus einem frühen HIV Subtyp C Isolat kloniert. Das hier neu klonierte Virus (HIV-ZAC) zeigt sowohl einen höheren viralen Titer nach der Transfektion und auch eine höhere Replikationsrate als das zuvor publizierte HIV-1 Suptyp C Virus aus Botswana. Deshalb könnte der von mir optimierte und neu charakterisierte provirale molekulare Klon, pZAC, zukünftig in der Zellkultur und bei phenotypischen HIV Resistenztests als wildtypisches HIV-1 Suptyp C Virus eingesetzt werden

Investigation of the molecular epidemiology of HIV-1 in Khayelitsha, Cape Town, using serotyping and genotyping techniques

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2005.There are currently an estimated 5.3 million people infected with human immunodeficiency virus / acquired immunodeficiency syndrome (HIV/AIDS) in South Africa. HIV-1 group M Subtype C is currently responsible for the majority of HIV infections in sub-Saharan Africa (56% worldwide). The Khayelitsha informal settlement, located 30 km outside Cape Town, has one of the highest HIV prevalence rates in the Western Cape. The objective of this study was to investigate the molecular epidemiology of HIV-1 in Khayelitsha using serotyping and genotyping techniques. Patient samples were received from the Matthew Goniwe general health clinic located at site C in Khayelitsha. Serotyping was performed through a competitive enzymelinked immunosorbent assay (cPEIA). RNA was isolated from patient plasma and a two step RT-PCR amplification of the gag p24, env gp41 IDR, env gp120 V3 and pol genome regions performed. Sequences obtained were used for detailed sequence and phylogenetic analysis. Neighbour-joining and maximum likelihood phylogenetic trees were drawn to assess the relationship between the Khayelitsha sequences obtained and a set of reference sequences obtained from the Los Alamos National Library (LANL) HIV database (http://www.hiv.lanl.gov/). Through serotyping and genotyping the majority of HIV strains were characterised as HIV-1 group M subtype C. One sample (1154) was characterised as a possible C / D recombinant strain. In 9 other samples HIV-1 recombination cannot be excluded, as only one of the gene regions investigated could be amplified and characterised in these samples. The gag p24 genome region was found to be more conserved than the env gp41 IDR, with the env gp41 IDR more conserved than the env gp120 V3. The variability of the env gp120 V3 region indicates that patients might be dually infected with variant HIV-1 subtype C strains or quasispecies. Conserved regions identified in the Khayelitsha sequences can induce CD4+ T-cell responses and are important antibody recognition target sites. These conserved regions can play a key role in the development of an effective HIV-1 immunogen reactive against all HIV-1 subtypes. The majority of subtype C viruses were predicted to use CCR5 as their major chemokine co-receptor. The pol sequences analysed indicate that mutations associated with minor resistance to Protease Inhibitors (PIs) might be present in the Khayelitsha community. The identification of resistant mutations is vital for people receiving antiretroviral treatment (ART). It can influence the success of their treatment and delay the onset of AIDS. Serotyping is a quick characterisation method, but not always accurate. With genotyping detailed molecular analysis can be performed. However, with genotyping the success of amplification often depends on viral load. In Southern Africa a subtype C candidate vaccine appears to be the best option for future vaccine considerations. The sporadic detection of non-subtype C and recombinant subtype C viruses remains a concern and will thus have to be closely monitored. Phylogenetic analysis can help to classify and monitor the spread and evolution of these viruses