Virology

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The development, optimisation and comparison of various virological assays and their uses in antiviral assessment of compounds wih potential anti-HIV activity.
(2009) Singh, Varish.; Parboosing, Raveen.
The development and optimization of anti-viral screening methods are essential to develop newer more effective, treatments against HIV. The XTT method is a widely described method for antiviral screening. Both continuous HIVinfected cells and experimentally infected T-cells have been used in the XTT assay. We compared these methods to screen several plant-derived extracts for cytotoxicity. Several considerations were taken into account when performing these tests (effect of media, solvents and plant enymes). Experiments were performed to investigate these effects. In addition, p24 and viral load quantification were compared as antiviral screening methods. The study showed that several modifications were necessary when performing the XTT assay on plant extracts, due to the effect of media, solvents and plant enymes. The XTT assays and p24 assays performed using experimentally infected cells are far more specific than those using chronically infected cells. The use of viral loads as an antiviral screening method consistently demonstrated the expected efficacy of AZT.
Molecular diagnosis and typing of HTLV-I in KwaZulu-Natal.
(1998) Tarin, Michelle Lucille.; York, Denis Francis.; Bhigjee, Ahmed Iqbal.
Two areas of the HTLV-I genome were targeted for an in-house molecular diagnostic test, namely the pol and env regions. The pol primers proved the most sensitive (100%)and specific (100%). Amplification using the env primer pair was not reproducible, and was not pursued further. The AmpliSensor assay (Acugen Systems, Lowell, MA) was also tested. The assay was very specific, but not as sensitive as our in-house PCR. To investigate the predominant HTLV-I subtype in the region, a 1535 by env gene was isolated from peripheral blood obtained from five local HTLV-I seropositive patients. Four of the patients presented with HAM/TSP, and the fifth presented with a skin disease. Nucleotide sequencing of the amplified products revealed the local strains to be very conserved, differing by 0.1% to 0.9% among themselves. No apparent difference was noted for the two clinical manifestations. Phylogenetic analysis was performed using repesentative strains from around the world. The local strains clearly fell within the cosmopolitan subtype. The local strains were most closely related to the North American strains suggesting an unexpected link between the two countries.
Molecular characterization of HIV-1 Subtype C strains from KwaZulu-Natal, South Africa, with a special emphasis on viral fitness and drug resistance.
(2004) Gordon, Michelle Lucille.; Cassol, Sharon.
As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management.
The epidemiology of dual HIV infection in the KwaZulu-Natal Anti-Retroviral Roll-out Programme.
(2007) Naidoo, Anneta Frances.; Parboosing, Raveen.
KwaZulu-Natal has the highest prevalence of HIV in South Africa. The prevalence of dual infection in a normal-risk population in this region is unknown. Dual HIV infection has important implications for diagnosis, treatment response and vaccine development. This cross-sectional study aimed to establish and optimize methods for subtyping and detection of dual infection in KZN. Samples were from chronically-infected patients on ARV treatment within the ARV Rollout Programme, from sites throughout KZN. Subtyping of the samples was performed using HMA. Four samples had indeterminate results by HMA and were then cloned and sequenced. Phylogenetic analysis showed that one of the four samples was a dual infection. This study showed 1/46(2%) samples to be dually infected which suggests that the prevalence of dual infection is low in the sample population. The low prevalence of dual infection reported could be due to the low-risk profile of the sample population. It was concluded that the low prevalence of dual infection is unlikely to have a considerable impact on HIV management.
Maedi-Visna virus : the development of serum and whole blood immunodiagnostic assays.
(1997) Boshoff, Christoffel Hendrik.; York, Denis Francis.; Conradie, Jan D.
This thesis describes the development of serum and whole blood immunodiagnostic assays for Maedi-Visna virus (MVV). All previously described recombinant MVV ELISA assays utilised either the core p25 or transmembrane (TM) proteins alone, or combined, but as individual proteins. The p25 and TM genes of MVV were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. Sera from 46 positive and 46 negative sheep were tested using the GST-TM and GST-TM-p25 ELISAs and a commercial p25 EIA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA. The GST-TM-p25 ELISA showed a sensitivity and specificity of 100%. The human AIDS lentivirus transmembrane (TM) glycoprotein portion of the envelope viral protein has been identified as the antigen most consistently recognised by antibodies. There is suggestive evidence that the same applies to MVV as the GST-TM fusion protein, expressed in E. coli, has comparable sensitivity to the GST-TM-p25 fusion protein, but lacks specificity. However, due to the hydrophobic nature of the MVV TM protein, purification of the expressed fusion protein required lengthy purification protocols. This was despite the fact that only a truncated version of the TM protein was expressed. This prompted investigating an alternative expression system that could possibly circumvent the above mentioned problems. The yeast Pichia pastoris is known to be suitable for the high-level expression of heterologous proteins which are secreted into the culture supernatant. These features made P. pastoris an attractive host for the expression of the hydrophobic TM protein of MVV. However, limited success was achieved as only low expression levels were obtained and detection and quantification was only accomplished by means of ELISA. Evaluation of the diagnostic performance of the P. pastoris expressed MVV TM-polypeptide was performed using a panel of 36 confirmed negative and positive sera, and evaluated using a TG-ROC analysis programme, which yielded an equal Se and Sp of 83%. The use of a novel rapid immunoassay system, which allows the detection of circulating antibodies in whole blood, has been investigated for use as a MVV diagnostic assay. The central feature of this immunoassay lies in a monoclonal antibody against a glycophorin epitope present on all sheep erythrocytes. A Fab'-peptide conjugate was constructed by coupling a synthetic peptide, corresponding to a sequence from MVV TM protein, to the hinge region of the Fab' fragment of the antisheep erythrocyte antibody. Within the limited number of 10 seronegative and 10 seropositive samples the autologous red blood cell agglutination assay had a sensitivity of 90% and a specificity of 80%. Despite the limitations and difficulties encountered, the use of such rapid whole blood immunodiagnostic assays for MVV holds promise.
An investigation into the serological and molecular diagnosis of Jaagsiekte Sheep Retrovirus (JSRV)
(2005) Padayachi, Nagavelli.; York, Denis Francis.
The Jaagsiekte Sheep Retrovirus (JSRV), an exogenous type B/D-retrovirus with about 10-15 endogenous counterparts in all normal sheep genomes, causes Jaagsiekte (JS) or ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. This sheep lung cancer has been identified as the best natural out-bred model that can be used to study human epithelial tumours. The close similarity between the pathology of the sheep disease and Human Bronchiolo-alveolar carcinoma are highly suggestive that the human disease could have a similar aetiology and mechanism to the sheep disease. However, in the case of sheep at the time of the study there was a need for an assay that could be used to screen for infected sheep. The isolation, cloning and subsequent sequencing of the first full-length exogenous and endogenous forms of JSRV contributed greatly towards JSRV research. Until recently the diagnosis of OPA was based mostly on clinical presentation with confirmation by micro and macro examination of the affected lungs by expert pathologists. In the absence of a specific humoral response no serology-based tests were available to diagnose the disease early in live animals. Control and management of the disease was primarily by regular flock inspections and prompt culling of the suspected cases. The objective of this research project was therefore to assess and investigate the serological and molecular diagnosis of JSRV. In an attempt to develop a serology based assay three proteins were identified as candidate diagnostic antigens, the group specific antigen JSRV p26, the transmembrane and the orf-X proteins. Genes coding for all three proteins were isolated, cloned and expressed. The JSRV p26 was sufficiently purified and its potential as a diagnostic antigen was evaluated in both a Western blot and ELISA. Our studies confirmed that there were no circulating antibodies to the JSRV capsid protein. Evidence suggested that the immune response was localised to the lungs. Lung lavage samples were therefore collected from infected and normal sheep and analysed for the presence of JSRV p26 antibodies using an in-house JSp26 peroxidase conjugate in an antigen capture assay. This assay lacked sensitivity but the results indicated that there was a specific localised immune response to JSRV in the lungs of OPA affected sheep. This was confirmed with an in-house antigen capture assay that we developed. JS antigen was detected in the lung and nasal fluid of affected sheep, but not in equivalent samples from normal sheep. Three molecular assays were investigated for their sensitivity and specificity, the LTR-gag PCR, U3/LTR hemi-nested PCR and the PCR that covered the V1/V2 region. The U3/LTR hemi-nested assay was 2 logs more sensitive than the LTR-gag PCR. However, it detected the endogenous JSRV5.9A1 loci at higher concentrations. This was overcome by designing a more specific primer P3M for the first step of the U3/LTR hemi-nested PCR and the use of the AmpliTaq Gold DNA polymerase. This assay proved to be both sensitive and specific enough to screen for the infectious exogenous JSRV in peripheral blood samples from individual sheep. It is now possible to use this assay to selectively eradicate the disease from a flock through a selective culling programme. Furthermore, the assay could be made quantitative by the inclusion of concentration standards.
The application of DNA hybridisation methods to a determination of the association of hepatitis B virus with cirrhosis and hepatoma.
(1987) Nair, Shamila.; Windsor, Isobel M.; Van den Ende, Jan.
Autopsy liver material from patients having died of chronic liver disease, cirrhosis, hepatocellular carcinoma (HCC) and causes unrelated to liver diseases was examined by dot blot hybridisation for the presence of HBV DNA. The results indicate that of the patients with chronic liver disease 6/9 were positive for HBV DNA in the liver tissue; of the patients with HCC 3/4 were positive for HBV DNA; of the patients with cirrhosis 4/4 showed the presence of HBV DNA in the liver. Thus by this technique 13/17 (76%) of these patients, all of whom were HBsAg positive, were shown to have HBV DNA present in liver tissue. However, autopsy liver samples were found to be unsuitable for Southern blot hybridisation. Biopsy liver/tumour tissue was examined for the presence of integrated or non-integrated HBV DNA by Southern blot analysis using the enzymes Eco R1 and Hind 111. 5/5 patients who were both HBsAg and HBeAg positive had extrachromosomal HBV DNA and 2/5 also showed the presence of integrated HBV DNA. 3/4 patients who were HBsAg positive and HBeAg negative had extrachromosomal HBV DNA and all three also had integrated HBV DNA. One control patient was negative for both markers and also for Southern blot hybridisation with the HBV DNA probe. These results support the hypothesis that HBV is a factor in the development of HCC, and indicate that the dot blot hybridisation method would be suitable for routine evaluation of patients with chronic liver disease or cirrhosis.
GB Virus C / Hepatitis G Virus (GBV-C/HGV) infection in KwaZulu Natal, South Africa : its diagnosis, distribution and molecular epidemiology.
(2003) Sathar, Mahomed Aslam.; York, Denis Francis.
Recently a new Flavivirus, GB Virus C also referred to as Hepatitis G virus (GBV-C/HGV) was identified in humans with indeterminate hepatitis . Whilst in non-African countries this discovery led to an enormous enthusiasm to elucidate an association with liver disease, very little was known about the prevalence and pathogenicity of GBV-C/HGV infection in KwaZulu Natal, South Africa, where Hepatitis B Virus (HBV) infection is endemic and infection with the Human immunodeficiency virus (HIV) is a catastropic health problem. Sera from patients with liver disease (chronic liver disease [n = 98]; alcoholic liver disease [n = 50]); high risk groups (haemodialysis patients [n = 70]; HIV positive mothers and their babies [n = 75]) and control groups (alcoholics without liver disease [n = 35] and blood donors from the four racial groups [n = 232]) were screened for GBV-C/HGV RNA and Anti-E2 antibodies by reverse transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay (ELISA), respectively. Overall 43.9% (43/98) of patients with chronic liver disease; 60 % (30/50) of patients with alcoholic liver disease; 47.1% (33/70) of haemodialysis patients; 60% (21/35) of alcoholics without liver disease and 31.9% (74/232) of blood donors (Africans] 44/76; 5.9%); Asians (5/52; 9.6%); Whites (15/49; 30.6%) and "Coloureds" [mixed origin] (9/54; 16.6%)]) were exposed to GBV-C/HGV infection as determined by the detection of Anti-E2 &/or RNA in serum. There was a significant difference in the prevalence of GBV-C/HGV infection (RNA &/or anti E2) between African blood donors and the other racial groups (p < 0.001), between blood donors and haemodialysis patients (p = 0.02) and or patients with chronic liver disease (p =0.04). There was no significant difference in the prevalence of GBV-C/HGV between African blood donors (45/76, 59.2%) and alcoholics with and without liver disease (30/50, 60% and 21/35, 60%, respectively). Anti-E2 antibodies and GBV-C/HGV RNA were almost mutually exclusive. GBV-C/HGV infected dialysis patients tended to have had more transfusions (p = 0.03) and had a longer duration of dialysis than non infected patients, indicating that the majority of patients on maintenance haemodialysis acquire their GBV-C/HGV infection through the transfusions they receive. There was no evidence for in utero and/or intrapartum transmission of GBV-C/HGY. However, there is some mother-to-infant transmission of GBV-C/HGV, though it is very probable that in KZN GBV-C/HGV is transmitted by as yet undefined non-parenteral routes. Sequence and phylogenetic analysis of the 5' non-coding region (5' NCR) and E2 gene segments of the GBV-C/HGV genome identified an additional "genotype" (Group 5) of GBV-C/HGV that is distinct from all other known GBV-C/HGV sequences (Groups 1-4). Although there is a high prevalence of Group 5 GBV-C/HGV isolates in KZN, there was no significant difference in liver biochemistry between GBV-C/HGV infected and noninfected patients with liver disease or between blood donors in each of the four racial groups. There was no significant differences in CD4 (461.12 ± 163.28 vs 478.42 ± 181.22) and CD8 (680.83 ± 320.36 vs 862.52 ± 354.48) absolute cell counts between HIV positive patients co-infected with GBV-C/HGV and those not infected with GBV-C/HGV, respectively. However, significantly higher relative CD3 [80.0 ± 4.17% vs 70.99 ± 19.79%] (p = 0.015), gamma delta T cells (yLT) [3.22± 1.30% vs 2.15 ± 29.12%] (p = 0.052) and lower CD 30 [35.45 ± 17.86% vs 50.59 ± 9.20%] (p = 0.041) status were observed in GBV-C/HGV positive compared to GBV-C/HGV negative HIV infected patients, respectively. Although there is a high prevalence of novel Group isolates of GBV-C/HGV in KZN, the lack of elevated liver enzymes and clinical hepatitis in blood donors and haemodialysis patients suggests that GBV-C/HGV is not associated with liver disease. HBV and not GBV-C/HGV modifies the course of alcoholic liver disease. The relatively higher number of CD3 cells and increased yLT expression, together with a decrease in CD 30 cells tends to suggest an association with protection and or delayed progression of HIV disease in GBV-C/HGV infected patients. Whilst GBV-C/HGV is not associated with liver disease, it may be an important commensal in HIV infected patients.
Molecular characterisation of endogenous loci related to jaagsiekte sheep retrovirus.
(2007) Hallwirth, Claus Volker.; York, Denis Francis.; Fan, Hung Y.
The study of retroviruses has been of pivotal significance to the field of biomedical science, where it has provided fundamental insights into the processes underlying both viral and non-viral carcinogenesis. Ovine pulmonary adenocarcinoma (OP A), a contagious lung cancer of sheep and goats, has emerged over the past three decades as an invaluable model of human epithelial cancers. It is one of the very few animal models of retrovirus induced neoplasia of epithelial tissues, whereas most other such animal models of human cancers pertain to the haematopoietic system. OP A represents a unique, naturally occurring, inducible, outbred animal model of peripheral lung carcinomas, and is caused by a betaretrovirus - jaagsiekte sheep retrovirus (JSRV) - that is receiving increasing attention in the fields of retrovirology and lung cancer research. JSRV exists in two highly homologous, yet molecularly distinct forms. The first is an exogenous form of the virus that is transmitted horizontally from one animal to another. This form is infectious and the direct cause of OP A. The other is an endogenous form, 15 to 20 proviral copies of which reside benignly in the genome of sheep and are transmitted vertically from one generation to the next. At the time this study commenced, no knowledge existed regarding the underlying pathogenic mechanism by which JSRV causes OPA. Even though the nucleotide sequence of exogenous JSRV had been elucidated seven years earlier, only limited sequence information was available on endogenous JSRVs. With a view towards identifying genetic regions or elements within exogenous JSRV that could potentially be implicated in its pathogenic function, this study began with the cloning of the first three full-length endogenous JSRV loci ever isolated from sheep. The DNA sequences of these full-length endogenous JSRV loci were determined and comprehensively analysed. Comparison with exogenous JSRV isolates revealed that the two forms of the virus are highly homologous, yet can be consistently distinguished in three short regions within the coding genes. Two of these reside in the gag gene, and one at the end of the env gene. These regions were named the variable regions (VRs) of sheep betaretroviruses. The JSRV VR3 in env was linked by our collaborators to the virus's ability to transform cells in tissue culture. The effects and biological significance of VRI and VR2 in gag are subtler and more difficult to determine. After identifying these regions, it became the objective of this study to develop relevant molecular tools that could be used to discern the significance of these variable regions in vivo, and to characterise these tools in vitro to assess their suitability for in vivo studies. The development of these tools entailed the design of a novel strategy that was implemented to precisely substitute the endogenous VRI and VR2 (individually and in combination) into an infectious molecular clone of exogenous JSRV. These chimeric constructs were shown to support retroviral particle release into the supernatant of transiently transfected cells in tissue culture. These particles were confirmed by independent experiments to have arisen specifically from transfection with the chimeric clones. Finally, the particles were shown to be capable of infecting cultured cells and of productively integrating their genomes into those of their host cells, rendering these particles fully competent retroviruses that can be used in the context of in vivo studies to determine the biological significance of VRI and VR2. This study has made a significant contribution to the further development of the OP A / JSRV model system of human epithelial lung cancers. It has also led to the design of a molecular substitution strategy that can be adapted to introduce any genetic region into a cloned DNA construct, regardless of the degree - or lack of interrelation - of the two DNA sequences, thereby creating a highly versatile molecular biological tool.
Regulation of TRIM E3 Ligases and Cyclophilin A and the impact on HIV-1 replication and pathogenesis.
(2011) Singh, Ravesh.; Ndung'u, Peter Thumbi.
No abstract available.
Impact of p2/NC cleavage site polymorphisms on HIV-1 subtype C viral fitness.
(2012) Wilson, Serron.; Gordon, Michelle Lucille.
Subtype C accounts for the majority of HIV infections and in South Africa, is the dominant subtype. The Gag cleavage sites of subtype C viruses show a high degree of natural variation compared to subtype B and group M sequences, with the p2/NC site having the highest degree of variation among all cleavage sites and between all subtypes. This study therefore aimed to determine the functional effect of this variation on viral fitness. A library of drug naïve subtype C sequences were screened using computational analysis to predict binding affinity between HIV protease and the Gag substrate at the p2/NC site. Ligands with high predicted affinity had hydrophobic cleavage sites with substantial diversity at positions P5-P3. Lower ranking ligands were mostly similar to the consensus subtype C. Three ligands were selected for fitness assays from each the high ranking and low ranking groups. Chimeric viruses expressing selected cleavage sites were generated by site directed mutagenesis. Replication capacity assays of these viruses showed moderate differences in fitness but failed to demonstrate a correlation with computational estimates of binding affinity. Enzymes assays were performed to further investigate substrate preferences and the binding mechanism of protease. To this end, recombinantly expressed HIV-1 protease was tested against a range of substrates the matching the p2/NC cleavage sites used in the replication capacity assay. Results of the enzyme assay did not correlate with either the computation studies or the replication capacity assay results, suggesting a sequence independent binding and recognition mechanism of HIV-1 protease. Taken together the results suggest that processing of Gag is determined by tertiary folding of the polyprotein and not amino acid sequence at the cleavage site.
The role of the protease cleavage sites in viral fitness and drug resistance in HIV-1 subtype C.
(2010) Giandhari, Jennifer.; Gordon, Michelle Lucille.
There is an increasing number of patients failing second line highly active antiretroviral therapy (AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR). Therefore, it is important to collect data on subtype C protease and gag sequences from patients as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens. In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM resistance genotyping kit and the gag region from these isolates were then characterised. These sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and subtype B, C and group M naïve sequences that were downloaded from the Los Alamos Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To investigate the effect of CS mutations on replication capacity, a mutation was introduced by site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit). Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23 in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro was found to have a lower replication capacity compared to the wild type. These findings emphasize the need for further investigation of gag mutations and their contribution to the evolution of HIV resistance to PIs.
Impact of immune-driven sequence variation in HIV-1 subtype C Gagprotease on viral fitness and disease progression.
(2011) Wright, Jaclyn.; Ndung'u, Peter Thumbi.
Understanding of the viral and host factors that determine time for progression to acquired immunodeficiency syndrome (AIDS) in individuals infected with human immunodeficiency virus type 1 (HIV-1) could aid in the design of an effective HIV-1 vaccine. Human leukocyte antigen (HLA) class I profile is strongly and consistently associated with differential rates of HIV-1 disease progression, however the mechanisms explaining this are not well understood. It has been hypothesised that “protective” HLA alleles select escape mutations in functionally important epitopes in the conserved group specific antigen (Gag) protein resulting in HIV-1 attenuation, which may result in slower disease progression. Many of the studies investigating the fitness cost of Gag escape mutations have concentrated on a few pre-selected mutations and have not assessed fitness consequences in the natural sequence background. Furthermore, the majority of studies have focussed on HIV-1 subtype B, while HIV-1 subtype C is the most prevalent subtype worldwide. Therefore, in the present study, a large population-based approach and clinically-derived Gag-protease sequences were used to comprehensively investigate the relationship between immunedriven sequence variation in Gag, viral replication capacity and markers of disease progression in HIV-1 subtype C chronic infection. The influence of Gag function on HIV-1 disease progression was further investigated in early HIV-1 subtype C infection. It was also hypothesised that Gag may contribute significantly to overall HIV-1 fitness and towards fitness differences between HIV-1 subtypes. Materials and Methods Recombinant viruses encoding Gag-protease, derived from antiretroviral naïve HIV-1 subtype C chronically (n=406) and recently (n=60) infected patients as well as a small subset of HIV-1 subtype B chronically infected patients (n=25), were generated by electroporation of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positively of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positivelyof an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positively with baseline viral load (p<0.0001) and negatively with baseline CD4+ T cell count (p=0.0004), but not with subsequent rate of CD4+ T cell decline (p=0.73). In HIV-1 subtype C recent infection, replication capacities of the early viruses did not correlate with subsequent viral set points (p=0.37) but were significantly lower in individuals with below median viral set points (p=0.03), and there was a trend of correlation between lower replication capacities and slower rates of CD4+ T cell decline (p=0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (p=0.04) but host HLA-B-specific polymorphisms were associated with higher viral set points (p=0.01), suggesting a balance between effective Gag CD8+ T cell responses and viral replication capacity in influencing viral set point. A moderate statistically significant correlation was found between the replication capacities of whole isolates and their corresponding Gag-protease recombinant viruses (p=0.04) and the replication capacities of the subtype C recombinant viruses were significantly lower than that of the subtype B recombinant viruses (p<0.0001). The subtype-specific difference in the consensus amino acids at Gag codons 483 and 484 was found in site-directed mutagenesis experiments to largely contribute to the fitness difference between subtypes, possibly by influencing budding efficiency. Discussion The data support that protective HLA alleles, in particular HLA-B*81, attenuate HIV-1 through HLA-restricted CD8+ T cell-mediated selection pressure on Gag. Results suggest that viral replication capacity determined by sequence variability in Gag-protease has an impact on HIV-1 disease progression, but also indicate that a balance between HLA-driven fitness costs and maintenance of effective CD8+ T cell responses is important in determining clinical outcome. Gag-protease was observed to significantly contribute to overall HIV-1 replication capacity and variability in this region between HIV-1 subtypes B and C is suggested to partly explain the difference in viral fitness between these subtypes. Specific mutations in Gag-protease associated with viral attenuation were identified and it was also observed that mutations in conserved Gag regions carried the greatest cost to HIV-1 replication capacity. Overall, the data support the concept of, and may assist in the rational design of, an HIV-1 vaccine in which immune responses are directed towards several conserved epitopes, particularly in Gag, with the aim to constrain immune escape (thereby maintaining effective CD8+ T cell responses) and attenuate HIV-1 (in the event of partial escape), resulting in slower disease course and reduced HIV-1 transmission at the population level.
The role of natural killer cells in preventing HIV-1 acquisition and controlling disease progression.
(2013) Naranbhai, Vivek.; Abdool Karim, Salim Safurdeen.; Carr, William Henry.
In sub-Saharan Africa, women carry a disproportionate burden of the Human Immunodeficiency Virus Type 1 (HIV-1) pandemic. The high risk of HIV acquisition in these women and the variability in their disease progression is not fully understood. Natural Killer (NK) cells, which are innate immune antiviral lymphocytes, present systemically and at mucosal surfaces, may play a role in preventing HIV acquisition and/or altering disease progression, as they are key early mediators of the response to viral infections and are equipped to kill infected cells. The purpose of this study was to evaluate the role of NK cells in HIV-1 acquisition and following acquisition, in disease progression. The study participants were selected women who were participating in a randomized controlled trial assessing the effectiveness of 1% Tenofovir gel in preventing HIV-1 (CAPRISA 004 trial). The study design was a case-control study nested within the cohorts followed up in the CAPRISA 004 trial. In this trial, 889 sexually-active women aged 18-40 years were randomized to receive Tenofovir or placebo gel and prospectively followed. Assessment of HIV infection was performed monthly by rapid HIV-1 antibody tests, supplemented by HIV-1 RNA polymerase chain reaction (PCR), p24 Western blotting and/or ELISA. Samples obtained prior to the first positive rapid antibody test were retrospectively tested by HIV specific PCR to identify window period infections. The date of infection in this study was estimated as the midpoint between the last negative and first positive antibody test, or 14 days prior to the first HIV-1 RNA-PCR positive result. Multi-parametric flow cytometry techniques developed and validated in healthy blood donors were used to asses the bidirectional relationship between NK cells and HIV-1. To simulate in vivo interaction between NK cells and autologous HIV infected cells, an in vitro infection and coculture assay was used in addition to conventional assays of NK cell recognition of HLA-deficient cell lines. These were supplemented with measurement of plasma cytokines by Luminex and microbial products by ELISA. In this study, 44 cases who acquired HIV-1 were sampled prior to infection and 39 controls who remained HIV-1 negative despite high behavioural exposure at the timepoint when their preceding sexual activity was highest. To understand how HIV infection affected NK cells during early HIV-1 infection, the first sample obtained after acquisition was studied and compared to preinfection samples from the same participant. The case and control groups were broadly similar in the proportions using tenofovir gel, proportions infected with HSV-2 and number of sexual partners but tended to be marginally older than cases (27.6 vs 23.3 years). By design control women had higher sexual activity than cases (mean 11 vs. 5.7 sex acts per month). The frequency of IFN-γ secreting NK cells from women who acquired HIV infection were significantly lower than from women who remained uninfected in response to 721 cells-an EBV transformed B cell line (background-adjusted median 13.7% vs. 21.6%; p=0.03) and to autologous HIV infected T-cells (background-adjusted median 0.53% vs. 2.09%; p=0.007). NK cells from HIV acquirers displayed impaired proliferation but enhanced spontaneous degranulation compared with non-acquirers after co-culture with HIV uninfected or infected autologous T-cell blasts. Adjusting for age, gel arm, HSV-2 infection status and levels of NK cell activation, IFN-γ+ NK cell responses to autologous HIV infected cells were associated with reduced odds of HIV acquisition (OR 0.582; 95% CI 0.35-0.98; p=0.04). In addition, even in the absence of ex vivo stimulation, HIV acquirers had higher levels of generalised innate immune activation measured by systemic cytokine concentrations (TNF-α, IL2, IL-7 and IL12p40), peripheral blood platelet concentrations (p=0.038), and non-specific ex vivo NK cell activation (p<0.001). Generalised NK cell activation measured directly ex vivo without stimulation was associated with acquisition. Further, if innate immune activation was assembled as a principal component in an unsupervised fashion but taking into account all the measures made, it was significantly associated with HIV acquisition (OR adjusted for age, tenofovir gel use, and HSV-2 status for PC with innate immune factor loadings 11.27; 95% CI 1.84- 69.09; p=0.009). The causes of preinfection innate immune activation could not be established in this study but the degree of activation could not be explained by microbial translocation as both HIV acquirers and non-acquirers had similar levels of plasma lipopolysaccharide (LPS), soluble CD14 (sCD14) and intestinal fatty-acid binding protein (I-FABP). Similarly, both HIV acquirers and non-acquirers had similar NK cell and cytokine responses to Toll-like Receptor (TLR)-2, 3 or 7/8 agonists 11. During early HIV-infection, NK cells demonstrated significantly higher activation (p=0.03), expression of Killer-cell immunoglobulin-like Receptors (KIR) (p=0.006) and expression of chemokine receptor 7 (CCR7, p<0.0001) compared with prior to acquisition. Although NK cells had reduced cytolytic potential following HIV acquisition, antiviral IFN-γ secretion appeared to be preserved. NK cell responses were not different between tenofovir and placebo gel recipients, but women who acquired HIV whilst using tenofovir gel had higher gag-specific IFN-γ CD4+ T-cell responses during early infection. Overall, the findings suggest that the frequency of NK cells producing IFN-γ specifically after co-culture with HIV-1 infected target cells was associated with protection from HIV-1 acquisition but, generalised, non-specific activation of NK cells and other innate immune components enhanced HIV acquisition. Since neither microbial translocation nor TLR responsiveness were associated with pre-existing immune activation further studies will be required to identify the drivers of generalised innate immune activation. Methods to dampen generalised innate immune activation and/or augment specific NK cell antiviral responses in women at risk for HIV-1 may reduce HIV-1 acquisition. During primary HIV-1 infection, NK cells underwent impairment of cytolytic function but not IFN-γ secretory function; this may affect their ability to affect disease progression. Although Tenofovir gel did not alter innate immune responses in women with breakthrough infection, it preserved HIV-specific Tcell immune responses, the consequences of which need further exploration. Understanding how Tenofovir gel mediated preservation of adaptive immune responses may lead to interventions that will reinforce protective host responses. In conclusion, innate immune responses by NK cells have been shown to impact HIV acquisition; HIV-specific IFN-γ responses by NK cells were protective while generalised NK activation was detrimental. The causes of innate immune activation are not known but these effects were independent of the impact of Tenofovir gel. Future prevention strategies targeting mucosal transmission of HIV should assess their impact on NK cell responses, to avoid general innate immune activation and enhance their ability to protect against HIV acquisition.
Molecular epidemiology of HIV-2 infection in KwaZulu-Natal Provnce, South Africa.
(2013) Singh, Lavanya.; De Oliveira, Tulio De Paiva Nazareth Andrade.
Infection with HIV-2 has important implications for the diagnosis, treatment and management of the infection. The objective of this study was to describe the seroprevalence and molecular epidemiology of HIV-2 in KwaZulu-Natal – the province with the highest HIV prevalence in South Africa, which in turn is the country with the highest HIV prevalence in the world. HIV-1 positive samples were screened using a rapid test for HIV-2. Samples showing antibody positivity were subject to molecular confirmation by PCR and / or serological confirmation by Western blot. There was a large difference in results (10.6% by Western blotting versus 0% by PCR). This discrepancy between molecular and serological confirmation by Western blot. There was a large difference in results (10.6% by Western blotting versus 0% by PCR). This discrepancy between molecular and serological confirmation was attributed to cross-reacting antibodies. The use of rapid tests and Western blots for HIV-2 diagnosis in South Africa, should, therefore, be interpreted with caution. Based on the results of this study, HIV-2 is most probably not present in KwaZulu-Natal.
An investigation of virologic failure and the spectrum of drug resistance mutations in a paediatric ART programme in rural KZN, SA.
(2014) Pillay, Sureshnee.; Danaviah, Sivapragashini.; Bland, Ruth Margaret.
Background Better understanding of drug resistance patterns in HIV-infected children on antiretroviral therapy (ART) is required to inform public health policies in high prevalence settings. The aim of this study was to characterise the acquired drug resistance in HIV-infected children failing first-line ART in a decentralised rural HIV programme. Methods Plasma samples were collected from 101 paediatric patients (<15 years of age) identified as failing ART. RNA was extracted from the plasma, reverse transcribed and a 1.3kb region of the protease gene was amplified and sequenced using Sanger sequencing protocols. Sequences were edited in Geneoius and drug resistance mutations were identified using the RegaDB and the Stanford, Rega and ANRS resistance algorithms. The prevalence and frequency of mutations were analysed together with selected clinical and demographic data in STATA v11. Results A total of 101 children were enrolled and 89 (88%) were successfully genotyped; 73 on a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen and 16 on a protease inhibitor (PI)-based regimen at the time of genotyping. The majority of patients on an NNRTI regimen (80%) had both nucleoside reverse-transcriptase inhibitor (NRTI) and NNRTI resistance mutations. M184V and K103N were the most common mutations amongst children on NNRTI-based and PI-based regimens. 23% had one or more thymidine analogue mutation (TAM) and 6% had ≥3 TAMs. Only one child on a PI-based regimen harboured a major PI resistance mutation. Conclusions Whilst the patterns of resistance were largely predictable, the few complex resistance patterns seen with NNRTI-based regimens and the absence of major PI mutations in children failing PI-based regimens suggest the need for wider access to genotypic resistance testing in this setting.
Nucleoside reverse transcriptase inhibitors-associated mutations in the RNase H region of HIV-1 isolates in South African adults and children failing highly active antiretroviral therapy.
(2012) Ngcapu, Sinaye.; Gordon, Michelle Lucille.
Background: The South African national treatment program includes NRTIs in both first and second line highly active antiretroviral therapy regimens. Recently, mutations in the RNase H domain have been associated with resistance to NRTIs. Here we investigated the prevalence and association of RNase H mutations with NRTI resistance in isolates of HIV-1 subtype C infected individuals. Methods: RNase H sequences from 134 NRTI treated (104 adults and 30 children) and 134 drug-naïve sequences (30 KZN isolates and 104 downloaded from the Los Alamos Database) were analyzed. Spearman’s rank correlation and a Bayesian network were used to explore the relationship between mutations occurring within the RNase H domain and NRTI treatment. Results: 130 of 134 samples clustered phylogenetically with HIV-1 subtype C, with one subtype A, two subtype B and two subtype D. All 30 sequences from HAART-naїve patients were classified as subtype C. Five mutations in the RNase H region had significantly higher frequency when comparing ART-naïve and NRTI-experienced patients. These were: (E438GKR, L517ISV, K527GENQR, E529DK and Q547HKR) (Table 1). Three mutations (E432D, A446SVY and Q507HK) showed decreased proportions in treatment-experienced isolates when compared to ART- naїve isolates. E438GKR was seen in 6.72% of treated versus only 0% of naїve isolates (p= 0.0034), L517IV was found in 17.16% of treated isolates versus 7.46% of naїve isolates (p= 0.0245). Similarly, K527GENQRS was found in 41.04% of treated isolates versus 26.12% of naїve isolates (p= 0.0138), and E529DK was more prevalent in treated (17.91%) when compared to 2.99% of naїve subtype isolates (p <0.001). Finally, Q547HKR was seen in 5.22% of treated versus 0% of naïve subtype C patients (p= 0.0144). Interestingly, samples of twenty treatment experienced individuals that did not show of the classical NRTI mutations in the RT domain harbored E438GKR, L517ISV, K527GENQR, E529DK and Q547HKR. Conclusion: Results obtained from this study suggested that drug resistance could be caused by mutations in the RNase H domain either alone (T470S), or in combination with mutations in the pol region (D67N and L491P). Phenotypic studies are required to understand the prevalence and impact of RNase H mutations, particularly E438GKR, T470S, L517ISV, K527GENQR, E529DK and Q547HKR on NRTI resistance in HIV-1 subtype C as suggested by our data. Further studies using site-directed mutagenesis may also reveal the impact of these mutations on viral fitness.
Allele-specific polymerase chain reaction (ASPCR) to detect resistance mutations in minor variants of HIV-1 subtype C in patients failing highly active antiretroviral therapy (HAART).
(2014) Maharaj, Shevani.; Gordon, Michelle Lucille.
The World Health Organization (WHO) has recommended Tenofovir disoproxil fumarate (TDF) as one of the preferred first-line antiretrovirals (ARVs). TDF and Abacavir (ABC) were introduced into the South African National Antiretroviral Treatment Guidelines in 2010. However, exposure to TDF and ABC can result in the development of the K65R and L74V resistance mutations, respectively. The K65R mutation occurs preferably in subtype C viruses, due to the unique polymorphisms found at codons 64 and 65 (which are not present in subtype B). This is a cause for concern in South Africa, where subtype C is the most common HIV-1 subtype. In addition, these mutations may be present in the minor viral population (i.e <20% of the viral population) and it has been shown that the presence of a resistance mutation in a frequency as low as <0.5% may be associated with an increase in the risk of virological failure. This study investigated the prevalence of K65R and L74V in the minor viral population, using Allele-specific PCR (ASPCR), in a cohort of subtype C infected patients that failed their first-line treatment regimen that did not include TDF or ABC. RNA was extracted from stored plasma samples from a subset of the South African Resistance Cohort Study (SARCS) and the pol region was reverse transcribed and amplified using a one-step RT-PCR kit (Invitrogen; California, USA). For both the K65R and L74V mutations, ASPCR was performed using specific and non-specific primers. A specific and non-specific standard curve was optimised for each mutation (using a mutant plasmid control) and these standard curves were used to perform an absolute quantification. Subsequently, the percentage of each mutation (in each sample) was calculated by dividing the quantity of mutant sequences in the sample by the quantity of total viral sequences in the sample and multiplying this ratio by 100. The Limit of Detection (LOD) of the K65R ASPCR was 0.72%. Of the 84 patients that were assayed, the K65R mutation was detected in 7 (8.33%) of the patients. Five of the 7 samples were detected above 1% (i.e 3 were approximately 2%, 1 was 9.48% and 1 was 100%) and 2 were detected below 1% (i.e 1 was 0.88% and the other was 0.93%). The limit of detection for the L74V ASPCR was 0.013%.We found the L74V mutation to be prevalent in 9 (10.7%) of 84 patients. In 4 of the 9 patients, the L74V mutation was found in ≥1% of the viral population (viz. 2.82%, 10.10%, 12.02% and 18.22%) and in the other 5 patients, the L74V mutation was detected in <1% of the viral population (2 were between 0.5% and1%, while 3 were detected between 0.013% and0.5%). In this study, ASPCR detected additional K65R and L74V mutations in the minor viral population of TDF and ABC-inexperienced patients that were missed by standard genotyping. These minorityK65R mutations could contribute to treatment failure in these patients when switched to TDF or ABC-containing ARV regimens. ASPCR is a useful tool for screening for minority mutations before starting or switching regimens.
Functional and clinical consequences of immune-driven sequence variation of gag-protease in HIV-1 subtypes A, C, D and recombinants.
(2016) Kiguoya, Marion Wangui.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
Abstract available in PDF file.
Impact of injectable hormonal contraceptives on innate immune environment in the genital tract in women at high risk for HIV-1 infection.
(2015) Ngcapu, Sinaye.; Abdool Karim, Quarraisha.; Passmore, Jo-Ann Shelley.
Abstract available in PDF file.

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