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Identification and characterization of HIV-1 specific neutralizing antibodies from HIV-1 seropositive patients and autoimmune (HIV-1 seropositive or seronegative) participants.Naidoo, Thenusha 17 January 2012 (has links)
Since the discovery of HIV-1, the production of an effective prophylactic or
therapeutic vaccine remains elusive. An effective vaccine must be able to elicit a
potent humoral and cellular immune response. Neutralizing antibodies target the
envelope glycoproteins on the surface of HIV-1 virions thereby preventing viral
entry. Unfortunately, to date only a handful of neutralizing antibodies have been
identified that are capable of neutralizing different viral strains within diverse
subtypes, and none have been isolated from HIV-1 subtype C infected patients. In
this study, we screened four different HIV-1 subtype C infected patient cohorts for
the presence of neutralizing antibodies against a panel of 5 subtype C and 1
subtype B pseudovirus/es in a pseudovirion based neutralizing antibody assay.
The CT cohort comprised 9 slow progressor plasma samples, the FV cohort
consisted of 11 antiretroviral drug naïve HIV-1 subtype C infected plasma
samples. Plasma samples from 10 antiretroviral treatment experienced HIV-1
subtype C infected patients failing first line therapy made up the DR cohort and the
JM cohort consisted of 10 serum samples from HIV-1 seropositive or seronegative
individuals with an autoimmune disorder. A pseudovirion neutralizing antibody
assay was successfully established, and all plasma and serum samples were heat
inactivated and screened using this assay. Analysis of the percentage
neutralization and IC50 data showed no correlation between the presence of
neutralizing antibodies and delayed disease progression in the SP cohort. High
levels of neutralizing antibodies were observed in the DR cohort, however future
studies are required to confirm if the measured neutralization is due to residual
antiretroviral drugs in the plasma or neutralizing antibodies. No samples within the FV cohort showed promising neutralizing antibody activity however the JM cohort
harboured 3 serum samples (TN5, TN6 and TN8) that exhibited a greater than
average breadth of neutralization and are worth investigating further in future
studies. Patients TN5, TN6 and TN8 were all HIV-1 positive with an additional
autoimmune disease. The availability of stored bone marrow samples for TN5,
TN6 and TN8 will allow for the generation of antibody phage display libraries and
isolation of monoclonal antibodies, with potentially broadly cross reactive activity.
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Development of an HIV-1 intergrase enzyme strand transfer assayFish, Muhammad Qasim 30 January 2012 (has links)
M.Sc.(Med.) (Molecular Medicine and Haematology), Faculty of Health Sciences, University of the Witwatersrand, 2011 / The Human Immunodeficiency Virus type 1 (HIV-1) integrase is an essential
enzyme required for viral replication. Integrase forms part of an ensemble of
proteins known as the preintegration complex and functions by a two-step
process. Firstly, the cleaving of the 3’ ends of the viral cDNA genome, known as
3’-end processing. The second step is the insertion of these ends into host DNA
by esterification, known as strand transfer. There is no human homologue to
integrase which makes it an ideal drug target. However, the strand transfer
inhibitor raltegravir is currently the only antiretroviral treatment available that
inhibits integrase. The aims of this study were two-fold: firstly to characterise a
cohort of South African patients so as to determine the viability of introducing
raltegravir as a new treatment option, and secondly, to set up high-throughput
integrase inhibitor screening assays (testing integrase enzymatic functionality).
An HIV-1 subtype C specific RT-PCR and PCR assay was established for integrase
genotyping using 51 integrase inhibitor-naïve patient plasma samples and 22
antiretroviral drug-naive primary viral isolates from South Africa. Seventy-one of
the 73 samples were classified as HIV-1 subtype C and two samples were unique
AC and CG recombinants in integrase. Amino acid sequence analysis revealed
there were no primary mutations (Y143R/C/H, Q148H/R/K, and N155H/S)
associated with reduced susceptibility to the integrase inhibitor raltegravir.
However, one sample had the T97A mutation, three samples had the E157Q and
V165I mutations, and the majority of samples contained the polymorphic
mutation, V72I. The expected finding of no major integrase mutations conferring
resistance to integrase inhibitors suggests that this new antiretroviral drug class
will be effective in our region where HIV-1 subtype C predominates. However,
the impact of E157Q and other naturally occurring polymorphisms warrants
further phenotypic investigation. The integrase sequence of viral isolate, FV3,
was closest to the consensus sequence, and thus chosen for preintegration
complex isolation for use in strand transfer assays. Isolation of preintegration
complexes following FV3 infections of several cell lines was unsuccessful as
determined by western blot analysis. Subsequently, the focus was changed to
isolation of HIV-1 subtype B recombinant integrase and its functional evaluation.
Expression of native integrase (INwt) and soluble integrase (INsol) was induced in E.
coli, and both proteins were purified by nickel chelating chromatography. The
purified recombinant proteins were used to develop three assays to test for
strand transfer activity, of which two were successfully established. Furthermore,
only INsol showed strand transfer activity in the high-throughput microtitre plate
assay and scintillation proximity assay (SPA)-bead strand transfer assay. Activity
of INsol was shown to be inhibited by the control compound, chicoric acid with an
IC50 of 101.5nM in the high-throughput microtitre plate assay, whereas INsol
activity as well as a dose response to chicoric acid with an IC50 of 248.5nM was
recorded in the SPA-bead strand transfer assay. Visualization of radiolabelled
enzymatic products of strand transfer by polyacrylamide gel electrophoresis of
urea sequencing gels was unsuccessful. Overall, the high-throughput microtitre
plate and SPA-bead strand transfer assays have been successfully established in
our laboratories, and are available to screen compound libraries for potential
antiretroviral drug candidates targeting integrase strand transfer.
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Differential timing of translocation of HIV-1 subtype B and C Vpu to the ER/Golgi an plasma membrane compartmentsBell, Catherine Macdonald 19 April 2010 (has links)
MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, 2009 / The HIV-1 Vpu protein functions largely to target CD4 molecules for proteasomal
degradation, and to enhance virion release. The subcellular localisation of Vpu is
related to these functions. Previous studies showed subtype B Vpu localisation at the
ER/Golgi complex, while subtype C Vpu localised to the plasma membrane (PM) at
48 hours post-transfection. To determine if subtype C Vpu can localize to the
ER/Golgi, we evaluated the cellular localisation of Vpu from two South African
subtype C isolates as compared to subtype B Vpu, over time. Codon optimized vpu
genes from subtype C isolates FV5 and FV15 (which have a six and two amino acid
insert in the N-terminal domain, respectively) and a representative subtype B vpu
were TA cloned into the pcDNA6.2/C-emGFP expression vector. The three VpuemGFP
recombinant plasmids were cotransfected with pDsRed-ER, pDsRed-Golgi,
or pDsRed-Mem into HEK 293T cells, and observed at 24, 48, and 60 hours posttransfection
under a confocal microscope to confirm the presence of Vpu at different
subcellular compartments. Cotransfection and microscopy conditions were
methodically optimised. At 24 hours post-transfection, the subtype C FV5 Vpu had
ER/Golgi localisation, but none at the PM. The subtype C FV15 Vpu had weaker
ER/Golgi localisation and no PM localisation. In contrast, the subtype B Vpu had
strong PM localisation. At 48 hours, FV5 and FV15 Vpu showed PM localisation,
while subtype B Vpu was clearly localised at the ER/Golgi. At 60 hours, FV5 Vpu was
observed at the PM, whereas FV15 and subtype B Vpu showed ER/Golgi localisation.
These findings illustrate the efficient translocation of Vpu between different cellular
compartments and for the first time, the difference in timing between subtype B and C
Vpu, as well as íntrasubtype differences. This difference in shuttling suggests
implications for the timing of viral assembly and release. Further investigations may
clarify the impact of this timing on the difference in disease pathogenesis noted
between infections with the different subtypes.
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Role of the leader sequence of human immunodeficiency virus type 1 in viral replication, genome dimerization, encapsidation, and proviral DNA synthesisShen, Ni, 1969- January 2002 (has links)
Human immunodeficiency virus type 1 (HIV-1) genome consists of two identical RNAs that appear noncovalently linked near their 5' ends. The 5' untranslated region is called leader region. The 3' part of leader, i.e. nucleotides U200 to G335 in HIV-1 genomic RNA, between the primer binding site and the gag gene, can fold into 3 stem-loops: the kissing-loop domain (KLD) or stem-loop 1 (SL1), the 5' splicing junction hairpin (SD) or SL2 and SL3. The KLD, from nucleotide (nt) 243 to 277, forms a stem-loop (kissing loop hairpin) seated on top of a small stem bulge (stem B and loop B). The kissing-loop hairpin, or dimerization initiation site (DIS) hairpin consists of stem C and loop C. Loop C contains the autocomplementary sequence (ACS) GCGCGC262 or GUGCAC262, also called DIS. / In the kissing-loop model of HIV-1 genome dimerization, HIV-1 RNA dimerization is initiated by base pairing between the ACS of one RNA monomer and that of an adjacent monomer. / To understand the role of the ACS in HIV-1 replication and HIV-1 genomic RNA dimerization, we replaced the central CGCG261 (or tetramer) of the HIV-1 Lai ACS by other tetramers. Genomic RNAs containing the UUAA tetramer (non-HIV-1 tetramer) were half dimeric, but UUAA genome packaging was unaffected. This was the first evidence that genomic RNA dimerization and packaging can be dissociated (Chapter 2). Destroying stem-loop C reduced genomic RNA dimerzation by ~50%, proviral DNA synthesis by ~85%, and reduced viral infectivity by ~3 logarithmic units. Destroying stem-loop B had similar effects on genome dimerization, reverse transcription, and viral infectivity. We also observed that mutations in stem-loop B and in the DIS hairpin were "non additive" (Chapter 2). / The existence of stem-loop C is supported by phylogenetic evidence, while that of stem-loop B is not, namely, its sequence is completely conserved. We investigated the role of stem B and loop B nucleotides in viral replication, and genomic RNA dimerization. The putative CUCG246/CGAG277 duplex was replaced by 9 alternative complementary sequences, 4 likely to base pair in long (~500 nts) RNAs, as assessed by the algorithm mfold. Among the 4 sequences, 3 preserved genome dimerzation, 1 did not significantly inhibit it, and 2 preserved viral replication. We also asked if 9 deletions or nucleotide substitutions within nucleotide 200 to 242 and/or 282 to 335 could influence genome dimerization. Delta200--226 and Delta236--242 genomic RNAs dimerized relatively poorly despite having neutral or positive influences on stem B, loop B and klh folding (Chapter 3). / Mutations within the Matrix, Capsid, p2 and nucleocapsid genes suppress several functional defects caused by KLD destruction. We tested the effect of these suppressor mutations on genome dimerization and infectiousness of viruses bearing moderate to crippling KLD mutations. Our conclusion is that these suppressor mutations can restore genomic RNA dimerization when DIS is weakened, but not when DIS is denatured or the KLD is destroyed (Chapter 4).
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Factors influencing the evolution of HIV-1 /Birk, Markus, January 1900 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.
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Role of the leader sequence of human immunodeficiency virus type 1 in viral replication, genome dimerization, encapsidation, and proviral DNA synthesisShen, Ni, 1969- January 2002 (has links)
No description available.
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Study of the correlation between the CD4 T cell repertoire in simian immunodeficiency virus infected macaques and disease progressionSalha, Marie-Danielle January 2002 (has links)
Note: no title page
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In Vitro Selection and Characterization of Drug-Resistant Variants of HIV-1Gao, Qing January 1994 (has links)
Note:
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Η έκφραση του ογκογονιδίου ets-2 σε υποπληθυσμούς Τ λεμφοκυττάρων και ο ρόλος του στην μεταγραφή του γονιδίου της ιντερλευκίνης-2 και του ιού HIV-1Παναγούλιας, Ιωάννης 25 July 2008 (has links)
Η έκφραση του ογκογονιδίου ets-2 δρα κατασταλτικά στην έκφραση του γονιδίου της ιντερλευκίνης-2 καθώς και στην έκφραση του ιού HIV-1 στα παρθενικά Τ λεμφοκύτταρα. / -
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Molecular and biological characterisation of the human immunodeficiency virus type 1Ball, Jonathan K. January 1994 (has links)
No description available.
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