Abstract
Abstract
Since its discovery in 1983, HIV-1 has spread rapidly resulting in a global
pandemic. Currently an estimated 40 million people are infected with the
virus. Infection leads eventually to the acquired immunodeficiency syndrome
(AIDS) and death. The symptoms of the syndrome are largely due to the
devastating effect HIV has on the immune system. HIV has been the focus of
an unprecedented amount of research however there are still many aspects
of HIV pathogenesis which remain incompletely characterized. One area
which continues to attract attention is HIV tropism. HIV requires two
receptors- CD4 and a co-receptor- for infection. In vivo, HIV variants mainly
use either CCR5 or CXCR4 as co-receptors and thus variants can be
classified as either R5 (CCR5 using), X4 (CXCR4 using) or (R5X4) (dual
tropic). However these classifications do not fully explain the differing abilities
of variants to infect particular cell types. It is thought that other factors, such
as receptor density or alternative receptors, may underlie observed
differences in tropism. In addition to studying viral variants to investigate
determinants of tropism, it is also possible to investigate post-mortem
material from HIV infected individuals to characterize nature and cellular
location of HIV variants. HIV can infect cells in the brain and this leads in
some cases to HIV associated dementia (HAD). The presence of HIV in the
brain poses a challenge to attempts to eradicate the virus as many drugs
have poor CNS penetration. There have been concerns that sub-optimal
levels of drugs in the CNS may support the evolution of drug resistant
variants. Alternatively, low levels of drugs may allow growth of drug sensitive
variants which could reseed the periphery if the drug regime was halted.
Characterisation of the provirus present in post mortem brain material could
lead to a better understanding of this issue. The virus in the brain appears to
mainly infect microglia and macrophages however it is not clear to what
extent other brain cells might be infected. Conflicting reports have emerged
regarding the infection of astrocytes and neurons. Thus a method for
separating the various brain cells which allows them to be tested for HIV
could be valuable.
In the first part of this project, methods to characterise the cellular tropism of
HIV were investigated. A problem with many previous methods is that
manipulation or amplification of the HIV genome has resulted in artefactual
changes in the sequence. Thus any results obtained regarding the cellular
tropism of variants may not accurately reflect the in vivo situation. A vector
was produced which could be used to produce recombinant HIV expressing
study subject derived env. The vector incorporated the EGFP to allow easy
identification of infected cells. Three methods for production of virus without
the need to amplify the genome in E. coli were designed. All methods were
capable of producing infectious virus expressing various env genes.
The second part of the project sought to investigate the distribution of drug
resistance variants in vivo. DNA was extracted from post mortem brain and
lymphoid tissues from HIV infected study subjects. Limiting dilution PCR was
used to obtain PCR products each derived from a single template molecule.
No differences were found in the distribution of drug-resistance mutations
between the two compartments for three out of four study subjects. In one
study subject there were considerably more drug resistance mutations in the
lymphoid derived sequences than in those amplified from brain tissue. This is
probably due to low levels of anti-HIV drugs in the CNS of this subject.
In the final part of the project, a method for the bulk isolation of microglia and
astrocytes from post-mortem HIV-infected brains was optimised. Cellular
debris was first removed using a density gradient. The cells were then fixed
using a gentle ethanol fixation technique. This inactivated the HIV, fixed and
permeabilised the cells while maintaining DNA integrity. FACS sorting was
carried out using CD68 and GFAP as markers of microglia and astrocytes
respectively. Separation of cells was carried out using two HIV-infected
brains. No provirus was detected in the separated cells however the brain
material used was from asymptomatic study subjects and had extremely low
proviral loads. It is hoped that in the future the technique could be used to
separate brain material which has a higher proviral load. This would allow
quantification of the level of HIV infection of these two cell types.
