Abstract
HIV-1 adsorption and entry into target cells is initiated by the binding of the viral envelope
glycoprotein gp120 to cell surface molecules. Subsequent conformational changes of gp120
and gp41 drive membrane fusion between the virus and target cell. Blocking viral entry into
the cell is considered a very attractive strategy for both microbicide and vaccine. Successful
development of such intervention strategies requires detailed knowledge of the interactions
between virus and target cells. The aim of these studies was to investigate the HIV-1 entry
process by dissecting the actions of neutralizing antibodies and inhibitors on initial
attachment to the target cell, receptor engagement and fusion.
This thesis is divided into three sections which report different aspects of my work.
The first part focuses on the main aspect of my work: The establishing of the required
methods and the detailed investigation of the interaction between HIV, target cell receptor
and attachment factors, and antibodies and inhibitors that interfere with HIV entry. A
particular focus therein was to identify the precise step in the attachment and entry process in
which antibodies and inhibitors take action.
My studies determined that the HIV attachment process can only be successfully blocked by
inhibitors that interfere with the engagement of CD4, such as CD4-binding site (CD4bs)
specific antibodies, soluble CD4 and CD4-specific inhibitors. Other neutralizing antibodies or
inhibitors could not efficiently block attachment to the target cells. Particularly noteworthy
were the MPER-specific neutralizing antibodies 2F5 and 4E10 which both had no impact on
attachment, yet displayed potent inhibitory activity even after the virus had bound to CD4.
Screening of antibody activities in patient plasma showed a high prevalence of antibodies
with activity that prevents virus attachment, suggesting that anti-CD4bs antibodies were
active. Notably, I was also able to detect presence of antibodies with a post-CD4-engagement
activity in several plasma.
In a separate line of experiments I investigated the thermodynamic requirements of HIV
entry. Here I was able to show that during spinoculation (low speed centrifugation-based
virus inoculation) virus fusion can also occur under suboptimal temperatures. While generally
temperatures above 30 °C are considered necessary for HIV entry, spinoculation allowed
entry at temperatures as low as 18 °C. Whether the decreased temperature dependence is due
Research Summary
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to local thermodynamic events generated by shear forces during centrifugation or whether
simply limitations of diffusion are overcome by sedimentation is still under investigation.
The second section of my thesis refers to a study I performed and published in collaboration
with colleagues from our research group. It focuses on the development and characterization
of a novel CD4-directed molecule which inhibits HIV infection (Schweizer et al., 2008). My
contribution to this study was the analysis of CD4 functionality upon engagement of these
CD4-inhibitors. I was able to show that, while the compounds bound to the D1 domain of
CD4, which is required for CD4 functionality as immune coreceptor, cells remained
functional at inhibitor concentrations that interfered with HIV entry,
The third section of my thesis refers to a second study I performed and published in
collaboration with colleagues from our research group. The aim of this study was to explore
the extent to which target and virus producer cell properties steer HIV sensitivity to
neutralization by antibodies and inhibitors (Mann et al., 2009). Within the frame of this study
I investigated the contribution of differentially expressed attachment and entry receptors on
different target cells with respect to the efficacy of HIV entry and its inhibition.
Berlinger, L