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Selected Publication:

Type of publication: Master Thesis
Type of document:

Year: 2010

Authors: Bachler, Barbara

Title: Isolating HIV-1 Envelope Mimotopes Using Polyclonal Sera from Pig-Tailed Macaques Infected with a CCR5-Tropic Subtype-C Simian-Human Immunodeficiency Virus.

Source: Master Thesis, Vet. Med. Univ. Wien, pp. 102.


Advisor(s):

Ruprecht Ruth M.
Klein Dieter

Reviewer(s):
Saalm├╝ller Armin

Vetmed Research Units:
Institute of Virology


Graduation date: 20.08.10


Project(s): Virus-host interactions


Abstract:
More than 25 years ago HIV, the causative agent of the AIDS epidemic was described. Since then, HIV research has intensely been focusing on understanding the virus life cycle and on finding potential drug or vaccine targets. Thus far, classical immunization approaches have not been able to prevent an HIV infection. To achieve sterilizing protection, a vaccine would have to elicit high titers of neutralizing antibodies capable of protecting against diverse HIV strains. The isolation and characterization of novel immunogens and antibody epitopes is an important step in antiviral vaccine development. In July 2009, the consequences of intrarectal inoculation of pig-tailed macaques (Macaca nemestrina) with a R5-tropic clade C simian-human immunodeficiency virus (SHIV), termed SHIV-1157ipd3N4 was reported. SHIV-1157ipd3N4 caused AIDS and the virus-induced pathogenesis in pig-tailed macaques was very similar to that seen in R5-tropic SIV or SHIV-infected rhesus macaques. Some of the infected animals showed neutralizing antibody responses not only against the homologous virus but also against heterologous clade B viruses, such as HIVSF162.LS and SHIVSF162P4. In this follow-up study, polyclonal serum of the SHIV-1157ipd3N4-infected pig-tailed macaques was used to isolate phagotopes mimicking epitopes (mimotopes) of broadly neutralizing antibodies against the HIV-1 envelope protein. Mimotope isolation was performed by various selection strategies using commercially available random peptide phage libraries. After three rounds of affinity selection positive clones were identified via phage ELISA. Three different strategies have been performed to identify either (1) highly specific gp160 mimotopes, (2) common gp160 mimotopes shared by three different SHIV- 1157ipd3N4-infected pig-tailed macaques or (3) phagotopes mimicking neutralizing antibody epitopes. The resulting motifs were analyzed for linear homologies to SHIV-1157ipd3N4 gp160. Potential conformational homologies were identified using a model of the SHIV-1157ipd3N4 gp120 monomer and 3DEX. Promising mimotopes were tested for cross-reactivity and binding characteristics in conformational phage ELISAs. Using the first strategy, we were able to select conformational mimotopes representing gp120 epitopes, known to be important for virus entry (receptor and co-receptor binding site) and highly specific only for the used target sample. When all three infected animals were used in a sequential biopanning, common motifs with a cross-reactivity profile were isolated. Interestingly, approximately 70% of those mimotopes represented immunodominant regions on gp41. Isolated peptide sequences from the third selection strategy showed linear homologies to the V2 loop, and conformational similarities to highly conserved regions in the CD4 binding site. In-vitro experiments confirmed broad cross-reactivity as well as conformational dependence. To evaluate the potential of inducing broadly neutralizing antibodies, the most promising mimotopes were included in a pilot mouse immunization study. Finding mimotopes which represent conserved, immunogenic regions of the HIV-1 Env and which have the potential to induce neutralizing antibody responses could be very useful for developing an antibody-based vaccine against HIV-1 in the future.

Keywords:
HIV-1, neutralizing antibodies, peptide-phage display, 3DEX


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