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Selected Publication:

Type of publication: PhD Thesis
Type of document:

Year: 2013

Authors: Bachler, Barbara

Title: Novel Biopanning Strategies: Characterization of HIV-1-specific Antibody Responses in Vaccine-protected Primates.

Source: PhD-Arbeit, Vet. Med. Univ. Wien, pp. 103.


Advisor(s):

Klein Dieter

Reviewer(s):
Jensen-Jarolim Erika

Vetmed Research Units:
VetCore


Graduation date: 14.03.14


Abstract:
Classical immunization approaches have failed to prevent HIV-1 infection and the type of immunity required to achieve sterilizing protection from HIV-1 acquisition is still not clearly defined. To improve the design of future vaccines, immunization/challenge studies in nonhuman primate (NHP) models are used to elucidate vaccine-induced as well as virus-specific immune responses. Four different immunization/challenge studies performed by our group yielded a cohort of rhesus macaques (RMs) that showed different levels of protection against heterologous simian-human immunodeficiency virus (SHIV) strains encoding HIV-1 clade C envelopes (SHIV-Cs). The overall aim of this PhD thesis was to dissect humoral immune responses in vaccinees with different levels of protection and to address two main questions: i) is there a difference in the antibody (Ab) repertoire between the vaccinees that resisted multiple SHIV-C challenges compared with monkeys where the vaccine failed to protect and ii) did the multiple exposures to live virus have any quantitative and/or qualitative effects on pre-existing vaccine-induced Ab responses in completely protected animals - although the SHIV-C challenges did not cause any viremia in these vaccinees? To answer these questions, we employed the peptide phage display technology and devised two novel selection strategies that allow the characterization of Ab responses at defined time points. First, to probe the paratopes associated with vaccine protection, we designed a ¿protection-linked (PL) biopanning¿ approach that differentiates between the vaccine-induced Ab repertoires in protected monkeys versus those in non-protected macaques. We screened three different protected RMs (positive selection) in combination with plasma from a non-protected vaccinee given the same immunogens (negative selection) and hypothesized that by depleting phages recognizing Abs that only bind but were not associated with protection, we could enrich for phages representing epitopes recognized only by protective Abs. The resulting recombinant phages were analyzed for homologies with the vaccine components (HIV-1 Env, HIV-1 Tat and SIV Gag-Pol), which led to the identification of phage inserts that reflected a conserved motif at the N-terminus (NT) of HIV-1 Tat (Tat-specific PL mimotopes). When we further investigated the binding profile of these Tat mimotopes, we observed broad cross-recognition among protected vaccinees. In contrast, Abs from non-protected RMs showed only low or undetectable binding to the same mimotopes. Of note, some of the Tat PL mimotopes were also recognized by a mouse neutralizing monoclonal Ab against Tat NT suggesting that these mimotopes represent a neutralizing epitope. Moreover, polyclonal IgG purified from partially protected RMs had anti-Tat neutralizing activity in vitro. Lastly, we observed a significant inverse correlation of Abs targeting the neutralizing NT of Tat with peak viremia indicating an association with these Ab responses and the observed protection in vivo. The second part of this work focused on the effects of heterologous live-virus exposures on vaccine-induced immune responses in protected macaques. Thus, we first measured anti-Env Ab responses before and after virus challenges in five vaccinees that had completely resisted all virus encounters. Although no viremia was ever detected in these animals, four out of five RMs showed measurable increases in anti-gp140 Ab titers after they had been exposed to live SHIVs. To further investigate these changes in Ab titers, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after ¿ but not before ¿ any SHIV challenge. With this ¿subtractive biopanning¿ approach, gp120 V3 mimotopes were isolated and subsequent detailed analysis of the anti-V3 Ab responses revealed that the virus challenges had not only boosted pre-existing binding and neutralizing Ab titers, but also had induced Abs targeting neo-antigens presented by the heterologous challenge virus. In summary, our data i) highlight the utility of our new PL biopanning approach to identify Ab responses with significant association to vaccine protection, regardless of the mechanism(s) or targets of the protective Abs, ii) argue for including HIV-1 Tat in a multicomponent AIDS vaccine, and iii) show that exposures to live virus that had failed to cause viremia can nevertheless result in changes in the Ab repertoire initially induced by vaccination. Combined, these results may have major implications for both the design of future vaccine candidates as well as the interpretation of immunogenicity data for clinical vaccine trials in humans.


Publication(s) resulting from University thesis:

Watkins, JD; Sholukh, AM; Mukhtar, MM; Siddappa, NB; Lakhashe, SK; Kim, M; Reinherz, EL; Gupta, S; Forthal, DN; Sattentau, QJ; Villinger, F; Corti, D; Ruprecht, RM (2013): Anti-HIV IgA isotypes: differential virion capture and inhibition of transcytosis are linked to prevention of mucosal R5 SHIV transmission. AIDS. 2013; 27(9):F13-F20
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Bachler, BC; Humbert, M; Palikuqi, B; Siddappa, NB; Lakhashe, SK; Rasmussen, RA; Ruprecht, RM (2013): Novel biopanning strategy to identify epitopes associated with vaccine protection. J Virol. 2013; 87(8):4403-4416
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Bachler, BC; Humbert, M; Lakhashe, SK; Rasmussen, RA; Ruprecht, RM (2013): Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia. Retrovirology. 2013; 10:63
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