Identification of a new epitope for HIV‐neutralizing antibodies in the gp41 membrane proximal external region by an Env‐tailored phage display library

Abstract

HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIVneutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development.Keywords: Elite controllers r Epitopes r HIV-1 r neutralizing antibodies r phage display Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Ursula Dietrich e-mail: ursula.dietrich@em.uni-frankfurt.de IntroductionEliciting broadly neutralizing antibodies (bnAbs) against HIV-1 is a major goal in HIV vaccine development [1]. However, despite the identification of a number of epitopes for bnAbs present in C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 500Mingkui Zhou et al. Eur. J. Immunol. 2013. 43: 499-509 patient sera [2], these epitopes failed so far to induce such antibodies after vaccination in vivo [3][4][5][6][7][8]. The mechanism behind the elicitation of bnAbs during natural infection remains unclear. Emerging evidence indicates that most HIV-1-specific antibodies elicited during early infection fail to neutralize a wide-spectrum of HIV-1 strains and emphasizes the importance of analyzing the Ab profile of patients showing delayed disease progression to allow for sufficient affinity maturation [9]. A subgroup of HIV-1-infected individuals controlling the infection over years in the absence of antiretroviral therapy and maintaining serum viral loads (VL) below the detection limit (<50 copies/mL) is termed "elite controllers" (ECs) [10]. Although virus control is certainly due to a potent HIV-specific immune response [10], few qualitative differences in immune responses have been identified between controllers and viremic individuals. The differences encountered so far mainly concern the cellular immune response an...

Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

“…Neutralization studies were performed as described previously [33]. Briefly, standardized HIV-1 pseudoviruses were used on TZM-bl cells [57], [58].…”

“…HIV controllers are a promising source for the identification of nAbs, as here they have time to develop and mature over years in a rather uncompromised immune system and in the absence of therapeutic selection pressure. We previously identified LTNP and EC with neutralizing activity in plasma and dissected the humoral immune response based on phage libraries displaying short peptides [32] or longer HIV-1 Env fragments [33]. This allowed the identification of new linear and conformational epitopes able to induce neutralizing antibodies upon vaccination in mice [32], [34], [35].…”

Targeting Cellular Cofactors in HIV Therapy

“…These antibodies represented by mAbs 2F5, 4E10, and Z13 are rarely found in patients due to their crossreactivity with autoantigens (for review see [95]). More recently, the antibodies m44, 10E8, and antibodies targeting the EC26-2A4 epitope combined broad neutralization capacity with lack of autoreactivity [96][97][98]. This may direct new strategies towards the development of more effective antibodies for prevention of HIV-1 entry.…”

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

“…Ag fragmentation to identify the recognized segments is a widely used epitope mapping method per se. [53][54][55][56][57][58] The major drawbacks of this approach are that short segments of Ag sequence not always contain all the information to give rise to the native epitopes, particularly if they are discontinuous or conformation-sensitive, and the use of long segments results in low resolution maps. 55 Despite these limitations, the epitopes for some mAbs and for a substantial fraction of the antibodies contained in polyclonal antisera can be readily elucidated through Ag fragmentation.…”

“…9,26 Remarkably, the whole process can take advantage of the phage display platform, because Ag fragments of different lengths can be displayed and eventually give rise to large libraries. [53][54][55]57 Sometimes the displayed fragments keep essential structure-determining elements like internal disulfide bonds, allowing the study of conformation-sensitive epitopes. 9 If no starting point can be defined for mapping a given mAb, competition with an already mapped antibody can still point to the antigenic region recognized by the latter as the area to search for the epitopes of competitor Abs.…”