1.5 V IRAL D IVERSITY AND THE I MPACT ON V ACCINE D EVELOPMENT
1.5.5 The Neutralizing Human Monoclonal Antibodies (Nmabs)
Currently, there are no immunogens that have been successful in stimulating the production of broadly neutralizing antibodies. This is despite the fact that several epitopes have been identified in conserved and variable regions of both gp120 and gp41that induce broadly neutralizing antibodies (Muster et al., 1993, Wyatt et al., 1998). Inducing nAbs to these epitopes using rationally designed immunogens has been difficult possibly due to subtle structural or conformational differences compared to native immunogens (Selvarajah et al., 2005, Law et al., 2007, Selvarajah et al., 2008). The human neutralizing monoclonal antibodies (nmAbs) with broad and potent neutralizing ability are b12, 2G12, 2F5, 4E10 and VRC01 (Roben et al., 1994, Stiegler et al., 2001, Zwick et al., 2001, Zhou et al., 2010). In addition, monoclonal antibodies PG9 and PG16 have been characterized relatively recently, and more recently- the PGT series of antibodies and CH01-04 nmAbs (Walker et al., 2009, Pejchal et al., 2011, Bonsignori et al., 2011). All of these nmAbs target different regions of Env (see Table 1.1) and they can work in one of two ways, either they bind to the mature trimer on the virion surface thereby acting as an entry inhibitor or they bind to the pre-hairpin
inter-mediate, that is after receptor and co-receptor binding thereby acting as a fusion inhibitor (Parren and Burton, 2001). More recently the focus has been on designing the immunogen based on the interaction between the antibody and its cognate site on Env using crystallographic and computed tomography studies (Liu et al., 2008a). This type of reverse engineering may be more effective when developing immunogens in the context of the native Env trimer-antibody interaction.
Table 1.1 List of nmAbs and the targeted Env sites
nmAb Target Site on Env Reference
b12 CD4 binding site Roben et al. (1994)
VRC01 CD4 binding site Zhou et al. (2010)
2G12 Discontinuous epitope on
gp120
Trkola et al. (1996) PGT Series- PGT 128 Glycans on gp120 Pejchal et al. (2011)
2F5 MPER Stiegler et al. (2001)
4E10 MPER Zwick et al. (2001)
PG9/PG16 V2-V3 Walker et al. (2009)
CH01-04 V2-V3 Bonsignori et al. (2011)
In 1992, b12 was one of the first nmAbs to be isolated and neutralizes about 40% of HIV-1 strains. b12 was selected from a phage-display library constructed from the bone marrow of an HIV-1-infected individual. b12 was mutated for high affinity binding to gp120, targets the CD4 binding site with high affinity and the epitope targeted by b12 is surrounded and protected by N-linked glycans (Roben et al., 1994). The CD4 binding site is one of the more conserved regions of Env gp120. b12 recognizes a discontinuous epitope on gp120 as they react with several residues in different regions of gp120 (Zhou et al., 2007). This nmAb occludes the CD4 binding site and acts primarily as an entry inhibitor. VRC01 is another nmAb recently characterized, and was isolated through binding on a rationally designed mimic of the CD4 binding site. VRC01 also targets a narrow site in the CD4-binding domain (Zhou et al., 2010) and neutralized 91% of Env pseudotyped primary isolates, potently
neutralized multiple HIV-1 subtypes and had the broadest neutralization activities compared to any antibody to date (Wu et al., 2010). In addition, this nmAb is highly desirable as it does not have autoreactive properties (Scheid et al., 2011, Li et al., 2011).
2G12 is an unusual nmAb as it targets a discontinuous epitope (conformational epitope) on gp120. 2G12 targets a unique conformational epitope of oligomannose-rich carbohydrate in gp120 that is non-immunogenic (Trkola et al., 1996) and it has an unusual shape. 2G12 represents proof that the carbohydrate-rich gp120 can be immunogenic. More recently however, the PGT series of nmAbs was discovered. Specifically, PGT128, which targets glycans, is highly potent and also displays tremendous breadth (Pejchal et al., 2011). Despite all the intense effort in immunogen design, it still remains unclear how to elicit such unique antibodies.
2F5 and 4E10 are nmAbs that target epitopes proximal to each other within the membrane proximal external region (MPER) of gp41 just N-terminal to the insertion of gp41 into the viral membrane (Stiegler et al., 2001, Zwick et al., 2001). The 2F5 epitope resides in amino acid (AA) position 662-668 (ELDKWAS) while the 4E10 epitope resides in amino acid position 671-676 (NWFDIT). Both these epitopes are in lipid-rich sites within the MPER.
These nmAbs do not interfere with receptor binding but they inhibit the fusion between the viral and target cell membranes most likely through steric hindrance. Both 2F5 and 4E10 exhibit potent and broad neutralization of diverse HIV-1 isolates, however, due to their auto- reactive properties and membrane-lipid affinity, their potential for clinical application is a subject of ongoing debate. Although the epitopes that 2F5 and 4E10 target remain attractive vaccine immunogens, it is uncertain whether elicitation of such nmAbs may be detrimental and unsafe due to the autoreactive properties that the antibodies possess and this therefore remains a topic of ongoing research.
Two potent nmAbs, PG9 and PG16 were recently obtained using a high-throughput neutralization screen of antibody-containing culture supernatants from approximately 30,000 activated memory B cells from a subtype A-infected African donor (Walker et al., 2009).
The nmAbs exhibit broad and potent neutralization profiles to genetically diverse HIV-1 isolates. PG9 and PG16 target conserved epitopes within the V2 and V3 loops, it is likely that their epitopes overlap because the two nmAbs are somatic variants. Interestingly, these nmAbs recognize epitopes on the native Env trimer, are able to bind monomeric gp120 and monomeric scaffold proteins presenting V1-V2 and neutralize about 80% of primary HIV-1 isolates (Walker et al., 2009, Pancera et al., 2010, McLellan et al., 2011). Both nmAbs do not have autoreactive properties and would therefore be desirable to elicit such antibodies.
Similarly, CH01-04 nmAbs that have recently been characterized, are also conformationally dependent on the quartenary structure and target the V2 and V3 loops (Bonsignori et al., 2011). Another nmAb, 2909, targets a quaternary neutralizing epitope on parts of V2 and V3, however 2909 is strain specific and does not bind to monomeric gp120 (Gorny et al., 2005, Honnen et al., 2007).
Overall, these nmAbs have provided the opportunity to define vulnerable epitopes that exist across diverse strains. In addition, their conformational properties in conjunction with their cognate epitopes on the native Env trimer have been well characterized. Crystallographic and structure-function analyses are crucial to designing immunogens for efficacious HIV-1 vaccines.