The negative impact that dimers present in gp preparations h

The negative impact that dimers present in gp120 preparations have on overall gp120 affinity for CD4i ligands, mainly by affecting the on-rate, was addressed in this report. Indeed, when his(6)-tagged gp120 proteins, purified with nickel beads (before FPLC, containing gp120 dimers) were used to measure the effect of V5 on binding affinities for CD4i 17b and A32 antibodies, an important binding enhancement when V5 was removed along with the V1V2V3 variable regions was observed. However, when gp120 dimers were removed by size-exclusion chromatography, these differences disappeared (Table 2), indicating that gp120 dimers can affect the overall affinity of gp120 preparations for different ligands and thus bias gp120-ligands affinities interpretation. Moreover, not all gp120 variants have the same amount of contaminating dimers; for example, it has been reported that V1, V2 and V3 loops contribute to gp120 dimer formation [(Finzi et al., 2010a) and Fig. 1] thus making impossible to calculate accurate affinity constants by SPR without FPLC purification of monomeric gp120s.
Conclusion
Acknowledgments We thank Maxime Veillette for helpful discussions. We thank Dr. James Robinson for 17b, 48d and A32 monoclonal HU 308 receptor and Dr. Joseph Sodroski for the CD4-Ig construct. This work was supported by a Canada Foundation for Innovation Program Leader grant #29866, by CIHR operating grants #119334 and #134117, by a FRQS Establishment of Young Scientist grant #26702 to A.F. A.F. is the recipient of a Canada Research Chair on Retroviral Entry. The authors have no conflicts of interest to report.
Introduction HIV-1 is the causative agent for AIDS. About 37 million people are living with HIV worldwide. There are many circulating HIV-1 strains, which are further divided into subtypes [1], [2]. When more than one strain infects an individual, circulating recombinant forms are produced. Partly because of this high diversity, a successful vaccine remains elusive. The virus infects cells of the immune system when the envelope glycoprotein of HIV-1 binds to CD4 receptors on T helper cells [3]. The envelope glycoprotein (Env) is the primary target of the humoral immune response elicited against HIV-1. Env is synthesized as a single polypeptide precursor (gp160) which is processed in the Golgi to produce two chains, gp120 and gp41. These associate in a non-covalent manner to form a trimer of heterodimers of surface-exposed gp120 and membrane-anchored gp41 chains. gp120 contains the conserved CD4 receptor binding site and gp41 drives the fusion of viral and host cell membranes [4], [5]. In natural infection, most of the antibodies produced at any given time are unable to efficiently neutralize the virus present [6], [7], [8]. Viral sequence diversity and the presence of misfolded/unfolded forms of Env shift the focus of the antibody response away from conserved sites [9]. A large amount of glycosylation on the surface of gp120 also occludes the exposure of conserved sites [10]. In an effort to focus the immune response on conserved epitopes of the CD4 binding site, we previously reported the design of a non-glycosylated, bacterially expressed outer domain fragment of HIV-1 Env which was folded and could bind its cognate ligands b12, CD4 and VRC01 [11], [12]. In rabbit immunization studies, it elicited neutralization of primarily Tier 1 viruses. We also designed a small fragment of gp120, called b122a, comprised of a compact beta barrel located on the lower part of the outer domain (Fig. S1A) [13]. When bacterially expressed, b122a was found to be only partially folded but able to bind the broadly neutralizing antibody b12 with micromolar affinity. Upon immunization in rabbits and subsequent boosting with gp120, the anti-sera showed broad, albeit moderate neutralization of a 16 virus panel. Competition experiments and serum depletion studies showed that the neutralization was mediated by CD4 binding site antibodies. Since b122a was only partially structured as assessed by CD and protease resistance [13], we attempted to enhance its ability to elicit neutralizing antibodies by stabilizing the structure of the gp120 fragment immunogen with additional disulfides, and by displaying it on the surface of simple virus-like particles (VLPs) to present the epitope in polyvalent fashion [14], [15], [16], [17]. The design, production, and immunogenic properties of these constructs are described here.