Extensive immunologic and viral studies have previously shown that many anti-V3 mAbs display cross-reactivity between V3 peptides and gp120 proteins from diverse viruses of the different clades of HIV-1 (Gorny et al. 1993
, Gorny et al. 2002
, Binley et al. 2004
, Pantophlet et al. 2008
). These studies demonstrated that, although by definition V3 is highly variable in its sequence, this region of gp120 contains immunologically conserved elements. Immunologic data are further supported by findings that, despite its sequence variability, V3 must retain certain conserved structures in order to interact with the chemokine receptors on the surface of target cells (Shioda, Levy, and Cheng-Mayer 1992
, Trkola et al. 1996
, Hill et al. 1997
, Labrosse et al. 2001
, Cardozo et al. 2007
). Moreover, the conformational conservation of V3 is confirmed by crystallographic (Stanfield et al. 2006
, Stanfield et al. 2004
, Bell et al. 2008
, Dhillon et al. 2008
, Burke et al. 2009
, Jiang et al. 2010
) and NMR(Sharon et al. 2003
) studies. This extensive literature was the basis of our initial use of the V3 region of gp120 as an epitope for inducing Abs with broad immunologic and antiviral activity.
In the present work, recombinant chimeric V3-CTB immunogens were successfully designed using structural data, molecular modeling, and protein engineering. The short V3-CTB form bound to mAb 447-52D, whose epitope it was intended to mimic optimally. The full-length V3-CTB bound to most anti-V3 mAbs, demonstrating the success of the design in presenting the V3 epitopes as exposed, correctly folded, Ab-accessible conformations. Use of these new immunogens to boost the immune response of rabbits showed that the full-length V3-CTB construct was able to induce V3-binding Abs and Abs that display cross-clade neutralizing activity against psVs and primary isolates. The full-length V3-CTB immunogen induced a much stronger and broader Ab response than did the short V3-CTB.
The two V3-scaffold constructs that we have designed and tested were based on the V3 loop found in clade B viruses. These were used because much of the immunologic and structural data was based on studies of the clade B V3 loop. However, the clade B V3 loop is relatively unusual among the HIV-1 group M virus clades because it contains a GPGR rather than a GPGQ motif at its center (Leitner T 2005
). Use of the clade B V3 as the epitope in both the short and full-length V3-CTB constructs studied here resulted in immunogens that were relatively limited in their ability to induce Abs that neutralize psVs and viruses carrying V3 loops that contain the GPGQ motif. Thus, for example, the neutralizing titers of the sera from animals boosted with full-length V3B
-CTB, against the chimeric psV carrying the homologous V3B
were 2–3 orders of magnitude higher than those against chimeric psVs carrying heterologous V3 loops bearing the GPGQ motif (). Our previously published work (Zolla-Pazner et al. 2009
) suggests that follow-up studies with full-length V3-CTB immunogens where V3B
is replaced by V3 sequences containing the GPGQ motif or other rationally designed V3 loops will give rise to more broadly reactive and perhaps higher titers of anti-V3 Abs.
As noted above, the use of CTB as a scaffold for the HIV-1 V3 epitope was based on scans of the Protein Data Base for molecules with surface-exposed beta-turns that could be structurally matched to the beta-hairpin structure at the base or in the crown of the V3 loop. Among the proteins found to have a suitable beta-turn, we further looked for proteins that would form high-order oligomers, could be easily expressed in bacteria, and for which some immunogenicity data was available. Wild type CTB emerged as the preferred scaffold because it has been used extensively as a component of vaccine in humans to protect against cholera (marketed as Dukoral (Lopez-Gigosos et al. 2007
)), and has been tested as a scaffold for other immunogens (Backstrom et al. 1994
, Matoba et al. 2006
We have previously shown that using an immunization regimen in which animals are primed with gp120 DNA and boosted with a scaffold immunogen carrying only the V3 epitope of gp120 is able to focus the immune response on this neutralizing epitope and induce cross-clade neutralizing Abs (Zolla-Pazner et al. 2008
, Zolla-Pazner et al. 2009
). In the previous studies, the V3-scaffold immunogen consisted of various V3 loops fused to the C-terminus of a truncated form of the murine leukemia virus gp70 (Kayman et al. 1994
). This construct carried one copy of V3 per molecule of gp70. In contrast, the V3-CTB immunogens designed and tested in this study carries five copies of V3 per pentamer of CTB. Although direct comparisons cannot be made because immunizations were not conducted in parallel using exactly the same immunization regimen, it would appear that full-length V3-CTB is a better immunogen than full length V3-gp70, giving Ab responses of greater breadth and potency. The most direct comparison can be made by analyzing the responses of rabbits #6–10, immunized in this study with clade B gp120 DNA and boosted with the full-length V3B
-CTB) to those immunized in an earlier study with the gp120 DNA from a clade A strain carrying a V3 loop with the GPGR motif (Ar) and boosted with V3B
-gp70)(Zolla-Pazner et al. 2008
). The GMT50
for neutralization of V3 chimeric psVs averaged 10-fold higher in the rabbits receiving the B/V3B
-CTB vs. the Ar/V3B
-gp70 regimen. This may be due to the difference in the V3 valency of the immunogens (one for V3-gp70 vs. five for V3-CTB), and/or to differences in modes of antigen presentation and induction of B cell maturation due to the differential binding of these immunogens to cell receptors: CTB binds to ganglioside GM1, mammalian cell wall glycosphingolipid widely distributed in all tissues, whereas gp70 binds to mouse cationic amino acid transporter (mCAT-1) (Albritton et al. 1993
). Another difference between the gp70 and CTB carriers is that the gp70-V3 proteins contained N-linked glycans at the base of the V3 loop and at the internal glycosylation site at position 6 of the V3 loop. The proximity of this position to residues known to be included in V3 epitopes might affect immunogenicity.
The inability of the short V3-CTB construct to elicit significant Ab titers underscores the challenge of designing effective recombinant immunogens that direct the immune response towards a highly restricted singular three-dimensional epitope. The poor performance of the short V3-CTB construct may be explained by several possible causes: 1) the rabbit Ab repertoire may not contain genes that are appropriate to develop 447-52D-like Abs, or 2) flexibility of an epitope loop may be required for immunogenicity. Further investigation would be needed to establish the minimal essential epitope(s) within the complete V3 that are sufficient for a robust immune response, although it is clear from previous studies that the length of V3 is not the only critical variable that contributes to the induction of neutralizing Abs (Yang et al. 2004
). The limited efficacy of previously described V3-CTB constructs and other HIV epitope-scaffold immunogens for eliciting neutralizing Abs (Bckstrom et al. 1995
, Backstrom et al. 1994
, Law et al. 2007
, Muster et al. 1994
, Eckhart et al. 1996
) also highlights the challenge of constructing effective recombinant immunogens that focus the immune response on neutralizing epitopes.
In contrast, the immunogenicity data obtained after boosting with the full-length V3-CTB underscores the potential that optimally designed immunogens can have in focusing the immune response on a neutralizing epitope. The full-length V3-CTB induced cross-clade neutralizing Abs in rabbits. Because the immunogen was rationally designed, this result serves as an important initial point for immunogen optimization for achieving the desired breadth and potency for a protective Ab response. Importantly, a variety of V3 loop sequences and structures can be placed on this scaffold to optimize the breadth and potency of the Ab response. Moreover, this approach may serve as a platform for designing other epitope-scaffold immunogens that will induce Abs specific for additional HIV-1 neutralizing epitopes and/or for protective epitopes against other pathogens.