The isolation of J3 represents a significant improvement on previous nAbs derived from immunized animals as in single-domain VHH form it has a comparable breadth and potency to the best nAbs obtained from a limited number of natural human infections. In contrast, previous nAb clones characterized from immunized animals have only exhibited limited breadth (Forsman et al., 2008
; Sundling et al., 2010
). A caveat to this is the observation that sera with 17b-like binding specificity can be induced after immunization of humans (Vaine et al., 2010
), and it should be noted that 17b and other Abs to CD4-induced epitopes are less broadly neutralizing as full-length mAbs than in Fab form (Labrijn et al., 2003
). However, given the previously reported decrease in neutralization ability seen with the Fab of b12 (Labrijn et al., 2003
), it appears the CD4-binding site of Env is not per se more easily targeted for neutralization by small Ab fragments as is the CD4-induced binding site, presumably because of the required presence of the coreceptor interacting with Env in the latter case. As J3 neutralizes via interaction with the CD4-binding site, it is unlikely that its breadth is a function solely of the smaller size of VHH. Therefore, whether the breadth and potency of J3 are conserved in a full-length heavy chain–only llama Ab will be investigated. Thus, this study reports for the first time an nAb response after experimental immunization with a breadth equivalent to that seen in the best Ab responses in natural infection.
Llama 8 was multiply immunized with trimeric Gp140 Env derived from a subtype A and CRF BC viruses in the presence of the adjuvant Stimune. The resulting phagemid VHH library was screened via a selection method based on neutralization function rather than pre-enriching a polyclonal mixture of VHH for the ability to bind to recombinant HIV-1 Env and/or compete with nAbs. However, to isolate a VHH specific for a particular binding site on Env, the original biopanning method remains advantageous, as it enables targeted selection of VHH via enriching for those VHH that compete with a known nAb to a specific region. As Env glycoproteins were used to elicit J3 and given that patient-derived nAbs have been shown to provide protection from infection when passively infused into NHPs (Mascola et al., 1999
; Burton et al., 2011
; Watkins et al., 2011
), these recombinant immunogens represent valid vaccine candidates for HIV. However, given the weak level of serum neutralization observed, they will require further modification to induce high titer J3-like broad cross-neutralizing serum responses.
J3 neutralizes 96% of all strains tested in this study, which included 100 representatives from a wide range of subtypes and CRFs. In fact, J3 neutralizes 100% of subtype A, B, D, and CRF AC, ACD, AG, BC, and CD and >85% of the remaining subtypes tested, C, G, and CRF AE. In an analysis of viruses previously tested against VRCO1/2 and b12, J3 neutralized 94.2% of viruses (this study) compared with VRCO1, which neutralized 88.4% of the subset of 69 viruses (Wu et al., 2010
). Sensitivity to the full-length human nAb and the llama VHH were similarly distributed across different subtypes in this subset. However, at the level of individual viruses, resistance to VRCO1 did not confer resistance to J3 and vice versa. No clear pattern of residues associated with J3 resistance was discernible from analysis of the available sequences for viruses that were not neutralized by J3. Although the evaluation of J3 against additional viruses may provide further insight into key residues for J3-mediated neutralization, it may be more thorough to evaluate the neutralization sensitivity of a panel of engineered viral mutants to define the functional J3 epitope in combination with escape mutant studies.
J3 binds to both the trimeric immunogens that llama 8 received as well as monomeric Gp120IIIB and Gp120YU2. J3 targets the binding site of Env for the HIV-1 cellular receptor CD4 as confirmed by the loss of binding seen with the CD4-binding site D368R Gp120YU2 mutant relative to wild-type Gp120YU2. In addition, J3 competes with sCD4 itself as well as with the well-characterized human mAb b12. Competition experiments with A12, a previously isolated anti–CD4-binding site VHH, showed that binding assays, while demonstrating a level of difference between the two VHH, were not able to detect the disparity between these two VHH in terms of neutralization ability. Together with the level of neutralization breadth observed, these findings indicate that J3 recognizes part of the Env CD4-binding site and very few if any of the adjacent amino acids, although this remains to be defined in detail.
This study describes the isolation of an extremely broad and potent HIV-1–neutralizing VHH, J3, from a screen of almost 3,000 clones. This method provides a relatively quick screening process compared, for example, with the 30,000 human B cell clones screened to isolate the PG9 and PG16 nAbs (Walker et al., 2009
). Thus, this method could be used alongside testing the immune sera for neutralization to evaluate new HIV-1 immunogens in the established llama model, accelerated by the advantage of heavy chain–only Abs that they do not require heavy and light chain random recombination. Furthermore, the independently functional nature of the VHH Ab fragments (Vanlandschoot et al., 2011
) coupled with their intrinsic stability (Gorlani et al., 2012
) makes them suitable candidates for anti–HIV-1 microbicide development. Thus, broad and potent neutralizing VHH isolated in this way, such as J3, can both aid progress toward an HIV-1 vaccine and simultaneously provide a useful reagent for anti-HIV therapeutics and/or prophylaxis.
Recently, the precedent for an anti–HIV-1 microbicide has been established by the successful phase III trial of an antiretroviral-containing gel (Abdool Karim et al., 2010
). Furthermore, immunological microbicides, such as mAbgel (Brinckmann et al., 2011
), are under investigation. A VHH with the breadth of J3 has potential as the active component of such a microbicide because of the intrinsic temperature and pH stability of VHH (Gorlani et al., 2012
) and the ease and low cost with which they can be manufactured as compared with full-length human nAbs. In addition, J3 neutralizes a range of SHIV strains as well, making it an ideal candidate for SHIV mucosal challenge experiments either via passive immunization or in a microbicide formulation. The potential use of microbicides is not mutually exclusive with that of a vaccine, as the two could be complementary in a situation whereby an effective microbicide could limit the probability infection and thus support a vaccine-induced immune response.
In conclusion, the isolation of such a potent and broad VHH fragment from an animal immunized with a relatively simple combination of recombinant protein immunogens argues for the potential of vaccination to elicit cross-reactive protective anti-HIV Abs, and further study of this VHH will provide insight into how to recapitulate the elicitation of such Abs, hopefully in conventional IgG format. VHH J3 neutralizes 96% of all strains tested to date, and these include a great variety of HIV-1 subtypes. This highly cross-reactive anti–HIV-1 VHH differs from previous anti–HIV-1 VHH not only in its breadth but in terms of the immunization procedure undertaken, the screening method by which it was isolated and its phylogenetically distinct and affinity-matured sequence. The breadth of neutralization achieved via targeting the CD4-binding site demonstrated by J3 has only previously been seen as a result of natural infection (Corti et al., 2010
; Wu et al., 2010
; Scheid et al., 2011
). The definition of the precise CD4-binding site epitope targeted by J3 will be clarified by structural studies and may be used to optimize future immunogens, which can be easily and efficiently evaluated using this redefined screening process for llama VHH libraries.