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A derivative of the D5 monoclonal antibody that targets the gp41 N-heptad repeat of HIV-1 with broad tier-2 neutralizing activity

HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the pre-hairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5’s six antibody complementarity-determining regions. One variant, D5_AR, demonstrated 6-fold enhancement in ID50 against lentivirus pseudotyped with HXB2 Env. Importantly, D5_AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5_AR IgG was greatly enhanced in target cells expressing FcγRI with ID50 values below 0.1 μg/mL; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5_AR have equivalent neutralization potency in IgG, Fab, and scFv formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR. Importance Despite advances in anti-retroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 pre-hairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date NHR antibodies have not been potent. Here, we engineered D5_AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5_AR was greatly potentiated in cells expressing FcγRI; FcγRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.


Introduction
Over 35 million people currently live with HIV/AIDS globally (1); despite the promise of anti-retroviral therapy, a preventative HIV-1 vaccine remains an urgent global health need.
Identifying and characterizing broadly neutralizing antibodies (bnAbs) is essential to informing the design of vaccine antigens that could elicit such antibodies in vivo (2)(3)(4). A major challenge to eliciting bnAbs is the high sequence variability of the viral glycoprotein Env; accordingly, significant efforts have been made to develop immunogens to focus the antibody response toward a handful of highly conserved regions that correspond to bnAb epitopes (5)(6)(7)(8). In contrast to these bnAb epitopes on the native conformation of Env, one highly conserved region of HIV-1 gp41, the N-heptad repeat (NHR), is only transiently exposed during viral entry. Upon binding cellular receptors, Env undergoes substantial conformational changes to form the pre-hairpin intermediate (PHI), in which the NHR and C-heptad repeat (CHR) regions are exposed before the cellular and viral membranes come together for membrane fusion (9)(10)(11).
Due to its high sequence conservation (~93%) (12) and critical role in viral entry (9), the NHR is a promising target for blocking HIV-1 infection. Several inhibitors of the NHR have been identified, most notably the FDA-approved fusion inhibitor enfuvirtide (13)(14)(15), validating the NHR as a therapeutic target in humans. In addition, cyclic D-peptides that bind the NHR disrupt the fusion process and inhibit HIV-1 infection (16,17). Of relevance for vaccine applications, several characterized monoclonal antibodies (mAbs) target the NHR (18)(19)(20). The first of these mAbs was D5 (18), which was isolated from a human B cell-derived phage display library using two synthetic mimetics of the gp41 PHI: 5-Helix (21) and IZN36 (22). D5 exhibits weak-but broad-neutralizing potency against laboratory-adapted and primary clinical isolates of HIV-1 by binding a conserved hydrophobic pocket of the NHR and preventing the fusion of the viral and cellular membranes (18). X-ray crystallography revealed that complementarity-determining region (CDR) loops in the antibody variable regions of both the heavy and light chains (VH and VL, respectively) contribute to the binding of D5 to the NHR (23). Informed by this insight, Montgomery et al. (24) sought to increase the neutralization potency of D5 by randomizing residues in five of the six CDRs (VH CDR 1, 2, 3 and VL CDR 1 and 3). Four D5 IgG variants, each with only one CDR mutated, had slightly increased neutralization potency (24).
Here we hypothesized that combining multiple CDR mutations from these D5 variants would create a more potent mAb against HIV-1. To test this hypothesis, we evaluated a panel of 16 variants: wild-type D5, four previously described CDR variants (24), and 11 recombined CDR variants ( Figure 1). The 11 recombined CDR variants are composed of combinations of the CDR sequences found in the four enhanced D5 variants. We determined that D5_AR, the most potent recombined variant, has increased neutralization efficacy in all tier-1 and tier-2 HIV-1 viruses tested compared to D5. As recently reported for D5 (25), and as previously characterized for membrane proximal external region (MPER) mAbs (26,27), the neutralization potency of D5_AR was enhanced at least 1,000-fold in target TZM-bl cells expressing the high-affinity immunoglobulin receptor FcγRI (TZM-bl/FcγRI cells). Although not expressed on CD4+ T cells, FcγRI is expressed on macrophages and dendritic cells (28), which are thought to be important in the early sexual transmission of HIV-1 at mucosal surfaces (29)(30)(31)(32)(33)(34)(35)(36). D5_AR demonstrates cross-clade tier-2 HIV-1 neutralizing activity and extremely potent activity when measured in cells expressing FcγRI, suggesting that the PHI may be a promising vaccine target for eliciting neutralizing antibodies with broad heterologous neutralization potency.
Next, we screened 11 additional D5 variants with lentivirus pseudotyped with HIV-1 HXB2 Env for neutralization potency using a single-round infectivity assay. Several recombinant D5 variants had little effect or even diminished the neutralization potency compared to D5 (Table   1). Nevertheless, we identified six D5 variants that modestly enhanced (>2.0-fold) the neutralization potency of D5 (Table 1). Among these, D5_H011_L0, in which both CDR2 and CDR3 of the heavy chain are mutated, demonstrated the greatest enhancement (4-fold) in ID50 (Table 1). We renamed this most potent D5 variant D5_AR for further characterization.
Our initial neutralization screen utilized antibody purified via Protein A affinity chromatography (Materials and Methods). During subsequent antibody purification, we removed aggregates through an additional size exclusion chromatography (SEC) step; comparison of neutralization potencies revealed that the presence of aggregates reduced the neutralization potencies of D5 and D5_AR ( Supplementary Information, Supplemental Figure 3). Experiments in Table 1 and Supplemental Table 1 were the only experiments reported here that used non-SEC-purified antibody preparations, which accounts for the difference in ID50 values for HXB2 reported in Table 1 and Figure 3.

D5 and D5_AR neutralize with similar potency as an scFv, Fab, and IgG
In the first description of D5, the IgG (~150 kD) and scFv (~25 kD) constructs neutralized similarly to one another (18). However, more recent studies reported that D5 scFv is more potent than D5 Fab (~50 kD), and that both were more potent than D5 IgG (20,23,24,42); in addition, there are reports that increasing the size of NHR inhibitors reduces neutralization potency (20,24,(42)(43)(44). These size-dependent findings would imply steric hindrance in accessing the PHI. Given our finding that SEC purification could impact the neutralization potency of D5 and D5_AR IgG (Supplemental Figure 3A), we decided to reinvestigate the question of size-dependent neutralization for D5 using antibody preparations free of aggregates. Notably, in agreement with the initial report (18) and in contrast to the previous reports (20,23,24,42), we found that D5 as an IgG, Fab, and scFv (all SEC-purified) did not exhibit a size-dependent pattern of neutralization ( Figure 2A). Additionally, we detected comparable neutralization potency for D5_AR as an scFv, Fab, and IgG ( Figure 2B). These results demonstrate that neither D5 nor D5_AR are impacted by steric hindrance and suggest the presence of protein aggregates could explain previous reports of size-dependent neutralization for D5 scFv, Fab, and IgG.

D5_AR neutralization potency is enhanced >1,000-fold in FcγRI-expressing cells
Recently, we reported the neutralization potency of D5 IgG to be greatly increased in TZM-bl cells expressing the cell-surface receptor FcγRI (TZM-bl/FcγRI cells) (25). FcγRI is the only known high-affinity IgG receptor in humans, capable of binding monomeric IgG (61). To determine whether D5_AR IgG is similarly potentiated, we tested its neutralization of an additional Consistent with an Fc-dependent mechanism, the Fab form of D5_AR did not exhibit potentiation ( Figure 5A). This observed potentiation was specific to FcγRI: enhanced neutralization was minimal or not observed in cell lines expressing other Fc receptors (FcγRIIa, FcγRIIb, and FcγRIIIa; Figure 5B). It is noteworthy that the ID50 values of D5_AR IgG in TZM-bl cells were approximately linearly related to the ID50 values in TZM-bl/FcγRI cells ( Figure 6).

Discussion
The PHI of HIV-1 is a validated drug target in humans (13)(14)(15), and antibodies that bind the NHR of gp41 that is exposed in the PHI can inhibit HIV-1 infection in vitro (18)(19)(20). The first of these NHR-binding antibodies, D5, has weak neutralization potency against tier-1 HIV-1 strains (18). Here we engineered and characterized a more potent D5 derivative, D5_AR, which exhibited enhanced neutralization of tier-1 strains and weak cross-clade neutralization of tier-2 HIV-1 viruses ( Figure 3). Using ten global HIV-1 reference strains, we determined that at modest concentrations (1-50 μg/mL), D5_AR neutralizes the majority of tier-1 and tier-2 HIV-1 strains tested from a variety of clades ( Figure 3). Taken together with the high sequence conservation of the NHR (12), these results support future vaccine design efforts to elicit neutralizing antibodies against the PHI.
Several previous reports had suggested that access to the D5 epitope was impacted by steric hindrance, as smaller antibody constructs were more potent than full-length IgG (20,23,24,42).
We reinvestigated this issue using antibody preparations that were free of observed protein aggregates ( Supplementary Information; Supplemental Figure 3). In contrast to earlier reports (20,23,24,42), here we confirm (18) that D5 and D5_AR are similarly potent when tested in neutralization assays in scFv, Fab, and IgG formats ( Figure 2). We hypothesize that the presence of higher-order protein aggregates (that can be removed by SEC) may explain the previous reports of size-dependent neutralization by D5. Given these findings for D5 and D5_AR, we conclude that steric hindrance is not an obstacle for at least some anti-PHI antibodies.
Previous work on antibodies targeting another epitope of gp41, the MPER, found that neutralization activity was potentiated as much as 5,000-fold in cells expressing FcγRI, an integral membrane protein that interacts with the Fc portion of γ immunoglobulins (26)(27)(28). Since the MPER is not fully exposed until after Env engages with cellular receptors (62,63), these results suggest that by binding the Fc region of MPER mAbs, FcγRI provides a local concentration advantage at the cell surface that enhances neutralization (26,27). Because the PHI, like the MPER, is fully exposed only during viral membrane fusion, we previously investigated the effect of FcγRI on D5 and found neutralization by this anti-PHI mAb is also enhanced ~5,000-fold (25).

TZM-bl/FcγRI cells enable sensitive detection of neutralization activity from anti-NHR
antibodies and could be used, in conjunction with TZM-bl cells, to monitor progress toward eliciting neutralizing antisera. We hypothesize that neutralizing activity detected by TZM-bl/FcγRI cells could be used as an indicator of low-affinity antibody precursors in serum that have the potential to mature to high-affinity neutralizing activity independent of FcγRI. This hypothesis is supported by our findings that the neutralizing activity of D5_AR in FcγRI-expressing cells were approximately linearly related to the neutralizing activity of D5_AR in cells without FcγRI ( Figure   6).
Although not normally expressed on CD4+ T cells, FcγRI receptors are expressed on some cells at mucosal surfaces where sexual HIV-1 transmission occurs, such as macrophages and dendritic cells (28). Mucosal macrophages and dendritic cells can be productively infected by HIV-1 (29-32) and can then mediate viral transmission to CD4+ T cells (33)(34)(35)(36). Importantly, Taken together, the findings presented here provide evidence that the NHR of the PHI is a promising target for future HIV-1 vaccine development, and pave the way for future studies of the in vivo significance of FcγRI-mediated potentiation of anti-PHI antibodies.

Mammalian expression of D5 constructs
The variable heavy (VH) chain regions of the D5 variants were ordered as gene fragments from Twist Biosciences (Supplemental Figure 1A). Gene fragments were resuspended to 10 ng/μL in H2O and PCR amplified using the following two primers: (HC_forward)

Purification and storage of D5 constructs
For the initial screening in neutralization assays (Table 1 and Supplemental Table 1) Fab and scFv constructs were eluted from affinity purification and then purified further via size exclusion chromatography using the Superdex 200 Increase 10/300 GL column (GE HealthCare) and 1X PBS. Samples were spin concentrated as described above.
For all samples, regardless of the purification procedure, concentrated elution samples were syringe filtered using a 0.22-μm filter and stored at 4 °C prior to use.

Transfection to produce HIV-1 pseudotyped lentiviruses
HEK293T cells were transiently co-transfected with a backbone plasmid as well as a HIV-1 Env plasmid for HIV-1 pseudotyped lentivirus production using the calcium phosphate transfection protocol previously described (77)(78)(79). HEK293T cells were passaged in T75 flasks and incubated at 37 °C at 5% CO2. The growth medium used for passaging and transfections was Virus-containing medium was harvested 48 h after medium swap and centrifuged at 300 x g for 5 min; the supernatant was sterile-filtered with a 0.45-μm polyvinylidene difluoride filter and stored in 1-mL aliquots at -80 °C.

Neutralization Assay
The neutralization assay was adapted from the TZM-bl assay protocol using HIV-1 Env-pseudotyped viruses as described previously (38,41). Briefly, TZM-bl cells, derived from the JC53-bl parental cell line, were used as reporter cells in this assay and were obtained through the NIH AIDS Reagent Program (Cat#8129) from Dr. John C. Kappes, and Dr. Xiaoyun Wu (80,(82)(83)(84)(85). TZM-bl cells are adherent HeLa cells that stably express CD4 and CCR5 and constitutively express CXCR4; they have integrated β-galactosidase and firefly luciferase reporter genes under the control of the HIV-1 LTR promoter. TZM-bl cells transduced to stably express FcγRI (26,27), were also used in these neutralization assays. TZM-bl cells were passaged in T25 flasks and incubated at 37 °C at 5% CO2 without shaking. The growth medium used for passaging and neutralization assays was Corning® DMEM with 10% fetal bovine serum, 1% penicillin streptomycin (Corning), and 1% L-glutamine (Corning).

Statistics and Data Analysis
Percent infectivity for the neutralization assays was calculated as follows: .
The ID50 values in Table 1 and Figure 3 represent the geometric mean of the biological replicates for the tested antibodies with the standard error of the mean reported. Fold difference in ID50 was calculated for each experiment by dividing the D5 ID50 by the D5 variant ID50. Because fold difference was calculated for each experiment, the reported fold differences in Table 1 and Figure   3 are the geometric mean and the standard error of the mean from all replicates.

Author Contributions
AAR, MVFI, CLB, and PSK conceived the experiments. AAR performed protein purification, antibody characterization, and neutralization assays. MVFI and BNB performed protein purification and neutralization assays. Additional neutralization assays were also conducted under the supervision of CCL and DCM. All authors contributed to revising the manuscript.

Purification method impacts D5's neutralization potency
After completing the initial neutralization screens of the D5 variants, a more in-depth neutralization profile was determined for D5_AR compared to D5. This profiling involved testing the constructs' neutralization potency against various viruses and in various antibody formats.
Notably, we found that the manner of purification and concentration of antibodies impacts the observed neutralization activity of the antibody. In our initial neutralization screens, we did not include a size exclusion chromatography (SEC) purification step after Protein A purification: we immediately spin-concentrated the elution and filtered the concentrated antibody for use in neutralization assays (Materials and Methods). For the in-depth neutralization analysis, we further purified antibodies via SEC after Protein A purification. After SEC purification, we concentrated the protein using spin concentrators. These ID50 values ( Figure 3) differed from the values that we previously measured (Table 1) when the antibodies were not SEC-purified (SEC-purified preparations had a 1.5-fold decrease in ID50 for D5 and a 2.3-fold decrease in ID50 for D5_AR) (Supplemental Figure 3). A side-by-side comparison revealed that there was indeed a difference in neutralization for D5-AR (Supplemental Figure 3A), although neutralization was still enhanced compared to D5. UV traces from SEC indicated that aggregates may explain this difference in reported neutralization (Supplemental Figure 3B), which likely explains the discrepancy in reported ID50 values for HXB2 in Table 1 and Figure 3 of the main text. Supplemental Table 1: Single-CDR mutants neutralize pseudotyped HIV-1 HXB2 in a fashion consistent with the report of Montgomery et al. (24). The ID50 for this work is represented by the geometric mean and standard error of the mean of replicate experiments (n=2). For each infection assay, the fold enhancement versus D5 was calculated (ID50, D5 / ID50, D5 variant); reported fold enhancement is the geometric mean (n=2).