HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism

Human immunodeficiency virus–1 infection remains a serious threat to health worldwide, and developing an effective HIV-1 vaccine is a global priority. A major challenge in achieving an effective vaccine is the inability of vaccines to induce broadly neutralizing Abs (bnAbs) that recognize conserved epitopes on a majority of circulating HIV-1 strains (1–4). bnAbs have not been elicited by vaccination, yet up to 50% of individuals chronically infected with HIV-1 develop bnAbs (5). Isolation of bnAbs from infected individuals revealed six conserved sites of vulnerability on the HIV-1 envelope protein (Env): the gp120 CD4 binding site (6–11), gp120 V1/V2 loop epitopes (12–14), gp120 V3-glycan epitopes (15–17), the gp41 membrane-proximal external region (MPER) (18–21), the gp41 fusion domain (22), and the gp120–gp41 interface (23, 24). At high concentrations, passive administration of bnAbs that target conserved Env epitopes (2F5, 4E10, 2G12, b12, VRC01, 3BNC117, or 10-1074) prevented simian HIV infection in monkeys (25–29).

Molecular mimicry of antigenic determinants between pathogens and host molecules can result in tissue damage, such as the Abs against a region of the hepatitis B virus that cross-react with myelin basic protein and can lead to tissue damage in the CNS (30–32). Similarly, Campylobacter jejuni lipo-oligosaccharides mimic host nervous system gangliosides and cause Guillain-Barre syndrome following infection (33, 34). Studies in mice demonstrated that these host ganglioside Abs are under tolerance control (33). Autoantibodies also were shown to penetrate the blood–brain barrier and affect intracellular enzyme function of neuronal cells during autoimmune disease (35). In retinopathy, anti-enolase Abs can alter the function of the enolase enzyme in neuronal cells (36). Furthermore, in multiple sclerosis, anti–heterogeneous nuclear ribonucleoprotein Abs were found to traffic into neurons and lead to apoptosis, contributing to multiple sclerosis pathology (37, 38).

HIV-1 bnAbs have unusual features, such as extensive somatic hypermutation and long third H chain complementarity regions (CDRH3s) that are associated with poly- or autoreactive BCRs, which can make Abs subject to immune tolerance control (39–41). We demonstrated previously that many HIV-1 bnAbs are autoreactive and/or polyreactive and the MPER-reactive bnAb, 2F5, binds to the tryptophan metabolism enzyme kynureninase (KYNU) (42, 43). A second MPER bnAb, 4E10, cross-reacts with the RNA splicing factor 3b subunit 3, as well as with anionic lipids. Because of the lipid reactivity, the 4E10 bnAb has anticoagulant activity; when administered to humans, it was biologically active and prolonged the partial thromboplastin time (44). Knock-in (KI) mice expressing the 2F5 or 4E10 bnAb V_HD_HJ_H and V_LJ_L rearrangements exhibit profound deletion of immature B cells in the bone marrow, demonstrating first-tolerance checkpoint control of these autoreactive HIV-1 bnAbs (45–50).

KYNU is a phylogenetically conserved enzyme of tryptophan metabolism that contains the 2F5 core epitope (ELDKWA) within the KYNU H4 domain; the 2F5 bnAb binds this site with nanomolar affinity (42). A key goal of HIV vaccine design is to induce gp41 MPER 2F5-like Abs, yet the effects of MPER Abs on KYNU function are unknown. Interestingly, dysregulated tryptophan metabolism was implicated in AIDS-related dementia; thus, it is of critical importance to determine whether MPER Abs that cross-react with KYNU disrupt tryptophan metabolism (51).

In this study, we determined the effect of 2F5 and 2F5-like anti-HIV gp41 Abs on KYNU enzymatic activity using an MPER peptide-liposome vaccine that activates anergic B cells in 2F5 bnAb double KI (dKI; V_HD_HJ_H + V_LJ_L) mice. This vaccine also elicited MPER-specific Abs in rhesus macaques (RMs) (50, 52). Thus, it is essential to determine whether Abs to the KYNU cross-reactive gp41 epitope inhibit KYNU or are deleterious in vivo. We demonstrate that immunization of 2F5 dKI mice and RMs with MPER peptide-liposomes elicited KYNU-reactive, 2F5 epitope–targeted Abs but that these Abs did not inhibit KYNU enzymatic activity in vitro or perturb tryptophan metabolism or induce tissue pathology in vivo.

Recombinant human kynureninase (catalog number 4877-KH; R&D Systems) and 10 nM 3-hydroxy-dl-kynurenine (catalog number H1771; Sigma-Aldrich) substrate are incubated in assay buffer (50 mM Tris; 0.05% [w/v] Brij-35, 5 μM pyridoxal phosphate [pH 8] in a F16 black MaxiSorp plate [catalog number 475515; Nunc]). Fluorescence is read at excitation and emission wavelengths of 315 and 415 nm, respectively, in kinetic mode for 5 min with a fluorescent plate reader (SpectraMax Gemini EM). Calibration standard 3-hydroxyanthranilic acid (catalog number H9391; Sigma-Aldrich) is used as a calibration control to calculate the conversion factor. Specific activity is calculated (Supplemental Fig. 2) (42).

rAbs are added at 0, 6, 60, 600, or 6000 ng, and immune sera are added at a 1:10 dilution to determine the inhibition or enhancement of KYNU enzyme activity compared with no inhibitor control.

Polyclonal anti-KYNU Ab was purchased (catalog number AF4887; R&D Systems), and the V(D)J gene fragments of Abs 2F5 and CH65 were synthesized and cloned (GenScript) into plasmids containing human or rhesus IgG1/IgK/IgL constant regions. Recombinant mAbs were produced in 293 F cells (Life Technologies) by cotransfection with plasmids expressing the Ig H and L chain genes and were purified from the culture supernatant by protein A column chromatography (53, 54). Human KYNU and human KYNU with a single aspartic acid–to–glutamic acid mutation genes were synthesized with a C-terminal 6-histidine tag and cloned into pcDNA3.1 (GenScript). Plasmids were transfected into 293F cells, and culture supernatants were purified by nickel and size-exclusion chromatography (55). Biotin-labeled SP62 (⁶⁵²QQEKNEQELLELDKWASLWN⁶⁷¹) peptide and biotin-labeled MPER656 (⁶⁵⁶NEQELLELDKWASLWNWFNITNWLWYIK⁶⁸³) peptide were synthesized (CPC Scientific).

Recombinant and serum Ab binding was determined using ELISA, as described (55, 56).

Mature 2F5 (m2F5) dKI and germline 2F5 (gl2F5) dKI mice were generated on the C57BL/6 background, as previously described (46, 50). Three groups of four m2F5 dKI mice were immunized six times, every 14 d, with i.p. injections (200 μl) of the MPER peptide-liposome (25 μg) formulated with glucopyranosyl lipid adjuvant (GLA; 0.625 μg), Alhydrogel (alum; 12.5 μg; lot: QD565), or a combination. MPER peptide-liposomes contained a version of the 2F5 epitope–containing MPER peptide 656 (⁶⁵⁶NEQELLELDKWASLWNWNITNWLWIK⁶⁸³) that was synthesized with the C-terminal hydrophobic membrane anchor tag GTH1 (YKRWIILGLNKIVRMYS) as previously described (57). All mice were 8–12 wk old at the start of the immunization study and were housed in the Duke University Vivarium in a pathogen-free environment with 12-h light/dark cycles at 20–25°C in accordance with all of the Duke University Institutional Animal Care and Use Committee–approved animal protocols.

Serum was collected and analyzed 10 d postimmunization, and animals were necropsied after completion of the study. Two groups of 6 (12 total) unimmunized aged-matched C57BL/6 control mice and unimmunized m2F5 (5 mice) and gl2F5 (6 mice) dKI mice were necropsied as unimmunized controls.

Serum and brain tryptophan, kynurenine, and kynurenic acid levels were determined for immunized and naive mice, as previously described (58).

Prepared stained tissue sections were delivered to a board-certified veterinary pathologist for assessment at Duke University’s Department of Pathology. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, cut on a microtome at 5 μm, and stained with H&E.

Twenty-three healthy adult Chinese-origin RMs were housed at BIOQUAL (Rockville, MD) in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. The protocol was approved by BIOQUAL’s Institutional Animal Care and Use Committee under Office of Animal Laboratory Welfare Assurance Number A-3086-01. BIOQUAL is International Association for Assessment and Accreditation of Laboratory Animal Care accredited. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and with the recommendations of The Weatherall Report on the use of Non-Human Primates in Research. All procedures were performed under anesthesia using ketamine hydrochloride, and all efforts were made to minimize stress, improve housing conditions, and to provide enrichment opportunities (e.g., social housing when possible, objects to manipulate in cage, varied food supplements, foraging and task-oriented feeding methods, interaction with caregivers and research staff). Animals were euthanized by sodium pentobarbital injection in accordance with the recommendations of the panel on Euthanasia of the American Veterinary Medical Association.

Macaques were immunized (250 μl at two sites) six times with MPER peptide-liposomes formulated with GLA (eight animals), GLA + alum (eight animals), or alum alone (seven animals) at 6-wk intervals. Blood was collected preimmunization and 2 wk after each immunization. Animals were necropsied at the completion of the study.

All animals were clinically monitored throughout the immunization study; this included complete blood count, serum chemistry, and hematology that were performed by the attending veterinarian using commercial automated hematology and serum chemistry analyzers (BIOQUAL). Prepared stained splenic tissue sections were delivered to a board-certified veterinary pathologist for assessment at Duke University’s Department of Pathology. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, cut on a microtome at 5 μm, and stained with H&E.

MPER-specific memory B cells of a macaque immunized three times with the MPER peptide-liposome vaccine formulated in GLA + alum were sorted by flow cytometry as described (21, 50). Briefly, ∼1 × 10⁷ PBMCs were decorated with B cell Ab panel: CD14 (BV570), CD3 (PerCPCy5.5), CD20 (FITC), CD27 (allophycocyanin-Cy7), and IgD (PE) (BD Biosciences) and Alexa Fluor 647– and Brilliant Violet 421–tagged MPER.03 peptides (KKKNEQELLELDKWASLWNWFDITNWLWYIRKKK). HIV gp41-specific memory B cells were gated as CD3⁻CD14⁻CD20⁺CD27⁺sIgD⁻MPER.03 (AF647)⁺ MPER.03 (BV421)⁺ and sorted into 96-well PCR AQ22 plates containing 20 μl of reverse-transcription reaction buffer that included 5 μl of 5× first-strand cDNA buffer, 1.25 μl of DTT, 0.5 μl of RNaseOUT (Life Technologies), 0.0625 μl of IGEPAL (Sigma-Aldrich), and 13.25 μl of ultrapure distilled water (Life Technologies).

RM V_HDJ_H and V_LJ_L segments were isolated by single-cell RT-PCR using the method described (54). The isolated V(D)J gene fragments were used for the construction of linear expression cassettes for production of recombinant mAbs in 293T cells for small-scale ELISA screening (59).

Cloanalyst (http://www.bu.edu/computationalimmunology/research/software/) was used to annotate isolated V_HDJ_H and V_LJ_L sequences with immunogenetic information and to test for clonal lineage membership (50, 54).

Select V(D)J gene fragments were synthesized, expressed, and purified as rhesus IgG1 recombinant mAbs, as described above.

RM serum and recombinant mAbs were screened by ELISA for binding to SP62 and select SP62 alanine mutants, recombinant KYNU and mutant KYNU (mutKYNU) (55).

The polyreactivity of rhesus mAbs was assessed with the AtheNA Multi-Lyte System (ZEUS Scientific) and HEp-2 cells immunofluorescence assay (Inverness Medical Professional Diagnostics), as described (50, 60).

Statistical analysis was performed in SAS version 9.4 (SAS Institute). All statistical analyses used the Wilcoxon–Mann–Whitney test with Benjamini–Hochberg false-discovery rate correction for multiple testing. GraphPad Prism version 6.01 was used for graphical representation.

We previously identified that KYNU cross-reacted with the HIV-1 gp41 membrane-proximal targeting bnAb 2F5 (42). 2F5 binds to the HIV-1 gp41 peptide SP62 (⁶⁵²QQEKNEQELLELDKWASLWN⁶⁷¹), but it does not bind this peptide when position 664 is mutated within the gp41 peptide SP62 Mut (⁶⁵²QQEKNEQELLELAKWASLWN⁶⁷¹, Fig. 1A). The KYNU H4 domain in most mammals (ELDKWA) exactly replicates this MPER epitope, and this motif is conserved within humans, mice, and rhesus monkeys (42). However, opossums carry a rare substitution in the KYNU H4 ELDKWA motif whereby aspartic acid (D) is replaced by glutamic acid (E) (42). The same replacement in human recombinant KYNU (mutKYNU) completely abrogates binding by the 2F5 bNAb (Fig. 1A).

KYNU catabolizes 3-hydroxy-kynurenine into 3-hydroxyanthranilic acid. We used a standard in vitro enzymatic assay to measure the production of 3-hydroxyanthranilic acid at saturating substrate concentrations (42) (Supplemental Fig. 1A). Addition of recombinant KYNU or mutKYNU resulted in identically increased rates of 3-hydroxyanthranilic acid production, whereas the addition of substrate alone or assay buffer did not (Fig. 1B). As a control for Ab-mediated enzyme inhibition, we added increasing concentrations of polyclonal anti-KYNU IgG Ab and demonstrated a concentration-dependent inhibition of 3-hydroxyanthranilic acid production (Fig. 1C). Increasing amounts of the CH65 influenza IgG Ab had no detectable effect on KYNU/mutKYNU enzyme activity (Fig. 1D, Supplemental Fig. 1E). KYNU and mutKYNU were identically inhibited by KYNU IgG, and neither was affected by CH65 rIgG (Supplemental Fig. 1B–E).

In contrast, at no concentration did the addition of 2F5 bNAb inhibit KYNU or mutKYNU enzymatic activity (Fig. 1E, 1F). Likewise, the 2F5 bnAb V region inserted into an RM IgG1 Fc backbone had no effect on KYNU or mutKYNU enzymatic activity (Supplemental Fig. 1D).

We immunized mice that express only the mature (m)2F5 bnAb rearranged H and L chain Ab genes (m2F5 dKI mice) with MPER peptide-liposomes formulated with alum, GLA (TLR4 agonist), or a combination of alum and GLA to determine which adjuvant elicited the highest titers of 2F5 bnAbs. After two immunizations, the GLA and GLA + alum groups had induced Abs that targeted the HIV gp41 MPER peptide (⁶⁵²QQEKNEQELLELDKWASLWN⁶⁷¹) with ELISA logAUC values of four and six, respectively, whereas no animal in the alum-alone group had titers of MPER-binding mAbs (Fig. 2A). Similarly, after two immunizations, GLA- and GLA + alum–immunized mice had titers of anti-KYNU Abs (Fig. 2A). Ab to MPER peptide and KYNU protein was boosted with subsequent immunizations for the three adjuvant groups, but GLA- or GLA + alum–immunized mice maintained higher Ab titers compared with alum-immunized mice (Fig. 2A). Abs to MPER and KYNU were strongly correlated with one another in all adjuvant groups, demonstrating the relatedness of the two responses (Fig. 2B). At bleed 6, plasma Ab neutralization was observed against the HIV virus MN in the TZM-bl–neutralization assay (Fig. 2C). The magnitude of neutralization titers corresponded with the HIV-binding Ab titers, with the GLA + alum and GLA adjuvant groups having the highest neutralization titers; only one animal in the alum-immunized group had ID₅₀ titers > 50 (Fig. 2C).

We then examined the effects of plasma IgG Ab from immunized m2F5 dKI mice on KYNU enzymatic activity. No significant inhibition of KYNU enzymatic activity was observed for any of the three immunization groups, including the GLA and GLA + alum groups, which had the highest titers of vaccine-elicited KYNU IgG Abs (Fig. 2D). Indeed, there was no significant difference in the inhibitory activity between pre- and postimmunization plasma IgG samples. Thus, vaccine-elicited 2F5 bnAbs from m2F5 dKI mice had no effect on KYNU activity.

Although we did not detect any inhibition of KYNU activity by the 2F5 bNAb or plasma IgG in immunized m2F5 dKI mice (Figs. 1E, 2D), we recovered tissue samples from naive C57BL/6 control mice, gl2F5 dKI mice that expressed the 2F5 unmutated ancestor, m2F5 dKI mice, and immunized m2F5 dKI mice and compared brain and serum levels of kynurenic acid, kynurenine, and tryptophan, the products of tryptophan metabolism upstream of KYNU (Fig. 3A). Ab that inhibited KYNU activity in vivo would result in increased levels of these metabolites. No significant differences were observed in the levels of kynurenic acid, kynurenine, or tryptophan in the brains of naive or immunized mice (Fig. 3B, Supplemental Fig. 2A). No significant differences were found in serum levels of kynurenic acid, but slightly higher levels of kynurenine and tryptophan were present in mice immunized with all forms of MPER peptide-liposome compared with control C57BL/6 mice (Fig. 3C, Supplemental Fig. 2A). The increases in serum kynurenine and tryptophan in immunized mice did not appear to be related to elicited KYNU Abs, because the alum-formulated liposome-immunized group with the lowest levels of KYNU Ab titers (Fig. 2A) exhibited levels of serum kynurenine and tryptophan that were similar to those animals receiving GLA and GLA + alum immunogens with higher levels of KYNU Abs (Fig. 3C).

A histopathology assessment of spleen tissue sections from m2F5 dKI mice was performed. Consistent with the deletional tolerance that is characteristic of m2F5 dKI mice (46), naive and immunized KI mice exhibited comparably low numbers of splenic B lymphocytes compared with C57BL/6 controls, resulting in small spleens and diminished white pulp areas (Fig. 3D). Furthermore, spleens were analyzed for vaccine-related lesions, and no pathologic abnormalities in the marginal zone, periarteriolar lymphoid sheath areas, or red pulp were observed in vaccinated animals with high titers of KYNU cross-reactive Abs (Fig. 3D, 3E). Immunized gl2F5 dKI mice without anti-KYNU Abs also exhibited B cell deletion and tolerance control (50), and our analysis revealed that, like m2F5 dKI mice, gl2F5 dKI mice had small spleens that were similarly lymphodepleted (Supplemental Fig. 2B). Thus, the lymphodepletion seen in m2F5 and gl2F5 dKI mice was due to the deletion of >95% of B cells in bone marrow as the result of immune tolerance.

We also performed a histopathology assessment of the brains from m2F5 dKI, gl2F5 dKI, and C57BL/6 control mice; there was no evidence of neural degeneration, demyelination, or inflammation in any of the brain sections studied (Supplemental Fig. 2C). We concluded that, despite the induced 2F5 bnAb levels and the high titers of KYNU binding IgG Ab resulting from the induced 2F5 bnAb levels elicited by immunizing m2F5 dKI mice with MPER peptide-liposome, it did not perturb tryptophan metabolism in the brain or serum and did not induce any pathological changes in peripheral lymphoid tissues (Fig. 3).

Three groups of RMs were immunized six times with MPER peptide-liposomes formulated with GLA, alum, or GLA + alum. In contrast to immunization of 2F5 KI mice, GLA alone was poorly immunogenic in RMs and elicited the lowest MPER IgG plasma Ab (Fig. 4A). In contrast, MPER liposomes formulated in alum or GLA + alum elicited higher titers of MPER plasma Ab (Fig. 4A, 4B). Immunization with alum or GLA + alum MPER peptide-liposomes also elicited higher titers of cross-reactive KYNU Abs in macaques compared with the GLA-alone group (Fig. 4C). The 2F5 core epitope is the DKW motif within the HIV-gp41 MPER peptide SP62 (⁶⁵²QQEKNEQELLELDKWASLWN⁶⁷¹), and plasma Ab binding to SP62-mutated peptides within the D, K, or W was reduced for all three groups, and it was significantly reduced for the SP62 D664A mutant in the GLA and GLA + alum groups (Fig. 4D; p < 0.05; Wilcoxon–Mann–Whitney U test). Thus, MPER peptide-liposome–induced plasma Ab was targeted near the 2F5 bnAb epitope in all RMs, with alum-alone and GLA + alum adjuvants eliciting the highest titers of MPER- and KYNU-reactive Abs.

In all three adjuvant groups, KYNU RM plasma IgG had no effect on KYNU enzyme activity in vitro (Fig. 5A). We analyzed the histopathology of immunized macaque spleen and found that it was normal in all groups (Fig. 5B). These data confirmed that the splenic lymphopenia of naive gl2F5 dKI mice and vaccinated m2F5 dKI mice is the consequence of B cell deletion due to immune tolerance and not a pathologic consequence of anti-KYNU Ab production.

Blood samples from RMs did not reveal any changes in electrolytes, glucose, renal and liver function, or complete blood counts related to MPER peptide-liposome vaccination (Supplemental Fig. 3A, 3B).

gp41 Env–reactive memory B cells (CD3⁻CD14⁻CD20⁺CD27⁺sIgD⁻) from PBMCs from an RM immunized with MPER liposome formulated in GLA and alum that were decorated with MPER peptide tetramers (MPER.03) labeled with two fluorophores were sorted for sequencing of the BCR (V_H + V_L) genes. MPER-reactive memory B cells represented 0.26% of total memory B cells (Fig. 6A). BCR genes were amplified by RT-PCR, sequenced, and fused to cassettes containing a CMV promoter, Ig C region, and polyA tail by overlapping PCR. H and L chain amplicon pairs were transiently transfected into 293F cells, and supernatants containing rAb were screened for binding by ELISA. Using this approach, we identified 31 MPER-reactive Abs that bound clade B MN gp41 recombinant protein, 27 that bound rHIV-1 gp140 Env (Con-S or B. JRFL gp140, Supplemental Fig. 4A), and 6 MPER-reactive rAbs that, in addition to MPER, bound human KYNU (Fig. 6B).

VDJ sequence analysis revealed that the isolated MPER Abs used diverse V_H gene segments, and three of the six KYNU–cross-reactive Abs used a VH gene segment orthologous to the human V_H2–5 gene used in the 2F5 bNAb (Supplemental Fig. 4B, Table I) (61). The remaining KYNU binders carried rearrangements of V_H4 family gene segments (Supplemental Fig. 4B, Table I).

The 31 VDJ rearrangements recovered from memory B cells that bound the MPER.03 tetramer exhibited varying levels of somatic hypermutation and diverged from the germline RM VH gene segments by 0.38–11.8%; rearrangements from KYNU-reactive Abs exhibited a similar range of mutation frequencies (1.1–5.2%) (Supplemental Fig. 4C, Table I). Most MPER bnAbs have long CDRH3s that are >15 aa, and the RM Abs had CDRH3 lengths ranging from 12 to as long as 21 aa (Supplemental Fig. 4D, Table I).

We selected four mAbs, based on gp41 and KYNU cross-reactivity, including two that used the RM V_H2–5 gene segment ortholog, for further study. All four mAbs bound to HIV-1 gp41 peptide (SP62; ⁶⁵²QQEKNEQELLELDKWASLWN⁶⁷¹), and three of the four mAbs had >60% reduction in binding to peptides containing alanine substitutions within the 2F5 bnAb DKW epitope (Fig. 6C). The mAb DH563 showed a more moderate 20–50% reduction on the DKW alanine mutants (Fig. 6C). Similarly, all four Abs bound to KYNU and had reduced binding to mutKYNU that contains ELEKWA instead of wild-type KYNU, ELDKWA (Fig. 6D). None of the mAbs were capable of neutralizing HIV-1 in the TZM-bl pseudovirus inhibition assay (data not shown).

In addition to KYNU cross-reactivity, three of the four Abs were cross-reactive with other host proteins associated with autoimmune disease: DH653 reacted with dsDNA, DH654 reacted with SSA, SSB, and Jo1 proteins, and DH656 reacted with Jo1 protein (Fig. 6E). Three of the four KYNU-reactive Abs also exhibited antinuclear Ab phenotypes by reacting with nuclear Ags in HEp-2 cells; DH654 decorated cytoskeletal proteins, DH655 bound to nuclear punctae (dots), and DH656 diffusely bound within the cytoplasm (Fig. 6F). Although these KYNU-binding Abs exhibited reactivity for HEp-2 cell components, none of them were capable of inhibiting KYNU enzymatic activity in vitro (Fig. 7). Thus, adjuvanted MPER liposome immunogens were capable of inducing polyclonal and polyreactive IgG Ab responses to MPER epitopes in RMs; although these responses included anti-KYNU Abs, they did not result in tissue pathology or inhibit KYNU enzyme function.

Mimicry of host proteins by microbial pathogens can subvert the host immune response (30–34, 62, 63). In this study, we demonstrated that the HIV-1 bnAb 2F5 had no inhibitory effect on KYNU enzyme activity. Moreover, a single-residue change within the 2F5 epitope in KYNU that abolished 2F5 binding also did not affect KYNU enzyme function. The 2F5 epitope (ELDKWA) within KYNU is in the H4 domain that mediates KYNU homodimerization (64), likely explaining the inability of anti-ELDKWA Abs to inhibit KYNU activity.

B cell development in 2F5 V_HDJ_H and V_LJ_L KI mice was blocked by clonal deletion in bone marrow, because of host cross-reactivity with lipids and KYNU resulting in bnAb immune tolerance control (46, 50). MPER bnAbs 2F5, 4E10, and 10E8 all interact with the virion membrane as a part of their epitopes (47, 65, 66). We developed a minimal peptide-liposome immunogen that was designed to present MPER bnAb epitopes in the same manner as on an HIV-1 virion (57). Immunization of 2F5 KI mice with the MPER peptide-liposome immunogen rescued anergic B cells that escaped deletion to produce high levels of bnAbs (46). Immunization of macaques showed that the MPER peptide-liposome can initiate Abs with 2F5-like characteristics (50).

In this study, bnAb 2F5 KI mouse plasma Abs recognized KYNU but, like the human 2F5 mAb, did not inhibit KYNU activity. Moreover, we examined brain and serum levels of tryptophan and its metabolites after MPER peptide-liposome immunization in vivo and did not detect tryptophan pathway metabolite perturbations that could be attributed to induction of 2F5 bnAb activity.

Immunization of RMs with MPER peptide-liposomes formulated in alum or GLA + alum elicited high-titers of Abs that corecognized the 2F5 epitope and KYNU. In macaques, plasma Ab inhibition of KYNU was not observed, and no significant pathology was observed in macaque spleen or blood hematology or chemistry tests. Isolation of mAbs by Ag-specific single-cell flow cytometry revealed that the 2F5 epitope (ELDKWA) in HIV-1 gp41 and KYNU could be recognized by Abs that used diverse Ig gene segments, and these mAbs did not inhibit KYNU activity. We did not detect plasma HIV-1 neutralization in the TZM-bl assay for any of the vaccine-induced MPER-KYNU cross-reactive Abs, indicating that additional immunizations that increase affinity maturation, selecting Abs with recognition of lipid, will be required to achieve neutralization activity (50).

It has been reported that elevated tryptophan metabolites can be associated with depression in animal models (67). Depression side-effects of elevated tryptophan metabolism would not have been detected in our studies, but in the absence of evidence that 2F5 bnAb or individual anti-KYNU Abs inhibited KYNU enzyme activity, our studies do not support a role for ELDKWA-targeted Abs in tryptophan metabolite alteration.

In summary, host mimicry is a strategy used by HIV-1 to escape Ab responses to functionally conserved bnAb epitopes. Efforts to induce bnAbs by vaccination and to use bnAbs as therapeutic Abs are critical to control the AIDS epidemic. Thus, determining the impact of potentially protective Abs on host protein function is an important safety consideration. Our study demonstrates that the human bnAb 2F5 and vaccine-elicited 2F5 epitope–targeted Abs elicited in mice and macaques did not inhibit KYNU enzymatic function in vitro or cause tissue or enzyme activity abnormalities in vivo despite cross-reacting with KYNU protein. Although cross-reactivity with host Ags may impede bnAb development and impact therapeutic Ab pharmacokinetics, the 2F5–KYNU interaction neither impairs host enzyme function nor appears to mediate adverse events. Thus, the 2F5 HIV-1 Env gp41 region appears to be a safe target on HIV-1 Env for vaccine development.

We thank Dawn J. Marshall, John F. Whitesides, Joshua Eudailey, Tarra A. Von Holle, Thaddeus C. Gurley, Lawrence C. Armand, Andrew Foulger, Giovanna Hernandez, Jamie Pritchett, Krissey Lloyd, and Christina Stolarchuk for expert technical assistance. We also thank Kelly Soderberg and Samantha Bowen for project coordination and management.

B.F.H., S.M.A., and H.-X.L. have patent applications submitted on vaccine candidates used in this study (see international patent application PCT/US06/13684).