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

This information is current as Todd Bradley, Guang Yang, Olga Ilkayeva, T. Matt Holl, of September 29, 2021. Ruijun Zhang, Jinsong Zhang, Sampa Santra, Christopher B. Fox, Steve G. Reed, Robert Parks, Cindy M. Bowman, Hilary Bouton-Verville, Laura L. Sutherland, Richard M. Scearce, Nathan Vandergrift, Thomas B. Kepler, M. Anthony Moody, Hua-Xin Liao, S. Munir Alam, Roger McLendon, Jeffrey I. Everitt, Christopher B. Newgard, Downloaded from Laurent Verkoczy, Garnett Kelsoe and Barton F. Haynes J Immunol 2016; 197:4663-4673; Prepublished online 14 November 2016; doi: 10.4049/jimmunol.1601484 http://www.jimmunol.org/content/197/12/4663 http://www.jimmunol.org/

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

Todd Bradley,*,†,1 Guang Yang,‡,1 Olga Ilkayeva,x,1 T. Matt Holl,‡ Ruijun Zhang,* Jinsong Zhang,* Sampa Santra,{ Christopher B. Fox,‖ Steve G. Reed,‖ Robert Parks,* Cindy M. Bowman,* Hilary Bouton-Verville,* Laura L. Sutherland,* Richard M. Scearce,* Nathan Vandergrift,*,† Thomas B. Kepler,# M. Anthony Moody,*,†† Hua-Xin Liao,* S. Munir Alam,*,† Roger McLendon,x Jeffrey I. Everitt,x Christopher B. Newgard,†† Laurent Verkoczy,*,†,x Garnett Kelsoe,*,‡ and Barton F. Haynes*,†,x

The HIV-1 envelope protein (Env) has evolved to subvert the host immune system, hindering viral control by the host. The tryptophan metabolic enzyme kynureninase (KYNU) is mimicked by a portion of the HIV Env gp41 membrane proximal region (MPER) and is cross- Downloaded from reactive with the HIV broadly neutralizing Ab (bnAb) 2F5. Molecular mimicry of host proteins by pathogens can lead to autoimmune disease. In this article, we demonstrate that neither the 2F5 bnAb nor HIV MPER-KYNU cross-reactive Abs elicited by immunization with an MPER peptide-liposome vaccine in 2F5 bnAb VHDJH and VLJL knock-in mice and rhesus macaques modified KYNU activity or disrupted tissue tryptophan metabolism. Thus, molecular mimicrybyHIV-1Envthatpromotestheevasionofhostanti–HIV-1Ab responses can be directed toward nonfunctional host protein epitopes that do not impair host protein function. Therefore, the 2F5 HIV Envgp41regionisakeyandsafetarget for HIV-1 vaccine development. The Journal of Immunology, 2016, 197: 4663–4673. http://www.jimmunol.org/

uman immunodeficiency virus–1 infection remains a seri- passive administration of bnAbs that target conserved Env epitopes ous threat to health worldwide, and developing an effec- (2F5, 4E10, 2G12, b12, VRC01, 3BNC117, or 10-1074) prevented H tive HIV-1 vaccine is a global priority. A major challenge simian HIV infection in monkeys (25–29). in achieving an effective vaccine is the inability of vaccines to Molecular mimicry of antigenic determinants between patho- induce broadly neutralizing Abs (bnAbs) that recognize conserved gens and host molecules can result in tissue damage, such as the epitopes on a majority of circulating HIV-1 strains (1–4). bnAbs Abs against a region of the hepatitis B virus that cross-react with have not been elicited by vaccination, yet up to 50% of individuals myelin basic protein and can lead to tissue damage in the CNS (30– by guest on September 29, 2021 chronically infected with HIV-1 develop bnAbs (5). Isolation of 32). Similarly, Campylobacter jejuni lipo-oligosaccharides mimic bnAbs from infected individuals revealed six conserved sites of host nervous system gangliosides and cause Guillain-Barre syn- vulnerability on the HIV-1 envelope protein (Env): the gp120 drome following infection (33, 34). Studies in mice demonstrated CD4 binding site (6–11), gp120 V1/V2 loop epitopes (12–14), that these host ganglioside Abs are under tolerance control (33). gp120 V3-glycan epitopes (15–17), the gp41 membrane-proximal Autoantibodies also were shown to penetrate the blood–brain external region (MPER) (18–21), the gp41 fusion domain (22), barrier and affect intracellular enzyme function of neuronal cells and the gp120–gp41 interface (23, 24). At high concentrations, during autoimmune disease (35). In retinopathy, anti-enolase Abs

*Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710; assisted with rhesus macaque Ab gene sequencing; J.Z., H.B.-V., C.M.B., and L.V. †Department of Medicine, Duke University Medical Center, Durham, NC 27710; provided 2F5 knock-in mice and carried out immunization and tissue collection; S.S., ‡Department of Immunology, Duke University Medical Center, Durham, NC 27710; xDe- L.L.S., and R.M.S. assisted with rhesus macaque immunizations and sample collection; partment of Pathology, Duke University Medical Center, Durham, NC 27710; {Beth S.M.A., C.B.F., and S.G.R. provided membrane-proximal external region peptide- Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; ‖Infec- liposome immunogen; R.P. performed immunoassays; N.V. performed statistical anal- tious Disease Research Institute, Seattle, WA 98102; #Department of Microbiology, yses; T.B.K. designed software and performed analysis of Ab sequences; M.A.M. Boston University, Boston, MA 02215; **Department of Pediatrics, Duke University produced fluorophore-labeled membrane-proximal external region proteins and oversaw Medical Center, Durham, NC 27710; and ††Department of Pharmacology and Cancer flow cytometry experiments; R.M. and J.I.E. performed histopathology analysis; and Biology, Duke University Medical Center, Durham, NC 27710 B.F.H. designed the study, oversaw all experiments, analyzed all data, and wrote and edited the manuscript. 1T.B., G.Y., and O.I. contributed equally to this work. The sequences presented in this article have been submitted to GenBank (http://www. ORCIDs: 0000-0002-2342-8232 (R.Z.); 0000-0002-2195-2997 (J.Z.); 0000-0002- ncbi.nlm.nih.gov/geo/) under accession numbers KX914888–KX914899. 8796-1714 (S.S.); 0000-0002-6903-2831 (C.M.B.); 0000-0002-1383-6865 (T.B.K.); 0000-0002-3890-5855 (M.A.M.); 0000-0001-6682-4588 (R.M.); 0000-0003-0273- Address correspondence and reprint requests to Dr. Todd Bradley, Prof. Garnett 6284 (J.I.E.); 0000-0001-7497-6645 (L.V.). Kelsoe, and Prof. Barton F. Haynes, Human Vaccine Institute, Duke University Medical Center, 2 Genome Court, MSRBII, DUMC 103020, Durham, NC 27710. Received for publication August 25, 2016. Accepted for publication October 14, E-mail addresses: [email protected] (T.B.), [email protected] (G.K.), 2016. and [email protected] (B.F.H.) This work was supported by the Center for HIV/AIDS Vaccine Immunology- The online version of this article contains supplemental material. Immunogen Discovery (Grant UMI-AI100645), a grant from the National Institutes of Health/National Institute of Allergy and Infectious Diseases/Division of AIDS, Abbreviations used in this article: bnAb, broadly neutralizing Ab; CDRH3, third and a Collaboration for AIDS Vaccine Discovery Grant from the Bill & Melinda chain H complementarity region; dKI, double KI; ELDKWA, 2F5 core epitope; Gates Foundation. Env, envelope protein; GLA, glucopyranosyl lipid adjuvant; gl2F5, germline 2F5; KI, knock-in; KYNU, kynureninase; m2F5, mature 2F5; MPER, membrane-proximal T.B. isolated and characterized Abs, designed assays, analyzed and interpreted data, and external region; mutKYNU, mutant KYNU; RM, rhesus macaque. wrote and edited the manuscript; G.Y., T.M.H., and G.K. designed and analyzed the kynureninase in vitro enzyme assay, interpreted data, and edited the manuscript; O.I. Ó and C.B.N. performed tryptophan metabolite analysis; R.Z. and H.-X.L. designed and Copyright 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601484 4664 AUTOREACTIVE gp41 HIV-1 Abs DO NOT INHIBIT KYNU can alter the function of the enolase enzyme in neuronal cells (36). natants were purified by nickel and size-exclusion chromatography (55). 652 671 Furthermore, in multiple sclerosis, anti–heterogeneous nuclear ribo- Biotin-labeled SP62 ( QQEKNEQELLELDKWASLWN ) peptide and biotin-labeled MPER656 (656NEQELLELDKWASLWNWFNITNWLWYIK683) nucleoprotein Abs were found to traffic into neurons and lead to peptide were synthesized (CPC Scientific). apoptosis, contributing to multiple sclerosis pathology (37, 38). Recombinant and serum Ab binding was determined using ELISA, as HIV-1 bnAbs have unusual features, such as extensive somatic described (55, 56). hypermutation and long third H chain complementarity regions Mouse immunization (CDRH3s) that are associated with poly- or autoreactive BCRs, which can make Abs subject to immune tolerance control (39–41). We Mature 2F5 (m2F5) dKI and germline 2F5 (gl2F5) dKI mice were generated on the C57BL/6 background, as previously described (46, 50). Three groups demonstrated previously that many HIV-1 bnAbs are autoreactive and/ of four m2F5 dKI mice were immunized six times, every 14 d, with i.p. or polyreactive and the MPER-reactive bnAb, 2F5, binds to the tryp- injections (200 ml) of the MPER peptide-liposome (25 mg) formulated tophan metabolism enzyme kynureninase (KYNU) (42, 43). A second with glucopyranosyl lipid adjuvant (GLA; 0.625 mg), Alhydrogel (alum; m MPER bnAb, 4E10, cross-reacts with the RNA splicing factor 3b 12.5 g; lot: QD565), or a combination. MPER peptide-liposomes con- tained a version of the 2F5 epitope–containing MPER peptide 656 subunit 3, as well as with anionic lipids. Because of the lipid reactivity, (656NEQELLELDKWASLWNWNITNWLWIK683) that was synthesized the 4E10 bnAb has anticoagulant activity; when administered to hu- with the C-terminal hydrophobic membrane anchor tag GTH1 mans, it was biologically active and prolonged the partial thrombo- (YKRWIILGLNKIVRMYS) as previously described (57). All mice were plastin time (44). Knock-in (KI) mice expressing the 2F5 or 4E10 8–12 wk old at the start of the immunization study and were housed in bnAb V D J and V J rearrangements exhibit profound deletion of the Duke University Vivarium in a pathogen-free environment with 12-h H H H L L light/dark cycles at 20–25˚C in accordance with all of the Duke Uni- immature B cells in the bone marrow, demonstrating first-tolerance versity Institutional Animal Care and Use Committee–approved animal checkpoint control of these autoreactive HIV-1 bnAbs (45–50). protocols. Downloaded from KYNU is a phylogenetically conserved enzyme of tryptophan Mouse tissue processing, chemistry, and pathology metabolism that contains the 2F5 core epitope (ELDKWA) within the KYNU H4 domain; the 2F5 bnAb binds this site with nano- Serum was collected and analyzed 10 d postimmunization, and animals were necropsied after completion of the study. Two groups of 6 (12 total) unimmu- molar affinity (42). A key goal of HIV vaccine design is to induce nized aged-matched C57BL/6 control mice and unimmunized m2F5 (5 mice) gp41 MPER 2F5-like Abs, yet the effects of MPER Abs on and gl2F5 (6 mice) dKI mice were necropsied as unimmunized controls.

KYNU function are unknown. Interestingly, dysregulated trypto- Serum and brain tryptophan, kynurenine, and kynurenic acid levels were http://www.jimmunol.org/ phan metabolism was implicated in AIDS-related dementia; thus, determined for immunized and naive mice, as previously described (58). Prepared stained tissue sections were delivered to a board-certified it is of critical importance to determine whether MPER Abs that veterinary pathologist for assessment at Duke University’s Department cross-react with KYNU disrupt tryptophan metabolism (51). of Pathology. The tissues were fixed in 10% neutral buffered formalin, In this study, we determined the effect of 2F5 and 2F5-like anti- embedded in paraffin, cut on a microtome at 5 mm, and stained with H&E. HIV gp41 Abs on KYNU enzymatic activity using an MPER peptide- RM immunization and pathology liposome vaccine that activates anergic B cells in 2F5 bnAb double KI (dKI; V D J +VJ ) mice. This vaccine also elicited MPER- Twenty-three healthy adult Chinese-origin RMs were housed at BIOQUAL H H H L L (Rockville, MD) in accordance with the standards of the American Association specific Abs in rhesus macaques (RMs) (50, 52). Thus, it is essen- for Accreditation of Laboratory Animal Care. The protocol was approved by by guest on September 29, 2021 tial to determine whether Abs to the KYNU cross-reactive gp41 BIOQUAL’s Institutional Animal Care and Use Committee under Office of epitope inhibit KYNU or are deleterious in vivo. We demonstrate Animal Laboratory Welfare Assurance Number A-3086-01. BIOQUAL is that immunization of 2F5 dKI mice and RMs with MPER peptide- International Association for Assessment and Accreditation of Laboratory Animal Care accredited. This study was carried out in strict accordance with liposomes elicited KYNU-reactive, 2F5 epitope–targeted Abs but the recommendations in the GuidefortheCareandUseofLaboratoryAn- that these Abs did not inhibit KYNU enzymatic activity in vitro or imals of the National Institutes of Health and with the recommendations of perturb tryptophan metabolism or induce tissue pathology in vivo. 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 Materials and Methods to provide enrichment opportunities (e.g., social housing when possible, ob- In vitro KYNU enzyme assay jects to manipulate in cage, varied food supplements, foraging and task- oriented feeding methods, interaction with caregivers and research staff). Recombinant human kynureninase (catalog number 4877-KH; R&D Sys- Animals were euthanized by sodium pentobarbital injection in accordance tems) and 10 nM 3-hydroxy-DL-kynurenine (catalog number H1771; Sigma- with the recommendations of the panel on Euthanasia of the American Vet- Aldrich) substrate are incubated in assay buffer (50 mM Tris; 0.05% [w/v] erinary Medical Association. Brij-35, 5 mM pyridoxal phosphate [pH 8] in a F16 black MaxiSorp plate Macaques were immunized (250 ml at two sites) six times with MPER [catalog number 475515; Nunc]). Fluorescence is read at excitation and peptide-liposomes formulated with GLA (eight animals), GLA + alum emission wavelengths of 315 and 415 nm, respectively, in kinetic mode for (eight animals), or alum alone (seven animals) at 6-wk intervals. Blood 5 min with a fluorescent plate reader (SpectraMax Gemini EM). Calibration was collected preimmunization and 2 wk after each immunization. Ani- standard 3-hydroxyanthranilic acid (catalog number H9391; Sigma-Aldrich) mals were necropsied at the completion of the study. is used as a calibration control to calculate the conversion factor. Specific All animals were clinically monitored throughout the immunization activity is calculated (Supplemental Fig. 2) (42). study; this included complete blood count, serum chemistry, and hema- rAbs are added at 0, 6, 60, 600, or 6000 ng, and immune sera are added at tology that were performed by the attending veterinarian using commercial a 1:10 dilution to determine the inhibition or enhancement of KYNU enzyme automated hematology and serum chemistry analyzers (BIOQUAL). Pre- activity compared with no inhibitor control. pared stained splenic tissue sections were delivered to a board-certified veterinary pathologist for assessment at Duke University’s Department Recombinant proteins and binding of Pathology. The tissues were fixed in 10% neutral buffered formalin, m Polyclonal anti-KYNU Ab was purchased (catalog number AF4887; R&D embedded in paraffin, cut on a microtome at 5 m, and stained with H&E. Systems), and the V(D)J gene fragments of Abs 2F5 and CH65 were syn- Isolation and characterization of MPER-reactive Abs thesized and cloned (GenScript) into plasmids containing human or rhesus IgG1/IgK/IgL constant regions. Recombinant mAbs were produced in 293 F MPER-specific memory B cells of a macaque immunized three times with cells (Life Technologies) by cotransfection with plasmids expressing the Ig the MPER peptide-liposome vaccine formulated in GLA + alum were sorted H and L chain genes and were purified from the culture supernatant by by flow cytometry as described (21, 50). Briefly, ∼1 3 107 PBMCs were protein A column chromatography (53, 54). Human KYNU and human KYNU decorated with B cell Ab panel: CD14 (BV570), CD3 (PerCPCy5.5), with a single aspartic acid–to–glutamic acid mutation genes were syn- CD20 (FITC), CD27 (allophycocyanin-Cy7), and IgD (PE) (BD Biosci- thesized with a C-terminal 6-histidinetagandclonedintopcDNA3.1 ences) and Alexa Fluor 647– and Brilliant Violet 421–tagged MPER.03 (GenScript). Plasmids were transfected into 293F cells, and culture super- peptides (KKKNEQELLELDKWASLWNWFDITNWLWYIRKKK). HIV The Journal of Immunology 4665 gp41-specific memory B cells were gated as CD32CD142CD20+CD27+ 2 + + Results sIgD MPER.03 (AF647) MPER.03 (BV421) andsortedinto96-well The 2F5 bnAb does not inhibit KYNU enzymatic activity PCR AQ22 plates containing 20 ml of reverse-transcription reaction buffer that included 5 mlof53 first-strand cDNA buffer, 1.25 mlofDTT, We previously identified that KYNU cross-reacted with the HIV-1 0.5 ml of RNaseOUT (Life Technologies), 0.0625 mlofIGEPAL(Sigma- gp41 membrane-proximal targeting bnAb 2F5 (42). 2F5 binds to m Aldrich), and 13.25 l of ultrapure distilled water (Life Technologies). the HIV-1 gp41 peptide SP62 (652QQEKNEQELLELDKWASLWN671), RM VHDJH and VLJL segments were isolated by single-cell RT-PCR using the method described (54). The isolated V(D)J gene fragments were but it does not bind this peptide when position 664 is mutated within used for the construction of linear expression cassettes for production of the gp41 peptide SP62 Mut (652QQEKNEQELLELAKWASLWN671, recombinant mAbs in 293T cells for small-scale ELISA screening (59). Fig. 1A). The KYNU H4 domain in most mammals (ELDKWA) Cloanalyst (http://www.bu.edu/computationalimmunology/research/software/) exactly replicates this MPER epitope, and this motif is conserved was used to annotate isolated VHDJH and VLJL sequences with immunoge- netic information and to test for clonal lineage membership (50, 54). within humans, mice, and rhesus monkeys (42). However, opossums Select V(D)J gene fragments were synthesized, expressed, and purified as carry a rare substitution in the KYNU H4 ELDKWA motif whereby rhesus IgG1 recombinant mAbs, as described above. aspartic acid (D) is replaced by glutamic acid (E) (42). The same RM serum and recombinant mAbs were screened by ELISA for binding to SP62 and select SP62 alanine mutants, recombinant KYNU and mutant replacement in human recombinant KYNU (mutKYNU) com- KYNU (mutKYNU) (55). pletely abrogates binding by the 2F5 bNAb (Fig. 1A). KYNU catabolizes 3-hydroxy-kynurenine into 3-hydroxyanthranilic Polyreactivity analysis of Abs acid. We used a standard in vitro enzymatic assay to measure the The polyreactivity of rhesus mAbs was assessed with the AtheNA Multi- production of 3-hydroxyanthranilic acid at saturating substrate

Lyte System (ZEUS Scientific) and HEp-2 cells immunofluorescence assay concentrations (42) (Supplemental Fig. 1A). Addition of recom- Downloaded from (Inverness Medical Professional Diagnostics), as described (50, 60). binant KYNU or mutKYNU resulted in identically increased Statistical analysis rates of 3-hydroxyanthranilic acid production, whereas the ad- dition of substrate alone or assay buffer did not (Fig. 1B). As a Statistical analysis was performed in SAS version 9.4 (SAS Institute). All statistical analyses used the Wilcoxon–Mann–Whitney test with Benjamini– control for Ab-mediated enzyme inhibition, we added increasing Hochberg false-discovery rate correction for multiple testing. GraphPad concentrations of polyclonal anti-KYNU IgG Ab and demonstrated

Prism version 6.01 was used for graphical representation. a concentration-dependent inhibition of 3-hydroxyanthranilic http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. 2F5 does not inhibit KYNU enzymatic activity. (A) ELISA binding of 2F5 to the HIV-1 gp41 peptide SP62 that includes the 2F5 epitope (solid red line), SP62 Mut (dashed red line), KYNU (solid blue line), and mutKYNU (dashed blue line). (B) In vitro KYNU enzymatic assay mea- suring levels of 3-hydroxyanthranilic acid being converted from 3-hydroxykynurenine by KYNU, measured as RFU over time, with the addition of KYNU, mutKYNU, substrate alone, and buffer alone. Sequences of KYNU and mutKYNU that contain the 2F5 nominal epitope were tested in the in vitro KYNU enzymatic assay. Vmax and spe- cific activity of wild-type KYNU and mutKYNU in the in vitro KYNU enzymatic assay using 0.2 mg of each enzyme. KYNU enzymatic assay by adding increasing amounts of polyclonal anti-KYNU Ab (C), CH65 Ab (D), 2F5 Ab (E), or 2F5 Ab with mutKYNU enzyme (F). 4666 AUTOREACTIVE gp41 HIV-1 Abs DO NOT INHIBIT KYNU acid production (Fig. 1C). Increasing amounts of the CH65 in- to MPER peptide and KYNU protein was boosted with subse- fluenza IgG Ab had no detectable effect on KYNU/mutKYNU quent immunizations for the three adjuvant groups, but GLA- or enzyme activity (Fig. 1D, Supplemental Fig. 1E). KYNU and GLA + alum–immunized mice maintained higher Ab titers mutKYNU were identically inhibited by KYNU IgG, and neither compared with alum-immunized mice (Fig. 2A). Abs to MPER was affected by CH65 rIgG (Supplemental Fig. 1B–E). and KYNU were strongly correlated with one another in all In contrast, at no concentration did the addition of 2F5 bNAb adjuvant groups, demonstrating the relatedness of the two re- inhibit KYNU or mutKYNU enzymatic activity (Fig. 1E, 1F). Like- sponses (Fig. 2B). At bleed 6, plasma Ab neutralization was wise, the 2F5 bnAb V region inserted into an RM IgG1 Fc backbone observed against the HIV virus MN in the TZM-bl–neutrali- had no effect on KYNU or mutKYNU enzymatic activity (Supple- zation assay (Fig. 2C). The magnitude of neutralization titers mental Fig. 1D). corresponded with the HIV-binding Ab titers, with the GLA + Vaccine-elicited gp41 bnAbs in mutated 2F5 KI mice do not alum and GLA adjuvant groups having the highest neutrali- inhibit KYNU zation titers; only one animal in the alum-immunized group . We immunized mice that express only the mature (m)2F5 bnAb had ID50 titers 50 (Fig. 2C). rearranged H and L chain Ab genes (m2F5 dKI mice) with MPER We then examined the effects of plasma IgG Ab from im- peptide-liposomes formulated with alum, GLA (TLR4 agonist), or munized m2F5 dKI mice on KYNU enzymatic activity. No a combination of alum and GLA to determine which adjuvant significant inhibition of KYNU enzymatic activity was ob- elicited the highest titers of 2F5 bnAbs. After two immunizations, served for any of the three immunization groups, including the the GLA and GLA + alum groups had induced Abs that targeted GLA and GLA + alum groups, which had the highest titers of Downloaded from the HIV gp41 MPER peptide (652QQEKNEQELLELDKWASLWN671) vaccine-elicited KYNU IgG Abs (Fig. 2D). Indeed, there was with ELISA logAUC values of four and six, respectively, whereas no no significant difference in the inhibitory activity between pre- animal in the alum-alone group had titers of MPER-binding mAbs and postimmunization plasma IgG samples. Thus, vaccine- (Fig. 2A). Similarly, after two immunizations, GLA- and GLA + elicited 2F5 bnAbs from m2F5 dKI mice had no effect on alum–immunized mice had titers of anti-KYNU Abs (Fig. 2A). Ab KYNU activity. http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 2. Immunization of m2F5 dKI mice with MPER peptide-liposomes. (A) Plasma Ab binding of SP62 and KYNU at preimmunization (bleed 0) and after seven immunizations (bleeds 1–7) with MPER peptide-liposomes formulated with alum, GLA, or GLA + alum adjuvants, as measured by ELISA. Binding for each individ- ual animal is displayed; horizontal line indicates the group mean. (B) Plot of plasma Ab-binding titers to SP62 and KYNU for each mouse in all adjuvant groups at bleed six. The y-axis values are log area under the curve measured in ELISA binding assays. The Spearman correlation is shown (p , 0.0001). (C) Plasma Ab HIV neu- tralization of MN virus measured in the TZM-bl neutralization assay at bleed six for mice im- munized with MPER peptide-liposomes for- mulated with alum, GLA, or GLA + alum adjuvants. (D) KYNU enzymatic assay showing the percentage change in KYNU activity for control Abs (far left panel) and mice from all three adjuvant groups before and after the fifth immunization (right three panels). Binding for each individual mouse is displayed. h, human Ab; Rh, RM Ab. The Journal of Immunology 4667 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Tryptophan metabolites and spleen histology of 2F5 VHDHJH +VLJL KI mice. (A) The first three steps of the tryptophan metabolic pathway; monitored metabolites are highlighted in red. Tryptophan metabolite levels in brain (B) and serum (C) of MPER peptide-liposome–immunized m2F5 dKI mice formulated with GLA + alum, GLA, or alum, unimmunized m2F5 dKI and gl2F5 dKI mice, and two groups of C57BL/6 control mice. Metabolite levels of individual animals graphed with animal group mean displayed by the solid line in each graph. The p values were determined using the Wilcoxon– Mann–Whitney test, comparing all immunized mice (GLA + Alum, GLA, Alum) versus all unimmunized groups. (D) Spleens from m2F5 dKI MPER peptide-liposome–immunized mice, unimmunized m2F5 dKI mice, and control C57BL/6 mice (H&E, original magnification 340). (E) Spleens from immunized m2F5 dKI mice and C57BL/6 mice (H&E, original magnification 3200). 4668 AUTOREACTIVE gp41 HIV-1 Abs DO NOT INHIBIT KYNU

Induction of KYNU Ab has no deleterious effects on lymphodepleted (Supplemental Fig. 2B). Thus, the lymphode- m2F5 dKI mice pletion seen in m2F5 and gl2F5 dKI mice was due to the deletion . Although we did not detect any inhibition of KYNU activity by of 95% of B cells in bone marrow as the result of immune the 2F5 bNAb or plasma IgG in immunized m2F5 dKI mice tolerance. (Figs. 1E, 2D), we recovered tissue samples from naive C57BL/6 We also performed a histopathology assessment of the brains control mice, gl2F5 dKI mice that expressed the 2F5 unmutated from m2F5 dKI, gl2F5 dKI, and C57BL/6 control mice; there was ancestor, m2F5 dKI mice, and immunized m2F5 dKI mice and no evidence of neural degeneration, demyelination, or inflamma- compared brain and serum levels of kynurenic acid, kynurenine, tion in any of the brain sections studied (Supplemental Fig. 2C). and tryptophan, the products of tryptophan metabolism upstream We concluded that, despite the induced 2F5 bnAb levels and the high titers of KYNU binding IgG Ab resulting from the induced of KYNU (Fig. 3A). Ab that inhibited KYNU activity in vivo 2F5 bnAb levels elicited by immunizing m2F5 dKI mice with would result in increased levels of these metabolites. No signifi- MPER peptide-liposome, it did not perturb tryptophan metabolism cant differences were observed in the levels of kynurenic acid, in the brain or serum and did not induce any pathological changes kynurenine, or tryptophan in the brains of naive or immunized in peripheral lymphoid tissues (Fig. 3). mice (Fig. 3B, Supplemental Fig. 2A). No significant differences were found in serum levels of kynurenic acid, but slightly higher Adjuvanted MPER peptide-liposome vaccine-elicited, levels of kynurenine and tryptophan were present in mice immu- nonpathogenic 2F5-epitope targeting KYNU-reactive nized with all forms of MPER peptide-liposome compared with Abs in RMs control C57BL/6 mice (Fig. 3C, Supplemental Fig. 2A). The in- Three groups of RMs were immunized six times with MPER Downloaded from creases in serum kynurenine and tryptophan in immunized mice peptide-liposomes formulated with GLA, alum, or GLA + alum. In did not appear to be related to elicited KYNU Abs, because the contrast to immunization of 2F5 KI mice, GLA alone was poorly alum-formulated liposome-immunized group with the lowest levels immunogenic in RMs and elicited the lowest MPER IgG plasma Ab of KYNU Ab titers (Fig. 2A) exhibited levels of serum kynurenine (Fig. 4A). In contrast, MPER liposomes formulated in alum or and tryptophan that were similar to those animals receiving GLA GLA + alum elicited higher titers of MPER plasma Ab (Fig. 4A, and GLA + alum immunogens with higher levels of KYNU Abs 4B). Immunization with alum or GLA + alum MPER peptide- http://www.jimmunol.org/ (Fig. 3C). liposomes also elicited higher titers of cross-reactive KYNU Abs A histopathology assessment of spleen tissue sections from in macaques compared with the GLA-alone group (Fig. 4C). The m2F5 dKI mice was performed. Consistent with the deletional 2F5 core epitope is the DKW motif within the HIV-gp41 MPER tolerance that is characteristic of m2F5 dKI mice (46), naive and peptide SP62 (652QQEKNEQELLELDKWASLWN671), and plasma immunized KI mice exhibited comparably low numbers of splenic Ab binding to SP62-mutated peptides within the D, K, or W was B lymphocytes compared with C57BL/6 controls, resulting in small reduced for all three groups, and it was significantly reduced for spleens and diminished white pulp areas (Fig. 3D). Furthermore, the SP62 D664A mutant in the GLA and GLA + alum groups spleens were analyzed for vaccine-related lesions, and no pathologic (Fig. 4D; p , 0.05; Wilcoxon–Mann–Whitney U test). Thus, by guest on September 29, 2021 abnormalities in the marginal zone, periarteriolar lymphoid sheath MPER peptide-liposome–induced plasma Ab was targeted near areas, or red pulp were observed in vaccinated animals with high the 2F5 bnAb epitope in all RMs, with alum-alone and GLA + alum titers of KYNU cross-reactive Abs (Fig. 3D, 3E). Immunized gl2F5 adjuvants eliciting the highest titers of MPER- and KYNU-reactive dKI mice without anti-KYNU Abs also exhibited B cell deletion Abs. and tolerance control (50), and our analysis revealed that, like m2F5 In all three adjuvant groups, KYNU RM plasma IgG had no dKI mice, gl2F5 dKI mice had small spleens that were similarly effect on KYNU enzyme activity in vitro (Fig. 5A). We analyzed

FIGURE 4. Adjuvanted MPER peptide- liposome–immunized RMs elicit KYNU- reactive Abs. Plasma Ab levels, as measured by ELISA, targeting MPER656 (A), SP62 (B), and KYNU (C) were analyzed after immunization with MPER peptide-lipo- somes formulated with GLA, alum, and GLA + alum adjuvants in RMs. Animals were immunized at weeks 0, 6, 12, 18, 24, and 58. (D) Average plasma Ab binding to wild-type SP62 and alanine-mutated versions (red; core 2F5 epitope is underlined) at week 60 for all vaccine groups. *p , 0.05, Wilcoxon–Mann– Whitney test. The Journal of Immunology 4669 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Adjuvanted MPER peptide-liposome–immunized RMs do not inhibit KYNU or display tissue abnormalities. (A) Percentage inhibition of KYNU enzymatic activity, as measured by the in vitro KYNU enzyme assay for positive- and negative-control Abs (far left panel) and for plasma Abs from RMs measured pre- and postimmunization with MPER peptide-liposomes formulated with GLA + alum, GLA, or alum (right three panels). (B) Spleens from macaques immunized with MPER peptide-liposomes formulated with GLA + alum or alum alone (H&E, original magnifications 340, 3100, and 3200). the histopathology of immunized macaque spleen and found that rAb were screened for binding by ELISA. Using this approach, we it was normal in all groups (Fig. 5B). These data confirmed that identified 31 MPER-reactive Abs that bound clade B MN gp41 the splenic lymphopenia of naive gl2F5 dKI mice and vaccinated recombinant protein, 27 that bound rHIV-1 gp140 Env (Con-S or B. m2F5 dKI mice is the consequence of B cell deletion due to immune JRFL gp140, Supplemental Fig. 4A), and 6 MPER-reactive rAbs tolerance and not a pathologic consequence of anti-KYNU Ab that, in addition to MPER, bound human KYNU (Fig. 6B). production. VDJ sequence analysis revealed that the isolated MPER Abs Blood samples from RMs did not reveal any changes in elec- used diverse VH gene segments, and three of the six KYNU–cross- trolytes, glucose, renal and liver function, or complete blood counts reactive Abs used a VH gene segment orthologous to the human related to MPER peptide-liposome vaccination (Supplemental VH2–5 gene used in the 2F5 bNAb (Supplemental Fig. 4B, Table I) Fig. 3A, 3B). (61). The remaining KYNU binders carried rearrangements of VH4 family gene segments (Supplemental Fig. 4B, Table I). Vaccine-elicited MPER recombinant mAbs in RMs target the The 31 VDJ rearrangements recovered from memory B cells that 2F5 epitope bound the MPER.03 tetramer exhibited varying levels of somatic gp41 Env–reactive memory B cells (CD32CD142CD20+CD27+ hypermutation and diverged from the germline RM VH gene seg- sIgD2) from PBMCs from an RM immunized with MPER lipo- ments by 0.38–11.8%; rearrangements from KYNU-reactive Abs some formulated in GLA and alum that were decorated with exhibited a similar range of mutation frequencies (1.1–5.2%) (Supple- MPER peptide tetramers (MPER.03) labeled with two fluorophores mental Fig. 4C, Table I). Most MPER bnAbs have long CDRH3s that were sorted for sequencing of the BCR (VH +VL) genes. MPER- are .15 aa, and the RM Abs had CDRH3 lengths ranging from 12 to reactive memory B cells represented 0.26% of total memory B cells as long as 21 aa (Supplemental Fig. 4D, Table I). (Fig. 6A). BCR genes were amplified by RT-PCR, sequenced, and We selected four mAbs, based on gp41 and KYNU cross- fused to cassettes containing a CMV promoter, Ig C region, and reactivity, including two that used the RM VH2–5 gene segment polyA tail by overlapping PCR. H and L chain amplicon pairs were ortholog, for further study. All four mAbs bound to HIV-1 gp41 transiently transfected into 293F cells, and supernatants containing peptide (SP62; 652QQEKNEQELLELDKWASLWN671), and three 4670 AUTOREACTIVE gp41 HIV-1 Abs DO NOT INHIBIT KYNU

FIGURE 6. Isolation of MPER-targeting KYNU cross-reactive Abs from RMs im- munized with MPER peptide-liposomes. (A) Memory B cells decorated with an MPER tetramer (MPER.03) conjugated with two fluorophores (Alexa Fluor 647 and Brilliant Violet 421) from PBMCs of an RM immunized with MPER peptide- liposomes 2 wk after the third immuni- zation. Single cells were sorted into 96-well plates for Ig gene amplification, and sequencing is shown within the black sort gate. (B) Individual wells with positive Ig sequences were amplified and transiently transfected for small-scale production of rAb and tested for MPER and KYNU binding by ELISA (OD at 450 nm is shown on the x-axis and y- axis). (C) Epitope mapping of MPER- and KYNU-reactive Abs on wild-type (WT) Downloaded from HIV-1 SP62 peptide and alanine mutants in the core 2F5 epitope (ELDKW) and 2 aa outside of the 2F5 core epitope (LS). The ratio of Ab binding to the alanine mutants (ELDKWLS) to the unmutated wild-type (mutant/WT). Red shading indicates a http://www.jimmunol.org/ decrease in binding. (D) Isolated Ab binding to KYNU and mutKYNU, as measured by ELISA. (E) Autoreactivity of MPER Abs isolated from RMs in the AtheNA autoantibody assay. Values . 150 are positive and are highlighted in yellow. Synagis and the MPER bnAb 4E10 were used as negative and positive controls, respectively. (F) Hep-C indirect

fluorescent Ab staining of Abs targeting by guest on September 29, 2021 the HIV-1 MPER and cross-reactive with KYNU (original magnification 340). Ab concentration was 50 mg/ml, and the exposure time was 5 s. Represen- tative sections are displayed. The cellular staining pattern for each Ab is shown below. of the four mAbs had .60% reduction in binding to peptides disease: DH653 reacted with dsDNA, DH654 reacted with SSA, containing alanine substitutions within the 2F5 bnAb DKW epi- SSB, and Jo1 proteins, and DH656 reacted with Jo1 protein tope (Fig. 6C). The mAb DH563 showed a more moderate 20–50% (Fig. 6E). Three of the four KYNU-reactive Abs also exhibited reduction on the DKW alanine mutants (Fig. 6C). Similarly, all four antinuclear Ab phenotypes by reacting with nuclear Ags in HEp-2 Abs bound to KYNU and had reduced binding to mutKYNU that cells; DH654 decorated cytoskeletal proteins, DH655 bound to contains ELEKWA instead of wild-type KYNU, ELDKWA (Fig. 6D). nuclear punctae (dots), and DH656 diffusely bound within the None of the mAbs were capable of neutralizing HIV-1 in the cytoplasm (Fig. 6F). Although these KYNU-binding Abs exhibited TZM-bl pseudovirus inhibition assay (data not shown). reactivity for HEp-2 cell components, none of them were capable In addition to KYNU cross-reactivity, three of the four Abs were of inhibiting KYNU enzymatic activity in vitro (Fig. 7). Thus, cross-reactive with other host proteins associated with autoimmune adjuvanted MPER liposome immunogens were capable of inducing

Table I. Immunogenetic characteristics of KYNU cross-reactive Abs

Ab VH (human) HCDR3 (aa) Mutation Frequency (%) VL (human) LCDR3 (aa) Mutation Frequency (%) DH653.2 2-A (2–5) 12 1.1 k2-S10 (2–28) 9 8.7 DH653 2-A (2–5) 12 3.4 k2-S10 (2–28) 9 7.5 DH656 2-A (2–5) 20 2.7 l3-B (3–19) 11 2.7 DH655 4-H (4–39) 14 4.9 l1-F (1–36) 11 1.1 DH654 4-L (4, 4) 16 4.9 l3-B (3–19) 11 2.7 DH673 4-L (4, 4) 18 2.6 l3-B (3–19) 11 4.6 DH653, DH654, DH655, and DH656 were studied as purified mAbs. DH656.2 and DH673 were identified by transient transfection. The Journal of Immunology 4671

FIGURE 7. MPER and KYNU cross- reactive Abs from RMs immunized with MPER peptide-liposomes do not inhibit KYNU enzymatic activity. KYNU en- zyme activity over time with no Ab and with increasing concentrations of mAbs isolated from RMs immunized with MPER peptide-liposomes that cross-reacted with KYNU. Downloaded from

polyclonal and polyreactive IgG Ab responses to MPER epitopes these mAbs did not inhibit KYNU activity. We did not detect

in RMs; although these responses included anti-KYNU Abs, they plasma HIV-1 neutralization in the TZM-bl assay for any of the http://www.jimmunol.org/ did not result in tissue pathology or inhibit KYNU enzyme vaccine-induced MPER-KYNU cross-reactive Abs, indicating that function. additional immunizations that increase affinity maturation, selec- ting Abs with recognition of lipid, will be required to achieve Discussion neutralization activity (50). Mimicry of host proteins by microbial pathogens can subvert the It has been reported that elevated tryptophan metabolites can be host immune response (30–34, 62, 63). In this study, we demon- associated with depression in animal models (67). Depression side- strated that the HIV-1 bnAb 2F5 had no inhibitory effect on effects of elevated tryptophan metabolism would not have been KYNU enzyme activity. Moreover, a single-residue change within detected in our studies, but in the absence of evidence that 2F5 the 2F5 epitope in KYNU that abolished 2F5 binding also did not bnAb or individual anti-KYNU Abs inhibited KYNU enzyme ac- by guest on September 29, 2021 affect KYNU enzyme function. The 2F5 epitope (ELDKWA) tivity, our studies do not support a role for ELDKWA-targeted Abs within KYNU is in the H4 domain that mediates KYNU homo- in tryptophan metabolite alteration. dimerization (64), likely explaining the inability of anti-ELDKWA In summary, host mimicry is a strategy used by HIV-1 to escape Abs to inhibit KYNU activity. Ab responses to functionally conserved bnAb epitopes. Efforts to B cell development in 2F5 VHDJH and VLJL KI mice was induce bnAbs by vaccination and to use bnAbs as therapeutic blocked by clonal deletion in bone marrow, because of host cross- Abs are critical to control the AIDS epidemic. Thus, determining reactivity with lipids and KYNU resulting in bnAb immune tol- the impact of potentially protective Abs on host protein function erance control (46, 50). MPER bnAbs 2F5, 4E10, and 10E8 all is an important safety consideration. Our study demonstrates that interact with the virion membrane as a part of their epitopes (47, the human bnAb 2F5 and vaccine-elicited 2F5 epitope–targeted 65, 66). We developed a minimal peptide-liposome immunogen Abs elicited in mice and macaques did not inhibit KYNU en- that was designed to present MPER bnAb epitopes in the same zymatic function in vitro or cause tissue or enzyme activity manner as on an HIV-1 virion (57). Immunization of 2F5 KI mice abnormalities in vivo despite cross-reacting with KYNU pro- with the MPER peptide-liposome immunogen rescued anergic tein. Although cross-reactivity with host Ags may impede bnAb B cells that escaped deletion to produce high levels of bnAbs (46). development and impact therapeutic Ab pharmacokinetics, the Immunization of macaques showed that the MPER peptide-liposome 2F5–KYNU interaction neither impairs host enzyme function can initiate Abs with 2F5-like characteristics (50). nor appears to mediate adverse events. Thus, the 2F5 HIV-1 Env In this study, bnAb 2F5 KI mouse plasma Abs recognized KYNU gp41 region appears to be a safe target on HIV-1 Env for vaccine but, like the human 2F5 mAb, did not inhibit KYNU activity. development. Moreover, we examined brain and serum levels of tryptophan and its metabolites after MPER peptide-liposome immunization in vivo Acknowledgments and did not detect tryptophan pathway metabolite perturbations We thank Dawn J. Marshall, John F. Whitesides, Joshua Eudailey, Tarra A. that could be attributed to induction of 2F5 bnAb activity. Von Holle, Thaddeus C. Gurley, Lawrence C. Armand, Andrew Foulger, Immunization of RMs with MPER peptide-liposomes formu- Giovanna Hernandez, Jamie Pritchett, Krissey Lloyd, and Christina Stolarchuk lated in alum or GLA + alum elicited high-titers of Abs that cor- for expert technical assistance. We also thank Kelly Soderberg and Samantha ecognized the 2F5 epitope and KYNU. In macaques, plasma Ab Bowen for project coordination and management. inhibition of KYNU was not observed, and no significant pathology was observed in macaque spleen or blood hematology or chemistry Disclosures tests. Isolation of mAbs by Ag-specific single-cell flow cytometry B.F.H., S.M.A., and H.-X.L. have patent applications submitted on vaccine revealed that the 2F5 epitope (ELDKWA) in HIV-1 gp41 and KYNU candidates used in this study (see international patent application PCT/ could be recognized by Abs that used diverse Ig gene segments, and US06/13684). 4672 AUTOREACTIVE gp41 HIV-1 Abs DO NOT INHIBIT KYNU

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Figure S1. In vitro KYNU enzyme activity assay, Related to Figure 1. (A) KYNU enzymatic assay measures 3-hydroxyanthranilic acid at saturating substrate concentrations (10nM). Calibration determined by recombinant 3-hydroxyanthranilic acid (Conversion factor). Calculation of specific activity shown. (B) KYNU enzymatic assay using 0.2µg of enzyme with increasing concentrations of 2F5 with a rhesus constant region backbone (Rhesus 2F5) with wild-type KYNU enzyme (C-E) KYNU enzymatic assay using 0.2µg of mutant KYNU enzyme of with increasing concentrations of (C) polyclonal anti-KYNU antibody (D) 2F5 with a rhesus constant region backbone or (E) CH65 antibody.

Figure S2. In vivo tryptophan metabolite analysis in mice. Related to Figure 3. (A) Statistical comparisons of kynurenic acid, kynurenine and trypophan levels in the brain and serum between different mouse groups, Related to Figure 3. (P-values determined by Wilcoxon-Mann-Whitney). Antigens tested were kynurenic acid, kynurenine and tryptophan (Trp) in brain (top) and serum (bottom). Groups were mutated 2F5 double KI (m2F5 dKI) mice not immunized, mice with the germline 2F5VHDHJH + VLJL knocked-in (gl2F5 dKI) not immunized, m2F5 dKI mice that have been immunized (All adjuvant groups combined; Combined Vaccinated) and two groups of unimmunized control C57/B6 mice (Control 1, Control 2; both combined, Combined Control). Values of individual animals shown in Figure 3. (B-C) Histology of spleen and brain from 2F5 double knock-in and control mice. (B) Representative HE stains at 100x magnification of spleens from a 2F5 double KI germline (gl2F5 dKI) mouse and C57B/6 control mouse. (C) Representative HE stains of brain sections from mutated 2F5 double KI (m2F5 dKI) and gl2F5 dKI and a C57B/6 control mouse. No abnormalities were found specific for the MPER peptide-liposome vaccine.

Figure S3. Blood comprehensive metabolic panel and complete blood counts from MPER peptide- liposome immunized rhesus macaques during immunization, Related to Figure 5. (A) ALK_Phos, alkaline phosphatase; Albumin; ALT, alanine amino transferase; AST, aspartate amino transferase; A_G_Ratio, albumin/globulin ratio; BUN, blood urea nitrogen; Calcium; Chloride; Cholesterol; Creatinine; Globulin; Glucose; PHOS, phosphorus; POTAS, potassium; Protein; Sodium; TOT_BIL, total bilirubin; CPK, creatine phosphokinase. Dashed lines indicate values within normal range. (B) HCT, hematocrit; HCB, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MONOS, monocytes; Nucleated RBC, nucleated red blood cells; Platelets; RBC, red blood cells; SEG, mature neutrophils; WBC, white blood cell; Basos, basophils; Eosin, eosinophils; Total Lymphs, total lymphocytes; Total Neuts, total neutrophils. Dashed lines signify values within normal range.

Figure S4. MPER peptide-liposome vaccine-elicited rhesus macaque antibodies bind the 2F5 bnAb epitope, Related to Figure 6. (A) Number of isolated and sequenced antibodies that reacted with multiple HIV specificities in ELISA. (B) Proportions of VH gene usage among vaccine-elicited MPER antibodies (red asterisk indicates the rhesus gene most similar to that of 2F5, VH2-5). (C-D) Distributions of (C) mutation frequency and (D) CDRH3 length among the MPER antibodies.