amino acid starvation enhances vaccine efficacy by ...sumbul afroz1, shama1, srikanth battu1, shaikh...

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

VACC INES

1School of Life Sciences, Department of Biotechnology and Bioinformatics, Uni-versity of Hyderabad, Hyderabad, 500046 Telangana, India. 2Department of Micro-biology and Immunology, College of Veterinary Medicine, Cornell University,Ithaca, NY, USA. 3Department of Pathobiological Sciences, School of VeterinaryMedicine, Louisiana State University, Baton Rouge, LA, USA.*Corresponding author. Email: [email protected]

Afroz et al., Sci. Signal. 12, eaav4717 (2019) 12 November 2019

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Amino acid starvation enhances vaccine efficacy byaugmenting neutralizing antibody productionSumbul Afroz1, Shama1, Srikanth Battu1, Shaikh Matin1, Sabrina Solouki2, Jessica P. Elmore2,Gillipsie Minhas1, Weishan Huang2,3, Avery August2, Nooruddin Khan1*

Specific reduction in the intake of proteins or amino acids (AAs) offers enormous health benefits, includingincreased life span, protection against age-associated disorders, and improved metabolic fitness and immunity.Cells respond to conditions of AA starvation by activating the amino acid starvation response (AAR). Here, weshowed that mimicking AAR with halofuginone (HF) enhanced the magnitude and affinity of neutralizing, antigen-specific antibody responses in mice immunized with dengue virus envelope domain III protein (DENVrEDIII), a potentvaccine candidate againstDENV.HF enhanced the formationof germinal centers (GCs) and increased theproductionofthe cytokine IL-10 in the secondary lymphoid organs of vaccinatedmice. Furthermore, HF promoted the transcriptionof genes associated with memory B cell formation and maintenance and maturation of GCs in the draining lymphnodes of vaccinated mice. The increased abundance of IL-10 in HF-preconditioned mice correlated with enhancedGC responses andmaypromote the establishment of long-livedplasma cells that secrete antigen-specific, high-affinityantibodies. Thus, these data suggest that mimetics of AA starvation could provide an alternative strategy to augmentthe efficacy of vaccines against dengue and other infectious diseases.

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INTRODUCTIONMetabolic regulation of the immune system is evolutionarily conservedand paramount for overall organismal health (1, 2). Nutrient uptakeand nutritional status affect immune cell proliferation, differentiation,and survival (3). In this regard, epidemiological and clinical studies in-dicate that malnutrition impairs host immune responses and increasesrates ofmorbidity andmortality after infection (4, 5). Similarly, reducedintake of nutrients without malnutrition, often termed as caloric or di-etary restriction (CR or DR), improves metabolic fitness and longevity(6) and provides enormous benefits against age-associated disorderssuch as neurological, cardiovascular, and skeletal problems (7, 8).Exceptions include prolonged energy restriction in small animals,which can reduce growth and development of lymphoid organsand impair antigen-specific immune responses (9, 10). The benefitsof CR during viral infections are largely dependent on body weightand may vary between different animals (7, 11–13). Small and agedanimals subjected to long-term CR are more susceptible to influenzainfection because of continuous body mass loss and accompanyingenergy deficits that impair recovery, despite increased splenocyteproliferation (11). However, short-term refeeding after energy restric-tion inmice restores bodyweight and fat composition, as well as naturalkiller cell function required to combat influenza infection (12).Emerging evidence suggests that CR has a beneficial impact on variousattributes of the immune system. These include enhancing thymopoiesisand maintenance of T cell diversity (14), natural killer and CD4+ andCD8+ T cell activity (15–17), and the apoptotic clearance of senescentT cells in aged mice (16, 18, 19). Furthermore, CR stimulates adaptiveimmunity against parasitic infection in experimental cerebral malaria(20).

Although the associated benefits of CR are mostly linked to reducedcalorie intake, accumulating evidence couples dietary amino acid (AA)

restriction to the benefits of CR (8). For instance, AA sensing pathwaysinfluence both innate and adaptive immunity (21). Innate immune cellsare auxotrophs for AAs and sense the availability of extracellular AAsthrough their intrinsic metabolic sensing pathway (8, 22). AA depletionresults in the accumulation of uncharged transfer RNAs (tRNAs),which are sensed by general control nonderepressible-2 (GCN2) kinase(23) and trigger its activation. Activated GCN2 phosphorylates eukary-otic translation initiation factor 2a (eIF2a), which results in the activa-tion of amino acid starvation response (AAR) pathway that coordinatesposttranscriptional and translational immune reprogramming (24, 25).The AAR can also be activated by halofuginone (HF), a derivative of theChinese herb Dichroa febrifuga, which creates a pool of unchargedtRNAs by inhibiting prolyl-tRNA synthetase (25),mimicking the effectsof AA starvation (26). HF has garnered substantial attention in the lasttwo decades owing to its therapeutic potential in various diseases in-cluding autoimmune disease, cancer, muscular dystrophy, and hepaticand renal ischemia-reperfusion injury (25, 27–31).

Activation of the AAR using HF or amino acid–restricted dietabrogates the production of interleukin-1b (IL-1b) through the GCN2pathway and provides protection against intestinal inflammation inan experimental mouse model (24, 32). Furthermore, HF inhibitsT helper 17 cell differentiation and provides protection against exper-imental autoimmune encephalomyelitis (33). In addition, a molecularsignature composed of GCN2 correlates with the CD8+ T cell responseto the yellow fever vaccine–17D (YF17D) (34, 35). Furthermore, theGCN2 pathway is crucial for optimal proliferation in response to anti-gen stimulation in vitro and for the appropriate trafficking of CD8+

T cells to lymphoid organs (36). These findings raise the intriguingpossibility that the activation of the AAR pathway with HF couldenhance the immunogenicity of vaccine antigens.

Although rates of dengue virus (DENV) infection are increasing,there is still no licensed vaccine to date that is effective against allDENV serotypes (37). Therefore, there is an urgent need to identifystrategies that can enhance the neutralizing antibody response to allthe four serotypes of DENV and promote long-term vaccine protec-tion. We used the recombinant envelope protein domain III (EDIII)of DENV as a model vaccine antigen in our study because it is

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considered as one of the most potent vaccine candidates againstDENV (38). Our results demonstrated that HF increased numbersof DENV EDIII–specific interferon-g (IFN-g) and IL-2–producingCD4+ and CD8+ T cells in mice. HF also increased the amountand affinity of DENV EDIII–specific immunoglobulin G (IgG) inthe serum of vaccinated mice and its neutralizing activity againstall the four serotypes of DENVs. These changes were accompaniedby enhanced production of IL-10 and expression of gene signaturesinvolved in antigen-specific memory B cell formation, proliferation,and differentiation of germinal center (GC) B cells in the draininglymph nodes of vaccinated mice. Collectively, these findings highlighta promising role for HF in enhancing robust antigen-specific immunityagainst vaccine antigens and further suggest a potential application ofmanipulating AAR through AA restriction mimicry for the design anddevelopment of new vaccines.

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RESULTSAA starvation mimetic HF enhances antigen-specificT cell responsesThe amino acid starvation sensor GCN2 promotes the developmentof protective immune responses triggered by the YF17D vaccine(35). To evaluate the effect of AA restriction on antigen-specific Tcell immunity, we used HF, a pharmacological activator of AAR,and DENV EDIII antigen, a potential vaccine candidate againstDENV. We first purified 6X His-tagged recombinant EDIII of allthe four DENV serotypes, with the purity andmolecular weights ver-ified on Coomassie-stained SDS–polyacrylamide gel electrophoresis(PAGE) gel, and the specificity of the proteins confirmed by immu-noblotting (fig. S1, A and B). The purified proteins were polymyxinbead treated to remove any endotoxin content before use in immu-nization studies. DENV-2 EDIII (DENVrEDIII) was administered inmice preconditioned withHF or dimethyl sulfoxide (DMSO) control(Fig. 1A). Because HF activation of the AAR pathway induces eIF2aphosphorylation, which promotes cellular translational arrest andATF4 (activating transcription factor 4) transcription factor expression(33, 39), we evaluated eIF2a phosphorylation and the expression ofATF4 target genes including eIF4Ebp1, Chop, Asns, and Gpt2 in thesplenocytes of immunized mice. We found that HF treatmentincreased phosphorylation of eIF2a and activation of ATF4 targetgenes (fig. S2, A and B). Furthermore, HF treatment promoted eIF2aphosphorylation in immune cells including B cells, T cells, and den-dritic cells (fig. S2, C andD)when administered inmice, with no changesin the bodyweight ofHF-treatedmice (fig. S2E). Next, we examined theeffect of AAR activation on the magnitude of DENVrEDIII-specificT cell responses after primary immunization. We found that HFtreatment enhanced [3H]thymidine incorporation by DENVrEDIII-restimulated splenocytes from immunized mice, suggesting that HFincreased the expansion of antigen-specific lymphocytes (Fig. 1B).We also found thatHF treatment enhancedDENVrEDIII-induced pro-duction of IFN-g byDENVrEDIII-restimulatedCD8+ andCD4+T cellsfrom blood, spleens, and lymph nodes at 4 weeks after secondary im-munization (Fig. 1, C to J). Similar results were observed for the pro-duction of antigen-specific IL-2 (fig. S3, A to D). The CD8+ and CD4+

T cell responses in peripheral blood mononuclear cells (PBMCs) weremore rapid and robust when HF- or DMSO-preconditioned mice wereimmunized with DENVrEDIII protein (Fig. 2A). Although there is areduction in T cell responses after 4 weeks, the enhanced DENVrEDIII-specific induction of IFN-g–secreting T cells was nevertheless evident


in the HF-treated group (Fig. 2A). The reduction in effector CD8+ andCD4+ T cell responses with time occurs during the contraction phase,allowing the maintenance of T cell homeostasis and avoiding non-specific immunopathology that may be generated due to a largenumber of activated T cells in the host (40, 41). Polyfunctional T cells,which are largely memory T cells, secrete multiple cytokines includ-ing IFN-g, IL-2, and tumor necrosis factor–a (TNF-a) and are associatedwith providing increased protection against the pathogens (40, 42). Ourresults indicate that HF treatment increased the frequency of antigen-specific polyfunctional double cytokine–producing (IFN-g and IL-2)CD8+ and CD4+ T cells found in immunized mice after DENVrEDIIIrestimulation (Fig. 2, B and C). This increase was most notable in theDENVrEDIII reactive polyfunctional T cell (IFN-g+IL-2+) subsets(Fig. 2D). Furthermore, splenocytes and lymph node cells from HFpretreated mice showed increased production of IFN-g, IL-12p40,and TNF-a uponDENVrEDIII stimulation (Fig. 3). Secretion of theseproinflammatory cytokines provides a third signal for effector T cellexpansion (43) and indicates enhanced antigen-specific cellular re-sponse under the conditions of AAR activation. Together, these resultspoint toward a role for AAR activation in enhancing themagnitude andquality of antigen-specific CD4+ and CD8+ T cell responses, perhaps, inpart, by augmenting the delivery of antigen to antigen-presenting cells(APCs) as previously reported (35).

HF enhances antibody responses against DENV envelopedomain III (DENVrEDIII) proteinThe activation of CD4+ T helper cells is a vital step in establishing anadaptive immune response, acting both as an effector and a modulatorof the immune response. CD4+ T helper cell differentiation into T fol-licular helper (Tfh) cells provides a vital signal that drives the activationand differentiation of high-affinity memory B cells and antibody-producing plasma cells (44, 45). Having observed the effect of HFon the induction of robust antigen-specific CD4+ T cell responses,we further determined the effects of HF on antigen-specific antibodyresponses. We assessed the amount of DENV-2 EDIII–specific totalIgG and IgG subtypes IgG2a, IgG2b, and IgG1 in the serum fromDENVrEDIII-immunized mice preconditioned with HF or vehiclecontrol after both primary and secondary immunization. We ob-served a substantial increase (more than twofold) in the productionof DENVrEDIII-specific total IgG and in all the IgG subtypes in theserum of HF-preconditioned mice (Fig. 4A). Secondary immunizationwith the same immunogen 2weeks later enhanced antibody amounts inall immunized groups, and again, significant enhancement was evidentin theDENVrEDIII +HF–immunized group (Fig. 4A). The productionof ovalbumin (OVA)–specific antibodies (total IgG and IgG subtypes)was also enhanced when HF-primed mice were immunized with OVAantigen as compared with vehicle control DMSO (fig. S4). Theseresults establish that activation of the AAR pathway may enhanceantigen-specific immune responses for diverse antigens.

To determine whether HF enhances antigen-specific responsesthrough GCN2, we evaluated the amount of DENVrEDIII-specificantibodies in the serum of HF- or DMSO-preconditioned wild-type(WT) or Gcn2−/− mice immunized with DENVrEDIII protein. Wefound that HF had no effect on antigen-specific total IgG or IgG sub-type responses inGcn2−/−mice, whereas we observed a notable increasein antibody responses in HF-preconditioned WT littermate controls(fig. S5). These data support the conclusion that HF enhances antigen-specific immune responses through GCN2, and not through off-targeteffects.

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Fig. 1. HF enhances antigen-specific T cell responses in vivo. (A) Experimental outline of HF or vehicle control DMSO treatment and DENV-2 envelope domain III(DENVrEDIII) protein immunization. i.p., intraperitoneally; s.c., subcutaneously. (B) Proliferation analysis by [3H]thymidine incorporation in antigen-specific T cells fromsplenocytes of mice 28 days after immunization that were restimulated with DENVrEDIII protein. Data are means ± SEM of 12 mice per treatment group from twoindependent experiments.(C to J) Flow cytometry analysis of DENVrEDIII-specific CD8+ (C to F) and CD4+ (G to J) T cell responses in blood (D and H), spleen (E and I), andlymph node (F and J) after treatment and immunization, as indicated. Fluorescence-activated cell sorting (FACS) plots (C and G) are representative of two independentexperiments. Quantification of the percentage of IFN-g–producing T cells (D to F and H to J) are means ± SEM of 12 mice per treatment group from two experiments.*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired Student’s t test (B) and Mann-Whitney U test (D to F and H to J).

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Fig. 2. HF increases the fre-quency of antigen-specific poly-functional T cells inmice. (A) Flowcytometry analysis of antigen-specific CD8+IFN-g+ and CD4+IFN-g+

T cell kinetics in PBMCs of micetreated with DMSO or HF and im-munized with DENV-2 envelopedomain III after 14 and 28 days.Data are means ± SEM of 12 miceper group from two independentexperiments. (B to D) Flow cy-tometry analysis of DENVrEDIII-specific polyfunctional CD8+ (B)and CD4+(C) T cells in blood, spleen,and lymph node of immunizedmice. The frequency of double cy-tokine (IFN-g and IL-2)–producingcells with means (bar) ± SEM (Band C) and pie charts of the fre-quency of all cytokine-producingT cells (D) are from 12 mice pergroup from two independent ex-periments. *P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001by two-way analysis of variance(ANOVA) with Bonferroni posthoc between DENVrEDIII + DMSO–and DENVrEDIII + HF–immunizedgroups (A), and Mann-WhitneyU test (B and C).

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HF augments the quality of antigen-specificantibody responsesT cell–dependent B cell activation is predominantly characterizedby the development of high-affinity antibodies, mostly the IgG sub-type (44), and the avidity of antigen-antibody interaction is one ofthe parameters that determine the quality of the antibody responses(46). We examined the binding response and avidity of DENV-2 EDIII(DENVrEDIII)–specific antibodies using surface plasmon resonance(SPR) assay. Serum samples taken frommice 28 days after the secondaryimmunization were assayed for binding to immobilized DENVrEDIIIon CM5 sensor surface. With DENVrEDIII secondary immunization,serum from HF-pretreated mice gave the highest antigen-binding re-sponse [in response unit (RU)] as compared with vehicle control pre-treated mice (Fig. 4B). SPR analysis of twofold serial dilution of theserum (ranging from 1:50 to 1:400) fromHF-preconditioned immu-nizedmice indicated stable association with DENVrEDIII comparedwith the sera fromunimmunized orDMSO-preconditioned controlmice(fig. S6). The qualitative analysis of the DENVrEDIII-specific antibodyresponse was evaluated in terms of avidity by measuring the disso-ciation rate constants. Serum from HF-preconditioned, DENVrEDIII-


immunized mice associated with DENVrEDIIIprotein faster and dissociated fromDENVrEDIIIslower, suggesting that HF induced enhancedhigh-affinity antibody responses (Fig. 4C).Increased amounts of antigen-specific, high-avidity antibody after DENVrEDIII vaccinationwithHF pretreatment correlated with improvedantibody function as assessed by the virus-neutralizing ability in vitro. Serum from miceimmunizedwithDENVrEDIII afterHF precon-ditioning neutralized DENV-2 virus more effi-ciently than serum from mice immunized withDENVrEDIII after vehicle control treatment(Fig. 4D). We also observed a notable increasein DENV serotype–specific total serum IgGwhen we immunized HF-preconditioned micewith a tetravalent formulation consisting ofdomain III proteins of all the four DENV sero-types (tDENVrEDIII) (Fig. 5A). In addition,the resultant antibodies were effective againstDENV-1, DENV-2, DENV-3, and DENV-4serotypes (Fig. 5B). Together, the above resultsindicate that activation of the AAR pathway en-hances both themagnitude andquality of antigen-specific B cell responses.

HF induces enhanced development ofGCs in draining lymph nodesGCs are crucial for humoral immunity and playa vital role in the development of immunologicalmemory. The explicit colocalization of antigen-specific T cells, B cells, and follicular dendriticcells (FDCs) is crucial for GC formation (47).The progression of B cell responses along theGC pathway is important in the generation oflong-lived plasma cells that secrete high-affinityantibodies (48). Because our results suggest thatHF enhances the production of high-avidityantibodies, we determined whether activation

of the AAR axis regulates the GC pathway. Thus, immunized micepreconditioned with HF or DMSO were euthanized on day 28 afterthe booster dose, inguinal lymph nodes were isolated, and the pres-ence of GCs was evaluated by immunohistology. We found that HF-preconditioned, DENVrEDIII-immunized mice developed morelymph node GCs than DMSO-preconditioned, DENVrEDIII-immunized mice (Fig. 6, A and B), which were also apparent in he-matoxylin and eosin (H&E)–stained sections of inguinal lymph nodes(fig. S7, A and B). To determine the persistence of GC responses inHF-preconditioned mice, we examined GC kinetics over a durationof 8 weeks by evaluating the frequency of GC B cells in the draininglymph nodes using flow cytometry (fig. S7C). We observed a sub-stantial increase in GC expansion within 10 days of antigen immu-nization, with increased frequency of B220+GL7+IgG+ GC-B cells inthe lymph nodes of DENVrEDIII-immunized mice primed with HF(Fig. 6C). The GC responses peaked at 28 days after antigen boost,correlated with the antigen-specific antibody responses, followed bya decline. The decline in GC responses at a later time point is perhapsa result of reduction in the number of CD4+/Tfh cells, which is a pre-requisite for establishing optimal GC reactions for effective protective

Fig. 3. HF enhances secretion of multiple cytokines after DENVrEDIII immunization. Enzyme-linkedimmunosorbent assay (ELISA) analysis of the amounts of IFN-g, IL-12p40, and TNF-a produced by spleno-cytes and lymph node cells from immunized mice after restimulation in vitro with DENVrEDIII for 72 hours.Spleen and lymph nodes were collected from mice at the 28th day after secondary immunization. Data aremeans ± SEM from 12 mice per treatment group from two independent experiments. *P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001 by two-tailed unpaired Student’s t test.

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Fig. 4. HF-mediatedAAR activation augments the antibody responses against DENVrEDIII. (A) ELISA analysis of DENVrEDIII-specific total IgG, IgG2a, IgG2b, and IgG1 in theserum of mice 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 12 mice per group from two independent experiments.OD, optical density; ns, not significant. (B andC) BIAcore SPR analysis of DENVrEDIII protein binding by pooled serum samples frommice preconditionedwithHF orDMSO28 daysafter immunization. Sensogram trace of the DENVrEDIII-specific antibody-binding affinity (B) and correlation of the maximal response unit (RUmax) with the dissociation constant(C) (top) are representative of two independent experiments. Quantified avidity scores (C) (bottom) are means ± SEM from two independent experiments on pooled serumsamples from 10 mice per group assayed in triplicate. (D) DENV-2 virus neutralization assay on serum samples collected from mice 28 days after immunization. The 50% focusreduction neutralization titer (FRNT50) data are means ± SEM of 10 mice from two independent experiments assayed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001 by one-way ANOVA with Bonferroni post hoc test (A), two-tailed unpaired Student’s t test (C), and Mann-Whitney U test (D).

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immunity, while avoiding the production of low-affinity self-reactiveantibodies that lead to autoimmunity (49, 50).

The pleiotropic cytokine IL-10 has a key role in the generation ofGC-B cell responses. IL-10 produced by CD4+ follicular T cells (51)and B cell–intrinsic IL-10 signaling (52) have been observed to directlypromote GC responses. Therefore, wemeasured the amount of IL-10 inthe DENVrEDIII-restimulated splenocytes and lymph node cells fromthe immunizedmice and found elevated IL-10 production from cells ofHF-preconditioned mice (Fig. 6D), highlighting an important role forIL-10 inAAR-mediated regulation ofGC responses.We speculated thatthe effects of AAR activation on the GC pathway might be due to tran-


scriptional programming of genes in antigen-specific B cells; thus,we carried out quantitative real-time polymerase chain reaction(qRT-PCR) analysis to look for the molecular signatures involvedin the maintenance of GCs in draining lymph nodes. We observedenhanced expression of genes associatedwith programming ofmemoryB cells in the lymph nodes of HF-preconditioned, DENVrEDIII-immunized mice. Memory B cells swiftly respond to booster immuni-zation, thereby driving the localization of antigen-specific plasma cellsto surviving niches in bone marrow and generating long-lived plas-ma cells that are the primary source of high-avidity antibodies in ser-um (53). HF preconditioning–induced AAR activation enhanced the

Fig. 5. HF enhances the antibody response to a tetravalent DENV. (A) ELISA of total IgG specific for all four DENV serotypes in the serum of mice immunized with atetravalent combination of DENVrEDIII protein 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 10 mice pergroup from two independent experiments. (B) DENV serotype neutralization assay on serum from tDENVrEDIII-immunized mice. The FRNT50 data are means ± SEM of10 mice from two independent experiments. *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni post hoc test (A) and Mann-Whitney U test (B).

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expression of genes involved in maintaining the integrity of GCs [Bcl2(54) and Tank (42)]; genes crucial in the formation andmaintenance ofB cell memory [Plcg2 (55) and Cd38 (56)]; genes involved in the prolif-eration, survival, and differentiation of GC B cells such as Il17ra (57),Il18r1, Pax5 (58), Ikzf1 (59), Irf7, andMx1; and type 1 interferon genesinvolved in the differentiation of B cells (Fig. 6E). These findings exem-plify the benefits of amino acid restriction in the production of robusthigh-avidity andneutralizing antibodymediated throughprogrammingof genes associated with memory B cell development and differentia-tion. Hence, this study suggests that the AAR pathway might be ma-nipulated for the design and development of vaccines for tailoringlong-lived protective immunity against the pathogens.

DISCUSSIONMetabolic control of the immune system is a crucial requirement for theregulation of cell survival and the development of immunity (1). Ac-cumulating evidence suggests that the immune system can sense andrespond to diverse changing environmental signals, including fluctua-


tions in the abundance of amino acids and othermetabolites. Metabolicsensors like GCN2 are able to sense depletion of even a single type ofamino acid in the cellular microenvironment and respond appropriatelythrough the activation of the AAR pathway, which, in turn, can dictateand tailor the fate of immune cells (60). Although activation of theGCN2-AAR pathway has broad anti-inflammatory effects (24, 32) andtherapeutically benefits various metabolic diseases (25), the impact ofAAR on the adaptive immune response in infectious disease remainspoorly characterized. GCN2 stimulates protective immunity to YF17D,one of the most successful vaccines developed (35). In the present study,we used a plant-derived biomolecule, HF, to activate the AAR pathway(33) and evaluated its role in tailoring the protective efficacy of vaccines inamousemodel usingDENVenvelopeproteindomain III (DENVrEDIII)as amodel antigen.Our results highlight a critical role of theAARpathwayin the regulation of T and B cell immunity to DENVrEDIII antigen.

HF enhanced T cell responses, including the frequency of IFN-gand IL-2, and double cytokine (IFN-g and IL-2)–producing CD8+ andCD4+ effector and polyfunctional T cells. The latter are a prerequisitefor antigen-specific protective T cell adaptive immunity (40, 42).

Fig. 6. HF pretreatment in DENVrEDIII-immunized mice enhances GC formation. (A and B) Confocal microscopy imaging of the GL7+ (red), B220+ (green), and IgG+

(blue) GC B cells in lymph node sections from mice treated with HF or DMSO and immunized with DENVrEDIII protein. Images (A) are representative of two independentexperiments. Quantified data (B) are means ± SEM of eight mice per condition from all experiments. (C) Flow cytometry analysis of lymph node GC–B cell frequency inDENVrEDIII-immunized mice at the indicated time points. Data are means ± SEM of eight mice per group at each time point from two independent experiments. (D) ELISAanalysis of IL-10 production by splenocytes and lymph node cells from mice 28 days after secondary immunization that were restimulated with DENVrEDIII. Data are means± SEM of 10 mice per group from two independent experiments. (E) qRT-PCR analysis of the indicated gene expression in lymph node cells restimulated with DENVrEDIIIfor 24 hours. Heat maps of statistically significant changes are from the analysis of 10 biological replicates from two independent experiments. *P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001 by two-tailed unpaired Student’s t test (B and D) and two-way ANOVA with Bonferroni post hoc test (C).

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Furthermore, the DENVrEDIII-specific T cell recall responses cor-related with the magnitude of antigen-specific antibody responses afterAAR activation. Effective vaccines establish protective humoral immu-nity, which depends on the production of long-lived plasma cells secret-ing high-affinity antibodies that are necessary for pathogen clearance(61). The activation of the AAR pathway enhanced the production ofhigh-avidity and greater virus-neutralizing antibody titers, which wereable to efficiently neutralize all the serotypes of DENV, one of the keygoals of DENV vaccine development. T cell activation promotes neu-tralizing antibody production by stimulating the development of GC inresponse to antigenic challenge (62), and our results demonstrate thatAAR activation enhanced the formation of GCs in the draining lymphnodes of HF-pretreated mice. HF preconditioning also promoted theproduction of the cytokine IL-10, which is involved in GC formation(52). Our data suggest that AAR activation might increase theestablishment of GCs through enhanced IL-10 production and therebyimprove antigen-specific humoral immunity. Supporting the antigen-driven nature of this response, we did not observe GC formation inthe lymph nodes from mice 5 days after HF preconditioning only, de-monstrating that the GC phenotype did not exist before antigen immu-nization or that HF priming merely resulted in the production of self-reactive antibodies.

Our results suggest that AAR activation stimulates a molecularprogram that aids in the differentiation, maintenance, and survival ofGC-B cells, thereby augmenting immunological memory. For example,B cell lymphoma 2 (Bcl2) is involved inmaintaining the integrity of GCby inhibiting apoptosis within theGC-B cells (54). The Plcg2 gene prod-uct conveys survival signals to GC andmemory B cells necessary for thegeneration of the secondary immune responses (55). In addition,signaling through IL-17RA is required to secure the localization of Tfhcells in the GC to induce its function, stabilizing the interaction of GCB cells with nearby T helper cells (57). Pax5 (paired box protein 5)is a master regulator of B cell development and is expressed in pro–B cells to mature GC-B cells (58). Pax5 works in coordination withother proteins critical for B cell function and drives the expressionof interferon regulatory factors such as IRF8, thereby contributingtoward sensing the advancement of GC reaction (58, 63). Our datademonstrated that HF treatment augmented the expression of all ofthese critical B cell survival factors.

Together, these data demonstrate that HF augmented GC forma-tion, which enhanced the magnitude and quality of antigen-specificantibody responses and correlated with transcriptional reprogrammingof lymphoid organs, after experimental DENV vaccination. Althoughthis work demonstrates the feasibility of using HF to enhance vaccineresponses, the mechanisms by which AAR promotes GC formationremain poorly understood and merit further investigation. Further-more, the detailed study of the T cell dependence of the effects of HFon antigen-specific antibody responses could be the next step.However,our data demonstrate that AAR mimicry–based nutraceuticals such asHF are potent agonists of vaccine responses poised for further develop-ment against human diseases.

MATERIALS AND METHODSPurification of recombinant DENV envelope domain IIIprotein (DENVrEDIII)The optimized envelope domain III sequences for all the four DENVserotypes (DENV-1, DENV-2, DENV-3, and DENV-4) were clonedinto bacterial expression vector pET-28a by GenScript (USA). The


cloned constructs were expressed in Escherichia coli strain Rossettaand induced with 0.6mM isopropyl-b-D-thiogalactopyranoside (IPTG)(DENV-2) or 1 mM IPTG (DENV-1, DENV-3, and DENV-4). The6X His-tagged recombinant Dengue envelope protein domain III(DENVrEDIII) was purified using the affinity chromatography techni-que under denaturing conditions as described earlier(64) using TalonSuperflow (GE Healthcare) high-affinity resins. The His-DENVrEDIIIproteins were eluted using 300 mM imidazole, run on SDS-PAGE andsubjected to Coomassie staining. The desired band sizes of 14.5 kDa(DENV-1, DENV-2, and DENV-4) and 15 kDa (DENV-3) were ob-served in the Coomassie-stained gel.

ImmunoblottingThe 6XHis-tagged purifiedDengue envelope domain III (DENVrEDIII)protein for all the four serotypes was confirmed by immunoblottingusing anti-His antibody (Cell Signaling Technology). For evaluationof HF-mediated AAR activation, splenocytes from immunized micewere lysed in lysis buffer [1% Triton X-100, 150 mM NaCl, 1 mMEDTA, 10% glycerol, 20 mM Hepes (pH 7.5), 100 mM NaF, 17.5 mMb-glycerophosphate, and supplemented with 1X Protease InhibitorCocktail (Sigma-Aldrich)] (24), incubated on ice for 30min, and clearedby centrifugation at 12,000 rpm for 15 min at 4°C. An equal amount ofprotein was separated on SDS-PAGE and transferred to nitrocellulosemembrane. For the detection of eIF2a phosphorylation, the membranewas blocked with 5% bovine serum albumin and probed with rabbitanti–eIF2a-P antibody (Cell SignalingTechnology). Protein bandswerevisualizedwith FemtoPlus ECL chemiluminescent substrate.Densitom-etry analysis of eIF2a-P bands with respect to eIF2a-T bands was per-formed using the National Institutes of Health ImageJ software.

Immunization of miceSix- to 8-week-old Balb/c mice were administered intraperitoneallywith HF (0.1 mg/kg body weight) or vehicle control (DMSO) (33) for4 days, followed by immunization with 20 mg per mouse per dose ofDENV-2 envelope domain III protein (DENVrEDIII) in 200 ml of 1Xphosphate-buffered saline (PBS), delivered subcutaneously at thebase of the tail, followed by resting for 10 days. The booster injectionwas given at the 14th day after primary immunization with an iden-tical dose of HF or DMSO (intraperitoneally) again for 4 days,followed by subcutaneous administration of 20 mg per mouse perdose DENVrEDIII protein. The mice were rested for 10 days andeuthanized at the 28th day after primary injection for further analysisof blood, spleen, and lymph node cells. In another set of experiments,Balb/c mice previously conditioned with HF or DMSO were immu-nized by subcutaneous injection of tetravalent DENV envelope domainIII protein (tDENVrEDIII) formulation (10 mg each ofDENV-1,DENV-2, DENV-3, and DENV-4 rEDIII) following the same regimen asmentioned above. In yet another experiment, Balb/c mice previouslyconditioned with HF or DMSO were injected subcutaneously with10 mg of OVA as antigen per mouse per dose following the same immu-nization schedule as mentioned above. Blood samples were collected onthe 14th and 28th days by tail bleeding for serum collection and PBMCisolation. In another set of experiments, WT or Gcn2−/− mice (on aC57Bl/6 background) were immunized with 20 mg of DENVrEDIIIantigen per mouse after HF or DMSO priming following the above-mentioned immunization schedule. Blood samples were collected atthe 14th and 28th days by tail bleeding for serum collection for the anal-ysis of antigen-specific antibody responses. Animals that were injectedwith PBS alone (without candidate antigen) served as negative controls.

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All animal experiments were performed according to the animal ethicalguidelines of the University of Hyderabad and the Institutional AnimalCare and Use Committee at Cornell University.

Phospho-eIF2a staining by flow cytometryTotal splenocytes isolated from HF- or DMSO-preconditioned micewere surface stained using PerCP Cy5.5 anti-mouse CD45R/B220(BD Pharmingen), APC anti-mouse CD11c (BD Pharmingen), andAlexa Fluor 700 anti-mouse CD3 (eBioscience) at 4°C for 30 min.The stained cells were washed with fluorescence-activated cell sorting(FACS) buffer and quickly fixed using 4% paraformaldehyde, followedby intracellular staining for eIF2a-P as described earlier (32). Fixed cellswere permeabilized using 1X PermWash Buffer (BioLegend) and incu-bated with rabbit anti-mouse monoclonal eIF2a-P antibody (CellSignaling Technology) or respective isotype control overnight at4°C. After washes with 1X FACS buffer, the cells were stained withanti-mouse Alexa Fluor 488 secondary antibody for 1 hour at roomtemperature, washed and resuspended in FACS buffer, acquired on a BDLSRFortessa (BD Biosciences) cytometer, and analyzed using FlowJo(Tree Star Inc.).

T cell assays by flow cytometryLymph node and spleen CD4+ and CD8+ T cells from immunizedmice(collected at day 28) and those from PBMCs (collected at day 14 andday 28) were evaluated for antigen-specific responses. PBMCs wereisolated by sucrose gradient density separationmethod usingHistopaque(Sigma-Aldrich) as described earlier (65). Briefly, isolated PBMCs,lymph node cells, and splenocytes were cultured for restimulation withDENVrEDIII protein (10 mg ml−1) in the presence of GolgiStop andGolgiPlug (BD Biosciences) at 37°C for 10 hours. The restimulatedcells were stained with Alexa Fluor 488–labeled anti-mouse CD8 (BDBiosciences) and PerCP (eBioscience) or fluorescein isothiocyanate–labeled anti-mouse CD4 antibodies (BD Biosciences) for 60 min at 4°C.The cells were further washed three times with FACS buffer, fixedwith 4% paraformaldehyde, permeabilized using 1X PermWash Buffer(BioLegend), and stained using APC-labeled anti-mouse IFN-g (BDBiosciences) and phycoerythrin-conjugated anti-mouse IL-2 antibody(BD Biosciences), diluted in 1X PermWash Buffer for 60 min at roomtemperature. Cells were washed with FACS buffer, and data wereacquired on a BD LSRFortessa (BD Biosciences) cytometer and ana-lyzed using FlowJo (Tree Star Inc.).

Cytokine secretion by enzyme-linked immunosorbent assayand proliferation assayThe cytokines secreted were evaluated in splenocytes and lymph nodecells isolated from immunized mice. Splenocytes (2 × 105) or lymphnode cells were seeded in triplicates in 96-well round-bottom plateand restimulated with DENVrEDIII (10 mg ml−1) for 72 hours. Thesupernatants were then collected and analyzed for IFN-g, IL-12p40,TNF-a, and IL-10 (BD Biosciences) cytokines through sandwichenzyme-linked immunosorbent assay (ELISA) following the protocolas per the manufacturer’s instructions. Lymphocyte proliferation was as-sessed in 72-hour splenocyte culture as mentioned earlier (66). Briefly,after 72 hours, splenocytes were pulsed with 1-mCi [3H]thymidine foran additional 16 hours. Cells were harvested, fixed with 10% trichlor-oacetic acid for 30 min at room temperature, followed by solubiliza-tion with 150 ml of 1 N NaOH for 30 min at 37°C with constantagitation. Next, 150 ml of 1 N HCl was immediately added; 300 ml ofeach sample was mixed with 5 ml of scintillation fluid (Sisco Research


Laboratories), and the counts were measured in a scintillation counter(PerkinElmer).

Antibody ELISAFor detection of antigen-specific antibodies in the serum of immunizedmice, 100 ml of DENVrEDIII protein (5 mg ml−1) diluted in carbonatebuffer was coated on 96-well Nunc MaxiSorp ELISA plates and in-cubated overnight at 4°C. Next, the plates were washed three timeswith 1X PBST (0.05% Tween 20) using a Thermo Fisher ScientificWellwash 4 MK 2 ELISA washer. The plates were blocked using4% skimmedmilk (Himedia) for 1 hour at room temperature. Serumsamples were diluted in 0.1% skimmed milk prepared in 1X PBS andincubated on blocked plates for 2 hours at room temperature. Afterwashing five times with 1X PBST, the plates were incubated with anti-mouse IgG (whole molecule)–peroxidase (1:5000; Sigma Aldrich),anti-mouse IgG2a–horseradish peroxidase (HRP) (1:2000; SantaCruz), anti-mouse IgG2b-HRP (1:2000; Santa Cruz), and anti-mouseIgG1-HRP (1:5000; Santa Cruz) diluted in 1X PBS for 1 hour at roomtemperature. Afterwashing six timeswith 1XPBST, the plateswere last-ly developedwith 100 ml per well of tetramethylbenzidine substrate (BDBiosciences). The reaction was stopped using 2N H2SO4. Plates wereanalyzed using a Tecanmicroplate reader at l = 450 nm and correctionat l = 570 nm (66).

BIAcore assaySPR binding and kinetics measurement were carried out at 25°C ona BIAcore T200 instrument (BIAcore/GE Healthcare) as describedearlier (42, 67, 68). The CM5 sensor chip (GE Healthcare) was immo-bilized with DENVrEDIII (for serotype 2) diluted in 10 mM sodiumacetate (pH 4.5) using standard amine coupling chemistry. Serumsamples collected at day 28 from immunized mice were injected at1:50 to 1:400 dilution by using twofold dilution steps at a flow rate of30 ml min−1. The contact time for the serum samples to interact withligand immobilized on the sensor chip was 60 s, and dissociation timefor the sample was 60 s. After each cycle, the sensor chip was regen-erated with 50 mMNaOHwith a specified contact time for 60 s. Theexperimental data were fit using 1:1 Langmuir model for determiningthe binding kinetics, and analysis was performed using BIAcore T200Evaluation software version 2.0. To analyze antigen (DENVrEDIII)–specific antibody-binding avidity, maximal response unit (RUmax)and dissociation rates (kd) were measured. Avidity scores were deter-mined as mentioned earlier (avidity score = maximum binding re-sponse/kd in RU.s) (61).

DENV stock preparationDENV-1 (Hawaii), DENV-2 (TR1751), DENV-3 (Thailand 1973), andDENV-4 (Columbia 1982) viral strainswere propagated inC636 cells asdescribed earlier (69). Viral titers were quantitated by focus-forming as-say in BHK-21 and Vero cells as previously described (70).

Focus reduction neutralization testsFocus reduction neutralization test (FRNT) was carried out in Verocells seeded at a density of 25,000 cells in 96-well plate 24 hoursbefore infection as described earlier (71, 72). Serum samples fromimmunized mice were heat activated at 56°C for 30 min and weretwofold serially diluted (from 1:12.5 to 1:3200) in serum-free DMEM.After decomplementation and dilution of serum, 50 ml of each dilutionwas thoroughly mixed with 50–plaque-forming unit DENV (DENV-1,DENV-2, DENV-3, and DENV-4) diluted in DMEM and incubated at

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37°C for 2 hours. Next, Vero cells were washedwith serum-freeDMEMand infected in duplicate with 45 ml of the neutralization mixturefollowed by another incubation for the next 2 hours. After incuba-tion, the viral inoculum was removed and overlaid with 200 ml of1.5% carboxymethylcellulose (Sigma-Aldrich) in serum-free DMEMand incubated at 37°C for 4 to 5 days, depending on the serotype.After incubation, the overlay medium was carefully removed followedby three washes with 1X PBS. The cells were further fixed, permea-bilized with 0.2% Triton X-100, and stained with anti-dengue mono-clonal antibody (GeneTex) for 1 hour at 37°C. After washing with 1XPBS, the stained cells were incubated with HRP-linked anti-mousesecondary antibody for 1 hour at 37°C. The cells were lastly washedwith 1X PBS and developed using DAB (3,3′-diaminobenzidine)substrate. Viral foci were counted manually, and the percentage offoci reduction against control serumwas calculated. Neutralizing an-tibody titer (FRNT50) values were determined as the reciprocal of thehighest dilution that resulted in 50% decrease in focus-forming unitsand analyzed using GraphPad Prism software.

Quantitative real-time polymerase chain reactionGC gene expression profiling was performed on DENVrEDIII (serotype2)–restimulated cells isolated from the lymph nodes of different im-munized mice. Total RNA was extracted using TRI Reagent (Sigma-Aldrich) as described earlier (72). Total purified RNA was reversetranscribed using Easy Script cDNA Synthesis Kit (abm) according tothe manufacturer’s instructions. qRT-PCR was performed using Ap-plied Biosystems QuantStudio 5. The complementary DNA (cDNA)was amplified using SYBR Green Mix (Kappa Biosystems) with gene-specific primers (table S1) following the thermocycler program of onecycle of 95°C for 10min, next 40 cycles of 15 s at 95°C, 30-s annealing at56°C, and 40-s extension at 68°C. The relative mRNA expression wasnormalized to housekeeping geneGapdh. The normalized expression ofstatistically significant genes is presented as heat map using the Gene-esoftware (www.broadinstitute.org). Genes with P < 0.05 (two-tailedunpaired Student’s t test) were considered significant. Activation of AARpathway genes was evaluated in splenocytes from immunized mice. ThemRNA expression of AAR genes was determined using gene-specificprimers (table S1) and normalized to housekeeping gene Hprt.

Histology and immunofluorescenceDraining lymph nodes were isolated and snap frozen in OCT (optimalcutting temperature) tissue embedding medium for sectioning asmentioned earlier (42, 66). Sections were either stained with H&E orfluorescently stained with Alexa Fluor 488–labeled anti-mouse B220(eBioscience), Alexa Fluor 647–labeled anti-mouse total IgG (CellSignaling Technology), or biotin-labeled anti-GL7 (eBioscience),followed by Alexa Fluor 555–labeled streptavidin (Invitrogen) antibo-dies. The slides were washed andmounted with coverslips using a DPXmounting medium. The images were captured using a 40× objective ona Zeiss confocal microscope.

GC staining by flow cytometryFor identification of GCs by flow cytometry, cells isolated from lymphnodes collected at the 5th (afterHForDMSOpriming), 14th, 28th (afterbooster), 42nd, and 56th days were passed through a 40-mmcell strainerto generate single-cell suspensions, after which they were stained withAlexa Fluor 488–labeled anti-mouse B220 (eBioscience), Alexa Fluor647–labeled anti-mouse total IgG (Cell Signaling Technology), or biotin-labeled anti-GL7 (eBioscience) for 1 hour at 4°C, followed byAlexa Fluor


555–labeled streptavidin (Invitrogen) for 1 hour at room temperature.The stained cells were fixed using 4% paraformaldehyde, washedwith FACS buffer, and data were acquired on a BD LSRFortessa (BDBiosciences) cytometer and analyzed using FlowJo (Tree Star Inc.).

Statistical analysisStatistical analysis was performed using GraphPad Prism 7 software.Normality test (Shapiro-Wilk normality test) was performed on allthe data shown. Accordingly, parametric two-tailed unpaired Student’st test was applied on normally distributed data, whereas nonparametricMann-Whitney U test was applied on data that were not normally dis-tributed to measure statistical significance. One- or two-way ANOVAwith Bonferroni post hoc test has been used for multiple comparisons.

SUPPLEMENTARY MATERIALSstke.sciencemag.org/cgi/content/full/12/607/eaav4717/DC1Fig. S1. Expression of dengue envelope protein domain III (DENVrEDIII) from all four DENVserotypes.Fig. S2. HF activated the AAR in immunized mice.Fig. S3. HF increased the frequency of IL-2–producing CD4+ and CD8+ T cells in DENVrEDIII-immunized mice.Fig. S4. HF augments antigen-specific antibody responses to OVA antigen.Fig. S5. HF-mediated augmentation in antigen-specific antibody responses is GCN2dependent.Fig. S6. HF enhances affinity of DENVrEDIII antigen–specific antibodies.Fig. S7. HF enhances GC formation in mice immunized with DENVrEDIII.Table S1. Gene-specific primers.

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Acknowledgments: We thank P. Kumar of the Proteomics Facility, University of Hyderabad,for technical assistance in SPR experiments, and A. Mishra for help in visualizing data. Wethank the FACS and confocal microscopy core facilities of the University of Hyderabad.Funding: This work was supported by grants from the Department of Science andTechnology, Nano Mission, government of India [DST no. SR/NM/NS-1040/2013(G)], and theUniversity Grants Commission, government of India [UGC no. MRP-MAJOR-BIOT-2013-40689]to N.K., and from the National Institutes of Health (AI129422 to A.A. and W.H. andAI137822 to W.H.). J.P.E. was supported by T32 EB023860. Author contributions: N.K.conceptualized the study. S.A. designed and performed most of the experiments withassistance from S.B. S. and S.M. expressed and purified the recombinant DENVrEDIII protein.S.S., J.P.E., and W.H. performed the experiments in WT andGcn2−/− C57Bl/6 mice. N.K., S.A.,S.B., and A.A. wrote the manuscript. S.B. made the figures. S.A., A.A., and N.K. interpretedthe data. G.M. assisted during the experiments. Competing interests: A.A. receives researchfunding from 3M Corp. All the other authors declare that they have no competinginterests. Data and materials availability: All data needed to evaluate the conclusionsin the paper are present in the paper or the Supplementary Materials.

Submitted 19 September 2018Accepted 18 October 2019Published 12 November 201910.1126/scisignal.aav4717

Citation: S. Afroz, Shama, S. Battu, S. Matin, S. Solouki, J. P. Elmore, G. Minhas, W. Huang,A. August, N. Khan, Amino acid starvation enhances vaccine efficacy by augmentingneutralizing antibody production. Sci. Signal. 12, eaav4717 (2019).

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productionAmino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody

Avery August and Nooruddin KhanSumbul Afroz, Shama, Srikanth Battu, Shaikh Matin, Sabrina Solouki, Jessica P. Elmore, Gillipsie Minhas, Weishan Huang,

DOI: 10.1126/scisignal.aav4717 (607), eaav4717.12Sci. Signal.

activation of this stress-induced kinase improves vaccination.increased germinal center formation, and increased neutralizing antibody responses. These data demonstrate how

in immune cells,αthe nutrient-sensing kinase GCN2, HF promoted phosphorylation of the translation initiation factor eIF2starvation mimetic halofuginone (HF) increased the adaptive immune response to a DENV vaccine. Known to activate

found that pretreatment of mice with the amino acidet al.in people who have not previously encountered DENV. Afroz mosquito-borne RNA virus that can potentially cause lethal disease. The efficacy of currently approved vaccines is poor

Nearly 50% of the world's population is considered at risk of infection with dengue virus (DENV), aStarving for DENV vaccines

ARTICLE TOOLS http://stke.sciencemag.org/content/12/607/eaav4717

MATERIALSSUPPLEMENTARY http://stke.sciencemag.org/content/suppl/2019/11/08/12.607.eaav4717.DC1

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is a registered trademark of AAAS.Science SignalingYork Avenue NW, Washington, DC 20005. The title (ISSN 1937-9145) is published by the American Association for the Advancement of Science, 1200 NewScience Signaling

Science. No claim to original U.S. Government WorksCopyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of

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