Research ArticleVACCINES

Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

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Science Signaling  12 Nov 2019:
Vol. 12, Issue 607, eaav4717
DOI: 10.1126/scisignal.aav4717

Starving for DENV vaccines

Nearly 50% of the world’s population is considered at risk of infection with dengue virus (DENV), a mosquito-borne RNA virus that can potentially cause lethal disease. The efficacy of currently approved vaccines is poor in people who have not previously encountered DENV. Afroz et al. found that pretreatment of mice with the amino acid starvation mimetic halofuginone (HF) increased the adaptive immune response to a DENV vaccine. Known to activate the nutrient-sensing kinase GCN2, HF promoted phosphorylation of the translation initiation factor eIF2α in immune cells, increased germinal center formation, and increased neutralizing antibody responses. These data demonstrate how activation of this stress-induced kinase improves vaccination.

Abstract

Specific reduction in the intake of proteins or amino acids (AAs) offers enormous health benefits, including increased 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, we showed 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 potent vaccine candidate against DENV. HF enhanced the formation of germinal centers (GCs) and increased the production of the cytokine IL-10 in the secondary lymphoid organs of vaccinated mice. Furthermore, HF promoted the transcription of genes associated with memory B cell formation and maintenance and maturation of GCs in the draining lymph nodes of vaccinated mice. The increased abundance of IL-10 in HF-preconditioned mice correlated with enhanced GC responses and may promote the establishment of long-lived plasma cells that secrete antigen-specific, high-affinity antibodies. Thus, these data suggest that mimetics of AA starvation could provide an alternative strategy to augment the efficacy of vaccines against dengue and other infectious diseases.

INTRODUCTION

Metabolic regulation of the immune system is evolutionarily conserved and paramount for overall organismal health (1, 2). Nutrient uptake and nutritional status affect immune cell proliferation, differentiation, and survival (3). In this regard, epidemiological and clinical studies indicate that malnutrition impairs host immune responses and increases rates of morbidity and mortality after infection (4, 5). Similarly, reduced intake of nutrients without malnutrition, often termed as caloric or dietary restriction (CR or DR), improves metabolic fitness and longevity (6) and provides enormous benefits against age-associated disorders such as neurological, cardiovascular, and skeletal problems (7, 8). Exceptions include prolonged energy restriction in small animals, which can reduce growth and development of lymphoid organs and impair antigen-specific immune responses (9, 10). The benefits of CR during viral infections are largely dependent on body weight and may vary between different animals (7, 1113). Small and aged animals subjected to long-term CR are more susceptible to influenza infection because of continuous body mass loss and accompanying energy deficits that impair recovery, despite increased splenocyte proliferation (11). However, short-term refeeding after energy restriction in mice restores body weight and fat composition, as well as natural killer cell function required to combat influenza infection (12). Emerging evidence suggests that CR has a beneficial impact on various attributes of the immune system. These include enhancing thymopoiesis and maintenance of T cell diversity (14), natural killer and CD4+ and CD8+ T cell activity (1517), and the apoptotic clearance of senescent T cells in aged mice (16, 18, 19). Furthermore, CR stimulates adaptive immunity against parasitic infection in experimental cerebral malaria (20).

Although the associated benefits of CR are mostly linked to reduced calorie intake, accumulating evidence couples dietary amino acid (AA) restriction to the benefits of CR (8). For instance, AA sensing pathways influence both innate and adaptive immunity (21). Innate immune cells are auxotrophs for AAs and sense the availability of extracellular AAs through their intrinsic metabolic sensing pathway (8, 22). AA depletion results 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 eukaryotic translation initiation factor 2α (eIF2α), which results in the activation of amino acid starvation response (AAR) pathway that coordinates posttranscriptional and translational immune reprogramming (24, 25). The AAR can also be activated by halofuginone (HF), a derivative of the Chinese herb Dichroa febrifuga, which creates a pool of uncharged tRNAs by inhibiting prolyl-tRNA synthetase (25), mimicking the effects of AA starvation (26). HF has garnered substantial attention in the last two decades owing to its therapeutic potential in various diseases including autoimmune disease, cancer, muscular dystrophy, and hepatic and renal ischemia-reperfusion injury (25, 2731).

Activation of the AAR using HF or amino acid–restricted diet abrogates the production of interleukin-1β (IL-1β) through the GCN2 pathway and provides protection against intestinal inflammation in an experimental mouse model (24, 32). Furthermore, HF inhibits T helper 17 cell differentiation and provides protection against experimental autoimmune encephalomyelitis (33). In addition, a molecular signature composed of GCN2 correlates with the CD8+ T cell response to the yellow fever vaccine–17D (YF17D) (34, 35). Furthermore, the GCN2 pathway is crucial for optimal proliferation in response to antigen stimulation in vitro and for the appropriate trafficking of CD8+ T cells to lymphoid organs (36). These findings raise the intriguing possibility that the activation of the AAR pathway with HF could enhance 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 all DENV serotypes (37). Therefore, there is an urgent need to identify strategies that can enhance the neutralizing antibody response to all the four serotypes of DENV and promote long-term vaccine protection. We used the recombinant envelope protein domain III (EDIII) of DENV as a model vaccine antigen in our study because it is considered as one of the most potent vaccine candidates against DENV (38). Our results demonstrated that HF increased numbers of DENV EDIII–specific interferon-γ (IFN-γ) and IL-2–producing CD4+ and CD8+ T cells in mice. HF also increased the amount and affinity of DENV EDIII–specific immunoglobulin G (IgG) in the serum of vaccinated mice and its neutralizing activity against all the four serotypes of DENVs. These changes were accompanied by enhanced production of IL-10 and expression of gene signatures involved in antigen-specific memory B cell formation, proliferation, and differentiation of germinal center (GC) B cells in the draining lymph nodes of vaccinated mice. Collectively, these findings highlight a promising role for HF in enhancing robust antigen-specific immunity against vaccine antigens and further suggest a potential application of manipulating AAR through AA restriction mimicry for the design and development of new vaccines.

RESULTS

AA starvation mimetic HF enhances antigen-specific T cell responses

The amino acid starvation sensor GCN2 promotes the development of protective immune responses triggered by the YF17D vaccine (35). To evaluate the effect of AA restriction on antigen-specific T cell immunity, we used HF, a pharmacological activator of AAR, and DENV EDIII antigen, a potential vaccine candidate against DENV. We first purified 6X His-tagged recombinant EDIII of all the four DENV serotypes, with the purity and molecular weights verified on Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) gel, and the specificity of the proteins confirmed by immunoblotting (fig. S1, A and B). The purified proteins were polymyxin bead treated to remove any endotoxin content before use in immunization studies. DENV-2 EDIII (DENVrEDIII) was administered in mice preconditioned with HF or dimethyl sulfoxide (DMSO) control (Fig. 1A). Because HF activation of the AAR pathway induces eIF2α phosphorylation, which promotes cellular translational arrest and ATF4 (activating transcription factor 4) transcription factor expression (33, 39), we evaluated eIF2α phosphorylation and the expression of ATF4 target genes including eIF4Ebp1, Chop, Asns, and Gpt2 in the splenocytes of immunized mice. We found that HF treatment increased phosphorylation of eIF2α and activation of ATF4 target genes (fig. S2, A and B). Furthermore, HF treatment promoted eIF2α phosphorylation in immune cells including B cells, T cells, and dendritic cells (fig. S2, C and D) when administered in mice, with no changes in the body weight of HF-treated mice (fig. S2E). Next, we examined the effect of AAR activation on the magnitude of DENVrEDIII-specific T cell responses after primary immunization. We found that HF treatment enhanced [3H]thymidine incorporation by DENVrEDIII-restimulated splenocytes from immunized mice, suggesting that HF increased the expansion of antigen-specific lymphocytes (Fig. 1B). We also found that HF treatment enhanced DENVrEDIII-induced production of IFN-γ by DENVrEDIII-restimulated CD8+ and CD4+ T cells from blood, spleens, and lymph nodes at 4 weeks after secondary immunization (Fig. 1, C to J). Similar results were observed for the production of antigen-specific IL-2 (fig. S3, A to D). The CD8+ and CD4+ T cell responses in peripheral blood mononuclear cells (PBMCs) were more rapid and robust when HF- or DMSO-preconditioned mice were immunized with DENVrEDIII protein (Fig. 2A). Although there is a reduction in T cell responses after 4 weeks, the enhanced DENVrEDIII-specific induction of IFN-γ–secreting T cells was nevertheless evident in the HF-treated group (Fig. 2A). The reduction in effector CD8+ and CD4+ T cell responses with time occurs during the contraction phase, allowing the maintenance of T cell homeostasis and avoiding nonspecific immunopathology that may be generated due to a large number of activated T cells in the host (40, 41). Polyfunctional T cells, which are largely memory T cells, secrete multiple cytokines including IFN-γ, IL-2, and tumor necrosis factor–α (TNF-α) and are associated with providing increased protection against the pathogens (40, 42). Our results indicate that HF treatment increased the frequency of antigen-specific polyfunctional double cytokine–producing (IFN-γ and IL-2) CD8+ and CD4+ T cells found in immunized mice after DENVrEDIII restimulation (Fig. 2, B and C). This increase was most notable in the DENVrEDIII reactive polyfunctional T cell (IFN-γ+IL-2+) subsets (Fig. 2D). Furthermore, splenocytes and lymph node cells from HF pretreated mice showed increased production of IFN-γ, IL-12p40, and TNF-α upon DENVrEDIII stimulation (Fig. 3). Secretion of these proinflammatory cytokines provides a third signal for effector T cell expansion (43) and indicates enhanced antigen-specific cellular response under the conditions of AAR activation. Together, these results point toward a role for AAR activation in enhancing the magnitude and quality of antigen-specific CD4+ and CD8+ T cell responses, perhaps, in part, by augmenting the delivery of antigen to antigen-presenting cells (APCs) as previously reported (35).

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 from splenocytes of mice 28 days after immunization that were restimulated with DENVrEDIII protein. Data are means ± SEM of 12 mice per treatment group from two independent 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), and lymph node (F and J) after treatment and immunization, as indicated. Fluorescence-activated cell sorting (FACS) plots (C and G) are representative of two independent experiments. Quantification of the percentage of IFN-γ–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).

Fig. 2 HF increases the frequency of antigen-specific polyfunctional T cells in mice.

(A) Flow cytometry analysis of antigen-specific CD8+IFN-γ+ and CD4+IFN-γ+ T cell kinetics in PBMCs of mice treated with DMSO or HF and immunized with DENV-2 envelope domain III after 14 and 28 days. Data are means ± SEM of 12 mice per group from two independent experiments. (B to D) Flow cytometry analysis of DENVrEDIII-specific polyfunctional CD8+ (B) and CD4+(C) T cells in blood, spleen, and lymph node of immunized mice. The frequency of double cytokine (IFN-γ and IL-2)–producing cells with means (bar) ± SEM (B and C) and pie charts of the frequency of all cytokine-producing T cells (D) are from 12 mice per group from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way analysis of variance (ANOVA) with Bonferroni post hoc between DENVrEDIII + DMSO– and DENVrEDIII + HF–immunized groups (A), and Mann-Whitney U test (B and C).

Fig. 3 HF enhances secretion of multiple cytokines after DENVrEDIII immunization.

Enzyme-linked immunosorbent assay (ELISA) analysis of the amounts of IFN-γ, IL-12p40, and TNF-α produced by splenocytes 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 are means ± 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.

HF enhances antibody responses against DENV envelope domain III (DENVrEDIII) protein

The activation of CD4+ T helper cells is a vital step in establishing an adaptive immune response, acting both as an effector and a modulator of the immune response. CD4+ T helper cell differentiation into T follicular helper (Tfh) cells provides a vital signal that drives the activation and differentiation of high-affinity memory B cells and antibody-producing plasma cells (44, 45). Having observed the effect of HF on the induction of robust antigen-specific CD4+ T cell responses, we further determined the effects of HF on antigen-specific antibody responses. We assessed the amount of DENV-2 EDIII–specific total IgG and IgG subtypes IgG2a, IgG2b, and IgG1 in the serum from DENVrEDIII-immunized mice preconditioned with HF or vehicle control after both primary and secondary immunization. We observed a substantial increase (more than twofold) in the production of DENVrEDIII-specific total IgG and in all the IgG subtypes in the serum of HF-preconditioned mice (Fig. 4A). Secondary immunization with the same immunogen 2 weeks later enhanced antibody amounts in all immunized groups, and again, significant enhancement was evident in the DENVrEDIII + HF–immunized group (Fig. 4A). The production of ovalbumin (OVA)–specific antibodies (total IgG and IgG subtypes) was also enhanced when HF-primed mice were immunized with OVA antigen as compared with vehicle control DMSO (fig. S4). These results establish that activation of the AAR pathway may enhance antigen-specific immune responses for diverse antigens.

Fig. 4 HF-mediated AAR activation augments the antibody responses against DENVrEDIII.

(A) ELISA analysis of DENVrEDIII-specific total IgG, IgG2a, IgG2b, and IgG1 in the serum 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 and C) BIAcore SPR analysis of DENVrEDIII protein binding by pooled serum samples from mice preconditioned with HF or DMSO 28 days after 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 serum samples 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% focus reduction 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).

To determine whether HF enhances antigen-specific responses through GCN2, we evaluated the amount of DENVrEDIII-specific antibodies in the serum of HF- or DMSO-preconditioned wild-type (WT) or Gcn2−/− mice immunized with DENVrEDIII protein. We found that HF had no effect on antigen-specific total IgG or IgG subtype responses in Gcn2−/− mice, whereas we observed a notable increase in 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-target effects.

HF augments the quality of antigen-specific antibody responses

T cell–dependent B cell activation is predominantly characterized by the development of high-affinity antibodies, mostly the IgG subtype (44), and the avidity of antigen-antibody interaction is one of the 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 from mice 28 days after the secondary immunization were assayed for binding to immobilized DENVrEDIII on CM5 sensor surface. With DENVrEDIII secondary immunization, serum from HF-pretreated mice gave the highest antigen-binding response [in response unit (RU)] as compared with vehicle control pretreated mice (Fig. 4B). SPR analysis of twofold serial dilution of the serum (ranging from 1:50 to 1:400) from HF-preconditioned immunized mice indicated stable association with DENVrEDIII compared with the sera from unimmunized or DMSO-preconditioned control mice (fig. S6). The qualitative analysis of the DENVrEDIII-specific antibody response was evaluated in terms of avidity by measuring the dissociation rate constants. Serum from HF-preconditioned, DENVrEDIII-immunized mice associated with DENVrEDIII protein faster and dissociated from DENVrEDIII slower, suggesting that HF induced enhanced high-affinity antibody responses (Fig. 4C). Increased amounts of antigen-specific, high-avidity antibody after DENVrEDIII vaccination with HF pretreatment correlated with improved antibody function as assessed by the virus-neutralizing ability in vitro. Serum from mice immunized with DENVrEDIII after HF preconditioning neutralized DENV-2 virus more efficiently than serum from mice immunized with DENVrEDIII after vehicle control treatment (Fig. 4D). We also observed a notable increase in DENV serotype–specific total serum IgG when we immunized HF-preconditioned mice with a tetravalent formulation consisting of domain III proteins of all the four DENV serotypes (tDENVrEDIII) (Fig. 5A). In addition, the resultant antibodies were effective against DENV-1, DENV-2, DENV-3, and DENV-4 serotypes (Fig. 5B). Together, the above results indicate that activation of the AAR pathway enhances both the magnitude and quality of antigen-specific B cell responses.

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 a tetravalent combination of DENVrEDIII protein 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 10 mice per group from two independent experiments. (B) DENV serotype neutralization assay on serum from tDENVrEDIII-immunized mice. The FRNT50 data are means ± SEM of 10 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).

HF induces enhanced development of GCs in draining lymph nodes

GCs are crucial for humoral immunity and play a vital role in the development of immunological memory. The explicit colocalization of antigen-specific T cells, B cells, and follicular dendritic cells (FDCs) is crucial for GC formation (47). The progression of B cell responses along the GC pathway is important in the generation of long-lived plasma cells that secrete high-affinity antibodies (48). Because our results suggest that HF enhances the production of high-avidity antibodies, we determined whether activation of the AAR axis regulates the GC pathway. Thus, immunized mice preconditioned with HF or DMSO were euthanized on day 28 after the booster dose, inguinal lymph nodes were isolated, and the presence of GCs was evaluated by immunohistology. We found that HF-preconditioned, DENVrEDIII-immunized mice developed more lymph node GCs than DMSO-preconditioned, DENVrEDIII-immunized mice (Fig. 6, A and B), which were also apparent in hematoxylin and eosin (H&E)–stained sections of inguinal lymph nodes (fig. S7, A and B). To determine the persistence of GC responses in HF-preconditioned mice, we examined GC kinetics over a duration of 8 weeks by evaluating the frequency of GC B cells in the draining lymph nodes using flow cytometry (fig. S7C). We observed a substantial increase in GC expansion within 10 days of antigen immunization, with increased frequency of B220+GL7+IgG+ GC-B cells in the 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 by a decline. The decline in GC responses at a later time point is perhaps a result of reduction in the number of CD4+/Tfh cells, which is a prerequisite for establishing optimal GC reactions for effective protective immunity, while avoiding the production of low-affinity self-reactive antibodies that lead to autoimmunity (49, 50).

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 independent experiments. 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 in DENVrEDIII-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) ELISA analysis 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 DENVrEDIII for 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).

The pleiotropic cytokine IL-10 has a key role in the generation of GC-B cell responses. IL-10 produced by CD4+ follicular T cells (51) and B cell–intrinsic IL-10 signaling (52) have been observed to directly promote GC responses. Therefore, we measured the amount of IL-10 in the DENVrEDIII-restimulated splenocytes and lymph node cells from the immunized mice and found elevated IL-10 production from cells of HF-preconditioned mice (Fig. 6D), highlighting an important role for IL-10 in AAR-mediated regulation of GC responses. We speculated that the effects of AAR activation on the GC pathway might be due to transcriptional 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 involved in the maintenance of GCs in draining lymph nodes. We observed enhanced expression of genes associated with programming of memory B cells in the lymph nodes of HF-preconditioned, DENVrEDIII-immunized mice. Memory B cells swiftly respond to booster immunization, thereby driving the localization of antigen-specific plasma cells to surviving niches in bone marrow and generating long-lived plasma cells that are the primary source of high-avidity antibodies in serum (53). HF preconditioning–induced AAR activation enhanced the expression of genes involved in maintaining the integrity of GCs [Bcl2 (54) and Tank (42)]; genes crucial in the formation and maintenance of B cell memory [Plcg2 (55) and Cd38 (56)]; genes involved in the proliferation, survival, and differentiation of GC B cells such as Il17ra (57), Il18r1, Pax5 (58), Ikzf1 (59), Irf7, and Mx1; and type 1 interferon genes involved in the differentiation of B cells (Fig. 6E). These findings exemplify the benefits of amino acid restriction in the production of robust high-avidity and neutralizing antibody mediated through programming of genes associated with memory B cell development and differentiation. Hence, this study suggests that the AAR pathway might be manipulated for the design and development of vaccines for tailoring long-lived protective immunity against the pathogens.

DISCUSSION

Metabolic control of the immune system is a crucial requirement for the regulation of cell survival and the development of immunity (1). Accumulating evidence suggests that the immune system can sense and respond to diverse changing environmental signals, including fluctuations in the abundance of amino acids and other metabolites. Metabolic sensors like GCN2 are able to sense depletion of even a single type of amino acid in the cellular microenvironment and respond appropriately through the activation of the AAR pathway, which, in turn, can dictate and tailor the fate of immune cells (60). Although activation of the GCN2-AAR pathway has broad anti-inflammatory effects (24, 32) and therapeutically benefits various metabolic diseases (25), the impact of AAR on the adaptive immune response in infectious disease remains poorly 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 in a mouse model using DENV envelope protein domain III (DENVrEDIII) as a model antigen. Our results highlight a critical role of the AAR pathway in the regulation of T and B cell immunity to DENVrEDIII antigen.

HF enhanced T cell responses, including the frequency of IFN-γ and IL-2, and double cytokine (IFN-γ and IL-2)–producing CD8+ and CD4+ effector and polyfunctional T cells. The latter are a prerequisite for antigen-specific protective T cell adaptive immunity (40, 42). Furthermore, the DENVrEDIII-specific T cell recall responses correlated with the magnitude of antigen-specific antibody responses after AAR activation. Effective vaccines establish protective humoral immunity, which depends on the production of long-lived plasma cells secreting high-affinity antibodies that are necessary for pathogen clearance (61). The activation of the AAR pathway enhanced the production of high-avidity and greater virus-neutralizing antibody titers, which were able to efficiently neutralize all the serotypes of DENV, one of the key goals of DENV vaccine development. T cell activation promotes neutralizing antibody production by stimulating the development of GC in response to antigenic challenge (62), and our results demonstrate that AAR activation enhanced the formation of GCs in the draining lymph nodes of HF-pretreated mice. HF preconditioning also promoted the production of the cytokine IL-10, which is involved in GC formation (52). Our data suggest that AAR activation might increase the establishment of GCs through enhanced IL-10 production and thereby improve antigen-specific humoral immunity. Supporting the antigen-driven nature of this response, we did not observe GC formation in the lymph nodes from mice 5 days after HF preconditioning only, demonstrating that the GC phenotype did not exist before antigen immunization or that HF priming merely resulted in the production of self-reactive antibodies.

Our results suggest that AAR activation stimulates a molecular program that aids in the differentiation, maintenance, and survival of GC-B cells, thereby augmenting immunological memory. For example, B cell lymphoma 2 (Bcl2) is involved in maintaining the integrity of GC by inhibiting apoptosis within the GC-B cells (54). The Plcg2 gene product conveys survival signals to GC and memory B cells necessary for the generation of the secondary immune responses (55). In addition, signaling through IL-17RA is required to secure the localization of Tfh cells in the GC to induce its function, stabilizing the interaction of GC B 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 with other proteins critical for B cell function and drives the expression of interferon regulatory factors such as IRF8, thereby contributing toward sensing the advancement of GC reaction (58, 63). Our data demonstrated that HF treatment augmented the expression of all of these critical B cell survival factors.

Together, these data demonstrate that HF augmented GC formation, which enhanced the magnitude and quality of antigen-specific antibody responses and correlated with transcriptional reprogramming of lymphoid organs, after experimental DENV vaccination. Although this work demonstrates the feasibility of using HF to enhance vaccine responses, the mechanisms by which AAR promotes GC formation remain poorly understood and merit further investigation. Furthermore, the detailed study of the T cell dependence of the effects of HF on antigen-specific antibody responses could be the next step. However, our data demonstrate that AAR mimicry–based nutraceuticals such as HF are potent agonists of vaccine responses poised for further development against human diseases.

MATERIALS AND METHODS

Purification of recombinant DENV envelope domain III protein (DENVrEDIII)

The optimized envelope domain III sequences for all the four DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) were cloned into bacterial expression vector pET-28a by GenScript (USA). The cloned constructs were expressed in Escherichia coli strain Rossetta and induced with 0.6 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (DENV-2) or 1 mM IPTG (DENV-1, DENV-3, and DENV-4). The 6X His-tagged recombinant Dengue envelope protein domain III (DENVrEDIII) was purified using the affinity chromatography technique under denaturing conditions as described earlier(64) using Talon Superflow (GE Healthcare) high-affinity resins. The His-DENVrEDIII proteins were eluted using 300 mM imidazole, run on SDS-PAGE and subjected to Coomassie staining. The desired band sizes of 14.5 kDa (DENV-1, DENV-2, and DENV-4) and 15 kDa (DENV-3) were observed in the Coomassie-stained gel.

Immunoblotting

The 6X His-tagged purified Dengue envelope domain III (DENVrEDIII) protein for all the four serotypes was confirmed by immunoblotting using anti-His antibody (Cell Signaling Technology). For evaluation of HF-mediated AAR activation, splenocytes from immunized mice were lysed in lysis buffer [1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 20 mM Hepes (pH 7.5), 100 mM NaF, 17.5 mM β-glycerophosphate, and supplemented with 1X Protease Inhibitor Cocktail (Sigma-Aldrich)] (24), incubated on ice for 30 min, and cleared by centrifugation at 12,000 rpm for 15 min at 4°C. An equal amount of protein was separated on SDS-PAGE and transferred to nitrocellulose membrane. For the detection of eIF2α phosphorylation, the membrane was blocked with 5% bovine serum albumin and probed with rabbit anti–eIF2α-P antibody (Cell Signaling Technology). Protein bands were visualized with FemtoPlus ECL chemiluminescent substrate. Densitometry analysis of eIF2α-P bands with respect to eIF2α-T bands was performed using the National Institutes of Health ImageJ software.

Immunization of mice

Six- to 8-week-old Balb/c mice were administered intraperitoneally with HF (0.1 mg/kg body weight) or vehicle control (DMSO) (33) for 4 days, followed by immunization with 20 μg per mouse per dose of DENV-2 envelope domain III protein (DENVrEDIII) in 200 μl of 1X phosphate-buffered saline (PBS), delivered subcutaneously at the base of the tail, followed by resting for 10 days. The booster injection was given at the 14th day after primary immunization with an identical dose of HF or DMSO (intraperitoneally) again for 4 days, followed by subcutaneous administration of 20 μg per mouse per dose DENVrEDIII protein. The mice were rested for 10 days and euthanized at the 28th day after primary injection for further analysis of blood, spleen, and lymph node cells. In another set of experiments, Balb/c mice previously conditioned with HF or DMSO were immunized by subcutaneous injection of tetravalent DENV envelope domain III protein (tDENVrEDIII) formulation (10 μg each of DENV-1, DENV-2, DENV-3, and DENV-4 rEDIII) following the same regimen as mentioned above. In yet another experiment, Balb/c mice previously conditioned with HF or DMSO were injected subcutaneously with 10 μg of OVA as antigen per mouse per dose following the same immunization schedule as mentioned above. Blood samples were collected on the 14th and 28th days by tail bleeding for serum collection and PBMC isolation. In another set of experiments, WT or Gcn2−/− mice (on a C57Bl/6 background) were immunized with 20 μg of DENVrEDIII antigen per mouse after HF or DMSO priming following the abovementioned immunization schedule. Blood samples were collected at the 14th and 28th days by tail bleeding for serum collection for the analysis of antigen-specific antibody responses. Animals that were injected with PBS alone (without candidate antigen) served as negative controls. All animal experiments were performed according to the animal ethical guidelines of the University of Hyderabad and the Institutional Animal Care and Use Committee at Cornell University.

Phospho-eIF2α staining by flow cytometry

Total splenocytes isolated from HF- or DMSO-preconditioned mice were surface stained using PerCP Cy5.5 anti-mouse CD45R/B220 (BD Pharmingen), APC anti-mouse CD11c (BD Pharmingen), and Alexa 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, followed by intracellular staining for eIF2α-P as described earlier (32). Fixed cells were permeabilized using 1X Perm Wash Buffer (BioLegend) and incubated with rabbit anti-mouse monoclonal eIF2α-P antibody (Cell Signaling Technology) or respective isotype control overnight at 4°C. After washes with 1X FACS buffer, the cells were stained with anti-mouse Alexa Fluor 488 secondary antibody for 1 hour at room temperature, washed and resuspended in FACS buffer, acquired on a BD LSRFortessa (BD Biosciences) cytometer, and analyzed using FlowJo (Tree Star Inc.).

T cell assays by flow cytometry

Lymph node and spleen CD4+ and CD8+ T cells from immunized mice (collected at day 28) and those from PBMCs (collected at day 14 and day 28) were evaluated for antigen-specific responses. PBMCs were isolated by sucrose gradient density separation method using Histopaque (Sigma-Aldrich) as described earlier (65). Briefly, isolated PBMCs, lymph node cells, and splenocytes were cultured for restimulation with DENVrEDIII protein (10 μg ml−1) in the presence of GolgiStop and GolgiPlug (BD Biosciences) at 37°C for 10 hours. The restimulated cells were stained with Alexa Fluor 488–labeled anti-mouse CD8 (BD Biosciences) 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, fixed with 4% paraformaldehyde, permeabilized using 1X Perm Wash Buffer (BioLegend), and stained using APC-labeled anti-mouse IFN-γ (BD Biosciences) and phycoerythrin-conjugated anti-mouse IL-2 antibody (BD Biosciences), diluted in 1X Perm Wash Buffer for 60 min at room temperature. Cells were washed with FACS buffer, and data were acquired on a BD LSRFortessa (BD Biosciences) cytometer and analyzed using FlowJo (Tree Star Inc.).

Cytokine secretion by enzyme-linked immunosorbent assay and proliferation assay

The cytokines secreted were evaluated in splenocytes and lymph node cells isolated from immunized mice. Splenocytes (2 × 105) or lymph node cells were seeded in triplicates in 96-well round-bottom plate and restimulated with DENVrEDIII (10 μg ml−1) for 72 hours. The supernatants were then collected and analyzed for IFN-γ, IL-12p40, TNF-α, and IL-10 (BD Biosciences) cytokines through sandwich enzyme-linked immunosorbent assay (ELISA) following the protocol as per the manufacturer’s instructions. Lymphocyte proliferation was assessed in 72-hour splenocyte culture as mentioned earlier (66). Briefly, after 72 hours, splenocytes were pulsed with 1-μCi [3H]thymidine for an additional 16 hours. Cells were harvested, fixed with 10% trichloroacetic acid for 30 min at room temperature, followed by solubilization with 150 μl of 1 N NaOH for 30 min at 37°C with constant agitation. Next, 150 μl of 1 N HCl was immediately added; 300 μl of each sample was mixed with 5 ml of scintillation fluid (Sisco Research Laboratories), and the counts were measured in a scintillation counter (PerkinElmer).

Antibody ELISA

For detection of antigen-specific antibodies in the serum of immunized mice, 100 μl of DENVrEDIII protein (5 μg ml−1) diluted in carbonate buffer was coated on 96-well Nunc MaxiSorp ELISA plates and incubated overnight at 4°C. Next, the plates were washed three times with 1X PBST (0.05% Tween 20) using a Thermo Fisher Scientific Wellwash 4 MK 2 ELISA washer. The plates were blocked using 4% skimmed milk (Himedia) for 1 hour at room temperature. Serum samples were diluted in 0.1% skimmed milk prepared in 1X PBS and incubated on blocked plates for 2 hours at room temperature. After washing 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; Santa Cruz), anti-mouse IgG2b-HRP (1:2000; Santa Cruz), and anti-mouse IgG1-HRP (1:5000; Santa Cruz) diluted in 1X PBS for 1 hour at room temperature. After washing six times with 1X PBST, the plates were lastly developed with 100 μl per well of tetramethylbenzidine substrate (BD Biosciences). The reaction was stopped using 2N H2SO4. Plates were analyzed using a Tecan microplate reader at λ = 450 nm and correction at λ = 570 nm (66).

BIAcore assay

SPR binding and kinetics measurement were carried out at 25°C on a BIAcore T200 instrument (BIAcore/GE Healthcare) as described earlier (42, 67, 68). The CM5 sensor chip (GE Healthcare) was immobilized with DENVrEDIII (for serotype 2) diluted in 10 mM sodium acetate (pH 4.5) using standard amine coupling chemistry. Serum samples collected at day 28 from immunized mice were injected at 1:50 to 1:400 dilution by using twofold dilution steps at a flow rate of 30 μl min−1. The contact time for the serum samples to interact with ligand immobilized on the sensor chip was 60 s, and dissociation time for the sample was 60 s. After each cycle, the sensor chip was regenerated with 50 mM NaOH with a specified contact time for 60 s. The experimental data were fit using 1:1 Langmuir model for determining the binding kinetics, and analysis was performed using BIAcore T200 Evaluation 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 determined as mentioned earlier (avidity score = maximum binding response/kd in RU.s) (61).

DENV stock preparation

DENV-1 (Hawaii), DENV-2 (TR1751), DENV-3 (Thailand 1973), and DENV-4 (Columbia 1982) viral strains were propagated in C636 cells as described earlier (69). Viral titers were quantitated by focus-forming assay in BHK-21 and Vero cells as previously described (70).

Focus reduction neutralization tests

Focus reduction neutralization test (FRNT) was carried out in Vero cells seeded at a density of 25,000 cells in 96-well plate 24 hours before infection as described earlier (71, 72). Serum samples from immunized mice were heat activated at 56°C for 30 min and were twofold serially diluted (from 1:12.5 to 1:3200) in serum-free DMEM. After decomplementation and dilution of serum, 50 μl of each dilution was thoroughly mixed with 50–plaque-forming unit DENV (DENV-1, DENV-2, DENV-3, and DENV-4) diluted in DMEM and incubated at 37°C for 2 hours. Next, Vero cells were washed with serum-free DMEM and infected in duplicate with 45 μl of the neutralization mixture followed by another incubation for the next 2 hours. After incubation, the viral inoculum was removed and overlaid with 200 μl of 1.5% carboxymethylcellulose (Sigma-Aldrich) in serum-free DMEM and incubated at 37°C for 4 to 5 days, depending on the serotype. After incubation, the overlay medium was carefully removed followed by three washes with 1X PBS. The cells were further fixed, permeabilized with 0.2% Triton X-100, and stained with anti-dengue monoclonal antibody (GeneTex) for 1 hour at 37°C. After washing with 1X PBS, the stained cells were incubated with HRP-linked anti-mouse secondary antibody for 1 hour at 37°C. The cells were lastly washed with 1X PBS and developed using DAB (3,3′-diaminobenzidine) substrate. Viral foci were counted manually, and the percentage of foci reduction against control serum was calculated. Neutralizing antibody titer (FRNT50) values were determined as the reciprocal of the highest dilution that resulted in 50% decrease in focus-forming units and analyzed using GraphPad Prism software.

Quantitative real-time polymerase chain reaction

GC gene expression profiling was performed on DENVrEDIII (serotype 2)–restimulated cells isolated from the lymph nodes of different immunized mice. Total RNA was extracted using TRI Reagent (Sigma-Aldrich) as described earlier (72). Total purified RNA was reverse transcribed using Easy Script cDNA Synthesis Kit (abm) according to the manufacturer’s instructions. qRT-PCR was performed using Applied 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 one cycle of 95°C for 10 min, next 40 cycles of 15 s at 95°C, 30-s annealing at 56°C, and 40-s extension at 68°C. The relative mRNA expression was normalized to housekeeping gene Gapdh. The normalized expression of statistically significant genes is presented as heat map using the Gene-e software (www.broadinstitute.org). Genes with P < 0.05 (two-tailed unpaired Student’s t test) were considered significant. Activation of AAR pathway genes was evaluated in splenocytes from immunized mice. The mRNA expression of AAR genes was determined using gene-specific primers (table S1) and normalized to housekeeping gene Hprt.

Histology and immunofluorescence

Draining lymph nodes were isolated and snap frozen in OCT (optimal cutting temperature) tissue embedding medium for sectioning as mentioned earlier (42, 66). Sections were either stained with H&E or fluorescently stained with Alexa Fluor 488–labeled anti-mouse B220 (eBioscience), Alexa Fluor 647–labeled anti-mouse total IgG (Cell Signaling Technology), or biotin-labeled anti-GL7 (eBioscience), followed by Alexa Fluor 555–labeled streptavidin (Invitrogen) antibodies. The slides were washed and mounted with coverslips using a DPX mounting medium. The images were captured using a 40× objective on a Zeiss confocal microscope.

GC staining by flow cytometry

For identification of GCs by flow cytometry, cells isolated from lymph nodes collected at the 5th (after HF or DMSO priming), 14th, 28th (after booster), 42nd, and 56th days were passed through a 40-μm cell strainer to generate single-cell suspensions, after which they were stained with Alexa Fluor 488–labeled anti-mouse B220 (eBioscience), Alexa Fluor 647–labeled anti-mouse total IgG (Cell Signaling Technology), or biotin-labeled anti-GL7 (eBioscience) for 1 hour at 4°C, followed by Alexa Fluor 555–labeled streptavidin (Invitrogen) for 1 hour at room temperature. The stained cells were fixed using 4% paraformaldehyde, washed with FACS buffer, and data were acquired on a BD LSRFortessa (BD Biosciences) cytometer and analyzed using FlowJo (Tree Star Inc.).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 7 software. Normality test (Shapiro-Wilk normality test) was performed on all the data shown. Accordingly, parametric two-tailed unpaired Student’s t test was applied on normally distributed data, whereas nonparametric Mann-Whitney U test was applied on data that were not normally distributed to measure statistical significance. One- or two-way ANOVA with Bonferroni post hoc test has been used for multiple comparisons.

SUPPLEMENTARY MATERIALS

stke.sciencemag.org/cgi/content/full/12/607/eaav4717/DC1

Fig. S1. Expression of dengue envelope protein domain III (DENVrEDIII) from all four DENV serotypes.

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 GCN2 dependent.

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.

REFERENCES AND NOTES

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. We thank the FACS and confocal microscopy core facilities of the University of Hyderabad. Funding: This work was supported by grants from the Department of Science and Technology, Nano Mission, government of India [DST no. SR/NM/NS-1040/2013(G)], and the University 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. and AI137822 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 with assistance 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. interpreted the data. G.M. assisted during the experiments. Competing interests: A.A. receives research funding from 3M Corp. All the other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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