E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells
© The Author(s). 2016
Received: 19 January 2016
Accepted: 12 July 2016
Published: 16 July 2016
The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses.
The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4+ and CD8+ cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature.
The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.
The E2 system is a delivery vehicle in which antigenic determinants are inserted on the surface of an icosahedral scaffold formed by the acyltrasferase component (E2 protein) of the multienzyme pyruvate dehydrogenase (PDH) complex from Geobacillus stearothermophilus [1, 2]. E2 naturally serves as a docking unit for other large PDH subunits. This property makes it an excellent scaffold for the presentation of N-terminal fused heterologous antigens. The scaffold can be refolded in vitro to produce pure or chimeric particles similar to virions in size and complexity, it is able to confer high immunogenicity to the displayed determinants, and it is suitable for vaccine formulations [3–6]. Various protein domains, such as the HIV-1 Envelope (Env) V3 loop, can be assembled into lipopolysaccharide (LPS)-free E2 recombinant vaccines, and we previously demonstrated that their systemic administrations are able to elicit potent binding antibodies and T-cell responses in mice, as well as autologous neutralizing antibodies in rabbits .
A shared feature of many pathogens is that the infection occurs or initiates at a mucosal surface. While systemic vaccination offers protection against pathogens such as polio and influenza viruses, induction of mucosal immunity is required for effective protection against pathogens such as HIV, human papillomavirus, herpes viruses, Vibrio cholera and the Mycobacterium species [8–13]. Antibodies patrolling the mucosal epithelium appear to play a crucial role in blocking HIV-1 mucosal challenge . Therefore, it is necessary to develop adequate mucosal vaccination protocols for this type of infection.
Understanding the immunological mechanisms of vaccination is of paramount importance for the rationale design of a vaccine. Recently, the use of systems biology approaches gave an important contribution to elucidate the fundamental mechanisms by which the innate immune system orchestrates protective immune responses triggered by vaccination [15–17]. High-throughput sequencing can be applied to explore cell transcriptome and to identify differences in gene expression and alternative splicing. In vaccinology, gene expression patterns induced by priming antigen presenting cells (APCs) could be used to predict antigen immunogenicity and the type of T-helper cell polarization, and/or help to select the appropriate adjuvant to administer in the vaccine formulation.
As proof-of-concept, here we report the immunogenicity of E2 scaffold in a model of mucosal vaccination, and show that intranasal administration of E2-based vaccines is able to induce mucosal and systemic immune responses. Through RNA-Sequencing (RNA-Seq) analysis, we attempt to identify the molecular signatures that could account for E2-primed immune responses, and analyze the gene expression profile of bone marrow-derived dendritic cells (BMDCs) pulsed with E2 scaffold compared to un-pulsed cells.
Purification of HIV-1 E2-based multimeric scaffolds
Expression and purification of E2 wild type (E2wt) and recombinant E2 multimeric particles displaying the HIV-1 SF162 Envelope V3 loop peptide [amino acid 291–336, reference strain HIV-HXB2; (Env(V3)-E2)], were performed following previously described protocols [7, 18]. The purified 1.5MDa E2 60-mer particles were subjected to endotoxin (LPS) removal by Triton X-114 (Sigma Aldrich, Milan, Italy) phase separation  and to detergent removal by using Thermo Scientific Pierce Detergent Removal Resin (Thermo Fisher Scientific, IL, USA). The final particles were tested for endotoxin using the Limulus Amebocyte Lysate (LAL) Assay (QCL-1000, Lonza, Basel, Switzerland), according to the manufacturer’s instructions and were less than 0.05EU/ml.
Antibodies, reagents and synthetic peptides
For flow cytometric analysis, fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8 (clone 53-6.7) and phycoerythrin (PE)-conjugated anti-mouse IFN-γ (XMG1.2) were purchased from Biolegend (San Diego, CA). For Intracellular Cytokine Staining (ICS) assay, the H-2d MHC class I-restricted synthetic IGPGRAFYA(311–318) peptide, corresponding to residues 311-318 of the HIV-1 Env V3 loop, was purchased from PRIMM srl (Naples, Italy). For analysis of fecal IgA, serum IgA and IgG, the HIV-1 SF162 clade B V3 peptide (PNNNTRKSITIGPGRAFYATGD) and the V3 scrambled peptide (PNNNTRKSIFYRGAPGITATGD) were purchased from Invitrogen (Carlsbad, CA) and Genscript (Piscataway, NJ), respectively. Mouse IL-4 and IFN-γ ELISA MAXTM Standard SET kits were obtained from BioLegend. Mouse CD4+ T cell isolation kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). For the quantitative measurement of IgA antibodies in fecal samples, RayBio® Mouse IgA ELISA Kit was purchased from RayBiotech, Inc. (Norcross, GA, USA).
Mucosal vaccination with E2-based vaccines
Female BALB/c (H-2d MHC) mice, 6 to 12 week-old, were purchased from Charles River Laboratory (Lecco, Italy) and housed under specific pathogen free conditions at the Animal Facility of the Institute of Food Science of C.N.R., Avellino, Italy (accreditation no. DM.161/99). Groups of five BALB/c mice were intranasally immunized with 50 μg of E2wt, Env(V3)-E2, or Env(V3)-E2 with 1 μg/mouse of cholera toxin (CT) adjuvant (Sigma Aldrich), at weekly intervals for 7 weeks. The intranasal (i.n.) administration was carried out by inoculating 20 μl of antigens to the nostril. Fecal pellets were collected at weekly intervals, weighed, homogenized at a concentration of 10 mg/ml in 1× phosphate buffered saline (PBS), 0.01 % sodium azide (Sigma Aldrich), and centrifuged at 10,000 g for 10 min to remove debris as previously described ; supernatants were recovered and stored frozen. One week after the final boost, mice were sacrificed to collect spleen and serum samples.
ELISA assay for detection of IgA and IgG antibodies
Analysis of fecal IgA, serum IgA and IgG antibodies (IgG, IgG1, and IgG2a) were performed by in house ELISA as previously described  using the synthetic HIV-1 SF162 V3 peptide, V3 scrambled peptide, Env(V3)-E2 or E2wt proteins as antigens. Briefly, 96-well ELISA plates (MaxiSorpTM, NUNC, NY) were precoated with 100 μl/well of 2 μg/ml of E2 antigens or V3 synthetic peptides in 1× PBS and incubated overnight at 4 °C. Plates were washed 4 times with PBS containing 0.05 % Tween-20 (Sigma Aldrich) and blocked by adding 200 μl/well of PBS containing 2 % bovin serum albumin (Sigma Aldrich) at room temperature (RT) for 2 h. After the incubation, plates were washed 4 times with washing buffer. Two-fold serial dilutions of sera (starting at 1:100 and ending to 1:12,800) and 100 μl of soluble fecal extracts were added to the plates and incubated at RT for 2 h. All samples were tested in duplicate. After washing, plates were incubated with 1:2,000 dilution of peroxidase-conjugated rabbit anti-mouse IgA, IgG, IgG1, or IgG2a antibodies (Santa Cruz Biotechnology, Dallas, USA). Results were expressed as absorbance values after blank subtraction or as reciprocal endpoint titer of the last dilution exhibiting OD450 ≥ 0.12. The highest dilution tested was 1:128,000. Serum titers of <1:100 were considered negative and were reported in figures with an arbitrary titer value of 1:10. Levels of total IgA antibodies in soluble fecal extracts (100 μl of 10 mg/ml) were quantified in duplicates using the RayBio® Mouse IgA ELISA Kit, according to the manufacturer's instructions. The Env(V3)-specific IgA antibodies were normalized by dividing the specific OD450 readings of IgA by the total IgA concentration in ng/ml , measured in fecal samples at week 4 for each animal from Env(V3)-E2 and Env(V3)-E2 + CT groups.
Detection of IL-4 and IFN-γ
Spleen cells were isolated from BALB/c mice mucosally immunized with E2wt or Env(V3)-E2 administered with CT. Splenocytes from each group were pooled and used as antigen presenting cells (APCs). Afterwards, CD4+ T cells were purified from pooled splenocytes using the CD4+ T cell isolation kit. 1 × 105 CD4+ T cells were incubated with 1 × 105 γ-irradiated APCs preloaded or not with 50 μg/ml of E2wt, or Env(V3)-E2. Cells were cultured overnight in 0.2 ml of RPMI 1640 (Lonza) medium supplemented with 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 % Fetal Calf Serum (FCS) (all from GIBCO, Milan, Italy). Supernatants (0.1 ml/well) were assayed in duplicate for detecting IL-4 and IFN-γ, using the mouse IL-4 and IFN-γ ELISA MAXTM Standard SET kits, according to the manufacturer's instructions.
Analysis of CD8 T-cell response
The antigen-specific CD8 T-cell response was assessed by intracellular cytokine staining (ICS) assay on spleen cells isolated from mice described above co-cultured in vitro (2.5 × 106 cells/ml) for 6 days with γ-irradiated (10,000 rad) LPS-blasts (1.25 × 106 cells/ml) prepulsed with 10 μg/ml of MHC-I restricted V3 peptide. IFN-γ production was evaluated on CD8+ gated T cells by FACS (Fluorescence Activated Cell Sorter) analysis using FACSCanto II flow-cytometer and DIVA (Data-Interpolating Variational Analysis) software (Becton Dickinson, Fullerton, CA), as previously described by Jaworski et al. .
BMDCs, RNA-Seq library production, sequencing and data analysis
Bone marrow-derived dendritic cells (BMDCs) were generated according to previously described protocols . Briefly, bone marrow cells were collected by flushing tibias of C57BL/6 mice (Charles River Laboratory) with complete medium (RPMI 1640, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 % FCS). Cells were seeded in bacteriological Petri dishes in complete medium supplemented with 200 U/ml of recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF, Peprotech, NJ, USA) for 7 days. Immature cells were harvested and incubated or not for 1 more day with 50 μg/ml of LPS-free E2wt antigen at a final concentration of 2.5 × 106 cells/ml. Total RNA was extracted from untreated or E2-pulsed BMDCs using Tri Reagent (Sigma Aldrich) according to manufacturer's protocol. RNA integrity was assessed as described in Costa et al. . Paired-end libraries (100 x 2 bp) prepared using TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA) were sequenced on Illumina HiSeq2000 platform. About 200 million paired-end reads were sequenced. Quality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). TopHat version 2.0.10  was used to map reads on reference mouse genome (mm9) and RefSeq mouse transcripts annotation with default parameters. More than 95 % of sequenced reads uniquely mapped to mm9. Such reads were used for further analyses. Coverage files were produced using BEDTools and loaded on UCSC Genome Browsers to analyze gene-specific features. Gene expression was measured using Cufflinks 2 . Cuffdiff and CummeRBund  were used to identify differentially expressed genes using normalized expression values (FPKM, Fragments Per Kilobase of transcript and Million of mapped reads). An arbitrary threshold of 0.05 FDR (False Discovery Rate) and 1 FPKM in at least one condition was used to filter out differentially expressed genes. Gene ontology and pathway analysis were performed using DAVID (Database for Annotation, Visualization and Integrated Discovery) .
Statistical analyses were performed using the unpaired two-tailed Student's t-test. Differences were considered statistically significant when P < 0.05.
Dissection of fecal antibody responses after mucosal immunization
To determine whether HIV-1 epitopes displayed on the surface of E2 scaffold could elicit antigen-specific mucosal immune responses, the V3 loop of HIV-1 Envelope gp120 glycoprotein was expressed as an N-terminal fusion to the catalytic core domain of E2 and purified according to previously described methodologies . The HIV-1 clade B primary isolate SF162 (Tier 1) V3 portion (46 aminoacids total) inserted into the E2 scaffold contained the epitope (G312PGR315) recognized by the human anti-V3 monoclonal neutralizing antibody (NmAb) 447-52D and the H-2d restricted CTL (cytotoxic T lymphocyte) epitope (I311GPGRAFYA319). The resulting vaccines were nonreplicative multimeric particles formed by HIV-1 antigens inserted on the surface of E260-mer scaffold protein, and we evaluated their immunogenicity as mucosal vaccines in mice. To this end, BALB/c mice of the H-2d haplotype (n = 5 per group) were intranasally immunized with Env(V3)-E2, administered with or without the adjuvant cholera toxin (CT). Vaccinations were conducted weekly for seven weeks. Fecal pellets were collected after each immunization.
Overall, this experiment demonstrates that intranasal administration of E2-based vaccines is able to induce anti-Env antigen-specific antibodies in fecal samples.
Analysis of systemic antibody and CD4 T cell responses
In order to examine the type of immune responses induced in vivo by E2 vaccination following the intranasal route, we analyzed the isotype ratios of serum IgG1/IgG2a and evaluated the induction of antigen-specific CD4+ T cells in vaccinated mice.
Analysis of V3-specific CD8 T-cell response
Analysis of transcriptome profile of E2-pulsed BMDCs
Specific immune responses are characterized by different patterns of cytokines produced by CD4+ T cells, an event known as polarization of helper T cells and there can be striking differences in the type of response preferentially stimulated by different carriers and/or adjuvants. In the context of a vaccine formulation, it is important to dissect the polarization induced by the E2 carrier in order to understand how to best deliver and eventually combine immunogens.
We previously described that subcutaneous administration of E2 particles expressing the Env(V3) hypervariable region elicited a sustained immune response by inducing both antibodies and antigen specific T cells . Here we report that mucosally administered Env(V3)-E2 were also able to elicit mucosal and systemic V3-specific humoral immune responses. Due to the limited sample volume obtained from mice we were not able to assess the neutralization activity of the antibodies induced by E2 immunization. However, it should be emphasized that preclinical studies of HIV-1 vaccine candidates have typically shown that neutralizing antibodies (NAbs) with a long heavy chain complementarity-determining region 3 (HCDR3) are found in non-human primate and rabbit animal models, with the latter being currently the favorite model for testing candidates at eliciting NAbs. We previously reported that our Env(V3)-E2 construct was able to induce autologous NAbs when administered subcutaneously in rabbits, with a modest level of neutralization of some Tier 1 viruses . For this reason we plan to assess in future work using larger animal models the induction of neutralizing antibodies by E2 mucosal immunization.
Here we also report that intranasal administration of Env(V3)-E2 particles elicits antigen-specific splenic CD8+ and CD4+ T cells. The IgG1/IgG2a ratio in the sera and the ability of Env-E2 specific CD4+ T cells to produce IL-4 suggest that a Th2-type of immune response is induced in mice also after mucosal immunization. Th2-type CD4 T-cell polarization has been reported to favor mucosal IgA responses . Indeed, we observed the presence of V3-specific IgA in feces and anti-V3 IgG in sera of vaccinated mice following E2 mucosal administration. Since the contribution of monomeric IgA, essentially transudated serum IgA, to total IgA in fecal samples has been previously found negligible in comparison to the mucosally secreted dimeric IgA (generally less than 0.1 %) , we can assume that the IgA in feces from vaccinated mice were mainly of the secretory type. However, we cannot exclude the presence of monomeric IgA. In contrast to previous results obtained by subcutaneous administration [5, 28], here, we observed the ability of antigen-specific CD4+ T cells to produce IFN-γ. These data indicate the presence of Th2 and Th1-polarized T cells following mucosal immunization in agreement with reports demonstrating a Th1 switch in mucosally primed mice . However, from our data, we cannot exclude that the production of these two cytokines may depend on the same cells.
Using an in vitro system, we measured the ability of E2 scaffold to induce gene expression changes in antigen presenting cells. We profiled the entire transcriptome of BMDCs stimulated with the E2 scaffold, showing a significant up-regulation of genes associated with Th2-polarizing DC capability . Notably, we observed a significant up-regulation of the gene encoding the Notch ligand Jagged 1 (encoded by Jag1). Its expression in DCs has been previously described to support Notch-mediated Th2 differentiation . In addition, we reported the up-regulation of Stat5 and Nfkb1, whose expression is required within DC in promoting Th2 immunity [38, 39], and of Irf4, known to regulate the transcription of key cytokines involved in the immune response . Irf4 expression in DC has been very recently described to play a role in the initiation of the Th2 cell response [41, 42]. Previous findings also revealed that Irf4 expression is significantly correlated with the expression of CD11b (encoded by Itgam), programmed death ligand-2 (encoded by Pdcd1lg2) and MHC class II major histocompatibility complex transactivator (encoded by Ciita) . In agreement with this study, we report here the up-regulation of these genes in E2-stimulated DCs.
The results obtained from the transcriptome analysis by RNA-Seq for E2-pulsed BMDCs confirm, and further strengthen, our previous observations and findings. Indeed, we have reported that subcutaneous vaccination of C57BL/6 mice with E2 polarizes the immune response towards a high IgG1/IgG2a isotype ratio and induces IL-4 producing CD4+ T cells, even in Th1-prone strains . We also observed this type of polarization when subcutaneous administration of E2 particles was performed in the absence or in presence of various types of adjuvants, such as the strong reactogenic CFA (Freund's Complete Adjuvant) or in formulation suitable for human use, as alum (Allhidrogel) or squalen-based oil in water emulsion (Addavax) . However, when we administered E2 with another carrier, namely the filamentous bacteriophage fd which we know to prompt Th1 polarization, in a priming-boosting strategy, we demonstrated a shift towards a mixed Th1/Th2 type of polarized response , indicating that it is also possible to modulate the Th2-type of the immune response induced by E2 administration.
E2 particles free of endotoxin, administered subcutaneously in the absence of added adjuvants, are also immunogenic, although the addition of adjuvants increases the magnitude of the response . However, in this case of mucosal administration we could not detect antigen-specific immune response in the absence of adjuvant administration. Indeed, RNA-Seq analysis indicates that endotoxin-free E2 particles are able to activate in DCs pathways of immune response as the chemokine and the Jak-STAT signaling pathways. Moreover, the up-regulated expression of Il12 and Il6 was found, while the expression levels of genes encoding other proinflammatory cytokines did not increase upon E2 stimulation. These levels of inflammatory response may explain why E2 particles free of endotoxin are immunogenic when administered subcutaneously in the absence of added adjuvants. However, it is not sufficient to prime the immune response in the absence of adjuvants in the case of mucosal delivery.
We previously have reported that Env(V3)-E2 particles were much more immunogenic when co-administered with other systems such DNA expression vectors, encoding Env proteins, delivered intradermally (via Gene gun) in rabbits . On the basis of these findings and of the known role played by Th2 cells in activating and enhancing B cell proliferation and humoral immune responses , in order to increase the mucosal immune response we plan, in future work, to test the mucosal delivery of E2 particles in combination with other strongly immunogenic delivery systems.
In the current preclinical study we showed that E2 scaffold is immunogenic when administered via mucosal route in the presence of adjuvant and thus may represent a relevant option for immunization against pathogens whose infections occur or initiate at a mucosal surface. Moreover, by using a genome-wide transcriptional approach, we attempted to dissect the mechanisms by which DCs may orchestrate the immune response following vaccination with the E2 scaffold.
APCs, antigen presenting cells; BMDC, bone marrow-derived dendritic cell; CFA, freund’s complete adjuvant; CT, cholera toxin; CTL, cytotoxic T lymphocytes; DCs, dendritic cells; E2wt, E2 wild-type; Env, envelope; EU, endotoxin unit; FACS, fluorescent activated cell sorter; FCS, fetal calf serum; FDR, false discovery rate; FITC, fluorescein isothiocyanate; FPKM, fragments per kilobase of transcript and million mapped reads; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICS, intracellular cytokine staining; IFN, interferon; Ig, immunoglobulin; IL, interleukin; IN, intranasal administration; LAL, Limulus Amebocyte Lisate; LPS, lipopolysaccharide; MHC, major histocompatibility complex; NAbs, neutralizing antibodies; OD, optical density; PBS, phosphate buffer saline; PDH, pyruvate dehydrogenase; PE, phycoerythrin; PRRs, pattern recognition receptors; RNA-Seq, RNA-Sequencing; Th, T helper
We would like to thank the IGB-IBP FACS Facility, at CNR, Naples; the Animal House Facility at CNR, Avellino; Dr. Marianna Aprile and Dr. Margherita Scarpato (from IGB “A. Buzzati-Traverso”, CNR, Naples) for the construction of the libraries for the RNA-Seq experiments; the NGS Facility of TIGEM for the Illumina platform.
This work was supported by NIH (US) PHS grants R01-AI074379, MIUR-PON 01_00117, “Progetto Invecchiamento” C.N.R., and Regione Campania PO FESR 2007/2013. The funding sources had no role in study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, and decision of publication.
Availability of data and materials
The datasets supporting the conclusions of this article are available in the G.E.O. (Gene Expression Omnibus) repository (identifier: GSE60231; http://www.ncbi.nlm.nih.gov/geo/).
The authors declare that they have no competing interests.
MT designed and performed experiments, analyzed data, and wrote the manuscript. FM performed mucosal vaccinations and antibody assays. LDA, RS and FC performed cellular assays and critically reviewed the manuscript. VC performed the RNA-Seq and the bioinformatic analysis. AP, SPM, SJK, and NLH contributed to study design and interpretation of data, and critically reviewed the manuscript. AC supervised bioinformatics analysis. MR supervised mucosal experiments and helped to draft the manuscript. PDB supervised the project and wrote the manuscript. All authors read and approved the final manuscript.
Consent for publication
Ethics approval and consent to participate
All experiments with mice were performed in accordance with European Union Laws and guidelines. All animal studies were approved by the national institutional review committee of Health Ministry, General Division of Animal Health and of Veterinary Medicine, and performed according with European regulations (EU Directive 2010/63/EU). Animals were housed under specific pathogen free conditions at the Animal Facility of the Institute of Food Science of C.N.R., Avellino, Italy (accreditation no. DM.161/99).
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