...nanoparticle vaccine against Zika virus elicits antibody...

来自 : 发布时间：2024-05-06

An RNA nanoparticle vaccine against Zika virus elicits antibody and CD8+ T cell responses in a mouse model AbstractThe Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV鈥檚 neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2Db-restricted epitope to which there was a CD8+ T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus. IntroductionThe ongoing outbreak of Zika virus (ZIKV), a flavivirus, in Latin America and the South Pacific is associated with an increased incidence of neurological complications, including Guillain-Barr茅 syndrome1, 2 and fetal abnormalities, including spontaneous abortion, microencephaly, and placental insufficiency3, 4. At present there are no approved vaccines or specific treatments for ZIKV infection. In February 2016, the World Health Organization declared the Zika outbreak a Public Health Emergency of International Concern. Hundreds of cases have already been reported in the United States, attributed to acquisition by travel to affected areas. Local transmission is now prevalent in US Territories, particularly in Puerto Rico5. Mosquito-borne and sexual transmission on mainland US soil led to unprecedented warnings by the CDC for pregnant women and their sexual partners6, 7. The expanding habitat of the Aedes mosquito species that serves as a vector for ZIKV could spread this epidemic even further. Vaccine development efforts to date have yielded DNA-based candidates, one of which has entered a clinical trial8, 9. However, deployment of DNA-based immunoprophylactics requires electroporation or jet-injection systems9, 10. This makes administration of the lead vaccine candidate a challenge in most of the seriously affected regions. Therefore, developing new analytical tools that can accelerate further vaccine candidate research is of the utmost importance.Zika viruses are broadly classified into Asian, East African, and West African lineages11. The current outbreak has been attributed to the Asian genotype12,13,14. The causative Western Hemisphere strains share a high degree of nucleotide identity within the clade ( 99%). The long-studied prototypical ZIKV reference strain MR 766 was first isolated in Uganda in 1947, and is a member of the East African cluster. It is less homologous (~89%) to the Western Hemisphere strains. The frequent cross-reactivity among Flavivirus species complicates serological detection of ZIKV infection or virus-specific antibodies, making studies of ZIKV-specific humoral immunity challenging15,16,17,18,19. Susceptible adult animal models of ZIKV pathogenesis that recapitulate neurotropic disease exploit IFN receptor gene knockouts/blockade in C57BL/6 and other mouse strains carrying the MHC H-2b haplotype20,21,22. Other IFN-deficient mouse strains are similarly susceptible to ZIKV infection21.The geographic co-distribution and co-circulation of many serologically similar classes of arboviruses23 pose a hurdle to the characterization of ZIKV-specific immune responses. Commercially available enzyme-linked immunosorbent assay (ELISA) kits that detect anti-flaviviral antibodies suffer from cross-reactivity to other Flavivirus strains. This confounds the study of multivalent flaviviral vaccines.In the C57BL/6 model of ZIKV infection, CD4+ T cell depletion does not abrogate protective efficacy of gene-based vaccines24. Therefore, cytotoxic CD8+ T cell responses likely play a role in protection against ZIKV infection, as for other flaviviruses25,26,27. MHC-restricted ZIKV epitopes remain to be defined. Even a single-peptide MHC class I determinant would aid current vaccine development efforts, as it would provide a parameter to screen vaccine performance in mice.We previously developed a modified dendrimer nanoparticle (MDNP)-based RNA replicon vaccine platform that provides single-dose protection in mouse models of lethal Influenza, Ebola, and Toxoplasma gondii challenges28, and in the current study applied it to ZIKV. The vaccine induced detectable anti-ZIKV IgG responses in C57BL/6 mice. Analyses of the cellular response to the vaccine revealed an immunodominant H-2Db-restricted epitope derived from the ZIKV envelope (E) protein. By means of T cell stimulation assays we could unambiguously distinguish between unvaccinated and vaccinated animals.ResultsGeneration of the ZIKV RNA nanoparticle vaccineThe premembrane (prM) and envelope (E) proteins of ZIKV isolate Z1106033 (derived from an Asian lineage virus, isolated from a patient in Suriname at the onset of the late-2015 expansion of the virus in the Americas)29 were encoded as a single open reading frame into an RNA replicon vector (Fig.聽1a). RNA was transcribed from the plasmid in vitro, and expression of the correct post-translationally cleaved envelope glycoprotein was confirmed by immunoblot in transfected hamster kidney cells (BHK21) after 3 days using a polyclonal antiserum against ZIKV E protein. Expression of the expected ~54鈥塳Da protein product was observed (Fig.聽1b). Additional bands (indicated by asterisks) were detected that may correspond to incompletely processed ZIKV E protein. The full-length prM-E polyprotein segment encoded in the replicon has a predicted molecular weight of 73.4鈥塳Da after signal peptidase processing to release the prM N-terminus. Incomplete signal peptidase cleavage of the M-E junction would yield a 62鈥塳Da intermediate polypeptide. As alphaviral replicons saturate and substantially disrupt the secretory pathway in BHK21 cells over time, retention of polyprotein processing intermediates in transfected cells is not surprising30, 31. Regardless, only the correctly processed ~54鈥塳Da polypeptide was observed to accumulate in the culture medium conditioned by the transfected cells, indicating that antigen release took place (Fig.聽1c), as expected: expression of the prM and E proteins of flaviviruses results in the release of sub-viral particles (SVPs) of slightly smaller size than capsid-containing virons32. As SVPs are antigenically similar to functional virions, their production underlies the performance of nucleic acid-based vaccines. The RNA replicon was formulated with a modified dendrimer nanomaterial to form MDNP vaccines, as described28 (Figure聽S1). C57BL/6 mice (n鈥?鈥?) were immunized with this formulation by intramuscular (i.m.) injection. A control group was immunized with a similar RNA replicon vaccine encoding the Zaire Ebola virus glycoprotein (ZEBOV). After immunization, serum was collected and IgG reactivity against a recombinant ZIKV envelope protein was determined by ELISA (Fig.聽1d). All immunized mice exhibited IgG reactivity against the ZIKV envelope protein, while only two of the control mice were seropositive above the detection limit of the assay. These two seropositives may have been the result of nonspecific cross-reactivity due to structural similarities between flavivirus and filovirus envelope proteins33,34,35,36. Nevertheless, the subsequently identified Class I MHC-restricted epitope distinguished between the two immunized groups unambiguously in a cytokine release assay (described below).Figure 1RNA nanoparticle vaccine design and function. (a) ZIKV polypeptide region encoded in the RNA replicon as the target antigen (numbers refer to amino acid positions), and predicted topology of the expressed truncated protein segment. (b) Immunoblot performed on lysates of replicon-transfected BHK21 cells 72鈥塰 post-transfection with an anti-ZIKV E rabbit polyclonal antibody. Asterisks indicate additional high-molecular weight bands possibly corresponding to incompletely processed E protein (*) or a combination of incompletely processed E protein and background band also present in untransfected control cells (**). (c) Immunoblot performed as in (b) but on conditioned supernatant removed from transfected cells. The asterisk indicates a common background band presumably produced by cross-reactivity of the polyclonal antibody used for detection against a culture medium serum component. The membrane was cropped above the 75鈥塳Da marker to eliminate contaminating bovine serum albumin (from the culture medium) bands from the blot. (d) Anti-ZIKV recombinant E protein IgG titers from C57BL/6 mice immunized with the indicated RNA nanoparticle vaccine.Full size image Peptide screening for CD8+ envelope gene-derived epitopesAn overlapping 15-mer peptide library (Table聽S1) spanning amino acids 105 to 713 of the ZIKV polyprotein (Fig.聽1a) was generated, and individual pools of 5鈥? peptides each were used in ex vivo stimulation experiments on splenocytes isolated from an IgG-positive ZIKV-immunized mouse. Four pools (pools #5, 6, 7, 11) were identified that induced interferon gamma (IFN纬) expression in CD8+ T cells (Table聽1). Peptides from each of these pools were then tested individually, and 7 peptides were found to induce a response (defined as IFN纬 expression in 0.2% of CD8+ T cells, as nearly all other peptides stimulated 0.1%; Table聽1). Three stimulatory contiguous peptides from pool #6 covered a 23 amino acid span corresponding to positions 284 to 306 of the ZIKV polypeptide. Two of these three contained a distinctive H-2Db-compatible 9-mer sequence, based on the presence of an asparagine anchor at position 5 and a hydrophobic C terminus (valine) at position 9. H-2Db- and H-2Kb-binding epitopes from all 7 individual 鈥榟its鈥?were computationally evaluated based on a prediction of the half maximal inhibitory concentration (IC50) using the artificial neural network (ANN) algorithm from the Immune Epitope Database Analysis Resource37, 38. The H-2Db-restricted peptide IGVSNRDFV was calculated to have an IC50 of approximately one order of magnitude lower than any other predicted sequence. The next best ANN-predicted epitope-containing peptide (IAPAYSIRCIGV), assigned to H-2Kb by the algorithm, was also selected for comparison in further experiments. The candidate peptides were prepared by solid-phase peptide synthesis (SPPS) on a flow-based peptide synthesizer39 using 2-chlorotrityl chloride resin and Fmoc-protected building blocks to yield peptides with free termini. In an in vitro Class I MHC peptide binding experiment utilizing RMA-S cells40, IGVSNRDFV significantly stabilized surface H-2Db molecules to the same degree as the well-known H-2Db-restricted human papillomavirus type 16 (HPV16) E749鈥?7 epitope41, which served as a positive control for H-2Db binding (Fig.聽2). The 12-mer peptide predicted to contain H-2Kb epitopes indeed exhibited H-2Kb stabilization, as did two 9-mer and one 8-mer derivatives thereof, though not to the same degree as the well-established H-2Kb ovalbumin SIINFEKL peptide42. Interestingly, an intermediate degree of H-2Db stabilization was also observed with these peptides.Table 1 15-mer Peptide candidates tested individually for CD8+ T cell cytokine release.Full size table Figure 2RMA-S cell peptide-binding assay. The binding of the indicated peptides to MHC class I molecules was determined by surface stabilization of H-2Kb or H-2Db molecules on RMA-S cells as measured by flow cytometry. Assays were performed in triplicate. The 鈥溾垝鈥?denotes that no peptide was added, and the 鈥?鈥?denotes the addition of a peptide known to bind to the measured MHC class I molecule (OVA SIINFEKL for H-2Kb, and HPV16 E7 RAHYNIVTF for H-2Db). Fluorescence intensities were normalized to the no peptide control and data are shown as the means鈥壜扁€塖D. ns, not statistically significant from the no-peptide control value according to an unpaired two-tailed t-test (p鈥?gt;鈥?.05).Full size image The H-2Db-restricted ZIKV envelope-derived peptide IGVSNRDFV stimulates ex vivo IFN纬 production in CD8+ T cells from ZIKV-immunized H-2b miceSplenocytes were cultured alone or in the presence of 0.2鈥壩糶/mL of the candidate ZIKV-derived peptides or the unrelated OVA-derived H-2Kb-restricted peptide SIINFEKL42. Only in ZIKV-immunized mice did we detect CD8+ T (~1.2-% of total) cells positive for intracellular IFN纬-staining when incubated with the candidate H-2Db-restricted peptide IGVSNRDFV (Fig.聽3). Incubation with the putative H-2Kb-restricted peptide IAPAYSIRCIGV resulted in no activation. This peptide is substantially longer than typical H2Kb epitopes; therefore, to evaluate the possibility that it failed to be correctly processed to the appropriate length in vitro, the additional 8-mers (IAPAYSIR and YSIRCIGV) and 9-mers (IAPAYSIRC and AYSIRCIGV) derived from this peptide tested in RMA-S binding assays were also tested separately. An additional two peptides derived from the screen 鈥榟its鈥?were selected based on the presence of anchor residues for H-2Db and H-2Kb. All peptides tested are summarized in Table聽2. No peptide other than IGVSNRDFV induced a detectible CD8+ T cell response (Figure聽S2).Figure 3Candidate ZIKV E H-2Db-binding peptide stimulates CD8+ T cells from ZIKV-immunized mice. Splenocytes from mice vaccinated with the indicated RNA nanoparticle vaccine were stimulated ex vivo with 0.2鈥壩糶/mL of the indicated peptide. After 7鈥塰, intracellular cytokine staining for IFN纬 was performed and measured by flow cytometry. Error bars represent SD, and N鈥?鈥? animals per group.Full size image Table 2 Individual peptides tested for CD8+ T cell cytokine release.Full size table DiscussionGiven the threat of the ZIKV outbreak and likelihood of continuing transmission worldwide, accelerated vaccine development is critical. We used a fully synthetic MDNP-based RNA replicon vaccine platform28 as a tool to generate a ZIKV vaccine candidate in the complete absence of reference virus stocks. We identified a Class I MHC-restricted 9-mer peptide to which a CD8+ T cell response was induced in immunized C57BL/6 mice, using an ex vivo stimulation assay. The identified IGVSNRDFV sequence is conserved across all clades of ZIKV. Our work facilitates the assessment of immune responses against the dominant ZIKV antigen without the need for recombinant production of the native glycoprotein.This work uncovered only a single Class I MHC class I-restricted epitope from the ZIKV E protein to which a CD8+ T cell response was elicited in immunized mice. Given the size of the ZIKV genome and the number and size of proteins it encodes, this is somewhat surprising, and our method may have missed other, perhaps minor epitopes. However, the CD8 T cell response in C57BL/6 mice against Sendai virus provides an example of a single dominant epitope (FAPGNYPAL) being responsible for a protective CD8+ T cell response, despite the presence of other predicted and experimentally verified Class I MHC binders43,44,45. Mutations in H-2Kb that abrogate FAPGNYPAL binding make Sendai virus more pathogenic46, 47. In addition, a single mutation in the epitope sequence rendered the virus lethal at a dose readily handled for wild type Sendai virus by C57BL/6 mice. In this regard, it is worth pointing out that FAPGNYPAL is an excellent H-2Db binder, owing to the asparagine residue at position 545, but apparently no CD8+ T cells are induced against it in the anti-Sendai response.A DNA ZIKV vaccine candidate is currently undergoing clinical testing9, but the self-limiting nature of an RNA-based vaccine possesses obvious safety advantages over DNA-based approaches, chiefly owing to a lack of potential genome integration. In the case of replicon RNAs, there is also a potential potency advantage, as self-replication in the cytoplasm can drive greater transgene expression than DNA vectors. Given the observation here that a fully synthetic MDNP-based RNA replicon nanoparticle preparation induced humoral and cellular immune responses that have been correlated with protective immunity elsewhere24, it will be interesting to pursue further characterization of this vaccine candidate in terms of protective potential in vivo in comparison to other nucleic acid and conventional protein/virus-like particle approaches. Notably, the generation of this MDNP vaccine candidate, and the elucidation of an immunodominant Class I MHC-restricted epitope, were performed without the need for ZIKV itself. This fully synthetic and chemically defined process required no more than access to the ZIKV coding sequence.MethodsRNA nanoparticle vaccine productionCloning and RNA synthesis was performed as previously described28. Briefly, Venezuelan equine encephalitis virus (VEEV) replicon RNAs were produced by cloning antigens into the VEEV replicon plasmid pTK126, based on the wild-type TRD strain (kindly provided by Tasuku Kitada, Weiss Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA) to replace the mVenus coding sequence located downstream of the VEEV subgenomic promoter sequence. Replicon RNAs were synthesized from these vectors after linearization with the restriction enzyme I-SceI. I-SceI cuts downstream of the VEEV 3鈥?untranslated region (UTR) and a short poly(A) tract of 40 base pairs (bp), and upstream of a T7 RNA polymerase promoter element preceding the VEEV 5鈥?UTR. RNAs were synthesized from the linearized plasmid vectors by in vitro transcription with MEGAscript kits (Life Technologies, Carlsbad, CA, USA), 5鈥?capped to produce cap-1 structured 2鈥?O-methylated 7-methylguanylate 5鈥?ends using the ScriptCap m7G Capping System and 2鈥?O-methyltransferase kits (CellScript Inc., Madison, WI, USA), and 3鈥?poly(A)-tailed using A-Plus Poly(A) Polymerase Tailing kits (CellScript) according to manufacturer protocols. MDNP formulation was performed as we described previously28. Nanoparticles were characterized with a Zetasizer Nano-ZS machine (Malvern, UK). The concentration of RNA was determined by theoretical mass balance calculations and confirmed by spectrophotometry (NanoDrop, Thermo Fisher Scientific, Waltham, MA, USA).ZIKV antigen detection by immunoblotAntigen expression was assayed in transfected baby hamster kidney cells (BHK21) maintained at 37鈥壜癈 and 5% (v/v) CO2 in Eagle鈥檚 minimal essential medium supplemented with 5% (v/v) FBS and 2鈥塵M sodium pyruvate (Invitrogen, Carlsbad, CA, USA). Cells growing in log phase were transfected with the indicated RNA at 50鈥?5% confluence using TransIT-mRNA transfection kits (Mirus Bio, Madison, WI, USA). After 72鈥塰, the cells were lysed, and proteins were extracted in CelLytic鈩?M Mammalian Cell Lysis/Extraction Reagent (Sigma-Aldrich, St. Louis, Missouri) supplemented freshly with 25鈥塙/渭L benzonase (EMD Millipore, Billerica, MA, USA), and cOmplete, Mini EDTA-free Protease Inhibitors (Roche Life Science, Mannheim, Germany) according to the manufacturer鈥檚 recommendations. Subsequently, lysates were mixed with Laemmli SDS buffer (Boston Bioproducts Inc., Ashland, MA, USA) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4鈥?2% gradient Bolt庐 Bis-Tris gels (Thermo Fisher, Waltham, MA, USA) before transfer to PVDF membranes for immunoblotting. Membranes were blocked with 10% milk in Tris-buffered saline with 0.1% Tween-20 (TBST), incubated with primary detection antibodies in blocking buffer (10% milk in TBST) for 1鈥塰 at room temperature, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies in blocking buffer for 1鈥塰 at room temperature. For ZIKV envelope antigen detection, membranes were incubated with a rabbit polyclonal anti-Zika primary antibody (product number GTX133314, GeneTex, Inc., Irvine, CA, USA) diluted 1:1000, followed by incubation with an anti-mouse HRP-conjugated secondary antibody diluted 1:10,000 (GE Healthcare, Pittsburgh, PA, USA). Enhanced luminol-based detection was performed using Western Lightning-ECL kits (Perkin-Elmer, Boston, MA, USA). For detection of secreted antigen, cells were transfected exactly as described, but medium was switched to reduced serum (1% FBS) 16 hrs post-transfection, and the conditioned culture medium was harvested 72鈥塰rs post-transfection. The conditioned medium was clarified by centrifugation to remove cellular debris, mixed 1:1 with Laemmli SDS buffer, and immunoblot was performed as described above for lysates except that the membrane was cropped at the 75鈥塳Da marker band to remove large contaminating BSA bands from the culture medium that were visible upon Ponceau S staining. Raw images of the immunoblot films without cropping or annotation are shown in Figure聽S3.Peptide synthesisA) Overlapping peptide pool screeningAn overlapping peptide library consisting of 15-mers with 5 amino acid overlaps was synthesized by Mimotopes Pty Ltd (Clayton, Australia), and individual lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) at a concentration of ~9鈥塵g/mL. These peptides were screened in ex vivo splenocyte stimulation assays as described below.B) Large scale synthesis and validation of target epitope sequences2-Chlorotrityl chloride resin (125鈥塵g, 1.6鈥塵mol/g, Chemprep, Miami, FL, USA) was swollen in dichloromethane (10鈥塵L) followed by the addition of the first amino acid (0.6鈥塵mol) and N,N-diisopropylethylamine (DIPEA; 500鈥壩糒). After agitation at room temperature for 1鈥塰, the solution was drained and the resin was washed three times with dimethylformamide (DMF), and capped with a mixture of dichloromethane (DCM)/MeOH/DIPEA (80:15:5 v/v/v). After 30鈥塵in, the resin was washed and transferred to the flow reaction vessel. Flow-based peptide synthesis was then performed following an established protocol39. Briefly, each coupling cycle involved four steps: washing (60鈥塻), deprotection (45鈥塻), washing (90鈥塻), and coupling. All reactions were conducted at 60鈥壜癈. Except for coupling, all cycles were conducted at a flow rate of 20鈥塵L/min. For the coupling step, 2鈥塵mol (10 eq.). Fmoc-protected amino acids were dissolved in 5鈥塵L of 0.4鈥塎 2-(1H-benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate (HBTU) solution in DMF, followed by the addition of 500鈥壩糒 DIPEA. The mixture was injected by a syringe pump at 60鈥塵L/h. Deprotection was conducted by switching the flow from DMF to 20% piperidine in DMF. The finished sequence was cleaved and deprotected using TFA/TIPS/water/phenol/EDT (90:2.5:2.5:2.5:2.5, v/v/v/v/v), precipitated in cold ether, and purified by high-performance liquid chromatography (HPLC; Gemini C18 column, 5鈥壩糾, 10鈥壝椻€?50鈥塵m; Phenomenex, Torrance, CA, USA). The corresponding fractions were analyzed via liquid chromatography鈥搈ass spectrometry (LC-MS; Waters Xevo system equipped with UPLC-C18 column, Manchester, UK; Figure聽S4), and the following masses were observed: IGVSNRDFV [M鈥?鈥塇]+ calculated鈥?鈥?006.523, observed鈥?鈥?006.515; IAPAYSIRCIGV [M鈥?鈥塇]+ calculated鈥?鈥?262.693, observed鈥?鈥?262.679.Mice and immunizationAll animal studies were performed at the Whitehead Institute for Biomedical Research, Cambridge, MA, and were in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) to comply with all applicable local, state, and federal regulations. Female C57BL/6 mice were primed on day 0 and boosted 5 weeks later with 40鈥壜礸 doses (based on RNA mass) of the nanoparticle vaccine by bilateral i.m. injection as previously described28. Blood was collected 4 weeks after the boost, and splenocytes were isolated and cryogenically stored in fetal bovine serum (FBS) with 10% DMSO before analysis.Serum ELISAHigh binding surface-treated polystyrene 96-well microplates (Corning, Corning, NY, USA) were coated overnight at 4鈥壜癈 with 0.5鈥壜礸/mL recombinant Zika E protein (MyBioSource Inc., San Diego, CA, USA) in 100鈥塵M carbonate/bicarbonate buffer (pH 9). Plates were blocked for 2鈥塰ours with blocking buffer (PBS with 10% FBS, Life Technologies) at room temperature, and serum was applied to wells in duplicate at a minimum 1:100 dilution in blocking buffer, and incubated for 2鈥塰ours at room temperature. Plates were washed with wash buffer (PBS with 0.05% Tween-20), and incubated at room temperature with anti-mouse IgG-HRP (GE Healthcare) diluted 1:3000 in blocking buffer for 1鈥塰. After 4 washes with wash buffer, the plates were developed with tetramethylbenzidine (TMB) substrate (Sigma, St. Louis, MO, USA) for 20鈥?0鈥塵in, and the reaction was stopped by the addition of one volume of 1鈥塎 HCl before measuring absorbance at 450鈥塶m. Endpoint titers were determined using an optical density (OD) 450鈥塶m cutoff of 0.08. Ex vivo splenocyte stimulation assayThe peptides were commercially provided in a 96-well format, and were pooled in equivalent volumes by combining individual columns for ex vivo splenocyte stimulation assays, which resulted in a final pooled concentration of ~1鈥壩糶/mL in each culture. After the identification of four pools that stimulated enhanced IFN纬 expression in CD8+ T cells, individual peptides were tested in identical stimulation assays at ~4鈥壩糶/mL. A total of 107 splenocytes in 100鈥壩糒 of complete media (RPMI 1620 supplemented with GlutaMAX, 8% FBS, 1鈥塵M nonessential amino acids, 1鈥塵M sodium pyruvate, 10鈥塵M HEPES, penicillin/streptomycin, Life Technologies; and 50鈥壩糓 2-mercaptoethanol, Sigma) were added to each well of a 96-well flat-bottom cell culture plate. After a 1-h pre-incubation, the cells were incubated with IL-2 (10鈥塙/mL), anti-CD28, anti-CD49d (0.5鈥壩糶/mL each, BioLegend), and GolgiStop (BD Biosciences) diluted 1:1500. For evaluation of the synthetic H-2Db and H-2Kb candidate peptides and the SIINFEKL control peptide, IL-2 and anti-CD28/CD49d were omitted, as costimulatory signals proved unnecessary for IFN纬 induction. Intracellular cytokine staining for IFN纬+CD8+ T cells was performed as described previously28, and populations were analyzed on a BD LSR II Flow Cytometer (BD Biosciences). In the screening, 鈥榟its鈥?were defined if the percentage of IFN纬+CD8+ T cells was 0.2% of total CD8+ T cells (background levels in control cultures were consistently 0.2%). A minimum of 10000 CD8+ T cells were analyzed per sample.Peptide-binding assay with RMA-S cellsMHC class I molecules (H-2Kb and H-2Db) lacking peptides are expressed on the surface of RMA-S cells upon culture at reduced temperature40. These cell surface empty class I molecules are thermolabile at 37鈥壜癈, but are stabilized by the addition of MHC binding peptides. RMA-S cells were cultured at 26鈥壜癈 for 18鈥塰 and washed with PBS. Then, 105 cells/well were seeded onto a 96-well cell culture plate. ZIKV or control peptides (OVA257鈥?64 for H-2Kb and HPV16 E749鈥?7 for H-2Db) were added to each well at 30鈥壩糶/106 cells. The cells were then incubated at 37鈥壜癈 for 2鈥塰 and washed twice with ice cold PBS. For H-2Kb detection, the cells were stained with a mouse FITC-conjugated anti-H-2Kb antibody (1:200 dilution in PBS with 1% BSA, Biolegend) for 45鈥塵in at 4鈥壜癈. For H-2Db detection, the cells were stained with a mouse anti-H-2Db antibody (1:500 dilution in PBS with 1% BSA, BD Biosciences) for 45鈥塵in, followed by incubation with a goat anti-mouse Alexa Fluor 488-conjugated secondary antibody (1:10000 dilution in PBS with 1% BSA, Thermo Fisher) for 30鈥塵in at 4鈥壜癈. The cells were then washed three times with ice cold PBS, and fluorescence intensity was measured with a BD Accuri C6 Flow Cytometer (BD Biosciences) and normalized to the no peptide control. References1.Cao-Lormeau, V.-M. et al. Guillain-Barr茅 Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. The Lancet 387, 1531鈥?539, doi:10.1016/S0140-6736(16)00562-6 (2016).2.Oehler, E. et al. 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Immunity 14, 231鈥?42, doi:S1074-7613(01)00105-4 (2001).Download referencesAcknowledgementsFunding was provided by the National Institutes of Health (grant R01 AI087879), a Fujifilm/MediVector, Inc. grant, and a grant from the Lustgarten Foundation to H.L.P., and in part by an award from the Bridge Project, a partnership between the Koch Institute for Integrative Cancer Research at MIT and the Dana-Farber/Harvard Cancer Center (DF/HCC) to D.G.A., as well as the Department of Defense Office of Congressionally Directed Medical Research鈥檚 Joint Warfighter Medical Research Program to D.G.A. This work was supported in part by Cancer Center Support (core) Grant P30-CA14051 from the National Cancer Institute. A.W.W. is an Arnold O. Beckman Postdoctoral Fellow and received additional support from an NIH F32 grant (GRANT11974346). J.L. was supported by an ASTAR NSS (PhD) scholarship. We thank the Whitehead Institute flow cytometry facility for instrument usage.Author informationAuthor notesHidde L. PloeghPresent address: Program in Cellular and Molecular Medicine, Boston Children鈥檚 Hospital, Boston, MA, 02115, USAAffiliationsWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USAJasdave S. Chahal,聽Tao Fang,聽Andrew W. Woodham,聽Jingjing Ling聽 聽Hidde L. PloeghThe Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02142, USAJasdave S. Chahal,聽Omar F. Khan聽 聽Daniel G. AndersonAuthorsJasdave S. ChahalView author publicationsYou can also search for this author in PubMed聽Google ScholarTao FangView author publicationsYou can also search for this author in PubMed聽Google ScholarAndrew W. WoodhamView author publicationsYou can also search for this author in PubMed聽Google ScholarOmar F. KhanView author publicationsYou can also search for this author in PubMed聽Google ScholarJingjing LingView author publicationsYou can also search for this author in PubMed聽Google ScholarDaniel G. AndersonView author publicationsYou can also search for this author in PubMed聽Google ScholarHidde L. PloeghView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsJ.S.C., T.F., A.W.W., O.F.K. and H.L.P. designed experiments; J.S.C., T.F., A.W.W., and O.F.K. performed experiments; J.S.C., T.F., A.W.W., O.F.K., and J.L. contributed reagents/analytic tools; J.S.C., T.F., A.W.W., and H.L.P. analyzed data; and J.S.C., T.F., A.W.W., D.G.A., and H.L.P. wrote the paper.Corresponding authorCorrespondence to Hidde L. Ploegh.Ethics declarations Competing Interests O.F.K., J.S.C., H.L.P., and D.G.A. have filed a patent on the MDNP-based vaccine platform. 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Sci Rep 7, 252 (2017). https://doi.org/10.1038/s41598-017-00193-wDownload citationReceived: 27 October 2016Accepted: 13 February 2017Published: 21 March 2017DOI: https://doi.org/10.1038/s41598-017-00193-w Ramalingam Karthik Raja, Phuong Nguyen-Tri, Govindasamy Balasubramani, Arun Alagarsamy, Selcuk Hazir, Safa Ladhari, Alireza Saidi, Arivalagan Pugazhendhi Arulandhu Anthoni Samy Applied Nanoscience (2021) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.