key: cord-0943182-98nv62rd
authors: Sankaradoss, Arun; Jagtap, Suraj; Nazir, Junaid; Moula, Shefta-E; Modak, Ayan; Fialho, Joshuah; Iyer, Meenakshi; Shastri, Jayanthi S.; Dias, Mary; Gadepalli, Ravisekhar; Aggarwal, Alisha; Vedpathak, Manoj; Agrawal, Sachee; Pandit, Awadhesh; Nisheetha, Amul; Kumar, Anuj; Bordoloi, Mahasweta; Shafi, Mohamed; Shelar, Bhagyashree; Balachandra, Swathi S.; Damodar, Tina; Masika, Moses Muia; Mwaura, Patrick; Anzala, Omu; Muthumani, Kar; Sowdhamini, Ramanathan; Medigeshi, Guruprasad R.; Roy, Rahul; Pattabiraman, Chitra; Krishna, Sudhir; Sreekumar, Easwaran
title: Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences
date: 2022-01-07
journal: Mol Ther
DOI: 10.1016/j.ymthe.2022.01.013
sha: ecf3a37461bbc6786f269e9fd417936260714bdc
doc_id: 943182
cord_uid: 98nv62rd

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a DENV-2 NS1 protein coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying of intracellular IFN-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios and immune gene profiling suggest a strong Th1-dominant immune response. Finally, passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design; offering a novel vaccine candidate with a possible broad spectrum protection and successful clinical translation either as a stand-alone or in a mix and match strategy.

DENV2 lineages within the cosmopolitan genotype are propagating simultaneously in all four sites in this study. 151 Both the lineages emerged in the middle of the 1980s. Most of the neighboring sequences are from Asian 152 countries. However, one cluster in the cosmopolitan-b lineage is found in Kenya ( Figure S1 ) and one sequence 153 of a traveler from Ethiopia and Djibouti. Our findings are also consistent with a previous report from Kenya 36 . 154 All the Indian sequences of DENV3 genotype III cluster with other Asian countries. Only genotype I of DENV4 155 is observed in India. All Indian DENV4-I sequences along with one sequence from Pakistan cluster separately 156 within genotype I from other Southeast Asian sequences. This data highlights that even though there is an 157 enormous intra-serotype variation recorded all over the world, India has distinct genotypes for all four DENV 158 serotypes with prominent intermixing between neighboring countries. Based on the implications of our findings 159 on region-specific genotypes, we proposed a vaccine design approach against circulating strains, which may be a 160 better representation rather than strains not found here. 161 EDIII and NS1 global and local genetic diversity and target for vaccine design 162 The E protein, primarily DIII domain of the E protein (EDIII), stimulates host immune responses by evoking 163 protective and neutralizing antibodies 20 . Mutations in the EDIII region could potentially impact the neutralization 164 of DENV and the host-receptor interaction. In addition to structural proteins, immune response to DENV 165 infections target non-structural protein NS1, which is currently used as diagnostic candidate for dengue 166 infection. We investigated EDIII and NS1 protein diversity in global strains as well as how our study sequences 167 are diverging from reference strains. To assess antigenic differences between the EDIII and NS1 of DENV1-4 168 strains from distinct parts of the world, we generated a maximum likelihood tree and selected representative 169 genotype variants from each branch to comprehensively represent global diversity within the serotype. We 170 performed a sequence alignment of these proteins and compared their EDIII and NS1 genetic diversity relative to 171 the respective reference strains. Variable sites were designated when at least one virus showed an amino acid 172 change at any of the amino acid positions in the alignment. While DENV genotypes are closely related, 8 Further, EDIII and NS1 amino acid sequences of DENV1-4 clinical isolates from our study were compared 176 for their similarity with the wild-type DENV strains. Even though India has its unique genotypes, considerable 177 genetic variations were found in the EDIII and NS1 protein across genotypes of the same serotype. Our diversity 178 analysis revealed 9, 7, 2, and 8 sites of EDIII variation ( Figure 2A) and 24, 24, 8, and 17 variable sites within 179 NS1 ( Figure S2 ) in DENV1, DENV2, DENV3, and DENV4 among Indian genotypes, respectively. Among all 180 four strains, DENV1 exhibited the highest EDIII diversity with a median of 4.85% (range, 2.91%-5.83%), 181 followed by DENV4 and DENV2 with 2.91% (range, 2.91%-3.88%) and 1.94% (range, 0.97%-3.88%), 182 respectively. In the case of NS1, the highest median diversity was 3.69%; observed in DENV1 (range, 1.99%-183 4.55%) and DENV4 (range, 3.12%-4.26%), followed by that of DENV2 with 2.27% (range, 1.99%-2.84%). The 184 percentage diversity of DENV3 EDIII sequences was found to be limited, ranging from 0% to 0.97%. This was 185 also observed for DENV3 NS1 with a median value of 1.42 (range, 1.14%-1.7%). A site was considered highly 186 variable when greater than 50% of the study isolates showed a mutation at that position. The number of highly 187 variable sites in EDIII and NS1 were 5, 3, 2, and 1 and 13, 13, 8 and 5 in DENV1, DENV4, DENV2, and DENV3 188 strains, respectively. 189 Ig-G like fold present in the EDIII protein is typically associated with structures that have an adhesion function 8 190 hence we investigated the effect of EDIII mutations on EDIII protein stability using FoldX. The predicted ΔΔG 191 values for DENV1-DENV4 mutations ranged from -1.4 to 2.6 kcal/mol (Figure 2 B, D, F & H) . Except for 192 DENV2 EDIII-I28V and DENV4 EDIII-A37T, all other EDIII non-synonymous mutations found in our clinical 193 isolates have a minor or no effect on protein stability (Table S3) . We also observed mutations in residue within 194 known B-cell and T-cell epitope regions. These mutations were spotted on the EDIII PDB structure (Figure 2 C , 195 

Furthermore, some of the mutations (DENV1 EDIII-E90G, DENV3 EDIII-I88T) were observed in 196 type-specific monoclonal antibody binding sites 37-40 which could impact the antibody binding and neutralization. 197 In addition to our study sequences, we also investigated EDIII mutations in all Indian DENV1-4 strain sequences 198 deposited in the ViPR database. The frequency of amino acid variations is shown in (Figure S3 ). In line with 199 previous reports, our analysis implicates the EDIII amino acid residues as sites under immune pressure 41 . 200 J o u r n a l P r e -p r o o f DENV DNA vaccine construction 201 We developed the DENV DNA vaccine candidate which is more adapted to the strains of dengue viruses found 202 in India and Africa. Consensus EDIII-NS1 vaccine sequence was designed by combining whole genome 203 sequences obtained from our study with published Indo-Africa specific DENV sequences that were retrieved from 204 the ViPR database. Generated consensus sequences were codon and RNA optimized, synthesized commercially, 205 cloned into pVAX1 expression vector and the generated plasmid was designated as DDV (Dengue DNA Vaccine) 206 ( Figure 3A & B) . 207 It is also noteworthy that approximately 26-50% of Indian DENV1-4 strains exhibited 100% identity with 208 consensus EDIII sequences represented in DDV. The remaining Indian sequences, for all the serotypes, exhibited 209 greater than 93% identity. Furthermore, DDV has 100% identity with African DENV2&3 strains, while African 210 DENV 1&4 strains exhibited >96% identity with their corresponding serotype DDV sequences. DDV also shares 211 >95.15 identities with the EDIII of the top 1000 international dengue sequences of the cognate serotype in the 212 ViPR database (Table S4) .

Epitope analysis for the EDIII construct 214 We predicted the structural stability of the EDIII constructs and checked for the 3D structural conservation at the 215 predicted B-cell discontinuous epitope regions ( Table S5 ). The homology models for the EDIII constructs were 216 subjected to energy minimization, rmsd (root mean square deviation with the template used for modelling) 217 calculation with the PDB structures and Ramachandran map (https://saves.mbi.ucla.edu) and energy analysis 218 (https://prosa.services.came.sbg.ac.at/prosa.php) which predicted that the constructs are stable structurally and 219 energetically ( Figure S4) . In order to estimate the population coverage of the vaccine constructs, we also predicted 220 the T-cell epitopes and the HLA subtypes predicted to bind to each of the epitopes. This analysis revealed that 221 90-98% of the world population could recognize the MHCI epitopes (using predicted strong binding epitopes) 222 and 90-99% of the population can recognize the MHCII epitopes (using both strong and weak binding epitopes). 223 We have chosen nine geographical regions with either frequent or sporadic dengue occurrence as per the CDC 224 J o u r n a l P r e -p r o o f report-https://www.cdc.gov/dengue/areaswithrisk/around-the-world.html. These regions are South Asia, 225 Southeast Asia, East Africa, West Africa, Central Africa, West Indies, Central America, South America and 226 Oceania. We see that the population coverage for epitopes is more than 75% of most of the regions except West 227 Indies and Central America which have lower population coverage for some of the serotypes (Table S6-8) . 228 In vitro antigen expression and localization 229 We first assessed encoded DENV EDIII and NS1 transgene expression at the RNA level in HEK293T cells 230 transfected with DDV. Using the total RNA isolated from the transfected 293T cells, we confirmed the EDIII and 231 NS1 mRNA expression by qRT-PCR ( Figure 3C ). In vitro, EDIII and NS1 protein expression in HEK-293T Figure 5C ). This suggests that T-cell responses elicited by DDV 282 are antigen-specific rather than owing to non-specific T cell activation. IL-4 is a strong Th2 cytokine known to 283 suppress the production of antiviral cytokines and cell-mediated immune response 45 . Thus, in addition to IFN-γ, 284 we evaluated IL-4 secreting cells upon DDV or pVAX1 vaccination via IL-4 ELISpot. Single-cell suspensions 285 were stimulated with the peptide pools (Pool 1-EDIII peptide mixture; Pool 2-NS1 peptide mixture; Pool3-EDIII 286 and NS1 peptide mixture) number of IL-4 producing cells were calculated. While a low number of spots were 287 detected for IL-4 ( Figure 6F ), DDV vaccinated animals showed significantly higher counts of IFN-γ spots. 288 PMA/IO was used as a non-specific positive control. As expected, stimulation with PMA/IO in all ELISpot assays 289 performed with cells from DDV vaccinated or pVAX1 animals induced high IFN-γ and IL-4 spots. 290 We further analyzed intracellular IFN-γ in CD8+ and CD4+ T-cells in both DDV and pVAX1 groups of animals. To assess the role of humoral immune response in mediating protection from DENV challenge, we passively 338 transferred serum from BALB/c mice immunized with either plasmid control or DDV into AG129 mice. Groups 339 of AG129 mice received anti-pVAX1 sera at 300 μl per mouse or anti-DDV immune sera, at two dosage levels, 340 100 and 300 μl per mouse. All these mice were challenged 2 hours after-passive transfer with a lethal dose of 341 DENV-2 (10 5 FIU/mouse). The control group did not receive immune sera but were subjected to the lethal 342 challenge dose. All groups were tracked for body weight changes, clinical signs, and survival for up to 14 days 343 post-challenge ( Figure 8A ).

The DENV-infection-only group showed an initial increase in body weight; however, with a steep decrease from 345 day 5 or 6. The maintenance of body weight or percentage increase were observed to be better in 300 μl immunized 346 mice compared to DENV-challenged group and pVAX1 control group ( Figure RepairPDB module, models for each mutation for all four serotypes were generated with the BuildModel module. 488 The The Gibbs free energy of folding (in kcal mol−1) was calculated for each mutation and provided an threshold Dengue DNA vaccine construction 492 The polyvalent DENV DNA vaccine construct encodes EDIII of all four serotypes and NS1 sequence of DENV2. 493 The consensus gene sequences were constructed using the predicted consensus sequences from sequences 494 obtained from our study (DENV1 n =40, DENV2 n =48, DENV3 n =22, DENV4 n =9) and Indo-Africa specific 

Homology modeling was carried out using Modeller tool (v) 87 , 100 models were predicted and the model with 512 the lowest DOPE score was used for the analysis. Energy minimization was carried out using Schrodinger module 513 (v). B-cell discontinuous epitopes were predicted for the PDB templates and the construct sequences using from 514 the DiscoTope webserver (Immune Epitope Database and Analysis Resource (IEDB)) using the default 515 parameters 88 . This tool uses solvent-accessible surface area and contact distances for predicting structural B-cell 516 epitopes. T-cell epitopes were predicted using the NetMHCpan EL 4.1 (MHCI) and a combination of 517 NetMHCIIpan 4.0, NN-align 2.3 and SMMalign (MHCII) from IEDB. The thresholds used were <0.5 for strong 518 binders and <2 for weak binders for MHCI and <2 for strong binders and <10 for weak binders. 

DENV binding antibody titer was analyzed by indirect ELISA. 96 well plates (Thermo Scientific) were coated 560 with recombinant protein in a coating buffer (0.1M NaHCO3) and incubated overnight at 4 C. The following day, 561 plates were blocked with 3% BSA in PBS for 2 hours at room temperature. Triplicate samples of serially diluted 562 plasma ranging from 1:100 to 1:5,00,000 were added to the plate and incubated for 2 hours at room temperature 563 or overnight at 4 C. After washing, secondary anti-IgG (Sigma), anti-IgG1(Invitrogen), and anti-IgG2a 

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Complete assembly of a 717 dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum

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Serotype and genotype diversity of dengue 724 viruses circulating in India: a multi-centre retrospective study involving the Virus Research Diagnostic 725 Laboratory Network

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The 740

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and NS1 peptide mixture; Pool 4-HIV-nef peptide mixture). (G-J) Flow cytometric analysis of Intracellular 994 cytokine staining for IFN-γ in C57BL/6J mice (n=5/group) splenocytes

Intracellular IFN-γ staining in CD8+ and CD4+ T cells, respectively in DDV vaccinated mice splenocytes

Pool 1-ConA; Pool 2-DENV EDIII and NS1 peptides). Data representative of three 998 independent experiments. Values depicted are mean ± SD. Percentage of IFN-γ producing T cell were compared 999 between groups with student unpaired t test

Figure 6. DDV elicits Th1-biased immune responses. (A) Th1/Th2 Assay schedule: BALB/c (n=5/group)

C57BL/6J mice (n=4/group were immunized by TA injection of 50 µg DDV or pVAX1 at day 0,15 and 30. two 1003 weeks after the 2 nd dose sera collected and assayed for IgG subclass antibodies. (B & C) DENV specific IgG 1004 subclasses and the ratio of IgG2a/IgG1 in BALB/c mice immunized with DDV or plasmid control

IL-4 responses to pooled EDIII-NS1 peptides were measured by ELISpot after vaccination with 1007 DDV or pVAX1 in BALB/c (n=4/group) (Pool 1-EDIII peptide mixture; Pool 2-NS1 peptide mixture; Pool 3-1008 EDIII and NS1 peptide mixture). (G) Number of IFN-γ and IL4 secreting cell ratios. PMA/IO was used as a 1009 non-specific positive control

P values determined by student unpaired t test

0005. # insignificant. Study design schematic 1011 diagram created with BioRender

Transcriptomic analysis of immune genes after vaccination with DDV. (A) C57BL/6J 1013 (n=5/group) mice were immunized with either pVAX1 or DDV and animals were sacrificed at 14 days after 2 nd 1014

Gene expression of immune genes were measured in lymph 1015 nodes. (B) Principal-component analysis (PCA) of immune gene expression following vaccination with DDV 1016 or pVAX1 control. (C) Volcano plots of fold change of DDV versus pVAX1 control (x axis) and log 10 p value of DDV versus pVAX1 control (y axis). A deviation of 1.5 log2 fold change in gene expression was set as the 1018 cut-off value. (D) Differentially expressed genes DDV and plasmid control in lymph nodes presented as 1019 heatmap of Z scores

Nanostring counts per 50ng RNA of selected IFN and inflammatory genes. Data representative of three 1021 independent experiments. Values depicted are mean ± SD. P values determined by student unpaired t test

Groups of AG129 mice were 1025 administered (i.p) BALB/c immune sera in two dosage levels, 100 µl (n=4/group) and 300 µl (n=6/group) per 1026 mouse. pVAX1 group of animals received 300 ul dosage (n=3/group). Two hours after passive transfer the mice 1027 were challenged with a lethal dose of DENV2 (105 FIU/mouse; n=7/group). All groups were monitored for 1028 body weight changes (B), clinical symptoms (C) and Survival (D). Data representative of three independent 1029 experiments. Values depicted are mean ± SD. Study design schematic diagram created with BioRender

The authors thank Dr. Krishnamurthy, MS. Raksha, Dr. Yogesh Chandra, and Dr. Swetha Reddy for their 652 technical assistance. We acknowledge the staff of the BLiSC Animal Care and Resource Centre and TheraIndx