Study Design:
This is a phase 1, open-label, human dengue virus challenge study using DENV-1 strain 45AZ5 to assess the protective efficacy of participants previously vaccinated with TDEN-PIV prime followed by TDEN-LAV boost. Unvaccinated dengue-naïve adult participants served as controls. All eligible participants were administered a single subcutaneous injection of 0.5 mL of 6.5 mL x 103 PFU/mL DENV-1 strain 45AZ5 in the triceps area of the arm on Day 0. Following the DENV-1 challenge, participants were followed for dengue-associated symptoms and unsolicited AEs daily, and safety labs collected every other day, and beginning Day 4, DENV RT-PCR daily, and until two consecutive negative tests for those who developed detectable RNA. For participants who remained without detectable RNA, daily follow-ups and DENV RT-PCR and every other day safety labs were continued till Day 16, then every 3 days till Day 28. Participants were followed up to 6 months post-DHIM for long term immunologic labs.
Participants:
We enrolled healthy male and non-pregnant, non-breastfeeding female adult participants between the ages 18-50 years, who have either been previously vaccinated with TDEN PIV prime followed by TDEN LAV boosted (Vaccinees) or were unvaccinated dengue-naïve adults (Controls).
Vaccinees were previous participants of TDEN vaccine studies (ADVP003 and ADVP004) conducted at the Clinical Trial Center, Walter Reed Army Institute of Research (CTC, WRAIR). Participants vaccinated as part of ADVP003, conducted in 2015-16, received PIV adjuvanted in Alum (Day 0) followed by LAV formulation 17 post-transfection (Day 28). TDEN MN antibody titers were monitored from days 0-180. Participants vaccinated as part of ADVP004, conducted in 2018, received PIV (Day 0) followed by LAV (randomized to day 90 or 180). TDEN MN antibody titers were monitored at regular intervals from days 0- 549 (18 months). All DENV-1 titers peaked at Day 28 following LAV. Participants who had evidence of tetravalent response following PIV-LAV inoculation were eligible for participation. Investigators at CTC, WRAIR contacted participants to discuss their willingness to participate in a follow-up dengue live virus human challenge (LVHC) to be conducted at the University of Maryland, Baltimore (UMB). Following institutional review board approval at UMB and the Army Human Research Protections Office, contact information of individuals who expressed willingness to participate was shared. Participants were contacted to inquire as to interest in participating and to screen for eligibility. Eligible participants were in general good health, without significant medical illness based on medical history, physical examination, vital signs, and clinical laboratories (complete blood count, chemistry, coagulation assays, pregnancy screen, HIV, Hepatitis serologies) as determined by the investigators. Participants were instructed to avoid travel to dengue endemic areas and avoid pregnancy or fathering a child through the trial duration. Baseline flavivirus antibodies were assessed (DENV 1-4, West Nile Virus, Japanese Encephalitis Virus, Yellow Fever Virus and Zika Virus).
Participants for the control group were screened as above. Eligible participants were seronegative for flavivirus antibodies and satisfied all inclusion criteria and possessed no exclusion criteria.
Study Site and Oversight:
This study was conducted at the UMB Center for Vaccine Development and Global Health (CVD) located in Baltimore, Maryland. The dengue mosquito vector, Aedes albopictus is present seasonally. UMB CVD and WRAIR investigators developed the protocol and conducted the trial under US FDA Investigational New Drug (IND) #26871 cross referencing #16332. All participants signed an informed consent and passed an assessment of understanding prior to all trial activities. The trial was registered with ClinicalTrials.gov (NCT04786457) on March 8, 2021.
Dengue Challenge Product Information, Administration and Dengue Surveillance: The DENV-1 strain 45AZ5 study product was manufactured at the WRAIR Pilot Bioproduction Facility under IND #16632. The parent DENV-1 strain 45AZ5 was manufactured at the Salk Institute as a vaccine candidate (Salk vaccine, lot 1-82) and underwent transfection into cultures of fetal rhesus lung (FRhL) cells.37 WRAIR challenge lot 1806, DENV-1-LVHC, is a lyophilized powder for injection consisting of: DENV-1 strain 45AZ5 virus, Eagle’s minimal essential medium (EMEM, modified) culture medium, human plasma albumin-USP, L-glutamine, streptomycin, neomycin, and lactose. The freeze-dried DENV-1-LVHC is rehydrated with 0.7 mL of water for injection (WFI). The route of administration is subcutaneous (SC) injection. The product dose was optimized as part of a prior study by DHIM consortium members, State University of New York (SUNY) Upstate Medical University.3 Quantitative DENV-1-specific RT-PCR was performed using previously published techniques.4,38
Safety Outcome:
The primary outcome measure was safety and tolerability of the DHIM-1 human challenge model. Local and systemic reactogenicity parameters were recorded until 7 days following inoculation of DHIM-1. The occurrence, intensity, and duration of dengue like symptoms were evaluated until 28 days post virus inoculation. A calculated score was generated on each participant to allow for statistical analysis of values tabulated between vaccinated and unvaccinated controls. Solicited dengue symptoms and measured laboratories (liver function tests, White blood cell count, and platelets) were scored daily (Day 0-16 or until resolution of symptoms and dengue RNAemia) and a score representing a summation of A) the duration of symptoms in days; B) the number of symptoms per day; and C) the maximum severity score per day and divided by 3 was tabulated (Supplementary Table 2). Laboratory values were graded according to protocol-modified FDA-CIBR guidelines.39
PBMC and Sera Isolation:
Participant whole blood was collected into sterile Eppendorf and EDTA tubes at study visits (Day 0 (pre-DHIM), 4, onset of viremia, and Days 16, 28, 90 and 180). Sera was processed as previously described.40 Blood was processed by density centrifugation within two hours of acquisition, utilizing lymphocyte separation medium (ICN Biomedical Inc, Aurora, OH) following standard techniques.
Antibody Response Measurements:
ELISA – Anti-DENV-1 IgM and IgG ELISA titers were determined at baseline, RNAemia onset, RNAemia peak, final day of RNAemia, as well as day 90 and 180 post-exposure. DENV antigen for ELISAs was produced in Vero cells, clarified by centrifugation at 3,000 rpm for 30 min at 4°C, and then pelleted through a 30% sucrose solution at 26,000 rpm for 4h at 4°C. Purified DENV antigen was resuspended in PBS, the protein concentration was determined using a NanoDrop One spectrophotometer, and then aliquots were frozen at -80C. ELISA plates were coated with 50 ng/well of purified DENV antigen in PBS overnight at 4C followed by washing and blocking with PBS containing 0.1% Tween-20 and 0.25% BSA (blocking buffer). Plates were washed with PBS containing 0.1% Tween-20 (PBS-T) followed by addition 50ul of serially diluted human sera in blocking buffer and incubated for 2 hrs at room temperature. Plates were washed with PBS-T followed by addition of 50ul of either peroxidase labeled goat anti-human IgG g-chain specific secondary antibody (Sigma-Aldrich Cat# A6029) or peroxidase labeled goat anti-human IgM secondary antibody (KPL, cat# 074-1003) and incubated for 1h at room temperature. Plates were washed with PBS-T followed by addition of TMB substrate (KPL, cat# 50-76-00), incubated at room temperature >30 min until color development stabilized and then stopped with 1N H2SO4. Plates were read on a Synergy Neo2 Hybrid Multi-Mode Microplate Reader at 450nm. Endpoint titers are reported as reciprocal dilutions. Dilution reported was >2-fold for IgM and >1.5-fold for IgG, above background. The lower limit of detection for IgM was 900 and for IgG was 100 and the upper limit for both assays was 218,700. Titers <900 for IgM and <100 for IgG were set to 100 and 10, respectively, for graphing and analyses.
Microneutralization Assay: A high throughput, qualified, microneutralization (MN) assay was used to assess baseline flavivirus immunity to dengue virus serotypes 1, 2, 3 and 4. Additionally, a modified version was used to detect yellow fever virus, Japanese encephalitis virus, West Nile virus and Zika virus, with a qualitative readout and seropositivity defined as a titer of 1:10 or greater, as previously described.41,42 Only participants that screened negative for all flavivirus were eligible for enrollment as a seronegative control participant. Previously vaccinated participants were documented as flavivirus naïve prior to their original vaccine study participation (ADVP003 or -004). Only participants documented to have a tetravalent dengue virus response following vaccination were eligible for DHIM-4, however, flavivirus screen was not repeated. Serial MNT were measured at Days 0, 16, 90 and 180 following DHIM-4 in both vaccinees and controls.
NS1 Antigen Quantification: Quantification of DENV NS1 antigen was done following instructions provided by the manufacturer of the Dengue virus NS1 ELISA EUROIMMUN kit (REF EQ 266a-9601-1). Briefly, a standard NS1 antigen curve (Relative Unit per milliliter, or RU/ml) was established using synthetic NS1 proteins provided by the kit. The NS1 protein in the samples was quantified using the equation y= 0.0081x + 0.0677, which had an R2 value of 0.9997. Prior to testing, all plasma samples stored at -80°C were thawed on ice and analyzed without a dilution of the sample. The assay was performed on a SpectraMax iD5 using a photometric measurement wavelength of 450nm and a reference wavelength of 620 nm. Samples from four timepoints for each participant were tested: Day 0 (baseline, pre-challenge) and at onset (first day with detectable RNA; or Day 8 if undetectable), peak (day with the highest RNAemia; or Day 16 if undetectable for RNA), and end of RNAemia (last day with detectable RNA; or Day 28 if undetectable). The results were interpreted as described in the kit instructions: ≥11 RU/ml as positive, <8 RU/ml as negative, and between 8 and 11 RU/ml as borderline.
Cell Mediated Immune Analysis:
DENV-1-LVHC peptide pools. Recombinant peptide pools spanning the entire DENV-1-LVHC proteome were generated and purchased from JPT Peptide Technologies GmbH. Peptides were 15mers overlapping by 11 amino acids, and were lyophilized into six, roughly equivalently sized pools across the proteome, as depicted in Supplementary Figure 3. Lyophilized pools were reconstituted in DMSO at 200 mcg/mL/peptide and used for stimulation at a final concentration of 1mcg/mL/peptide.
IFNγ ELISpots- IFNγ ELISPOT assays were similarly performed as previously described.43 In brief, cryopreserved PBMC were thawed and let to rest overnight before plating 100,000-200,000 cells per well of a PVDF (polyvinylidene difluoride) membrane plate coated with anti-IFN-γ antibody. Cells were co-incubated overnight with DHIM-1 peptide pools A-F. The cells were then removed, and IFN-γ-producing spots were developed using a biotinylated secondary anti-IFN-γ antibody, streptavidin-conjugated horseradish peroxidase, and TMB (3,3',5,5'-Tetramethylbenzidine) substrate. The number of spot-forming cells (SFC) per well were counted using an ImmunoSpot S6 Universal-V Analyzer (Cellular Technology Limited). Negative (0.5% DMSO in media) and positive (anti-CD3) controls were run for each sample, and all conditions were run in replicate. Data from replicates were averaged and then normalized to SFC/million PBMC for reporting purposes.
Conventional Flow Cytometry - Flow cytometry-based intracellular cytokine staining assays were also performed as previously described.43 In brief, cryopreserved PBMC were thawed, counted, and plated at 1 million cells per well of a 96-well assay plate. After overnight rest, cells were washed and co-incubated with DHIM-1 peptide pools A-F along with co-stimulating antibodies against CD28 and CD49d and fluorescently labeled antibodies to CCR7 and CXCR5. After 1 hour, Golgi transport inhibitors were added and the co-culture allowed to incubate for an additional 6 hours. The plates were stored at 4°C overnight, and the following day stained with a viability marker and fluorescently labeled antibodies to surface proteins (CD3, CD4, CD8, CD14, CD19, CD27, CD45RA). Cells were then fixed and permeabilized, followed by staining with fluorescently labeled antibodies to IFN-γ, TNF-a, and IL-2. Data were acquired on a LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo vX software (BD Biosciences).
Analytical Mass Cytometry:
PBMC were thawed, washed, and rested for 24h in complete media (cRPMI) at 37 degrees C. 2X 106 PBMC from participants at selected timepoints were stimulated with either one of six separate peptide pools spanning the entire DENV-1 genome (denoted, A-F, as described), or Staphylococcus enterotoxin B (positive control), or cRPMI (negative control). Incubation was permitted for 3h, then a Golgi Plug®/Stop® cocktail was administered. Cells were harvested 16h later, placed into MaxPar® cell staining buffer, and barcoded with anti-CD45 monoclonal antibodies (mAbs) conjugated with either 154Gd, 156Gd, or 89Y (Fluidigm, San Francisco, CA). Up to three differently barcoded samples (for stimulant condition restricted to single time point and participant) were combined and stained with Cell ID Cisplatin, followed by washes, then treated with human IgG at room temperature for 30 minutes to block non-specific Fc receptor binding. A panel of mAb targeting human cell surface human markers including anti-CD3 170Er, anti-CD4 174yYb, anti-CD8 146Nd, and anti-CD69 162Dy was applied to the cells and incubated for 30 minutes at 4 degrees. Cells were washed then treated with Invitrogen fixation and permeabilization buffer per manufacturers protocol, followed by incubation with intracellular antibodies including anti-IFN-γamma 165Ho for 30 minutes, followed by washing steps and fixation in 1% PFA, and storage at 4 degrees. Prior to acquisition cells were treated with Cell-ID™ Intercalator-Ir per for 1 hour at 4 degrees, then rinsed with water. Acquisition was performed on a Helios instrument at the University of Maryland Flow and Mass Cytometry core. Normalization and de-barcoding were performed with Fluidigm Helios software and subsequent analysis performed with FlowJo (version v10.7.2) software. Responses observed in media control samples were subtracted from measurements against respective peptide pools per individual timepoint/participant.
Antibody Dependent Enhancement Assays:This assay has been described previously.31 In vitro ADE of DENV-1 infection was quantified in BHK cells expressing human CD32a. Beginning at 1:8, two-fold serial dilutions of heat-inactivated sera were incubated with DENV-1 West Pac 74 (GenBank accession U88535) (in sufficient amount to infect approximately 1% of BHK[CD32a] cells) at 1:1 for 1h at 37°C. Duplicate wells of a 96-well plate containing confluent BHK(CD32a) cells were then infected with 30µL of this mixture. Cells were infected and incubated for 48h in a 37°C, 5% CO2, humidified incubator. The medium was removed, a single cell suspension was prepared, and then processed according to the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD, cat# 554714) instructions. The cells were immunostained with primary monoclonal antibody 4G2 and secondary PE goat-anti-mouse Ig (BD Pharmingen, cat# 550589) and DENV-1 antigen positive cells were quantified on an Attune NxT flow cytometer. Fold-infection relative to control serum is reported as fold-ADE.
Gene Expression Analysis:
RNA sequencing library preparation and sequencing: Whole blood was collected from all study participants using PAXgene RNA collection tubes (BD), sub-aliquoted, and frozen at -20o C until analyzed. RNA was extracted from the collection tubes using the Qiagen PAXgene Blood RNA isolation Kit and sequencing libraries created using Illumina Stranded Total RNA Prep with Ribo-Zero Plus and IDT-Ilmn RNA UD Indexes SetA. Final library QC and quantification was performed using a Bioanalyizer (Agilent) and DNA 1000 reagents. Libraries were pooled at an equimolar ratio and sequenced on a 200 cycle Novaseq 6000 instrument using v1.5 S2 reagent set.
RNA sequencing gene expression analysis. Raw FASTQ files were filtered to contain only pair-end reads and to remove any Illumina adaptor contamination and low-quality reads using Trimmomatic (version 0.39). Filtered paired-end FASTQ files were mapped to the human reference transcriptome (Ensembl, Home sapiens, GRCh38) using Kallisto version 0.46.2.44 Transcript-level counts and abundance data were imported and summarized in R (version 4.2.1) using the TxImport package (version 1.16.1)45 and TMM normalized using the package EdgeR (version 3.30.3)46,47. Differential gene expression analysis performed using linear modeling and Bayesian statistics in the R package Limma (version 3.44.3).48 Genes with a log2 fold change of >1 and a Benjamini-Hochberg adjusted p-value < 0.05 were considered significant. Gene module scores were calculated by summing the TMM normalized abundance (TPM) of the genes highlighted as belonging to a previously defined DENV-infection associated gene set.4 B cell receptor (BCR) clonotype abundance was assessed using MiXCR (version 3.0.3) and the RNA-seq/non-targeted genomic analysis pipeline.49,50
Statistical Analysis:
Safety analysis was performed on all participants who received study product administration and analyzed by treatment group. Efficacy analysis included all enrolled participants who underwent DHIM. All statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA) except for RNAseq gene expression. In vitro ADE assays utilized Spearman correlations with P <0.05 required for significance followed by a correction for multiple comparisons using a False Discovery Rate two-stage step-up method of Benjamini, Krieger and Yekutieli with Q = 5% (GraphPad Prism 8.1.0).