Vaccination and CHIKV infection in non-human primates

2.5 to 3.5 year-old female and male Mauritian cynomolgus macaques (Macaca fascicularis) were purchased from Worldwide Primates, Inc. (Miami, FL). All animals screened negative for TB, SIV, simian T lymphotropic virus, herpes B virus, T. Cruzi, SRV, and CHIKV. Cynomolgus macaques were vaccinated intramuscularly (i.m) with a low dose [LD, 1.3 × 106 plaque forming unit (PFU)] or high dose (HD, 1.3 × 108 PFU) of the EILV/CHIKV, 3.1 × 105 PFU of CHIKV 181/25, 5 × 105 PFU of the inactivated wild-type (WT) CHIKV Martinique strain, or PBS. Prior to challenge, each NHP was implanted intraabdominally with a DST micro-T temperature logger (Star-Oddi, Garðabær, Iceland) programed to take a temperature reading every 15 min. Macaques were challenged with 1.0 × 105 PFU of the WT-CHIKV La Réunion (LR) strain at days 6 or 370 post vaccination (PV). Macaques were bled via the femoral vein at various time points post vaccination and virus challenge. All blood collections, vaccinations, and virus challenge were performed under anesthesia by i.m. injection of ketamine (10 mg/kg body weight). For data logger surgery, ketamine (5 mg/kg body weight), Dexmedetomidine (0.02 mg/kg body weight), and Glycopyrrolate (0.01–0.02 mg/kg body weight) were given pre-operatively i.m. to prepare for intubation, followed by Buprenorphine ER (0.2 mg/kg body weight) and Meloxicam ER (0.6 mg/kg body weight) given subcutaneously (S.Q.) during skin preparation and Isoflurane (0.5–5%) inhalation during operation. Atipamezole (0.02 mg/kg body weight) was given i.m. at the end of surgery to reverse the sedatives. Two weeks post virus challenge, macaques were anesthetized by i.m. injection of ketamine (20 mg/kg body weight) and intracardiac blood collection was performed. Euthanasia was done by intracardiac injection of Euthasol (2 to 4 ml).

Passive immunization studies in mice

Six to seven week-old C57BL/6 (B6) mice deficient in the IFN-α/β receptor (AB6) were bred and maintained at the University of Texas Medical Branch (UTMB). For passive immunization studies, AB6 mice were transferred intraperitoneally (i.p.) injection with diluted sera from mock or vaccinated macaques 24 h before challenge i.p. with 1 × 103 PFU WT CHIKV Martinique strain. Mice were anesthetized by isoflurane (3%) inhalation during adoptive transfer and infection. Infected animals were monitored daily for clinical signs and weight loss and were euthanized by CO2 inhalation if they became moribund. Surviving animals were euthanized via CO2 inhalation 21 days post infection.

Guinea pig sensitization study

Six to eight week-old female Dunkin Hartley albino Guinea pigs were purchased from Charles River (Wilmington, MA). Guinea pigs were anesthetized via i.p. injection with ketamine (20 mg/kg body weight)/ xylazine (2 mg/kg body weight), shaved a portion of the abdomen and then taped 1 pint cartons with screened lids, containing 50 females Ae. albopictus mosquitoes, to the animal for 30 min. The feeding was repeated for 3 times on a 14 day interval. On day 43, guinea pigs were anesthetized via isoflurane inhalation (3%) and injected intradermally (i.d.) at the mosquito probing/feeding area with 20 µg Ae. albopictus SGE, 1.3 × 108 PFU of purified EILV/CHIKV, or PBS-G (mock). 30 min to 2 h following injection, inoculation sites were assessed via measurements of site swelling and imaging. Animals were euthanized via CO2 inhalation on day 44.

Ethic statement

All animal studies were approved by the Institutional Animal Care and Use Committee at UTMB (protocol # 1704031A and #1904041). Both male and female cynomolgus macaques and mice were used in this study. Due to animal availability during the pandemic, single sex (either female or male) macaques were used in the long-term and short-term protection studies respectively. Female guinea pigs were used for skin hypersensitivity study. It’s unknown whether the findings are relevant to male guinea pigs.

Virus stocks

To prepare for the purified EILV/CHIKV stocks, C7/10 cells were seeded at 2 × 107 cells per 150 mm dish 24 h prior the infection and inoculated with 2 × 106 PFU of EILV/CHIKV in phosphate-buffered saline (PBS), supplemented with 1% FBS for 1 h at 28 oC. Cells were then washed and further incubated for 18 h in VP-SF media (Invitrogen) supplemented with 10% TPB and glutamine. HEPES buffer pH 7.5 was added to the harvested virus-containing media before filtered the media through 0.22 µm filter and passed at room temperature through Cellufine sulfate (AMSBIO) column equilibrated with PBS. Column was washed with PBS, and virus was eluted in PBS. It was immediately loaded on the top of freshly prepared sucrose gradient (1.5 ml of 50%, 2 ml of 40% and 7 ml of 30% prepared on PBS) and centrifuged at 36,000 rpm, 4 oC for 3.5 h. A well visible band was collected, diluted with PBS, and aliquots were stored at -80 oC. Viral titers were evaluated in C7/10 cells as described previously22. CHIKV 181/25 was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at UTMB and was amplified once in Vero cells to obtain a stock for animal studies. CHIKV LR and Martinique strain stocks were kindly provided by Dr. Trevor Brasel at UTMB, which were passaged and titrated in Vero cells. Formalin-inactivation of WT-CHIKV was performed as described previously21. Briefly, a CHIKV Martinique stock was diluted 1:10 in DMEM media and inactivated by the addition of 20 µl of 10% formaldehyde (Sigma, St. Louis, MO) to a final concentration of 0.1% (v/v), or mock-inactivated by the addition of an equivalent volume of PBS. Virus suspensions were incubated at 37°C with mixing twice daily for 3 days, followed by incubation at 4°C with mixing twice daily for 4 days. Formaldehyde was neutralized by the addition of 3.5% (w/v) sodium metabisulfite (Sigma) to a final concentration of 0.035% (w/v). Inactivation was confirmed by plaque assay.

Blood collection and peripheral blood mononuclear cell (PBMC) isolation

Blood was collected by femoral venipuncture into EDTA in serum separator/lithium heparin vacutainer tubes (BD Biosciences). Blood tubes were centrifuged at 3000 rpm at 4°C for 15 min first and the upper layer was collected. For PBMC isolation, heparin-treated blood was diluted 1:1 with sterile PBS and carefully layered onto Ficoll-Paque (GE Healthcare Bio-Sciences, Sweden). The tubes were centrifuged at 400 x g for 30 min at 20°C, and then the mononuclear cells were transferred to a sterile centrifuge tube. Cells were washed twice with PBS and resuspend in RPMI medium supplemented with 10% FBS, and 1% l-glutamine (Thermo Fisher). Cells were cryopreserved and stored in freezing medium (10% DMSO/ 90% FBS) in liquid nitrogen tank.

Plaque assay

Vero cells were seeded in 12-well plates and incubated at 37 °C, 5% CO2 for 16 h. Serum samples were serially diluted in DMEM with 2% FBS and 100 μl of diluted samples were used to infect the monolayers. Plates were incubated at 37°C with 5% CO2 for 1 h. After the incubation, 0.4% agarose overlay medium was added to the infected cells. The plates were incubated at 37 °C with 5% CO2 for 48 h. Plates were fixed with 10% formaldehyde for 30 min. Cells were stained with 0.25% crystal violet for 3 min, then rinsed with H2O. The plaques were counted and calculated as PFU /mL.

Quantitative PCR (Q-PCR)

Blood cells were re-suspended in Trizol (Invitrogen) for RNA extraction. Complementary (c) DNA was synthesized by using a qScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The sequence of the primer sets for CHIKV capsid protein, cytokines and PCR reaction conditions were described previously22,57,58. The PCR assay was performed in the CFX96 real-time PCR system (Bio-Rad). Gene expression was calculated using the formula 2^ -[Ct(target gene)-Ct(β-actin)] as described before59.

NanoString sample preparation

Targeted transcriptomics was performed on PBMC samples from macaques. Nonhuman primate (NHP) V2_Immunology reporter and capture probe sets (NanoString Technologies) were hybridized with ~100 ng of each RNA sample for ~24 h at 65°C. The RNA:probeset complexes were subsequently loaded onto an nCounter microfluidics cartridge and assayed using a NanoString nCounter SPRINT Profiler. Samples with an image binding density <0.2 were re-analyzed with a higher concentration of RNA to meet quality control criteria.

Transcriptional analysis

Analysis was performed on samples at 7 days post-vaccination or 4 days post infection. nCounter .RCC files were imported into NanoString nSolver 4.0 software. To compensate for varying RNA inputs and reaction efficiency, an array of housekeeping genes and spiked-in positive and negative controls were used to normalize the raw read counts. As both sample input and reaction efficiency were expected to affect all probes uniformly, normalization for run-to-run and sample-to-sample variability was performed by dividing counts within a lane by the geometric mean of the reference/normalizer probes from the same lane (i.e., all probes/count levels within a lane are adjusted by the same factor). The ideal normalization genes were automatically determined by selecting those that minimize the pairwise variation statistic and are selected using the widely used geNorm algorithm. The data were analyzed with NanoString nSolver Advanced Analysis 2.0 package for differential expression and to generate principal component analysis (PCA), heatmap, and cell-type trend plots. Human annotations were added for each mRNA to perform immune cell profiling within nSolver and generate the cell-type scores. Normalized data (fold change values and Benjamini–Hochberg adjusted p-values) were exported as an .xlsx file (Supplementary Data 2) and imported into GraphPad Prism version 10.0.1 to generate heatmaps. Figures were prepared using Adobe Illustrator 2024. Any differentially expressed transcripts with a Benjamini–Hochberg false discovery rate (FDR) corrected p-value <0.05 were deemed significant unless otherwise stated. Enrichment of differentially expressed transcripts was performed using Ingenuity Pathway Analysis (Qiagen). Samples were filtered for immunity-specific canonical signaling pathways.

Peptide pools

Fifteen amino acid (aa) peptides overlapping 12 aa spanning the structural proteins of CHIKV were purchased from Sigma and were divided into 4 overlapping peptide pools, including core (peptides 1-53), E3 (peptides 54-77), E2 (peptides 78-211), and E1 (peptides 212-343).

Intracellular cytokine staining (ICS) and flow cytometry

Cryopreserved PBMCs were thawed and washed once with RPMI medium containing 10% FBS. Cells were incubated with individual CHIKV peptide pools (2 μg/ml) for 6 h in the presence of BD GolgiPlug (BD Bioscience). Cells were harvested and stained with antibodies for CD3, CD4, or CD8, fixed in 2% paraformaldehyde (PFA), and permeabilized with 0.5% saponin before adding anti-IFN-γ (e-Biosciences). PBMCs were also stained for the following cell surface markers: CD56 (clone: SP34-2; BD Biosciences) and CD3 (clone: TULY56; eBioscience) for 30 min at 4°C, washed and fixed in 2% PFA. Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).

CHIKV IgG ELISA

ELISA plates were coated with 50 ng/well recombinant CHIKV E2 protein (SinoBiological, USA) overnight at 4°C. The plates were washed twice with PBS, containing 0.1% Tween-20 (PBS-T) and then blocked with 2% BSA for 1.5 h at room temperature (RT). Sera were diluted 1:200 in blocking buffer and 50 µl were added per well for 1 h at RT. Plates were washed five times with PBS-T. Goat anti-monkey IgG (Fitzgerald, USA) coupled to horseradish peroxidase (HRP) was added as the secondary antibody at a 1:25000 dilution for 1 h at RT. Color was developed with TMB substrate (Thermo Scientific) and the intensity was read at an absorbance of 450 nm.

Plaque reduction neutralization assay

Fifty percent plaque-reduction neutralization tests (PRNT50) were performed on Vero cells. Briefly, NHP sera were heat-inactivated at 56°C for 30 min then serially diluted (2-fold dilutions) in 2% FBS MEM media. CHIKV 181/25 stock was diluted to 800 PFU/ml. Then 150 µL of the diluted sera were mixed with 150 µL of virus stock, incubated at 37°C for 60 min. Afterwards, 250 μl of each sera/virus mixture was added to Vero cells, and incubated for 1 h at 37°C, with rocking every 15 min. Then MEM/0.4% agarose overlay media was added to each well, and incubated at 37°C with 5% CO2 until plaques appeared (about 48 h). Plates were fixed with 10% formaldehyde and stained with 0.25% crystal violet. The plaques were counted and the PRNT titers calculated as the highest dilution of serum that inhibited 50% of plaques.

B cell ELISPOT assay

ELISpot assays were performed as previously described60 with some modifications. Briefly, PBMCs were stimulated with 1 µg/ml R848 and 10 ng/ml recombinant human IL-2 (Mabtech In, OH). Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 108 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech). The stimulated PBMCs were harvested, washed, and added in duplicates to assess total IgG ASCs or CHIKV- specific B cells. Plates were incubated overnight at 37˚C, followed by incubation with biotin conjugated anti-human IgG (Mabtech) for 2 h at room temperature, then 100 µL/well streptavidin-ALP was added for 1 h. Plates were developed with BCIP/NBT-Plus substrate until distinct spots emerge. After washing, the plates were scanned using an ImmunoSpot 4.0 analyzer and the spots were counted with ImmunoSpot software (Cellular Technology Ltd, Cleveland, OH).

IFN-γ ELISPOT

Monkey anti-IFN-γ capture Ab pre-coated plates (Mabtech) were washed twice and blocked with RPMI media containing 10% FBS for 30 min. PBMCs were stimulated in duplicates with CHIKV peptide pools (2 μg/ml) for 24 h at 37°C. PBMCs stimulated with anti-CD3 (1 μg/ml, e-Biosciences) or medium alone were used as controls. This was followed by incubation with biotin-conjugated anti-IFN-γ for 2 h at room temperature, and then conjugated streptavidin-HRP for 60 min. Plates were developed with TMB substrate, followed by washing and scanning using an ImmunoSpot 4.0 analyzer. The spots were counted with ImmunoSpot software (Cellular Technology Ltd, Cleveland, OH) to determine the spot-forming cells (SFC) per 106 PBMCs.

Cytokine bead arrays

Sera of infected NHPs were inactivated with 5 megarads Mrads in a Cobalt-60 irradiator at Galveston National Lab (GNL, UTMB) before used for cytokines, chemokines, and growth factors measurement using LegendPlex bead-based multiplex technology and NHP inflammation 13-plex kits (BioLegend). Serum samples were processed in duplicate at a 1:4 dilution according to the manufacturer’s instructions. Following bead staining and washing, at least 3,900 bead events were collected on an Aurora cytometer (Cytek) using SpectroFlo software. The raw .fcs files were exported and analyzed with BioLegend’s cloud-based Qognit data analysis software. The concentration data based on standard curves were exported to Microsoft Excel to calculate average fold-change values over the mock group average baseline.

Histopathology

At day 14 PC, spleens were collected from all animals scheduled for euthanasia and placed in 10% formalin (Thermo Fisher Scientific, Waltham, MA, USA) before embedment in an optimal cutting temperature compound. H&E staining was performed at the Histopathology Laboratory Core of UTMB. Slides were examined by a board-certified pathologist at the Experimental Pathology Laboratories, Inc. (EPL®) in Sterling, Virginia.

Aedes albopictus salivary gland protein extract

Around a hundred salivary glands from 6-day old sugar-fed Ae. albopictus females, (strain: Lake Charles, LA) were dissected in droplets of PBS as described before61. Dissected salivary glands were then combined and ground in protein lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). The resulting salivary gland extract was kept on ice and for storage frozen at −80 C.

Statistical analysis

Survival curve comparison was performed using GraphPad Prism software 10.0.1, which uses the log-rank test. Values for viral load, cytokine production, antibody titers, memory B cell frequency, and T cell responses were compared using Prism software (GraphPad) statistical analysis and were presented as means ± SEM. P values of these experiments were calculated with a non-paired Student’s t-test.