Ethics statement
This study was carried out in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Tech.
Cell culture and viruses
We maintained BHK-21 (ATCC CCL-10, hamster kidney fibroblast), Vero (ATCC CCL-81, African green monkey kidney epithelial), HEK293T (ATCC CRL-3216, human kidney epithelial), 3T3 (ATCC CRL-1658, mouse embryo fibroblast) at 37℃ in 5% CO2. C6/36 (ATCC CRL-1660, Aedes albopictus) cells were maintained at 28℃ in 5% CO2. All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% nonessential amino acids, and 0.1% gentamicin (herein referred to as DMEM-5). All CHIKV vaccine candidates were modified from a chimeric virus clone31 originally derived from a wild-type (WT) clone of strain SL-CK174. WT CHIKV SL15649 clone, courtesy of Mark Heise (the University of North Carolina at Chapel Hill), was used in challenge studies.
Generation of CHIKV vaccine candidates
The construction of CHIKV-SFV/DomC, which expresses nanoluciferase (nLuc; Promega, WI, USA) was described previously30. Platinum SuperFi 2X master mix (ThermoFisher, MA, USA) was used to generate vector backbone segments around nLuc and cytokine fragments (Sino Biological, PA, USA). Each cytokine fragment was individually assembled into the vector backbone using NEBuilder HiFi DNA Assembly Master Mix at a 1:2 (vector: insert) molar ratio to generate each vaccine candidate. Each Hifi assembly was treated with DpnI before electroporation into homemade NEB stable electrocompetent cells. Cells were then plated onto LB agar with 50 µg/mL carbenicillin and grown at 30°C overnight. Single isolated colonies were grown in liquid cultures, and plasmids were extracted using the NucleoSpin Plasmid Transfection-grade kit (Macherey-Nagel, PA, USA). Insertion of each cytokine was confirmed with Sanger sequencing. Primer and clone sequences are available upon request. For virus rescue, plasmids for each vaccine candidate were transfected into HEK-293T cells using JetOPTIMUS (Polyplus, Illkirch, France) according to the manufacturer’s protocol. Viral supernatants were collected, aliquoted, and stored at − 80°C until titration by plaque assay.
Virus and antibody titers
Plaque assays and plaque reduction neutralization tests (PRNTs) were performed in Vero cells as previously described75. For mouse sera collected from immunocompetent C57BL/6 mice, viral titers were assessed by C6/36 pre-amplification followed by transfer to Vero cells as previously described41.
Enzyme-linked immunosorbent assays (ELISAs)
Cytokine expression was assessed using Bio-Techne’s DuoSet kits for mouse IFNγ (DY485-05) and IL-21 (DY594-05) according to the manufacturer’s protocol.
Viral growth kinetics
Twenty-four well plates were grown to 80–90% confluency for BHK-21 and 3T3 fibroblasts. Cells were infected at a multiplicity of infection (MOI) of 0.01 PFU/cell for each vaccine candidate and assessed against the parental CHIKV-SFV/DomC and the original WT strain SL-CK1. Virus stocks were diluted in Roswell Park Memorial Institute medium (RPMI 1640) with 25 mM HEPES and 1% FBS (herein referred to as viral diluent). The viral inoculum was added to each well in triplicate, rocked, and incubated at 37℃ in 5% CO2 for 1 h. Back-titers of viral inocula were also assessed to confirm MOI. After adsorption, cells were washed 1–2 times with phosphate-buffered saline (PBS), and DMEM-5 was added to each well. A 0 h-time point was collected to assess baseline viral titers, and supernatants were harvested every 24 h until 50–75% CPE was observed. Samples were stored at -80℃ until titration by plaque assay.
Animal studies
Immunocompetent mice. Four-week-old C57BL/6 mice were obtained from The Jackson Laboratory (ME, USA). Groups of male mice (n = 10 per group) were infected in the hind-left footpad with 104 PFU of one of the CHIKV vaccine candidates or mock-infected with viral diluent. Mice were weighed and monitored daily for signs of disease. Footpad widths were measured daily using digital calipers. Mice were anesthetized using isoflurane and then bled via the maxillary vein each day for three days post-infection. Thirty-one- or seventy-one-days post-vaccination (dpv), five mice per group were infected with 103 PFU of WT CHIKV and monitored as previously described.
Immunocompromised mice. Four-week-old C57BL/6 male and female mice (n = 5 per group/sex) were vaccinated with the candidates and challenged with WT CHIKV as earlier described; however, before viral inoculations, mice were intraperitoneally (i.p.) inoculated with 0.1 mg of MAR1-5A3, an anti-interferon-α/β receptor (IFNAR) antibody (Leinco Technologies, Fenton, MO)76. Euthanasia criteria included weight loss greater than 15% of initial body weight. Mice were bled through the maxillary vein 30 dpv to assess neutralizing antibodies. For viral dissemination studies, groups of male mice were treated with 0.1 mg of MAR1-5A3 prior to vaccine inoculation as described above and were euthanized 1, 2, and 3 dpv to collect footpad and calf tissues, and popliteal and inguinal lymph nodes. Tissues were collected in 2 mL tubes with a metal bead, and viral diluent was added at a 10% weight/volume ratio. Tissues were homogenized with a Tissue Lyser II (Qiagen, Germantown, MD) at 25 freq/s for 2 min at a time until tissues were fully homogenized. After collection of clarified tissue supernatant, 1x DNA/RNA shield was added to the remaining footpad tissues and re-homogenized. RNA was extracted from the footpad with the Quick-RNA Miniprep Plus kit (Zymo Research, Irvine, CA, USA) per the manufacturer’s instructions.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
For determining the differential expression of genes (DEGs) between mice vaccinated with either cytokine-expressing vaccine candidates and the parental virus CHIKV-SFV/DomC, primers for several IFNγ and IL-21 stimulated genes were ordered from Integrated DNA Technologies (IDT, Leuven, Belgium). GBP1 (forward:5’ ACCTGGAGACTTCACTGGCT-3’, reverse: 5’- TTTATTCAGCTGGTCCTCCTGTATCC-3’) and GBP2 (forward: 5’- CTGCACTATGTGACGGAGCTA-3’, reverse: 5’- CGGAATCGTCTACCCCACTC-3’) were tested on CHIKV-SFV/DomC-IFNγ and IL-12Rβ2 (forward: 5’-TGACAGCTGCTGGTGAAAGT-3’, reverse: 5’-ATGTTGGAGGGTAAATAGCC-3’) was tested on CHIKV-SFV/DomC-IL-21 samples, with CHIKV-SFV/DomC as the comparator for both. RT-qPCR was performed on the footpad RNA using NEB Luna Universal One-Step RT-qPCR kit with SYBR-Green (NEB, MA, USA). The conditions for the reactions in a Bio-Rad CFX-96 were: 55°C for 10 min for reverse transcription, 95°C for 1 min for initial denaturation and polymerase activation, followed by 45 cycles of 95°C for 10 s for denaturation, and 60°C for 30 s for annealing/extension, followed by a melt curve from 60°C to 95°C for 0.05 s with 0.5°C increments. All samples were normalized to mouse GADPH which was utilized as the reference gene. The fold change gene expression was calculated using the Delta Delta Ct (ΔΔCt) method of relative quantification77.
Statistical Analysis
Statistical comparisons were performed in Prism 9 (GraphPad, CA, USA). Unpaired t-tests and 2-way ANOVA with Dunnett’s multiple comparisons test were performed for ELISAs and growth curves, respectively. For animal weight and footpad swelling, statistical comparisons were made with a mixed-effects analysis with Dunnett's multiple comparisons test. PRNT50 was calculated using nonlinear regression curve fitting and values were compared through ordinary one-way ANOVA.