Ethics statement
Animal studies were approved and conducted in compliance with all the regulations stated by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia (UGA; under AUP A2019 01-004-A2). Vaccination and challenge studies were conducted under ABSL-2 conditions at the Davison Life Sciences Complex, UGA. Animal studies and procedures were performed according to the Institutional Animal Care and Use Committee Guidebook of the Office of Laboratory Animal Welfare and PHS policy on Humane Care and Use of Laboratory Animals. Animal studies were carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org).
Cells, eggs, and mice
Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells (HEK293T) were a kind gift from Robert Webster, St Jude Children’s Research Hospital, Memphis, TN, USA. Cells were used for the rescue of the different viruses used in this study. Cells were maintained at 37°C, 5% CO2, in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA) and 1X antibiotic/antimycotic solution (ATB/ATM, 100 IU/mL of penicillin, 100 µg/mL for streptomycin, and 0.25 µg/mL of amphotericin B, Gibco, ThermoFisher Scientific). Specific pathogen-free (9-11 days old) embryonated chicken eggs (ECEs) used for virus propagation and stock titration were obtained from Charles River (Wilmington, MA).
Male and female DBA/2J mice (5 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME) and raised until 7 weeks of age. Mice were housed in negative pressure caging in the Davison Life Sciences Complex, University of Georgia and were provided food and water ad libitum for the duration of the experiment.
Recombinant Plasmids
The PB1 plasmids containing the re-arranged M (PB1BM2) and re-arranged NS (PB1BNS2) and the M and NS plasmids encoding early stops codons have been previously described 18. The PB1 plasmid containing the temperature sensitive mutations and C-terminal HA tag (pDP-PB1att) was previously described 21,29. The plasmid pDP_B/Bris-PB1_Nluc (B/Bris) was produced carrying a chimeric PB1 gene segment from B/Brisbane/60/2008 (B/Bris) WT with an in-frame C-terminal nanoluciferase ORF (Nluc) between the C-terminal end of the PB1 ORF and the 3’UTR. The plasmid pDP2018-FluB-IGIP-HA was produced by subcloning the ORF of the B/Bris BHA into a reverse genetics vector intermediate carrying a synthetic DNA fragment (Genscript, Piscataway, NJ, USA) encoding the 5′ untranslated region (UTR) and signal peptide sequence of BHA (B/Bris), IGIP mature peptide sequence, additional spacer sequences, and sequences encoding the transmembrane domain, cytoplasmic tail, and 3’ UTR of B/Bris. Plasmids were propagated in E. coli Top10 cells (ThermoFisher Scientific). Plasmid were purified using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). Plasmid sequences were confirmed by Sanger sequencing (Psomagen, Rockville, MD, USA)
Rescue of recombinant FluB-RAM/IGIP, FluB-RANS/IGIP, and Flu-att/IGIP viruses.
Rescue of recombinant FluB-RAM, FluB-RANS, and Flu-att viruses were previously described 21,29,30. Rescue of FluB-RAM/IGIP, FluB-RANS/IGIP, and FluB-att/IGIP was performed similarly. Briefly, plasmids encoding wild type gene segments from B/Bris (PB2, PA, NP, NA and either NS and/or M, respectively) were mixed with the corresponding set of plasmids to produce the FluB-RAM/IGIP (pSCG-PB1BM2, pDP2018-FluB-HA-IGIP and pSCG-BM1-∆M2), FluB-RANS/IGIP (pSCG-PB1BNS2, pDP2018-FluB-HA-IGIP and pSCG-BNS1-∆NEP), and FluB-att/IGIP (pDP-PB1att and pDP2018-FluB-HA-IGIP) viruses. Recombinant viruses were propagated and titrated in 11-day-old SPF ECEs incubated at 33ºC for 48 h. First passage in ECEs (E1) virus stocks were stored at −80°C until further use.
Stability of recombinant viruses through Serial Passages in ECEs
Six additional serial passages were performed in 11-day-old ECEs from the E1 stock until E7 as previously described 18. Aliquots from each passage were stored at −80°C until needed. RNA was extracted from allantoic fluids collected at each passage and from the original virus stock using the QIAmp Viral RNA isolation kit (QIAGEN). The PB1, HA, M, and/or NS gene segments were amplified by RT-PCR using SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher Scientific). Sanger sequencing (Psomagen) was then performed from the resulting RT-PCR products to assess the stability after passaging by confirming the rearrangement of the PB1 gene segments, the presence of the IGIP coding sequence, and the early stop mutations within either the M or the NS gene segments, respectively.
Virus Growth Kinetics
MDCK cells were seeded in 6-well plates and incubated overnight at 37ºC, 5% CO2. The next day, cells were inoculated with 0.01 MOI of either B/Bris WT or recombinant viruses contained in 500 µL, each in triplicate wells. Inoculated MDCK cells were incubated for 1 h at 35ºC, 5% CO2, gently rocking the plates every 15 min. Next, the virus inoculum was removed, cells were washed twice with PBS and replenished with 2 mL of fresh Opti-MEM I (ThermoFisher Scientific) supplemented with 1X ATB/ATM and 1 µg/mL of L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated Trypsin. Plates were incubated at either 33º, 35º, or 37ºC, 5% CO2. Supernatants (200 µL) were collected at 0, 12, 24, 48, 72, and 96 h post-inoculation (hpi) and stored at −80 °C until processed. Samples were titrated by TCID50 in MDCK cells. Virus titers were calculated using the Reed and Muench protocol and plotted as the mean TCID50/mL ± SD 31.
Vaccine Safety and Efficacy
Please note that the vaccine safety and efficacy study described herein was part of a larger study that included a set of recombinant viruses whose efficacy profiles have been previously described 18,29. Following a prime-boost strategy 20 days apart, 7-week-old DBA/2J mice were primed intranasally (i.n.) with 50 µL of inoculum at a virus dose of 106 EID50/mouse. Male and female mice, housed separately, were allocated into 8 groups (½ males and ½ females) as follows: Mock-vaccinated (mock-vac, PBS, n=24); FluB-RAM/IGIP (n=12); FluB-RANS/IGIP (n=12); FluB-att/IGIP (n=12), FluB-RAM (n=12); FluB-RANS (n=12); FluB-att (n=12); and B/Brisbane/60/2008 (B/Bris wt, n=12, control virus). Mice were monitored daily for clinical signs, body weight changes, and mortality for up to 12 days following vaccination (dpv) and boost (dpb). On day 19 dpb, a subset of mice from each group (n=4/group, ½ females, except where noted) was anesthetized with isoflurane, terminally bled to collect sera, and subsequently humanely euthanized (Figure 2A, study design).
Mice from the vaccine safety study (n=8/group, ½ females) were challenged i.n. with a lethal dose (107 EID50/mouse, ~1,000 MLD50) of the B/Brisbane/60/2008 PB2-F406Y (B/Bris/F406Y) strain 21 contained in 50 µL of PBS. A subset of mice in the mock group (n=8, ½ females) remained unchallenged and served as negative controls. Mice were monitored twice daily to record clinical signs and mortality for up to 14 days post-challenge (dpc). Bodyweight was recorded for up to 12 dpc. At 14 dpc, survivors were anesthetized with isoflurane, terminally bled to collect sera, and subsequently humanely euthanized (Figure 2A, study design).
Hemagglutination Inhibition (HI) Assay
RDE-treated sera collected at 19 dpb (n = 4/group, ½ females) and 14 dpc (n=8/group, ½ females) was used for HI assays performed in V-bottom microtiter plates, using 4 hemagglutination units (HAU) of viral antigen (B/Bris WT) per 25 µL, as recommended by the OIE and as previously described 32, using a suspension of turkey red blood cells (0.5%). HI titers were plotted using Prism v9 (GraphPad, San Diego, CA, USA). The limit of detection was a dilution of 1/10, and samples with undetectable titers were assigned a dilution value of 1/8 for statistical purposes.
Recombinant B/Brisbane/60/2008 and B/Wisconsin/01/2010 viruses expressing nano luciferase (Nluc), B/Bris/Nluc and B/Wis/Nluc
The recombinant Victoria lineage B/Bris/Nluc and Yamagata lineage B/Wis/Nluc viruses expressing PB1-Nluc were generated for a modified virus neutralization assay as previously described 19. Rescue of B/Bris/Nluc was achieved with 7 B/Bris WT plasmids co-transfected with pDP_ B/Bris-PB1_Nluc (7 + 1). Rescue of B/Wis/NLuc was performed using 5 plasmids from B/Bris WT co-transfected with pDP_ B/Bris-PB1_Nluc, and the plasmids encoding the HA and NA from B/Wisconsin/01/2010. The recombinant viruses were propagated in 11-day-old ECEs and titrated by TCID50 in MDCK cells. Virus identities were confirmed by Sanger sequencing. Nluc activity from infected cells was detected using the Nano-Glo Luciferase Assay System (Promega, Madison, WI) following manufacturer’s instructions.
Virus Neutralization Assay based on Nluc activity (VNluc)
Two-fold dilutions in PBS of sera treated with receptor destroying enzyme (RDE; Denka Seiken, VWR, PA, USA) collected at 19 dpb (n=8/group, ½ females) was used for VNluc assays. 100 TCID50 contained in 50 µL of either B/Bris/Nluc (homologous) or B/Wis/Nluc (heterologous) were added to each of the corresponding wells containing serum dilutions. Serum/virus mixes were incubated at 37°C for 1 h. Thereafter, the serum/virus mixes were added to MDCK cell monolayers and set to incubate at 4°C for 15 min and then at 35°C for 45 min. After incubation, the serum/virus mixes were removed from the cell monolayers and 200 µL of Opti-MEM (Gibco) supplemented with 1X ATB/ATM and 1 µg/mL of TPCK-Trypsin was added to each well. Plates were set to incubate for 72 h at 35°C, under 5% CO2. Virus neutralization titers were determined by measuring luminescence activity using the Nano-Glo Luciferase Assay System (Promega, Madison, WI) following the manufacturer’s instructions.
Microarray for IgG and IgA Determination
Sera collected at 19 dpb and 14 dpc, and nasal washes collected at 14 dpc were analyzed through protein microarrays to determine anti-HA, -NA, and -NP IgG and IgA levels from multiple Victoria- and Yamagata-like FLUBVs (Table 1). Purified FLUBV protein antigens were purchased from Sino Biological (Wayne, PA) (Table 1). Microarrays were carried out as described previously 19,33. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. Due to low MFI signals, those from HA1 B/Florida/4/2006 (Catalog# 11053-V08H1, HEK293), HA1 B/Brisbane/60/2008 (Catalog# 40016-V08B, E. coli), and NA B/Brisbane/60/2008 (Catalog# 40203-VNAHC, HEK293) were not utilized in the analyses. MFIs were plotted and analyzed using Prism v9 (GraphPad).
Statistical analyses:
Virus growth kinetics were analyzed using the Gompertz growth non-linear regression model followed by Area Under the Curve (AUC) analysis, and Brown-Forsythe and Welch ANOVA plus Dunnett’s T3 post hock analysis to identify differences in growth rate between vaccine candidates. Two-way ANOVA was employed to determine virus growth differences by timepoint. VNluc assay curves were analyzed using multiple t-tests followed by the Holm-Sidak method to correct for multiple comparisons. Pre-challenge and post-challenge mean HI titers were analyzed by 2-way ANOVA followed by post hock Sidak’s multiple comparison test to determine differences between sex and vaccine treatment groups. Survival curves were analyzed using the Log-rank test. Brown-Forsythe and Welch ANOVA or Two-way ANOVA were performed to analyze the microarray data to compare responses between sex and/or vaccine groups, followed by a Dunnett’s T3 or a Tukey’s multiple comparisons test, respectively. The level of significance for all the analysis was considered at p<0.05. P values were adjusted to account for multiple comparisons. All the analyses were performed using Prism v9.3.1.