Bacterial strains, plasmids, and growth conditions

The bacterial strains and plasmids used in the present study are listed in Table 1. E. coli BL21 (DE3) cells were cultured in the Luria Bertani (LB) Media (Himedia, India) under continuous shaking at 37 °C. The L. lactis (NZ9000) cells were cultured in M17 media (Himedia, India) supplemented with 0.5% (w/v) glucose (Merck, Germany) at 30 °C under static conditions.

Table 1 List of the bacterial strains, plasmids, and viruses used in this studyVirus and cell culture

The murine melanoma cells (B16F10 cells) were purchased from NCCS (Pune, India) and murine macrophage cells (J774A.1) were procured from ATCC (USA). Both cells were maintained in complete Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL Penicillin, and 100 μg/mL Streptomycin (P/S) (HiMedia) under 5% CO2 at 37 °C.

The Influenza Type A/PR/8/1934 (H1N1) virus was purchased from ATCC (ATCC ® VR95 ™) and propagated in embryonated chicken eggs as per the published method with some modifications in our lab [10]. Briefly, 10–2 dilution of the virus stock was propagated in embryonated 10–11 days-old chicken eggs for 48 h at 37 °C under 60% humidity. The allantoic fluid was subjected to viral titer determination by haemagglutination assay and TCID50 /mL value estimation as per the published methods [10, 62, 63].

Cloning, expression, and purification of rMuIFNλ3 in E. coli

The pCMV3 untagged vector containing the full-length cDNA clone of MuIFNλ3 (IL28B) was procured from Sino Biological Inc., Japan (Catalog No: MG51306-UT). The target gene sequence (582 bp) was PCR amplified from the vector and cloned into pHis-TEV expression vector (BioBharti, India), having an N-terminal 6X-Histidine tag in-frame, and subsequently transformed into chemically competent E. coli BL21 (DE3) cells (BioBharati, India). The expression of the recombinant MuIFNλ3 protein was optimized by induction with 1.0 mM IPTG (Isopropyl ß-d-1-thiogalactopyranoside) (Sigma Aldrich, USA) when OD600 of seed culture reached ~ 0.4–0.5. Induced E. coli cells were further incubated for 6–7 h under continuous shaking at 37 °C. The cells were then collected after centrifugation at 5000×g at 4 °C, and the cell pellet was resuspended in 10 mL cell lysis buffer (6 M Guanidine hydrochloride, 50 mM NaH2PO4, and 300 mM NaCl; pH ~ 8.0). The resuspended pellet was sonicated with 5 s pulses at 35% amplitude and an intermediate stop of 10 s for 10–15 min (Sonics and Materials Inc., USA). The supernatant of the lysate was collected by centrifugation at 15,000×g for 10 min at 4 °C and the supernatant was subjected to Ni- NTA column chromatography to purify the His-tagged recombinant protein as per manufacturer’s instruction (Qiagen, USA). The eluted protein fraction was dialyzed, and the protein concentration was measured by the bicinchoninic acid (BCA) method using a commercial kit (Thermo Fisher Scientific, USA). The size and the purity of the protein were further confirmed by SDS-PAGE as well as by Western blot analysis using mouse monoclonal anti-His antibody (Thermo Fisher Scientific, USA). For Western blot analysis, the protein was transferred to PVDF (Polyvinylidene fluoride) membrane and blocked overnight with 3% BSA and probed with mouse monoclonal anti-His antibody (1:5000) for 1 h at room temperature (RT). The membrane was washed thrice with TBS (50 mM Tris–Cl, 150 mM NaCl, pH 7.5) and twice with TBS-T (TBS with 0.1% Tween-20) solution for 5 min each and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H & L) secondary antibody (1:5000) (Thermo Fisher Scientific, USA) for 1 h at RT. Finally, 3,3′-Diaminobenzidine (DAB) solution (Sigma Aldrich, USA) was used as a substrate to develop the blot.

Assessing the immunoreactivity of rMuIFNλ3 protein

To assess the immunogenicity of rMuIFNλ3, New Zealand White rabbits were used to raise polyclonal antibodies as per the standard protocol described elsewhere [10]. Briefly, 100 μg of rMuIFNλ3 protein emulsified in Complete Freund’s Adjuvant (CFA) (Sigma Aldrich, USA) was administered on day 7 (Fig. 1e), followed by secondary immunization on days 14 and 21 with 50 μg of rMuIFNλ3 protein emulsified in Incomplete Freund’s Adjuvant (IFA) (Sigma Aldrich, USA). Seven days after the last immunization, blood was collected from the marginal ear veins, serum was separated, and immunoreactivity against rMuIFNλ3 protein was checked by Western blot analysis.

Assessing the in vitro cell cytotoxicity of purified rMuIFNλ3 protein

To determine the cytotoxicity of the purified rMuIFNλ3 protein, we performed the standard MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide] assay in murine B16F10 cells and J774A.1 cells as described previously with minor modifications [64]. In brief, cells were grown in DMEM media containing 10% FBS and 1% P/S and seeded at a density of 1 × 104 cells in a 96-well tissue culture plate (Thermo Fischer Scientific, USA) and allowed to grow up to more than 80% confluency. Further, the cells were treated with rMuIFNλ3 protein starting with a concentration of 130 µg/mL and incubated for 24 h at 37 °C under 5% CO2. Following incubation, cells were then washed with 1 X PBS, and 100 µL MTT solution (0.1 mg/mL in DMEM) was added to each well and incubated at 37 °C under 5% CO2 for 4 h. The formazan crystals (MTT metabolic product) thus formed were dissolved in 100 µL Dimethyl sulfoxide (DMSO), and the absorbance was (A595 nm) in Epoch 2 spectrophotometer (Biotek). Finally, the 50% cytotoxic concentration (CC50) was determined from the dose–response curve (percentage cell survival vs. protein concentration).

Bioengineering L. lactis cells expressing rMuIFNλ3

The MuIFNλ3 gene sequence was PCR-amplified from the pCMV3 vector and cloned into a nisin-inducible pSec plasmid backbone in-frame with an N-terminal signal peptide sequence (spUSP45). The schematic of the cloning strategy and protein expression is provided in Fig. 2a, b. The modified pSec plasmid encoding the MuIFNλ3 gene sequence was then transformed into a food-grade LAB vector of L. lactis sub sp. cremoris (strain NZ9000) by electroporation. The recombinant L. lactis (rL. lactis) colonies that appeared on M17 agar plates supplemented with 0.5% (w/v) glucose and 20 µg/mL chloramphenicol were screened by colony PCR using a gene-specific primer set, followed by gene sequencing.

Detection of MuIFNλ3 protein in the culture supernatant of rL. lactis

The selected clones were tested for nisin inducibility using varied concentrations of nisin (1–15 ng/mL), induction time, and the effect on bacterial growth [10, 23]. Using optimized nisin concentration (12 ng/mL), the culture supernatant was harvested at 3 h post-induction by centrifuging the cells at 5000×g for 10 min. Ice-chilled Trichloroacetic acid (TCA) (5% w/v) (Merck) was added to the supernatant and incubated overnight at 4 °C. The precipitated protein pellet was then washed with chilled acetone (80% v/v), air-dried, and resuspended in IEF buffer [8 M Urea and 0.4 (w/v) dithiothreitol (DTT)] and subjected to Western blot analysis using polyclonal rabbit anti-MuIFNλ3 antibody (1:500 dilution) as primary antibody and HRP-conjugated goat anti-rabbit IgG (H & L) as the secondary antibody (1:2500 dilution) (Thermo Fisher Scientific, USA).

Quantification of MuIFNλ3 secreted by rL. lactis culture

Indirect ELISA was performed to quantify the MuIFNλ3 protein secreted by the nisin-induced rL. lactis cells as described previously [10, 23]. Briefly, the TCA precipitated protein pellet from 100 mL culture supernatant of both nisin-induced and uninduced rL. lactis cells were reconstituted in PBS buffer and coated onto a 96-well ELISA plate (Nunc, USA) using carbonate-bicarbonate buffer (pH ~ 9.6) overnight at 4 °C. The plate was then washed thoroughly with PBS-T (0.05% Tween-20 in PBS) and blocked with 5% BSA at 37 °C for 1 h. Next, probing was done by adding 100 μL of rabbit polyclonal anti-MuIFNλ3 antibody (1:500 dilution) to each well and incubating for 2 h at RT, followed by washing and incubating with HRP-conjugated goat anti-rabbit IgG (H & L) secondary antibody (1:2500 dilution) (BioBharati, India) for 1 h at RT. Finally, the plate was washed thrice with PBS-T, followed by the addition of 200 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to each well. Finally, the reaction was stopped by the addition of 50 µL stop solution [1 M sulphuric acid (Merck)] in each well. The absorbance was measured at 450 nm in Epoch 2 microplate reader (BioTek). A varied concentration of purified rMuIFNλ3 protein (1 to 1000 ng) expressed by E. coli was used as a standard for protein quantification [10, 23].

Assessing the immunoregulatory effect of MuIFNλ3 pre-treatment of B16F10 cells

To study the functionality and immunoregulatory effect of MuIFNλ3, murine B16F10 cells were treated with different forms of MuIFNλ3 protein. For E. coli purified rMuIFNλ3, the purified protein was directly added to the cells (1000 ng/mL) while treating the cells with MuIFNλ3 secreted by rL. lactis, we used a transwell plate module (Merck) according to the previously published method [10] (Fig. 7). Briefly, B16F10 cells (5 × 104 /well) were seeded in a 24-well plate with 1 mL of complete DMEM and incubated for 24 h at 37 °C under 5% CO2. The rL. lactis cells were grown until OD600 reached ~ 0.3 to 0.5, followed by centrifugation at 2400×g for 5 min. The cell pellet was then resuspended with fresh growth media containing nisin (12 ng/mL) and added to the upper chamber of the transwell setup. Following incubation for 4 h at 37 °C, the upper chamber was removed, and cells in the bottom compartment were kept for 12 h at 37 °C under 5% CO2 in complete DMEM. The untreated cells [treated with 1X PBS or wild-type (WT) L. lactis] cells served as controls. Next, the cells were washed, and total RNA was extracted from cells using the TRIzol method according to the manufacturer’s instructions (Invitrogen, USA), and cDNA was synthesized using the Superscript MuLV cDNA Synthesis Kit (BioBharati, India) with oligo-dT primers, following the manufacturer’s protocol. The transcriptional analysis of murine MX-1, ISG-15, IRF-7, IL-6, IL-4, and IL-10 was performed by semi-quantitative PCR analysis, using MuGAPDH as an endogenous control. The details of the specific primers used for target gene amplification are provided in Table 2.

Fig. 7

Schematic of in vitro experimental setup using a transwell system. Representative assay setup in transwell system showing murine B16F10 cells grown in bottom wells and nisin-induced rL. lactis cells were added in the upper inserts. After 4 h, cells were washed and incubated further for 12 h followed by infection with the virus (1.0 MOI). Residual infectivity of the virus was determined by cell survivability assay/CPE observation, M-gene quantification, presence of viral NP, and HA assay. In addition, before virus infection, the expression of various antiviral genes and cytokines was checked

Table 2 List of the primers used for cloning and gene expression in this studyOptimization of A/PR/8/1934 (H1N1) virus infection in B16F10 cells

To establish an in vitro cell culture model for testing the effect of MuIFNλ3 pre-treatment of IAV infections, we used murine B16F10 cells. In addition to expressing receptors for MuIFNλ3, B16F10 cells are also reported to be susceptible to IAV infections [25]. Prior to in vitro protection study, TCID50 /mL of the virus was determined for B16F10 cells. For this, the cells were grown in a 96-well plate till 80% confluency. The media was changed to fresh virus growth media consisting of incomplete DMEM media supplemented with 0.2% BSA, 1 mM MgCl2, 0.9 mM CaCl2, and 0.5 μg/mL N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-trypsin (Sigma Aldrich, USA). To this, a ten-fold serially diluted A/PR/8/1934 (H1N1) virus (in PBS) was added and incubated for 1 h at 37 °C. After 1 h, the virus infection/growth media was removed, cells were washed with PBS, and fresh DMEM media was added, supplemented with 1% FBS and 0.5 μg/mL TPCK-trypsin. After 24–72 h incubation, the Reed-Muench method was employed, and the TCID50 /mL was calculated [10, 65].

In a parallel setup, the B16F10 cells were grown in 24-well plates till 80% confluency was reached and were subsequently infected with 0.1 MOI and 1.0 MOI of the virus as described above and incubated for the next 24 h to monitor CPEs. The percentage of cell survival against virus infection was determined by the standard MTT assay, as described in the previous section.

Viral M-gene quantification

To check the optimal infection, influenza Type A/PR/8/1934 (H1N1) virus replication in B16F10 cells was quantified by assessing the viral M-gene transcripts using RT-qPCR. For this, the B16F10 cells were infected with 0.1 MOI and 1.0 MOI of the A/PR/8/1934 (H1N1) virus for 24 h. The TRIzol reagent was then used to extract the total RNA from the infected cells according to the manufacturer’s instructions (Invitrogen, USA). Approximately 500 ng of the total RNA was used for cDNA synthesis using the Superscript MuLV cDNA Synthesis Kit (BioBharti, India) with random hexamer primers following the manufacturer’s protocol. For M-gene quantification, 5 µL of 2 X SYBR green PCR mixture (Applied Biosystems, USA), 0.3 µL of the M-gene-specific primers, 2.4 µL nuclease-free water, and 2 µL of the 1:2 dilution of the cDNA template were mixed. The PCR conditions were set at one cycle at 50 ℃ for 2 min, 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. The M-gene transcript number of each group was calculated from Ct value using the standard curve plotted for the M-gene cloned into the pMD20 vector. For the standard curve, Ct values were plotted against the viral M-gene transcript number (nmolecules) as per the formula:

$$\mathrm_\mathrm=\frac}_}\times}_}}\times}_}\times^}$$

where mtemplate [ng] = quantity of the M-gene plasmid, Nbases [bp] = fragment length of the M-gene, k = average mass of one base (340 [Da/bp]), and NA. = Avogadro constant [mol−1] [10, 66].

Indirect immunofluorescence assay (IFA) to detect viral nucleoprotein

To detect viral NPs in the infected cells, IFA was carried out using the rabbit polyclonal anti-influenza NP antibody (Sino Biologicals, Japan). Briefly, B16F10 cells were grown on coverslips inside the 24-well plate and infected with the virus as described in the earlier section. Finally, the cells were washed and fixed with 4% paraformaldehyde (PFA) for 15 min at RT, then permeabilized with ice-cold acetone for 30 min. Fixed cells were incubated with a blocking solution (3% BSA in PBS-T) for 1 h at 37 ℃ and treated with the anti-NP antibody in a 1:1000 ratio. After 24 h, the cells were then incubated for 1 h at RT in the dark with FITC-labelled anti-rabbit IgG (H&L, 1:1000 dilution; Invitrogen, USA). Following washing with PBS-T and counter-staining with 4′,6-diamidino-2-phenylindole-dihydrochloride (DAPI), the cells were mounted on a glass slide using VectaShield mounting media (Vector Laboratories, USA). All images were visualized in the Leica SP8 confocal microscope using oil immersion ×63 objective (NA 1.4), and the LAS-X software was then used to acquire and process the images.

Assessing in vitro antiviral effect of MuIFNλ3 protein secreted by rL. lactis cells

To determine the antiviral effect of the rMuIFNλ3, B16F10 cells (5 × 104 /well in the 24-well plate) were co-cultured with rL. lactis in a transwell plate system, as described previously (Fig. 7). Subsequently, the cells were infected with influenza A/PR/8/1934 (H1N1) virus using 1.0 MOI of the virus by following the protocol described earlier. After 36–48 h incubation at 37 °C, the cells were processed for MTT assay, quantification of viral M-gene transcript, and IFA for detecting viral NP as per the methods described in the previous section.

Further, a viral haemagglutination (HA) assay was performed to evaluate the residual viral titer in the culture supernatant of B16F10 cells. In brief, a two-fold serial dilution of the culture supernatant (in PBS) was prepared, and 50 µL of each dilution was dispensed into a 96-well “U” bottom culture plate. After that, 50 µL of 0.5% fresh chicken erythrocyte solution (cRBC) (supplemented with 5% heat-inactivated FBS) was added to each well, and the plate was incubated for 30 min at RT. The reciprocal of the highest dilution of the sample that shows complete hemagglutination (button formation) was considered as the HA titer (HAU/50 μL) [62, 63].

Tissue-specific immune responses in mice mucosally administered with live rL. lactis cells expressing MuIFNλ3

To determine the effect of mucosal administration of live rL. lactis cells expressing MuIFNλ3, 6–8 weeks old female BALB/c mice were divided randomly into four groups: Group-1: Control (received PBS only), Group 2: WT L. lactis (received live WT L. lactis cells), Group 3: rL. lactis (received live rL. lactis expressing MuIFNλ3) Group 4: rMuIFNλ3 (received a subcutaneous injection of IFA-emulsified rMuIFNλ3 protein purified from E. coli). Further details of the dose and immunization schedules are provided in Fig. 6a and Table 3.

Table 3 Details of different experimental groups of mice for in vivo studyPreparation of rL. lactis cells for mucosal (oro-nasal) administration

The rL. lactis cells were grown in M17 medium supplemented with 0.5% (w/v) glucose and 20 μg/mL chloramphenicol at 30 °C under static conditions till OD600 ~ 0.3. The culture was induced with 12 ng/mL nisin and grown for ~ 3 h. For the vector control, WT L. lactis cells were used. The nisin-induced cells were harvested by centrifugation at 2400×g for 5 min and washed 2–3 times with PBS. The number of cells was adjusted to 1 × 109 CFU/mL in 100 μL of PBS and administered oro-nasally to the mice for three consecutive days over 3 weeks. For systemic administration, 1 μg of IFA-emulsified rMuIFNλ3 protein was administered subcutaneously on indicated time points, followed by secondary administration on days 14 and 21 with 1 µg of IFA-emulsified rMuIFNλ3 protein.

Assessing the in vivo viability of rL. lactis

To check the in vivo viability of the modified vector harboring recombinant plasmid, fresh fecal samples were collected from each study group and processed as per the established protocols with some modifications [67]. In brief, homogenized fecal pellets were plated onto an M17 agar plate supplemented with 0.5% glucose and with or without 20 µg/mL chloramphenicol and incubated at 30 °C in static conditions. Colonies that appeared in the next 24 h were randomly selected and subjected to colony PCR using MuIFNλ3-specific primers set (Table 2).

Transcriptional analysis of antiviral genes in lung and intestinal tissue

For transcriptional analysis, on day 7 post-last treatment, mice from the different treatment groups were euthanized by CO2 inhalation, and approximately 0.10 gm of the lung and intestine tissues were aseptically collected. For RNA extraction, the tissues were thoroughly washed with 1X PBS, minced, and the total RNA was extracted using the TRIzol method, per the manufacturer’s instructions (Invitrogen, USA). For each sample, 1000 ng of total RNA in a final volume of 20 μL reaction mixture was transcribed to cDNA using the Superscript MuLV cDNA Synthesis Kit (BioBharati, India). The transcriptional analysis was performed by semi-quantitative PCR for MX-1, ISG-1, IRF-7, IL-4, IL-6, and IL-10 genes, and MuGAPDH was taken as an endogenous control.

Histopathological assessment of lung and intestinal tissue of experimental mice

To see the effect of MuIFNλ3 on lung or intestinal tissue, on day 28, approximately 0.10 gm of the tissue was collected from the different treatment groups and subjected to histopathological analysis. Briefly, the tissue was sliced to a thickness of ~ 0.5 cm and fixed in a 10% formalin solution. Then, the sections were washed under running water and dehydrated using an ascending acetone gradient (70%, 90%, and 100%). The dehydrated tissues were cleaned and methodologically impregnated with melted paraffin at 62 ℃. The paraffin blocks were further sectioned and proceeded for H & Estaining [23].

Statistical analysis

The experimental data of the gene expression study was analyzed for any significant difference by calculating the band intensity for each gene using ImageLab software (Version 3.0.1). The GraphPad Prism software (Version 8) was used to plot the graphs and analyze the experimental data using the non-parametric Mann–Whitney U test. A p-value less than 0.05 (p < 0.05) was considered statistically significant.