Lactic acid bacterial surface display of scytovirin inhibitors for anti-ebolavirus infection

Scytovirin (SVN) is a lectin from cyanobacteria which has a strong inhibitory activity against Ebola virus infection. We engineered scytovirin as the inhibitor for surface display of lactic acid bacteria to block Ebola virus infection. Two different bacterial strains (Lactobacillus casei and Lactococcus lactis) were successfully engineered for scytovirin expression on the bacterial surface. These bacteria were found to be effective at neutralizing pseudotyped Ebolavirus in a cell-based assay. This approach can be utilized for prophylactic prevention, as well as for treatment. Since lactic acid bacteria can colonize the human body, a long-term efficacy could be achieved. Furthermore, this approach is also simple and cost-effective and can be easily applied in the regions of Ebola outbreaks in the developing countries.


Introduction
Ebola virus (EBOV) is an enveloped negative-stranded RNA virus which can cause severe Ebola viral disease (EVD) in humans and nonhuman primates (Jacob et al., 2020).EBOV belongs to the family Filoviridae along with Marburg virus that also causes a similar disease.Ebolavirus in Zaire was first identified in 1976 in Africa (Bowen et al., 1980), now five Ebola species have been recognized (Kuhn et al., 2019): Zaire ebolavirus (ZEBOV or EBOV), Sudan ebolavirus (SUDV), Tai Forest ebolavirus (TAFV), Reston ebolavirus (RESTV) and Bundibugyo ebolavirus (BDBV).Ebolavirus has produced more than 20 outbreaks in humans with high mortality rates from 25 to 90% (Feldmann and Geisbert, 2011;WHO, n.d.).The recent large Ebola outbreak in 2014 in West Africa infected more than 28,000 people and more than 11,000 died (Cenciarelli et al., 2015).It is evident that this emerging and reemerging viral pathogen represents a great threat to human health.However, we do not have any medicines to treat this lethal viral disease until December 2019, when a vaccine (rVSVΔG-ZEBOV-GP) was approved by FDA for limited use against Zaire Ebolavirus, and in 2020, two antibody drugs (mAb114 and REGN-EB3) were approved for treating this viral infection.It is apparent that more medicines are required for fighting against this deadly, infectious viral disease.
Because scytovirin demonstrated potent antiviral activity against Ebola infection, we used it to develop a novel live microbicide for Ebola infection control.This approach displayed scytovirin on the surface of Lactic acid bacteria (LAB) for delivery against Ebola virus infection.These bacteria can be delivered into mucosal surfaces of the body (e.g., mouth, nose, and GI tract) that are the ports of viral entry where they can colonize and replicate.Previous studies using LAB expressing the antiviral lectin CV-N demonstrated that the bacteria can pass through the GI tract unharmed and stably produce lectins to inhibit HIV (Lagenaur et al., 2011, Li et al., 2011, Brichacek et al., 2013).Therefore, this approach for application of viral inhibition should be safe, long-lasting and effective.Furthermore, this approach is cost-effective and easy-to-use, so it is especially valuable for use in those outbreak regions in Africa.Here, we reported the in vitro data we have achieved successfully in this research direction.

Scytovirin constructs for surface display on LAB
To express the SVN protein on the surface of LAB, constructs were created which included the Lactate dehydrogenase promoter (Pldh), signal peptide (SP), cell membrane anchor protein (ANC), E-tag marker (E), and the protein marker (GFP).The constructs were built up based on our previous plasmid pWZ486 (Wei et al., 2019) derived from pTRKH3-ldhGFP (Addgene).The SVN-E-tag-GFP fusion sequence replaced the CD4 gene sequence in pWZ486 using the SacI/XhoI restriction sites.The constructs pLSVN3 and pLSVN7 were verified by DNA sequencing and PCR using the specific primers (Table 1) marked in the construct maps.

Electroporation
Plasmids were transformed into the Lactic acid bacteria (LAB) by electroporation as previously described (Wei et al., 2019;Welker et al., 2019).Briefly, overnight cultures of LAB cells were diluted (1:50) into fresh MRS media with 1% glycine and incubated at 37°C without shaking for 2 h.Cells were harvested and treated with 50 mM EDTA (pH 8.0) for 15 min, followed by two washes with ice-cold electroporation buffer (0.5 M sucrose) and resuspended in electroporation buffer (1/100 volume of the initial culture).50 μL of cells were mixed with plasmid DNA and incubated on ice for 15 min.The mixture was added to an ice-cold 0.2 cm GenePulser (Biorad) cuvette and pulse was immediately applied at the conditions of 10 KV/ cm, 200 Ω, and 25 μF.Cells were suspended in 1 mL MRS broth with 2 mM CaCl2 and 20 mM MgCl2 and then incubated at 37°C for 4 h.Cells were pooled on MRS plates with 5 μg/mL erythromycin and cultured as described above.To verify transformation, single colonies were picked and added to 25 μL PCR master mix (Promega, M791B) containing 1 μM forward and reverse primers (Table 1).PCR amplification occurred in a SimpliAmp thermocycler (Applied BioSystems, A24811) under the following conditions: Stage 1; 95°C for 5 min, Stage 2 (35 cycles); 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, Stage 3; 72°C for 7 min.PCR products were visualized on 1% agarose gel electrophoresis.

Flow cytometry
For the pLSVN3 construct transformed into L. casei, the fusion protein was detected based on the GFP fluorescence.Bacteria were washed three times with PBS and analyzed on a BD FACSAria using a 488 nm laser.For the pLSVN7 construct transformed into L. lactis, the bacteria were first stained for 1 h with two primary antibodies: mouse monoclonal anti E-tag (Novus NBP2-67081) and rabbit polyclonal anti SVN (A64238-050, Epigentek).Goat anti mouse conjugated with AlexaFluor488 (A-11011, ThermoFisher) and goat anti rabbit conjugated with AlexaFluor594 (A-11012, ThermoFisher) were used as secondary antibodies, respectively.Bacteria were analyzed on a Beckman Coulter CytoFLEX FX at 488 nm and 561 nm.Unstained bacteria and stained wild-type bacteria were used in all experiments for an appropriate gating strategy.

Confocal microscopy
Confocal microscopy was performed using the same fluorescent fusion protein or antibody combinations as described for flow cytometry (above).Following the antibody staining, bacteria were pelleted, resuspended in 10 μL PBS, and transferred to a microscope slide with cover slip.Images were captured using a Nikon A1R-Ti2 (Nikon Instruments, NY, USA) inverted confocal system.

Pseudotyping viruses
The Ebola pseudotyped viruses were made from a HIV-1 backbone plasmid, pSG3 ΔEnv (NIH HIV Reagent Program).The Ebola Envelope gene (GP, Zaire ebolavirus, GenBank: AIO11753.1),was synthesized by GenScript and cloned into pcDNA3.1(+).Both plasmids were co-transfected into 293 T cells in a 10 cm plate using transfection reagent polyethyleneimine (PEI).Three days post transfection, the medium was harvested and centrifuged at 500 g to remove cell debris, and then the supernatants were stored at −80°C (Platt et al., 2009;Wang et al., 2022).The viral titers were determined by reverse transcriptase assay.

Reverse transcriptase assay
The titers of pseudotyped viruses were determined by Reverse transcriptase assay (RTA) (Wei et al., 2019).500 μL of pseudotyped virus stock was spun at 14,000 g for 2 h at 4°C to precipitate the virus.The viral pellet was resuspended in a Triton X-100-based suspension  buffer and vortexed, followed by three rapid freeze-thaw cycles to lyse the viruses.50 μL of reaction mix [Oligo-dT Poly-A and 3 H-dTTP (PerkinElmer)] was added, and the samples were incubated at 37°C for 1 h in a heating block.Then, the samples were pipetted onto DEAE Filter mat circle papers (PerkinElmer), followed by three 10 min washes in 2X SSC buffer, and one 10 s wash in 100% ethanol.The filters were dried at room temperature and analyzed using a scintillation counter to measure the incorporation of 3 H-dTTP into cDNA.The average CPM values from duplicates were determined.

Virus adsorption
Pseudotyped Ebola virus stocks were mixed with wild-type bacteria or engineered bacteria (~5×10 7 /mL) in 1.5 mL microcentrifuge tubes.The mixtures of bacteria and viruses were incubated for 1 h at room temperature.Then the tubes were spun for 1 min at a 13,000 g to remove the bacteria and bound pseudotyped virus.The supernatants were collected, and the viral titers determined by RTA.

Virus neutralization
For the neutralization assay, pseudotyped Ebola virus was mixed with wild-type or engineered bacteria in the same manner as the adsorption assay described above.After centrifugation to remove the bacteria and bound pseudovirus, the remaining supernatants were applied to TZM-bl cells which were used as the target cells due to their ability to express luciferase when infected (Platt et al., 2009).The TZM-bl cells were set at a density of 6.0 × 10 3 per well in a 96-well plate.Each neutralization assay was performed in triplicate with 5,000 RT units of pseudovirus per well used as the starting titer.Two days post-infection, the supernatants were removed, the cells were washed once with PBS, lysed in 1x Passive Lysis Buffer, and frozen at −80°C.The plates were then thawed, and luciferase activity was measured using beetle luciferin substrate (Promega) in a Veritas Luminometer.

Statistical analyses
Statistical analyses were conducted for virus adsorption and virus neutralization data using GraphPad Prism software (version 9.0).The significances were determined by using unpaired two-tailed Student's t-test at p-value ≤0.05.

SVN gene cloning and expression
The scytovirin gene was initially synthesized by adding an E-tag at the N-terminus and cloned into pET28a with SacI/XhoI sites.The SVN plasmid was transformed to E. coli BL21 (DE3) cells for protein expression.The SVN fusion protein was observed under induction of 1 mM IPTG by Coomassie blue staining.The induced band of ~16kD fusion protein was noticed clearly (Figure 1A).To further confirm this protein, Western blotting was carried out by using E-tag, His-tag, and SVN specific antibodies.Positive bands were observed from all three different specific antibodies (Figure 1B), suggesting that the ~16kD SVN-E-tag fusion protein is expressed correctly.Figure 1C shows the construct design including the two His-tags encoded by the pET28a vector, as well the SVN ribbon structure with two highly similar domains (D1 and D2) for carbohydrate binding (Moulaei et al., 2007).Thus, the SVN-E-tag expressing construct can be further utilized for following bacterial engineering.

SVN gene engineering for surface display on Lactobacillus casei
The SVN-E-tag construct was cloned into our previous plasmid (pWZ486) through SacI/XhoI sites to produce a new construct designated as pLSVN3 (Figure 2A).This GFP-E-tag-SVN fusion protein with the anchor has been modeled and shown in Figure 2B, which should be flexible for capturing viral particles.The construct was created first in E. coli DH5α cells and demonstrated the PCR fragment of ~1800 bp of GFP-E-tag-SVN sequence was correctly amplified using primers 154 and 037 (Figure 2C).To engineer the L. casei strain, the SVN construct pLSVN3 was transformed by electroporation into the L. casei for protein expression.The transformants were verified by PCR.A ~ 1800 bp PCR band from the amplification with primers 154 and 037 revealed that the pLSVN3 plasmid was transformed into the L. casei cells (Figure 2D).A ~ 75kD band corresponding to the fusion protein size (GFP-E-SVN-ANC) was detected by Western blotting using specific GFP and SVN antibodies (Figure 2E), suggesting that the SVN-GFP based fusion protein was produced from the L. casei cells.The smaller size band (~43kD) that appeared is the partial fragment of the fusion protein complex without the anchor domain (ANC).Flow cytometry analysis revealed the rate of SVN-GFP positive cells was 33.5% (Figure 2F a and b).To determine whether these protein inhibitors are displayed on the bacterial surface, confocal microscopy was used to visualize these SVN-GFP fused proteins.The pictures from confocal microscopy exhibited the fusion protein in green (GFP) displayed on the surface of bacteria (Figure 2F b).The biological functional studies were conducted for virus binding and neutralization.The engineered bacteria showed a moderate binding activity to the pseudotyped Ebola particles, reduced 37.2% of virus load, but the wild-typed (WT) bacteria also showed weak binding to the pseudotyped viruses because of the unspecific binding, reduced 21.8% (Figure 3A).The virus inhibition assay indicated that SVN-expressing bacteria had a moderate inhibition activity against pseudotyped Ebola virus infection which was 39.1%, but the WT-bacteria also had 23% inhibition (Figure 3B).These moderate functions may be due to the lower positive rate of SVN-expressing cells and the GFP interference of SVN binding in the SVN-GFP-ANC fusion protein complex.

SVN gene engineering for surface display on Lactococcus lactis
Lactococcus lactis is another type of LAB which has a round shape and is widely used in the production of buttermilk and cheese.Thus, it is a very safe host strain candidate for anti-Ebola infection.We created the construct to remove the protein marker GFP but keep the E-tag marker from the construct pLSVN3, and this new construct was designated as pLSVN7 (Figure 2A).The construct pLSVN7 was transformed into the L. lactis using electroporation method.Positive bacterial colonies in the erythromycin selection plates were picked for further evaluation.The PCR method was first used to confirm the pLSVN7 plasmid was in the bacterium of L. lactis using primers 048 and 199.The expected ~700 bp band was identified (Figure 4A).Western blotting further demonstrated that SVN fusion protein (E-tag-SVN-ANC) was shown and in the correct molecular weight of 55kD both in anti-E-tag and anti-SVN specific antibodies.The 16kD SVN-E-tag fusion protein from the E. coli BL-21 (DE3) lysate as the positive control was also shown in the Western blots (Figures 4B,C).These Western blotting data demonstrated that the E-tag-SVN-ANC fusion protein was expressed in L. lactis.Next, the Confocal microscopy method was used to verify the surface display.Two colors of fluorescence were used for labeling E-tag (green) and SVN (red), respectively.The results demonstrated that the SVN fusion protein is clearly expressed on the surface of the bacterium.The single color, green or red and the merged color of green and red indicated the overlapping presence of fusion protein surface expression (Figure 5).Furthermore, Flow cytometry analysis was also conducted, and the data presented in Figure 6, shows that the positive rate of bacteria has reached 92.4%.The results demonstrated that the SVN-fusion protein is unambiguously expressed on the surface of these bacteria.
Functional studies were performed against the pseudotyped Ebola viruses.First, we used the viral particles adsorption method to evaluate the bacterial ability for capturing the viruses.We then tested

Discussion
Bacterial therapy is a promising approach for human health and the common use of probiotics demonstrates its great potential (Yadav et al., 2020;Tegegne and Kebede, 2022).Using commensal bacteria for treating viral diseases has received broad attention since the bacterial microbicide has its advantages such as easy-to-use, cost-effective, and long-term   Confocal analysis of SVN-engineered Lactococcus lactis.Antibodies used for staining: AF-488 (green) for E-tag; AF-594 (red) for SVN.For more details, please see the Method section.efficacy (Ramachandran and Shanmughavel, 2009;Obiero et al., 2012).Especially, natural lectins as inhibitors can reduce unnecessary immune responses and enhance specific inhibiting activities for therapeutic applications (Akkouh et al., 2015;Mitchell et al., 2017;El-Maradny et al., 2021;Fernandez Romero et al., 2021;Mazur-Marzec et al., 2021;Naik and Kumar, 2022).For developing this method to use bacteria for antiviral diseases, bacterial engineering is a critical step.In this report, we engineered two types of Lactic acid bacteria (LAB) for surface display of the Ebola inhibitor scytovirin (SVN).Both bacterial strains successfully displayed scytovirin on the surface, suggesting the applicability of our constructs for surface expression of SVN fusion protein in lactic acid bacteria.The fluorescence of the SVN lectin fusion molecules when stained with GFP or SVN antibodies perfectly overlapped on the surface of the bacterium indicating a high display of inhibitors.The positive rate of engineered bacteria is higher in L. lactis (92.4%) than in L. casei (33.5%), suggesting that lower positive rate in L. casei is due to the higher genetic instability because certain bacteria may lose the SVN plasmid or SVN gene during the replication, especially when the antibiotic pressure is reduced.Another possibility is the genetic recombination between the SVN plasmid and host genome by which the plasmid composition could be changed or damaged.We found that LAB were capable of quickly developing resistance to erythromycin (5 μg/mL) selective pressure, especially when grown in stationary, liquid culturing conditions, suggesting that the bacteria lose the plasmid (data not shown).Thus, increased erythromycin concentration is usually needed to get more genetically stable strains.In general, using a genomic integration method for engineering would be better than plasmid transformation for making genetically stable strains, which is essential for clinical or therapeutic use.In this research, L. lactis was found to be highly stable and would be assessed for in vivo (mice) colonization, and for protective efficacy against challenge with infectious Ebola viruses conducted in the BSL-4 containment.
In the construction of fusion protein plasmids, the GFP protein marker seems unnecessary.Small protein tags such as E-tag appear to be adequate for detection and evaluation during the studies.Removing GFP reduced the size of the fusion protein by ~27kD, nearly 3x the size of SVN, maximizing the exposure of SVN inhibitor and preventing possible steric hindrance from GFP. Eliminating GFP from the fusion protein also presents another advantage for in vivo applications by avoiding the potential for immunogenicity and cytotoxicity caused by the GFP protein (Ansari et al., 2016).
In this report, our data from the in vitro study with pseudotyped Ebola virus serves as a proof of concept of using engineered commensal bacteria for blocking Ebola infection.Commensal bacteria have previously been demonstrated to inhibit HIV-1 infection (Nahui Palomino et al., 2017), and it is likely they provide a similar baseline level of protection at mucosal surfaces against other viral infections.This study indicates that this baseline level of protection is around 20-25% for WT L. lactis.Our data shows that lectin displaying bacteria can bind and neutralize significantly more viruses compared to the wild-type commensal bacteria, suggesting that engineered bacteria could be used prophylactically to improve upon the health benefit already provided by commensal bacteria by decreasing the likelihood of contracting a viral disease.This is contingent upon the engineered bacteria successfully colonizing various mucosal surfaces in the body and stably producing recombinant protein.To investigate this, we will test different routes of administration in mice (oral, nasal, rectal, etc.) to determine the stability of the L. lactis strain under different physiological conditions, as well as determining the efficacy of the bacteria to prevent in vivo infection prior to advancing to clinical trials.In conclusion, the commensal bacterial based anti-Ebola approach is promising and will be beneficial for combating this deadly viral disease.

FIGURE 1
FIGURE 1 Expression of SVN-E-tag-His-tag in bacterial Escherichia coli K12 BL21 cells.(A) Coomassie blue staining, the induced fusion protein band was marked with the red arrow.(B) Western blots showing the specific positive bands.The specific antibody for anti-SVN is a scytovirin polyclonal antibody.(C) The SVN construct in pET28a and the SVN 3D structure, showing the two similar domains (D1 and D2) in magenta.H, His-tag, E, E-tag.

FIGURE 3
FIGURE 3 Functional studies of SVN-engineered L. casei bacteria.(A) Virus adsorption assay.(B) Virus neutralization assay.Wild-type (WT) L. casei as the negative bacterial control; virus only used as the positive control; DMEM medium or cells as the negative control.

FIGURE 6
FIGURE 6Flow cytometry analysis of SVN-engineered Lactococcus lactis.The 1st antibody (AF-488 conjugated, green) used is for staining E-tag; The 2nd antibody used (AF-594, red) is for staining SVN.

TABLE 1
List of strains, cell lines, plasmids, and primers used in this study.