Using FHR-1 as a "model" dimerization domain containing FHR, structural analysis of the Sbi-III-IV:C3d:FHR-1 tripartite complex, using SAXS, indeed suggests the formation of a dimer mediated by FHR-1 domain 1 and 2 and provides details of the role of the extended unfolded nature of domain III in the binding of FHR-1 (**Figure 4**). The molecular basis of the preferential binding of FHR-1 over FH cannot easily be explained on the basis of differences in amino acid sequence between the two complement regulators, since their C3d binding regions (SCR 4– 5 of FHR-1 and SCR 19–20 of FH) share 99% sequence identity. However, our SAXS analyses, and binding studies using C3d(g) or iC3b as ligands (**Figures 3**, **4**), indicate that the C-terminal regions of FHR proteins are readily exposed, unlike those of FH that exist in a "latent" conformation with the C-terminal part of the protein folded back and partially blocked (47–50). The dimeric physiological state of FHR-1 and the other FHRs tested in this study is also likely to enhance their ability, due to increased avidity, to assemble a tripartite complex.

Analysis of the hydrodynamic volume of the Sbi-III-IV:C3d complex using switchSENSE highlighted a significant contraction of the normally extended conformation Sbi-III-IV structure (34) caused by the K173A substitution in domain III (**Table 2**). SAXS analysis confirms these findings, showing a partially kinked N-terminal structure of domain III in K173A with reduced conformational freedom (**Figures 4E–G**). The contraction of the Sbi-III-IV structure caused by the K173A substitution suggests that the normally flexible and extended conformation of domain III plays an important role in the recruitment of FHRs, especially FHR-5 into the tripartite complex after the initial interaction between Sbi-IV and C3b. Previous structural analyses of the Sbi's domain III, using NMR, revealed that this domain is indeed natively unfolded (51).

Based on the structural and functional information described here we decided to construct a Sbi-III-IV-Ag85b fusion construct that could be used to test its effect on the immune response against this model antigen in vivo. We chose Mycobacterium tuberculosis Ag85b, a fibronectin-binding protein with mycolyltransferase activity (52), because it is known to be immunogenic and previously suggested as a vaccine candidate (53). Indeed, there is evidence that Ag85b can elicit both humoral and cellular immune reactions in patients with TB, but there is conflicting evidence of its efficacy as a vaccine (54, 55), suggesting adjuvants may improve its overall immunogenicity. This target also gives scope to allow further testing in animal models of disease (56). Structural analysis, using Circular Dichroism and SAXS confirmed that secondary structural elements of both parent proteins have been preserved in the fusion protein construct and that the crucial functional Sbi domains are accessible for interactions with complement (**Figure 5** and **Figure S5**). We also show that the Sbi-III-IV-Ag85b fusion construct can induce AP activation and is opsonized with C3 breakdown products (**Figure 5C**).

With AP activation in human and mouse serum confirmed (**Figures 5**, **6**), we opted to use straightforward immune response, IgG titer, analysis to demonstrate the potential of Sbi-III-IV to trigger complement in vivo and act as a vaccine adjuvant in a mouse model, in a similar manner to many previous studies (57). Our data herein firstly indicates that Sbi-III-IV can activate mouse complement in an analogous manner to that of the human complement system. This obviously allows direct analysis of these pro-vaccine compounds in both mouse and human model systems (a huge advantage to previous C3d based adjuvants) (13), indeed Sbi-III-IV has acted as a C3 activator in all species tested thus far (data not shown). As predicted from the in vitro work, the opsonisation of fusion proteins or co-immunized antigen by mouse complement breakdown fragments results in a significant increase in the immunogenicity of Ag85b, with increased IgG titres noted in the presence of fused or co-immunized Ag85b (**Figure 6**). The adjuvant function both increased the intensity of the response and the rate of the response when compared to Ag85b immunized alone. We will need to further explore the potency of this response to that of common adjuvants and with a mix of target antigens to fully assess the utility of Sbi-III-IV as a universal vaccine adjuvant. For instance, comparison of the action of Sbi-III-IV to the Glaxo-Smith-Kline's adjuvant systems, particularly AS01 (58), or to MF59 (59) may be of key interest and recent approaches may provide ideal pre-clinical model systems to facilitate this (60, 61) before progression to clinical studies. This is because our data provides evidence of a 4-fold increase in humoral response whilst AS01 has been demonstrated to have a much more significant effect of T cell effector function (58). The work is ongoing but the data herein demonstrate the initial proof of concept.

In summary, we have demonstrated that Sbi-III-IV triggers consumption of complement component C3 via activation of the alternative complement pathway, by acting as a competitive antagonist of FH via the recruitment of FHRs into dimeric tripartite complexes that can protect C3b bound to Sbi (**Figure 6D**). It is likely this provides a stable nidus for alternative pathway mediated C3 convertase generation, i.e., local fluid phase C3bBb generation that overwhelms any local complement regulators, providing the potential for bystander lysis, or opsonisation of surfaces. Our ability to harness this potential, targeting complement opsonisation to the surface of an antigen (in this case from Mycobacterium) and therefore use Sbi-III-IV as a vaccine adjuvant clearly demonstrates Sbi-III-IV has great potential for use with a range of antigens across multiple species, including humans, although more work remains to make that a reality.

#### MATERIALS AND METHODS

#### Proteins, Antibodies, and Sera

Factor H (FH), C3b, factor B (FB), factor D (FD), factor I (FI), properdin (FP), FI-depleted serum, goat anti-human C3 polyserum, and goat anti-human FB polyserum were purchased from Complement Technologies (Tyler, TX). FHR-11−2, FHR-1, −2, and −5 used in the tripartite complex reconstruction and binding competition assay were produced using Chinese Hamster ovary cell culture [as previously described Nichols et al. (62)]. Horse radish peroxidase (HRP) conjugated rabbit anti-goat immunoglobulin polyserum and HRP-conjugated Streptavidin were acquired from Sigma Aldrich. HRP-conjugated goat anti-rabbit immunoglobulin G (Thermo Fisher, catalog no.<PHONE_NUMBER>), HRP-conjugated rabbit antimouse immunoglobulin G (Thermo Fisher, catalog no. 31452) and biotin-conjugated FH monoclonal antibody OX24 (catalog no. MA5-17735) were purchased from Thermo Fisher Scientific. The goat anti-human FH polyclonal serum (catalog no. 341276- 1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore. Human C3 was purified from mixed pool citrated human plasma (TCS Bioscience, PR100) using polyethylene glycol 4,000 precipitation, anion, and cation exchange chromatography as previously described (63). A pET15b-C3d construct was acquired from Prof. David E. Isenman and transformed into Escherichia coli (E. coli) stain BL21 (DE3), recombinant C3d was then expressed, and purified using a previously described protocol (64). Lyophilized normal human serum (NHS) was purchased from Euro Diagnostica (catalog no. PC300). Additional proteins and antibodies are described in the specific experimental section.

#### Sbi-III-IV Constructs

The expression and purification of the N-terminally 6×His tagged recombinant Sbi-III-IV from a pQE30:sbi-III-IV construct were described previously (24).

# Sbi-III-IV Mutagenesis

Mutations in the Sbi-III-IV sequence were introduced using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies), the primers used are listed in **Table S1**. The mutated pQE30:sbi-III-IV plasmids were sequenced to confirm the success of the mutagenesis. SDS-PAGE profiles of all the Sbi-III-IV mutant proteins used in this study are shown in **Figure S1**.

#### Sbi-III-IV Induced C3 Consumption Assay

Lyophilized NHS was re-suspended in chilled dH2O to a 2× concentration. Equal volumes of 2×NHS and Sbi (10µM) were combined. Sbi treated sera were then incubated in a thermocycler at 37◦C for 30 min. Treated serum samples were collected at time intervals, 0.5 µl of serum was loaded on an SDS-PAGE gel analyzed under reducing condition. The proteins were Western blotted, and the blots were probed with anti-C3d, anti-Sbi, anti-C3a or anti-factor B antibodies.

A hemolytic assay was modified from a previously published procedure (65) to measure Sbi induced consumption of C3. Briefly, rabbit erythrocytes (TCS Bioscience) were resuspended in GVB buffer (5 mM veronal, 145 mM NaCl, 10µM EDTA, 0.1 % (w/v) gelatin) by washing three times via centrifugation at 600 g for 6 min. The concentration of rabbit red cells to be used in each experiment was determined by adding a stock of 5 µl of erythrocytes to 245 µl of water to give complete lysis and then re-adjusting cell concentration until an optical density reading of 0.7 (A405) was reached. Lysis experiments were conducted in two steps, first, 15 µl of NHS, 5 µl of Mg+-EGTA (70 mM MgCl2, and 100 mM EGTA), 20 µl of protein in E2 buffer was mixed and pre-incubated at 37◦C for 30 min. Subsequently, 5 µl of rabbit erythrocyte was added and incubated for an additional 30 min at 37◦C. At the end of the incubation, 150 µl of quenching buffer (GVB supplemented with 10 mM EDTA) was added. The cells were pelleted by centrifugation at 1,500g for 10 min, and absorbance (A405) of 100 µl of supernatant measured. Postconsumption lysis percentage was calculated as 100×((A405 test sample-A405 0% control)/(A405 100%-A405 0% control)).

#### *In vitro* Complement Activation Assay in Mouse Serum

Mouse serum was collected from male Cr2−/− mice by cardiac puncture and allowed to clot fully on ice for 4 h followed by separation of serum by centrifugation at 2,000 g in a refrigerated centrifuge. Serum was then mixed with Sbi-III-IV or Sbi-III-IV-Ag85b, ensuring that the amount of Sbi-III-IV in each preparation was equivalent. The reaction was stopped at 0, 30, 60, and 120 min, by the addition of reducing sample buffer, boiled for 5 min and analyzed on a 10% SDS-PAGE gel. After transfer to nitrocellulose the blots were probed with Rabbit anti-C3d (1/1000, DAKO, A0063) and Goat anti-Rabbit-HRPO (1/2000, 111-035-046-JIR, Stratech), developed with ECL substrate (Pierce), and exposed to X-Ray film for 2 min.

# Binding Kinetics and Hydrodynamic Diameter Analysis

A switchSENSE DRX 2,400 instrument (Dynamic Biosensors) was used to characterize the binding kinetics and protein size changes based on switchSENSE technology (66, 67). Purified Sbi-III-IV-cys, K173A, R231A, and their ligand C3d were sent to Dynamic Biosensor's protein analyzing facility for binding kinetic and hydrodynamic diameter analysis. In the case of a protein binding event, based on the real-time measurements of the switching dynamics in a range of ligand concentrations, binding rate constants (kON and kOFF) and dissociation constants (KD) can be analyzed (67). Alternatively, under saturated binding conditions, the switching dynamic of the protein (or protein complex) can be compared with the switching dynamics of bare DNA and with a biophysical model with which the size of the immobilized protein (or protein complex) can be determined. For determination of Sbi-III-IV:C3d binding kinetic parameters, 130, 100, 70, and 40 nM of C3d were applied sequentially onto the Sbi-III-IV immobilized microchip. All Sbi:C3d complexes' hydrodynamic diameters were estimated at a C3d concentration of 130 nM.

#### Fluorometric C3b Breakdown Assay

Fluorometric C3b breakdown assay was performed using a black 96 well microplate (Thermofisher, M33089) in a TECAN Spark 20 M temperature-controlled fluorescence plate reader. Excitation was at 386 nm and emission was recorded at 475 nm with a 20 nm bandwidth. The control C3b breakdown rate, performed in PBS, contained 100 µl of 1µM C3b, 160 nM FH, 17 nM of FI and 10µM ANS, and was scanned every 5 s for 15 min. To study the interruption of C3b breakdown, 32 nM of FHR was either added alone or in combination with 1µM of Sbi-III-IV. Data were collected at 25◦C, normalized by Excel using the equation "Percentage of C3b=((FX-(F15min))/(F15min-F0min))∗ 100" and plotted by Graphpad Prism.

# Small Angle X-ray Scattering Analysis

Synchrotron radiation X-ray scattering from solutions of the Sbi-III-IV:C3d:FHR-1 tripartite complex, the Sbi-III-IV(K173A):C3d complex, and the Sbi-III-IV-Ag85b fusion protein were collected at the EMBL P12 beamline of the storage ring PETRA III (DESY, Hamburg, Germany). Images were collected using a photon counting Pilatus-2M detector and a sample to detector distance of 3.1 m and a wavelength (λ) of 0.12 nm covering the range of momentum transfer (s) 0.1 < s< 4.5 nm−1 ; with s=4πsinθ/λ. Different solute concentrations were measured using a continuous flow cell capillary. To monitor radiation damage, 20 successive 50 ms exposures were compared and frames displaying significant alterations were discarded. The data were normalized to intensity of the transmitted beam and radially averaged; the scattering of the buffer was subtracted, and the different curves were scaled for solute concentration. The forward scattering I(0), the radius of gyration (Rg) along with the probability distribution of the particle [p(r)] and the maximal dimension (Dmax) were computed using the automated SAXS data analysis pipeline SASFLOW (68).

For the Sbi-III-IV-Ag85b fusion protein data quality was improved with SEC-SAXS mode and the parallel analysis of light scattering data in a similar manner as described in Gräwert et al. (69). Frames comprising solely the monomeric version of the fusion protein were averaged and used for further processing after background subtraction.

The molecular masses (MM) were evaluated by comparison of the forward scattering with that from a reference solution of BSA and based on the Porod volumes of the constructs. With SAXS, the former estimation of MM is within an error of 10%, provided the sample and standard concentrate are determined accurately. DAMMIF was used to compute the ab initio shape models. For this, 10 independent models fitting the experimental scattering curves were generated and compared to each other. More detailed modeling was obtained with Coral. Here, existing partial crystal structure of the Sbi-IV:C3d complex was extended with 60 additional beads placed at the N-terminus of Sbi-IV to mimic the missing Sbi-III domain. Here too, 10 independent runs were performed, and the degree of variation addressed. Further analysis of the flexibility of the samples was addressed with Ensemble Optimization Method (EOM). For this, ensembles of models with variable conformations are selected from a pool of randomly generated models such that the scattering from the ensemble fits the experimental data, and the distributions of the overall parameters (e.g., Dmax) in the selected pool are compared to the original pool.

The proteins in the Sbi-III-IV:C3d:FHR-1 tripartite complex were combined 1:1:1 at a concentration of 45µM. The Sbi-III-IV(K173A):C3d complex were formed at a 1:1 ratio at 240µM (12 mg/ml). PDB structure 2wy8 (Sbi-IV:C3d complex) was used to model the complex using and compared with SAXS data previously recorded (34). The Sbi-III-IV-Ag85b fusion protein was provided at 29, 72, and 145µM concentrations (1.45, 3.6, and 7.2 mg/ml, respectively). The samples were dialysed against PBS, which was also used for background subtraction. From all samples concentration series were measured to exclude any concentration dependent alterations.

#### Surface Plasmon Resonance Analysis

Tripartite complexes were analyzed by surface plasmon resonance (SPR) technology using a Biacore S200 (GE Healthcare). All experiments were conducted at 25◦C on CM5 chips, using HBST (10 mM HEPES, 150 mM NaCl, and 0.005% Tween 20, pH 7.4) as running buffer, which was optionally supplemented with 1 mM of MgCl2 (HBST+) to be compatible with AP amplification condition. On the chip surface 800 RU of C3b was opsonized via AP C3 convertase through a method described before (70, 71). The iC3b surface was produced by injecting of repetitive cycles of FH and FI across a C3b opsonized surface, the completeness of the conversion was confirmed by the inability of FB binding. A separate chip surface was made by amine coupling 600 RU of recombinant C3d (CompTech, USA). In all SPR experiments, response differences were derived using the signal from a flow cell to subtract the parallel reading from a reference flow cell that blocked with carbodiimide, N-hydroxysuccinimide and ethanolamine. Analytes were injected in duplicate (at 30 µl/min for 200 s) followed by running buffer for 300 s and a regeneration phase involving injection of regeneration buffer (10 mM sodium acetate, 1 M NaCl pH 4.0) for 60 s. To analyze Sbi-III-IV binding and the assembly of tripartite complex, concentration series of Sbi-III-IV WT or K173A were flowed cross separately or co-injected with FH, FHR-1, FHR-2, FHR-5, or FH19−20 at a fixed concentration (100, 12.5, 20, 25, or 20 nM, respectively).

The C3 convertase DAA assay was performed on a CM5 chip amine coupled with 500 RU C3b, using HBST+ as running buffer throughout. A mixture of analytes for building C3 convertase were flowed across, including FB and FD in addition to various FH reagent combinations (FH or FH and FHR-1 or FH, FHR-1 and −5). The various FH reagents combinations were also flow across separately in order to derive the sensorgram for C3 convertase. To examining Sbi bound C3 convertase, 2µM of wild-type Sbi-III-IV was added to the mixture of analytes for building C3 convertase. The various FH reagent combinations spiked with Sbi were flowed across separately in order to derive the sensorgram for Sbi bound C3 convertase. Each injection cycle includes Injection of the C3 convertase mixture for 200–300 s, followed by running buffer for 300–400 s and two consecutive 60s regeneration phases.

#### FH/FHR-1 Competition Assay

C3b was diluted in carbonate buffer (pH 9.5) and coated on to wells of a Nunc MaxiSorp plate (0.25 µg/well) for 16 h at 4 ◦C. The wells were blocked with PBST (PBS with 0.1% Tween 20) supplemented with and 2% BSA for 1 h at 37◦C, and then washed with PBST buffer. Doubly diluted concentration series (9-600 nM) of FHRs, FH19−20, FHR-11−2 in PBST-2% BSA were then added to the wells, together with a constant concentration of FH (25 nM) and Sbi-III-IV (1,000 nM). The plate was incubated for 1 h at 37◦C, then washed with PBST. Fifty microliter of monoclonal anti-FH antibody OX-24 (specific to the FH SCR domain 5) diluted with PBS-2% BSA (0.6µg/ml) was added to the wells and the plate incubated for a further 1 h. The wells were washed with PBST, and 50 µl sheep anti-mouse IgG (1:5000 dilution in PBST-2% BSA) was added to the wells for 1 h at 37◦C. The wells were washed again and the conjugate was detected using TMB ELISA substrate solution, which was added to the wells for 5 min. The color reaction was stopped by 10% H2SO4 and the plate was read at A450 using a plate reader.

#### Design and Purification of the Sbi-III-IV-Ag85b Fusion Construct

The DNA sequence coding for Sbi-III-IV (sbi448−798) was fused to the 5' end of the DNA sequence for Ag85b (ag85b121−975) via a linker region of 84 bp (**Figure S5A**). The fusion gene was commercially synthesized and ligated into the pET15b vector, containing an ampicillin resistance cassette and a T7 promoter. The pET15b:sbi-III-IV-ag85b plasmid was verified using sequencing, and the resulting construct encoded an Nterminally his-tagged Sbi-III-IV-Ag85b protein. E. coli BL21 (DE3) cells harboring the pET15b:sbi-III-IV-ag85b plasmid were grown in LB broth supplemented with 100µg/ml ampicillin to an A600 = 0.4–0.6. Protein expression was induced with 0.5 mM IPTG and by incubating the cells at 17◦C for 16 h. Bacteria were harvested, lysed using sonication (80% amplitude, for six 10 s bursts) in the presence of protease inhibitor cocktail (set VII-Calbiochem, Merck), and the protein initially purified using nickel-affinity chromatography (His-Trap column, GE Healthcare) with a gradient of 0–0.5 M imidazole in 50 mM Tris, 150 mM NaCl, pH 7.4. It was further purified using size-exclusion chromatography (Hi-Load 16/60 Superdex S200 column, GE Healthcare) equilibrated in 20 mM Tris, 150 mM NaCl, pH 7.4. Fractions containing protein were pooled and concentrated. Protein concentration was measured at A280.

### Analysis of Sbi-III-IV-Ag85b Fusion Protein AP Complement Activity

Alternative pathway (AP) activity of Sbi III-IV-Ag85b-treated NHS samples was analyzed using the ELISA-based WIESLAB R (Euro Diagnostica) complement system AP assay. Sbi-III-IV-Ag85b was mixed with normal human serum (NHS) at a 1:1 volume ratio and incubated for 30 min at 37◦C in a thermal cycler. Treated serum was then diluted with AP diluent (blocking the activation of the other two complement pathways) by 1 in 20. From this point the manufacturer's instructions were followed. A blank (AP diluent), positive control (NHS) and negative control (heat-inactivated NHS) were also recorded. Complement activation was converted to residual AP activity (%) using the equation: (sample - negative control)/(positive control - negative control) × 100.

### Analysis of C3 Fragment Deposition on Sbi-III-IV-Ag85b Fusion Protein

The method used is similar to that described for WT Sbi-III-IV. Lyophilised NHS (Euro Diagnostica) was re-suspended in chilled dH2O. Sbi-III-IV-Ag85b (100µM) was mixed with NHS in a 1:1 ratio, and incubated for 1 h at 37◦C in a thermocycler. Samples were taken at regular intervals (0, 5, 15, 30, and 60 min), and separated by SDS-PAGE followed by Western blot analysis using either rabbit anti-Sbi (1.5:5000 dilution), rabbit anti-C3d (1.5:5000 dilution) or mouse anti-Ag85b (1:1000 dilution) polyclonal antibodies and detected using HRP-conjugated secondary antibodies (1:2500 goat anti rabbit or 1:1000 goat anti mouse). NHS-only was used as a negative control.

#### Measurement of Immune Response to Sbi-III-IV-Ag85b Fusion Protein

Eight week old male C57bl/6 mice (wild-type, C3−/− and Cr2−/−) were bled by tail vein venesection at day−2. Mice were then immunized at day 0 with molar equivalent doses of Ag85b alone (2 µg, a sub-optimal dose without adjuvant or boost, data not shown), Sbi-III-IV-Ag85b (fusion protein), Sbi-III-IV alone or a mixture of Sbi-III-IV and Ag85b, as appropriate. Mice were then bled weekly thereafter and plasma stored at −80◦C until required for batch analysis. Mice were boosted at day 28 and sacrificed at day 42.

For analysis of IgG response to Ag85b by ELISA, 96 well plates (NUNC maxisorb) were coated with 1µg/ml Ag85b (Abcam, UK) or 1.35µg/ml Sbi-III-IV-Ag85b in carbonate buffer at 50 µl per well and incubated at 4◦C for 16 h. Plates were washed with 0.01% PBS-Tween and a 1% BSA blocking solution was applied for 1 h at 20◦C. Serum samples were diluted to 1/50 or 1/100 in 0.01% PBS-Tween, added at 50 µl per well and incubated for 1 h at 20◦C. Plates were washed and secondary antibody (sheep anti mouse IgG-HRPO, 515-035-071-JIR, Stratech, UK) was added at 1/100 dilution, 50 µl per well and incubated for 1 h at 20◦C. TMB substrate (50 µl per well) was added and allowed to develop for 6 min. The reaction was stopped by the addition of 100 µl 10% H2SO4 per well and plates were read at A450. A mouse monoclonal anti-Ag85b (Abcam, ab43019) used as a positive control. The mean absorbance ± SEM of each mouse group is shown. Data for each mouse, at time 0, has been normalized to the day 0 average reactivity to Ag85b in all mice screened.

# DATA AVAILABILITY

The raw data generated or analyzed in this published article (and its **Supplementary Information** files) will be made available by the authors, without reservation, to any qualified researcher.

# ETHICS STATEMENT

Mice were housed at The Comparative Biology Center, Newcastle University. All experiments were conducted in accordance with institutional guidelines and approved by the UK Home Office under the auspices of project license P35D9C60C.

# AUTHOR CONTRIBUTIONS

JvdE, AGW, and KM conceived the idea of the project. YY, KM, and JvdE designed the experiments. YY, CB, AAW, RK, and JP performed and analyzed the experiments. KW and WK conducted and analyzed the switchSENSE experiments. MG and DS conducted SAXS experiments and oversaw the structural analysis. RK, HD, JP, and KM conducted the in vivo experiments. AS and AGW significantly contributed to the discussions about the overall project. YY, CB, KM, and JvdE wrote and edited the manuscript, with significant contributions from AAW, MG, and DS.

# ACKNOWLEDGMENTS

This research was funded by the Biotechnology and Biological Sciences Research Council (BBSRC Follow On Fund BB/N022165/1, awarded to JvdE and KM). AAW was supported by a Ph.D. scholarship granted Raoul and Catherine Hughes and the University of Bath Alumni. KM, HD, and RW were also supported by the MRC and Newcastle University's Confidence in Concept funding. AS thanks the Royal Society URF and Alumni Fund at the University of Bath for funding. MG was supported by the EMBL interdisciplinary Postdoc Programme under Marie Curie COFUND Actions as well as the Horizon 2020 programme of the European Union, iNEXT (H2020 Grant # 653706).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.03139/full#supplementary-material

# REFERENCES

on a self surface. Nat Struct Mol Biol. (2011) 18:463–70. doi: 10.1038/ nsmb.2018

protein Sbi in complex with ligand C3d. Mol Immunol. (2011) 48:452–62. doi: 10.1016/j.molimm.2010.09.017

a dynamically switchable biosurface. J Am Chem Soc. (2012) 134:15225–8. doi: 10.1021/ja3061276

**Conflict of Interest Statement:** KW and WK are employed by Dynamic Biosensors GmbH. KM is a member of the scientific advisory board of Gemini Therapeutics, Inc., Cambridge, Massachusetts, USA.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Yang, Back, Gräwert, Wahid, Denton, Kildani, Paulin, Wörner, Kaiser, Svergun, Sartbaeva, Watts, Marchbank and van den Elsen. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nanoparticle-Based Vaccines Against Respiratory Viruses

Soultan Al-Halifa1,2, Laurie Gauthier 1,2,3,4, Dominic Arpin1,2,3,4, Steve Bourgault 1,2,4 \* and Denis Archambault 3,4 \*

1 Département de Chimie, Université du Québec à Montréal, Montreal, QC, Canada, 2 Quebec Network for Research on Protein Function, Engineering and Applications, PROTEO, Quebec, QC, Canada, 3 Département des Sciences Biologiques, Université du Québec à Montréal, Montreal, QC, Canada, 4 Faculté de Médecine Vétérinaire, Centre de Recherche en Infectiologie Porcine et Avicole (CRIPA), Université de Montréal, St-Hyacinthe, QC, Canada

The respiratory mucosa is the primary portal of entry for numerous viruses such as the respiratory syncytial virus, the influenza virus and the parainfluenza virus. These pathogens initially infect the upper respiratory tract and then reach the lower respiratory tract, leading to diseases. Vaccination is an affordable way to control the pathogenicity of viruses and constitutes the strategy of choice to fight against infections, including those leading to pulmonary diseases. Conventional vaccines based on live-attenuated pathogens present a risk of reversion to pathogenic virulence while inactivated pathogen vaccines often lead to a weak immune response. Subunit vaccines were developed to overcome these issues. However, these vaccines may suffer from a limited immunogenicity and, in most cases, the protection induced is only partial. A new generation of vaccines based on nanoparticles has shown great potential to address most of the limitations of conventional and subunit vaccines. This is due to recent advances in chemical and biological engineering, which allow the design of nanoparticles with a precise control over the size, shape, functionality and surface properties, leading to enhanced antigen presentation and strong immunogenicity. This short review provides an overview of the advantages associated with the use of nanoparticles as vaccine delivery platforms to immunize against respiratory viruses and highlights relevant examples demonstrating their potential as safe, effective and affordable vaccines.

Keywords: respiratory viruses, nanocarriers, nanovaccine, mucosal sites, immune response

# INTRODUCTION

Lower respiratory tract infections (LRTIs) constitute a major public health burden worldwide. LRTIs represent a leading cause of human mortality and morbidity, causing annually over 3 million deaths worldwide (1). Among these infections, about 80% of LRTI cases are caused by viruses (2). In most cases, these pathogens enter the host via airborne transmissions (e.g., droplets or aerosols), replicate efficiently in the respiratory tract and cause clinical manifestations, ranging from fever to bronchiolitis and pneumonia (3). In addition, LRTIs associated with viruses represent an important source of economic loss for livestock and poultry industry as these infections predispose animals to secondary bacterial infections (4–6).

Viruses infecting the human lower respiratory tract include the influenza virus, the respiratory syncytial virus (RSV), the parainfluenza virus and the adenovirus (7, 8). Seasonal influenza virus epidemics result in a significant burden of disease in children and elderlies and account for 3–5

#### Edited by:

Delphyne Descamps, INRA Centre Jouy-en-Josas, France

#### Reviewed by:

Prabakaran Ponraj, Sanofi Genzyme, United States Randy A. Albrecht, Icahn School of Medicine at Mount Sinai, United States

#### \*Correspondence:

Steve Bourgault<EMAIL_ADDRESS>Denis Archambault<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 06 November 2018 Accepted: 07 January 2019 Published: 24 January 2019

#### Citation:

Al-Halifa S, Gauthier L, Arpin D, Bourgault S and Archambault D (2019) Nanoparticle-Based Vaccines Against Respiratory Viruses. Front. Immunol. 10:22. doi: 10.3389/fimmu.2019.00022 million cases of severe illness and for nearly 290,000–650,000 deaths worldwide each year (9). RSV and parainfluenza virus infections are the leading cause of hospitalization for acute respiratory infections in young children, causing 45 and 40% of pediatric hospitalizations, respectively (10, 11). Adenovirus infections account for 3–5% of LRTIs cases in children and can be fatal for immunocompromised patients (12). In general, respiratory viruses represent a major health problem in infants, young children, immunocompromised patients and the elderly population. According to Global Burden of Diseases (GBD), 74% of deaths associated with LRTIs represent these vulnerable patient groups (13).

Vaccination remains the most cost-effective strategy to fight against infectious diseases. Conventionally, vaccine formulations consist of attenuated viruses, killed pathogens (inactivated) or subunit protein antigens, which elicit a specific immune response. These vaccine formulations have allowed the prevention, or the control, of several important diseases including rubella, yellow fever, polio and measles, and, in the case of smallpox, even eradication (14, 15). Considerable efforts have been devoted for the development of efficient vaccines against LRTIs, including inactivated/fragmented trivalent or quadrivalent seasonal vaccines against influenza type A and type B viruses such as Influvac R (16), Vaxigrip R (17), and Fluzone R (18) as well as live attenuated vaccines such as Nasovac R and Flumist R for nasal administration in young children (19, 20). Nevertheless, live-attenuated vaccines against influenza virus suffer from safety concerns due to their nature and represent a risk for elderly and immunosuppressed humans (21). Besides, killed pathogen vaccines and virus-derived subunit vaccines induce weaker immune responses and often require the use of an adjuvant to boost efficiency (22).

Several promising vaccines are currently evaluated in the clinics for different respiratory viruses (23). These new vaccine formulations aim to be safer and more efficient compared to traditional vaccines based on attenuated viruses, killed pathogens and subunits. Nevertheless, the high level of antigenic drift (genetic mutations) of some viruses, such as the influenza virus, reduces the efficacy of vaccines and needs to be addressed (24). Therefore, while improving safety and efficiency, vaccines should also be less sensitive to antigenic drift. The concept of "universal vaccine" is critical for viruses like the influenza virus, and new formulations to induce broad-spectrum immunity are being investigated. In the next sections, we discuss the advantages of using nanoparticle formulations against respiratory viruses and we highlight relevant examples of the use of nanoparticles as safe, effective, and affordable vaccines.

#### NANOPARTICLES, AN ALTERNATIVE APPROACH TO CONVENTIONAL VACCINES

The use of particles as nanoplatforms displaying relevant antigenic moieties is appealing as an alternative approach to conventional vaccines. These nano-sized materials can be obtained from biological sources and/or can be synthetic. Currently, there is a large variety of particles evaluated as antigen carriers, including inorganic and polymeric nanoparticles, viruslike particles (VLPs), liposomes and self-assembled protein nanoparticles (**Figure 1A**). The advantages of these materials reside primarily in their size (at least one dimension should be at the nanometer level), since many biological systems such as viruses and proteins are nano-sized (25). Nanoparticles can be administered via sub-cutaneous and intramuscular injections, or can be delivered through the mucosal sites (oral and intranasal), and penetrate capillaries as well as mucosal surfaces (26, 27). Recent progresses have allowed the preparation of nanoparticles with unique physicochemical properties. For instance, size, shape, solubility, surface chemistry, and hydrophilicity can be tuned and controlled, which allows the preparation of nanoparticles with tailored biological properties (28). Moreover, nanoparticles can be designed to allow the incorporation of a wide range of molecules including antigens which makes them highly interesting in vaccinology (29, 30).

Incorporation of antigens in nanoparticles can be achieved by encapsulation (physical entrapment) or by conjugation (covalent functionalization) (21). Studies have demonstrated that nanoparticles could protect the native structure of antigens from proteolytic degradation and/or improve antigen delivery to antigen-presenting cells (APCs) (31). In addition, nanoparticles incorporating antigens can exert a local depot effect, ensuring prolonged antigen presentation to immune cells (32). Interestingly, nanoparticles have also shown intrinsic immunomodulatory activity (33). For instance, nanoparticles such as carbon nanotubes (CNTs), carbon black nanoparticles, poly(lactic-co-glycolic acid) (PLGA) and polystyrene nanoparticles, titanium dioxide (TiO2) nanoparticles, silicon dioxide (SiO2) nanoparticles, and aluminum oxyhydroxide nanoparticles have been reported to induce NLRP3-associated inflammasome activation (34). In fact, once internalized by APCs, these nanoparticles provide signals that trigger lysosomal destabilization and the production of reactive oxygen species (ROS), leading to the release of lysosomal contents, including the cysteine protease cathepsin B. This protease is sensed by NLRP3, which subsequently activates the formation of the inflammasome complex (35–39). Subsequently, interleukins are produced as downstream signaling events, leading to the recruitment and/or activation of immune cells (35, 40–45). Taken together, these properties advocate that nanoparticles are promising antigen carriers and immune cell activators for vaccination.

# NANOPARTICLES AND THE RESPIRATORY TRACT IMMUNE SYSTEM

The respiratory mucosa represents the primary site for invasion and infection by a virus whose replication occurs in the ciliated cells of the upper respiratory tract (URT). Subsequently, infection spreads to the low respiratory tract (LRT) by virus-rich secretions and by infected cell debris from the URT (46). Nasal-associated lymphoid tissue (NALT), the first site for inhaled antigen recognition located in the URT, is an important line of defense

against respiratory viruses. NALT is present in rodents, birds and primates (47). This structure is characterized by aggregates of lymphoid cells located in the nasopharyngeal cavity (48). In human, the Waldever's ring, made of adenoid and tonsil, is considered as the equivalent of NALT structure, which contains various narrow epithelial channels. NALT comprises aggregates of lymphoid follicles (B-cell areas), interfollicular areas (T-cell areas), macrophages and dendritic cells (DCs) (**Figure 1B**), which, when activated, support the clearance of infectious agents (46, 48, 49). Accordingly, NALT is considered as an inductive site for humoral and cellular immune responses and represents a promising target for vaccines against respiratory viruses. Ideally, nanovaccines would follow a path similar to respiratory viruses in order to efficiently deliver antigens to NALT and trigger a specific mucosal immune response. Therefore, formulation, size and antigen exposition are critical aspects when designing nanovaccines targeting NALT. Most respiratory viruses have an average diameter size ranging between 20 and 200 nm (50–53). Thus, in addition of being safe and immunogenic on its own, a nanovaccine should have a size similar to viruses while incorporating relevant antigens (54).

Over the last decade, a number of nanoparticles have been designed to mimic respiratory viruses in terms of size, shape and surface property in order to target NALT as well as to raise humoral and cellular immune responses (21, 55, 56). First, beside a nanoparticle size of 20–200 nm in diameter to match the size of most respiratory viruses, nanoparticles should be preferably positively charged. In fact, positively charged polymeric, phospholipidic, metallic, inorganic, and proteinbased nanoparticles have shown stronger immune responses compared to their negatively charged counterparts (21, 57). Second, the incorporation of antigens/epitopes within or on the surface of the nanoparticles can be challenging and requires advanced approaches in chemical and/or biological engineering (21). The most common strategy is to encapsulate or entrap antigens/epitopes within the nanoparticles. In this case, Al-Halifa et al. Nanovaccines Against Respiratory Viruses

nanoparticles are used to protect the antigen/epitopes and deliver them to NALT (58–60). Nanoencapsulation can be achieved by using different procedures, including nanoprecipitation and oil in water (o/w) emulsion (61). Alternatively, antigens can be attached and exposed on the nanoparticle surface. This strategy aims at mimicking viruses. Conjugation of antigenic epitope can be performed directly on the nanoparticles using different chemical reactions like the disulfide bond and the thiolate-gold bond formation (62–64). Otherwise, it can be achieved by first preparing an epitope-functionalized selfassembling unit, which upon self-assembly form nanoparticles decorated with the antigen (65–67). Third, the formulation and administration strategies are also critical aspects to consider. Vaccines administered via subcutaneous or intramuscular injection induce systemic immunity and usually, a weak mucosal response is observed. On the other hand, mucosal vaccination, either oral or intranasal delivery, induces humoral, and cellular immune responses at the systemic level and the mucosal surfaces, which is more effective in the protection against respiratory viruses (68, 69). Studies have demonstrated that vaccination via the intranasal route provides a better protection when compared to subcutaneous immunization in the context of respiratory pathogens and mucosal immunity. Intranasal vaccination led to higher antigen-specific lymphocyte proliferation, cytokine production (interferon-γ, interleukins) and induction of antigenspecific IgA antibody (70–74). A promising formulation strategy is the intranasal spray, which delivers conveniently and safely the nanovaccines directly to the respiratory mucosa (75–77). However, the number of clinical trials using nanovaccine formulations for intranasal delivery, including spray dried nanovaccines, is limited. This is mostly associated with the difficulty of keeping the nanovaccine integrity during the entire formulation process (76). Moreover, the immune response is particularly sensitive to the nature, size, shape, and surface properties of the nanoparticles as well as to the density and the potency of the antigens. Thus, it is very challenging to predict the effect of a given nanovaccine on the immune system. In addition, nanoparticles have some limitations associated with their synthesis, or preparation, and their properties. These include limited antigen loading, low synthesis yield, poor targeting capability to immune cells, limited manufacturability, and, in some cases, toxicity (78–80). These drawbacks can lead to side effects and/or poor immunogenicity, which precludes their clinical usage. Besides, little is known about the interactions between nanoparticles and immune cells. In fact, their adjuvant effect and their ability to activate the immune system still remain unclear and need to be better understood at the molecular level (81). Nonetheless, nanoparticle formulations have recently revealed promising results against respiratory virus infections (**Table 1**) and relevant examples will now be discussed.

#### POLYMERIC NANOPARTICLES

A polymer consists of a large molecule constructed from monomeric units. Depending on the construction, polymers can be linear, slightly branched or hyperbranched (3D network) (104). Polymeric nanoparticles can be either obtained from the polymerization of monomeric units or from preformed polymers. These nanoparticles are attractive in the medical field due to their adjustable properties (size, composition, and surface properties), which allow controlled release, ability to combine both therapy and imaging (theranostics), and protection of drug molecules (105–107). For example, poly(lactic-co-glycolic acid) (PLGA) is a biodegradable and biocompatible polymer approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for use in humans. This is due to its ability to undergo hydrolysis in vivo, resulting in lactic acid and glycolic acid metabolites, which are efficiently processed by the body (108). PLGA can be engineered to form nanoparticles capable of encapsulating different types of biomolecules and release them sustainably over time (108–111). These nanoparticles can encapsulate antigens and prevent their degradation over 4 weeks under physiological conditions, which is critical for mucosal vaccination (112). Moreover, PLGA-NPs promote antigen internalization by APCs and facilitate antigen processing and presentation to naïve lymphocytes (113, 114). For instance, spherical PLGA-NPs (200–300 nm of diameter) were used to encapsulate an inactivated Swine influenza virus (SwIV) H1N2 antigens (KAg) via water/oil/water double emulsion solvent evaporation (83). It was observed that pigs vaccinated twice with this preparation and challenged with a virulent heterologous influenza virus strain, have a significantly milder disease in comparison to non-vaccinated animals. This observation correlated closely with the reduced lung pathology and the substantial clearance of the virus from the animal lungs. Other polymeric nanoparticles, such as chitosan, a natural polymer composed of randomly distributed β-(1–4)-linked d-glucosamine and N-acetyl-d-glucosamine, and N-(2-hydroxypropyl)methacrylamide/N-isopropylacrylamide (HPMA/NIPAM), were also investigated as intranasal vaccines against respiratory viruses (85–90, 115–121). Overall, polymeric nanoparticles have many advantages, including biocompatibility (122), antigen encapsulation and stabilization (123, 124), controlled release of antigens and intracellular persistence in APCs (125, 126), pathogen-like characteristics, and suitability for intranasal administration (126, 127). Nevertheless, the effect of the polymer properties (core chemistry, size, shape, surface properties) on the transport within the URT remains unknown. More studies are needed to better understand the effect of changing nanoparticle properties on their biological activities and to, ultimately, predict the fate of these nanocarriers upon their intranasal administration.

# SELF-ASSEMBLING PROTEIN NANOPARTICLES AND VLPs

Self-assembling protein nanoparticles (SAPNs) are structures obtained from the oligomerization of monomeric proteins. The protein building blocks are mostly obtained through recombinant technologies and are considered safe for biomedical applications (128). SAPNs can be engineered to have a diameter ranging from 20 to 100 nm, similar to the sizes of many TABLE 1 | Nanoparticle-based vaccines against respiratory viruses delivered via the intranasal route.

aPoly-γ -glutamic acid.

bQuillaia saponin, cholesterol, phospholipid, and associated antigen.

cDilauroylphosphatidylcholine.

viruses and therefore, are considered as nanovaccine candidates against viruses, including respiratory viruses (128, 129). For example, SANPs, designed to elicit an immune response against RSV, have been explored using the nucleoprotein (N) from the virus nucleocapsid. The N protein is a major target of antigen-specific cytotoxic T-cell response. The selfassembly of N protein protomers led to the formation of supramolecular nanorings of 15 nm diameter (93). This platform was modified by fusing the FsII epitope targeted by monoclonal neutralizing antibody (palivizumab) to the Nprotein, in order to form chimeric nanorings with enhanced immune response and virus protection against RSV. The results showed reduced virus load in the lungs of challenged mice (94). Similarly, chimeric nanorings displaying 3 repeats of the highly conserved ectodomain of the influenza virus A matrix protein 2 (M2e), were prepared by recombinant technologies (95). When administrated via the intranasal route, these M2efunctionalized nanorings induced local production of mucosal antibodies and led to mice protection (95). These N-nanorings are interesting for intranasal delivery of antigen due to their similarities with respiratory viruses in term of size and structure (sub-nucleocapsid-like superstructures). Other examples of SAPNs as potential nanovaccines against respiratory viruses include the capsid protein of the papaya mosaic virus (PapMV), the purified coronavirus spike protein and ferritin, which are selfassembling proteins that form rod-shaped and nearly spherical nanostructures, respectively (96, 130–140). Recently, assemblies composed of four tandem copies of M2e and headless HA proteins were prepared and stabilized by sulfosuccinimidyl propionate crosslinking, showing the possibility of generating protein nanoparticles almost entirely composed of the antigens of interest (141).

VLPs are spherical supramolecular assemblies of 20–200 nm diameter, which result from the self-assembly of viral capsid proteins. These particles are free from genetic materials and have the advantage of mimicking perfectly the structure and the antigenic epitopes of their corresponding native viruses. Therefore, this repetitive antigen display promotes efficient phagocytosis by APCs and subsequent activation (142–146). Recently, Lee and colleagues demonstrated that intranasal delivery of influenza-derived VLPs expressed in insect cells and exposing 5 repeats of the M2e epitopes, confers cross protection against different serotypes of influenza viruses by inducing humoral and cellular immune responses (99).

SAPNs and VLPs are thus attractive but their formulation into stable and spray dried vaccines for intranasal injection can be challenging and may require the use of surfactants and saccharides (147). In the last decades, self-assembling peptides (SAPs) have also been investigated as intranasal nanovaccines against respiratory viruses due to their straightforward chemical synthesis and their storage stability upon lyophilization (97).

#### INORGANIC NANOPARTICLES

There are many inorganic nanoparticles suitable for biomedical applications, including superparamagnetic nanoparticles (iron oxide nanoparticles), quantum dots and plasmonic nanoparticles (gold and silver nanoparticles). Inorganic materials are mostly used as tools with improved therapeutic efficacy, biodistribution and pharmacokinetics. However, inherently, plain inorganic core nanoparticles would not be suitable in biological fluids due to particle aggregation. Therefore, in the medical field, these nanoparticles are often coated with organic molecules via adsorption or chemical reactions. In fact, these biocompatible nanoparticles can be described as complex hybrids materials with an inorganic core and an organic outer shell (148, 149). Among inorganic nanoparticles, the most commonly used for vaccination are gold nanoparticles (AuNPs). AuNPs are readily internalized by macrophages and dendritic cells, and induce their activation (150, 151). Large scale production is possible with strict control on particle size and ease of functionalization using the strong affinity between thiol groups and gold. Thiol groups can be attached to AuNP surface by forming thiolate–Au bonds (152–155). Furthermore, no immune response is elicited toward inert carriers like AuNPs (156). Thus, these nanoparticles are an appealing platform for nanovaccine engineering via antigen functionalization.

A wide range of molecules, including adjuvants and antigens can be conjugated on AuNPs at high density, resulting in improved immunogenicity and antigen presentation (157, 158). AuNPs can be formulated for intranasal administration and can diffuse into the lymph nodes, triggering robust antigenspecific cytotoxic T-cell immune responses (159, 160). Tao and coworkers have demonstrated that the peptide consensus M2e of influenza A viruses with a non-native cysteine residue at the C-terminal end could be attached on the AuNPs via thiolate– Au chemistry. The resulting M2e-AuNPs was administered by the intranasal route to mice with CpG (cytosine-guanine rich oligonucleotide) adjuvant, triggering a fully protective immune response against the influenza virus PR8 strain (161). More recently, it was demonstrated that this formulation could induce lung B cell activation and robust serum anti-M2e IgG response, with stimulation of both IgG1 and IgG2a

#### REFERENCES

subclasses (161). Additionally, this vaccination strategy led to protection against infection by the pandemic influenza virus strain, A/California/04/2009 (H1N1pdm) pandemic strain, influenza virus A/Victoria/3/75 (H3N2) strain and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) (64). Although gold nanoparticles constitute an attractive platform for antigen conjugation, they can accumulate in organs such as liver and spleen for a long period, which could be ultimately associated with toxicity (162). Coating with biocompatible materials reduces their toxicity, although it can lead to alterations of the physicochemical and biological properties. Therefore, safety issues of AuNPs still need to be addressed.

#### CONCLUSION AND PERSPECTIVES

Engineered nanoparticles have demonstrated their potential as vaccine delivery platforms. They can be envisaged as both antigen nanocarriers and adjuvants. In all cases, intranasal administration of nanovaccines allows a convenient and safe delivery of the antigen to NALT, inducing mucosal and systemic immunity. Nonetheless, additional studies are still needed before their clinical translation. While intranasal vaccination of nanoparticles generates specific IgA antibody in the URT and leads to high survival rates in animal models, there are still limited studies on non-human primates, thus making nanoparticle's fate difficult to predict in a human URT. In addition, nanoparticle vaccines are generally functionalized with specific antigen(s), which result in an immune response targeted against these antigenic determinants. Considering antigenic drifts, the growing human population that needs to be vaccinated and the different type of viruses, the cost to address all these aspects would be too prohibitive to produce affordable vaccines. Consequently, the development of broad spectrum vaccines constitutes a critical need and we consider that nanovaccine engineering will contribute to achieve this objective.

#### AUTHOR CONTRIBUTIONS

SA-H, LG, DoA, SB, and DeA have participated in writing and preparation of the manuscript, and approved it for publication.

#### FUNDING

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2016-06532 and RGPIN-2018-06209) and the International Development Research Center (IDRC; 108517) to DeA and SB.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Al-Halifa, Gauthier, Arpin, Bourgault and Archambault. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures

Andrés Pizzorno1,2†, Olivier Terrier 1 \* † , Claire Nicolas de Lamballerie1,3, Thomas Julien1,4 , Blandine Padey 1,4, Aurélien Traversier 1 , Magali Roche3 , Marie-Eve Hamelin2 , Chantal Rhéaume2 , Séverine Croze5 , Vanessa Escuret 1,6, Julien Poissy 7 , Bruno Lina1,6 , Catherine Legras-Lachuer 3,8, Julien Textoris 9,10, Guy Boivin2 and Manuel Rosa-Calatrava1,4 \*

#### Edited by:

Delphyne Descamps, INRA Centre Jouy-en-Josas, France

#### Reviewed by:

Rong Hai, University of California, Riverside, United States Sascha Trapp, INRA Centre Val de Loire, France

#### \*Correspondence:

Olivier Terrier<EMAIL_ADDRESS>Manuel Rosa-Calatrava<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 18 September 2018 Accepted: 10 January 2019 Published: 29 January 2019

#### Citation:

Pizzorno A, Terrier O, Nicolas de Lamballerie C, Julien T, Padey B, Traversier A, Roche M, Hamelin M-E, Rhéaume C, Croze S, Escuret V, Poissy J, Lina B, Legras-Lachuer C, Textoris J, Boivin G and Rosa-Calatrava M (2019) Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures. Front. Immunol. 10:60. doi: 10.3389/fimmu.2019.00060 1 Virologie et Pathologie Humaine—VirPath Team, Centre International de Recherche en Infectiologie, INSERM U1111, CNRS UMR5308, ENS Lyon, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France, 2 Research Center in Infectious Diseases of the CHU de Quebec and Laval University, Quebec City, QC, Canada, 3 Viroscan3D SAS, Lyon, France, 4 VirNext, Faculté de Médecine RTH Laennec, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France, 5 ProfileXpert, SFR-Est, CNRS UMR-S3453, INSERM US7, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France, 6 Laboratoire de Virologie, Centre National de Référence des virus Influenza Sud, Institut des Agents Infectieux, Groupement Hospitalier Nord, Hospices Civils de Lyon, Lyon, France, 7 Pôle de Réanimation, Hôpital Roger Salengro, Centre Hospitalier Régional et Universitaire de Lille, Université de Lille 2, Lille, France, 8 Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université Claude Bernard Lyon 1, Université de Lyon, Villeurbanne, France, 9 Service d'Anesthésie et de Réanimation, Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France, 10 Pathophysiology of Injury-Induced Immunosuppression (PI3), EA 7426 Hospices Civils de Lyon, bioMérieux, Université Claude Bernard Lyon 1, Hôpital Edouard Herriot, Lyon, France

Influenza virus infections remain a major and recurrent public health burden. The intrinsic ever-evolving nature of this virus, the suboptimal efficacy of current influenza inactivated vaccines, as well as the emergence of resistance against a limited antiviral arsenal, highlight the critical need for novel therapeutic approaches. In this context, the aim of this study was to develop and validate an innovative strategy for drug repurposing as host-targeted inhibitors of influenza viruses and the rapid evaluation of the most promising candidates in Phase II clinical trials. We exploited in vivo global transcriptomic signatures of infection directly obtained from a patient cohort to determine a shortlist of already marketed drugs with newly identified, host-targeted inhibitory properties against influenza virus. The antiviral potential of selected repurposing candidates was further evaluated in vitro, in vivo, and ex vivo. Our strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of 1,309 FDA-approved bioactive molecules, 31 of which were validated for their significant in vitro antiviral activity. Our in vivo and ex vivo results highlight diltiazem, a calcium channel blocker currently used in the treatment of hypertension, as a promising option for the treatment of influenza infections. Additionally, transcriptomic signature analysis further revealed the so far undescribed capacity of diltiazem to modulate the expression of specific genes related to the host antiviral response and cholesterol metabolism. Finally, combination treatment with diltiazem and virus-targeted oseltamivir neuraminidase inhibitor further increased antiviral efficacy, prompting rapid authorization for the initiation of a Phase II clinical trial. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identification of novel anti-infectious drugs, with potential major implications for the management of antimicrobial resistance and the rapid response to future epidemic or pandemic (re)emerging diseases for which we are still disarmed.

Keywords: influenza viruses, antivirals, inhibitors of viral infection, transcriptome, host targeting, drug repurposing

### INTRODUCTION

Besides their well-known pandemic potential, annual outbreaks caused by influenza viruses account for several million respiratory infections and 250,000 to 500,000 deaths worldwide (1). This global high morbidity and mortality of influenza infections represents a major and recurrent public health threat with high economic burden. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for innovative treatment strategies presenting fewer obstacles for their clinical use (2).

For decades, the strategy for antiviral development was mostly based on serial screenings of hundreds of thousands of molecules to identify "hits" and "leads" that target specific viral determinants, a quite costly and time-consuming process. However, the dramatic reduction in successful candidate identification over time (3), along with a concomitant increase of regulatory complexity to implement clinical trials, have fostered rising interest in novel strategies. Indeed, new approaches, focused on targeting the host instead of the virus, as well as on marketed drug repurposing for new antiviral indications (3–5) have been recently proposed in the context of global health emergencies posed by Ebola (6) and Zika (7) viruses. Such innovative strategies are strongly supported by a shift of paradigms in drug discovery, from "one-drug-one-target" to "one-drug-multiple-targets" (8). In that sense, different in silico approaches based on structural bioinformatic studies (9, 10), systems biology approaches (11), and host gene expression analyses (12) have been applied to decipher multi-purpose effects of many US Food and Drug Administration (FDA)-approved drugs. Additionally, as successfully demonstrated in antiretroviral therapy (13), targeting host instead of viral determinants may confer a broad-spectrum antiviral efficacy, and also reduce the risk of emergence of drug resistance against influenza viruses (14). As a result, the last decade has witnessed several host-directed experimental approaches against influenza infections, notably nitazoxanide, DAS181 or acetylsalicylic acid (15–17).

In line with this emerging trend, we previously postulated that host global gene expression profiling can be considered as a "fingerprint" or signature of any specific cell state, including during infection or drug treatment, and hypothesized that the screening of databases for compounds that counteract virogenomic signatures could enable rapid identification of effective antivirals (18). Based on this previous proof-of-concept obtained from in vitro gene expression profiles, we further improved our strategy by analyzing paired upper respiratory tract clinical samples collected during the acute infection and after recovery from a cohort of influenza A(H1N1)pdm09 infected patients and determined their respective transcriptomic signatures. We then performed an in silico drug screening using Connectivity Map (CMAP), the Broad Institute's publicly available database of more than 7,000 drug-associated gene expression profiles (19, 20), and identified a list of candidate bioactive molecules with signatures anti-correlated with those of the patient's acute infection state (**Figure 1A**). The potential antiviral properties of selected FDA-approved molecules were firstly validated in vitro, and the most effective compounds were further compared to oseltamivir for the treatment of influenza A(H1N1)pdm09 virus infections in both C57BL/6 mice and 3D reconstituted human airway epithelia. Altogether, our results highlight diltiazem, a calcium channel blocker with so far undescribed capacity to stimulate the epithelial antiviral defense, as a promising repurposed host-targeted inhibitor of influenza infection. Moreover, our results plead in favor of the combination of diltiazem with the virus-targeted antiviral oseltamivir for the improvement of current anti-influenza therapy, and possibly decreasing the risk of antiviral resistance. This study confirms the feasibility and interest of integrating clinical virogenomic and chemogenomic inputs as part of a drug repurposing strategy to accelerate bedside-to-bench and bench-to-bedside drug development.

# MATERIALS AND METHODS

#### Ethics Approval and Consent to Participate

Adult patients were recruited by general practitioners in the context of a previously published randomized clinical trial Escuret et al. (21) (ClinicalTrials.gov identifier NCT00830323) and all of them provided written informed consent. The study protocol was approved by the Lyon Ethics Committee (Comité de Protection des Personnes Lyon B) on September 9th, 2009 and conducted in accordance with the Declaration of Helsinki.

All animal procedures were approved by the Institutional Animal Care Committee of the Center Hospitalier Universitaire de Québec (CPAC protocol authorization #2012-068-3) according to the guidelines of the Canadian Council on Animal Care.

#### Clinical Samples

A previously published randomized clinical trial (ClinicalTrials.gov identifier NCT00830323) was conducted in Lyon and Paris (France) during the peak circulation of the influenza A(H1N1)pdm09 virus, with the aim to assess the efficacy of oseltamivir-zanamivir combination therapy compared with oseltamivir monotherapy (21). Briefly, patients tested positive for influenza A infection by the QuickVue rapid antigen kit (Quidel) were randomized in one of the two treatment

FIGURE 1 | From nasal wash clinical samples to a shortlist of 35 candidate molecules. (A) Overview of the in silico strategy used in this study. A detailed description of the strategy is described in the Online Methods section. (B) Hierarchical clustering and heatmap of the 1,117 most differentially deregulated genes between "infected" (red) and "cured" (light green) samples. Raw median centered expression levels are color coded from blue to yellow. Dendrograms indicate the correlation between clinical samples (columns) or genes (rows). (C) Functional cross-analysis of candidate molecules obtained from Connectivity Map (CMAP). Three lists of candidate molecules were obtained using different set of genes in order to introduce functional bias and add more biological significance to this first screening: a Main List based on the complete list of differentially expressed genes, and two other lists (List #1 and #2) based on subsets of genes belonging to significantly enriched Gene Ontology (GO) terms. (D) Venn Diagram comparing the total 160 molecules obtained from the three lists described in (C), with monensin as the only common molecule. Only the candidates selected for in vitro screening and validation are depicted.

groups and nasal wash specimens were collected within 2 h of the first visit and every 24 h until 96 h after treatment initiation. Nasal swabs were also performed on days 5 and 7. In voluntary patients, an optional supplementary nasal wash was performed at least 3 months after influenza infection (recovery phase). H1N1 subtype was further confirmed by PCR. For nine of these patients, transcriptomic data were obtained from paired samples collected during influenza infection without treatment and in the recovery phase.

# Sample Processing, RNA Preparation and Hybridization

Nasal wash samples were collected in RNAlater R Stabilization Solution (Thermo Fisher Scientific). Total RNA was extracted using RNeasy Micro kit (Qiagen) following the manufacturer's instructions. RNA quality was assessed using a Bioanalyzer2100 (Agilent technologies, Inc, Palo Alto, CA, USA). To account for samples having low amount and/or partially degraded RNA (RNA Integrity Numbers between 1 and 8), we applied two types of corrections: (i) cRNA labeling was performed after a linear amplification protocol, as previously described (22) and (ii) raw signals obtained after hybridization of labeled cRNA on microarray and data acquisition were processed using the MAXRS algorithm (23). Labeled cRNA were hybridized on Affymetrix HG-U133plus2 microarrays according to manufacturer's instructions in a GeneChip R Hybridization Oven 640 (Affymetrix) and microarrays were subsequently scanned in an Affymetrix 3000 7G scanner.

### Data Normalization and MAXRS Computational Analysis

The MAXRS algorithm (23) is particularly suited to gene expression analysis under low hybridization conditions. Briefly, this method takes advantage of the specific design of Affymetrix probe sets, which are composed of an average of 11 different probes that target the same locus, and is based on the observation that for most of the probe sets the same probe shows the highest fluorescence intensity in almost all arrays. For each microarray (m = 1..M) and for each probeset (t = 1..T), fluorescence intensity values on microarray m of all probes (p = 1..Pt) belonging to the probeset t are sorted in increasing order. These ranks are denoted as rmtp. Then, we calculated across all microarrays the rank sum (RStp) for each probeset t for each probe p belonging to the probeset t. Finally, for each probeset t, we kept the three probes p with the highest RStp. The mean intensity of these three probes is attributed to the probeset t. As it is common practice with many modern preprocessing algorithms, and because of the low global fluorescence signal intensity, mismatched probes were excluded from MAXRS analysis.

After pre-processing the raw dataset with the MAXRS algorithm, a normalization step was performed using Tukey median-polish algorithm (24). Differential expression was assessed by applying a Student t-test for each probeset, and multiple testing was corrected using the Benjamini-Hochberg algorithm in the qvalue library (25). For further downstream analysis, genes were selected according to two criteria: (i) absolute fold change >2, and (ii) corrected p-value < 0.05. Data were generated according to the Minimum Information About a Microarray Experiment guidelines and deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) (26) under accession number GSE93731.

# Functional Analysis

Functional enrichment analysis was performed on a selection of differentially-expressed genes with DAVID tools (27), using the Gene Ontology (GO) (28). To further select genes for the CMAP query, we selected 6 Biological Process (BP) terms (GO\_BP: GO:0009615-response to virus; GO:0006955-immune response; GO:0042981-regulation of apoptosis; GO:0006952-defense response; GO:0009611-response to wounding; GO:0042127 regulation of cell proliferation) that shared >90% of genes with all significantly enriched GO\_BP terms, and 3 relevant Cellular Component terms (GO\_CC: GO:0031225-anchored to membrane; GO:0005829-cytosol; GO:0005654-nucleoplasm). To visualize and compare the different lists of compounds, Venn diagrams were obtained using the webtool developed by Dr. Van de Peer's Lab at Ghent University (http://bioinformatics.psb. ugent.be/webtools/Venn/).

#### Cells and Viruses

Human lung epithelial A549 cells (ATCC CCL-185) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fœtal calf serum and supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U/mL), and streptomycin (100µg/mL) (Lonza), maintained at 37◦C and 5% CO2. MucilAir R human airway epithelia (HAE) were obtained from Epithelix SARL (Geneva, Switzerland) and maintained in air-liquid interphase with specific MucilAir R Culture Medium in Costar Transwell inserts (Corning, NY, USA) according to the manufacturer's instructions.

Influenza viruses A/Lyon/969/09 and A/Quebec/144147/09 were produced in MDCK (ATCC CCL-34) cells in EMEM supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U/mL), streptomycin (100µg/mL) (Lonza) and 1µg/mL trypsin. Viral titers in plaque forming units (PFU/ml) and tissue culture infectious dose 50% (TCID50/mL) were determined in MDCK cells as previously described (29, 30).

# Viral Growth Assays

For viral growth assays in the presence of molecules, A549 cells were seeded 24 h in advance in multi-well 6 plates at 1.8 × 105 cells/well. Three treatment protocols were evaluated. (1) In pretreatment protocol, cells were washed with DMEM and then incubated with different concentrations of candidate molecules diluted in DMEM supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U/mL), streptomycin (100µg/mL) (Lonza) and 0.5µg/mL trypsin. Six hours after treatment, cells were washed and then infected with A/Lyon/969/09 (H1N1)pdm09 virus at a multiplicity of infection (MOI) of 0.1. (2) In pre-treatment plus post-treatment protocol, cells were initially treated and infected in the same conditions as explained above. One hour after viral infection, a second identical dose of candidate molecules in supplemented DMEM was added. (3) In post-treatment protocol, cells without pre-treatment were infected in the conditions described and treatments with candidate molecules at the indicated concentrations were initiated 24 h p.i. In all cases, supernatants were collected at 48 h p.i. and stored at −80◦C for TCID50/ml viral titration.

#### Viability and Cytotoxicity Assays

Cell viability was measured using the CellTiter 96 R AQueous One Solution Cell Proliferation Assay (MTS, Promega). A549 cells were seeded into 96-well plates and treated with different concentrations of molecules or solvents. Cells were incubated at 37◦C and 5% CO2 and then harvested at different timepoints, following the same scheme as in viral growth assays. Results were presented as a ratio of control values obtained with solvents. Treatment-related toxicity in HAE was measured using the Cytotoxicity Detection KitPLUS (LDH, Roche) according to the manufacturer's instructions. Briefly, duplicate 100 µLaliquots of basolateral medium from treated and control HAEs were incubated in the dark (room temperature, 30 min) with 100 µL of lactate dehydrogenase (LDH) reagent in 96-well plates. After incubation, "stop solution" was added and the absorbance was measured in a conventional microplate ELISA reader. The photometer was set up for dual readings to determine nonspecific background at 750 nm, and absorbance was measured at 490 nm. Percent cytotoxicity was calculated as indicated by the manufacturer, using mock-treated and 1% triton-treated epithelia as "low" and "high" controls, respectively. Percent viability is presented as 100–percent cytotoxicity.

#### Mouse Model of Viral Infection

All protocols were carried out in seven to 9-weeks old female C57BL/6N mice (Charles River, QC, Canada). Animals were randomized in groups of 15 according to their weight to ensure comparable median values on each group, and then housed in micro-isolator cages (5 animals per cage) in a biosafety 2 controlled environment (22◦C, 40% humidity, 12:12 h photoperiods), with ad libitum access to food and water.

On day 0, mice were lightly anesthetized with inhaled 3% isoflurane/oxygen, and then infected by intranasal (i.n.) instillation of influenza A/Quebec/144147/09 (H1N1)pdm09 virus in 30 µl of saline, as specified in each case. Control animals were mock-infected with 30 µl of saline. Candidate molecules were evaluated in two different treatment protocols: (i) treatments were started on the same day of infection (day 0, 6 h prior to infection), or (ii) treatments were started 24 h after infection (day 1). Regardless of treatment initiation time, all treatments were performed per os (150-µl gavage) once daily for 5 consecutive days (5 drug administrations in total). Mortality, body weight and clinical signs such as lethargy and ruffled fur were daily monitored on 10 animals/group for a total of 14 days. Animals were euthanized if they reached the humane endpoint of >20% weight loss. The remaining 5 animals/group were euthanized on day 5 p.i. to measure lung viral titers (LVTs).

Vehicle (saline) or oseltamivir were used as placebo and positive treatment control, respectively. The oseltamivir dose (10 mg/kg/day) was adjusted to confer ∼50% protection in the selected experimental conditions and is considered a good correlate of half the normal dose of 150 mg/day given to humans (31). The doses of repurposed candidate molecules were selected to be in the non-toxic range for mouse studies, according to published preclinical data for their first therapeutic indication. To validate this choice in our specific model, potential drug toxicity was evaluated in mock-infected animals treated with the same regimens as virus-infected mice.

#### Pulmonary Viral Titers

In order to evaluate the effect of different treatments on viral replication, 5 animals per group were euthanized on day 5 p.i. and lungs were removed aseptically. Mice were randomly selected from the 3 cages of each group to minimize cage-related bias. Lungs were homogenized in 1 ml of PBS using a bead mill homogenizer (Tissue Lyser, Qiagen) and debris was pelleted by centrifugation (2,000 g, 5 min). Triplicate 10-fold serial dilutions of each supernatant were plated on ST6GalIMDCK cells (kindly provided by Dr. Y. Kawaoka, University of Wisconsin, Madison, WI) and titrated by plaque assays (29). The investigator was blinded to group allocation.

#### Viral Infection in Reconstituted Human Airway Epithelium (HAE)

For HAE infection experiments, apical poles were gently washed with warm PBS and then infected with a 100-µL dilution of influenza A/Lyon/969/09 (H1N1)pdm09 virus in OptiMEM medium (Gibco, ThermoFisher Scientific) at a MOI of 0.1. Basolateral pole sampling as well as 150-µL OptiMEM apical washes were performed at the indicated time points, and then stored at −80◦C for PFU/mL and TCID50/mL viral titration. Treatments with specific dilutions of candidate molecules alone or combined with oseltamivir in MucilAir R Culture Medium were applied through basolateral poles. Control HAE were mock-treated in the same conditions with MucilAir R Culture Medium without molecules. All treatments were initiated on day 0 (5 h after viral infection) and continued once daily for 5 consecutive days (5 drug administrations in total). Variations in transepithelial electrical resistance (1 TEER) were measured using a dedicated volt-ohm meter (EVOM2, Epithelial Volt/Ohm Meter for TEER) and expressed as Ohm/cm2 .

#### High Throughput Sequencing and Bioinformatics Analysis

cDNA libraries were prepared from 200 ng of total RNA using the ScriptseqTM complete Gold kit-Low Input (SCL6EP, Epicenter), according to manufacturer's instructions. Each cDNA library was amplified and indexed with primers provided in the ScriptSeqTM Index PCR Primers kit (RSBC10948, Epicenter) and then sequenced as 100 bp paired-end reads. Prior to sequencing, libraries were quantified with QuBit and Bioanalyzer2100, and indexed libraries were pooled in equimolar concentrations. Sequencing was performed on an Illumina HiSeq 2500 system (Illumina, Carlsbad, CA), with a required minimum of 40 million reads sequenced per sample. Conversion and demultiplexing of reads was performed using bcl2fastq 1.8.4 (Illumina). The FastQC software (http://www.bioinformatics. babraham.ac.uk/projects/fastqc) was used for quality controls of the raw data. Reads were trimmed using the Trimmomatic (32) software, with a minimum quality threshold of Q30. Trimmed reads were pseudo-aligned to the Homo sapiens genome (GRCh38.p11) using the Kallisto software (33). Statistical analysis was performed in R3.3.1 with the package EdgeR 3.14.0 (34). Differential expression was calculated by comparing each condition to the mock using a linear model. The Benjamini-Hochberg procedure was used to control the false discovery rate (FDR). Transcripts with an absolute fold change >2 and a corrected p-value < 0.05 were considered to be differentially expressed. Enriched pathways and GO terms were assessed with DAVID 6.8 (27). For visualization purposes, a heatmap and stacked barplots were constructed in R3.3.1 on mean-weighted fold changes and association between conditions were assessed by Spearman correlation analysis.

#### Statistical Analysis

All experimental assays were performed in duplicate at a minimum, and representative results are shown unless indicated otherwise. No statistical methods were used to predetermine sample size in animal studies, which were estimated according to previous studies and the known variability of the assays. No mice were excluded from post-protocol analyses, the experimental unit was an individual animal and equal variance was assumed. Kaplan-Meier survival plots were compared by Log-Rank (Mantel-Cox) test and hazard ratios (HR) were computed by the Mantel-Haenszel method. Weight loss and viral titers of all groups were compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test. The testing level (α) was 0.05. Statistical analyses were performed on all available data, using GraphPad, Prism 7.

# RESULTS

#### Generation of Clinical Virogenomic Profiles

We determined in vivo transcriptional signatures of infection from paired nasal wash samples of nine untreated patients, collected during acute A(H1N1)pdm09 pandemic influenza infection ("infected") and at least 3 months later to ensure a recovery non-infected state ("cured") (21). The nine patients from whom transcriptomic data could be obtained constitute a representative sample of the whole studied cohort, except for the male sex ratio (**Table S1**). We combined two strategies to tackle the characteristic low RNA amount/quality of this type of clinical samples. Firstly, cRNA labeling was performed after a linear amplification of initial RNA, as previously described (35). Secondly, raw signals obtained after hybridization of labeled cRNA on microarray and data acquisition were processed using the MAXRS algorithm (23) to overcome low hybridization conditions. This approach, initially developed for the analysis of heterologous hybridizations, takes advantage of the specific design of the Affymetrix R microarray used in our study, with several probes targeting the same locus (23).

After normalization, differentially expressed genes were selected based on two criteria: (i) an absolute fold change >2, and (ii) a Benjamini-Hochberg corrected p-value < 0.05. We therefore identified a total of 1,117 commonly deregulated probes, with almost equal proportion of up-regulated (48.4%; n = 541) and down-regulated probes (51.7%; n = 576). Remarkably, despite considerable inter-patient variability among recovery state samples, a substantial homogenization of transcriptional profiles was observed in the context of infection, as shown in the heatmap presented in **Figure 1B** and by the median Spearman's ρ correlation values for both groups (0.60 "cured" vs. 0.90 "infected"). These virogenomic signatures of infection constituted the input for the subsequent in silico query for the identification of candidate compounds.

#### In silico Cross-Analysis of Chemogenomic vs. Virogenomic Clinical Profiles

We then performed an in-silico search for molecules that reverse the virogenomic signature of infection, using the CMAP database (Build 02) as previously described (18).  HG-U133plus2 probesets were mapped to the U133A probesets using the Ensembl BioMarts online tool (36, 37), and connectivity scores and p-values were obtained using the CMAP algorithm (19, 20). With the global set of 1,000 most differentially expressed genes as input (**Figure 1C**, Main List), we obtained a preliminary list of 60 candidate compounds. In parallel, we used two other subsets of genes belonging to significantly enriched Gene Ontology (GO) terms obtained from microarray analyses to introduce functional bias and add more biological significance to our first screening. Hence, by using 6 Biological Process terms (GO\_BP) that shared more than 90% of genes (**Figure 1C**, Functional cross-analysis #1), a second list of 109 compound candidates was obtained. A third list of 19 compounds was obtained using 3 relevant Cellular Component terms (GO\_CC) (**Figure 1C**, Functional cross-analysis #2). The comparison of the 160 compounds from the three distinct lists (12.2% of compounds of CMAP, **Table S2**) highlighted monensin as the only common compound (**Figure 1D**).

To rationally reduce the number of drug candidates, bioactive drugs were excluded if not compatible with a final use as antiviral, mostly for safety (e.g., teratogens, intercalating agents), and/or pharmacological (e.g., documented low bioavailability) reasons, based on clinical data and the PubMed/PubChem databases. Thus, the number of candidates was initially decreased to 139 and then to 110 (**Figure S1**). We subsequently determined a shortlist of 35 bioactive molecules (<3% of CMAP, **Table 1**) for in vitro screening, based on two main criteria: (i) molecules representative of the different pharmacological classes identified, and (ii) molecules evenly distributed in the three lists obtained after in silico screening (Main List, List #1 and List #2, **Figure 1D**), which comprise a panoply of documented pharmacological classes, including anti-fungal agents (e.g., monensin, flucytosine), anti-inflammatory agents (e.g., felbinac, apigenin, prednisone) and adrenergic agonists/antagonists (timolol, methoxamine,

TABLE 1 | Shortlist of the 35 selected molecules and their documented pharmacological classes.

Shortlist of the 35 selected candidates representative of the 110 molecules obtained from the in silico screening (Figure 1 and Figure S1). Documented pharmacological classes were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov). (\*) indicate molecules previously evaluated for their antiviral properties according to the literature, and numerals (# ) those belonging to anti-microbial or anti-inflammatory related pharmacological classes.

tolazoline), as represented in the Venn diagram (**Figure 1D**, **Table 1**). Interestingly, at least 14 (40%) molecules from our short-list belong to a pharmacological class related with antimicrobial or anti-inflammatory activities (**Table 1**, #), and 11 (31.4%) have already been reported in the literature for their antiviral properties against influenza or other viruses (**Table 1**, ∗ ), notably the nucleoside inhibitor ribavirin (38, 39) and the ionophore monensin (40).

#### Inhibitory Effect of the Selected Molecules on A(H1N1)pdm09 Viral Growth in vitro

In vitro screening of the antiviral potency of the 35 selected molecules was performed in A549 human lung epithelial cells seeded in 6-well plates. Firstly, we evaluated the impact of 6 h pre-treatment with a 10-fold drug concentration range, using the original CMAP concentration as reference. Six hours after treatment, cells were washed and infected with influenza A(H1N1)pdm09 virus at a MOI of 0.1. Viral titers in supernatants collected from treated samples at 48 h post infection (p.i.) were normalized with those measured in mock-treated controls (>105 TCID50/mL). Potential treatmentinduced cell toxicity was evaluated in the same experimental conditions using the MTS assay and expressed also as the percentage of cell viability compared to non-infected controls (**Figure 2**). Based on antiviral activity and cell viability profiles obtained (**Figure 2A**, blue triangles), we defined as "inhibitors"

CMAP/100, CMAP/1,000, and CMAP/10,000) was used. CMAP × 10 was only tested in the context of pre-treatment, by anticipation of a lower efficacy of molecules in this experimental setup. All experimental assays were performed in triplicate and mean values are represented. (A, right) Zoom panels depicting molecules defined (Continued)

FIGURE 2 | as "inhibitors" according to the following two criteria: (i) induce a 75% or higher reduction on viral production, and (ii) have minor impact on cell viability, with relative values in the 90–110% range. For clarity purposes, with the exception of diltiazem, etilefrine, monensin, and ribavirin.  Dose-response curves for all the 35 molecules are presented in Table S3. (B) Venn diagram of the 10 molecules identified in pre-treatment (10/35; 28.57%) and matching the "inhibitor" criteria, mainly when used at 10-fold CMAP concentration. EC50 curves for monensin and ranitidine are represented. (C) Venn diagram of the 30 "inhibitor" molecules identified in pre-treatment/treatment (30/35; 85.7%). EC50 curves for monensin, diltiazem, and etilefrine are represented.

compounds that fulfilled the following two criteria: (i) induce >75% reduction on viral production, and (ii) have minor impact on cell viability, with relative values in the 90–110% range (**Figure 2A**, squares in left panels and zooms in right panels). A total of 10 compounds (28.6%) matched both criteria, mainly when used at a 10-fold CMAP concentration (**Figure 2B**), yet only a limited number of them exhibited classic dose-dependent inhibition. Whenever possible, as in the case of monensin or ranitidine for example, EC50 values were calculated, which were mostly in the micromolar range (**Figure 2B**).

In a second round of screening, we tested the same 6 h pre-treatment but with serial 10-fold dilutions from the initial CMAP concentration to CMAP/10,000, followed by one additional treatment immediately after infection (**Figure 2A**, green circles in left and right panels). In these conditions, 30 compounds (85.7%) met our criteria to be considered as inhibitors of viral production (**Figure 2C**), with half of them showing a classic dose-dependent inhibition effect. Calculated EC50 values were in the nanomolar range and hence significantly lower than those calculated in the context of pre-treatment only. Dose response curves and calculated EC50 for all the 35 compounds are presented in **Figure S2** and **Table S3**, respectively.

#### Efficacy of Selected Molecules for the Treatment of Influenza A(H1N1)pdm09 Virus Infection in Mice

Based on EC50 and cytoxicity data from the in vitro screening, we selected 8 molecules to investigate their potential as inhibitors of influenza A(H1N1)pdm09 in C57BL/6 mice. Oseltamivir, the standard antiviral for the treatment of influenza infections was used as control. All treatments were performed per os, starting 6 h before infection and being continued once daily for 5 consecutive days (5 drug administrations in total) (**Figure 3**). While animals treated with oseltamivir or monensin showed clinical improvement compared to the saline (placebo) group in terms of survival and weight loss (oseltamivir only), treatment with Lanatoside C, prednisolone, flucytosine, felbinac, and timolol showed no clinical benefit at the selected concentrations (**Figure S3A**). In contrast, diltiazem and etilefrine not only significantly improved survival and maximum mean weight losses (**Figures 3A,B**), but also showed at least 1-log reductions in LVTs on day 5 p.i. (**Figure 3C**). Importantly, no signs of toxicity were observed for any of the drugs at the regimens tested (**Figure S3B**).

### Diltiazem Retains Its in vivo Efficacy When Administered 24 h After Viral Infection

To best mimic the therapeutic setting, we next evaluated the efficacy of the same 5-day oral regimen with diltiazem or etilefrine but when initiated 24 h after viral infection (**Figure 4**). As with oseltamivir and monensin, diltiazem treatment completely prevented mortality and reduced weight loss in influenza A(H1N1)pdm09 infected mice, which otherwise showed only 50% (5/10) survival for the etilefrine and saline groups (**Figures 4A,B**). Interestingly, 1- to 1.5-log reductions in LVTs compared to the saline group were observed at day 5 in groups of mice treated with diltiazem or etilefrine (**Figure 4C**). We then used a more stringent approach by increasing the viral inoculum to evaluate the same delayed (24 h post infection) 5-day diltiazem regimen in the context of a 100% lethal A(H1N1)pdm09 infection (**Figures 4D–F**). Whereas, treatment with oseltamivir and diltiazem successfully rescued 40% (4/10) and 20% (2/10) of mice, respectively, half-dose treatment with diltiazem (45 mg/kg) rescued 30% (3/10) of mice from death, also showing significant improvement in mean weight loss (**Figures 4D,E**). Calculated hazard ratios (HR) for the saline group compared to these three treatment groups were 8.41 (CI95: 1.65–43.02), 2.85 (0.56–14.47), and 7.62 (1.49–38.96), respectively. Noteworthy, LVTs at day 5 p.i. were comparable among all treated and untreated groups (**Figure 4F**), suggesting mainly a protective effect of diltiazem toward severe influenza infection rather than a direct role in decreasing viral production.

#### Diltiazem Significantly Reduces Viral Replication in Infected Reconstituted Human Airway Epithelia (HAE)

To further complement in vivo data, we characterized the inhibitory properties of diltiazem using a biologically relevant reconstituted airway epithelium model, derived from human primary bronchial cells (MucilAir R , Epithelix). HAE were infected with influenza A(H1N1)pdm09 at a MOI of 0.1, and treatments on the basolateral medium were initiated 5 h p.i. and continued once daily for 5 consecutive days. Viral replication at the apical surface of mock-treated (MucilAir R Culture Medium without molecules) HAE peaked at 48 h p.i. (∼1 × 108 PFU/ml) and was detectable at important levels for at least 7 days. As expected, trans-epithelial electrical resistance (TEER) values, measuring tight junction and cell

layer integrity, sharply decreased and bottomed out at 72 h p.i. in the untreated control, correlating with the first virus detection on the basolateral medium (**Figure 5A** and **Table S6**). A similar pattern was observed in infected HAE treated with oseltamivir 0.1µM or diltiazem 9µM (CMAP), which conferred no significant advantage over the untreated control. Conversely, oseltamivir 1µM and diltiazem 90µM treatments (10-fold CMAP) strongly inhibited viral replication, delaying the peak of viral production by 24 h. Both treatments induced >3-log reductions in apical viral titers at 48 h p.i. compared to the untreated control, and >2-log reductions when comparing peak titers (48 h p.i. untreated vs. 72 h p.i. treated). Moreover, whereas oseltamivir treatment stabilized TEER during the time-course of infection, diltiazem treatment partially buffered the TEER decrease observed in the untreated control (**Figure 5A** and **Table S6**). No virus was detected on the basolateral medium for these two treated groups, and absence of treatment-induced toxicity was confirmed by measuring the release of intracellular lactate dehydrogenase (LDH). Interestingly, we observed that inhibitory and protective properties demonstrated by diltiazem were progressively reversible when basolateral medium was replaced with fresh medium without drugs. Overall, these results are in accordance and strongly support the inhibitory and protective effects of diltiazem observed in vitro and in mice, respectively.

### Diltiazem-Oseltamivir Combination Confers Improved Efficacy When Compared to Monotherapy in Infected HAE

We anticipated that the combination of two antiviral compounds that target different viral/cellular determinants could induce better virological and physiological responses when compared to antiviral monotherapy. We therefore evaluated the diltiazemoseltamivir combination in the same conditions described above, notably a 5-day treatment course with treatment initiation at 5 h p.i. The diltiazem 90 µM/oseltamivir 1µM combination conferred >3-log reduction in apical peak viral titers when compared to the untreated control, even greater than that observed with same dose monotherapy. TEER values remained stable during combined treatment, comparable to those observed with oseltamivir 1µM monotherapy (**Figure 5B** and **Table S6**). Remarkably, although not effective as monotherapy in the low concentrations tested above, the diltiazem 9 µM/oseltamivir 0.1µM combination contrariwise delayed the peak of viral production, significantly reduced apical viral titers, and slightly buffered TEER values compared to the untreated control (**Figure 5B** and **Table S6**). Once again, no treatment-related toxicity was observed for any of the combinations tested. These results plead in favor of the potential of diltiazem for the improvement of current anti-influenza therapy with neuraminidase inhibitors.

FIGURE 4 | Efficacy of post-infection oral treatment with diltiazem and etilefrine in mice infected with influenza A(H1N1)pdm09 virus. C57BL/6N mice (n = 15/group) were intranasally inoculated with 1 × 105 (A–C) or 4 × 106 (D–F) PFU of influenza A/Quebec/144147/09 virus on day 0 and treated by gavage with saline (gray), oseltamivir 10 mg/kg/day (red), monensin 10 mg/kg/day (blue, A only), diltiazem 45 mg/kg/day (light green, B only), diltiazem 90 mg/kg/day (dark green), or etilefrine 3 mg/kg/day (orange, (A) only). A mock-infected, saline-treated group (black dotted line, n = 6) was included as control. Treatments were initiated on day 1 (24 h after infection and administered once daily for 5 consecutive days. (A,D) Survival rates (n = 10/group), (B,E) mean weight changes (±SEM, n = 10/group or remaining mice), and (C,F) median (±CI95, n = 5/group) lung viral titers on day 5 p.i. are shown. \*\*\*p < 0.001, compared to the infected saline-treated group by one-way ANOVA with Tukey's post-test. Data are representative of two independent experiments.

electrical resistance (1 TEER±SEM) in MucilAir® human airway epithelium infected on the apical pole with influenza A/Lyon/969/09 (H1N1) pdm09 virus at a MOI of 0.1 and subjected to (A) single or (B) combined treatments by the basolateral pole. Treatments with culture medium (mock, gray), oseltamivir 0.1µM (red, dotted line), oseltamivir 1µM (red, solid line), diltiazem 9µM (green, dotted line), diltiazem 90µM (green, solid line), oseltamivir 0.1 µM/diltiazem 9µM (brown, dotted line) or oseltamivir 1 µM/diltiazem 90µM (brown, solid line) were initiated 5 h after infection and administered once daily for 5 consecutive days. \*\*p < 0.01 and \*\*\*p < 0.001 compared to the infected Mock-treated group by one-way ANOVA with Tukey's post-test. Data are representative of three independent experiments.

# Diltiazem Treatment Induces a Significant Reversion of the Viral Infection Signature

Since the rationale behind our approach relies on attaining antiviral activity through a drug-induced global and multi-level inversion of the infection signature, we advantageously used the MucilAir R HAE model coupled with high-throughput sequencing in order to characterize and compare the specific transcriptional signatures induced by infection and/or diltiazem treatment (**Figure 6** and **Figure S4**). HAE were mock-infected or infected with influenza A(H1N1)pdm09 virus and then mock-treated or treated in the same experimental conditions in which the antiviral effect of diltiazem has been previously validated (MOI of 0.1, 90µM diltiazem). At 72 h p.i., cells were lysed and total RNA was extracted. cDNA libraries were then produced, amplified, and subjected to high-throughput sequencing. Taking the mock-infected / mock-treated ("mock") as baseline, we initially performed DAVID functional gene enrichment (absolute fold change >2, Benjamini-Hochberg corrected p < 0.05) on the specific transcriptional signature of diltiazem with the objective of gaining insight on the putative host pathways involved in its antiviral effect. The lists of upregulated (n = 194) and down-regulated (n = 110) transcripts in the mock-infected/diltiazem ("mock + diltiazem") condition were analyzed using DAVID 6.8 to highlight associations with specific GO terms. Although no enriched BP was identified among down-regulated transcripts, the list of up-regulated

transcripts associated with diltiazem treatment highlighted 7 particularly enriched BP. While 4 of these BP (GO:0009615; GO:0045071; GO:0051607; GO:0060337) are directly linked

to antiviral response/cellular response to virus, the remaining 3 (GO:0055114; GO:0008299; GO:0006695) are involved in cholesterol biosynthesis/metabolism (**Figure 6A**). We then compared the common differentially expressed transcript levels between the three infection/treatment conditions. These transcriptional signatures revealed a marked anti-correlated profile between the "mock + diltiazem" and the infected / mock-treated ("H1N1") conditions (**Figure 6B**), supported by a median Spearman's ρ correlation value of −0.82 (**Figure 6C**). Most important, the infected/diltiazem ("H1N1 + diltiazem") condition yielded ρ correlation values of 0.40 and −0.72 when compared to either "mock + diltiazem" or "H1N1," respectively, therefore confirming a partial reversion of the infection virogenomic signature during effective antiviral treatment with diltiazem (**Figure 6D** and **Figure S4**), as expected.

#### DISCUSSION

The existing urge for alternative strategies to cope with the limited efficacy of currently approved antivirals for the prevention and treatment of influenza infections (2, 41, 42), mostly in the case of patients with severe influenza and acute respiratory distress syndrome (ARDS) (43, 44), represented the central driving force of this study. Here, we developed and validated for the first time an innovative approach based on clinical genomic signatures of respiratory viral infections for the rapid discovery, in vitro, in vivo, and ex-vivo evaluation, as well as the repurposing of FDA-approved drugs for their newly identified host-targeted inhibitory and protective properties against influenza infections.

Targeting host components on which viral replication depends instead of viral determinants represents a real change of paradigm in antiviral development, with pioneering results mainly observed in the context of antiretroviral therapy (13, 45). Nevertheless, and despite strong putative advantages such as the achievement of broad-spectrum antiviral efficacy and the minimization of viral drug resistance, this approach usually fails to overcome two major limiting factors of classic compound screening. Firstly, it remains target-centered per se, therefore leading to the identification of drugs with limited efficacy due to the complex network and high redundancy of the host cellular pathways. Secondly, the need of high-throughput screenings often entails the measurement of a very limited number of viral parameters, usually in non-physiologically and hence poorly relevant conditions and/or cellular models.

Based on our initial proof-of-concept study on the in silico screening of the CMAP database (19, 20) with no initial a priori on specific host targets (18), we moved our approach up to the clinical trial setting, by determining exploitable and more relevant virogenomic profiles directly from standard clinical samples of influenza-infected patients. Since the low amount of often degraded RNA obtained from these samples represented a major challenge, we implemented an original combination of sample preparation techniques for low input but high quality samples with data processing initially designed for expression analysis of non-model species (22, 23).

Another substantial development was the integration of several lists of candidate molecules issued from different transcriptomic signatures with enriched relevant DAVID Gene Ontology terms, and their final selection based on their pharmacological classes and potential compatibility as antivirals. Our refined strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of a total of 1,309 FDA-approved bioactive molecules. This drastic positive selection step constituted a major advantage, since it enabled the implementation of relevant and integrated in vitro, in vivo and ex-vivo evaluations in a time- and costeffective manner. Most important, the use of patient (in vivo) virogenomic profiles led to the identification of molecules with highly improved in vitro activity and significant in vivo antiviral efficacy as compared with compounds previously obtained from our initial study based on cell culture (in vitro) virogenomic profiles (18). These results truly highlight the added value of using relevant clinical virogenomic signatures to optimize the computational screening for active drugs.

Two of the molecules identified in this study with transcriptomic profiles that counteract clinical virogenomic signatures (e.g., ribavirin and monensin) have already been validated for their anti-influenza properties (38, 40), and then supported the relevance of our compound selection strategy. Nevertheless, although different modes of action have been postulated for the anti-influenza activity of the synthetic guanosine analog ribavirin (39), the exact mechanisms remain uncharacterized so far. Similarly, it has been postulated that monensin, an antibiotic isolated from Streptomycesspp, may have a role as a ionophore that interferes with intracellular transport of several enveloped viruses, including influenza (40). In that sense, even if we cannot rule out that some of the molecules identified in silico exert a direct effect on a specific pathway or cellular target, the fact that these molecules have been identified with a high anti-correlation rate in CMAP strongly supports a potential multi-target inhibitory effect, probably resulting in deep modifications of host gene expression. In fact, both monensin and ribavirin were previously reported to modulate the host cellular gene expression profile, notably through the up-regulation of the cholesterol and lipid biosynthesis genes (46) or the virus-induced ISRE signaling and antiviral ISGs genes (47), respectively.

The two most promising molecules highlighted in this study are etilefrine, an alpha and beta- adrenergic receptor agonist, currently indicated as a cardiotonic and anti-hypotensive agent (48) and mainly diltiazem, a voltage-gated Ca2+ channel antagonist that is currently used to control angina pectoris and cardiac arrhythmia (49). In addition to their strong inhibitory effect on the viral growth of circulating A(H1N1)pdm09 viruses, with in vitro EC50 values in the nanomolar range (**Figure 2**), both molecules also demonstrated antiviral properties against oseltamivir-resistant A(H1N1)pdm09 and prototype H3N2 and B influenza strains (**Table S4**). Interestingly, virus pre-incubation with diltiazem or etilefrine before infection did not affect final viral titers compared to PBS-incubated controls, hence suggesting that the observed antiviral effect of these molecules is not mediated by direct drug-virus interactions at early stages of viral entry (**Table S5**). Our in vivo results (**Figures 3**, **4**), obtained without previous treatment optimization in terms of dosage or administration route, also suggest that these drugs harbor a protective role toward influenza infection, particularly in the case of diltiazem, which conferred increased survival in mice even in a model of severe influenza infection (**Figures 4D–F**). Moreover, the inhibitory and protective properties of diltiazem were validated in the reconstituted human airway epithelium model, also showing enhanced efficacy when combined with oseltamivir (**Figure 5**).

Finally, a very recent study by Fujioka and colleagues (50) confirmed the antiviral activity of diltiazem anticipated by our approach. In that study, based on the role of Ca2+ channels on the attachment of influenza viruses to the host cell, the authors discuss whether the diltiazem induced modulation of Ca2+ channel activity might not fully explain such observed antiviral activity, consistent with a multi-level (off-target) effect of diltiazem. In this context, in which not all Ca2+ channel inhibitors confer significant antiviral activity, the newly described capacity of diltiazem to partially reverse the global virogenomic signature of infection and modulate specific genes related to the host antiviral response and cholesterol metabolism (**Figures 6** and **Figure S4**) suggests a putative explanation for its inhibitory effect observed in vitro, ex vivo and in mice. Nevertheless, although RT-qPCR mRNA quantification performed on a set of genes representative of the two hubs further validated these observations (**Figure S5**), further investigations are underscored to shed light on the specific mechanisms underlying such potential multi-level mode of action of diltiazem.

Overall, the results presented here set a solid baseline for our drug repurposing strategy and for the use of diltiazem as a host-targeted antiviral in clinical practice. Moreover, the increased antiviral efficacy observed in reconstituted human airway epithelium (**Figure 5B** and **Table S6**) plead in favor of the combination of diltiazem with the virus-targeted antiviral oseltamivir for the improvement of current anti-influenza therapy, and possibly decreasing the risk of development of viral resistance. In that regard, our results prompted a French multicenter randomized clinical trial aimed at assessing the effect of diltiazem-oseltamivir bitherapy compared with standard oseltamivir monotherapy for the treatment of severe influenza infections in intensive care units, hence completing the bedsideto-bench and bench-to-bedside cycle of our innovative approach. Additionally, retrospective signature analysis of sequential respiratory samples from patients included in both study arms and stratified according to their clinical response to treatment will provide valuable data to pursue the investigations on the specific mediators of the diltiazem-related antiviral response.

#### REFERENCES

This trial (FLUNEXT TRIAL PHRC #15-0442, ClinicalTrials.gov identifier NCT03212716) is currently ongoing.

Finally, our study underscores the high value of clinical specimens and the advantages of exploiting virogenomic and chemogenomic data for the successful systematic repurposing of drugs already available in our modern pharmacopeia as new effective antivirals. We propose that our approach targeting respiratory epithelial cells, the principal influenza infected cell type in the lung, could be extended to other respiratory viruses and eventually to other pathogens involved in acute infections. Importantly, drug repurposing presents several financial and regulatory advantages compared to the development of de novo molecules (5), which are of particular interest not only in the context of antimicrobial resistance but also against both emerging or recurrent pathogens for which we are still disarmed.

#### AUTHOR CONTRIBUTIONS

AP, OT, JT, GB, and MR-C designed and coordinated the study. VE, JP, and BL acquired clinical data from the cohort. AP, OT, TJ, BP, AT, MR, M-EH, CR, CN, SC, CL-L, and JT performed experiments and provided technical support. AP, OT, MR, JP, JT, GB, and MR-C interpreted data. AP, OT, and MR-C wrote the manuscript. JT, GB, and MR-C revised the manuscript.

#### ACKNOWLEDGMENTS

The authors want to thank Jacques Corbeil and Frederic Raymond (Research Center in Infectious Diseases of the CHU de Quebec and Laval University, Quebec) for their help and useful advice. This work was funded by grants from the French Ministry of Social Affairs and Health (DGOS), Institut National de la Santé et de la Recherche Médicale (INSERM), the Université Claude Bernard Lyon 1, the Région Auvergne Rhône-Alpes (CMIRA N◦ 14007029 and AccueilPro COOPERA N◦ 15458 grants), and Canadian Institutes of Health Research (N◦ 229733 and 230187). GB is the holder of the Canada Research Chair on influenza and other respiratory viruses. Funding institutions had no participation in the design of the study, collection, analysis and interpretation of data, or in the writing of the manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00060/full#supplementary-material

combination with EGFR inhibitors. Mol Cancer Ther. (2014) 13:2559–71. doi: 10.1158/1535-7163.MCT-13-1086

**Conflict of Interest Statement:** AP, OT, JT, GB, and MR-C are co-inventors of a patent application filed by INSERM, Université Claude Bernard Lyon 1, Laval University and Hospices Civils de Lyon for the repurposing of diltiazem and etilefrine as anti-influenza agents (FR15/52284 - PCT/ep2016/056036- WO2016146836).

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Pizzorno, Terrier, Nicolas de Lamballerie, Julien, Padey, Traversier, Roche, Hamelin, Rhéaume, Croze, Escuret, Poissy, Lina, Legras-Lachuer, Textoris, Boivin and Rosa-Calatrava. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Evaluation of the Antiviral Activity of Sephin1 Treatment and Its Consequences on eIF2α Phosphorylation in Response to Viral Infections

Maxime Fusade-Boyer 1 , Gabriel Dupré1 , Pierre Bessière1 , Samira Khiar 2 , Charlotte Quentin-Froignant 1,3, Cécile Beck 4 , Sylvie Lecollinet 4 , Marie-Anne Rameix-Welti 5,6, Jean-François Eléouët 7 , Frédéric Tangy 2 , Barbora Lajoie8 , Stéphane Bertagnoli 1 , Pierre-Olivier Vidalain9 , Franck Gallardo3 and Romain Volmer 1 \*

#### Edited by:

Cynthia Calzas, Institut National de la Recherche Agronomique (INRA), France

#### Reviewed by:

Rong Hai, University of California, Riverside, United States Owen Kavanagh, York St John University, United Kingdom

> \*Correspondence: Romain Volmer<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 05 October 2018 Accepted: 16 January 2019 Published: 12 February 2019

#### Citation:

Fusade-Boyer M, Dupré G, Bessière P, Khiar S, Quentin-Froignant C, Beck C, Lecollinet S, Rameix-Welti M-A, Eléouët J-F, Tangy F, Lajoie B, Bertagnoli S, Vidalain P-O, Gallardo F and Volmer R (2019) Evaluation of the Antiviral Activity of Sephin1 Treatment and Its Consequences on eIF2α Phosphorylation in Response to Viral Infections. Front. Immunol. 10:134. doi: 10.3389/fimmu.2019.00134 1 Université de Toulouse, ENVT, INRA, UMR 1225, Toulouse, France, 2 Viral Genomics and Vaccination Unit, CNRS UMR-3569, Institut Pasteur, Paris, France, 3 NeoVirTech SAS, Institute for Advanced Life Science Technology, Toulouse, France, 4 UMR 1161 Virology, INRA, ANSES, Ecole Nationale Vétérinaire d'Alfort, ANSES Animal Health Laboratory, EURL for Equine Diseases, Maisons-Alfort, France, 5 UMR INSERM U1173 2I, UFR des Sciences de la Santé Simone Veil—UVSQ, Montigny-le-Bretonneux, France, 6 AP-HP, Laboratoire de Microbiologie, Hôpital Ambroise Paré, Boulogne-Billancourt, France, 7 Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Université Paris-Saclay, Jouy-en-Josas, France, 8 Laboratoire de Génie Chimique CNRS, INPT, UPS Université de Toulouse III, Faculté des Sciences Pharmaceutiques, Toulouse, France, 9 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Equipe Chimie & Biologie, Modélisation et Immunologie pour la Thérapie, CNRS UMR 8601, Université Paris Descartes, Paris, France

The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1—GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.

Keywords: PKR, GADD34, PPP1R15A, virus, antiviral, eIF2α, host, broad-spectrum

# INTRODUCTION

Most clinically available antiviral drugs act by directly targeting viral components to inhibit a critical step in the viral life cycle, such as entry, replication, or viral egress (1). These molecules have several advantages, as they can be very potent inhibitors and should have minor side effects because they are, in theory, virus specific. However, viruses evolve constantly and the selective pressure of the treatment can give rise to mutants that are resistant to these drugs. This is illustrated

for example by the emergence of influenza virus strains resistant to viral neuraminidase inhibitors (2).

By contrast, antiviral molecules targeting host functions that are necessary for the virus life cycle are less likely to lead to the emergence of resistant viral mutants (3). Moreover, broadspectrum antiviral molecules can be developed if the targeted host cell function regulates the replication of a wide range of viruses. Numerous host factors have been identified as required for viral replication through whole-genome genetic screens, providing impetus to develop antiviral molecules targeting these host factors (4). The numerous pathways experimentally identified as potential targets for antiviral therapy include viral entry or egress, viral assembly, viral protein synthesis or maturation, and the immune response against viruses (3, 5, 6). Currently, approved drugs targeting the host include the widely used type I interferons, which boost the antiviral innate immune response, ribavirin, which modulates the pool of intracellular nucleosides and is reported to modulate the innate immune response, and finally maraviroc, inhibiting human immunodeficiency virus entry by targeting C-C chemokine receptor type 5 (CCR5) (1). In an effort to limit toxicity, it is necessary to target a host cell function that is not crucial to the cell physiology and/or that is more specific to infected cells.

The phosphorylation of serine 51 of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) inhibits initiation of protein translation in response to various cellular stresses (7). Four protein kinases have been shown to specifically phosphorylate eIF2α. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylates eIF2α in response to endoplasmic reticulum stress, due to the accumulation of unfolded proteins in the endoplasmic reticulum or to perturbations of the endoplasmic reticulum membrane lipid composition (8, 9). The haem-regulated inhibitor kinase (HRI) phosphorylates eIF2α in response to iron deficiency and has been demonstrated to regulate the differentiation of red blood cells (10). The general control non-derepressible-2 (GCN2) phosphorylates eIF2α in response to amino-acid deficiency (11). Finally, the interferon-induced double-stranded RNA-activated protein kinase (PKR) phosphorylates eIF2α in response to the accumulation of viral RNA harboring a double-stranded or other nucleic acids secondary structures produced during viral replication (12). Increased eIF2α phosphorylation attenuates translation of most mRNAs and is a physiological response to adapt to the various cellular stresses described above. Activation of PKR is for example an antiviral response aiming at reducing the translation of viral proteins in infected cells. The importance of PKR in antiviral defense is illustrated by the broad-array of viral countermeasures selected during evolution to inhibit PKR activation or eIF2α phosphorylation (12). It should however be noted that increased eIF2α phosphorylation seems to benefit to some viruses, including viruses belonging the Togaviridae family (13), Reoviridae family (14), and hepatitis C virus (15), most likely because translation of their mRNAs relies on secondary structures from which initiation can proceed even in the presence of high levels of eIF2α phosphorylation (12). As a consequence, developing means to increase eIF2α phosphorylation could be an antiviral intervention only for viruses whose mRNA translation is inhibited by increased eIF2α phosphorylation.

Dephosphorylation of eIF2α allows the cell to resume initiation of protein translation and is achieved by a binary complex between the catalytic phosphatase subunit PP1 and a regulatory subunit composed of either GADD34 (or PPP1R15A) (16) or CReP (or PPP1R15B) (17). The regulatory subunits GADD34 and CReP target the phosphatase PP1 specifically to the phosphorylated eIF2α substrate. CReP is constitutively expressed. By contrast, GADD34 expression is induced by eIF2α phosphorylation and therefore should be specifically expressed in stressed cells. GADD34 thus provides a negative feedback on eIF2α phosphorylation (8).

The guanabenz derivative Sephin1 was shown to increase eIF2α phosphorylation in cells stimulated with drugs causing PERK activation via the accumulation of unfolded proteins in the endoplasmic reticulum lumen (18). Sephin1 was described as a specific inhibitor of GADD34, although the identity of its target is currently subject of debate [see section Discussion and (19–21)]. We reasoned that inhibition of GADD34 could have antiviral effects by potentiating eIF2α phosphorylation in infected cells. Moreover, given that GADD34 is induced in cells with increased eIF2α phosphorylation, a GADD34 inhibitor should specifically act in stressed cells, such as infected cells, thus enhancing drug selectivity.

In the current work, we provide evidence that Sephin1 exhibited antiviral effects against specific viruses belonging to various viral families. In addition, Sephin1 increased eIF2α phosphorylation in response to activators of PKR, suggesting that Sephin1 may act by increasing eIF2α phosphorylation in virus-infected cells.

#### MATERIALS AND METHODS

#### Reagents and Cellular Treatments

Cells were treated for 16 h with 2.5µg/ml tunicamycin (Sigma, USA) or with 1µg/ml of intracellularly delivered Poly(I:C) (HMW)/LyoVec (Invivogen, France). Sephin1 was purchased from Tocris (United-Kingdom) or synthesized according to the protocol described in Das et al. (18). Purity was verified by nuclear magnetic resonance. Sodium arsenite (Sigma, USA) was added to cells in culture at a final concentration of 500µM for 1 h before lysis. Cells were treated for 24 h with 1,000 U/ml of bacterially produced recombinant human interferon α A (PBL assay science, USA).

#### Cells and Viruses

Human HEK293, HEK293T, human ARPE-19, and rabbit RK13 cells were grown at 37◦C in DMEM containing glutamate supplemented with 10% FBS, 1x penicillin-streptomycin. Human HEp-2 cells were grown at 37◦C in MEM containing glutamate supplemented with 10% FBS, 1x Penicillin-Streptomycin. Wildtype mouse embryonic fibroblasts (MEF WT) and MEF in which the endogenous eIF2α gene has been genetically replaced by a nonphosphorylable (S51A) allele (MEF S51A) have been described previously and were kindly provided by David Ron, University of Cambridge, United Kingdom (22, 23). Human respiratory syncytial virus (hRSV), derived from the strain Long, genetically modified to express firefly luciferase or the fluorescent protein mCherry were previously described and used to infect HEp-2 cells (24). Enterovirus D68, kindly provided by Caroline Tapparel, Université de Genève, Switzerland (25), was used to infect human RD cells cultured at 33◦C, as previously described (26). Human adenovirus serotype 5 (hAdV), belonging to serotype 5, genetically modified to express the bacterial partitioning system-based AnchOR3 was used to infect human HEK cells, as recently described (27). Measles virus strain Schwartz genetically modified to express the firefly luciferase (28) was used to infect human HEKT cells, as previously described (29). Myxoma virus strain T1 was used to infect RK13 cells as previously described (30). Human cytomegalovirus (hCMV) derived from the TB40/E strain and genetically modified to express the bacterial partitioning system-based AnchOR3 was used to infect human ARPE-19 cells, as recently described (31). The AnchOR3 system is distributed by NeoVirTech SAS, France and is available upon request. Influenza A/Puerto Rico/8/1934 (H1N1) and A/turkey/Italy/977/1999(H7N1) were used to infect A549 or MDCK cells, as previously described (32, 33). Japanese encephalitis virus genotype 3 strain Nakayama (34) was used to infect HEK293T cells. Briefly, HEK293T cells were infected with JEV at a MOI of 0.01 for 48 h and JEV RNAs in cell supernatants were quantified by real-time RT-PCR as described in Yang et al. (35). Theiler's murine encephalomyelitis virus (TMEV) genetically modified to express a mutant L protein and the fluorescent protein Cherry (36) was used to infect MEF WT and MEF S51A. Cellular viability was measured with Vita-Blue Cell Viability Reagent (Biomake) according to the manufacturer's protocol. This assay is based on a fluorescent dehydrogenase enzymes substrate, which correlates with cellular metabolic activity.