Recombinant subunit vaccines are an attractive technology as they have previously been utilized for a variety of pathogens [159] and are economical to produce on a commercial scale. In the field of HCV vaccine research, attempts to generate a subunit vaccine have focused exclusively on targeting the HCV E proteins. The most successful candidate has been the recombinant E1E2 protein (rE1E2) derived from a Gt1a isolate. Initially, HCV-1 derived rE1E2 was used to vaccinate seven chimpanzees which resulted in a humoral response [160]. Following homologous HCV challenge, five out of seven vaccinated chimpanzees exhibited sterilising immunity with the remaining two developing acute infections that were subsequently cleared [160,161]. This was further validated in small animal models and that immunisation rE1E2 derived from a single isolate could induce cross-reactive nAbs [161]. Following this work, a phase I human trial was performed in which rE1E2 derived from the Gt1a HCV-1 isolate was administered at different doses (4 μg, 20 μg and 100 μg of rE1E2) with the MF59 adjuvant to healthy volunteers [162]. Importantly this study showed that rE1E2 can be safely tolerated in humans, a finding of significance given that a successful HCV vaccine candidate will likely require these proteins or derivatives of them. Polyfunctional CD4+ T cell responses were detected and the magnitude of this response was inversely related to dosage. Humoral responses and nAbs were initially reported [162,163] and further testing using a diverse HCVcc panel with isolates from Gt1-7 showed that 3/16 volunteers had elicited cross reactive nAbs [164]. Additionally, post vaccination sera from 5 volunteers competed with nAbs AP33, AR3B, AR4A, AR5A and IGH526, showing that the humoral response targeted both E1, E2 and the E1E2 heterodimer [165]. The ability for rE1E2 from a single isolate to induce nAbs in humans is significant and encouraging although work should be undertaken to enhance the efficacy at which nAbs are generated. A key issue with rE1E2 was purification which relied on lectin-based affinity methods that were low affinity and non-specific [166]. Furthermore, efforts to improve rE1E2 production were made by removing the transmembrane domains of the E protein. However, this truncated E1E2 was found to be a weak immunogen that elicited low levels of

nAbs [167]. In light of these issues, both flag-tagged and Fc-tagged rE1E2 have been investigated and elicit nAbs in mice at comparable levels to wild type rE1E2 [166,168].

Another subunit vaccine approach has been the use of truncated soluble E2 (sE2) which results in a secreted form of the E2 ectodomain. A candidate of particular note is the H77 derived sE2384-661 construct with deleted HVR1, HVR2 and IgVR (sE2Δ123) which can be expressed in a correct conformation which interacts with CD81 and conformation sensitive antibodies [169–171]. Vietheer et al. [170] reported that the expression of sE2Δ123 in mammalian cells results in biochemically and immunogenically distinct isoforms of this protein that exist as monomeric, dimeric, pentameric and a high molecular weight aggregate (HMW1; estimated at 51 protomers). Immunisation of guinea pigs with different isoforms showed that HMW1 elicited greater titres of cross-reactive nAbs that could neutralise isolates from Gt2a, 4a, 5a, 6a and 7a expressed in a HVCcc system [170]. Additionally, HMW1 had exhibited a reduced activity with non-neutralising antibodies, suggesting that this aggregate focuses the humoral immune response away from these epitopes, allowing for greater focusing to nAb epitopes [170]. Whilst this initial report is promising, it was noted that sE2Δ123 reduced homologous neutralisation activity, a likely result of the removal of HVR1 which was also observed by Law and colleagues [172].

#### *5.2. Virus-Like Particles*

Virus-like particles (VLPs) are a promising area of vaccine research and have been investigated extensively to design a HCV vaccine [173]. To date, there are two widely used commercial vaccines that utilize VLP technology, the hepatitis B virus (HBV) and human papilloma virus vaccines. VLPs are formed when the structural proteins of a virus assemble in a genome-independent manner to produce a particle that resembles the native virus whilst lacking the ability to replicate and are more immunogenic than soluble protein due to their repetitive structure and ability to drain into tissue lymph nodes [107,174].

HCV-LPs were first described by Baumert et al. [175] in which VLPs were expressed in the insect cell line Sf9 following transduction with a recombinant baculovirus containing the core E1 and E2 HCV structural proteins. These HCV-LPs elicited HCV-specific IgG production, IFN-γ secreting CD8+ T cell and CD4+ T cell mediated cellular responses in mice and baboons [176–178]. Immunisation of four chimpanzees with four doses of HCV-LPs resulted in CD4+ and CD8+ T cell responses in all animals [179]. Subsequent challenge with homologous HCV resulted in a reduced viremia in vaccinated animals compared to controls with two animals testing negative for HCV two years post challenge [179]. Interestingly, humoral responses in chimpanzees were not detectable in three of these animals and appear to have been absent during the reduction of viremia. Further work on this candidate has not been progressed since this first study in chimpanzees. However, it is an important example for future HCV vaccine work, as it showed that viral clearance can be achieved using an experimental VLP vaccine candidate. Despite work on this candidate ceasing, HCV-LPs continued to be investigated. Recently, a HCV-LP system has been established through the transduction of the human hepatoma cell line Huh 7 with recombinant adenovirus encoding the structural genes of HCV [180]. Initially designed using the H77 isolate, further development has expanded by generating HCV-LPs using the structural sequences of different isolates representing Gt1b, 2a and 3a subtypes to produce HCV-LPs with a conformation close to that the wild type virus [180–183]. Trials of this candidate in mice showed the induction of nAbs and activation of both CD4+ and CD8+ T cell responses and this has recently been reported in a vaccinated Landrace pigs [184]. To date, assessment of the humoral response has demonstrated that this vaccine elicits nAbs with activity towards homologous strains. Heterologous neutralisation data have not been reported for this candidate. Testing the sera from animals immunized with this quadrivalent vaccine against a diverse panel of HCV isolates would be of particular interest not only for this candidate but to inform future vaccine design.

#### *5.3. Viral Vector Vaccines*

Viral vectors have the capacity to induce high level cellular immune responses [185], making them an attractive approach for HCV candidates. Viral vectors of particular interest are human adenovirus 6 (HuAd6), chimpanzees Ad 3 (ChAd3) and modified vaccinia ankara (MVA) [186]. The only HCV vaccine candidates to be tested in phase I and phase I/II human trials has been ChAd3 and MVA which each encoded HCV NS3, NS4a, NS4b, NS5a and inactivated NS5b (NSmut) derived from the Gt1b BK isolate [187,188]. These candidates were tested together in a heterologous primer/boost strategy in which healthy human volunteers were primed with ChAd3-NSmut and boosted with MVA-NSmut 8 weeks later [188]. The results of this phase I study showed that prime/boost ChAd3/MVA-NSmut induced both CD4+ T cell and CD8+ T cell responses directed towards epitopes on all five HCV non-structural proteins, importantly the effect of the MVA-NSmut enhanced the T cell responses and these were detectable at week 70 [188]. Given the promising cellular responses observed in the phase I trial, this approach was tested in patients with chronic HCV infections although this was unable to reverse T cell exhaustion [189], ruling out the use of ChAd3/MVA-NSmut as a therapeutic vaccine. Recently ChAd3/MVA-NSmut was tested in a large phase I/II trial involving 548 high risk individuals was concluded (clinicaltrials.gov identifier: NCT01436357). At the time of writing, this trial has concluded, and data have yet to be published. However, a press release statement from the National Institute of Allergy and Infectious Disease stated that 14 vaccinated individuals became chronically infected with HCV, suggesting that this candidate does not provide protection against HCV infection [190]. Understanding the reasons for this failure will be vital to inform our understanding of vaccine design. Given that the ChAd3/MVA-NSmut candidate exclusively targeted T-cell epitopes, the failure of this vaccine candidate could further emphasize the need for both humoral and cellular immune responses in clearance of acute infections.

 (not applicable) for vaccine candidates that are not designed to elicit HCV-specific B cell responses. N.D (not determined) in the study.

#### **6. Conclusions**

In the 30 years since the discovery of HCV, it has been apparent that this virus is highly complex and presents a major public health challenge. Encouragingly, efforts toward a vaccine continue and investigation into a range of approaches have yielded interesting results. The vast improvements in in vitro analysis of HCV have greatly aided our understanding of how nAbs work, informing rational vaccine design. In this review, we have highlighted the potential advantages of adopting reference panels of patient-derived E1E2 sequences in order to assess vaccine candidates for their potential to induce nAbs capable of targeting circulating HCV strains. Adopting this approach will provide a more rigorous tool for evaluating the nAb response produced by novel HCV vaccine candidates a characteristic that will likely be essential in a successful HCV vaccine.

**Author Contributions:** Writing—original draft preparation, J.D.D.; review and editing, R.A.U., A.W.T. and J.K.B.; funding acquisition, J.K.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Medical Research Council [grant number MR/R010307/1]; and the NIHR Nottingham Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime**/**GP5-Mosaic rVaccinia (VACV) Vaccine Boost**

#### **Junru Cui, Caitlin M. O'Connell, Connor Hagen, Kim Sawicki, Joan A. Smyth, Paulo H. Verardi, Herbert J. Van Kruiningen and Antonio E. Garmendia \***

Department of Pathobiology and Veterinary Science, College of Agriculture, Health and Natural Resources, University of Connecticut, Storrs, CT 06269, USA<EMAIL_ADDRESS>(J.C<EMAIL_ADDRESS>(C.M.O<EMAIL_ADDRESS>(C.H<EMAIL_ADDRESS>(K.S<EMAIL_ADDRESS>(J.A.S<EMAIL_ADDRESS>(P.H.V<EMAIL_ADDRESS>(H.J.V.K.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +1-860-486-3945

Received: 22 January 2020; Accepted: 23 February 2020; Published: 28 February 2020

**Abstract:** Background: Porcine reproductive and respiratory syndrome (PRRS) viruses are a major cause of disease and economic loss in pigs worldwide. High genetic diversity among PRRSV strains is problematic for successful disease control by vaccination. Mosaic DNA and vaccinia (VACV) vaccines were developed in order to improve protection against heterologous PRRSV strains. Methods: Piglets were primed and boosted with GP5-Mosaic DNA vaccine and recombinant GP5-Mosaic VACV (rGP5-Mosaic VACV), respectively. Pigs vaccinated with rGP5-WT (VR2332) DNA and rGP5-WT VACV, or empty vector DNA and empty VACV respectively, served as controls. Virus challenge was given to separate groups of vaccinated pigs with VR2332 or MN184C. Necropsies were performed 14 days after challenge. Results: Vaccination with the GP5-Mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both VR2332 and MN184C PRRSV strains. In contrast, vaccination of animals with the GP5-WT vaccines induced responses only to VR2332. Furthermore, vaccination with the GP5-Mosaic based vaccines resulted in protection against challenge with two heterologous virus strains, as demonstrated by the significantly lower viral loads in serum, tissues, porcine alveolar macrophages (PAMs), and bronchoalveolar lavage (BAL) fluids, and less severe lung lesions after challenge with either MN184C or VR2332, which have only 85% identity. In contrast, significant protection by the GP5-WT based vaccines was only achieved against the VR2332 strain. Conclusions: GP5-Mosaic vaccines, using a DNA-prime/VACV boost regimen, conferred protection in pigs against heterologous viruses.

**Keywords:** PRRSV Mosaic T-cell DNA vaccine VACV; PRRS; cross protection; heterologous virus challenge

#### **1. Introduction**

Porcine reproductive and respiratory syndrome (PRRS) is a major disease in pigs that causes significant economic losses to industry across the world. In the United States alone, PRRS causes over \$600 million in losses a year [1]. PRRS causes reproductive failure in pregnant sows and respiratory disease in young pigs. The causal virus, PRRSV, is a positive-sense, single-stranded RNA virus with a lipid envelope, and belongs to the Family *Arteriviridae,* Order *Nidovirales* [2–4]. The genome is approximately 15 Kb long and encodes at least 22 different viral proteins including fourteen non-structural and eight structural proteins [5]. PRRSV induces both humoral and cellular immune responses in pigs and some of the viral proteins inducing such responses have been identified; however, protection against reinfection is incomplete [6–9]. Killed virus (KV) vaccines and modified

live virus (MLV) vaccines for PRRS have been licensed for more than two decades. These vaccines generally reduce the severity of clinical signs and the transmission of virus; however, they do not induce sterilizing immunity. In general, the efficacy of MLV vaccines is superior to that of KV vaccines [10–13]. Importantly, however, highly variable and generally sub-optimal levels of protection against heterologous PRRSV strains have been reported [10,14–18]. The potential for reversion to the virulence of MLV vaccines and subsequent transmission to susceptible pigs is a concern [19–21].

The major challenge for the development of efficacious, broadly protective PRRS vaccines is the extraordinary genetic variation among disease producing PRRSV strains. Currently, two major PRRSV genotypes are recognized, Genotype 1 (European) and Genotype 2 (North American), which exhibit nearly 40% sequence dissimilarity [22,23]. Genotype 1 and Genotype 2 PRRSVs can be further divided into four subgroups and nine different lineages, respectively, based on phylogenetic analysis of ORF5 [24]. The co-existence of multiple variants within one farm, or even within individual pigs, possibly indicates the occurrence of quasispecies variation of PRRSV [25]. Thus, the development of vaccines that can induce protection against different subgroups and lineages is highly desirable. Multi-subunit vaccines [26], consensus vaccines [27], molecular breeding of different viral structural proteins [28], Mosaic T-cell vaccines [29,30], a polyvalent vaccine containing five different live-attenuated PRRSV strains [31] and DNA prime-MLV boost [32] are just some of the vaccines that are or have been under evaluation recently.

Studies on potential vaccines against human immune deficiency virus type 1 (HIV-1), have shown that Mosaic sequences designed from naturally occurring HIV sequences could effectively address genetic diversity when used as vaccines [33,34]. Mosaic vaccines have been shown to elicit broader immune responses successfully and to confer cross protection in non-human primates and these Mosaic HIV vaccines have been trialed in humans [34–38]. We previously reported that a PRRSV Mosaic vaccine based on 748 GP5 sequences of the Genotype 2 PRRSV strain was immunogenic [29] and induced reactivity to four divergent Genotype-2 PRRSV strains that had at least 10% or more difference in their GP5 sequence—as shown by the expression of interferon gamma (IFN-γ) by peripheral blood mononuclear cells—and, further, that vaccinated pigs were protected against challenge with VR2332 [30]. The present study demonstrates further that vaccination of pigs with the GP5-Mosaic by using a DNA prime and rVaccinia boost approach protects them against strains that are more than 10% divergent in their sequences (VR2332 and MN184C).

#### **2. Materials and Methods**

#### *2.1. Viruses and Cells*

The viruses used in the study included the VR2332 NA reference strain (ATCC VR-2332) and the MN184C strain provided kindly by Drs. Kelly Lager and Kay Faaberg at U.S. Department of Agriculture-Agriculture Research Service (USDA-ARS). The viruses were propagated in MARC-145 cells [39] grown in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM L-glutamine, 100 U penicillin/mL, 100 μg streptomycin/mL and 10% fetal bovine serum (FBS). The Reed–Muench formula was used to calculate virus titers [40]. Viruses were purified over continuous cesium chloride gradients, quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and stored at −80 ◦C for later use. Titrated viruses were used for ex vivo recall immune response assay and challenge experiments.

#### *2.2. Transfer Vector Construction*

A combination of gene synthesis (DNA 2.0, Menlo Park, CA, USA) and standard sub-cloning was utilized to generate three transfer vectors. Each transfer vector contained a cassette with the tetR gene (based on GenBank: X00694), and either a natural or Mosaic version of Genotype 2 PRRSV ORF 5 [29,30], followed by the EMCV IRES (based on GenBank: NC\_001479.1) and the EGFP gene (based on plasmid pEGFP-1, GenBank: U55761). The tetR gene and the ORF 5–IRES-EGFP genetic segment were placed under back-to-back synthetic VACV early/late promoters [41]. The tetO2 operator sequence was inserted after the putative VACV D6R promoter, as described in [42]. The cassettes were flanked by 600 bp of the VACV D5R gene to the left and 600 bp of the VACV D6R gene to the right (based on GenBank: NC\_006998.1). Restriction endonuclease analysis was used to confirm plasmid identity.

#### *2.3. Generation of Recombinant VACVs and Preparation of High-Titer Stocks*

Standard homologous recombination was used to generate Tetracycline-inducible recombinant VACVs after the transfection of transfer vectors with FuGene HD Transfection Reagent (Promega, Madison, WI, USA) into BS-C-1 cells, infected 1 h before with an IPTG-inducible VACV strain (based on the WR clone <IP_ADDRESS>.) in the presence of 0.1 mM IPTG and 1 μg/mL doxycycline. Recombinant EGFP-positive tetracycline-inducible VACVs were plaque purified in the absence of Isopropyl βd-1-thiogalactopyranoside (IPTG) and in the presence of 1 μg/mL doxycycline. The elimination of the parental virus was confirmed by PCR analysis of viral DNA that was purified using a Nucleospin® Blood kit (Macherey-Nagel, Düren, Germany). High-titer stocks were generated by infecting HeLa S3 cells with recombinant virus at a multiplicity of infection (MOI)of 0.1 in the presence of 1 μg/mL doxycycline. Infected cells were harvested and homogenized 4 days post-infection. Homogenates were clarified by centrifugation at 750× *g* for 10 min, purified over a sucrose cushion [43], and resuspended in 1X PBS pH 7.3 (with no doxycycline).

#### *2.4. Protein Sequence Alignment and Phylogenetic Analysis*

MEGA 7 (Molecular Evolutionary Genetics Analysis) (www.megasoftware.net) (NIH, Bethesda, MD, USA) was utilized to evaluate protein sequence alignments between GP5 sequences of VR2332, MN184C, Mosaic 1 and Mosaic 2 and to create a phylogenetic tree.

#### *2.5. Vaccination and Collection of Samples*

Cross-bred three- to four-week-old male and female piglets, which were free of PRRSV and porcine circovirus-2, were used in this study. The experimental design is summarized in Table 1. GP5-Mosaic, GP5-WT and vector-control vaccines were administered by both intradermal and intramuscular injection. Briefly, 1 mL vaccine containing 500 μg DNA and 100 μg Quil-A® as adjuvant was injected intradermally (0.1 mL) on the back of ear and intramuscularly (0.9 mL) in the neck region at day 0 and again on day 14. The animals were further boosted at day 28 with a 1 mL suspension containing 108 PFU VACV expressing GP5-Mosaic, GP5-WT or VACV and 100 μg Quil-A® as adjuvant. Pigs were challenged at day 35 with VR2332 and at day 37 with MN184C to their respective groups. A total of 106 TCID50 of each virus was administered by intranasal and intramuscular challenge to each animal. Blood was collected at days 0, 7, 14, 21, 28, the challenge day, and 4, 7, 10, and 14 days after challenge.

a One mL vaccine containing 500 μg DNA and 100 μg Quil-A® as adjuvant was injected intradermally (0.1 mL) on the back of ear and intramuscularly (0.9 mL) in the neck region at day 0 and again at day 14. VACV 108 PFU and 100 μg Quil-A® as adjuvant was administered at day 28. b Pigs were challenged at day 35 with 106TCID50 VR2332 (*n* = 4/group) and day 37 with 106TCID50 MN184C (*n* = 4/group) both intranasally and intramuscularly.

The pigs were euthanized at 14 days post-challenge. The lungs were evaluated macroscopically, weighed, and bronchoalveolar lavage (BAL) was then performed. Tissue samples were collected from each lung lobe, tracheobronchial lymph nodes (TBLN), spleen, inguinal lymph nodes (ILN) and tonsils: portions of each were either fixed in 10% neutral buffered formalin or kept frozen. Fixed tissues were routinely processed to paraffin, and 4–5 μm sections were stained with hematoxylin and eosin and evaluated histologically. Lung lesion scores were calculated as reported before [44]. All animal work was performed under a protocol approved by the University of Connecticut Institutional Animal Care and Use Committee, the animal protocol number under which the study was conducted is IACUC A17-003.

#### *2.6. Measurement of IFN-*γ *Immune Response to PRRSV*

Peripheral blood mononuclear cells harvested from blood collected at days 0 and on the challenge day, were seeded in 24-well flat-bottom plates (5 × 105 cells/well). They were then stimulated, in duplicate, by adding 200 TCID50 VR2332 or MN184C/well, or mock treated for 48 h at 37 ◦C in a 5% CO2 atmosphere. The cells were then collected and total RNA was extracted for quantitative real-time PCR analysis.

#### *2.7. Serum Neutralization Test*

Virus neutralization testing was carried out as previously reported [30]. Briefly, each serum was mixed with equal volumes of DMEM containing 100 TCID50 of VR2332 or MN184C and the mixtures were incubated at 37 ◦C for 1 h. The serum–virus mixtures were then added to 96-well plates containing 80–90% confluent MARC-145 cells and incubated for 48 h at 37 ◦C in a 5% CO2 atmosphere (final serum dilution 1:4). The VR2332 or MN184C virus, negative serum, and uninfected cells served as the virus control, negative serum control and cell control, respectively. The neutralizing capacity of serum was quantified by measuring the viral copy numbers by RT-qPCR in supernatants 48 h after the addition of pre-incubated serum–virus mixtures to the cells.

#### *2.8. Quantitative Real-Time PCR*

TRIzol LS Reagent or TRIzol Reagent (Invitrogen, Grand Island, NY, USA) was used to extract total RNA from serum/ culture supernatants or tissues, respectively. The extracted RNA was quantified in a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cDNA was synthesized from each sample RNA using a 20 μL reaction mixture and random primers (Invitrogen, Grand Island, NY, USA) and the following reaction conditions: 26 ◦C for 10 min, 42 ◦C for 45 min and 75 ◦C for 10 min. The cDNA was then amplified by SYBR Green real-time PCR. To this effect, SYBR qPCR Master Mix (Bimake, Huston, TX, USA), the cDNA template and 5 -ATG ATG RCC TGG CAT TCT-3 and 5 -ACA CGG TCG CCC TAA TTG-3 were utilized as the forward and reverse primers for ORF7, respectively. Real time PCR was then performed as follows: 2 min at 95 ◦C, followed by 40 cycles at 95 ◦C for 15 s and 61 ◦C for 1 min using Bio-Rad CFX96 Touch System (Bio-Rad, Hercules, CA, USA). For use in quantification, a standard curve was generated using serial dilutions of viral RNA which contained 102–107 copies/μL. Both positive and negative reference samples were run concurrently with the test samples. Viral loads were determined by plotting the Ct values against the standard curve. Melting curves were analyzed to verify the specificity of the PCR.

To test for IFN-γ expression, total RNA was extracted by TRIzol Reagent from virus-stimulated or mock-treated PBMCs; this was then used as template for cDNA synthesis and this, in turn, was used for real-time PCR following the protocol described above. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward primer: 5 -CGT CCC TGA GAC ACG ATG GT-3 and reverse primer: 5 -CCC GAT GCG GCC AAA T-3 ) was used as internal control to calculate the fold changes in the expression of IFN-γ (forward primer: 5 -TGG TAG CTC TGG GAA ACT GAA TG-3 and reverse primer: 5 -GGC TTT GCG CTG GAT CTG-3 ) by the delta–delta method [44].

#### *2.9. Lung Lesion Scoring*

The scoring of lung lesions was done systematically, as previously described [45], by a board-certified veterinary pathologist, blinded to the treatment groups. Nine sections, representing all six lung lobes, were scored, with one section from each division of the left cranial lung lobe and two sections from each diaphragmatic lobe.

#### *2.10. Statistical Analysis*

Two-way ANOVA or Student's t-test was used to evaluate the differences in measurements between the samples within or between groups. The data were analyzed using GraphPad Prism (version 7.0) (GraphPad Software, San Diego, CA, USA).

#### **3. Results**

#### *3.1. Sequence Alignment and Analysis of GP5*

The GP5 amino acid (aa) sequences in Mosaic 1, Mosaic 2, MN184C and VR2332 (the prototype of Genotype 2 PRRSV) were all the same size, with no deletions or insertions. Sequence alignments showed aa identity from 85% to 93%. To further investigate the antigenic relationship of these strains, a phylogenetic tree was constructed using the neighbor-joining method b. The four sequences clustered into two subgroups (Figure 1), with the two most distant being Mosaic 1 and MN184C. Mosaic 1 and Mosaic 2 were, relatively, more closely related to VR2332 and MN184C, respectively.

**Figure 1.** Phylogenetic analysis of the GP5 Amino acid sequence of the two Mosaic sequences, MN184C and VR2332. The analysis was done via the neighbor-joining method using MEGA7.0.

#### *3.2. GP5-Mosaic Vaccines Induced Both Humoral and Cellular Response*

GP5-Mosaic-vaccinated pigs had significantly higher levels of neutralizing antibodies in their serum compared to the vector-control animals, both to VR2332 (*p* < 0.001) and MN184C (*p* < 0.05) The virus neutralizing capability of serum from GP5-WT-vaccinated animals was greater against VR2332 (*p* < 0.01) and against MN184C (*p* > 0.05) when compared to serum from vector control animals (Figure 2A); vaccination with the GP5-Mosaic vaccine resulted in broader recall cellular responses than those induced with the GP5WT vaccine. Thus, a significantly higher (*p* < 0.05) relative fold-change in IFN-γ mRNA expression was detected upon stimulation of PBMCs derived from GP5-Mosaic-vaccinated pigs, with either VR2332 or MN184C strains, when compared to those in equally stimulated PMBCs from vector control pigs (Figure 2B). In contrast, the expression of IFN-γ mRNA in GP5-WT-vaccinated pigs was significantly higher only if their PBMCs were stimulated with

VR2332, when compared to those in the equally stimulated PBMCs of the control pigs at the same time points (Figure 2B). No changes were detected with the PBMCs of vector-control pigs with any type of stimulation.

**Figure 2.** Vaccination-induced humoral and cellular responses. (**A**) The virus neutralization was expressed as viral copy numbers (log10 scale), as measured by RT-qPCR in cell supernatants after the infection of MARC-145 cells with pre-incubated serum–virus mixtures. (**B**) The expression of IFN-γ mRNA as fold changes, by either VR2332 or MN184C-stimulated PBMCs collected on the challenge day. Each dot represents the value of one animal. The variation is expressed as standard error of the means. There were three independent replications. Significant differences were calculated by a two-way ANOVA or Student's *t* test (\* *p* < 0.05, \*\*\* *p* < 0.0001).

#### *3.3. GP5-Mosaic Vaccination Induced Cross-Protection in Pigs*

Viral loads in serum of both GP5-WT and GP5-Mosaic-vaccinated groups were significantly lower than those in the vector-control group at 7, 10 and 14 days after challenge with VR2332 (\* *p* < 0.05, \*\* *p* < 0.01) (Figure 3A). Therefore, the capacity of reducing VR2332 virus loads in serum was relatively similar between the GP5-Mosaic and the GP5-WT vaccines. Furthermore, there was a steady decrease in viral loads from 4 days to 14 days after challenge in both GP5-WT and GP5-Mosaic vaccinated groups, while viral loads increased during the course of challenge and reached their peak at 10 days post-challenge (DPC), then declining slightly at 14 DPC in the vector-control group (Figure 3A). In contrast, the viral loads in serum of animals receiving the GP5-Mosaic-vaccine were significantly lower than those of animals receiving the GP5-WT vaccine or those of vector-control animals at 7 and 14 DPC with MN184C (\* *p* < 0.05) (Figure 3B), which was different compared to VR2332-challenged groups. Viral load levels in GP5-WT vaccinated animals, increased during the infection with MN184C and reached their peak at 10DPC, which is comparable to the vector-control group. The viral loads in GP5-Mosaic vaccinated animals decreased steadily from 4 to 10 DPC and remained unchanged until 14 DPC (Figure 3B). Furthermore, the viral loads in lung, TBLN, spleen, ILN and tonsils were significantly lower in GP5-Mosaic-vaccinated animals than those in the vector-control group (\* *p* < 0.05) after challenge with MN184C, while those of the GP5-WT group were similar or slightly lower compared to the vector-control group (Figure 3C). Viral loads in the lungs, TBLN, and tonsil were significantly lower in GP5-Mosaic-vaccinated animals than those in the vector-control group (\* *p* < 0.05) after challenge with VR2332, while those of the GP5-WT group were significantly lower in the lungs, spleen and ILN compared to the vector-control group (\* *p* < 0.05). There was no significant difference in VR2332 virus loads between the GP5-Mosaic and GP5-WT groups (Figure 3D).

**Figure 3.** Virus clearance in sera and tissues. (**A**) Viral copy numbers in serum from 0 to 14 DPC upon VR2332 challenge. (**B**) The viral copy numbers in serum from 0 to 14 DPC upon MN184C challenge. Each dot represents the mean value of each group. The variation bars are expressed as the standard error of the mean. Three separate experiments were performed for each. A two-way ANOVA or Student's *t* test (\* *p* < 0.05, \*\* *p* < 0.01, was used to calculate significant differences; days post-challenge (DPC). (**C**) The viral copy numbers in tissues at necropsy upon VR2332 challenge. (**D**) The viral copy numbers in tissues at necropsy upon MN184C challenge. Each bar represents the mean value of each group. The bars are the standard error of the mean. Three separate experiments were performed for each. Significant differences were calculated by Student's *t* test (\* *p* < 0.05).

Tests in PAMs and BAL fluids showed a similar pattern in which the viral loads in both the GP5-Mosaic and GP5-WT groups were lower than those in the vector-control group after challenge with VR2332 (Figure 4A,C), meanwhile only the viral loads of GP5-Mosaic-vaccinated animals were significantly lower than those in the vector-control animals after challenge with MN184C (\* *p* < 0.05 and \*\* *p* < 0.01, respectively), furthermore there was no apparent difference in MN184C viral loads between GP5-WT and vector-control animals (Figure 4B,D).

**Figure 4.** Virus clearance in bronchioalveolar lavage fluids (BAL) and PAMs. (**A**) The viral copy numbers in BAL fluids at necropsy upon challenge with VR2332. (**B**) The viral copy numbers in BAL fluids at necropsy upon challenge with MN184C. Each bar represents the mean value of each group. Bars represent the standard error of the mean. Three independent experiments were performed for each. Significant differences were calculated by Student's *t* test (\* *p* < 0.05). (**C**) The viral copy numbers in PAMs at necropsy upon challenge with VR2332. (**D**) The viral copy numbers in PAMs at necropsy upon challenge with MN184C. Each dot represents the mean value of each animal. The bars represent the standard error of the mean. Three independent experiments were performed for each. Student's *t* test was used to calculate significant differences (\* *p* < 0.05, \*\* *p* < 0.01).

#### *3.4. Lower Lung Lesion Scores Detected in GP5-Mosaic-Vaccinated Animals*

The lung lesion scores after challenge with MN184C were significantly lower in GP5-Mosaic-vaccinated animals than those in both GP5-WT and vector-control animals when evaluated using nine sections of lung (Figure 5 right panel, *p* < 0.05). In contrast, after VR2332 challenge, lung lesion scores were lower on average in both the GP5-Mosaic and GP5-WT groups than those in vector-control animals. There were no significant differences between the lung lesion scores of GP5-WT and GP5-Mosaic-vaccinated groups after challenge with VR2332 (Figure 5).

**Figure 5.** Lung lesion scores. The lung lesion scores of GP5-Mosaic-vaccinated animals were significantly lower (*p* < 0.05) than those of GP%-WT or vector-control animals after challenge with MN184C. Each figure represents the mean value of each individual. The bars represent the standard error of the mean. Three independent experiments were performed for each. Student's *t* test was utilized to calculate significant differences (days post-challenge (DPC)).

#### **4. Discussion**

There is extraordinary diversity among PRRSV strains. The diversity among PRRSV strains is as high as, or may even surpass that of, HIV. Recently, several vaccine candidates, including a synthetic consensus PRRSV strain [27], a chimeric PRRSV strain containing multiple ORFs DNA shuffled [46–48] and an intranasal live virus vaccine with adjuvant [49] induced various levels of heterologous protection in pigs. We have developed GP5-Mosaic vaccines that incorporate sequences derived from naturally circulating viruses. The data obtained from our GP5-Mosaic vaccinated pigs supports the Mosaic vaccine approach as an effective strategy to address PRRSV diversity. It has also been demonstrated that Mosaic T-cell vaccines have great potential for other viruses with extraordinary diversity, such as HIV [33–38]. In an earlier study, we demonstrated the immunogenicity of GP5-Mosaic vaccines in swine and their ability to induce cross-reactivity, as shown by their broad recall responses ex-vivo, and conferred the partial protection they provide in pigs [29,30]. The ability of the GP5-Mosaic vaccines to induce cross-protection in pigs against heterologous PRRSV strains was confirmed in the present study.

A sequence analysis of two GP5-Mosaic vaccines and the two Genotype 2 PRRSV strains utilized in the study, VR2332 and MN184C, that belong to lineage 5 and to lineage 1, respectively, revealed a 15% difference in the GP5 aa sequences. The aa sequences were used to generate a phylogenetic tree using the neighbor-joining method [24]. The GP5-Mosaic 1 was closer to VR2332 (93% aa identity), while GP5-Mosaic 2 was closer to MN184C (90% aa identity), which suggested that the two GP5-Mosaic sequences broaden the coverage to strains belonging to different lineages. Based on this analysis, a broader protection could be expected with GP5-Mosaic vaccines. The data of the present study supports clearly the capacity of the GP5-Mosaic vaccine to cross-protect pigs against VR2332 and MN184C that share only 85% aa identity.

VACV provides a very good platform for the development and testing of vaccines, including animal vaccines, human vaccines and cancer immunotherapy [50]. In our study, a GP5-Mosaic DNA vaccine prime/GP5-Mosaic rVaccinia (VACV) boost regime was used to immunize pigs. This type of prime-boost regime has previously been demonstrated to increase vaccination efficacy substantially [51,52]. In addition, Quil-A®, used as adjuvant for the priming of the GP5-Mosaic DNA vaccine, was previously shown to further increase the immune responses [53–55]. Therefore, the prime-boost vaccination regime used here for testing GP5-Mosaic vaccine efficacy, in terms of broad protection, proved appropriate for testing the hypothesis.

PBMCs collected on the challenge day from GP5-Mosaic-vaccinated pigs responded to a broader range of virus strains, as measured by IFN-γ mRNA expression, than those from GP5-WT vaccinates. Thus, PBMC from GP5-Mosaic-vaccinated pigs expressed higher levels of IFN-γ mRNA in response to stimulation with VR2332 or MN184C strains, respectively, compared to those of the vector-control pigs. In contrast, PBMC from GP5-WT vaccinated pigs expressed higher levels of IFN-γ mRNA only when stimulated with VR2332. These data were consistent with those of our previous study where the GP5-Mosaic vaccine induced broad recall cellular responses, as measured by significantly higher levels of IFN-γ mRNA expression in response to the four diverse Genotype 2 PRRSV strains tested, including VR2332, NADC9, NADC30 and SDSU73 [30]. The significant induction of IFN-γ is an important asset of a PRRSV vaccine, as IFN-γ reportedly plays a critical role in the control of and protection from PRRSV infection [56]. In addition, both the GP5-Mosaic and the GP5-WT vaccines induced relatively high levels of neutralizing antibodies in VR2332-challenged pigs. These data indicate that the GP5-Mosaic vaccine preserves virus-neutralizing epitopes that appear to contribute to the overall efficacy of this vaccine.

In terms of vaccine-induced protection, both GP5-Mosaic and GP5-WT-vaccinated pigs had significantly lower serum viral loads at 7, 10 and 14 DPC than the vector-control group after the VR2332 challenge. In contrast, only the GP5-Mosaic-vaccinated group showed significantly lower viral loads in sera at 7 and 14 DPC than the vector-control group after the MN184C challenge. These data demonstrate that the GP5-Mosaic vaccine is capable of cross-protecting pigs against two divergent PRRSV strains (15% difference) while the GP5-WT vaccine only provided protection in pigs against the homologous strain. Furthermore, a similar pattern was found with respect to viral loads in tissues, where the GP5-Mosaic-vaccinated pigs had significantly lower viral loads in their lungs, TBLN, spleen, ILN and tonsils compared to the vector-control group after challenge with MN184C, while the GP5-WT group only showed slightly lower (numerically, but not significant) viral loads. In the VR2332 challenge experiment, both the GP5-Mosaic and GP5-WT vaccinates had significantly lower viral loads in tissues generally—the viral loads in the GP5-Mosaic vaccinates were significantly lower in their lungs, TBLN and tonsils, while viral loads in the GP5-WT group were significantly lower in their lungs, ILN and spleen, compared to the vector-control group. Similar protective effects were also demonstrated in the GP5-Mosaic vaccine vaccinates, where lower viral loads were detected in the BAL fluids or PAMs after MN184C challenge, further confirming the cross-protective capability of the GP5-Mosaic vaccine. Indeed, this is finding may be particularly relevant since PAMs are the primary target of PRRSV infection. Lung lesion scoring is an essential and crucial method to evaluate vaccine efficacy, as infection by PRRSV tends to increase susceptibility to coinfection with other virus or bacterial pathogens [57–59] which may result in more severe lesions and mortality. Pigs vaccinated with the GP5-Mosaic vaccine had significantly lower lung lesion scores than both the GP5-WT and vector-control groups after challenge with MN184C, while both the GP5-Mosaic and GP5-WT groups had lower lung lesion scores compared to the vector-control group in the VR2332 challenge experiment.

#### **5. Conclusions**

Taken altogether, our results clearly demonstrate that the GP5-Mosaic vaccine provided cross-protection against two heterologous PRRSV strains, VR2332, the prototype Genotype 2 strain, and MN184C, a highly virulent strain, while the GP5-WT vaccine provided only homologous protection against VR2332. In summary, our results demonstrate that our GP5-Mosaic vaccine is able to induce broader protection against PRRS and warrants further research for field applications.

**Author Contributions:** Conceptualization, P.H.V. and A.E.G.; Methodology, J.C., C.M.O., J.A.S., P.H.V. and A.E.G.; Formal analysis, J.C. and A.E.G.; Investigation, J.C., C.M.O., C.H., K.S., J.A.S., P.H.V., H.J.V.K., and A.E.G.; Data curation, J.C. and A.E.G.; Writing—original draft preparation, J.C.; Writing—review and editing, J.C., C.M.O., J.A.S., P.H.V. and A.E.G.; Supervision, A.E.G.; Project administration, A.E.G.; Funding acquisition, J.A.S., P.H.V. and A.E.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by USDA/NIFA Grant Number 2011 67015-30176 and NC229.

**Acknowledgments:** The authors thank Kay Faaberg and Kelly Lager kindly providing the PRRSV strains.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector**

### **Da Shi** †**, Xiaobo Wang** †**, Hongyan Shi** †**, Jiyu Zhang, Yuru Han, Jianfei Chen, Xin Zhang, Jianbo Liu, Jialin Zhang, Zhaoyang Ji, Zhaoyang Jing and Li Feng \***

State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China<EMAIL_ADDRESS>(D.S<EMAIL_ADDRESS>(X.W<EMAIL_ADDRESS>(H.S<EMAIL_ADDRESS>(J.Z<EMAIL_ADDRESS>(Y.H<EMAIL_ADDRESS>(J.C<EMAIL_ADDRESS>(X.Z.);

<EMAIL_ADDRESS>(J.L<EMAIL_ADDRESS>(J.Z<EMAIL_ADDRESS>(Z.J<EMAIL_ADDRESS>(Z.J.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +86-0451-5105-1720

† These authors contributed equally to this work.

Received: 27 August 2019; Accepted: 16 October 2019; Published: 2 November 2019

**Abstract:** Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected *N* gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.

**Keywords:** porcine epidemic diarrhea virus; RNA interference; processivity factor; intestine epithelial cells; *N* gene

#### **1. Introduction**

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious intestinal infectious disease. PED is an important disease in swine-producing countries. PED causes the death of newborn piglets and weight loss in pigs of all ages from PEDV-induced severe symptoms, such as serious diarrhea, vomiting, and dehydration, which seriously damage the swine industry [1]. Following reports in 1978 [1], PED had an outbreak in swine-farming countries in Asia, North America, South America, and Europe [2–5]. Starting from the end of 2010, highly virulent PEDV variants that differed from the classic European strain CV777 were widespread in China, resulting in high mortality of newborn piglets and huge economic losses [6–9]. PEDV can be divided into genotypes G1 and G2 based on phylogenetic analysis of full-length *S* gene sequences [10]. PEDV strains detected in China since 2010 mostly belonged to genotype G2, which differed genetically from the CV777 vaccine strain belonging to subtype G1 [11]. However, many studies demonstrated that

commercially available PEDV vaccines derived from classical strains of PEDV do not provide effective protection against highly virulent PEDV variant infections in China [3]. Thus, PEDV infection remains a major veterinary problem. Understanding it is essential to developing novel antiviral drugs that will specifically inhibit PEDV propagation.

RNA interference (RNAi) is a short, double-strand RNA-induced process that targets and degrades the messenger RNA (mRNA) of specific sequences [12,13]. Post-transcriptional gene silencing can be mediated by exogenous small interfering RNAs (siRNAs), endogenous microRNAs (miRNAs), and short hairpin RNAs (shRNAs). Effective gene knockdown is achieved by shRNAs by inducing the endogenous RNAi process [14,15]. Transcribed shRNAs are exported from the nucleus by Exportin-5 and processed by the RNase III Dicer into small double-stranded RNA (dsRNA) molecules of 19 to 23 bp called siRNAs. The complementary guide strand of processed siRNA is incorporated into the RNA-induced silencing complex to mediate the cleavage of target mRNAs [16,17]. RNAi evolves in the host defense system directed at infectious viruses and transposable elements. Effective silencing of transgene expression and endogenous genes in vivo was shown by a number of groups [6,7,18]. These findings raised the possibility that RNAi could be another therapeutic approach for inhibiting virus infection. ShRNAs were employed for therapy against human viral diseases, as well as cancer and neurogenerative diseases [8]. PEDV primarily infects villous epithelial cells throughout the small intestine and causes serious injury of intestine epithelial cells (IECs), including superficial villous enterocyte swelling and severe diffuse atrophic enteritis [19]. Infection of PEDV variant strains in the field complicates the development of prophylactic and therapeutic strategies to protect suckling pigs from diarrhea. As an RNA virus, PEDV could be an ideal target for studying its biology and therapeutics using RNAi.

The PEDV nucleocapsid (N) protein, which is abundantly expressed in infected cells, has multiple functions. It is one of the structural proteins that forms complexes with genomic RNA, enhancing viral transcription and assembly. Therefore, the N protein may be a potential drug target for antiviral therapy against PEDV infection. In this study, we compared and analyzed *N* gene sequences from 25 different PEDV G1 and G2 isolates from different countries. Three novel shRNAs targeting conserved and unexploited regions in the *N* gene were tested for inhibition of PEDV CV777 and LNCT2 replication. Cell viability, viral titer, and protein expression were examined as indicators of the efficacy of targeted gene silencing by the shRNAs. All three shRNAs effectively inhibited PEDV replication and *N* expression of the G1 and G2 subtypes.

#### **2. Materials and Methods**

#### *2.1. Viral Propagation and Titer Assays*

Swine intestinal epithelial cells (IECs) were donated by Dr. Yanming Zhang (Northwest A&F University, China), and derived from the mid-jejunum of neonatal, unsuckled, one-day-old piglets. Primary intestinal epithelial cells were isolated by the tissue explant adherent method and purified by trypsin digestion with citric acid, forming an undifferentiated porcine intestinal epithelial cell line that was immortalized. Cells were maintained in a Dulbecco's minimum essential medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA)/nutrient mixture F-12 (Ham) (1:1) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco), 5 ng/mL epidermal growth factor (Life Technologies, Carlsbad, CA, USA), 5 mg/mL insulin-transferring selenium supplements (Life Technologies), and 1% penicillin–streptomycin (Life Technologies). Cell culture media were changed every two days, and cells were passaged every 3–4 days by trypsinization with 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) (Life Technologies). Vero E6 (African green monkey kidney cells, American Type Culture Collection (ATCC)) were grown and maintained in DMEM supplemented with 10% heat-inactivated FBS and penicillin–streptomycin, and incubated at 37 ◦C with 5% CO2.

The PEDV G1 CV777 vaccine strain (GenBank accession: AF353511.1) was preserved at Harbin Veterinary Research Institute (Harbin, China). PEDV G2 strain LNCT2 (GenBank accession: KT323980.1) was isolated in Vero E6 cells in our laboratory. Vero E6 cells were cultured and used to amplify PEDV as previously described [9]. After 70% of virus-infected cells showed cytopathic effects (CPEs), cultures were collected for three freeze–thaw cycles. Viral titration used 96-well microplates with Vero E6 cells. Viral cultures were 10-fold serially diluted with virus replication medium containing trypsin (10 μg/mL). Confluent Vero E6 cells from microplates were washed three times with phosphate-buffered saline (PBS) and inoculated at 0.1 mL per well into eight wells. Following adsorption for 1 h at 37 ◦C, the inocula were removed, and cells were washed three times with PBS. Subsequently, 0.1 mL of fresh virus replication medium was transferred into each well, and cells were incubated 4–5 days at 37 ◦C. The 50% tissue culture infective dose (TCID50) was expressed as the reciprocal of the highest dilution showing CPE by the Reed and Muench method. Assays were performed in triplicate in three independent experiments.

#### *2.2. Plasmid Construction*

Plasmids expressing GFP-tagged N and myc-Tagged N were described previously [20]. The design of shRNAs targeting the PEDV strain LNCT2 genome *N* gene (Figure 1A) used methods from the literature [21] and the web-based Block-iTTM RNAi Designer program [22]. A Basic Local Alignment Search Tool (BLAST) search [23] was performed to exclude possible homologous sequences. Three individual targeting sites were selected and chemically synthesized (Sangon Biotech, Shanghai, China) (Table 1). Control shRNA was designed at the same time to have no homology with PEDV or the IEC cell genome. All sequences were arranged as *Bbs*I + sense + loop + antisense + termination signal + *BamH*I and cloned into the pGPU6-Hygro vector to make the shRNA-expressing plasmids shR-N307, shR-N463, shR-N1071, and shR-NC (Figure 1B,C). Expression of siRNAs was driven by the U6 promoter.

**Figure 1.** Schematic description of the target viral genome, and small interfering RNA (siRNA)-expressing cassette. (**A**) Genomic structure of porcine epidemic diarrhea virus (PEDV) and position of target short hairpin RNA (shRNA) at *N* gene. ShR-N307, shR-N463, and shR-N1071 indicate initial shRNA target sites; (**B**) sequences and design map for shRNA constructs; (**C**) structure of predicted shRNA.

#### *2.3. Plasmid DNA Preparation*

Plasmid DNA was transformed into electrocompetent DH5α *Escherichia coli* and purified with EndoFree Plasmid Maxi Kits (QIAGEN, Hilden, Germany).

#### *2.4. Cell Transfection and Antiviral Activity In Vitro*

We validated the inhibitory effects of the shRNAs against N by target gene expression in an in vitro transfection system. One day before transfection, IEC cells were seeded into 12-well plates at 5 × 104 cells per well without antibiotics and grown at 37 ◦C overnight with 5% CO2. The 60–80% confluent cells were transiently transfected with the indicated plasmid using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. At 48 h after transfection, *N* expression was analyzed by Western blot or inverted fluorescence microscope. To examine the inhibitory effects of shRNAs against N on target gene expression during PEDV replication, IEC cells were transfected with or without 1 μg, 2 μg, or 4 of μg shR-N307, shR-N463, and shR-N1071 or 4 μg of shR-NC for 24 h and infected with 100 TCID50/mL PEDV strain CV777 or LNCT2. At 48 h post-infection (hpi), antigen slides were prepared to detect PEDV N protein expression by Western blot. In parallel experiments, cells from individual wells were collected and frozen and thawed twice, followed by centrifugation at low speed (1000× *g*), and supernatants were serially diluted, inoculated with Vero E6 cells, and titrated by the Reed and Muench method.

#### *2.5. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blots*

IEC cells grown in 12-well plates were transfected and infected as described. Cells were harvested at the indicated time points after transfection or virus infection, washed once with cold PBS, and lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) to determine protein concentrations. Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting onto nitrocellulose membranes. Membranes were washed twice in Tris-buffered saline (TBS) and incubated 2 h in Superblock blocking buffer (ThermoFisher, Waltham, MA, USA). Membranes were incubated with anti-N monoclonal antibody (mAb) 3G2 (prepared by our laboratory, diluted 1:1000), anti-myc monoclonal (Sigma, diluted 1:1000) or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Sigma, diluted 1:10,000). Proteins were revealed by IRDye 800CW goat anti-mouse immunoglobulin G (IgG) (H + L) (1:10,000) (LiCor BioSciences, Lincoln, NE, USA), and blots were visualized using an Odyssey infrared imaging system (LiCor BioSciences).

#### *2.6. Cell Viability Assays*

Cell viability assays were performed using cell counting kit-8 (CCK-8) (CK04; Dojindo, Shanghai, China), according to the manufacturer's protocol. In brief, IEC cells were seeded in 96-well plates at 10,000 per well and incubated at 37 ◦C for 24 h. Cells were transfected or not with shRNAs, and plates were incubated for 48 h before 10 μL of CCK-8 was added to wells for incubation for 2 h. Optical density at 450 nm was measured. Viability of treated cells was expressed as a percentage relative to untreated cells.

#### *2.7. Immunofluorescence Assays*

IEC cells were seeded in 12-well plates, and confluent cell monolayers were transfected with shR-N307, shR-N463 and shR-N1071, or shR-NC (4 μg) with Lipofectamine 3000 (Invitrogen) before PEDV infection. Cells were infected with PEDV strain CV777 or LNCT2 at 100 TCID50/mL. PEDV infection was analyzed using immunofluorescence assays (IFAs) at 48 hpi. Cells were fixed with 4% paraformaldehyde at 4 ◦C for 30 min and washed with PBS. Fixed cells were permeabilized with 0.2% Triton X-100 for 15 min at room temperature and blocked with blocking buffer (PBS with 5% bovine serum albumin (BSA)) for 2 h. Preparations were labeled with the mouse anti-PEDV N mAb (1:100 dilution) at 37 ◦C for 2 h followed by labeling with Alexa Fluor 488 goat anti-mouse IgG antibody (1:200 dilution) (ThermoFisher) for 1 h at 37 ◦C. Cell nuclei were stained with 4 ,6-diamidino-2-phenylindole (DAPI) (0.05 μg/mL) (D9542; Sigma) for 15 min and analyzed using an AMG EVOS F1 florescence microscope.

#### *2.8. Statistical Analysis*

All statistical data are expressed as means ± standard deviation (SD) of three independent experiments and analyzed using the Student's *t*-test. A *p*-value <0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. IEC Cells and PEDV Infection*

Phylogenetic analysis of CV777 and LNCT2 was based on complete genomic sequences (Figure 2A). CV777 clustered with PEDV G1 genotype (classic strains), whereas LNCT2 clustered with PEDV G2 genotype (epidemic strains), with >98% nucleotide identity to these strains. To determine PEDV propagation in IEC cells, CPEs and IFAs were used to monitor after infection (Figure 2B). At 48 hpi, CV777 and LNCT2 caused similar CPEs in IEC cells, characterized by rounding, aggregation, and rupturing. The morphology of negative control cells remained unchanged and did not exhibit any signs of CPEs. Inoculation of CV777 and LNCT2 with IEC cells resulted in positive immunofluorescent staining with mAb that recognized the virus N protein. Controls were negative for immunostaining.

**Figure 2.** Phylogenetic analysis and virus-induced cytopathic effect (CPE) in intestine epithelial cells (IECs). (**A**) Phylogenetic trees of PEDV based on complete genomic DNA. Black squares, strains in United States of America (USA); black triangles, strains in South Korea; black rhombuses, strains in China; red circle, G1 CV777 strain from this study; red rhombus, G2 LNCT2 strain from this study. (**B**) Production and growth properties of PEDV CV777 and LNCT2 in IEC cells. CPE 48 h post-infection (hpi) (upper panels, bar: 400 μm) and immunofluorescence assay (IFA) 48 hpi (lower panels, bar: 200 μm) of CV777 and LNCT2 in IEC cells. IEC cells were infected with PEDV CV777 or LNCT2 at 100 50% tissue culture infective dose (TCID50)/mL. CPE and IFA were examined at 48 hpi, and cell images were captured.

#### *3.2. Selection of Targeted Sites for N Gene-Specific shRNAs*

The prevalent strains that caused the outbreak of PEDV in China in 2010 and in North America in recent years belonged to the G2 genotype. PEDV is a positive-stranded RNA virus with higher mutation rates than DNA viruses [24]. Specific shRNAs were designed to target the conserved gene. We used the *N* gene of LNCT2 as the target sequence. Using nucleotide substitutions in the *N* gene and previous reports [25], we chose three unused regions that were well conserved in *N* genes among 25 isolates of PEDV G1 and G2 genogroups. The sites were at +307 to +327 nt, +463 to +483 nt, and +1071 to +1091 nt relative to the 5 ATG initiation codon (Table 1).

#### *3.3. Cell Viability Is Not A*ff*ected by Plasmids with shRNAs*

ShRNAs induce large amounts of cell death when transfected at large volume, resulting in interference with experimental results. To detect if the three shRNAs we used led to cytotoxicity at 4 μg, IEC cells were seeded in 96-well microplates and transfected with shR-N307, shR-N463, shR-N1071, or shR-NC (4 μg) using Lipofectamine 3000 (Invitrogen), or transfected with transfection reagent alone as mock. After transfecting for 48 h, CCK-8 solution (10 μL) was added to wells, and plates were incubated at 37 ◦C for 2 h. Absorbance was measured at 450 nm using a microtiter plate reader (Bio-Rad, Hercules, CA, USA). Viable cells that were mock transfected with transfection reagent alone were the reference of 100% cell viability. ShR-N307, shR-N463, and shR-N1071 exhibited no obvious cytotoxicity in transfected Vero E6 and IEC cells at 4 μg (Figure 3).

**Figure 3.** ShRNA treatment did not affect cell viability. Cell viability was detected by cell counting kit-8 (CCK-8) assays after transfection of IEC cells with shRNAs for 48 h. Absorption at 450 nm was recorded and expressed as a percentage of relative cell viability. Values are means ± SD (*n* = 3). Significant differences were assessed by Student's *t*-test; ns, no significant difference compared to control; *p* > 0.05.

#### *3.4. E*ffi*cient Inhibition of PEDV Myc-N or AcGFP-N Expression by shRNA Expression Cassettes*

We designed three shRNAs specifically targeting regions shown in Figure 1, named shR-N307, shR-N463, and shR-N1071. Western blots determined the inhibitory effects of the shRNAs on *N* gene expression. PMyc-N was co-transfected into IEC cells with increasing doses of shR-N307, shR-N463, or shR-N1071 using Lipofectamine 3000. At 48 h after transfection, N protein expression was analyzed by Western blots using mouse anti-Myc mAb. Western blots demonstrated that shR-N307, shR-N463, and shR-N1071 inhibited Myc-tagged N protein expression in a dose-dependent manner (Figure 4A).

**Figure 4.** Inhibitory effects of shR-N307, shR-N463, and shR-N1071 on the PEDV N protein. (**A**) Western blots for effect of shR-N307, shR-N463, and shR-N1071 on *N* gene expression. PMyc-N was co-transfected with increasing doses of shR-N307, shR-N463, or shR-N1071 into IEC cells. After 48 h, transfected cells were lysed. Equal amounts of cell lysates were resolved by 12.5% SDS-PAGE. Reaction products were probed with anti-Myc or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Densitometric data for N/GAPDH from three independent experiments are shown as means ± SD. \* *p* < 0.05. The *p* value was calculated using Student's *t*-test; (**B**) shR-N307, shR-N463, and shR-N1071 influence on pAcGFP-N expression in cultured IEC cells. PAcGFP-N was co-transfected with increasing doses of shR-N307, shR-N463, or shR-N1071 into IEC cells. PAcGFP-N expression plasmid was the unrelated control. Images show enhanced GFP (EGFP) expression at 48 h post-transfection.

The *N* gene was fused with the *AcGFP* gene to make pAcGFP-N. The effect of shRNAs on *N* gene expression was monitored by *AcGFP* expression. Plasmid pAcGFP-N was transfected into IEC cells alone or with the indicated concentrations of shR-N307, shR-N463, or shR-N1071 expression cassettes. At 48 h post-transfection, the effects of shRNA on enhanced GFP (EGFP) expression were monitored by fluorescence microscopy. The shR-N307, shR-N463, and shR-N1071 expression cassettes targeting *N* gene inhibited the expression of *AcGFP* to some extent compared with cells transfected with plasmid pAcGFP-N alone. The shR-N307, shR-N463, and shR-N1071 expression cassettes targeting the *N* sequence were similar, with weak fluorescence observed from cells transfected with 4 μg of shRNAs (Figure 4B).

#### *3.5. ShRNA-Mediated Inhibition of N Expression and PEDV Production in Infected IEC Cells*

To examine the inhibitory effects of shRNAs against N on target gene expression during CV777 and LNCT2 replication, IEC cells were transfected with indicated plasmids for 24 h and then infected with PEDV CV777 orLNCT2 strain (100 TCID50/mL). At 48 hpi, PEDV-infected cells were lysed with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and Western blots were used to analyze *N* expression using mAb 3G2. Treatment with shR-N307, shR-N463, and shR-N1071 significantly reduced *N* expression compared to shR-NC- or mock-transfected IEC cells. The inhibitory effects of the three shRNAs on *N* expression were elevated with increased shRNA concentration (Figures 5A and 6A). These results show that *N*-targeted shRNA efficiently inhibited target gene expression during PEDV replication.

**Figure 5.** Dose-dependent inhibitory effects of shR-N307, shR-N463, and shR-N1071 against N on target gene expression and PEDV CV777 replication in infected IEC cells. IEC cells with shRNA (1, 2, 4 μg) for 24 h before infection with PEDV CV777 for 48 h. (**A**) PEDV N protein by Western blot with anti-N protein monoclonal antibody (mAb). Densitometric data for N/GAPDH from three independent experiments, shown as means ± SD. \* *p* < 0.05. The *p* value was calculated using Student's *t*-test; (**B**) PEDV CV777 titers in shRNA-transfected IEC cells. ShRNA transfection and viral infection were as in panel A. Viral titers in supernatants collected at 48 hpi were determined using the Reed–Muench method. Error bars represent standard errors of the mean from three independent experiments. \* *p* < 0.05. The *p* value was calculated using Student's *t*-test.

**Figure 6.** Dose-dependent inhibitory effects of shR-N307, shR-N463, and shR-N1071 directed against N on target gene expression and PEDV LNCT2 replication in infected IEC cells. IEC cells with shRNA (1, 2, 4 μg) for 24 h were infected with PEDV LNCT2 for 48 h. (**A**) PEDV N protein was detected by Western blot with anti-N protein mAb. Densitometric data for N/GAPDH from three independent experiments are shown as means ± SD. \* *p* < 0.05. The *p* value was calculated using Student's *t*-test; (**B**) PEDV LNCT2 titers in shRNA-transfected IEC cells. ShRNA transfection and viral infection were as in panel A. Viral titers in supernatants collected at 48 hpi were determined using the Reed–Muench method. Error bars represent standard errors of the mean from three independent experiments. \* *p* < 0.05. The *p* value was calculated using Student's *t*-test.

To determine if downregulation of *N* expression by shRNAs decreased PEDV CV777 and LNCT2 strain replication, IEC cells were transfected with indicated plasmids for 24 h and infected with CV777 or LNCT2 (100 TCID50/mL). Cell cultures were collected at 48 hpi, and viral titers were determined. Transfection with shR-N307, shR-N463, or shR-N1071 significantly reduced PEDV CV777 and LNCT2 replication compared to shR-NC- or mock-transfected IEC cells (*p* < 0.05) (Figures 5B and 6B). Although differences in inhibition of PEDV CV777 and LNCT2 strain replication induced by 1 μg, 2 μg, or 4 μg of shR-N307, shR-N463, and shR-N1071 were seen, PEDV replication gradually reduced with increased shRNA concentrations.

#### **4. Discussion**

In this study, we demonstrated that shRNAs against PEDV N protein broadly inhibited PEDV G1 and G2 strains. This report found that shRNAs inhibit swine coronavirus replication in epithelia. Most coronaviruses such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and PEDV infect epithelial cells in the respiratory and/or enteric tracts. The control of coronavirus diseases is important for human public health security. Further developing RNAi as a potential therapeutic agent against coronavirus infection is worthwhile.

The PEDV N protein is predominantly produced in susceptible cells, which makes it a major target for early and accurate diagnosis [26]. The N protein forms complexes with coronavirus genomic RNA and enhances the viral transcription and assembly. Because of the importance of N protein in viral replication and the coronavirus *N* gene long used as a major target for shRNA design, we focused on the inhibition of PEDV infection using RNAi targeting the *N* gene of PEDV and systematically evaluated suppression efficiency. Alterations of viral genomes such as nucleotide substitution, insertion, and deletion have the potential to decrease the inhibitory effect of shRNA. We analyzed 25 *N* gene sequences derived from PEDV genotypes G1 and G2 and only found single-nucleotide substitutions. Considering the nucleotide substitutions in the *N* gene, we chose three conserved and unexploited regions for the design of shRNAs. PEDV is primarily transmitted through the fecal–oral route and infects intestinal villous epithelial cells in vivo [27]. Current in vitro cell cultures of PEDV include Vero E6 cells, MARC-145 cells (another monkey kidney cell line), and HEK293 cells [28]. Most are non-porcine intestinal epithelial cells and, thus, not ideal in vitro cellular models for studying the interaction between PEDV infection and the host response due to interspecific variation. The IEC cell line represented a better model of normal porcine intestinal epithelium than transformed cell lines, and provided a unique opportunity to explore host–pathogen interactions in an in vitro system [29]. Using this cell model, we showed that shRNA expression did not affect the viability of IEC cells. Co-transfection of recombinant plasmid pMyc-N and different concentrations of shRNAs highlighted the success of the gene knockdown at the protein level. GFP was an important reporter for *N* gene expression. For example, if the *N* gene was silenced by the shRNAs, the translation of GFP protein in frame with the *N* gene in the recombinant plasmid would also be inhibited. This hypothesis was consistent with our findings. Our shRNAs shR-N307, shR-N463, and shR-N1071 strongly reduced GFP expression, and the inhibition was dose-dependent. In addition, the model system was exploited to further study the pathological functions of PEDV genes. We assessed the capacity of shR-N307, shR-N463, and shR-N1071 for inhibiting the gene and protein expressions of PEDV CV777 and LNCT2 strains in vitro. We observed that shR-N307, shR-N463, and shR-N1071 were able to knock down target gene expression of PEDV CV777 and LNCT2 strains at 48 hpi. We also showed that shR-N307, shR-N463, and shR-N1071 against the *N* gene suppressed PEDV CV777 and LNCT2 strain replication.

Currently, siRNA import into cells requires vectors such as plasmids and recombinant lentiviruses that express shRNAs efficiently and stably. As recombinant lentiviruses have a higher adaptability and replication ability in host cells, and as their genes can be integrated into cellular genomes, the silencing effect of lentiviruses is more efficient than plasmids [30]. However, this kind of gene integration may induce host genome mutations and cause cellular injury. In addition, excessive expression of shRNAs competitively inhibits cell endogenous miRNAs and causes cytotoxicity [31]. Therefore, RNAi mediated by a plasmid is currently safer for high gene silencing efficiency. However, off-target effects from using plasmid-based siRNA are still a shortfall [32]. To solve this problem, designing improved siRNA sequences, as well as developing novel and progressive vectors, is necessary.

In conclusion, plasmids expressing three shRNAs targeting different sites of the *N* gene of PEDV were constructed and transfected into IEC cells. Infection of PEDV CV777 (G1) or LNCT2 (G2) occurred post transfection. Detection by Western blot and viral titer assays was used to measure levels of viral replication in cells. The results demonstrated that PEDV G1 and G2 strains were susceptible to RNAi pathways targeting the *N* gene. All shRNA tests led to the silencing of *N* gene expression and suppressed proliferation of PEDV G1 and G2 strains in IEC cells. In sum, our data showed the potential for the shRNA expression vectors to precisely and effectively interfere with the replication of PEDV G1 and G2 strains in vitro. To our knowledge, this is the first report of the inhibition of PEDV G1 and G2 strain infection with shRNAs in IEC cells. Therefore, this report enriches the antiviral spectrum of RNAi treatment. Although IEC cells are a non-transformed porcine intestinal epithelial cell line, these cells lack the complexity of the cell types found in the architecture of the intestinal epithelium and, thus, do not satisfactorily mimic the natural infection process. This method merits further investigation in animal studies to define its therapeutic potential. Determining if the technology could be used in vivo for anti-PEDV therapy is still under investigation.

**Author Contributions:** Conceptualization, L.F.; data curation, D.S., X.W., and H.S.; funding acquisition, L.F.; METHODOLOGY, D.S., X.W., and H.S.; project administration, L.F.; resources, J.C.; software, X.Z., J.L., J.Z. (Jialin Zhang), Z.J. (Zhaoyang Ji), and Z.J. (Zhaoyang Jing); supervision, L.F.; writing—original draft, D.S., X.W., H.S., and L.F.; writing—review and editing, J.Z. (Jiyu Zhang) and Y.H.

**Funding:** This work was supported by grants from the National Natural Science Foundation of China (31602072 and 31572541), the China Postdoctoral Science Foundation (2017M610136), and the Natural Science Foundation of Heilongjiang Province of China (C2017079 and C2018066).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **A Single Dose of Dendrimer B2T Peptide Vaccine Partially Protects Pigs against Foot-and-Mouth Disease Virus Infection**

**Rodrigo Cañas-Arranz 1, Mar Forner 2, Sira Defaus 2, Patricia de León 1, María J. Bustos 1, Elisa Torres 1, Francisco Sobrino 1,\*, David Andreu 2,\* and Esther Blanco 3,\***

Received: 16 December 2019; Accepted: 8 January 2020; Published: 10 January 2020

**Abstract:** Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals whose control relies on efficient vaccination. We have reported that dendrimer peptide B2T, with two copies of FMDV B-cell epitope VP1 (136–154) linked through maleimide units to T-cell epitope 3A (21–35)], elicits potent B- and T-cell specific responses and confers solid protection in pigs to type-O FMDV challenge after two doses of peptide. Herein we now show that B2T evokes specific protective immune responses after administration of a single dose of either 2 or 0.5 mg of peptide. High titers of ELISA and neutralizing antibodies against FMDV were detectable at day 15 post-immunization. Likewise, activated T cells and induced IFN-γ response to in vitro recall with FMDV peptides were also detected by the same day. Further, in 70% of B2T-vaccinated pigs, full protection—no clinical signs of disease—was observed upon virus challenge at day 25 post-immunization. These results strengthen the potential of B2T as a safe, cost-effective candidate vaccine conferring adequate protection against FMDV with a single dose. The finding is particularly relevant to emergency scenarios permitting only a single shot immunization.

 Introduction**

Vaccination remains the most effective approach to prevent human and animal diseases [1]. In animal health, development of safe, cost-effective and marker vaccines, capable of telling infected from vaccinated animals (DIVA), remains a challenge for many diseases, particularly viral ones [2,3]. Conventional vaccines based on inactivated or attenuated viruses entail risks such as accidental escape or incomplete inactivation of infectious viruses, as well as possible reversion of attenuated into virulent forms. Subunit or epitopic vaccines represent an alternative that solves most such problems by excluding the infectious agent and allowing targeting to well characterized viral epitopes relevant for protection [4–6].

Foot-and-mouth disease virus (FMDV) is the prototype member of the *Aphthovirus* genus within the *Picornaviridae* family [7] and the etiological agent of FMD, a highly transmissible infection of pigs and other cloven-hoofed animals, with huge economic impact worldwide [8,9]. FMD underscores paradigmatically the challenge of finding alternative strategies to the classic vaccines still used to prevent this highly contagious disease [10]. The massive amplification and shedding of FMDV in

infected pigs turns this species to a key epidemiological factor for the spread of the virus over the course of outbreaks in many regions of the world [11]. In addition, the growing numbers of domestic pigs worldwide, particularly in Asian countries, make the development of pig-suited FMD vaccines a strategic task.

Conventional FMDV vaccines based on chemically inactivated virus have allowed FMD control and eradication in some countries, although their manufacturing process—not upgraded over recent decades—poses significant biosafety concerns that have been related to occasional escape episodes of diverse consequence [12–14]. This risk plus other limitations, such as the need for a strict cold chain to preserve stability, and the use of updated vaccine strains, because of the high potential antigenic diversity of the virus, underlie the adoption of non-vaccination policies in FMDV-free countries, a controversial and by no means risk-free practice, as borne out by not infrequent outbreaks in those locations. In crisis scenarios of this kind [15], vaccines incorporating outbreak-relevant epitopes, eliciting protective responses and generated as a quick response to the epidemic, can become an invaluable emergency resource for FMD containment [16]. Among such emergency vaccines, those based on synthetic peptides [6] are particularly appealing because of their (i) total lack of biological hazard; (ii) possibility of displaying various epitopes on a single platform; (iii) DIVA compliance; (iv) efficient synthetic production and characterization as pharmaceuticals, and (v) no cold-chain required; easy transport and storage [17].

The main B-cell antigenic site in FMDV, located at the GH loop of capsid protein VP1 (residues ca. 140–160), is structurally continuous [18,19]. Linear peptides reproducing this loop, either alone or in combination with T-cell FMDV epitopes, have been shown to confer limited protection in natural hosts [20–23].

A substantial enhancement in immunogenicity can be achieved by multiple display of B- and/or T-cell epitopes on a single molecular scaffold [17] inspired on the multiple antigenic peptide (MAP) platform of Tam [24]. In an initial realization in this regard, a peptide spanning residues 21–35 of FMDV protein 3A [thereafter T3A], which delimit an immunodominant T-cell epitope in domestic pigs [25], was N-terminally elongated into a Lys tree to which four copies of a B-cell epitope (residues 140–158 of VP1; containing the RGD motif that mediates binding to integrins, the cell receptors) were covalently linked in a dendrimeric (branched) fashion. The sequence of the B-cell epitope corresponded to that of the epidemiologically relevant O/UKG/11/01 isolate, belonging to serotype O the most prevalent worldwide [26]. This multivalent construct (named B4T) elicited high titers of FMDV-neutralizing antibodies, activated specific T cells, and fully protected pigs against FMDV challenge [27]. Interestingly, a simpler version (i.e., two B-cell epitope branches) of the peptide vaccine candidate, termed B2T, also elicited potent specific responses and conferred solid protection in pigs to challenge [28] (Table 1). In both B4T and B2T trials, animals were immunized with two 2-mg doses of peptide (3 weeks apart from each other) before challenge.

As mentioned above, emergency FMD vaccines, eliciting protective responses upon a single shot, are particularly valuable in containing uncontrolled FMDV outbreaks [14]. The modular nature of B2T affords considerable versatility in the sequences that can be integrated into the constructions, either as part of a single molecule or as mixtures of different molecules. Thus, incorporation of VP1 GH-loop sequences from different FMDV isolates can modulate/enhance the protective spectrum of the candidates. This is particularly relevant because of the high FMDV antigenic diversity reflected in seven serotypes and many variants within each of them, which makes matching of vaccine strains with circulating virus a critical issue for vaccine efficacy [11,15]. On the other hand, the T3A sequence mentioned above is highly conserved among FMDV serotypes and therefore can evoke heterologous responses, again contributing to broaden the protective response conferred by B2T. In addition to versatile, readily adaptable responses to new virus threats, emergency vaccines require fast and cost-effective manufacturing programs, which are ideally met by the expediency and flexibility inherent to chemical synthesis production.

Herein we have explored the possibility of: (i) Eliciting protective responses in pigs upon administration of a single B2T dose, and (ii) reducing the amount of antigen required to elicit protective responses. Thus, we report results with both the 2 mg dose of previous experiments [28] and with a reduced 0.5 mg inoculum. Remarkably, the latter dose elicits a rapid immune response involving high titers of FMDV neutralizing antibodies and specific IFN-γ secreting T cells at 15 days post-immunization (dpi). In addition, solid protection is observed in 80% of pigs vaccinated with 0.5 mg B2T, with no clinical signs upon viral challenge at day 25 dpi. Taken together, our results highlight the value of B2T as candidate FMD vaccine for pigs in emergency scenarios.

#### **2. Results**

#### *2.1. A Single B2T Dose Elicits Rapid Humoral Specific Responses Including FMDV Neutralizing Antibodies*

Domestic pigs, in two different groups of five animals each, were immunized once with 2 or 0.5 mg of B2T (pigs 1 to 5 and 6 to 10, respectively), and two additional non-immunized animals were kept as controls (pigs 11 and 12 inoculated with PBS). Total FMDV-specific IgG antibodies were determined by ELISA at 0, 15, and 21 dpi. Both 2 and 0.5 mg doses elicited consistent and comparable IgG titers (log10) at 15 (4.3 ± 0.4 vs. 3.7 ± 0.3) and 21 (4.2 ± 0.2 vs. 4.3 ± 0.3) dpi (Figure 1A). Upon FMDV challenge (day 25) these titers were not boosted up, remaining similar in both B2T-immunized groups (4.3 ± 0.1 vs. 4.6 ± 0.5). Non-immunized control pig 11 that survived 10 days post-challenge (dpc) showed anti-FMDV titers >2log10 units lower than those of the immunized and challenged groups (Figure 1A).

Regarding induction of FMDV neutralizing antibodies, as observed with the ELISA results, no major differences were noticed between virus neutralization titers (VNT) in the groups immunized with either 2 or 0.5 mg B2T at day 15 (1.5 ± 0.2 vs. 1.4 ± 0.4) or day 21 pi (1.9 ± 0.4 vs. 2 ± 0.5) (Figure 1B). On the other hand, and in contrast to ELISA antibody titers, post-challenge VNT increased at 10 dpc (35 dpi) in both groups (3.1 ± 0.3 vs. 3.2 ± 0.4). As expected, no neutralizing antibodies were detected in the two control animals before challenge. The non-vaccinated animal that survived the challenge (pig 11), showed VNT (3.1) similar to those in immunized groups (Figure 1B), showing that neutralizing antibody levels in B2T-immunized/challenged pigs—including those found to be protected—were as high as those in infected and recovered animals.

**Figure 1.** B- and T-cell responses in pigs immunized with a single dose and different amounts of B2T. Time course of the specific antibody responses in sera collected on the indicated days pi. (**A**) Total anti peptide B IgG titers analyzed by ELISA. Each point depicts mean antibody titers (calculated as described in Methods) ± SD for each group of pigs (*n* = 5). (**B**) Virus neutralization titers, VNT, expressed as the reciprocal log10 of the last serum dilution that neutralized 100 TCID50 of FMDV isolate O/UKG 11/01. Each symbol represents the value for an individual pig (numbering is included). Horizontal lines indicate the geometric mean for each animal group. In no case individual spontaneous reactivity was observed in the titers determined at day 0. Dotted lines denotes the assay detection limit. In (**A**,**B**) arrows point FMDV challenge (day 25 pi). (**C**) Specific T-cell responses measured by an ex vivo IFN-γ ELISPOT at days 15 pi. The frequency of FMDV-specific IFN-γ secreting cells was determined as detailed in Methods. Horizontal bars represent the mean frequencies of IFN-γ release spots of triplicates of peripheral blood mononuclear cells (PBMCs) from pigs stimulated in vitro with B2T (circles) or T (T3A) (squares) peptides (pig numbering is included).

#### *2.2. B2T Elicits Early FMDV-Specific IFN-*γ *Responses*

Specific T cell responses elicited by B2T at 15 dpi were determined by ELISPOT analysis of IFNγ-expressing PBMCs. High frequencies of spot-forming cells were found at day 15 in pigs immunized with either dose of B2T in response to in vitro recall with homologous peptide (284.8 ± 321.1 vs. 645.6 ± 467.9). On average, pigs in the 0.5 mg group showed higher frequencies of IFNγ-expressing

PBMCs than those in the 2 mg group (Figure 1C). All responses were specific, as no peptide-driven IFN-γ-producing cells were detected in the two non-immunized pigs. PBMC stimulation with T3A peptide paralleled those observed with B2T (241.3 ± 365.8 vs. 614.4 ± 403.1) (Figure 1C), supporting the recognition of T3A as a T-cell epitope.

#### *2.3. A Single Dose of B2T Peptide Confers Clinical Protection against FMDV Challenge*

At 25 dpi, pigs in all three groups were challenged with FMDV. Animals were examined daily for clinical signs (see Methods) and considered protected when lesions were not observed or appeared only at the inoculation site [29]. As expected, PBS-inoculated control pigs 11 and 12 showed full FMD signs upon challenge, developing vesicular lesions on all four feet at 4 dpc and on the snout at 7 and 5 dpc, respectively (Table 2). The acute FMDV infection caused myocarditis, leading to heart failure and sudden death, of pig 12 at 5 dpc. In contrast, only 3 out of 10 peptide-immunized animals developed lesions outside the inoculation site: two pigs (3 and 5) in the 2 mg group and one (pig 10) in the 0.5 mg group. The remaining immunized animals did not develop any clinical signs, the cumulative lesion score of immunized groups being significantly lower than that of the non-immunized one (Table 2). In general, a correlation between lower body temperature and protection was observed, being the protected animals immunized with 0.5 mg B2T those that did not develop fever.

**Table 2.** Evidences for protection in immunized pigs.

a VNT and IFNγ spots/106 PBMCs determined at day 21 and 15 post-immunization, respectively. b Temperature ( ◦C) and (in parenthesis) day pi when maximum temperature registered. No fever: ≤39.0–39.5 ◦C. c Animals were monitored up to 10 days pc for lesions. Lesion score (maximum value of 7): 1 point/vesicle in foot (up to 4 points); 1 point/mouth, tongue or snout lesion; 1 point/>2 lesions of diameter ≥10 mm. In parenthesis, day pc when lesion(s) was first observed. d Animals were considered fully protected (++) if lesion score ≤ 1; partially protected (mild/delayed disease) (+) if lesion score ≤ 2, or non-protected (−) if lesion score > 2. e Detection of FMDV RNA in serum samples. ND: RNA not detected. The amount of RNA in positive animals are expressed as viral RNA copies (VRC)/mL serum. Detection limit: 5 × 103 VRC/mL serum. The values from RNA positive animals are presented as VRC/mL serum. The day(s) post-challenge when RNA was detected is shown in brackets. f Animal died on day 5 pc.

Detection of FMDV RNA in serum samples from challenged pigs by RT-qPCR showed the presence of viral RNA in non-immunized control pigs 11 (days 3 and 5 pc) and 12 (day 3 pc), as well as in the two immunized but non-protected pigs 10 (days 3 and 5 pc) and 3 (only at day 5 pc), which showed the higher lesion score at day 7 pc (Table 2). Hence, virus detection in sera associated with the severity of the lesions of challenged pigs. Further work is necessary to assess the potential contribution of viruses circulating in non-protected animals, and to a lesser extent in protected animals, to the spread of the disease under field conditions.

In conclusion, immunization with a single 2 mg dose and, remarkably, even more with a 0.5 mg dose of B2T dendrimer afforded substantial protection against conventional FMDV challenge.

#### **3. Discussion**

Protection after a single inoculation is a must for effective FMD vaccines, as it reduces both the cost of the vaccine as well as the logistics and labor expenses associated with the double immunization schedules. The application of a single dose vaccination program to the pig population can favor disease eradication, considering that pigs serve as amplifiers of FMDV and therefore a rapid control of the infection in swine is essential for disease control [11]. This is particularly relevant in settings such as diseases outbreaks occurring in areas where FMD is not enzootic and livestock remains unvaccinated [30]. To our knowledge, with the exception of adenovirus-vectored vaccines expressing FMDV empty capsids [31], few studies exist on the capability of FMD subunit vaccines alternative to classical ones to confer protection in relevant hosts after a single administration [32]. In this context, our optimization of the minimal amount of B2T dendrimer still conferring protection is pertinent, both for understanding the response mechanisms to B2T and for scaling down production costs, a relevant issue for FMD vaccine development.

We had previously shown that two 2 mg doses of B2T conferred solid protection to FMDV challenge in swine, and that protection correlated with the induction of strong B- and T-cell specific responses [28]. Here, we explore the feasibility of B2T as a protective, single dose subunit vaccine alternative to conventional FMD formulations. To this end, the original 2 mg dose and a four-fold lower one (0.5 mg) have been compared.

The challenge protocol followed in our experiment was as recommended by the OIE manual, for evaluation of protection against podal generalization (PPG) test, except that virus was inoculated 3 days earlier than indicated [33]. Under such conditions, full protection, considered as the absence of lesions at points other than the inoculation site, was observed in 70% of pigs vaccinated with B2T; 3 out of 5 in the 2 mg dose group, and 4 out of 5 in the 0.5 mg group. This level of protection correlated with an average increase in virus neutralizing (VN) antibodies that was not observed for B-specific IgG antibodies detected by ELISA (Figure 1), suggesting the recall upon viral infection of a subset of FMDV-specific memory B cells, as well as of memory T cells capable to promote B-cell maturation. This increment of VN antibodies in protected animals, matching that of a non-immunized pig, might reflect virus multiplication to a limited extent not resulting in detectable viremia (Table 2). Similar levels of VN antibodies boost upon virus challenge have been reported in pigs and cattle immunized with live-vectored vaccines or FMDV-like particles [34,35].

Non-protected (pigs 3 and 10) and partially protected (pig 5) animals showed lower levels of VN antibodies at the day of challenge, albeit animals with lower VNT yet protected were also observed (Figure 1B); the lack of correlation in a fraction of protected animals immunized with different FMDV vaccines has been described [21,36]. Experiments aimed at assessing the amount of antibodies that may target the FMDV receptor [37] as well as at examining cross-neutralization among other type O FMDVs will be foreseen to further characterize the antibody response evoked by the dendrimer peptide vaccines.

In addition, and supporting T3A recognition as a T-cell epitope, specific IFN-γ releasing activated T cells were detected rather early, at 15 dpi, in 7 of the 10 immunized pigs. The average frequencies of IFN-γ releasing cells in PBMCs from pigs in the 0.5 mg dose group were higher than those in the 2 mg group (Figure 1C and Table 2), possibly reflecting differences in the in vitro dose-effect of peptide stimulation and/or on the MHC allele composition of the animals analyzed. High doses of antigen might also facilitate its capture by APC/DC subsets with suboptimal costimulatory capacity that favor the development of regulatory T cells, which could limit the activation of B cells and subsequent antibody production as well as the development of IFN-γ producing cells [38]. In any case, additional studies of the effector mechanisms triggered by the dendrimers will be necessary to understand the effect on the protective responses of different peptide vaccine doses. One animal that was low responder at 15 dpi (pig 7), turned out to be protected upon challenge and pig 5, also low responder, was partially protected. Among responder animals, no clear correlation between IFN-γ releasing cell frequency at 15 dpi and protection to FMDV challenge was observed, i.e., non-protected pigs 3 and 10

showed high responses. Thus, as previously observed [21], VNT and T-cell stimulation do not fully correlate with the protection conferred by FMD peptide vaccines.

A potential problem of epitopic vaccines such us the dendrimeric peptides used in this study is the selection in non-protected animals of escape viruses capable to avoid antibody neutralization [21] and/or T cell recognition. In our experiment, we could not rule out that possibility since the limited number and size of the vesicles developed by non-protected animals did not allow virus recovery for sequencing analyses.

Our evidence of the level of full protection conferred by a single B2T dose upon challenge at 25 dpi is particularly remarkable with regard to the 0.5 mg dose group (80% of the pigs), as it opens the way to significant savings in manufacturing costs. Incidentally, on a related note, we have found that long-lasting (up to 19/20 weeks post-boost) reduced susceptibility to FMDV infection can be attained with two B2T doses and, remarkably, a similarly lasting protective response with a single dose of B2T (Cañas-Arranz et al., submitted).

Attempts to use B2T and B4T dendrimer peptides as vaccines in cattle showed a trend toward a reduced capacity to confer protection relative to swine, including the need for a third immunization to elicit protective levels on neutralizing antibodies [39,40]. Failure in conferring protection in cattle has been described for linear FMDV peptide vaccines containing a heterologous T cell epitope [41]. Further work on the requirements of the interactions between neutralizing epitopes and B cells, as well as the identification of new, effective T helper epitopes frequently recognized by individuals will contribute to the improvement of the effectiveness of this kind of vaccines for cattle and, eventually, other host species.

In summary, our findings portray the B2T peptide as a safe, potentially cost-effective candidate to be included in FMD vaccine formulations conferring single-shot protection in pigs. Formulation of dendrimers with alternative adjuvants as well as the incorporation of peptides corresponding to other T cell epitopes previously identified in pigs [42,43] can contribute to improve their immunogenicity in swine.

#### **4. Methods**

#### *4.1. Peptides*

Peptides identified as B- and T-cell epitopes of FMDV O/UKG/11/01 (Table 1) were assembled by Fmoc-solid phase synthesis, purified by reverse-phase liquid chromatography, and characterized by mass spectrometry. Dendrimeric B2T construct was conjugated in solution using two previously synthesized precursors: (i) B epitope (VP1, residues 136–154) plus a C-terminal Cys residue (free thiol form) and (ii) T epitope (3A, residues 21–35) elongated at the N-terminal with two Lys residues followed by an additional Lys branching derivatized as two maleimide groups. The B2T peptide was efficiently obtained after the thiol-maleimide ligation at pH = 6, RP-HPLC purification and MS characterization [27,28,44].

#### *4.2. Virus*

A virus stock derived from FMDV isolate O/UKG/11/01 [45] by two amplifications in swine kidney cells (IB-RS-2 cells) was used. The resulting virus maintained the consensus sequences at the capsid region [46].

#### *4.3. Animals and Experimental Design*

The immune response to B2T dendrimer peptide was assessed in ten 9-weeks-old white cross-bred Landrace female pigs (Agropardal SA breed), free of antibodies to FMDV. The study was approved (CBS2014/015 and CEEA2014/018) by the INIA Committees on Ethics of Animal Experiments and Biosafety, and by the National Committee on Ethics and Animal Welfare (PROEX 218/14). Pigs were randomly assigned to two groups of five animals each and immunized once (days 0) by intramuscular injection with 2 mL of Montanide ISA 50V2 emulsion (Seppic, Paris, France) containing 2 or 0.5 mg of B2T peptide. Two additional non-vaccinated pigs were kept as infection controls (#11 and 12). Animals were housed in separate units of the high-containment facility and challenged at day 25 with 1.6 × 104 plaque forming units (pfu) of FMDV O/UKG/11/01, by inoculation at two sites of both main claws of the left-hindfoot pad (0.1 mL/site). Animals were monitored for clinical signs of disease during the 10 days (Table 2), and then euthanized at day 35.

#### *4.4. Viral RNA Detection after Challenge*

Serum samples were examined for the presence of viral RNA by real time RT-qPCR. Briefly, the cDNA obtained in a RT reaction using primer A (5 -CACACGGCGTTCACCCA(A/T)CGC-3 ) [47] was amplified by qPCR using the "Light Cycler RNA Master SYBR Green I" kit (Roche, Basel, Switzerland) and LightCycler equipment following the instructions of the manufacturer. The amplicon synthesized spanned a conserved region of 290 pb length in the 3D protein coding sequence, and was amplified using primers A (5 -CACACGGCGTTCACCCA(A/T)CGC-3 ) and B (5 -GACAAAGGTTTTGTTCTTGGTC-3 ). The values for the quantification of the samples were obtained from a standard curve from a RNA transcript derived from the infectious clone pMT-28 codifying the genomic RNA from FMDV C-S8c1 [48].

#### *4.5. Virus Neutralization Test (VNT)*

Serial two-fold dilutions of sera were incubated with 100 infection units—50% tissue culture infective doses (TCID50) of FMDV O/UKG 11/01, for 1 h at 37 ◦C. End-point titers were calculated as the reciprocal of the serum dilution that neutralized FMDV infection in 50% of the wells [28].

#### *4.6. Detection of Specific Anti-FMDV Antibodies by ELISA*

Total anti-FMDV antibodies were determined by ELISA. Briefly, 96-well plates (Nunc, Rochester, NY, USA) were coated with peptide B (1 μg) overnight at 4 ◦C. Duplicate three-fold dilution series of each serum sample were prepared in 50 μL, starting at 1/100. Pre-immune sera from peptide-immunized pigs and sera from non-immunized animals were used as negative controls. Specific antibodies were detected with HRP-conjugated protein A (Thermo Fisher, Rochester, NY, USA), diluted 1/4000. Color development was obtained after addition of 100 μL/well of TMB (Sigma Aldrich, St. Louis, MO, USA) and stopped by an equal volume of 1M H2SO4. Plates were read at 450 nm, titers expressed as the reciprocal of the last dilution giving the absorbance recorded in the control wells (serum at day 0) plus 2 SD.

#### *4.7. PBMC Isolation and IFN-*γ *Detection by ELISPOT*

Porcine peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma, St. Louis, MO, USA) and cryopreserved prior to assay. Vials were defrosted and resuspended in complete RPMI 1640 and incubated overnight at 37 ◦C, 5% CO2. Cell counting and viability were tested by Trypan blue staining. For the IFN-γ ELISPOT assay 2.5 × 105 PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore, Burlington, MA, USA) coated with 5 μg/mL of anti-pig IFN-γ antibody (clone P2G10, Becton Dickinson, Franklin Lakes, NJ, USA). For in vitro antigen recall, PBMCs were stimulated with 25 μg/mL of the peptide used for pig immunization [49]. As positive control, PBMCs were incubated with 5 μg/mL of phytohaemagglutinin (Sigma) using cells incubated without antigen as negative control. After 48 h at 37 ◦C, 5% CO2, plates were washed and incubated with 2 μg/mL of biotinylated anti-mouse IFN-γ antibody (clone P2C11, BD) and HRP-streptavidin (BD). Antibody was visualized with 3-amino-9-ethyl carbazole (BD). The frequency of peptide-specific T cells in was expressed as the mean number of spot-forming cells/106 PBMCs, with background values (number of spots in negative control wells) subtracted from the respective counts of stimulated cells.

#### *4.8. Statistical Analyses*

Differences among the peptide-immunized groups in FMDV-antibody titers and number of IFN-γ producing cells were analyzed by two-way ANOVA, followed by Tukey's post-hoc comparisons tests. Values are cited in the text as means ± SD. All *p* values are two sided, and *p* values < 0.05 were considered significant. Statistical analyses were conducted using GraphPad Prism Software 5.0 (Graphpad Software, San Diego, CA, USA).

**Author Contributions:** Conception, design and funding of the work (D.A., E.B. and F.S.). Data Acquisition and analysis (R.C.-A., M.F., S.D., P.d.L., M.J.B. and E.T.). Interpretation of data for the work (R.C.-A., M.F., S.D., P.d.L., D.A., F.S. and E.B.). Drafting the work (R.C.-A., M.F., S.D., P.d.L., F.S. and E.B.). Revising the data critically for important intellectual content (D.A., E.B. and F.S.). All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Spanish Ministry of Science, Innovation and Universities (grant AGL2017-89097-C2 to FS and DA; AGL2016-76445-R to EB), Comunidad de Madrid co-financed with ECFEDER funds (S2013/ABI-2906-PLATESA and P2018/BAA-4370 to FS and EB) and Generalitat de Catalunya (2009SGR492 to DA). Work at Centro de Biología Molecular "Severo Ochoa" and at UPF was supported by Fundación Ramón Areces and by the Maria de Maeztu Program of the Spanish Ministry of Science, Innovation and Universities, respectively. R.C.-A. and M.F. were holders of a PhD fellowship from the Spanish Ministry of Science, Innovation and University (FPI programme). Article processing free of charge.

**Acknowledgments:** We thank P. Pallarés for her advice and support.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

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