A Multiepitope Mosaic DNA PRRSV Vaccine is Immunogenic in Pigs and Confers a Partial Level of Protection Against Challenge Virus

A Multiepitope Mosaic DNA PRRSV Vaccine is Immunogenic in Pigs and Confers a Partial Level of Protection Against Challenge Virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Multiepitope Mosaic DNA PRRSV Vaccine is Immunogenic in Pigs and Confers a Partial Level of Protection Against Challenge Virus Chesney Romer, Sahar Nouri Gharajalar, Jiaqi Zhu, Yangchao Lou, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8620504/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract A Multiepitope Mosaic DNA vaccine was tested in pigs for immunogenicity and protection from PRRS virus challenge. Mosaic coding sequences were synthesized and cloned into pHCMV-1 and their expression was confirmed. Vaccination challenge trial in PRRSV-free piglets followed using Mosaic DNA in lipid nanoparticles. Group 1 received Empty/LNP and served as negative vehicle controls; Group 2 received pooled Mosaic DNA/LNP vaccine; Group 3 received a commercial MLV vaccine and served as positive controls. A virus challenge was implemented by 40 days after vaccination began and euthanasia was implemented 11 or 12 days later. Virus specific antibodies detected in Mosaic DNA/LNP vaccinated pigs by ELISA were significantly higher than those in Empty/LNP controls before and after challenge. Virus neutralizing antibodies were detected in Mosaic DNA/LNP vaccinated pigs but not in controls. IFN \(\:ᵧ\) expression was detected in Mosaic DNA/LNP vaccinated animals and Empty/LNP group remained unresponsive. Viral loads in serum of the Mosaic DNA/LPN group, while not statistically significant, were lower than Empty/LPN pigs. The viral levels remained high in sera of control animals. Lower, but not statistically significant, viral loads were detected in, bronchoalveolar lavage, and tissues in the Mosaic DNA/LNP vaccine group than in the controls confirming level of protection. Biological sciences/Biotechnology Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology PRRSV swine pig Mosaic vaccine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Porcine reproductive respiratory syndrome (PRRS) is a high impact disease that is distributed worldwide affecting the swine industry globally. Significant direct losses are derived from reproductive failure, and infertility in sows and respiratory disease in piglets 1 , 2 . In the United States alone annual losses due to PRRS can amount over a billion dollars yearly 3 . The disease is caused by PRRSV, a member of the Arteriviridae Family within the order Nidovirales 4 , 5 . The virion is enveloped and contains a 15 kb positive sense single stranded RNA genome that codes for 8 structural proteins and 14 non-structural proteins 6 – 10 . The control of PRRS is highly challenging due primarily to the extensive genetic and antigenic heterogeneity of PRRSV. Based on whole genome sequence the virus was classified into two main genotypes, the Genotype 1 (European type) currently designated as Betaarterivirus Suid 1 and the Genotype 2 (North American type) or Betaarterivirus Suid 2, with differences of up to 40%, between genotypes and up to 20% divergence within the same genotype 11 , 12 . Recent phylogenetic analysis based on ORF5 sequencing thousands of Genotype 2 strains yielded up to 11 different lineages and 21 sub-lineages further illustrating the expanding and extensive genetic diversity of this virus 13 . Restriction fragment length polymorphisms (RFLP) of ORF5 have been in use to differentiate wild type field isolates from vaccine virus and a recent extensive analysis of thousands of sequences yielded over 200 distinct patterns some of which were used for cluster analysis 14 . However, the accuracy of RFLP to assign genetic relatedness among PRRSV 2 strains was questioned 13 . The constant evolution of this virus, as with other RNA viruses, may be explained in part to lack of proofreading mechanisms in the viral RdRp and extensive recombination events that contribute to the heterogeneity of isolates and potentially influence in variations in pathogenicity 14 – 18 . The infection with PRRSV is characterized by immune evasion, persistence and immunosuppression which make its control even more complex 2 , 19 . Briefly, infection results in down modulation of innate immunity affecting type I IFN -production, there is a decoy of host protective humoral immunity presumably caused by glycan shielding of neutralizing epitopes and early induction of non-neutralizing antibodies 20 , 21 . In addition, infection with PRRSV results in upregulation of IL10 and TGF-B and enhancement of Tregs and an immunosuppressive state ensues 18–20,22−24 . Vaccines are the main means to control PRRS. However, a major drawback with vaccines used in the field is that the protection afforded is limited against homologous strains with weak or no ability to provide cross-protection to circulating heterologous strains which are continuously mutating 25 , 26 . This emphasizes the urgent need of broadly protective vaccines to address the extensive genetic and antigenic diversity of PRRSV. Research efforts towards the development of broadly protective vaccines for PRRS using different platforms for delivery are ongoing 27 , 28 . Some of these include, modified MLV vaccines 29 , 30 consensus sequence vaccines 31 , subunit recombinant vaccines 27 , 31 – 33 , molecular breeding by DNA shuffling, mosaic T-cell epitope vaccines 34 – 39 , DNA vaccines, VLP vaccines, nanoparticle vaccines, and others 25 , 27 , 34 , 40 , 52 . The main goal in PRRS vaccinology is to achieve vaccines that provide expanded vaccine breadth and depth to thus handle hypervariable circulating PRRSV strains. Previously, we investigated a GP5-Mosaic vaccine that was developed using the Mosaic Vaccine Tool Suite originally developed for HIV at the Los Alamos National Laboratory (Los Alamos, NM) 41 , 42 . The GP5-Mosaic vaccine when delivered using a DNA expression vector conferred partial protection in pigs 38 , 39 . Furthermore, priming pigs with DNA GP5-Mosaic vaccine and boosting with recombinant vaccinia virus carrying the GP5 mosaic sequence resulted in partial protection against two strains of different lineages 39 . We report here testing the immunogenicity of a multiepitope DNA vaccine expressing 7 structural mosaic PRRSV proteins in pigs. The vaccine induced responses and partial protection from challenge are described and discussed. Materials and Methods Viruses and cells A series of PRRSV strains from different lineages or sub-lineages were used in the study. These include: VR2332 (L5), SDSU73 (L8B), NADC30 (L1C), Isolate 8981 (Accession 569974) (55), USA/MN/01775GA/2021 (ISU 1) (L5), USA/IN/65239GA/2014 (ISU-2) (L5) (the latter two isolates were kindly provided by Dr. Jianqiang Zhang VDL, ISU Ames, Iowa). The viruses were propagated and titrated in MARC 145 cell cultures using DMEM media containing 10% fetal bovine serum, penicillin streptomycin, glutamine, and stored at -80C for further use. Mosaic Vaccine Development and characterization Published whole genome sequences compiled from strains circulating during the last 10 years were utilized as input for computation of mosaic sequences. Each gene corresponding to the structural proteins E, GP2, GP3, GP4, GP5, M, and N was annotated. Duplicate sequences (identical sequences) were eliminated, and only unique sequences of each gene were utilized for mosaic antigen design. The number of input sequences was as follows: E = 207, GP2 = 273, GP3 = 278, GP4 = 345, GP5 = 343, M = 346, and N = 344. Two mosaic sequence versions from each protein were submitted for synthesis of their coding sequences and cloning into the expression vector pHCMV-1 (GenScript, Piscataway, New Jersey). Expression of each mosaic protein was performed by transformation to E. coli BL21, IPTG induction, and preparation of cell lysates using NEB Express® E. coli Lysis Reagent (NEB,USA). Separation of the cells lysates was carried out by SDS-PAGE using 8–16% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad, USA), followed by transfer onto nitrocellulose membranes (Bio-Rad, USA). The membranes were blocked for 2 hours in PBS containing 5% fat-free milk and then reacted for 1 hr at room temperature with PRRSV ORF specific primary antisera as follows: anti E mouse positive serum, anti GP2 swine positive serum, rabbit anti-PRRSV-GP3 polyclonal antibody, GP4 -positive mouse serum, rabbit anti-PRRSV-GP5 polyclonal antibody, rabbit anti-PRRSV-M polyclonal antibody, anti N- positive swine serum. After washing, membranes were incubated for 1 hr with HRP- conjugated Goat anti-mouse IgG (for GP4, and E), HRP- conjugated Goat anti-swine IgG(for GP2,and N), and HRP- conjugated Goat anti rabbit IgG(for M, GP3, and GP5) based on the primary antibody source used for each structural protein. Then, chemiluminescence Western blotting was used for detection. Preparation and Characterization of Lipid Nanoparticles for Plasmid Encapsulation The lipid components used to develop lipid nanoparticle (LNP) included DLin-MC3-DMA (ionizable lipid), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000). These lipids were first dissolved in a chloroform/methanol mixture (5:3, v/v) to form a uniform organic solution. Then, the lipid mixture was rotary evaporated at 45°C to form a thin lipid film along the flask walls. After complete solvent removal, the film was hydrated with RNase-free water and subjected to probe sonication for 15 minutes. Then, the LNP dispersion was mixed with a plasmid DNA solution at a 1:1 volume ratio and sonicated in a water bath for 10 minutes. The final mixture was then filtered through a 0.45 µm PES membrane. The particulate properties of the LNPs were measured using dynamic light scattering (DLS; Malvern Zetasizer Nano ZS). The average empty LNPs were 98.7 nm with a polydispersity index (PDI) of 0.241. Plasmid-loaded formulations showed particle sizes ranging from 122.2 nm to 128.7 nm, with PDI values between 0.274 and 0.262, indicating a relatively uniform size distribution. The zeta potential values ranged from − 30 to -40 mV, consistent with stable, negatively charged. Experimental Animal Vaccination and Challenge A vaccination-challenge trial was performed using 3- to 4-week-old, Yorkshire castrated males and female PRRS-free piglets (Parsons farm, MA) to assess primarily vaccine immunogenicity and to determine if protection from challenge could be afforded by the Mosaic DNA/LNP vaccine. Group 1 received three doses two weeks apart of Empty/LPN and served as vehicle negative control; Group 2 received three doses under the same schedule of pooled Mosaic DNA/LNP vaccine; Group 3 was vaccinated with 1 dose of Boehringer-Ingelheim Modified Live Virus Vaccine (MLV) (Ingelheim, Germany). An additional group remained unvaccinated to serve as a negative control for serologic and lung pathology assessments. These negative animals were euthanized before challenge was implemented in the other three groups. A virus challenge with VR2332 (1x10 5 TCID 50 / animal) was administered by IM injection and IN instillation on day 40 after vaccination began to groups 1 to 3. The experimentally vaccinated piglets were bled periodically throughout the vaccination phase, before and after challenge for testing. The piglets were euthanized 11 or 12 days after the challenge. Final blood samples were collected right prior to euthanasia. The anesthetic combination of Telazol+Ketamin+Xylazin (IM)was administered prior to euthanasia. Overdose of intracardiac Pentobarbital Sodium was administered for euthanasia after total anesthesia. Assessment of virus-specific antibody responses An in-house whole PRRS virus ELISA was developed for broad detection of vaccine or infection induced antibody responses. Archival pig sera were validated as positive and negative controls by IDEXX X-3 ELISA, and several Genotype 2 PRRSV virus strains were tested as potential antigens. ELISA conditions were determined by a series of checkerboard titrations until optimization was reached. Using our in-house validated ELISA, sera from vaccinated pigs were tested for virus-specific antibodies. Briefly, 96-well flat bottom microwell plates (NUNC, Thermo Fisher, Agawam, MA) were coated overnight at 4°C with Genotype 2 PRRSV isolate 8981 (0.625 µg/mL) diluted in Carbonate/Bicarbonate buffer (pH 9.2) as antigen. Excess antigen was removed and 5.0% skim milk made in PBS containing 0.05% Tween 20 (PBS-T) was added to the wells to block for 2 hours at room temperature. The wells were then rinsed once with PBS. Sera diluted in PBS were added to triplicate wells and incubated at room temperature for 1 hour. The wells were washed 5 times with PBS-T. Secondary anti-porcine IgG antibody HRP conjugate diluted in PBS was added to the wells and incubated for 1 hour at room temperature. The wells were washed as mentioned above and TMB Microwell peroxidase substrate was added and incubated for 15 minutes at room temperature in the dark. Then 4N H 2 SO 4 was added to the wells as the stop solution and the plates were read in a BioTek (Agilent Technologies, Santa Clara, CA) Synergy plate reader at 450nm, and at 630nm. To identify reaction values as positive or negative, a cutoff line was created based on previous literature. Briefly, to the average of all negative values 3 standard deviations of all negative values were added (43–47) Values above the cutoff line were recorded as positive, and values below the cutoff line were negative. Virus Neutralization Test Sera collected from the experimental animals on days 0, the challenge day, and 4 days after challenge were tested for virus neutralizing antibodies in MARC 145 cell cultures. Briefly, test sera from each group were pooled, diluted (1:5) with DMEM media. Then, the diluted serum samples were mixed with an equal volume of VR2332 virus containing 800 TCID 50 /ml (final serum dilution 1:10). After incubation at 37°C for 2 h, the serum-virus mixtures were added to 48-well plates seeded with MARC-145 cell monolayers and incubated at 37°C for 48 h in a 5% CO 2 atmosphere. VR2332 virus infected cells, and uninfected cells were used as virus and cell controls, respectively (34). The virus neutralizing capacity of test sera was determined by the Reed-Muench method of titration after 48h supernatants from the original 48-well plate diluted ten-fold were incubated in multiple wells of freshly seeded MARC-145 cells for 5 days. Measurement of Virus Loads in Serum and Tissues To measure virus loads in PRRSV-challenged pigs, viral RNA was extracted from serum, and bronchoalveolar lavages (BAL), using TRIzol LS Reagent and from tissues using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s instructions. Total RNA from each sample was then quantified by spectrophotometry (NanoDrop 1000, Thermo Scientific, USA). cDNA was synthesized utilizing the iScript cDNA synthesis kit (Bio Rad Hercules, CA) in a 10µl reaction mixture. The reaction conditions were as follows: 25°C for 5min, 46°C for 20min and 95°C for 1min. The real time PCR was performed using the SYBR Green PCR master mix(BIO-RAD, USA), cDNA, F:5 / -AAA TGG GGC TTC TCC GGG TTT T-3 / and R:5/-GCA CAG TAT GAT GCG TAG GC-3 / as forward and reverse primers for ORF7, respectively. The cycling conditions included an initial denaturation for 95°C for 2min, followed by 40 cycles consisting of 15s at 95°C, and 1min at 61°C using BIO-RAD CFX96 Touch system (Bio Rad Hercules CA). A standard curve was constructed by plotting CT values of serial ten-fold dilutions of viral RNA ranging from 10 2 to 10 7 copies/µl. For each assay, positive and negative reference samples were run. The viral loads in samples were determined by plotting the threshold-cycle number against the standard curve. For specificity of the PCR, melting curves were monitored. Expression of Interferon Gamma To measure interferon-gamma (IFN-γ) in response to recall stimulation with PRRSV, peripheral blood mononuclear cells (PBMCs) from experimentally vaccinated animals were harvested from blood collected on days 0, challenge day, and 4dpc. PMBCs seeded in 24-well flat-bottom plates in duplicate(5⋅10 5 cells/well) were stimulated with 200 TCID 50 of VR2332, SDSU73, NADC30, USA/MN/01775GA/2021, or USA/IN/65239GA/2014 for 48h at 37°C in a 5% CO 2 atmosphere. Uninfected untreated cells or 12.5 µg/mL concanavalin A (Con A) stimulated cells, were used as negative and positive controls, respectively. Total RNA was extracted using TRIzol Reagent. cDNA synthesis and real-time PCR protocols were as described above utilizing the primers F5′ -TGG TAG CTC TGG GAA ACT GAATG- 3′ and R5′ -GGC TTT GCG CTG GAT CTG- 3′ for IFN-γ and F5′ - CGT CCC TGA GAC ACG ATG GT- 3′ and R5′ -CCC GAT GCG GCC AAA T- 3′ for GAPDH. For calculating IFN-γ fold changes, GAPDH was used as internal control for the delta-delta method. Statistical analysis Virus-specific serum antibody levels detected by ELISA were compared between groups by a two-way Anova test to determine significant differences. Differences in IFN γ expression and viral loads between groups were calculated by the students t test. Athics decleration All animals studies were performed under an approved IACUC protocol by University of Connecticut. The informed consent was obtained from the farm owner, for the use of theses animals in the study. All procedures including anestesia and euthanasia followed guidelines and regulations. RESULTS Mosaic Proteins are Expressed in E. coli BL21 Expression of each structural Mosaic protein was examined by chemiluminescence western blotting after transforming recombinant plasmids carrying mosaic inserts into E. coli BL21 and following IPTG induction. The expression of two versions of mosaic proteins for each was detected by western blotting with specific antibodies as described in materials and methods. The migration of bands was consistent with their expected molecular weight in kDa (Fig. 1 ). Based on these results, the recombinant plasmids were regarded to be functional and ready for further use. Detection of Virus-Specific Antibodies Before Challenge and Significant Raises in Levels after Challenge Confirm the Immunogenicity of the Mosaic Vaccine Testing of day 0 sera by our in-house ELISA revealed an unexpected reactivity despite of the fact that all day 0 sera tested negative by IDDEX x-3 and the positive and negative control sera performed correctly in both tests. The consistent performance of the latter supported the soundness of the in-house ELISA test. The background issue was resolved effectively by adsorbing diluted test serum with Farrowsure Gold B one of the commercial vaccines used in the herd of origin. Thereafter, all whole-virus ELISA tests were performed after adsorption of serum with Farrowsure Gold B vaccine as described above. ELISA results thus obtained were compared between groups and are shown in Figs. 2A and 2B. All groups on day 0 tested negative as determined by the cutoff line with no significant differences (Figs. 2A, 2B). However, on the day of challenge (day 40), significantly higher antibody levels were detected in the Mosaic DNA/LNP vaccine group than in the Empty LNP group (p < 0.0007) which remained negative as expected (Fig. 2A). The trend of higher antibody levels in Mosaic DNA/LNP vaccinated animals persisted on days 4 (p < 0.0002), and 9 (p < 0.0001) after challenge. However, on the day of necropsy, there were no statistically significant differences between Mosaic DNA/LNP vaccinated animals and those receiving Empty/LNP (Fig. 1 A). Throughout the entire course of the study, there were no statistically significant differences in antibody levels between the Mosaic DNA/LNP vaccinated animals and the MLV vaccinated animals (Fig. 2B). Mosaic DNA/LNP Vaccine Induced Virus Neutralizing Antibodies Day 0 pooled serum samples were uniformly negative in virus neutralization test in all groups as expected (Fig. 3 ). However, under the assay conditions utilized here there was a significant virus neutralization as measured by a decrease in virus titer (TCID 50 ) with sera collected right before challenge, from both the MLV vaccine and the Mosaic DNA/LNP vaccine groups, while the virus titer remained high with sera from the Empty/LNP group indicating lack of virus-neutralizing activity. Compared to the Empty LNP group, the viral titers on challenge day (day 40) were 10 times and 4.6 times lower in sera from MLV and Mosaic DNA LNP vaccine groups respectively. There was a further decrease in virus titer from challenge day to 4 dpc with sera of the MLV group while an unexpected rise in titer was detected with sera of the Mosaic DNA/LNP group. However, despite this increase, the titer was still 2.1 times lower when compared with the titer with sera from pigs receiving Empty/LNP which remained steadily high. The lower virus titers detected with serum from MLV and Mosaic DNA/LNP vaccinated pigs confirm their virus neutralizing capacity. Mosaic DNA/LNP Vaccination Activated Relatively Broad Recall IFN γ Responses In vitro stimulation of PBMN cells with PRRSV strains of diverse lineages and sub-lineages of PRRSV (13) resulted in significant IFN γ responses in both MLV and Mosaic DNA/LNP vaccinated animals. PBMN cells from MLV vaccinated pigs collected right before challenge and 4 days after challenge responded significantly higher (p < 0.05) to stimulation with five different PRRSV strains than their corresponding day 0 PBMNCs (Figs. 1 A, 1 B, 1 C). PBMN cells isolated from Mosaic DNA/LNP vaccinated pigs on challenge day and 4 days after challenge responded to three virus strains significantly higher than the corresponding day 0 PBMNCs (p < 0.05) (Figs. 1 A, 1 B, 1 C). In contrast, PBMNCs from pigs receiving Empty LNP did not respond to any strain at any of these time points. Consequently, the PBMNC responses of MLV and Mosaic DNA/LNP vaccinated pigs at challenge day and 4 days after challenge were significantly higher (p < 0.05) than those of pigs receiving Empty LNP for 5 of 5 test strains for MLV and 3 of 5 test strains for Mosaic DNA/LNP vaccinated pigs respectively as shown in Figs. 4 A, 4 B, and 4 C. The IFN γ responses to recall stimulation of PBMNCs support that cellular immunity was activated by both the MLV and the Mosaic DNA/LNP vaccines. The LNP vehicle alone had no effect on these responses. Mosaic DNA/LNP Vaccine Induced Relatively Low Levels of Protection There were no differences between groups in viral copy numbers in serum collected 4 dpc. However, viral loads in sera of MLV vaccinated pigs were significantly lower than those in Mosaic DNA-LNP and empty LNP animals 9 days after challenge and at necropsy day (*p≤0.05) (Fig. 5 ). While there were no significant statistical differences between Mosaic DNA/LNP and Empty/LNP groups 9 days after challenge and at necropsy day, the viral loads in Mosaic DNA/LNP pigs in those days were 15% and 38.5% respectively lower in average than those detected in Empty/LNP treated animals. The viral load in sera of Mosaic DNA/LNP group had a clear descending pattern resulting in a 28% decrease from day 9 after challenge to necropsy day. In contrast the viral loads in sera of the Empty/LNP group remained steadily high at these time points. A consistent trend of lower viral loads, although not statistically significant, was also detected in bronchoalveolar lavage (p < 0.06), lungs (p < 0.065), tonsils (p < 0.077), and to lesser extent in tracheobronchial lymph node (p < 0.09) in the Mosaic DNA/LNP group compared to those detected in the corresponding fluids or tissues from Empty/LNP controls. Table 1 summarizes the actual differences in mean viral loads in tissues between our experimental vaccine and the control group. Table 1 Percent decrease in mean viral loads in tissues from Mosaic DNA-LNP compared to Empty LNP groups as detected by qRT PCR. Tissue/fluid Percent decrease in viral loads Bronchoalveolar lavage 97.92 Lung 78.5 Tonsil 98.52 Lymph node 35.89 Lung tissue examination yielded no significant difference in lesion scores between Mosaic DNA/LNP and Empty/LNP (data not shown) Discussion The immunogenicity and a partial level of protection from PRRSV challenge induced by a multiepitope Mosaic DNA vaccine PRRSV are discussed here. The Mosaic vaccine was designed using algorithms from the Mosaic DNA Vaccine Tool Suite developed at Los Alamos National Laboratory 41 , 42 . To that end hundreds of natural sequences of PRRSV strains circulating in the past ten years were used as input with which mosaic sequence outputs were obtained for each structural viral protein. Mosaic sequences were synthesized and individually cloned into the eukaryotic/prokaryotic DNA expression vector pHCMV-1. Expression of the corresponding PRRSV structural mosaic proteins was verified in E coli BL21. A cocktail of all pHCMV-1 coding for mosaic proteins was them formulated into lipid nanoparticles (Mosaic DNA/LNP) for vaccine delivery. The Mosaic DNA/LNP vaccine was tested in a vaccination-challenge trial in pigs to assess vaccine immunogenicity and determine protection afforded from challenge. Meantime given the nature of the vaccines an accompanying ELISA test was developed in-house using whole virus as antigen to detect responses to multiple viral antigens. Testing sera from experimentally vaccinated and challenged animals for antibody responses by the whole-virus in-house ELISA revealed unexpected results as some animals at day 0 showed reactivity contrary to IDEXX x3 tests which confirmed that all experimental animals were negative upon arrival. The reasons for this seemingly non-specific reactivity were not understood initially and needed further investigation. Routine vaccination of the sows in the herd of origin with Farrowsure Gold B a polyvalent bacterial and viral vaccine was thought to be a potential source of such background. In fact, adsorption of diluted test serum with Farrowsure Gold B vaccine prior to their addition to ELISA microplates resulted in a significant drop in the background below the cutoff line confirming negativity (data not shown). Furthermore, a pool of serum from four animals as well as serum from one animal collected on day 0 which reacted strongly in our in-house whole-virus ELISA, were tested in a hemagglutination inhibition assay against porcine parvovirus (a component of the Farrowsure vaccine) and demonstrated a positive result. The potential relationship of this seropositivity to parvovirus with the observed background is unknown at this point. In general, the multiplicity of pathogens both viral and bacterial contained in the vaccines used in the source farm may have increased the likelihood of non-specific reactivities. Potential background reactivities should be kept in mind in vaccine studies. The levels of antibody detected in adsorbed sera of the Mosaic DNA/LNP group measured right before challenge, were above the cutoff line and were significantly higher than those in similarly adsorbed sera from the Empty/LNP group which remained negative as expected. A significant raise in levels of antibodies in Mosaic DNA/LNP vaccinated pigs and to a much lesser extent in pigs given Empty/LNP detected after challenge further confirmed the priming and inductive effects of virus-specific antibody responses by the Mosaic DNA/LNP vaccine. The apparent equalization of antibody levels detected at necropsy could be attributed to a response against the challenge infection by Empty/LNP treated animals. The levels of antibody in the Mosaic DNA/LNP group and MLV group remained essentially similar by ELISA. Importantly, there was virus-neutralizing activity in sera collected right before challenge from the Mosaic DNA/LNP group but not in sera from the Empty/LNP group. There was a visible drop in neutralization activity in sera from Mosaic DNA/LNP at 4 dpc and the reasons are unclear at this point. The relatively short period between challenge and necropsy did not allow the examination of this trend at later time points which should be kept in mind in future vaccine studies. Collectively these results are significant as they confirmed that the virus neutralizing antibody being detected in the Mosaic DNA/LNP group was vaccine-induced. The detection of neutralizing antibodies confirms that relevant neutralizing B cell epitopes were preserved in mosaic proteins. which are required for protective immunity against PRRS 54 . The in vitro IFN γ recall responses to stimulation with diverse strains of PRRSV detected both in PBMN cells from MLV and Mosaic DNA/LNP vaccinated pigs and not in pigs receiving Empty/LNP is significant and is a good measure of vaccine induced broad cellular responses 24 , 53 . PBMNCs of pigs vaccinated with MLV responded to five different PRRSV strains while those from Mosaic DNA/LNP vaccinated pigs responded to three different strains. The reasons for the difference in IFN γ responses between MLV and Mosaic DNA/LNP vaccinated pigs could be attributed at least in part to intrinsic differences between vaccines. MLV vaccines by having a level of replication within the host are in general good inducers of cellular immunity 48 , 51 , 52 . On the other and DNA vaccines do induce cellular responses but usually require modifications to adjuvant the responses 49 , 50 . With the Mosaic DNA/LNP vaccine under the conditions utilized contrary to our expectations the in vitro IFN γ responses were limited to three of the triggering strains but these belong to three different lineages/sub lineage and are phylogenetically distant which may ultimately prove biologically significant. Other variables related to DNA vaccine delivery and differences in protein expression, and other modulatory conditions or use of incorporated adjuvants can influence the outcomes 49 , 50 . The viral loads in Mosaic DNA/LNP group, while not reaching statistical significance, were consistently lower in serum, bronchoalveolar lavage, lungs, tonsils, tracheobronchial lymph nodes than those detected in the corresponding samples from Empty/LNP controls. These results confirmed the immunogenicity of the Mosaic DNA/LNP vaccine these also indicate that the vaccine-induced responses could afford relatively low but measurable levels of protection. Differences in vaccine-induced protection between MLV and Mosaic DNA/LNP could be explained at least in part by a likely more efficient protein expression by the former due to its ability to closely mimic natural infection within the host 48 This in turn resulted in measurable differences in serum neutralizing antibodies, IFN γ expression and protection between MLV and Mosaic DNA/LNP as expected Together the antibody, IFN γ responses, and to some extent viral load data obtained from Mosaic DNA/LNP vaccinated support the potential of this vaccine as a priming platform that can be subsequently followed by recombinant mosaic vaccinia boosting 39 . Declarations Acknowledgements USDA NIFA support Grant award number 2021-67016-34564 Our sincere thanks to Dr. Jianqiang Zhang VDL, ISU Ames, Iowa for providing valuable PRRSV strains. Our gratitude to Dr. Junru Cui for valuable input on the manuscript. Data availability All data analyzed in this study are included in this article or are available from the corresponding author on request. Competing interests The authors declare no competing interests. Generation of new sequencing data No raw sequencing data were produced. Authorship contribution statement Supervision: AEG Conceptualization AEG, PV Funding acquisition: AEG, PV, HVK Methodology: CR, SNG, JZ, YL, XW, NS, EB, DHL, HVK, PV, AEG Formal analysis: CR, SNG, DH. Lee, AEG Writing – original draft: CR, SNG, AEG Review & Editing: CR, SNG, PV, AEG References Bøtner, A. Diagnosis of PRRS. Vet. Microbiol. 55 , 295–301. https://doi.org/10.1016/S0378-1135(96)01333-8 (1997). Done, S. H. & Paton, D. J. Porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression. Vet. Rec . 136 , 32–35 (1995). PMID: 7709569 2. Osemeke, O. et al. 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Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions. Front. Immunol. 10 , 579. 10.3389/fimmu.2019.00579 (2019). Vu, H. L. X. et al. Immune Evasion of Porcine Reproductive and Respiratory Syndrome Virus through Glycan Shielding Involves both Glycoprotein 5 as Well as Glycoprotein 3. J. Virol. Vol . 85 , 5555–5564. 10.1128/JVI.00189-11 (2011). Nedumpun, T. et al. Induction of porcine reproductive and respiratory syndrome virus (PRRSV)-specific regulatory T lymphocytes (Treg) in the lungs and tracheobronchial lymph nodes of PRRSV-infected pigs. Vet. Microbiol. 216 , 13–19. https://doi.org/10.1016/j.vetmic.2018.01.014 (2018). Ferrari, L. et al. A highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strongly modulates cellular innate and adaptive immune subsets upon experimental infection. Vet. Microbiol. 216 , 85–92. https://doi.org/10.1016/j.vetmic.2018.02.001 (2018). Cai, H. et al. Progress in PRRSV Infection and Adaptive Immune Response Mechanisms. Viruses 15 , 1442. https://doi.org/10.3390/v15071442 (2023). Zhang, H. et al. Research Progress on the Development of Porcine Reproductive and Respiratory Syndrome Vaccines. Vet. Sci. 10 , 491. https://doi.org/10.3390/vetsci10080491 (2023). Zhou, L., Ge, X. & Yang, H. Porcine Reproductive and Respiratory Syndrome Modified Live Virus Vaccine: A Leaky Vaccine with Debatable Efficacy and Safety. Vaccines 9 , 362. https://doi.org/10.3390/vaccines9040362 (2021). Nan, Y. et al. Improved Vaccine against PRRSV: Current Progress and Future Perspective. Front. Microbiol. 28 , 8:1635. https://doi.org/10.3389/fmicb.2017.01635 (2017). Jeong, J. et al. A modified-live porcine reproductive and respiratory syndrome virus (PRRSV)-1 vaccine protects late-term pregnancy gilts against heterologous PRRSV-1 but not PRRSV-2 challenge. Transbound. Emerg. Dis. 65 (5), 1227–1234. 10.1111/tbed.12862 (2018). Cao, Q. M. et al. Recombinant porcine reproductive and respiratory syndrome virus expressing membrane-bound IL-15 as immunomodulatory adjuvant enhances NK and γδ T cell responses and confers heterologous protection. J Virol . 13;92(13):e00007-18 (2018). https://doi.org/10.1128/JVI.00007-18 PMID: 29643245. Vu, H. L. X. et al. A Synthetic Porcine Reproductive and Respiratory Syndrome Virus Strain Confers Unprecedented Levels of Heterologous Protection. J. virol. 89 , 12070–12083. https://doi.org/10.1128/JVI.01657-15 (2015). PMID: 26401031 20. Jiang, Y. et al. Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus. Vaccine 25 , 547–560. https://doi.org/10.1016/j.vaccine (2007). 2006.07.032 PMID: 16920232 22. Shen, G. et al. Immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing GP5/GP3 of porcine reproductive and respiratory syndrome virus and swine IL-18. Vaccine 25 , 4193–4202. https://doi.org/10.1016/j.vaccine.2007.03.010 (2007). PMID: 17418456 23. Jiang, W., Jiang, P., Wang Xianwei, Li, Y., Du, Y. & Wang Enhanced immune responses of mice inoculated recombinant adenoviruses expressing GP5 by fusion with GP3 and/or GP4 of PRRS virus. Virus Res. 136 , 50–57. https://doi.org/10.1016/j.virusres.2008.04.016 (2008). Zhou, L. et al. DNA shuffling of the GP3 genes of porcine reproductive and respiratory syndrome virus (PRRSV) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous PRRSV strain. Virology . 434, 96–109 (2012). https://doi.org/10.1016/j.virol.2012.09.005 PMID: 23051709 25. Zhou, L., Ni, Y. Y., Piñeyro, P., Cossaboom, C. M. & Subramaniam, S. Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes. PLoS ONE . 8 (6), e66645. https://doi.org/10.1371/journal.pone (2013). Ni, Y. Y. et al. Attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of virus envelope genes from genetically divergent strains. J. Virol. 87 , 304–313. https://doi.org/10.1128/JVI.01789-12 (2013). PMID: 23077307 27. Tian, D. et al. Chimeric porcine reproductive and respiratory syndrome virus containing shuffled multiple envelope genes confers cross-protection in pigs. Virology 485 , 402–413. https://doi.org/10.1016/j.virol.2015.08.021 (2015). Cui, J. et al. A GP5 Mosaic T-cell vaccine for porcine reproductive and respiratory syndrome virus is immunogenic and confers partial protection to pigs. Vaccine Rep. 6 , 77–85. https://doi.org/10.1016/j.vacrep.2016.11.003 36 (2016). Cui, J. et al. Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime/GP5-Mosaic rVaccinia (VACV) Vaccine Boost. Vaccines 8 (1), 106. 10.3390/vaccines8010106 (2020). Jiang, Y. et al. DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity. Vaccine 24 , 2869–2879. https://doi.org/10.1016/j.vaccine.2005.12.049 (2006). Fischer, W. et al. Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat. Med. 13 , 100–106. https://doi.org/10.1038/nm1461 (2007). PMID: 17187074 30. Barouch, D. H. et al. Mosaic HIV1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat. med. 16 , 319–323. https://doi.org/10.1038/nm.2089 (2010). PMID: 20173752 33. De Lima, M., Pattnaik, A. K., Flores, E. F. & Osorio, F. A. Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus. Virology 353 (2), 410–421. 10.1016/j.virol.2006.05.036 (2006). Kannangai, R. et al. A peptide enzyme linked immunosorbent assay (ELISA) for the detection of human immunodeficiency virus type-2 (HIV-2) antibodies: an evaluation on polymerase chain reaction (PCR) confirmed samples. J. Clin. Virol. 22 (1), 41–46 (2001). Luong, H. Q., Lai, H. T. L. & Vu, H. L. X. Evaluation of Antibody Response Directed against Porcine Reproductive and Respiratory Syndrome Virus Structural Proteins. Vaccines 8 (3), 533. 10.3390/vaccines8030533 (2020). Mehdi, F. et al. Development of a Fast SARS-CoV-2 IgG ELISA, Based on Receptor-Binding Domain, and Its Comparative Evaluation Using Temporally Segregated Samples From RT-PCR Positive Individuals. Front. Microbiol. 11 , 618097 (2021). Pushpakumara, P. D. et al. G. Development and validation of an assay for detection of Japanese encephalitis virus specific antibody responses. Ansari AA, ed. PLOS ONE . 15(10),e0238609 (2020). 10.1371/journal.pone.0238609 . eCollection 2020. Madapong, A., Saeng-chuto, K., Boonsoongnern, A., Tantituvanont, A. & Nilubol, D. Cell-mediated immune response and protective efficacy of porcine reproductive and respiratory syndrome virus modified-live vaccines against co-challenge with PRRSV-1 and PRRSV-2. Sci. Rep. 10 , 1649. https://doi.org/10.1038/s41598-020-58626-y 1 (2020). Suradhat, S. et al. R. A novel DNA vaccine for reduction of PRRSV-induced negative immunomodulatory effects: A proof-of-concept. Vaccine 33 , 3997–4003 (2015). Du, Y. et al. Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon alpha/gamma in immediate and long-lasting protection against HP-PRRSV challenge. Virus Genes . 45 , 474–487. 10.1007/s11262-012-0790-1 (2012). Epub 2012 Jul 28. Kick, A. R. et al. The T-Cell Response to Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Viruses 11 10.3390/v11090796 (2019). Ren, J. Q. et al. Construction and immunogenicity of a DNA vaccine co-expressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus. BMC Vet. Res . 10,128 (2014). http://www.biomedcentral.com/1746-6148/10/12 Zuckermann, F. A. et al. Assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (PRRSV) vaccines based on measurement of serologic response, frequency of gamma-IFN-producing cells and virological parameters of protection upon challenge. Vet. Microbiol. 123 , 69–85. 10.1016/j.vetmic.2007.02.009 (2007). Epub 2007 Feb 16. Osorio, F. A. et al. Passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity. Virology 302 , 9–20. 10.1006/viro.2002.1612 (2002). Lee, S. M., Schommer, S. K. & Kleiboeker, S. B. Porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes. Vet. Immunol. Immunopathol. 102 , 217–231. 10.1016/j.vetimm.2004.09.009 (2004). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8620504","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":591926214,"identity":"1e06b205-8e40-4a4d-b97f-45c4a2dea4f5","order_by":0,"name":"Chesney Romer","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Chesney","middleName":"","lastName":"Romer","suffix":""},{"id":591926215,"identity":"c71be695-3a05-4a0d-9023-dd7ab1f1d534","order_by":1,"name":"Sahar Nouri Gharajalar","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Sahar","middleName":"Nouri","lastName":"Gharajalar","suffix":""},{"id":591926216,"identity":"13d3b29c-7219-4577-ac66-6e73d68e4769","order_by":2,"name":"Jiaqi Zhu","email":"","orcid":"","institution":"Columbia University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Zhu","suffix":""},{"id":591926217,"identity":"a3991283-a181-4907-a1a0-06f72b78c037","order_by":3,"name":"Yangchao Lou","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Yangchao","middleName":"","lastName":"Lou","suffix":""},{"id":591926218,"identity":"72061127-02b7-4974-94d7-22e8e9dadaa1","order_by":4,"name":"Xinhao Wang","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Xinhao","middleName":"","lastName":"Wang","suffix":""},{"id":591926219,"identity":"2cf676ae-c4fd-4be9-ab45-04e4078524f4","order_by":5,"name":"Noah Sneed","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Noah","middleName":"","lastName":"Sneed","suffix":""},{"id":591926220,"identity":"95884644-7bda-477e-a14c-72781bb50ab2","order_by":6,"name":"Evangelina Burdick","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Evangelina","middleName":"","lastName":"Burdick","suffix":""},{"id":591926221,"identity":"c3aff142-29d0-4b37-9410-6243efe3f122","order_by":7,"name":"Dong-Hun Lee","email":"","orcid":"","institution":"Konkuk University","correspondingAuthor":false,"prefix":"","firstName":"Dong-Hun","middleName":"","lastName":"Lee","suffix":""},{"id":591926222,"identity":"bcd5a5b4-c144-41d3-916d-57c9a7044bca","order_by":8,"name":"Herbert Van Kruiningen","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Herbert","middleName":"Van","lastName":"Kruiningen","suffix":""},{"id":591926223,"identity":"9dbd884b-0609-4f35-81c3-bba192acebc6","order_by":9,"name":"Paulo Verardi","email":"","orcid":"","institution":"University of Connecticut","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"","lastName":"Verardi","suffix":""},{"id":591926226,"identity":"b7891425-0a04-40f9-90c4-850687368c5f","order_by":10,"name":"Antonio Garmendia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYNACAziLmYeBvQHGScChnBldC88BYrSg8CTgKrFr4Z/df/BxQYENg7z72acbPu6wluGf+cbww8cddxj42XMMsGmRuHOY2XiGQRqD4Zl0s5szz6TzSNzOMZaceeYZg2TPG6xaGG4ks0nzGBxmMGxIY7vN23aYh+F2jhkzkMFgcAO7LfJwLf3P2G7/BWqRv3kGosUehxYDmBZ5CaAtjEAtBjd4oLZIYNdieCPZ2JjHII3HQOIZ283etnQewzNpxZIzgXolzjwrwKZF7kbiw8c8f2zk5PvT2G78bLO2lzt+eOOHj22H5fjbkzdg9T4U8BgcQBfBpxwSDg0ElYyCUTAKRsFIBQAu2VwusiqL4QAAAABJRU5ErkJggg==","orcid":"","institution":"University of Connecticut","correspondingAuthor":true,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Garmendia","suffix":""}],"badges":[],"createdAt":"2026-01-16 15:24:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8620504/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8620504/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102848343,"identity":"75d227d7-1062-49b2-b0c5-941fc6d85738","added_by":"auto","created_at":"2026-02-17 13:44:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":193957,"visible":true,"origin":"","legend":"\u003cp\u003eChemiluminescence detection of Western blot reactions showing two versions of PRRSV structural mosaic proteins expressed in \u003cem\u003eE.coli\u003c/em\u003e B21. Molecular mass markers (M) are in kDa on the left lane.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8620504/v1/b9f06e66221b3efe82adb299.png"},{"id":102848340,"identity":"2b80e099-d39c-4678-8ae6-3cf1b83919bd","added_by":"auto","created_at":"2026-02-17 13:44:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":241386,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of virus-specific antibody responses after adsorption of experimental serum with Farrowsure Gold B vaccine. (A) Comparison between antibody responses detected in the Empty LNP and the Mosaic DNA/LNP groups at different time points. (B) Comparison between antibody responses in the Mosaic DNA/LNP and the MLV Vaccine groups. Cut off calculations were made for data in each individual ELISA microwell plate run (red lines).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8620504/v1/3a3239460f8acd9f30c3294f.png"},{"id":102848341,"identity":"1bd9c505-d7e2-4c74-a8a2-c1b3636757fb","added_by":"auto","created_at":"2026-02-17 13:44:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43262,"visible":true,"origin":"","legend":"\u003cp\u003eResidual viral titers after 48 hours of virus neutralization by serum collected at different time points during vaccination and after challenge\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8620504/v1/c251e3d39e14a51955bd8092.png"},{"id":103049692,"identity":"0a140b99-6369-45e1-bddc-7acaea4fd105","added_by":"auto","created_at":"2026-02-20 07:44:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76130,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of IFN-γ by PBMNCs from MLV, Mosaic DNA-LNP, and Empty/LNP groups on different days during vaccination and after challenge. A IFN-γ mRNA fold changes in PBMCs on day 0; B. IFN-γ mRNA fold changes in PBMCs right before challenge; C. IFN-γ mRNA fold changes in PBMCs on day 4 after challenge. Error bars represent group standard error of the mean.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8620504/v1/df6fb7efdaf424b9cd797007.png"},{"id":102963120,"identity":"cbffcb23-b7ef-4713-b658-eb5cab147893","added_by":"auto","created_at":"2026-02-19 04:13:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62100,"visible":true,"origin":"","legend":"\u003cp\u003eVirus clearance in sera. Viral copy numbers in serum from day 40 before challenge to necropsy day after challenge with VR2332. Differences were calculated by Student’s t test(* p£0.05). The bars represent the standard error of mean.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8620504/v1/d85851a1a6df07f7910faad6.png"},{"id":103056399,"identity":"964d2bf0-58d0-45d1-86d1-fda8aa90f583","added_by":"auto","created_at":"2026-02-20 09:09:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1595257,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8620504/v1/808dfa17-b401-4bb9-a6a7-a73ee98f10bf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Multiepitope Mosaic DNA PRRSV Vaccine is Immunogenic in Pigs and Confers a Partial Level of Protection Against Challenge Virus","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePorcine reproductive respiratory syndrome (PRRS) is a high impact disease that is distributed worldwide affecting the swine industry globally. Significant direct losses are derived from reproductive failure, and infertility in sows and respiratory disease in piglets \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In the United States alone annual losses due to PRRS can amount over a billion dollars yearly \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The disease is caused by PRRSV, a member of the \u003cem\u003eArteriviridae\u003c/em\u003e Family within the order \u003cem\u003eNidovirales\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The virion is enveloped and contains a 15 kb positive sense single stranded RNA genome that codes for 8 structural proteins and 14 non-structural proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe control of PRRS is highly challenging due primarily to the extensive genetic and antigenic heterogeneity of PRRSV. Based on whole genome sequence the virus was classified into two main genotypes, the Genotype 1 (European type) currently designated as Betaarterivirus Suid 1 and the Genotype 2 (North American type) or Betaarterivirus Suid 2, with differences of up to 40%, between genotypes and up to 20% divergence within the same genotype \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Recent phylogenetic analysis based on ORF5 sequencing thousands of Genotype 2 strains yielded up to 11 different lineages and 21 sub-lineages further illustrating the expanding and extensive genetic diversity of this virus \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRestriction fragment length polymorphisms (RFLP) of ORF5 have been in use to differentiate wild type field isolates from vaccine virus and a recent extensive analysis of thousands of sequences yielded over 200 distinct patterns some of which were used for cluster analysis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the accuracy of RFLP to assign genetic relatedness among PRRSV 2 strains was questioned \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The constant evolution of this virus, as with other RNA viruses, may be explained in part to lack of proofreading mechanisms in the viral RdRp and extensive recombination events that contribute to the heterogeneity of isolates and potentially influence in variations in pathogenicity \u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe infection with PRRSV is characterized by immune evasion, persistence and immunosuppression which make its control even more complex \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Briefly, infection results in down modulation of innate immunity affecting type I IFN -production, there is a decoy of host protective humoral immunity presumably caused by glycan shielding of neutralizing epitopes and early induction of non-neutralizing antibodies \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In addition, infection with PRRSV results in upregulation of IL10 and TGF-B and enhancement of Tregs and an immunosuppressive state ensues \u003csup\u003e18\u0026ndash;20,22\u0026minus;24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVaccines are the main means to control PRRS. However, a major drawback with vaccines used in the field is that the protection afforded is limited against homologous strains with weak or no ability to provide cross-protection to circulating heterologous strains which are continuously mutating \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This emphasizes the urgent need of broadly protective vaccines to address the extensive genetic and antigenic diversity of PRRSV. Research efforts towards the development of broadly protective vaccines for PRRS using different platforms for delivery are ongoing \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Some of these include, modified MLV vaccines \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e consensus sequence vaccines \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, subunit recombinant vaccines \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, molecular breeding by DNA shuffling, mosaic T-cell epitope vaccines \u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, DNA vaccines, VLP vaccines, nanoparticle vaccines, and others \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The main goal in PRRS vaccinology is to achieve vaccines that provide expanded vaccine breadth and depth to thus handle hypervariable circulating PRRSV strains.\u003c/p\u003e \u003cp\u003ePreviously, we investigated a GP5-Mosaic vaccine that was developed using the Mosaic Vaccine Tool Suite originally developed for HIV at the Los Alamos National Laboratory (Los Alamos, NM) \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The GP5-Mosaic vaccine when delivered using a DNA expression vector conferred partial protection in pigs \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Furthermore, priming pigs with DNA GP5-Mosaic vaccine and boosting with recombinant vaccinia virus carrying the GP5 mosaic sequence resulted in partial protection against two strains of different lineages \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. We report here testing the immunogenicity of a multiepitope DNA vaccine expressing 7 structural mosaic PRRSV proteins in pigs. The vaccine induced responses and partial protection from challenge are described and discussed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eViruses and cells\u003c/h2\u003e \u003cp\u003eA series of PRRSV strains from different lineages or sub-lineages were used in the study. These include: VR2332 (L5), SDSU73 (L8B), NADC30 (L1C), Isolate 8981 (Accession 569974) (55), USA/MN/01775GA/2021 (ISU 1) (L5), USA/IN/65239GA/2014 (ISU-2) (L5) (the latter two isolates were kindly provided by Dr. Jianqiang Zhang VDL, ISU Ames, Iowa). The viruses were propagated and titrated in MARC 145 cell cultures using DMEM media containing 10% fetal bovine serum, penicillin streptomycin, glutamine, and stored at -80C for further use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMosaic Vaccine Development and characterization\u003c/h3\u003e\n\u003cp\u003ePublished whole genome sequences compiled from strains circulating during the last 10 years were utilized as input for computation of mosaic sequences. Each gene corresponding to the structural proteins E, GP2, GP3, GP4, GP5, M, and N was annotated. Duplicate sequences (identical sequences) were eliminated, and only unique sequences of each gene were utilized for mosaic antigen design. The number of input sequences was as follows: E\u0026thinsp;=\u0026thinsp;207, GP2\u0026thinsp;=\u0026thinsp;273, GP3\u0026thinsp;=\u0026thinsp;278, GP4\u0026thinsp;=\u0026thinsp;345, GP5\u0026thinsp;=\u0026thinsp;343, M\u0026thinsp;=\u0026thinsp;346, and N\u0026thinsp;=\u0026thinsp;344.\u003c/p\u003e \u003cp\u003eTwo mosaic sequence versions from each protein were submitted for synthesis of their coding sequences and cloning into the expression vector pHCMV-1 (GenScript, Piscataway, New Jersey). Expression of each mosaic protein was performed by transformation to \u003cem\u003eE. coli\u003c/em\u003e BL21, IPTG induction, and preparation of cell lysates using NEB Express\u0026reg; \u003cem\u003eE. coli\u003c/em\u003e Lysis Reagent (NEB,USA). Separation of the cells lysates was carried out by SDS-PAGE using 8\u0026ndash;16% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Protein Gels (Bio-Rad, USA), followed by transfer onto nitrocellulose membranes (Bio-Rad, USA). The membranes were blocked for 2 hours in PBS containing 5% fat-free milk and then reacted for 1 hr at room temperature with PRRSV ORF specific primary antisera as follows: anti E mouse positive serum, anti GP2 swine positive serum, rabbit anti-PRRSV-GP3 polyclonal antibody, GP4 -positive mouse serum, rabbit anti-PRRSV-GP5 polyclonal antibody, rabbit anti-PRRSV-M polyclonal antibody, anti N- positive swine serum. After washing, membranes were incubated for 1 hr with HRP- conjugated Goat anti-mouse IgG (for GP4, and E), HRP- conjugated Goat anti-swine IgG(for GP2,and N), and HRP- conjugated Goat anti rabbit IgG(for M, GP3, and GP5) based on the primary antibody source used for each structural protein. Then, chemiluminescence Western blotting was used for detection.\u003c/p\u003e\n\u003ch3\u003ePreparation and Characterization of Lipid Nanoparticles for Plasmid Encapsulation\u003c/h3\u003e\n\u003cp\u003eThe lipid components used to develop lipid nanoparticle (LNP) included DLin-MC3-DMA (ionizable lipid), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000). These lipids were first dissolved in a chloroform/methanol mixture (5:3, v/v) to form a uniform organic solution. Then, the lipid mixture was rotary evaporated at 45\u0026deg;C to form a thin lipid film along the flask walls. After complete solvent removal, the film was hydrated with RNase-free water and subjected to probe sonication for 15 minutes. Then, the LNP dispersion was mixed with a plasmid DNA solution at a 1:1 volume ratio and sonicated in a water bath for 10 minutes. The final mixture was then filtered through a 0.45 \u0026micro;m PES membrane. The particulate properties of the LNPs were measured using dynamic light scattering (DLS; Malvern Zetasizer Nano ZS). The average empty LNPs were 98.7 nm with a polydispersity index (PDI) of 0.241. Plasmid-loaded formulations showed particle sizes ranging from 122.2 nm to 128.7 nm, with PDI values between 0.274 and 0.262, indicating a relatively uniform size distribution. The zeta potential values ranged from \u0026minus;\u0026thinsp;30 to -40 mV, consistent with stable, negatively charged.\u003c/p\u003e\n\u003ch3\u003eExperimental Animal Vaccination and Challenge\u003c/h3\u003e\n\u003cp\u003eA vaccination-challenge trial was performed using 3- to 4-week-old, Yorkshire castrated males and female PRRS-free piglets (Parsons farm, MA) to assess primarily vaccine immunogenicity and to determine if protection from challenge could be afforded by the Mosaic DNA/LNP vaccine. Group 1 received three doses two weeks apart of Empty/LPN and served as vehicle negative control; Group 2 received three doses under the same schedule of pooled Mosaic DNA/LNP vaccine; Group 3 was vaccinated with 1 dose of Boehringer-Ingelheim Modified Live Virus Vaccine (MLV) (Ingelheim, Germany). An additional group remained unvaccinated to serve as a negative control for serologic and lung pathology assessments. These negative animals were euthanized before challenge was implemented in the other three groups.\u003c/p\u003e \u003cp\u003eA virus challenge with VR2332 (1x10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/ animal) was administered by IM injection and IN instillation on day 40 after vaccination began to groups 1 to 3. The experimentally vaccinated piglets were bled periodically throughout the vaccination phase, before and after challenge for testing. The piglets were euthanized 11 or 12 days after the challenge. Final blood samples were collected right prior to euthanasia. The anesthetic combination of Telazol+Ketamin+Xylazin (IM)was administered prior to euthanasia. Overdose of intracardiac Pentobarbital Sodium was administered for euthanasia after total anesthesia.\u003c/p\u003e\n\u003ch3\u003eAssessment of virus-specific antibody responses\u003c/h3\u003e\n\u003cp\u003eAn in-house whole PRRS virus ELISA was developed for broad detection of vaccine or infection induced antibody responses. Archival pig sera were validated as positive and negative controls by IDEXX X-3 ELISA, and several Genotype 2 PRRSV virus strains were tested as potential antigens. ELISA conditions were determined by a series of checkerboard titrations until optimization was reached. Using our in-house validated ELISA, sera from vaccinated pigs were tested for virus-specific antibodies. Briefly, 96-well flat bottom microwell plates (NUNC, Thermo Fisher, Agawam, MA) were coated overnight at 4\u0026deg;C with Genotype 2 PRRSV isolate 8981 (0.625 \u0026micro;g/mL) diluted in Carbonate/Bicarbonate buffer (pH 9.2) as antigen. Excess antigen was removed and 5.0% skim milk made in PBS containing 0.05% Tween 20 (PBS-T) was added to the wells to block for 2 hours at room temperature. The wells were then rinsed once with PBS. Sera diluted in PBS were added to triplicate wells and incubated at room temperature for 1 hour. The wells were washed 5 times with PBS-T. Secondary anti-porcine IgG antibody HRP conjugate diluted in PBS was added to the wells and incubated for 1 hour at room temperature. The wells were washed as mentioned above and TMB Microwell peroxidase substrate was added and incubated for 15 minutes at room temperature in the dark. Then 4N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added to the wells as the stop solution and the plates were read in a BioTek (Agilent Technologies, Santa Clara, CA) Synergy plate reader at 450nm, and at 630nm. To identify reaction values as positive or negative, a cutoff line was created based on previous literature. Briefly, to the average of all negative values 3 standard deviations of all negative values were added (43\u0026ndash;47) Values above the cutoff line were recorded as positive, and values below the cutoff line were negative.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVirus Neutralization Test\u003c/h2\u003e \u003cp\u003eSera collected from the experimental animals on days 0, the challenge day, and 4 days after challenge were tested for virus neutralizing antibodies in MARC 145 cell cultures. Briefly, test sera from each group were pooled, diluted (1:5) with DMEM media. Then, the diluted serum samples were mixed with an equal volume of VR2332 virus containing 800 TCID\u003csub\u003e50\u003c/sub\u003e/ml (final serum dilution 1:10). After incubation at 37\u0026deg;C for 2 h, the serum-virus mixtures were added to 48-well plates seeded with MARC-145 cell monolayers and incubated at 37\u0026deg;C for 48 h in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. VR2332 virus infected cells, and uninfected cells were used as virus and cell controls, respectively (34). The virus neutralizing capacity of test sera was determined by the Reed-Muench method of titration after 48h supernatants from the original 48-well plate diluted ten-fold were incubated in multiple wells of freshly seeded MARC-145 cells for 5 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of Virus Loads in Serum and Tissues\u003c/h3\u003e\n\u003cp\u003eTo measure virus loads in PRRSV-challenged pigs, viral RNA was extracted from serum, and bronchoalveolar lavages (BAL), using TRIzol LS Reagent and from tissues using TRIzol Reagent (Invitrogen, USA) following the manufacturer\u0026rsquo;s instructions. Total RNA from each sample was then quantified by spectrophotometry (NanoDrop 1000, Thermo Scientific, USA). cDNA was synthesized utilizing the iScript cDNA synthesis kit (Bio Rad Hercules, CA) in a 10\u0026micro;l reaction mixture. The reaction conditions were as follows: 25\u0026deg;C for 5min, 46\u0026deg;C for 20min and 95\u0026deg;C for 1min. The real time PCR was performed using the SYBR Green PCR master mix(BIO-RAD, USA), cDNA, F:5\u003csup\u003e/\u003c/sup\u003e-AAA TGG GGC TTC TCC GGG TTT T-3\u003csup\u003e/\u003c/sup\u003e and R:5/-GCA CAG TAT GAT GCG TAG GC-3\u003csup\u003e/\u003c/sup\u003e as forward and reverse primers for ORF7, respectively. The cycling conditions included an initial denaturation for 95\u0026deg;C for 2min, followed by 40 cycles consisting of 15s at 95\u0026deg;C, and 1min at 61\u0026deg;C using BIO-RAD CFX96 Touch system (Bio Rad Hercules CA). A standard curve was constructed by plotting CT values of serial ten-fold dilutions of viral RNA ranging from 10\u003csup\u003e2\u003c/sup\u003e to 10\u003csup\u003e7\u003c/sup\u003e copies/\u0026micro;l. For each assay, positive and negative reference samples were run. The viral loads in samples were determined by plotting the threshold-cycle number against the standard curve. For specificity of the PCR, melting curves were monitored.\u003c/p\u003e\n\u003ch3\u003eExpression of Interferon Gamma\u003c/h3\u003e\n\u003cp\u003eTo measure interferon-gamma (IFN-γ) in response to recall stimulation with PRRSV, peripheral blood mononuclear cells (PBMCs) from experimentally vaccinated animals were harvested from blood collected on days 0, challenge day, and 4dpc. PMBCs seeded in 24-well flat-bottom plates in duplicate(5\u0026sdot;10\u003csup\u003e5\u003c/sup\u003e cells/well) were stimulated with 200 TCID\u003csub\u003e50\u003c/sub\u003e of VR2332, SDSU73, NADC30, USA/MN/01775GA/2021, or USA/IN/65239GA/2014 for 48h at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Uninfected untreated cells or 12.5 \u0026micro;g/mL concanavalin A (Con A) stimulated cells, were used as negative and positive controls, respectively. Total RNA was extracted using TRIzol Reagent. cDNA synthesis and real-time PCR protocols were as described above utilizing the primers F5\u0026prime; -TGG TAG CTC TGG GAA ACT GAATG- 3\u0026prime; and R5\u0026prime; -GGC TTT GCG CTG GAT CTG- 3\u0026prime; for IFN-γ and F5\u0026prime; - CGT CCC TGA GAC ACG ATG GT- 3\u0026prime; and R5\u0026prime; -CCC GAT GCG GCC AAA T- 3\u0026prime; for \u003cem\u003eGAPDH.\u003c/em\u003e For calculating IFN-γ fold changes, \u003cem\u003eGAPDH\u003c/em\u003e was used as internal control for the delta-delta method.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eVirus-specific serum antibody levels detected by ELISA were compared between groups by a two-way Anova test to determine significant differences. Differences in IFN\u003csub\u003eγ\u003c/sub\u003e expression and viral loads between groups were calculated by the students t test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAthics decleration\u003c/h2\u003e \u003cp\u003e All animals studies were performed under an approved IACUC protocol by University of Connecticut. The informed consent was obtained from the farm owner, for \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe use of theses animals in the study.\u003c/span\u003e All procedures including anestesia and euthanasia followed guidelines and regulations.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMosaic Proteins are Expressed in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eBL21\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExpression of each structural Mosaic protein was examined by chemiluminescence western blotting after transforming recombinant plasmids carrying mosaic inserts into E. \u003cem\u003ecoli\u003c/em\u003e BL21 and following IPTG induction. The expression of two versions of mosaic proteins for each was detected by western blotting with specific antibodies as described in materials and methods. The migration of bands was consistent with their expected molecular weight in kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on these results, the recombinant plasmids were regarded to be functional and ready for further use.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of Virus-Specific Antibodies Before Challenge and Significant Raises in Levels after Challenge Confirm the Immunogenicity of the Mosaic Vaccine\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTesting of day 0 sera by our in-house ELISA revealed an unexpected reactivity despite of the fact that all day 0 sera tested negative by IDDEX x-3 and the positive and negative control sera performed correctly in both tests. The consistent performance of the latter supported the soundness of the in-house ELISA test. The background issue was resolved effectively by adsorbing diluted test serum with Farrowsure Gold B one of the commercial vaccines used in the herd of origin. Thereafter, all whole-virus ELISA tests were performed after adsorption of serum with Farrowsure Gold B vaccine as described above. ELISA results thus obtained were compared between groups and are shown in Figs.\u0026nbsp;2A and 2B. All groups on day 0 tested negative as determined by the cutoff line with no significant differences (Figs.\u0026nbsp;2A, 2B). However, on the day of challenge (day 40), significantly higher antibody levels were detected in the Mosaic DNA/LNP vaccine group than in the Empty LNP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0007) which remained negative as expected (Fig.\u0026nbsp;2A). The trend of higher antibody levels in Mosaic DNA/LNP vaccinated animals persisted on days 4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0002), and 9 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) after challenge. However, on the day of necropsy, there were no statistically significant differences between Mosaic DNA/LNP vaccinated animals and those receiving Empty/LNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Throughout the entire course of the study, there were no statistically significant differences in antibody levels between the Mosaic DNA/LNP vaccinated animals and the MLV vaccinated animals (Fig.\u0026nbsp;2B).\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMosaic DNA/LNP Vaccine Induced Virus Neutralizing Antibodies\u003c/h2\u003e \u003cp\u003eDay 0 pooled serum samples were uniformly negative in virus neutralization test in all groups as expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, under the assay conditions utilized here there was a significant virus neutralization as measured by a decrease in virus titer (TCID\u003csub\u003e50\u003c/sub\u003e) with sera collected right before challenge, from both the MLV vaccine and the Mosaic DNA/LNP vaccine groups, while the virus titer remained high with sera from the Empty/LNP group indicating lack of virus-neutralizing activity. Compared to the Empty LNP group, the viral titers on challenge day (day 40) were 10 times and 4.6 times lower in sera from MLV and Mosaic DNA LNP vaccine groups respectively. There was a further decrease in virus titer from challenge day to 4 dpc with sera of the MLV group while an unexpected rise in titer was detected with sera of the Mosaic DNA/LNP group. However, despite this increase, the titer was still 2.1 times lower when compared with the titer with sera from pigs receiving Empty/LNP which remained steadily high. The lower virus titers detected with serum from MLV and Mosaic DNA/LNP vaccinated pigs confirm their virus neutralizing capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMosaic DNA/LNP Vaccination Activated Relatively Broad Recall IFN\u003csub\u003eγ\u003c/sub\u003e Responses\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e stimulation of PBMN cells with PRRSV strains of diverse lineages and sub-lineages of PRRSV (13) resulted in significant IFN\u003csub\u003eγ\u003c/sub\u003e responses in both MLV and Mosaic DNA/LNP vaccinated animals. PBMN cells from MLV vaccinated pigs collected right before challenge and 4 days after challenge responded significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to stimulation with five different PRRSV strains than their corresponding day 0 PBMNCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). PBMN cells isolated from Mosaic DNA/LNP vaccinated pigs on challenge day and 4 days after challenge responded to three virus strains significantly higher than the corresponding day 0 PBMNCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In contrast, PBMNCs from pigs receiving Empty LNP did not respond to any strain at any of these time points.\u003c/p\u003e \u003cp\u003eConsequently, the PBMNC responses of MLV and Mosaic DNA/LNP vaccinated pigs at challenge day and 4 days after challenge were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those of pigs receiving Empty LNP for 5 of 5 test strains for MLV and 3 of 5 test strains for Mosaic DNA/LNP vaccinated pigs respectively as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. The IFN\u003csub\u003eγ\u003c/sub\u003e responses to recall stimulation of PBMNCs support that cellular immunity was activated by both the MLV and the Mosaic DNA/LNP vaccines. The LNP vehicle alone had no effect on these responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMosaic DNA/LNP Vaccine Induced Relatively Low Levels of Protection\u003c/h2\u003e \u003cp\u003eThere were no differences between groups in viral copy numbers in serum collected 4 dpc. However, viral loads in sera of MLV vaccinated pigs were significantly lower than those in Mosaic DNA-LNP and empty LNP animals 9 days after challenge and at necropsy day (*p\u0026le;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). While there were no significant statistical differences between Mosaic DNA/LNP and Empty/LNP groups 9 days after challenge and at necropsy day, the viral loads in Mosaic DNA/LNP pigs in those days were 15% and 38.5% respectively lower in average than those detected in Empty/LNP treated animals. The viral load in sera of Mosaic DNA/LNP group had a clear descending pattern resulting in a 28% decrease from day 9 after challenge to necropsy day. In contrast the viral loads in sera of the Empty/LNP group remained steadily high at these time points.\u003c/p\u003e \u003cp\u003eA consistent trend of lower viral loads, although not statistically significant, was also detected in bronchoalveolar lavage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.06), lungs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.065), tonsils (p\u0026thinsp;\u0026lt;\u0026thinsp;0.077), and to lesser extent in tracheobronchial lymph node (p\u0026thinsp;\u0026lt;\u0026thinsp;0.09) in the Mosaic DNA/LNP group compared to those detected in the corresponding fluids or tissues from Empty/LNP controls. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the actual differences in mean viral loads in tissues between our experimental vaccine and the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercent decrease in mean viral loads in tissues from Mosaic DNA-LNP compared to Empty LNP groups as detected by qRT PCR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTissue/fluid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePercent decrease in viral loads\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBronchoalveolar lavage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTonsil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e98.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLung tissue examination yielded no significant difference in lesion scores between Mosaic DNA/LNP and Empty/LNP (data not shown)\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe immunogenicity and a partial level of protection from PRRSV challenge induced by a multiepitope Mosaic DNA vaccine PRRSV are discussed here. The Mosaic vaccine was designed using algorithms from the Mosaic DNA Vaccine Tool Suite developed at Los Alamos National Laboratory \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. To that end hundreds of natural sequences of PRRSV strains circulating in the past ten years were used as input with which mosaic sequence outputs were obtained for each structural viral protein. Mosaic sequences were synthesized and individually cloned into the eukaryotic/prokaryotic DNA expression vector pHCMV-1. Expression of the corresponding PRRSV structural mosaic proteins was verified in \u003cem\u003eE coli\u003c/em\u003e BL21. A cocktail of all pHCMV-1 coding for mosaic proteins was them formulated into lipid nanoparticles (Mosaic DNA/LNP) for vaccine delivery. The Mosaic DNA/LNP vaccine was tested in a vaccination-challenge trial in pigs to assess vaccine immunogenicity and determine protection afforded from challenge. Meantime given the nature of the vaccines an accompanying ELISA test was developed in-house using whole virus as antigen to detect responses to multiple viral antigens.\u003c/p\u003e \u003cp\u003eTesting sera from experimentally vaccinated and challenged animals for antibody responses by the whole-virus in-house ELISA revealed unexpected results as some animals at day 0 showed reactivity contrary to IDEXX x3 tests which confirmed that all experimental animals were negative upon arrival. The reasons for this seemingly non-specific reactivity were not understood initially and needed further investigation. Routine vaccination of the sows in the herd of origin with Farrowsure Gold B a polyvalent bacterial and viral vaccine was thought to be a potential source of such background. In fact, adsorption of diluted test serum with Farrowsure Gold B vaccine prior to their addition to ELISA microplates resulted in a significant drop in the background below the cutoff line confirming negativity (data not shown).\u003c/p\u003e \u003cp\u003eFurthermore, a pool of serum from four animals as well as serum from one animal collected on day 0 which reacted strongly in our in-house whole-virus ELISA, were tested in a hemagglutination inhibition assay against porcine parvovirus (a component of the Farrowsure vaccine) and demonstrated a positive result. The potential relationship of this seropositivity to parvovirus with the observed background is unknown at this point. In general, the multiplicity of pathogens both viral and bacterial contained in the vaccines used in the source farm may have increased the likelihood of non-specific reactivities. Potential background reactivities should be kept in mind in vaccine studies.\u003c/p\u003e \u003cp\u003eThe levels of antibody detected in adsorbed sera of the Mosaic DNA/LNP group measured right before challenge, were above the cutoff line and were significantly higher than those in similarly adsorbed sera from the Empty/LNP group which remained negative as expected. A significant raise in levels of antibodies in Mosaic DNA/LNP vaccinated pigs and to a much lesser extent in pigs given Empty/LNP detected after challenge further confirmed the priming and inductive effects of virus-specific antibody responses by the Mosaic DNA/LNP vaccine. The apparent equalization of antibody levels detected at necropsy could be attributed to a response against the challenge infection by Empty/LNP treated animals. The levels of antibody in the Mosaic DNA/LNP group and MLV group remained essentially similar by ELISA.\u003c/p\u003e \u003cp\u003eImportantly, there was virus-neutralizing activity in sera collected right before challenge from the Mosaic DNA/LNP group but not in sera from the Empty/LNP group. There was a visible drop in neutralization activity in sera from Mosaic DNA/LNP at 4 dpc and the reasons are unclear at this point. The relatively short period between challenge and necropsy did not allow the examination of this trend at later time points which should be kept in mind in future vaccine studies. Collectively these results are significant as they confirmed that the virus neutralizing antibody being detected in the Mosaic DNA/LNP group was vaccine-induced. The detection of neutralizing antibodies confirms that relevant neutralizing B cell epitopes were preserved in mosaic proteins. which are required for protective immunity against PRRS \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e IFN\u003csub\u003eγ\u003c/sub\u003e recall responses to stimulation with diverse strains of PRRSV detected both in PBMN cells from MLV and Mosaic DNA/LNP vaccinated pigs and not in pigs receiving Empty/LNP is significant and is a good measure of vaccine induced broad cellular responses \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. PBMNCs of pigs vaccinated with MLV responded to five different PRRSV strains while those from Mosaic DNA/LNP vaccinated pigs responded to three different strains. The reasons for the difference in IFN\u003csub\u003eγ\u003c/sub\u003e responses between MLV and Mosaic DNA/LNP vaccinated pigs could be attributed at least in part to intrinsic\u003c/p\u003e \u003cp\u003edifferences between vaccines. MLV vaccines by having a level of replication within the\u003c/p\u003e \u003cp\u003ehost are in general good inducers of cellular immunity \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. On the other\u003c/p\u003e \u003cp\u003eand DNA vaccines do induce cellular responses but usually require modifications to\u003c/p\u003e \u003cp\u003eadjuvant the responses \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. With the Mosaic DNA/LNP vaccine under the\u003c/p\u003e \u003cp\u003econditions utilized contrary to our expectations the \u003cem\u003ein vitro\u003c/em\u003e IFN\u003csub\u003eγ\u003c/sub\u003e responses were limited\u003c/p\u003e \u003cp\u003eto three of the triggering strains but these belong to three different\u003c/p\u003e \u003cp\u003elineages/sub lineage and are phylogenetically distant which may ultimately prove\u003c/p\u003e \u003cp\u003ebiologically significant. Other variables related to DNA vaccine delivery and differences\u003c/p\u003e \u003cp\u003ein protein expression, and other modulatory conditions or use of incorporated adjuvants\u003c/p\u003e \u003cp\u003ecan influence the outcomes \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe viral loads in Mosaic DNA/LNP group, while not reaching statistical significance, were consistently lower in serum, bronchoalveolar lavage, lungs, tonsils, tracheobronchial lymph nodes than those detected in the corresponding samples from Empty/LNP controls. These results confirmed the immunogenicity of the Mosaic DNA/LNP vaccine these also indicate that the vaccine-induced responses could afford relatively low but measurable levels of protection. Differences in vaccine-induced protection between MLV and Mosaic DNA/LNP could be explained at least in part by a likely more efficient protein expression by the former due to its ability to closely mimic natural infection within the host\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e This in turn resulted in measurable differences in serum neutralizing antibodies, IFN\u003csub\u003eγ\u003c/sub\u003e expression and protection between MLV and Mosaic DNA/LNP as expected Together the antibody, IFN\u003csub\u003eγ\u003c/sub\u003e responses, and to some extent viral load data obtained from Mosaic DNA/LNP vaccinated support the potential of this vaccine as a priming platform that can be subsequently followed by recombinant mosaic vaccinia boosting\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUSDA NIFA support Grant award number 2021-67016-34564\u003c/p\u003e\n\u003cp\u003eOur sincere thanks to Dr. Jianqiang Zhang VDL, ISU Ames, Iowa for providing valuable PRRSV strains. Our gratitude to Dr. Junru Cui for valuable input on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data analyzed in this study are included in this article or are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cbr\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eGeneration of new sequencing data\u003c/p\u003e\n\u003cp\u003eNo raw sequencing data were produced.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eSupervision: AEG\u003c/p\u003e\n\u003cp\u003eConceptualization AEG, PV\u003c/p\u003e\n\u003cp\u003eFunding acquisition: AEG, PV, HVK\u003c/p\u003e\n\u003cp\u003eMethodology: CR, SNG, JZ, YL, XW, NS, EB, DHL, HVK, PV, AEG\u003c/p\u003e\n\u003cp\u003eFormal analysis: CR, SNG, DH. Lee, AEG\u003c/p\u003e\n\u003cp\u003eWriting – original draft: CR, SNG, AEG\u003c/p\u003e\n\u003cp\u003eReview \u0026amp; Editing: CR, SNG, PV, AEG\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eB\u0026oslash;tner, A. Diagnosis of PRRS. \u003cem\u003eVet. Microbiol.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e, 295\u0026ndash;301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0378-1135(96)01333-8\u003c/span\u003e\u003cspan address=\"10.1016/S0378-1135(96)01333-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDone, S. H. \u0026amp; Paton, D. J. Porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression. \u003cem\u003eVet. Rec\u003c/em\u003e. \u003cb\u003e136\u003c/b\u003e, 32\u0026ndash;35 (1995). 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Immunopathol.\u003c/em\u003e \u003cb\u003e102\u003c/b\u003e, 217\u0026ndash;231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vetimm.2004.09.009\u003c/span\u003e\u003cspan address=\"10.1016/j.vetimm.2004.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PRRSV, swine, pig, Mosaic, vaccine","lastPublishedDoi":"10.21203/rs.3.rs-8620504/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8620504/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA Multiepitope Mosaic DNA vaccine was tested in pigs for immunogenicity and protection from PRRS virus challenge. Mosaic coding sequences were synthesized and cloned into pHCMV-1 and their expression was confirmed. Vaccination challenge trial in PRRSV-free piglets followed using Mosaic DNA in lipid nanoparticles. Group 1 received Empty/LNP and served as negative vehicle controls; Group 2 received pooled Mosaic DNA/LNP vaccine; Group 3 received a commercial MLV vaccine and served as positive controls. A virus challenge was implemented by 40 days after vaccination began and euthanasia was implemented 11 or 12 days later. Virus specific antibodies detected in Mosaic DNA/LNP vaccinated pigs by ELISA were significantly higher than those in Empty/LNP controls before and after challenge. Virus neutralizing antibodies were detected in Mosaic DNA/LNP vaccinated pigs but not in controls. IFN\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:ᵧ\\)\u003c/span\u003e\u003c/span\u003e expression was detected in Mosaic DNA/LNP vaccinated animals and Empty/LNP group remained unresponsive. Viral loads in serum of the Mosaic DNA/LPN group, while not statistically significant, were lower than Empty/LPN pigs. The viral levels remained high in sera of control animals. Lower, but not statistically significant, viral loads were detected in, bronchoalveolar lavage, and tissues in the Mosaic DNA/LNP vaccine group than in the controls confirming level of protection.\u003c/p\u003e","manuscriptTitle":"A Multiepitope Mosaic DNA PRRSV Vaccine is Immunogenic in Pigs and Confers a Partial Level of Protection Against Challenge Virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 13:44:20","doi":"10.21203/rs.3.rs-8620504/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"232334799022698075827607469942225654533","date":"2026-03-04T02:14:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303435331935601413851770511176954202297","date":"2026-02-15T11:47:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-12T05:07:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T05:03:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-12T04:43:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-30T18:05:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-30T17:54:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d55be75-14c7-470e-8522-780dd27d9971","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62984097,"name":"Biological sciences/Biotechnology"},{"id":62984098,"name":"Health sciences/Diseases"},{"id":62984099,"name":"Biological sciences/Immunology"},{"id":62984100,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-02-17T13:44:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 13:44:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8620504","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8620504","identity":"rs-8620504","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}