Free Cholesterol Bioavailability and Atherosclerosis

Purpose of Review As both a cholesterol acceptor and carrier in the reverse cholesterol transport (RCT) pathway, high-density lipoprotein (HDL) is putatively atheroprotective. However, current pharmacological therapies to increase plasma HDL cholesterol (HDL-c) concentration have paradoxically failed to prevent or reduce atherosclerosis and cardiovascular disease (CVD). Given that free cholesterol (FC) transfer between surfaces of lipoproteins and cells is reversible, excess plasma FC can be transferred to the cells of peripheral tissue sites resulting in atherosclerosis. Here, we summarize potential mechanisms contributing to this paradox and highlight the role of excess free cholesterol (FC) bioavailability in atherosclerosis vs. atheroprotection. Recent Findings Recent findings have established a complex relationship between HDL-c concentration and atherosclerosis. Systemic scavenger receptor class B type 1 (SR-B1) knock out (KO) mice exhibit with increased diet-induced atherosclerosis despite having an elevated plasma HDL-c concentration compared to wild type (WT) mice. The greater bioavailability of HDL-FC in SR-B1 vs. WT mice is associated with a higher FC content in multiple cell types and tissue sites. These results suggest that dysfunctional HDL with high FC bioavailability is atheroprone despite high HDL-c concentration. Summary Past oversimplification of HDL-c involvement in cholesterol transport has led to the failures in HDL targeted therapy. Evidence suggests that FC-mediated functionality of HDL is of higher importance than its quantity; as a result, deciphering the regulatory mechanisms by which HDL-FC bioavailability can induce atherosclerosis can have far-reaching clinical implications.

Introduction
While levels of cholesterol have been strongly associated with the risk of atherosclerosis and cardiovascular disease (CVD), it is essential to many biological functions as well, from cell structure and membrane fluidity to brain development. Cholesterol is a precursor in biosynthesis of bile, steroid hormones, vitamin D [1], hedgehog protein, myelin, and other substances [2,3], and contributes to the formation of lipid rafts, synapses, and dendrites. Cholesterol primarily localizes to membranes, and is involved in the restriction of the passage of positive hydrogen and sodium ions across the cell membranes in nerve conduction [4][5][6], and facilitates proper cell signaling [7]. Through interaction with other lipids in the bilayer, cholesterol is also involved in regulation of the rigidity and permeability of cell membranes [8,9]. As a result of its involvement in a diverse number of crucial physiological functions, defective cholesterol metabolism can result in an increased risk of a number of pathological disorders. These include atherosclerosis, CVD [10], and central nervous system diseases (Smith-Lemli-Opitz syndrome [11], Niemann-Pick type C disease [12], Huntington's disease [13], and Alzheimer's disease) [14].

Cholesterol Metabolism
The current understanding of cholesterol metabolism begins with the metabolite acetyl coenzyme A (acetyl-CoA). Through enzymatic reactions mediated by the integral endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase enzyme, as well as mevalonate kinase, cholesterol is synthesized in the ER [10]. Acetyl CoA and acetoacetyl-CoA are dehydrated to form HMG-CoA, which is reduced to mevalonate by HMG-CoA reductase enzyme, a step that is blocked by HMG CoA reductase inhibitor, or statins ( Fig. 1). Cholesterol biosynthesis is controlled by a negative feedback mechanism involving sterol regulatory element-binding proteins 1 and 2 (SREBP1, 2). Low intracellular cholesterol concentrations increase cholesterol biosynthesis, which is then suppressed by elevated cellular cholesterol concentrations. Mechanistically, elevated cholesterol concentrations induce changes in the HMG CoA reductase membrane domain that make it susceptible to degradation (Fig. 1). Upon degradation of HMG CoA reductase, cholesterol biosynthesis is inhibited, resulting in decreased levels of cholesterol, which then promotes HMG CoA reductase production. Similarly, when intracellular cholesterol levels are high, SREBP is localized in the ER membrane and bound to sterol regulatory element binding protein cleavage activating protein (SCAP). At these cholesterol levels, SCAP undergoes a conformational change and binds to insulin-induced gene proteins (INSIG), keeping SREBP-SCAP in the ER. However, in low cholesterol levels, INSIG-SCAP binding is disrupted, allowing for SREBP to be transported from the ER to the Golgi apparatus by SCAP [15][16][17]. In the Golgi, SREBP is cleaved, Fig. 1 Cholesterol biosynthesis. Initiation: Acetyl-CoA and acetoacetyl-CoA are dehydrated by HMG-CoA synthase in order to form HMG-CoA, which is then reduced to form Mevalonate by HMG-CoA reductase. Statins, which are commonly used to lower cholesterol, inhibit HMG CoA reductase. Mevalonate is condensed to squalene, a biochemical precursor for cholesterol. HMG-CoA reductase negative feedback regulation: HMG-CoA reductase in the ER is regulated in a negative feedback loop by intracellular cholesterol content. As cholesterol content increases, HMG-CoA reductase undergoes membrane domain changes leading to its ubiquitination and proteasomal degradation, resulting in the inhibition of cholesterol biosynthesis. SREBP negative feedback regulation: When intracellular cholesterol is high, the SREBP-SCAP complex remains in the ER. However, when cholesterol levels decrease, SREBP-SCAP is then transported to the Golgi, where SREBP undergoes cleavage, allowing it to enter the nucleus and activate genes involved in cholesterol biosynthesis, increasing HMG-CoA, as well as cholesterol uptake via the LDL receptor. The subsequent induction of cholesterol biosynthesis results in an increase in intracellular cholesterol, and once again inhibits SREBP-SCAP transport to the Golgi, repressing SREBP activation processed, then sent to the nucleus where it activates genes related to cholesterol biosynthesis, leading to anincrease in HMG CoA, as well as cholesterol uptake through the lowdensity lipoprotein receptor (LDLR). The resulting increase in cholesterol then once again inhibits Golgi transport of SREBP-SCAP, resulting in inhibition of SREBP activation (Fig. 1). A genetic defect of cholesterol biosynthesis linked to mevalonate kinase deficiency, identified in 1986, is the only recognized disorder that belongs to the first half or 'pre-squalene' portion of human cholesterol biosynthesis, although a targeted mutation in mouse squalene synthase gene is associated with embryonic lethality and defects of neural tube closure [18][19][20][21][22]. AMP-activated protein kinase can phosphorylate and inhibit HMG CoA reductase activity [23]. Whole-body cholesterol is obtained endogenously and from dietary intake. The liver is the primary endogenous source and synthesizes 20 ~ 25% of total cholesterol daily; the adrenal glands, intestines, and reproductive organs synthesize smaller amounts. Since tissues can synthesize enough cholesterol for their various biological functions, cholesterol from food ingestion is not required for maintenance of homeostasis.
In cholesterol metabolism, dietary cholesterol, lipids, and triglycerides (TG) in the intestine are assembled into chylomicrons that are transported to the liver. Free fatty acids released from chylomicron-TG particles are taken up by muscle and adipose tissues. In the liver, cholesterol, endogenous-and dietary-TG, and other lipids are assembled into apolipoprotein-B (APOB)-containing lipoproteins and transported to peripheral tissues through the circulation. As the lipid-to-protein ratio increases, the lipoprotein particle sizes and densities, respectively, increase and decrease. Lipoproteins are classified based on their density, with sizes consisting of very low-density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Each lipoprotein type contains different surface-proteins, which determine their specific functions. LDL particles have the highest cholesterol content, defined as the sum of free cholesterol (FC) and cholesteryl ester (CE), and are the major lipoprotein for the transport of cholesterol from the liver to peripheral tissues. VLDL-TG lipolysis by lipoprotein lipase (LPL) converts VLDL to IDL. IDL-TG undergoes lipolysis by hepatic lipase (HL), which converts IDL to LDL. Both these processes release free fatty acids into the circulation [24,25]. A CE transfer protein (CETP) exchanges HDL-CE for the TG of APOB-containing lipoproteins [26]. These lipoproteins, if chemically modified, are taken up by macrophages, forming foam cells [27]. Excess plasma LDL transferred to the subendothelial space of the arterial wall is atherogenic but can be reduced by LDL-lowering therapies, especially statins. HDL particles vary in sizes, densities, and lipid-protein composition [28]. Spherical α-HDL particles are CE-rich, larger, but less dense vs. pre-β-HDL, which are lipid-poor, smaller, and denser [29,30]. Results from Gofman, Miller, and Miller, as well as the Framingham Heart Study, revealed an inverse correlation between plasma HDL-cholesterol concentrations and CVD risk [31][32][33]. From this seminal discovery, HDL was identified as a key player in the reverse cholesterol transport (RCT), i.e., the transfer of peripheral tissue cholesterol, including the arterial wall, to the liver for disposal. As both a cholesterol acceptor and carrier, HDL facilitates cholesterol efflux from peripheral, extra-hepatic, and arterial tissues, including cholesterol-loaded monocytederived macrophages and foam cells, back to the liver to protect the cells from toxicity induced by FC and oxysterol. In the liver, FC is excreted either as biliary cholesterol or converted to bile acids or recycled.

The Biliary Pathway of FC Efflux
FC efflux involves macrophage-ATP binding cassette transporter A1 (ABCA1) and the major HDL-apolipoprotein, APOA1 (Fig. 2). The ABCA1-APOA1 interaction mediates cholesterol and phospholipid transfer to APOA1, thereby forming nascent HDL particles (called pre-β HDL) that contain FC, phospholipid, and APOA1. Subsequently, pre-β HDL binds the sub-family G member 1 (ABCG1), which increases pre-β HDL FC content, forming mature HDL (α-HDL) [27]. Lecithin-cholesterol acyltransferase (LCAT) converts α-HDL-FC to CE and frees spaces on the HDL surface for FC uptake. The RCT pathway involves direct and indirect routes [34][35][36][37]. In the direct route, cholesterol influx to the liver is mediated by the HDL receptor scavenger receptor Class B Type 1 (SR-B1) (Fig. 2). The liver then extracts α-HDL-CE, FC, and phospholipids, thereby generating lipid-poor APOA1 and cholesterol-depleted HDL, both of which can re-enter the HDL/APOA1 cycle [26]. Following SR-B1-induced cholesterol uptake, CE is hydrolyzed to FC for bile acid production, which is either secreted into the intestine (to break down fats) excreted with the feces, which reduces whole-body FC), or recycled to the liver. SR-B1I, an SR-B1 alternative splicing isoform with the C-terminus cytoplasmic domain almost completely altered, is also expressed in caveolae and reduces plasma HDL cholesterol. SR-B1I mediates both selective cellular CE uptake and HDL-dependent cholesterol efflux from cells with around fourfold less efficiency as compared to SR-B1 [38]. In the indirect route, CETP-mediated lipid exchange transfers HDL-CE to VLDL [26] in exchange for TG, and the liver extracts cholesterol (CE and FC) from these particles via the LDL receptor (LDLR), which binds its APOB100 component (Fig. 2). Unlike the SR-B1-dependent mechanism, LDLR-mediated cholesterol uptake leads to lysosomal-induced degradation of lipoproteins to release cholesterol. This indirect route transfers ~ 70% of total CE back to the liver. Additionally, hepatic LDLR is also responsible for the direct uptake of APOE-containing HDL particles, which occur as both HDL 2 and HDL 3 and may be derived either by transfer from TG-rich lipoproteins or from tissue sources (liver and monocyte-macrophages). In addition to the two RCT routes, CE can be delivered back to the liver via the holo-particle uptake; however, the exact molecular mechanism in this transfer has not yet been identified.

The Non-biliary Pathway of FC Efflux
HDL/SR-B1 interaction and the subsequent selective CE uptake are central to the HDL-mediated hepato-biliary RCT pathway [39][40][41][42][43][44][45][46][47][48][49]. According to this widely accepted mechanism, elevated plasma HDL concentrations might be expected to irreversibly remove excess cholesterol and thereby prevent cholesterol influx into the arterial wall. Accordingly, increasing HDL concentrations seemed a promising antiatherogenic approach. However, currently, no studies using HDL therapy have been atheroprotective. Pharmacological induction of plasma HDL failed to prevent or reduce CVD [50][51][52]. Clinical trials failed to capture the efficacy of HDL-raising therapies on CVD [50,53]. Human genetic studies suggested that plasma HDL concentrations do not necessarily reflect its overall abundance, subspecies distribution, cholesterol efflux capacity, or are predictive of CVD risk. As such, further studies are required to completely understand the regulator mechanisms controlling cholesterol efflux. Besides the HDL-mediated hepato-biliary RCT pathway [54][55][56][57][58][59], cholesterol efflux can occur via a non-biliary pathway, or the non-hepatic transintestinal cholesterol excretion (TICE) pathway [60]. TICE involves the direct secretion of plasma lipoprotein-cholesterol by the small intestine [61][62][63][64] via an unknown mechanism [44,65]. Hepatic HDL binds SR-B1 and facilitates the HDLmediated hepato-biliary RCT pathway, sending CE back to the liver for biliary disposal [41]. Hepatocyte-specific SR-B1 knock out (KO) mice exhibit more severe atherosclerosis and early death due to coronary artery occlusions and fibrotic myocardial lesions [66]. In mice, hepatic SR-B1 overexpression increases CE selective uptake from HDL-CE and macrophageto-feces RCT [67,68]. SR-B1 is also expressed in enterocytes and small intestine-specific SR-B1 overexpression in mice reduces plasma HDL-C concentrations but does not change cholesterol absorption [69]. Treatment of mice with small intestine-specific SR-B1 overexpression with ezetimibe, which blocks cholesterol absorption, did not affect fecal neutral sterol Macrophages localize to fat deposits on the walls of blood vessels, forming lipid laden foam cells -both foam cell and hepatic ABCA1 contribute to HDL formation. ABCA1 and APOA1 interaction induces transfer of FC and phospholipids to APOA1, forming nascent HDL. LCAT mediated maturation: LCAT converts HDLFC to CE and allows for more FC uptake on the HDL surface; FC is also transferred to HDL via ABCG1. LCAT mediated conversion prevents reverse uptake of FC by the macrophages from mature HDL, which then enters either the direct (SR-B1) or indirect (CETP) route. Cholesterol influx: Hepatic SR-B1 selectively extracts FC, CE, and phospholipids from mature HDL, generating APOA-1 which can reenter the cycle. CE is hydrolyzed into FC and incorporated into bile acids which are secreted into the intestines. CETP-mediated lipid exchange: CETP transfers HDL-CE to VLDL and LDL in exchange for triglycerides. CE and FC associated with VLDL/LDL are then delivered to the liver by the LDL receptor. Created with BioRender. com excretion, suggesting that SR-B1 overexpression in the intestine does not increase TICE. In their review, Temel et al. have proposed a new integrated model of the non-biliary TICE and RCT pathway as well as strategies to stimulate intestinal cholesterol excretion for the prevention or treatment of CVD [60].

SR-B1 Is One of the Key Components in the Biliary Pathway of FC Efflux
SR-B1 is a member of the scavenger receptor class B family, which also includes CD36 and CD36 antigen-like II (LIMPII) [70]. This 82-kDa membrane glycoprotein was identified by its homology to CD36. Like LIMPII and CD36, SR-B1 has 2 transmembrane domains linked to 2 intracellular N-and C-termini and an extracellular glycosylated central domain separated by a large extracellular loop. SR-B1 binds various ligands including acetylated, oxidized, and native VLDL, LDL, HDL [71], as well as Lp(a), modified serum proteins such as maleylated bovine serum albumin, advanced glycation end-product modified proteins, anionic phospholipids, lipid vesicles containing negatively charged phospholipids, and apoptotic cells [71][72][73][74]. SR-B1 binds apolipoproteins on the HDL surface, including APOA1 [75][76][77][78][79]. This association of SR-B1/ APOA1 requires specific structural domains (the amino-or the carboxy-terminal amphipathic helices) [30] and conformation states of APOA1 on HDL particles [80]. Relative content of APOA1 and -2 in spherical HDL particles also affects the SR-B1/APOA1 association [81][82][83][84]. Moreover, the conformation and organization of APOA1 on HDL particles is crucial to SR-B1 binding to HDL, facilitating the transport of lipids (CE, phospholipids, TG, and lipid soluble vitamins). Binding of SR-B1 to the reconstituted discoidal APOA1 complexes is stronger than to α-HDL, whereas its binding to α-HDL is stronger than to pre-β-HDL [29,30]. While forming complexes with these lipoproteins/receptors including mature α-HDL particles, SR-B1 facilitates cholesterol transfer between HDL-cholesterol (HDL-C) and cell membranes, mediates a selective CE uptake from HDL-CE without particle uptake, and promotes the elimination of excess cholesterol via biliary cholesterol secretion. Unlike ABCA1 and ABCG1, which mediates cholesterol transport in a passive, concentration-dependent, and energy-independent manner, SR-B1 mediates bidirectional cholesterol flux.

Hepatic and Extra-hepatic SR-B1 Is Atheroprotective
SR-B1 binds a multi-PDZ domain containing adaptor protein PDZK1, which can regulate SR-B1 protein expression levels [85]. In PDZK1 KO mice, the SR-B1/PDZK1 complex controls SR-B1 expression in a tissue-specific and post-transcriptional dependent manner. SR-B1 expression is reduced by 95% in the liver and 50% in the proximal intestine of the PDZK1 KO, as compared to that of the WT mice. However, SR-B1 expression level is unaltered in steroidogenic organs (adrenal, ovary, and testis). In hepatocytes, expression of a SR-B1 splice variant with an alternative C-terminal cytoplasmic domain (SR-B1I) is not affected in PDZK1-KO mice, suggesting that the PDZK1/SR-B1 complex is required for hepatic SR-B1 expression [86].
Plasma cholesterol concentrations were increased in SR-B1 targeted heterozygous (HET, 31%) and homozygous KO (KO, 125%) mice due to the formation of large particles containing APOA1, while alternatively, cholesterol content in adrenal glands were decreased in HET (42%) and KO (72%) [87]. Since plasma APOA1 concentration was unaltered, the data suggests that the increased plasma cholesterol is due to defective HDL-C uptake [87]. Among SR-B1 KO mice, SR-B1 deficiency [87] differentially alters lipid metabolism in arterial and hepatic cells [88]. SR-B1 KO vs. WT mice fed a regular diet (4.3% fat, no cholesterol) exhibit a 1.8-fold increase in total serum cholesterol (TC) due to a 3.2-fold increase in HDL-FC [88]. Changes in TC concentrations in SR-B1 KO mice are due to an increased HDL-C. No significant difference in cholesterol profile between SR-B1 HET and WT was noted, except for a slightly increased VLDL-C concentration. After 20 weeks of feeding a high cholesterol diet (HCD, 15% fat, 0.25% cholesterol), plasma HDL-C of SR-B1 KO mice was greatly increased with minor changes in VLDL-C concentrations, and compared to the WT mice, the KO mice developed more severe atherosclerosis [88]. HDL compositions were also different in SR-B1 KO vs. WT, 9.6% and 4.5% for FC, 32.7% and 18.8% for CE, and 23.6 and 40.6% for phospholipid respectively. The protein contents of HDL from WT and SR-B1 KO mice were similar. Collectively, the data suggests that a HCD increases TC, HDL-FC, and HDL-CE and reduces phospholipids in SR-B1 KO mice without changing the serum TG concentrations and protein content [88]. Four hours after an initial injection of 3 H-labeled HDL, 3.3 ± 0.8% of the injected dose was recovered in the liver of SR-B1 KO as compared to 31.3 ± 4.5% for WT mice. After correction for the HDL-CE pool size, the HDL-CE mass influx into the liver was 4.1 ± 0.7 μg/h in KO compared to 22.0 ± 3.8 μg/h in WT mice. This data suggested that the RCT pathway is deficient in SR-B1 KO mice, leading to the accumulation of HDL-FC [88]. This data also revealed the role of hepatic SR-B1 in the overall HDL-C metabolism and atherosclerosis [88]. The role of hepatic SR-B1 in whole particle uptake was confirmed using 125 I-labeled tyramine cellobiose-labeled HDL. In SR-B1 KO mice, neither the hepatic cholesterol content (hepatic concentrations of FC, CE, and TG) nor the expression of key regulators of hepatic cholesterol homeostasis (HMG-CoA reductase, LDLR, and cholesterol 7α-hydroxylase) was altered, but biliary cholesterol content decreased ~ 40% [87,89]. Conversely, expression of genes mediating inflammation and the dysregulation of cholesterol homeostasis in the arterial wall were increased, resulting in an enhanced lipid deposition in their aortas.
Mechanistically, hepatic SR-B1 mediates selective hepatic uptake of CE in HDL-CE via the RCT pathway, thereby trafficking CE to bile for excretion, while mediating cholesterol transfer for steroid hormone synthesis in the adrenal glands, ovaries, and testes [71,79]. RCT is comprised of two steps (i) formation of an HDL/SR-B1 complex in an APOA1-dependent manner, and (ii) the transfer of CE from HDL-CE to the plasma membranes, internalized via a non-endosomal pathway and hydrolyzed by neutral CE via mechanisms yet to be characterized. CE hydrolysis produces FC that is used to produce bile, which is partly secreted into the intestine (to break down fats) and to the feces for excretion or recycled to the liver. Bile acid biosynthesis, the main pathway of cholesterol catabolism, is tightly regulated by the expression of the liver-specific enzyme cholesterol-7α-hydroxylase (CYP7A1) [90]. In some species, excess cholesterol consumption leads to enhanced CYP7A1 expression and increased bile acid biosynthesis. Conversely, mice and hamsters that lack liver-X-receptor-α are unable to induce CYP7A1 in response to dietary cholesterol, and therefore are susceptible to cholesterol accumulation in plasma lipoproteins and liver, suggesting that modulation of the cholesterol-induced bile acid biosynthesis is important for the maintenance of cholesterol homeostasis. Reduced bile acid synthesis or dysfunctional biliary cholesterol secretion is associated with cholelithiasis and atherosclerosis [90]. The major cholesterol transport proteins including ABC transporters mediating biliary secretion of bile acids (ABCB11), phospholipids (ABCB4), and cholesterol (ABCG5/G8), and others including SR-B1, the phosphatidylserine flippase ATP8B1, and NPC1L1, modulate biliary cholesterol secretion [91]. Among SR-B1 KO mice, there was reduced expression of ABCG5 (70%) and ABCG8 (35%), suggesting that ABCG5/G8 is involved in SR-B1-mediated HDL-FC uptake and biliary cholesterol secretion.
The conditional SR-B1 KO mice, achieved by flanking exon 1 with Cre recombinase loxP sites to produce a hypomorphic allele (hypomSR-B1 KO) [92], exhibit twofold increase in plasma TC concentrations and 2.5-fold increase in atherosclerosis, as compared to the WT [92]. Hepatocytespecific SR-B1 KO mice induced a hypomorphic SR-B1 allele inactivation in hepatocytes (hypomSR-B1-KO liver ) and showed higher plasma TC concentration and FC/ TC ratio as compared to the WT. The lipoprotein-cholesterol profile in these mice was similar to those of SR-B1 KO mice [87]. Plasma TC concentrations were increased twofold in hypomSR-B1 and control mice fed a HCD, while hypomSR-B1-KO liver and SR-B1 KO mice developed severe hypercholesterolemia due to accumulation of FCrich, VLDL-sized particles. Atherosclerosis in hypomSR-B1 mice was increased 2.5-fold compared to the control but was reduced 32-and 48-fold compared to hypomSR-B1-KO liver and SR-B1 KO, respectively [87]. Plasma lipid concentrations or the capacity of VLDL-sized lipoproteins to induce macrophage cholesterol loading were similar for hypomSR-B1-KO liver and SR-B1 KO mice, although atherosclerosis was reduced in hypomSR-B1-KO liver due to a decrease in macrophage content compared to SR-B1. These results suggest that SR-B1 in extra-hepatic tissues is also atheroprotective [92,93]. In ApoE KO mice, SR-B1 deletion (SR-B1/ApoE dKO) produces severe hypercholesterolemia (≈1000 mg/dl) with a significant increase in lipoprotein-FC and HDL particles in the size range of LDL and VLDL [94,95], with defects in morphology and function like those seen in human congenital heart disease. When fed a regular diet, SR-B1/ApoE dKO mice display atherosclerosis in the aortic sinus and the mice possess lipid-rich coronary artery occlusions, myocardial fibrosis, and cardiac dysfunction (enlarged hearts, reduced ejection fraction and contractility, and abnormal echocardiography) at 4 ~ 5 weeks after birth. Accelerated atherosclerosis in SR-B1/ApoE dKO mice, partly due to abnormal lipoproteins [96,97], leads to coronary artery occlusion, MI, and premature death 5 ~ 8 weeks after birth [66,88,94]. Coronary arterial lesions in SR-B1/ApoE dKO mice show evidence of cholesterol clefts and fibrin deposition, suggesting hemorrhage and clotting as seen in human atherosclerotic plaques. By 6 and 8 weeks of age, mortality was 50% and 100%, respectively [66]; however, longer-term high fat diet is required to induce coronary atherosclerosis in the SR-B1/ApoE-dKO mice. To induce myocardial infarction (MI), additional hypoxic or mental stress is needed and only occurs in 40% of the SR-B1/ApoE dKO mice [66,93]. Similarly, Fuller et al. [98] showed that compared to LDLR KO, SR-B1/LDLR dKO mice exhibit abnormal lipoprotein profiles, severe coronary atherosclerosis, MI, and decreased survival rate (average lifespan = 9.4 weeks) when fed a modified Western diet containing high cholesterol (1.25%). Altogether, these data indicated that SR-B1 in hepatic and extra-hepatic tissues is atheroprotecive.

Macrophage SR-B1 Is Atheroprotective
In humans, SR-B1 is abundantly expressed in tissues involved in a selective CE uptake with strong effects on HDL metabolism (adrenal glands, liver, steroidogenic tissues, etc.). SR-B1 is expressed in various cell types including hepatocytes, macrophages, endothelial cells, and enterocytes. Single-nucleotide polymorphisms (SNPs) in conserved regions of human SR-B1 extracellular domain are associated with impaired macrophage cholesterol efflux and platelet function, and increased CVD risk [99][100][101][102][103]. In mice on both LDLR KO and APOE KO background, the depletion of macrophage SR-B1 promotes atherosclerosis via a mechanism independent of its function as a hepatic HDL receptor [104,105]. In atherosclerotic lesions, macrophages of SR-B1 KO mice are proinflammatory and susceptible to apoptosis [106]. SR-B1 in macrophages localizes to autophagosomes, forming cholesterol domains, increasing the recruitment of Barkor and the VPS34/Beclin-1 complex. By activating PPARα, macrophage SR-B1 upregulates autophagy regulator transcription factor EB (TFEB) and the VPS34/LC3/ ATGs expression and thereby activating autophagy. Accordingly, SR-B1 depletion leads to the defective autophagy by inhibiting VPS34 activity in macrophages, increasing foam cell formation (1.8-fold), apoptosis (2.5-fold), and oxidized LDL-induced inflammatory cytokine expression, whereas TFEB or VPS34 overexpression reverses these processes [107].
However, despite this seemingly atheroprotective function, EC-specific SR-B1 KO mice generated on an ApoE KO background showed a marked reduction of atherosclerosis in both males and females in both mixed and C57BL/6 background, while plasma TC, TG, HDL, and lipoprotein profiles were unaltered [66]. Transgenic mice expressing endothelial SR-B1, both C57Bl/6 N and ApoE-KO backgrounds, possess a slight decrease in plasma lipoprotein profiles [108]. TG and HDL-C concentrations are 1.2-and 1.35-fold lower, respectively, in the EC-specific SR-B1 deficient mice (C57Bl/6 N background) as compared to sibling controls. However, no differences were noted in plasma cholesterol between the EC-specific SR-B1 deficient mice and female non-transgenic siblings [108]. Endothelial SR-B1 expression had little effect on plasma lipoprotein concentrations in mice fed a regular diet, but it protected mice from diet-induced atherosclerosis by reducing plasma cholesterol, while increasing plasma HDL-C concentrations. In 8-monthold APOE-KO mice fed a regular diet, EC-specific SR-B1 expression decreases aortic lesions by 24%. Mice expressing SR-B1 in ECs and in the liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice expressing SR-B1 only in the liver. This elevation was mostly due to increased HDL-C. In EC culture studies, SR-B1 was present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. Since EC SR-B1 deficiency does not affect the expression of genes that regulate inflammation in the aorta, or leukocyte-EC adhesion in basal or TNF-mediated proinflammatory conditions, a decrease in atherogenic lipoprotein profiles and atherosclerosis caused by EC SR-B1 expression suggests that EC SR-B1 might play a role in cholesterol efflux across ECs. SR-B1 expression is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. In EC-intracellular vesicles, LDL particles colocalize with SR-B1, which interacting with dedicator of cytokinesis 4 (DOCK4), which is a receptor that recruits the guanine nucleotide exchange factor to its eight-amino-acid cytoplasmic domain. DOCK4 couples the SR-B1/LDL complex with activated Rac1, promoting the SR-B1/LDL internalization and thereby transporting LDLcholesterol (LDL-C) into the arterial wall. Consequently, EC SR-B1 promotes atherosclerosis by facilitating LDL-C accumulation in the arterial wall [115••] (Fig. 3). Loss of EC PDZK1 (PDZK1 ∆EC ) had no effect on lesion severity [115••], suggesting that EC SR-B1 mediates atherosclerosis via a PDZK1-independent mechanism without altering circulating lipids or vascular inflammation. Similarly, HDL action in CVD results in reduced activation of eNOS and an increase in ROS formation through low levels of PON1, an increase in MDA, and thus, oxidation of lipoproteins such as HDL and LDL [114] (Fig. 3). Ox-LDL can bind lectin-type oxidized LDL receptor 1 (LOX-1), resulting in promotion of atherosclerosis formation.

Platelet SR-B1 Is Atheroprotective
In the atherosclerotic plaque development, the role of platelets is essential [116]. Activated platelets help to recruit immune cells, such as monocytes to injury sites on arterial walls. Macrophage stimulating factors expressed by platelets induce the monocytes and transformed them to activated macrophages. The activated macrophages induce apoptosis to the surrounding smooth muscle cells in the arterial wall. The arterial wall become weak and produce growth factors and free radicals which transform the normal, contractile smooth muscle cells to a proliferative stage [117]. As a result of these changes, those smooth muscle cells then migrate to the intimal layer of the arterial wall, proliferate, and narrow the lumen by creating plaques. The abnormal activation and aggregation of platelets is associated with increased atherosclerotic plaque formation and thus inhibiting that pathway can reduce risk of CVD [118]. Both the activated and resting platelets express SR-B1 and oxidized HDL binds to SR-B1 leading to inhibition of platelet activation and aggregation [119]. Additionally, natural HDL 3 binds to SR-B1 on platelets and inhibits their activation [120].

Potential Roles for SR-B1 And S1P in HDL-Independent Mechanisms of FC Efflux
Sphingosine 1-phosphate (S1P) plays a role in various biological functions including anti-apoptosis, anti-inflammation, pro-thrombosis, vasorelaxation, EC barrier functions, and the egression and activation of lymphocytes [121]. Low plasma S1P concentrations are associated with CVD. As a major carrier and acceptor, HDL binds and transports ~ 50-70% of plasma S1P. Plasma concentrations of HDL-S1P affect some HDL-mediated functions. Low HDL-S1P concentration is linked to atherosclerosis, CVD, MI, and renal insufficiency due to impaired HDL functions. Pharmacological increases in plasma HDL-S1P concentrations suppresses these adverse effects. However, the pathophysiological and molecular mechanisms by which changes in HDL-S1P concentrations are linked with these pathologies remain to be elucidated. Binding to HDL alters bioactivity and receptor presentation of S1P and thereby modulates S1P signaling. In the HDL-S1P complex, Apolipoprotein M (APOM) is a major S1P binding partner. The APOM-S1P complex modulates biological properties of S1P and metabolism of APOM-containing lipoproteins, which regulate plasma S1P concentration. S1P-APOM strengthens S1P Fig. 3 Overview of SR-B1-dependent signaling and mechanisms in endothelial cells. Healthy HDL action in endothelial cells: HDL can stimulate eNOS activation through several mechanisms. HDL binding to SR-B1 induces efflux of cholesterol and leads to adaptor protein PDZK1 interaction-dependent signaling; activation of nonreceptor tyrosine kinase c-SRC leads to activation of PI3K, resulting in activation of Akt and eNOS. S1P sequestered in HDL can interact with S1P3 receptors, also resulting in P13K/Akt/eNOS activation. SR-B1/PDZK1 signaling also results in LKB1 and CAMK mediated activation of AMPK, inducing Akt and eNOS activation. HDL particles carry active PON1 enzyme which hydrolyze biologically active lipid peroxidases, thus suppressing oxidation of lipids and lipoproteins. This results in diminished levels of MDA and oxidative stress. Transendothelial LDL transport: In ECs, guanine nucleotideexchange factor (GEF) DOCK4 couples with SR-B1 and is required for transcytosis of LDL. LDL binding with SR-B1 leads to interac-tion between SR-B1 and DOCK4, as well as activation of its associated Rho GTPase Rac1, resulting in caveolin-1 mediated transcytosis. Caveolae formation internalizes SR-B1 along with LDL, leading to trafficking of LDL to the subendothelial space where it contributes to foam cell and atherosclerotic plaque formation. HDL action in cardiovascular disease: HDL inhibits eNOS activation and induces ROS formation in cardiovascular disease. HDL in CVD has significantly reduced levels of PON1 activity, resulting in high levels of MDA and oxidation of lipoproteins and lipids. Oxidized lipoprotein can then activate LOX-1 which in turn stimulates PKCβ and inhibits Akt signaling. eNOS activity is reduced as a result. LOX-1 activation also regulates NADPH oxidase activity, inducing ROS generation. Decreased generation of the anti-atherosclerotic NO by eNOS and increased ROS generation results in promotion of EC inflammation and atherogenesis. Created with BioRender.com anti-atherosclerotic properties while potentially weakening S1P pro-atherosclerotic properties, suggesting that APOM-S1P is an anti-atherogenic target. S1P binding to its receptors (S1PRs) facilitate lipid transfer [122]. S1P/S1PRs regulate molecular pathways that control cellular homeostasis in multiple cell types including cardiac and vascular cells. Different cardiac S1PRs are implicated in different pathologies: S1PR1 is downregulated in experimental models of heart failure (HF), while its overexpression alleviates HF after MI; S1PR2 and 3 protect cardiomyocytes from ischemia/reperfusion damage. Dysregulated S1P/S1PRs is implicated in CVD, including atherosclerosis, coronary artery disease, MI, and HF. Similarly, SR-B1 also binds various S1PRs, required for the S1P-HDL-mediated pleiotropic effects of HDL [70,123]. Using protein-fragment complementation assays and confocal microscopy approaches in primary rat aortic vascular smooth muscle and HEK293 cells, Lee et al. found that upon interaction with HDL, S1P triggers a transient SR-B1/ S1PRs, thereby activating S1PRs and increasing intracellular calcium 25 . Both S1P and SR-B1 can bind S1PRs and HDL, and can be either anti-or pro-atherogenesis, suggesting that their paradoxical effects in atherogenesis could depend on their interacting partners. Although APOM is a major binding partner of S1P forming the HDL-S1P complex, other coexisting receptors can bind to S1P, particularly when plasma S1P concentration is acutely elevated. In such a context, biological properties modulated by S1P-signaling can be driven by HDL-independent mechanisms. One example is that the S1P-APOM association strengthens S1P-associated antiatherosclerotic properties while potentially weakening S1P pro-atherosclerotic properties; whereas the S1P-Albumin association exerts both beneficial and detrimental effects on atherogenesis.
Adding to its well-accepted function as a cell surface HDL receptor that facilitates a selectively CE uptake [124][125][126], SR-B1 has also been implicated in various important biological functions including intracellular signaling, lipid accumulation, foam cell formation, and cellular apoptosis. For instance, SR-B1 modulates the susceptibility to LPS-induced tissue injury and plays a role in adaptive and innate immunity [127,128]. SR-B1 recruits the nonreceptor tyrosine kinase src to its C-terminal cytoplasmic tail, increasing src autophosphorylation leading to the phosphorylation and activation of AMP-activated protein kinase (AMPK). Although in other tissues, the PDZK1-SR-B1 association is required for SR-B1-HDL signaling, in brain microvascular ECs, PDZK1 binding is not required for HDL-C uptake. Internalized HDL partially co-localizes with SR-B1. However, in brain microvascular ECs, SR-B1 facilitates lipid transfer from and to HDL via a mechanism independent of HDL internalization and degradation [124][125][126]. Inhibition of eNOS promotes HDL internalization but raising NO concentrations does not affect HDL concentrations, suggesting that SR-B1-mediated HDL transcytosis in brain microvascular ECs is distinct from HDL-FC uptake [129]. This observation also supports SR-B1's role beyond cholesterol efflux.

FC Bioavailability Is Increased in SR-B1 KO Mice
Defective cholesterol transfer triggered by an increased FC bioavailability has been implicated in atherosclerosis. In mice, most lipoprotein-FC is rapidly cleared from the circulation (t 1/2 < 5 min) without esterification. The remainder is taken up by various tissues and cells, especially erythrocytes. In SR-B1 KO vs. WT mice, diet-induced atherosclerosis is greater despite elevated plasma HDL concentrations. Similarly, a large population-based study showed that an increased plasma HDL concentration, in individuals who are heterozygous for the SR-B1 P376L mutation, is associated with increased CVD risks. In their study, Liu et al. noted a marked increase of HDL-FC mol% in plasma of the SR-B1 KO (41.1 mol%) as compared to that of the WT (16.0 mol%) mice [130 ••]. The SR-B1 KO show higher FC bioavailability for transfer to multiple tissues as compared to the WT mice. Plasma clearance of autologous HDL-FC mass is much slower in the SR-B1 KO as compared to the WT mice. Whereas FC efflux capacity is similar between the two genotypes, FC transfer from the HDL of SR-B1 KO vs. WT mice to LDL and to J774 macrophages was about 4-times greater. Moreover, in the SR-B1 KO, higher FC mol% in ovaries, erythrocytes, heart, and macrophages are associated with previously reported infertility in females, impaired cell maturation, cardiac dysfunction, and atherosclerosis. The FC contents of other tissues is similar between the SR-B1 KO and WT mice, and these tissues are not associated with any overt pathology. In addition, there were sex-dependent differences in tissue lipid composition and plasma FC clearance rates. This study highlights the importance of FC bioavailability in atherosclerosis and other pathologies [130••].

Conclusion
Given the association of high plasma cholesterol concentrations to increased risk of CVD and atherosclerosis, past clinical studies have largely focused on increasing HDL-c content to induce RCT. As the primary receptor for HDL-c in the RCT pathway, SR-B1 is a crucial mediator of cholesterol transport. Although widely expressed in multiple tissues and cell types, SR-B1 expression and function is highly context-dependent [131]. Despite primarily being studied as an HDL-selective receptor, SR-B1 colocalizes with LDL particles in EC intracellular vesicles, allowing for active caveolae-mediated transcytosis of LDL. This observation challenges the long-held concept that LDL-C passively crosses permeable EC membranes and suggests that the inhibition of endothelial SR-B1-mediated delivery of LDL to arterial walls may be an effective therapy for CVD. Similarly, this study demonstrates the complexity surrounding SR-B1 context-dependent functionality; although it has long been thought to be primarily an atheroprotective mediator of RCT via HDL-c binding, SR-B1 can contribute negatively towards atherosclerosis progression in CVD through a multitude of pathways (Fig. 3).
This long-term focus on SR-B1-HDL-mediated cholesterol efflux through the hepatic RCT pathway underlies the current oversimplification of the physiological functions involving the SR-B1-HDL complex. Although current studies have largely focused on the role of HDL quantity in atheroprotection, the clinical failure of therapeutics that increase HDL-c concentration to have an effect upon CVD progression challenges the notion that HDL quantity is the primary mediator of atheroprotection [45][46][47]. Similarly, a recent study has demonstrated that a systemic SR-B1-KO in mice will dramatically increase HDL-c, but paradoxically raise CVD [130 ••]. These studies have revealed that plasma HDL-c concentration associates in a U-shaped relationship with CVD risk; while low HDL-c results in high risk of CVD related to hypertriglyceridemia [132,133], those in the highest percentile for plasma HDL-c levels are also at risk for CVD. To explain this phenomenon, current studies should begin focusing on the quality and functionality of HDL-c rather than the quantity. Here, we review one proposal examining the FC bioavailability in HDL, resulting in a dysfunctional, atherogenic HDL-c that transfers FC back into macrophages, resulting in plaque formation. This hypothesis is supported by the study in SR-B1 KO mice and can be further examined clinically through the measurement of plasma HDL-c, and determination of HDL-FC and other phospholipid concentrations.
Other aspects of the SR-B1-HDL complex remain uncertain as well. Given the reduction in ABCG5/G8 expression in the SR-B1 KO mice, ABCG5/G8 may play a role in SR-B1-dependent biliary cholesterol secretion as well [130••]. While certain polymorphisms of ABCG5/G8 have been linked to reduction of HDL-C concentration [134], another meta-analysis examining common ABCG5/G8 polymorphisms has suggested that they demonstrate little association to markers of cholesterol homeostasis such as LDL-c levels [135]. Determination of a role for ABCG5/G8 in the SR-B1-HDL complex-dependent cholesterol secretion can further our understanding of this pathway. Similarly, given its reduction in SR-B1 KO mice, it is possible that low expression of ABCG5/G8 may contribute to FC bioavailability and the dysfunctionality of HDL-c. Therefore, the FC bioavailability proposal firstly demonstrates the previous exaggerated focus on HDL-c's involvement in RCT. Clinical validation of dysfunctional HDL with high FC bioavailability would lead to studies to elucidate the biological mechanisms underlying FC concentration for translational applications. Given the failures of therapies targeting HDL-c concentration in lowering CVD risk, future studies examining HDL-c functionality are likely to have more success in determining HDL-c importance in CVD risk.

Declarations
Human and Animal Rights and Informed Consent. This article does not contain any studies with human or animal subjects performed by any of the authors.
Conflict of Interest LF reports the following patents planned, issued, or pending: (1) Compositions and methods for regulating angiogenesis and cholesterol and treating dyslipidemia, atherosclerosis, cancer, and inflammatory conditions (US20160115211A1); (2) compositions and methods for increasing lymphangiogenesis (Attorney Docket Number 10063-057WO1); (3) compositions and methods for treating neovascularization and ischemic retinopathies by targeting angiogenesis and cholesterol transport; and (4) Methods and compositions for treatment of aberrant hematopoiesis (Attorney Docket No. 16315-P002WO). JPC reports consulting fees from JanOne. The other authors declare that they have no conflict of interest.
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s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the Npro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔNpro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV Erns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems.

Introduction
The successful development and application of a chimeric marker vaccine in combination with a DIVA (differentiation of infected from vaccinated animals) test strategy has been approved for many economically important animal pathogens such as bovine herpes virus type 1 (BoHV-1), suid herpesvirus type 1 (SuHV-1, syn. Aujeszky's disease virus [ADV] or pseudorabies virus [PRV]) and Pestivirus C (syn. classical swine fever virus [CSFV]) [1][2][3]. The serological DIVA test strategy encompasses the implementation of differentiating serology tests (e.g., ELISAs) that clearly discriminate between antibodies acquired by field infection and vaccination. It has been pointed out that modern DIVA capable vaccines will improve disease eradication in comparison to standard vaccines, for example, by administration as an emergency vaccine in a pathogen-free country [4].
The genus Pestivirus of the Flaviviridae family encompasses a number of pathogen species of great economic importance [5] including CSFV and bovine viral diarrhea virus have already shown the heterologous compatibility of pestivirus proteins resulting in the feasibility to generate pestiviral chimeras and their application for different purposes, for example, characterization of new viruses or viral proteins [43], development of serological assays for seroprevalence studies [44] and the development of marker vaccines [3,45,46]. As an example, the Suvaxyn CSF Marker ® vaccine (Zoetis Belgium SA, Louvain-la-Neuveb, Belgium) that contains live BVDV, which has been modified to replace the E2 gene of BVDV with the corresponding gene of CSFV, was market authorized in 2015. Numerous vaccination-challenge trials confirmed the efficacy and the safety of this chimeric pestivirus vaccine against CSFV genotypes 1 and 2 [47].
Here, the construction of a BVDV-1 double marker vaccine that is based on the genetic background of the atypical BuPV expressing the heterologous E1 and E2 glycoproteins of BVDV-1 is described. The double marker principle, that is defined by the presence of BVDV-1 E2-specific antibodies and the concurrent absence of BVDV-1 E rns -and NS3specific antibodies upon immunization, and the protective capacity of the vaccination were investigated in a BVDV-1 vaccination-challenge trial in cattle.

Cells and Viruses
All cell lines used in the presented study were provided by the Collection of Cell Lines in Veterinary Medicine, CCLV, Friedrich-Loeffler-Institut (FLI), Greifswald-Insel Riems. The diploid bovine esophageal cell line KOP-R (RIE244) was used for electroporation of infectious RNA, virus rescue, growth kinetics and neutralization assays. Madin-Darby bovine kidney (MDBK) cells (RIE261) were used for virus titration and the IFN-incompetent MDBK41 cells (RIE728) were used for virus propagation of BuPV_∆N pro _E1E2 CP7.
The cytopathic BVDV-1b strain CP7 was isolated from a case of fatal mucosal disease [48]. SE5508, a ncp BVDV-1b strain, was isolated from a clinically healthy immunotolerant calf and used for the heterologous challenge infection and the BVDV neutralization assay.

Generation of BuPV_∆N pro _E1E2 CP7 Virus and Virus Recovery
The generation of the infectious full-length cDNA clone of BuPV (pA/BV) (GenBank accession number: NC_023176) has been described in detail in the publication of Dalmann et al. [28]. The construct BuPV_∆N pro _E1E2 CP7 was generated by the deletion of most of the coding genomic region of N pro (nucleotides [nt] 428-922, amino acids [aa]  and by substitution of the glycoproteins E1 and E2 encoding genomic region with E1 and E2 of BVDV-1b strain CP7 (GenBank accession number: BVU63479). The deletion and substitution were achieved. For this purpose, the genomic region of CP7_E1E2 was amplified and extended by overlapping sequences of BuPV using Phusion High-Fidelity Polymerase (New England Biolabs GmbH, Frankfurt am Main, Germany) according to the manufacturer's instruction. These amplification products served as megaprimers and were used to delete and substitute BuPV_E1E2 in the BuPV backbone by Fusion PCR methods [49].
Plasmids were amplified in Escherichia coli DH10B™ cells (Thermo Fisher Scientific, Darmstadt, Germany). Using the Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany), the plasmid DNA was purified. The correct sequence was confirmed by Sanger termination cycle sequencing using a Big Dye Terminator Mix (Applied Biosystems, Thermo Fisher Scientific). Primer sequences and protocols are available on request. Sequencing products were purified with a NucleoSEQ Spin kit (Macherey-Nagel, Düren, Germany) and sequenced on an ABI 3500 Genetic Analyzer (Applied Biosystems). To recover virus, the cDNA construct was linearized by SmaI digest and in vitro transcribed with the T7 RiboMax Large Scale RNA Production System (Promega, Walldorf, Germany). The in vitro transcribed RNA was electroporated into SK6 cells [50]. The supernatant of electroporated cells was harvested 72 h later and was tested for the presence of infectious virus. Recovered virus was serially passaged on MDBK41 cells.

Growth Kinetics
To determine the growth kinetics of the vaccine candidate BuPV_∆N pro _E1E2 CP7 in comparison to wild type BuPV in vitro, KOP-R cells were inoculated at a multiplicity of infection (m.o.i.) of 1. Supernatants were collected at 0, 24, 48 and 72 h post-infection (p.i.) and titrated on MDBK cells. The titers were calculated and expressed as 50% tissue culture infectious dose per ml (TCID 50 /mL). Growth kinetics were repeated twice.

Ethics, Animals and Experimental Design
The animal study was conducted in compliance with governmental animal welfare guidelines and regulations and was approved by the State Office of Agriculture, Food safety and Fishery in Mecklenburg-Western Pomerania under the registration number 7221.3-1-034/15.
Fifteen conventionally reared female Holstein-Friesian calves obtained from local farms were included in the vaccination and challenge study after being tested negative for the presence of BVDV-and BoHV-1-specific antibodies. The animals were allocated into three different groups for the trial with a group size of five animals each. The animals were housed in the biosafety level-2 facility of the FLI. Their age ranged from four to seven months with an equal distribution in each group. Animals were vaccinated once or twice at an interval of four weeks. Four weeks after the final immunization, both groups as well as the non-vaccinated control group were intranasally (i.n.) infected with the BVDV-1 challenge strain SE5508. For BVDV-1 vaccination, the animals were either vaccinated intramuscularly (i.m) once with live virus BuPV_∆N pro _E1E2 CP7 adjusted to 5 × 10 5 TCID 50 or in two vaccination cycles with an inactivated vaccine at day 0 (first vaccination, V1) and at day 28 (second vaccination, V2) with 1 mL containing an equivalent of 5 × 10 5 TCID 50 viral particles adjuvanted with 20% polygen. Back titrations verified a dose of 1 × 10 6 TCID 50 of the live vaccine and 2.8 × 10 5 TCID 50 for each vaccination with the binary ethylenimine (BEI)-inactivated vaccine (titration before inactivation). For the BEI-inactivation, 210 mg 2-bromoethylammoniumbromide (BEA [Merck, Darmstadt, Germany]) was solved in 9 mL aqua dest. in the presence of 1 mL 2N NaOH and heated at 37 • C for 1 h to allow cyclisation of BEA to BEI. The final concentration of BEI was adjusted to 4 mM. Inactivation of the virus preparation was accomplished through a continuous inverting at 37 • C for 24 h. Sodium thiosulphate was finally added in a concentration of 4% to inactivate the excess of BEI. Complete inactivation was verified by inoculation on KOP-R cells. Two consecutive blind passages of supernatants were performed 3 days after inoculation.
For the i.n. BVDV-1 challenge infection at day 56, 2 mL (1 mL per nostril) with 2 × 10 7 TCID 50 of the SE5508 strain were administered with a diffusor. Data of the control group have already been published in Koethe et al., 2020 [46].

Clinical Evaluation
Rectal body temperatures were measured daily and clinical examinations were carried out daily during the course of the trial. The calves were examined for adverse reactions immediately after vaccination and challenge infection. Signs of clinical disease, focusing on respiratory and digestive disorders, and the general health status (depression, feed intake and behavior) were controlled.

Nasal Swabs and EDTA-Blood Samples
Over the period of 10 days after the respective first vaccination and for 14 days after the challenge infection, nasal swabs and EDTA-blood samples were collected daily. To monitor the serological response, serum samples were taken weekly. The samples were subjected to hematological, virological and serological analyses.

Hematological Investigations
Blood samples were taken by jugular venipuncture and were collected in potassium EDTA-coated sterile blood collection tubes (Monovette) (Sarstedt, Nürnbrecht, Germany). To determine the differential blood cell counts, an Abbott CellDyn 3700 analyzer (Abbott, Wiesbaden, Germany) was used.

Virus Isolation
The isolation of virus using cell culture was essentially performed as described by Zemke et al., 2010 [51]. Virus isolation was conducted on nasal swabs and on concentrated and purified Peripheral Blood Mononuclear Cells (PBMC) after ammonia lysis of red blood cells. Monolayers of KOP-R cells were inoculated in four replicates per animal and specimen. Virus replication was confirmed by indirect immunofluorescence staining of the viral NS3 protein using mab mix WB103/105 (c.c.pro, Oberdorla, Germany) and an Alexa488 goat anti-mouse IgG conjugate (Thermo Fisher Scientific). Evaluation was carried out using the fluorescence microscope Nikon Eclipse Ti (Nikon, Düsseldorf, Germany). One blind passage of the supernatants was performed after 3 to 4 days of inoculation.

Serology
Sterile blood collection tubes (Monovette) with clot activator were used for weekly intravenous serum sampling. After centrifugation at 3,000 rpm, serum aliquots were stored at −20 • C. All sera were incubated at 56 • C for 30 min prior to serological testing.
To perform serological investigations, the following ELISAs were used: ID Screen ® BVD p80 antibody competition ELISA (IDVet, Grabels, France), Monoscreen Ab ELISA BVDV (E0) (Bio-X Diagnostics S.A., Rochefort, Belgium) and IDEXX BVDV Total Ab ELISA (IDEXX, Liebefeld-Bern, Switzerland). To process the samples, the manufacturer's instructions were followed. For the ID Screen ® BVD p80 antibody competition ELISA, the relative blocking values (%S/N) were calculated and samples with %S/N ≤ 40% were considered as positive, between 40% and 50% as suspicious and ≥ 50% as negative according to the recommendation of the manufacturer. Sera from all animals were tested in a standard neutralization assay (NA) against homo-and heterologous BVDV-1 (CP7 and SE5508) and BuPV. The NA was conducted as described by Zemke et al., 2010 [51]. Titers were expressed as reciprocal of the highest dilution that caused 50% neutralization per ml (ND 50 /mL).

Statistics
To compare whether the means of the cumulated scoring values are significantly different between the three groups (unvaccinated control group, BuPV_∆N pro _E1E2 CP7 live 1× and BuPV_∆N pro _E1E2 CP7 inactivated 2×) a one-way analysis of variance (oneway ANOVA) was performed based on the results for virus isolation from nasal swabs as well as for virus isolation from leukocytes. The cumulated scoring values were normally distributed assessed by the Shapiro-Wilk test. Homogeneity of variances was determined using Bartlett's test and no outliers were observed. Thus, the assumptions for the one-way ANOVA were fulfilled. Dunnett's test was used as post-hoc test to determine which group means were significantly different from the unvaccinated control group. For all statistical tests, a significance level of 5% was assumed. Statistical analysis was performed in R (version 4.0.3) [52].

Construction and Characterization of BuPV_∆N pro _E1E2 CP7
BuPV_∆N pro _E1E2 CP7 was constructed synthetically on the genetic background of BuPV. The E1 and E2 glycoprotein sequences of BuPV were substituted with the E1 and E2 encoding genomic region of BVDV-1b CP7 strain to obtain a BVDV-1 vaccine candidate. In line with a comprehensive vaccine strategy and the published BVDV-1 and BVDV-2 chimeric marker candidate vaccines [46], the viral IFN antagonist N pro was deleted with retaining only the first 12 aa to ensure a correct processing of the polyprotein in combination with a stable attenuation of the vaccine candidate. To discriminate vaccinated from BVDVfield infected animals, the genetic sequences of the glycoproteins E rns and the nonstructural protein NS3 of BuPV were retained ( Figure 1).

Construction and Characterization of BuPV_ΔN pro _E1E2 CP7
BuPV_ΔN pro _E1E2 CP7 was constructed synthetically on the genetic background of BuPV. The E1 and E2 glycoprotein sequences of BuPV were substituted with the E1 and E2 encoding genomic region of BVDV-1b CP7 strain to obtain a BVDV-1 vaccine candidate. In line with a comprehensive vaccine strategy and the published BVDV-1 and BVDV-2 chimeric marker candidate vaccines [46], the viral IFN antagonist N pro was deleted with retaining only the first 12 aa to ensure a correct processing of the polyprotein in combination with a stable attenuation of the vaccine candidate. To discriminate vaccinated from BVDV-field infected animals, the genetic sequences of the glycoproteins E rns and the nonstructural protein NS3 of BuPV were retained ( Figure 1). After RNA transfection, the chimeric virus was rescued on KOP-R cells and passaged on MDBK41 cells. The co-expression of BuPV E rns and BVDV-1 E2 were confirmed in infected cells by immunofluorescence staining using appropriate specific antibodies (data not shown).
Growth kinetics of BuPV_ΔN pro _E1E2 CP7 were performed with an m.o.i. of 1. Delayed virus growth with reduced final titers for BuPV_ΔN pro _E1E2 CP7 on bovine cells in comparison to the parental BuPV was detected ( Figure S1). Furthermore, the genomic and sequence integrity of the rescued virus and the genetic stability of the viral sequence after 15 serial passages in cell culture could be confirmed by Sanger sequencing and highthroughput sequencing approaches (data on request).

Vaccination of Cattle by Administration of BuPV_ΔN pro _E1E2 CP7
The potential of BuPV_ΔN pro _E1E2 CP7 as a new BVDV marker vaccine candidate was analyzed by testing two different immunization strategies. The virus was administered i.m. once as a live vaccine (one shot: 1 × 10 6 TCID50) or twice as a BEIinactivated, polygen adjuvated virus preparation (two shots, each corresponding to 2.8 × 10 5 TCID50). No virus could be re-isolated from nasal swabs or purified leukocytes after vaccination in both groups. The rectal body temperatures and the leukocyte counts remained stable and no adverse reactions or clinical signs like nasal discharge or diarrhea were observed in both groups. The quantity of BVDV-E2 specific antibodies produced upon vaccination was specified in a serum neutralization assay against the E1/E2homologous CP7, the heterologous challenge virus BVDV-1 SE5508 strain and the parental BuPV. After RNA transfection, the chimeric virus was rescued on KOP-R cells and passaged on MDBK41 cells. The co-expression of BuPV E rns and BVDV-1 E2 were confirmed in infected cells by immunofluorescence staining using appropriate specific antibodies (data not shown).
Growth kinetics of BuPV_∆N pro _E1E2 CP7 were performed with an m.o.i. of 1. Delayed virus growth with reduced final titers for BuPV_∆N pro _E1E2 CP7 on bovine cells in comparison to the parental BuPV was detected ( Figure S1). Furthermore, the genomic and sequence integrity of the rescued virus and the genetic stability of the viral sequence after 15 serial passages in cell culture could be confirmed by Sanger sequencing and high-throughput sequencing approaches (data on request).

Vaccination of Cattle by Administration of BuPV_∆N pro _E1E2 CP7
The potential of BuPV_∆N pro _E1E2 CP7 as a new BVDV marker vaccine candidate was analyzed by testing two different immunization strategies. The virus was administered i.m. once as a live vaccine (one shot: 1 × 10 6 TCID 50 ) or twice as a BEI-inactivated, polygen adjuvated virus preparation (two shots, each corresponding to 2.8 × 10 5 TCID 50 ). No virus could be re-isolated from nasal swabs or purified leukocytes after vaccination in both groups. The rectal body temperatures and the leukocyte counts remained stable and no adverse reactions or clinical signs like nasal discharge or diarrhea were observed in both groups. The quantity of BVDV-E2 specific antibodies produced upon vaccination was specified in a serum neutralization assay against the E1/E2-homologous CP7, the heterologous challenge virus BVDV-1 SE5508 strain and the parental BuPV.
Initial vaccination with the inactivated BuPV_∆N pro _E1E2 CP7 resulted in the detection of BVDV-1b-specific neutralizing antibodies with titers of up to 1:44 against the homologous CP7 strain. The second vaccination clearly boosted the antibody levels leading to titers of up to 1:20,000. In comparison, the neutralizing titers against BuPV reached levels of only up to 1:46 after the second vaccination. Vaccination with the modified live vaccine candidate resulted in BVDV-1b neutralizing antibody titers of up to 1:98, and 1:4 against BuPV. No BVDV NS3-specific antibodies were detected by ELISA in the inactivated or in the live BuPV_∆N pro _E1E2 CP7-vaccinated animals fulfilling the requirements for a marker vaccine. All animals of the control group remained negative in all applied tests (Figure 2A-C and Figure 3B). reached levels of only up to 1:46 after the second vaccination. Vaccination with the modified live vaccine candidate resulted in BVDV-1b neutralizing antibody titers of up to 1:98, and 1:4 against BuPV. No BVDV NS3-specific antibodies were detected by ELISA in the inactivated or in the live BuPV_ΔN pro _E1E2 CP7-vaccinated animals fulfilling the requirements for a marker vaccine. All animals of the control group remained negative in all applied tests (Figures 2A-C and 3B).  modified live vaccine candidate resulted in BVDV-1b neutralizing antibody titers of up to 1:98, and 1:4 against BuPV. No BVDV NS3-specific antibodies were detected by ELISA in the inactivated or in the live BuPV_ΔN pro _E1E2 CP7-vaccinated animals fulfilling the requirements for a marker vaccine. All animals of the control group remained negative in all applied tests (Figures 2A-C and 3B).    CP7-vaccinated animals showed only a mild elevation of the body temperatures above 39 • C over a period of six days starting at day 4 p.chall. with mean body temperatures of 39.08-39.24 • C compared to a pre-challenge mean of 38.75 • C. Besides the elevated rectal body temperature, only minor additional clinical effects were in general observed after the SE5508 challenge infection. The control animals showed a slightly reduced feed intake and a marginally reduced general condition for one day (7 or 8 d p.chall.). Diarrhea was not observed and respiratory distress did not exceed the pre-challenge observations. The vaccinated animals of both groups remained completely asymptomatic.  Nasal shedding of infectious virus and a transient viremia may occur during an acute infection with BVDV-1. Therefore, virus isolation from nasal swabs and from blood was conducted daily starting on day 1 p.chall. until day 14 p.chall. In the control animals, long and pronounced nasal shedding of challenge virus and viremia were found. Virus was secreted from the nose of every animal of this group on five to eight consecutive days, whereas virus was isolated from nasal swabs on two to five coherent days in the live vaccinated and only on a maximum of two coherent days in the group that received the inactivated vaccine. In addition, virus was isolated over a period of 11 days from the leukocytes in the control group. In contrast, only on two or three consecutive days virus was isolated from the leukocytes of a few animals of the vaccinated groups, respectively (Table 1). Clearly, the two shots vaccination with the inactivated BuPV_ΔN pro _E1E2 CP7 protected one out of five animals completely from nasal shedding and two out of five from viremia. When shedding and viremia occurred, both events were only detectable on a single or maximum two days. In summary, both the nasal shedding of infectious virus In addition to the symptoms observed during clinical examination, all control animals showed a severe leukopenia. The triphasic decrease (3, 6 and 12 d p.chall.) in leukocyte counts showed a maximum mean level in reduction of 54% on day 3 p.chall. (Figure 4B). The live BuPV_∆N pro _E1E2 CP7 vaccinated animals also showed a leukopenia with a triphasic decrease (3, 5 and 11 d p.chall.) and revealed a comparable mean level in leukocyte counts reduction of 52%. The leukocyte counts normalized in the control and live vaccinated group on day 8 and day 7 p.chall, respectively. In contrast to the unvaccinated control group, a short term (one day) rebound in leukocyte counts was detected for the live vaccinated animals as early as by day 8 p.chall. Animals of the inactivated BuPV_∆N pro _E1E2 CP7 vaccinated group showed a biphasic decrease in leukocyte counts (6 and 12 d p.chall.). The Vaccines 2022, 10, 88 9 of 16 maximum mean level in leukocyte counts reduction was 41% on day 6 p.chall. Moreover, a rebound in leukocyte counts was shown for the inactivated BuPV_∆N pro _E1E2 CP7 vaccinated group on day 9 p.chall. with restoration of the pre-challenge values (Figure 4).
Nasal shedding of infectious virus and a transient viremia may occur during an acute infection with BVDV-1. Therefore, virus isolation from nasal swabs and from blood was conducted daily starting on day 1 p.chall. until day 14 p.chall. In the control animals, long and pronounced nasal shedding of challenge virus and viremia were found. Virus was secreted from the nose of every animal of this group on five to eight consecutive days, whereas virus was isolated from nasal swabs on two to five coherent days in the live vaccinated and only on a maximum of two coherent days in the group that received the inactivated vaccine. In addition, virus was isolated over a period of 11 days from the leukocytes in the control group. In contrast, only on two or three consecutive days virus was isolated from the leukocytes of a few animals of the vaccinated groups, respectively (Table 1). Clearly, the two shots vaccination with the inactivated BuPV_∆N pro _E1E2 CP7 protected one out of five animals completely from nasal shedding and two out of five from viremia. When shedding and viremia occurred, both events were only detectable on a single or maximum two days. In summary, both the nasal shedding of infectious virus and the viremia were significantly reduced in both vaccinated groups in comparison to the unvaccinated control group (p < 0.001).

Marker Serology
The challenge infection boosted the presence of BVDV E2-specific antibodies, as determined by a commercial E2-based ELISA ( Figure 3A). In the control group, BVDV E2-specific antibodies were first detected two weeks p.chall. In line, the presence of NS3specific antibodies in the sera of unvaccinated control and vaccinated animals was first observed at two weeks p.chall. confirming the absence of BVDV NS3-specific antibodies after vaccination and the applicability of the ELISA to discriminate BVDV NS3 and BuPV NS3 antibodies. Furthermore, the commercially available competitive BVDV E0 (E rns ) antibody ELISA could also discriminate between vaccinated and challenge-infected animals ( Figure 3A-C). However, certain levels of cross reactivity against BuPV E rns were observed upon vaccination.

Discussion
Vaccines assisted for decades in control and eradication programs for economically important animal pathogens such as BoHV-1 or SuHV-1. For BVDV, eradication programs have been implemented in several countries worldwide and the availability of a DIVAcompatible vaccine could contribute significantly in regions of high virus prevalence to drive back field virus prevalence [20]. Furthermore, a marker vaccine could be applied as an emergency vaccine in BVDV-free countries, where control and eradication are major objectives when the disease is introduced and where serological methods are essential to demonstrate freedom of disease once the outbreak is under control. However, none of the currently commercially available BVDV vaccines allows the establishment of a serological DIVA strategy and the differentiation of vaccinated from field-infected animals by serological methods. They only serve as genetic DIVAs as the vaccine virus can be differentiated using RT-PCR and/or sequencing methods [53].
The overall aim of this study was to develop a marker vaccine prototype for BVDV-1 using a novel approach based on a synthetic backbone strategy of a related pestivirus with chimeric protein expression that has successfully been employed previously [46]. The synthetic backbone strategy allows the rapid exchange, deletion or insertion of sequences or genes. The modified novel approach encompassed the utilization of the atypical pestivirus BuPV as the genetic background because of its spatial restriction to a single pig farm complex in Australia [25], the presence of major antigenic difference between BuPV and BVDV-1 [29], and the compatibility of BuPV and BVDV envelope proteins [30]. To allow attenuation of the vaccine candidate, the gene of the IFN antagonist N pro protein was partially deleted. To test the safety and efficacy of the novel potential marker vaccine candidate BuPV_∆N pro _E1E2 CP7, calves were experimentally vaccinated and subsequently challenged with a respective heterologous BVDV-1 field strain. In accordance with the prospective marker principle, immunization of cattle with the vaccine candidate should induce seroconversion of BVDV-1 E2-specific antibodies while BVDV-specific NS3-and E rns -antibodies should only be detectable after BVDV-1 challenge infection. This feature makes the novel vaccine candidate to our knowledge the first "double marker vaccine" against BVDV.
After virus recovery, virus growth kinetics revealed a 100-1,000-fold less efficient growth of BuPV_∆N pro _E1E2 CP7 in comparison to the wildtype BuPV. The observed in vitro attenuation of BuPV_∆N pro _E1E2 CP7 on bovine cells is most likely due to a combination of two factors-the expression of a heterologous heterodimer of E1 and E2 and the absence of the immunomodulatory N pro protein. A recent study characterizing a CSF mutant virus has already shown that the deletion of N pro and the absence of the immunosuppressive function led to an attenuation in vivo [54]. Despite that, most of the N pro encoding sequences were deleted in our construct in order to further increase the safety for pregnant dams, a concept already applied for the commercial BVDV vaccine Bovela ® (Boehringer Ingelheim Vetmedica GmbH, Ingelheim/Rhein, Germany) and the previously described marker vaccine candidates BVDV-1b_synCP7_∆N pro _E rns Bungo and BVDV-1b_synCP7_∆N pro _E rns Bungo_E1E2CS [46]. Regarding the factor of the heterologous heterodimer of E1 and E2 that might negatively influence the in vitro growth of BuPV_∆N pro _E1E2 CP7, we selected the combined replacement of both proteins because of the high genetic diversity between BuPV and BVDV-1 and as the formation of E1-E2 heterodimers is essential for virus entry [55]. The overall amino acid homology of the polyprotein between BVDV-1 (strain CP7) and BuPV is 52.6%, whereas the amino acid sequence identities within the E1 of BVDV-1 and BuPV only amount to 44.9% and within the E2 to 41.2%. Previous complementation studies showed that only the homologous co-expression of BuPV-E1 and E2 in a BVDV backbone resulted in recovery of infectious virus [30]. The heterodimer E1E2 is stabilized through the formation of disulfide bonds [33]. The glycoprotein E1 of BVDV_CP7 and BuPV differ in the number of cysteine residues (BVDV_CP7 E1: 6 residues vs. BuPV E1: 3 residues). Therefore, a single substitution of either E1 or E2 in chimeras may hamper the heterodimer formation. This is in contrast to the commercially available Suvaxyn CSF Marker ® vaccine (CP7_E2alf) where only the genetic sequence of the E2 protein of BVDV-1 was substituted with CSFV E2alf (strain Alfort/187). The E2 of CSFV and BVDV-1 share 58.3% amino acid identities and the structure of BVDV E2 is similar to that of CSFV, which might explain the interchangeability [56]. Resolving and comparing the crystal structure of BuPV E2 with BVDV and CSFV would help to explain whether a functional interchangeability of the glycoproteins E1 and E2 is only a matter of structure.
The utilization of BuPV as the backbone for the marker vaccine may rise safety concerns as the vaccination of cattle might lead to spillover infection of the vaccine candidate BuPV_∆N pro _E1E2 CP7 to other susceptible species. Previous studies have already determined the susceptibility and the clinical outcome of BuPV infection in various clovenhoofed species and interspecies transmission from chronically infected pigs. Cattle and sheep appeared to be less susceptible to infection in comparison to pigs, and BuPV was failing to establish immunotolerance and persistence in the bovine fetus. The authors of the study argued that the transmission of BuPV is likely to be inefficient in ruminants and will probably not sustain in these species [57]. Moreover, no nasal shedding of the vaccine candidate occurred after immunization in the present study minimizing the potential for any spillover infections to contact cattle or pigs. The deletion of most of the encoding sequencing of the IFN antagonist N pro in the BVDV-1 CP7 genetic background reduced virus titer and induced a strong IFN response in cell culture [46]. The abrogation of its immunosuppressive function through the deletion of N pro in CSF mutant viruses led to an attenuation in vivo [54]. Therefore, the deletion of N pro in the presented double marker vaccine is an important factor of safety. Nevertheless, safety aspects of the BuPV_∆N pro _E1E2 CP7 vaccine candidate need to be further investigated by vaccination of pregnant cows and in utero infection of growing fetuses to test for any possible induction of persistence or abortion and further determination of the susceptibility of pigs.
The application of numerous inactivated or killed and modified-live vaccines (MLV) against BVD have been described before. MLVs are considered to be efficacious and induce a strong humoral and cellular immunity and provide a solid fetal protection as immunogenic proteins are amplified through viral replication. However, there are reports about adverse effects that can be attributed to the immunosuppressive effects of live vaccines and safety issues with vaccination in pregnancy [18,58]. The development of inactivated or killed vaccines (KV) were prompted because of the safety concern for MLV. Furthermore, the detection of live vaccine viruses in tissue samples of newborn calves whose mothers were immunized within the first trimester of gestation [19,53] and the interference with the BVD ear notch diagnostics underline the need for a further safe and efficient vaccine to support BVDV control programs. The application of KV induces immune responses that are limited basically to antibody production towards the structural proteins in the absence of a profound induction of CD8+ cytotoxic T cells (CTL) [59]. However, the control and clearance of BVDV infection are facilitated by both antibody and CTL responses preventing clinical disease and fetal infection [60]. The higher efficacy of MLVs in comparison to KV against BVD can be explained by (i) production of antibodies against the structural E2 and nonstructural protein NS3, and (ii) the induction of BVDV-specific CD8 + T cells. A recent study demonstrated the presence of novel potent IFN-γ inducing CD8 + T cell epitopes in the structural BVDV antigens (Ag) of E rns , E1 and E2 and in the non-structural Ags of N pro , NS2-3, NS4A-B and NS5A-B, and showed that these epitopes are highly conserved among more than 200 strains from BVDV-1 and -2 genotypes [61]. In the light of these observations, the usage of BuPV as a heterologous genetic backbone for the development of a live BVD vaccine may reduce the efficacy because only the properties of structural BVD E1 and E2 Ag as CD8 + T cell epitopes are retained. In contrast, a study has shown that the application of a single shot of Suvaxyn CSF Marker ® vaccine, the licensed CP7_E2alf MLV, resulted in a significant reduction of proinflammatory cytokine levels upon challenge, especially TNF-α and IL-6, and suggested an important role of cell-mediated immunity in both short-and long-term protection against CSFV [62]. Whether the application of live BuPV_∆N pro _E1E2 CP7 stimulates the cell-mediated immunity and reduces proinflammatory cytokine levels upon BVDV challenge would be interesting to study.
The advantage of inactivated vaccines is the risk-free application at any age or stage of pregnancy. Furthermore, an inactivated vaccine is highly favored to be applied as an emergency vaccine in BVD-free countries as an accidental dissemination is impeded in contrast to live vaccines. On the downside, these vaccines are less efficient in comparison to MLV and at least one booster vaccination is required to achieve full protection. To balance the safety concerns and the efficacy, the chimeric vaccine candidate BuPV_∆N pro _E1E2 CP7 was tested as inactivated preparation applied twice in addition to the application as a live vaccine with only one shot. In comparison to the single live vaccination, the inactivated booster vaccination even protected from moderate rectal body temperature elevation (39.94 vs. 39.24 • C) and delayed the leukopenia (3 vs. 6d p.i.) and their severity (48.8 vs. 58.6% mean leukocyte count). Nevertheless, both vaccination regimes, that is, the one shot live and the two shots of the inactivated chimeric vaccine BuPV_∆N pro _E1E2 CP7, provided clinical protection against a virulent BVDV-1 challenge infection in the bovine host. Vaccination was efficient to confer a significant reduction of the nasal viral shedding and systemic replication shown by cell bound viremia in extent and duration. After double vaccination infectious challenge virus could not be recovered from blood samples of two out of five animals, however an actual sterile immunity was not induced as anti-NS3 and anti-E rns specific antibodies were detectable in all vaccinated animals following challenge infection.
Twenty-eight days after the first vaccination, both groups developed neutralizing antibodies against the heterologous challenge strain. Most importantly, no antibodies specific for BVDV-1 NS3 or E rns were found upon vaccination, facilitating the differentiation of immunized cattle from field-virus infected animals using a double marker principle which would be especially suitable under field conditions. Hence, the vaccination success could easily be monitored by using different commercially available and field-proven ELISA tests that can differentiate BuPV E rns from BVDV E rns and BuPV NS3 from BVDV NS3-specific antibodies, respectively, without interference of any cross-reactions. The challenge infection induced BVDV E rns -and BVDV NS3-specific antibodies in the controls and in the vaccinated groups, confirming the applicability of the double marker vaccine strategy to discriminate vaccinated from BVDV-1-infected animals. Furthermore, local and systemic field virus replication is significantly reduced which allows us to expect efficient suppression of field virus spread and prevention of fetal infection.

Conclusions
The application of the pestivirus BuPV as the genetic backbone for the design of a BVDV-1 double marker vaccine and the substitution of BuPV_E1E2 with BVDV-1_E1E2 resulted in the induction of a robust immune response that protected from clinical disease after BVDV-1 challenge infection. A clear serological discrimination of vaccinated from infected cattle was confirmed through the detection of BVDV-1 E2 in the absence of BVDV NS3 and BVDV E rns -specific antibodies. Independent of the applied vaccination regime (one shot live or two shots inactivated), BuPV_∆N pro _E1E2 CP7 provided protection from fever, and the animals experienced a shorter period of leukopenia and a subsequent rebound of leukocyte numbers. Most importantly, viremia and nasal shedding were significantly reduced in extent and in duration in the vaccines compared to the control group. However, sterile immunity could not be achieved with this type of vaccine. A first orientation study about potencies and risks of the live vaccine virus was conducted to gain basic insights into characteristics and safety aspects. Overall, double immunization with the inactivated marker virus was more efficient to reduce viremia and virus shedding in comparison to the one-shot live vaccine and might be preferred in future applications. Furthermore, the application of the inactivated double marker vaccine is advantageous in countries with control programs like Germany because vaccinated animals remain seronegative in the standard commercial NS3-specific ELISA.