There were no significant differences in 25(OH)D2concentrations amongst the 3 genotypes. Lleyn ewes had significantly higher 25(OH)D3 and 25(OH)D concentrations than both Scottish Blackface ewe genotypes, whereas these vitamin D parameters did not differ significantly between the UBF and IBF ewes. Concentrations of 25(OH)D3 and 25(OH)D were positively associated with subsequent birth weights of singleton and of twin lamb litters. No significant associations between vitamin D status and number of lambs born or weaned per ewe were found. This study demonstrates that concentrations of cutaneously-derived 25(OH)D3, but not of orally consumed 25(OH)D2, differed between breeds. The positive association between ewe vitamin D status and offspring birth weight highlights the need for further investigations.There is a growing interest in the influence of vitamin D on ovine non-skeletal health. The aim of this study was to explore the relationship between pre-mating vitamin D status, as assessed by serum concentrations of 25-Hydroxyvitamin D [25(OH)D; comprising D Vitamin D2and its precursor can be found in fungal resources, such as wild mushroom (e.g. Chantarellus tubaeformis)1, whereas the vitamin D3 content of certain animal species, such as wild salmon, is high2. During exposure to ultraviolet B radiation , 7-Dehydrocholesterol in the skin is converted to previtamin D3 and then vitamin D3. After vitamin D enters the systemic circulation, it is hydroxylated in the liver to the main circulating form, 25-Hydroxyvitamin D (25(OH)D). This metabolite is further hydroxylated in the kidneys to the biologically active form, 1,25-Dihydroxyvitamin D 2D)3. Compared to 1α,25-(OH)2D, the concentration of 25(OH)D is a more reliable indicator for determining vitamin D status, due to its longer half-life, a higher serum concentration and the fact that it is less tightly regulated by parathyroid hormone5.The two forms of vitamin D, namely D6. The quality and the quantity of ultraviolet B radiation are affected by latitude and season, as when the sun is low in the sky, more ultraviolet B radiation is scattered and absorbed when it travels through the ozone layer, compared to when the sun is directly overhead7. In regions where the latitudes are above 39°N, such as the UK (from 49 to 60°N), the low level of ultraviolet B radiation results in no previtamin D3 being synthesized from 7-Dehydrocholesterol in human skin during exposure to sunlight, from October to March8. This could also lead to low vitamin D status in sheep farmed in such high latitude locations, although literature quantifying optimal ultraviolet radiation levels for sheep is difficult to find.Endogenous photobiosynthesis of vitamin D depends on exposure to sunlight which contains sufficient ultraviolet B radiation3. However, in the last 2 decades, numerous studies have reported that vitamin D deficiency is associated with many non-skeletal health problems, such as autoimmune diseases, hypertension and cancer10. The presence of vitamin Dreceptors in the reproductive tract of women11 and females of other species, such as sheep12, goat13, mouse15 and rat16, and in ovine male reproductive tracts18 indicates that vitamin D may influence reproductive performance. Several human studies have found that vitamin D deficiency before and during pregnancy is associated with reduced reproductive success and increased risk of the newborn being small for gestational age, lighter in weight at birth, or having reduced head circumference22. A recent ovine study suggests a role for vitamin D3 in spermatogenesis18.The principal function of vitamin D is the maintenance of skeletal health through regulating the processes of intestinal absorption and renal excretion of calcium and phosphorus, bone formation and mineral mobilization23, whereas Lleyn sheep are native to Wales. We investigated the hypothesis that there is a breed-dependent effect on vitamin D status of sheep in North West Scotland (56°N) and that there is a positive relationship between ewe reproductive performance and vitamin D status. To investigate this hypothesis, serum 25(OH)D2 and 25(OH)D3 concentration of three populations of ewes were determined using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The relationship between vitamin D status of contrasting genotypes, all in the same flock, and breeding outcomes was investigated.The Scottish Blackface sheep Fig.  is a har24. The study flock comprised approximately 200 genetically unimproved Scottish Blackface (UBF) ewes, 200 genetically improved Scottish Blackface (IBF) ewes and 200 Lleyn ewes. The UBF ewes had been selected in each generation to remain close to the average genetic merit in the flock before selection commenced in 1998, while IBF ewes were a lineage progressively selected for superior genetic merit in both ewe and lamb traits25. The Lleyn ewes had been selected for high genetic merit since 2010 using the Carcass+ Index  as part of the Signet Sheepbreeder performance recording service (www.signetfbc.co.uk/sheepbreeder/).The experiment was conducted at Scotland’s Rural College (SRUC) Hill and Mountain Research Centre, Crianlarich, Scotland 26. Ewes in each system had shared the same pastures. From mating 2015, the flock was subject to two management systems, both of which used the PLF management approach previously developed26. Ewes were assigned to either a predominantly ‘Hill Grazing’ or ‘Park Grazing’ management system , the research flock was drawn equally from two ‘pre-study’ management system groups, one group having been managed conventionally (CON), and the other subjected to a Precision Livestock Farming (PLF) management protocol. Each system had different criteria for winter feeding, worming and culling27. The first feeding phase was from early January (6th/7th Jan 2016) to scanning (22nd/23rd Feb 2016), while the second feeding phase was from scanning to lambing  and pregnancy diagnosis, were assigned to one or other in each phase.Ewes were mated in single-sire mating groups. There were 4 single-sire mating groups per genetic line. The mating group size in 2015 ranged from 44 to 50 ewes in each single-sire group. From late-November, ewes were joined with rams selected from their own genotype category for two reproductive cycles of 17 days. Ewes were ultrasound pregnancy-scanned to determine pregnancy status and foetal numbers in mid-February. Two supplementary feeding levels  and at weaning (mid-August).Data were collected for all ewes in the flock and included production (e.g. ewe weight and CS), and reproduction (e.g. pregnancy and lambing) records, as well as individual and group health treatments. Ewes were gathered at pre-mating, mid-pregnancy scanning and pre-lambing. At these handling events, ewes were weighed, and condition scored using a 5 point scoring systemAll experiments had local ethical approval and were conducted in accordance with UK legislation. The experimental protocols involving animals were approved by the SRUC Animal Welfare and Ethical Review Body.In mid-November 2015, during pre-mating handling, blood samples were taken via jugular venepuncture into 6 ml silicone coated red-top blood collection tubes . Ewes were between 1.5 and 6.5 years-old at the time of sampling (Supplementary Table 2 and 25(OH)D3 serum concentrations.Blood samples of 88 ewes per genotype were analysed to determine pre-mating ewes’ 25(OH)D2 stock solution  and 30 µl of 25(OH)D3 stock solution  into 1 ml artificial serum [50 mg bovine serum albumin  were dissolved in 1 ml of phosphate buffered saline (prepared in house)], then 1 in 2 serial dilution with artificial serum. The concentrations of calibration standards were 230.8, 115.4, 57.7, 28.9, 14.4, 7.2, 3.6 and 1.8 nmol/l for 25(OH)D2; and 356.6, 178.3, 89.2, 44.6, 22.3, 11.2, 5.6 and 2.8 nmol/l for 25(OH)D3. These calibration standards were used to generate standard curves for quantification of the concentration of 25(OH)D2 and 25(OH)D3 in sheep serum by HPLC-MS/MS analysis.Eight calibration standards were freshly prepared, by adding 20 µl of 25(OH)D3-25(OH)D2  and 2 µl of 23,24,25,26,27-13C5-25(OH)D3 , as internal standards. After adding 20 µl of 1 M NaOH, each serum sample or calibration standard was then protein precipitated by the addition of 200 µl of acetonitrile29. The supernatant of the serum sample or the calibration standard was purified by solid phase extraction using a Discovery DSC-18 SPE-96 Plate . Briefly, the plate was activated with 3 ml of ethyl acetate, 3 ml of methanol and 3 ml of distilled water. After addition of a mixture of supernatant (approximately 300 µl) from protein precipitation and 1 ml 0.4 M K2HPO4, the plate was washed with 3 ml of distilled water and 2 ml of 40% methanol sequentially and eluted with 1.5 ml of acetonitrile29. After evaporating to dryness, samples were derivatized by 2 additions of 25 µl of 0.1 mg/ml DMEQ-TAD ethyl]−3H-1,2,4-triazole-3,5(4 H)-dione; Abcam, UK) in ethyl acetate30. After evaporation to dryness, derivatized extracts were reconstituted in 25 µl of 60:40 (vol:vol) methanol and 0.1% formic acid:water for HPLC-MS/MS analysis.After serum samples (0.5 ml) were thawed at room temperature, 100 µl of each sample, or calibration standard, was spiked in a 1.5 ml microtube with 2 µl of 6,19,19-dThe HPLC-MS/MS analyses were conducted using an UltiMate 3000 HPLC system interfaced to an amaZon ETD tandem mass spectrometer . Chromatographic separations were achieved using an ACE UltraCore 2.5 SuperC18 column , maintained at 40 °C. Gradient elution was performed . The standard curve was generated based on the ratio of the peak area of the standard to that of the corresponding internal standard . Generalized Linear Models (GLM) were used to investigate the effects of multiple independent variables. Stepwise regression was used to determine the fixed effects and relevant covariates to include in the final model for each response variate. These models were then applied in Linear Mixed Models (LMM), alongside appropriate random effects, to investigate: i) differences in vitamin D concentrations between breeds and among genotypes; and ii) any associations between vitamin D status and ewe breeding outcomes, as well as ewe litter weight at birth, marking and weaning D3 and total 25(OH)D (addition of 25(OH)D2 and 25(OH)D3)] between breeds and amongst genotypes, the ewe breed/genotype, ewe age, pre-study system, ewe pre-mating weight, ewe pre-mating CS, sire of the ewe, number of lambs weaned and weaned litter weight in the last breeding cycle were the factors considered in the maximal GLM models.To investigate differences in vitamin D concentrations [25(OH)D2/25(OH)D3/25(OH)D concentration, ewe genotype, ewe age, management system (Hill grazing or Park grazing), pre-study system (CON or PLF), ewe pre-mating weight, ewe pre-mating CS, number of lambs weaned in the last breeding cycle (i.e. in 2015) and first winter feeding level were fitted in the maximal models.In the GLM models used to examine ewe breeding outcomes, the number of lambs born in 2016 and the number of lambs weaned that year, were tested, in turn, as response variates, whilst 25(OH)D2/25(OH)D3/25(OH)D concentration, ewe genotype, ewe age, management system, pre-study system, first winter feeding level, ewe pre-mating weight, ewe pre-mating CS and ram group were the factors considered in the maximal models in the GLM analyses. These analyses were conducted separately for single- and twin-bearing ewes.When analysing the association of vitamin D status with litter weight at birth, marking and weaning, 25(OH)DThe final fixed models, selected by stepwise regression, and random effects, fitted for each response variate, are shown in Supplementary Table 2 and 25(OH)D3 concentrations indicated that the latter differed between breeds. The unadjusted average serum concentrations of 25(OH)D2, 25(OH)D3 and total 25(OH)D for the UBF, IBF and Lleyn ewes are shown in Table Analysis of two hundred and sixty-four sheep serum samples for pre-mating ewes’ 25(OH)D3 concentration, or both 25(OH)D2 and 25(OH)D3 concentrations were beneath the lower quantification limit (7.2 and 5.6 nmol/l for 25(OH)D2 and 25(OH)D3, respectively) of the assay as applied in the current experiment.The results for five UBF ewes were excluded from the data reported here, as either their measured serum 25(OH)DEighteen ewes (6.9%) had 25(OH)D concentrations <25 nmol/l, 238 ewes (91.9%) had concentrations between 25 and 75 nmol/l, and 3 ewes (1.2%) had concentrations >75 nmol/l  or genotype (P = 0.263) after adjusting for age and ewe pre-mating weight .Serum 25(OH)D P = 0.26 after ad3 concentration between the Scottish Blackface and Lleyn ewes D3 than UBF and IBF ewes .When breed, ewe pre-mating weight, number of lambs weaned and weaned litter weight in the last breeding cycle were accounted for in the LMM, there was a significant difference in 25(OH)DSimilarly, Lleyn ewes had a significantly higher total 25(OH)D concentration than UBF and IBF ewes D3 and 25(OH)D concentrations in UBF, IBF and Lleyn ewes grouped according to subsequent litter sizes at birth and at weaning, are presented in Table 2 (P = 0.06), 25(OH)D3 (P = 0.57) and 25(OH)D (P = 0.534) concentrations amongst ewes that, in 2016, were barren or had singles or had twins.The unadjusted pre-mating 25(OH)D2 (P = 0.130) concentration was not associated with number of lambs weaned, when this trait was adjusted for genotype, age, management system, pre-study system, ewe pre-mating weight, ewe pre-mating CS, number of lambs weaned in the last breeding cycle and first winter feeding level. In addition, the LMM models with 25(OH)D3 and 25(OH)D concentrations, fitted with genotype, management system, pre-study system, ewe pre-mating weight, ewe pre-mating CS, number of lambs weaned in the last breeding cycle and first winter feeding level, showed that 25(OH)D3 (P = 0.287) and 25(OH)D (P = 0.978) status at pre-mating was not a significant determinant of the number of lambs weaned in August 2016.Pre-mating 25(OH)DAmong the 259 ewes in this study, 54 (21%) did not produce any lambs; the remainder produced one or two lambs. The overall lambing percentage was 117%, with 112% for UBF ewes, 120% for IBF ewes and 119% for Lleyn ewes.One hundred and six ewes gave birth to singletons. The associations between the pre-mating vitamin D status of ewes and their subsequent singletons’ weights at birth, marking and weaning were investigated, based on all those ewes, including 13 ewes (12%) that lost their lambs between birth and weaning.2 concentration and the birth weight of singletons D concentrations were positively associated with lamb birth weight of singletons D2 (P = 0.246), 25(OH)D3 (P = 0.408), or 25(OH)D (P = 0.196) concentrations proved to be a significant factor. When genotype, first winter feeding level and ewe pre-mating weight were considered in the LMM models, 25(OH)D2 (P = 0.542), 25(OH)D3 (P = 0.722) and 25(OH)D (P = 0.535) status had no effect on lamb weaning weight.For the singleton marking weights, when genotype, management system, first winter feeding level and ewe pre-mating weight were included in the LMM model, none of the 25(OH)DNinety-nine ewes gave birth to twins. The associations between the pre-mating vitamin D status of ewes and their subsequent twin litter weights at birth, marking and weaning were investigated, based on all 99 ewes, including 1 ewe (1%) having no lamb weaned and 17 (17%) ewes having one lamb weaned.2 concentration was not a significant determinant of litter birth weight D concentrations were positively associated with litter birth weight of twins D2 (P = 0.540), 25(OH)D3 (P = 0.265), and 25(OH)D (P = 0.267) ewe pre-mating serum concentrations showed a significant association with the litter marking weight of the ewes’ twin offspring. When genotype, management system, first winter feeding level, ewe pre-mating weight and ewe pre-mating CS were adjusted for in the LMM analyses, none of 25(OH)D2 (P = 0.418), 25(OH)D3 (P = 0.355), and 25(OH)D (P = 0.297) had a significant association with the ewes’ twin litter weaning weights.At marking, when genotype, management system, first winter feeding level, ewe pre-mating weight and ewe pre-mating CS were included in the LMM, none of 25(OH)D3, but not D2, status in a commercially operated sheep farm. Sheep obtain vitamin D2 from their diet8. In this study, the sheep shared the same pastures, and thus the fact that serum 25(OH)D2 concentration did not differ significantly amongst the 3 genotypes indicates that genotype was not a determinant of vitamin D2 status within the flock. The concentrations of 25(OH)D2 recorded for the UBF, IBF and Lleyn ewes . Differences could be due to the different blood sampling seasons and contrasting geographical regions with different vegetation in the pasture, which influence herbage vitamin D2 content33.This study has identified a clear relationship between genotype and vitamin D3 in this study indicate the extent to which vitamin D has been obtained via endogenous photobiosynthesis, since they did not receive any supplementary foodstuffs which contained vitamin D3, for at least 5 months prior to blood sampling. Lleyn ewes had significantly higher 25(OH)D3 concentrations, therefore the black heads and legs of the Blackface ewes might have compromised vitamin D3 photobiosynthesis in those black hair covered areas that are contiguous with dark skin pigmentation34. The efficiency of the conversion of 7-Dehydrocholesterol to previtamin D3 in Scottish Blackface ewes might be compromised because melanin in the pigmented skin competes with 7-Dehydrocholesterol for ultraviolet B photons35, although the face and limbs might not be the main sites for vitamin D photobiosynthesis in sheep8. All ewes were shorn in late June only, so had full fleece cover prior to blood sampling. Breed differences in terms of 25(OH)D3 could also mean that the Lleyn ewes have a superior capability to retain vitamin D3 as biologically inert forms such as tachysterol and lumisterol in their body than their BF counterparts35.The sheep serum concentrations of 25(OH)D37 indicate that vitamin D deficiency has occurred in geographical locations close to our study site. In regions above latitudes of 55°N, sunlight only provides sufficient intensity of ultraviolet radiation (290–315 nm) to induce a conversion of 7-Dehydrocholesterol to previtamin D338 between mid-March and mid-September39. In the Handel et al. (2016) study31, the serum 25(OH)D3 concentrations in the Soay sheep from St Kilda (57.8°N 8.6°W) were measured in samples taken in August, whereas in this study (56°N 4°W) sheep samples were taken in mid-November. This might explain why the serum 25(OH)D3 concentrations of sheep in this study were much lower than those of Soay sheep 39.Reports of rickets outbreaks in sheep in Scotland34, our results show that 25(OH)D2 contributed nearly half of the total 25(OH)D concentration for all three genotypes at the November sampling timepoint. Nevertheless, Lleyn ewes showed significantly higher total 25(OH)D concentrations than Scottish Blackface ewes. This observation of a breed effect was in agreement with Willems et al.40, albeit that different sheep breeds were investigated. The concentration of 25(OH)D has also been reported to be positively associated with skin thickness, which decreases with age41. Unlike Handel et al.31, the current study did not find an age-related effect on the vitamin D metabolites investigated, probably due to a narrower age range of ewes (from 1.5 to 6.5 years old at blood sampling time) investigated, whilst Handel et al.31 studied a more age-diverse (from 0.5 to 13 years old) population of feral sheep. The age range considered in this study more or less corresponds with and affirms the ‘Adult’ category (combining ages from 1 to 6 years) used by Handel et al.31 to distinguish that cohort of Soay ewes from same-breed lambs and geriatric sheep in their study.Although the vitamin D content in grass is limited, and it is argued that grazing animals obtain vitamin D mainly from endogenous photobiosynthesis42, ewe age43 or poor nutrition44, could also contribute and larger studies would be required to fully assess the effect of vitamin D status on foetal survival. In contrast to our findings, the Soay sheep study demonstrated that vitamin D status  was positively associated with annual reproductive success in ewes, i.e. the number of their lambs that survived to one year old31. According to the benchmark  recommended for most species8, the majority of the ewes in our study were vitamin D insufficient, and in general, their 25(OH)D levels were lower than those reported for Soay sheep in St Kilda, Scotland31. However, our sheep were sampled in November, not August, and the general reproductive competence suggests they were not severely compromised45. We suggest that these ewes have adapted well to the environmental conditions in north west Scotland, and that their adaptation could include capability to thrive with a vitamin D status below the ‘sufficiency’ range posited by Nemeth et al.8, at least for part of the year47, as a prerequisite for survival.In the current study, the breeding outcomes (i.e. number of lambs born or weaned) were not associated with pre-mating ewe status in respect of the vitamin D metabolites investigated. Nevertheless, 9 ewes that lost foetus(es) between scanning and lambing (based on scanning results), had lower concentrations for one or both vitamin D metabolites investigated, compared to the corresponding average unadjusted vitamin D concentrations Table . This in49, and would produce lambs with heavier birth weights51.Ewes in our study that received the standard feeding level in the first winter feeding period had better body condition at that stage, compared to those with poorer body condition that received the corrective feeding level. These ewes with better condition scores in winter then went on to have greater litter sizes at birth and weaning in the subsequent spring and summer, respectively than their counterparts with relatively poorer body conditions. This is plausible and not unexpected as ewes with good body condition would have increased ovulation rates, reduced risk of embryo loss3 and total 25(OH)D concentrations produced heavier lambs for both singleton and twin litter categories. These findings were in line with previous reports of vitamin D status effects in pregnant women53. One possible explanation is that high 25(OH)D status could promote local metabolism from 25(OH)D to the active form of vitamin D, 1α,25(OH)2D, in the placenta, which supports placental development in early pregnancy54, and in turn enhances placental transportation of calcium for foetal growth56. However, this correlation (improved vitamin D status associated with lower incidence of low infant birthweight) has been a controversial issue in human studies, as several authors report different results59. Environmental conditions are more difficult to standardise in human studies, or those involving wild or feral animals, compared to those conducted on livestock within a research farm environment, which could account for differences in findings across trials and species. Our results suggest that ewes having higher pre-mating vitamin D status might, if there is a pay-off in terms of foetal growth, be favoured in a hill environment, where lamb birth weight is an important determinant of lamb survival in harsh conditions61. Equally, our results suggest that aforementioned ‘sufficient’ concentrations8 may be in excess of what high latitude- and altitude-adapted sheep either could accrue or in fact need to reproduce competently and successfully.In this study, ewes with higher serum 25(OH)D62, which apparently was not influenced by pre-mating vitamin D status of ewes in the current study.Our results showed that ewes’ pre-mating vitamin D status was associated with the lamb birth weight, but not the weight during early growth (marking at around 8 weeks old) or at weaning (around 18 weeks of age). In the early postnatal stage, especially between birth and marking, lamb growth relies mainly on the mother’s milk production3 and total 25(OH)D concentrations in Lleyn ewes were significantly higher than those in both unimproved and improved Scottish Blackface ewes kept at a Scottish commercial hill farm. The vitamin D status in the 3 genotypes did not have any effects on the number of lambs born or weaned per ewe. The concentrations of 25(OH)D3 and 25(OH)D were positively associated with litter weight for singleton and twin lambs at birth, but not at marking or weaning. The relationships between vitamin D status and reproductive outcomes in sheep are deserving of further study.Our findings show that the 25(OH)DInvestigation of relationship between vitamin D status and reproductive fitness in Scottish hill sheep


Hesx1 is expressed in the preimplantation mouse embryo, where it is required during developmental diapause. Absence of Hesx1 leads to reduced expression of epiblast and primitive endoderm determinants and failure of diapaused embryos to resume embryonic development after implantation. Genetic deletion of Hesx1 impairs self-renewal and promotes differentiation toward epiblast by reducing the expression of pluripotency factors and decreasing the activity of LIF/STAT3 signaling. We reveal that Hesx1-deficient ESCs show elevated ERK pathway activation, resulting in accelerated differentiation toward primitive endoderm, which can be prevented by overexpression of Hesx1. Together, our data provide evidence for a novel role of Hesx1 in the control of self-renewal and maintenance of the undifferentiated state in ESCs and mouse embryos.The role of the homeobox transcriptional repressor HESX1 in embryonic stem cells (ESCs) remains mostly unknown. Here, we show that  •Hesx1-deficient embryos fail to reinitiate normal development after diapause•Hesx1 expression in ESCs is controlled by core pluripotency factors•Hesx1 regulates ESC differentiation toward epiblast and primitive endoderm•Hesx1 promotes LIF/STAT3 signaling and inhibits ERK signaling Hesx1-defficient ESCs to reveal that Hesx1 expression is regulated by intrinsic and extrinsic pluripotency-stimulating signals. They show that Hesx1 promotes ESC self-renewal and inhibits ESC differentiation toward both the epiblast and primitive endoderm cell fates. They report the expression of Hesx1 in preimplantation embryos, where it is required to resume normal embryonic development following diapause.Pozzi and colleagues have used  Hesx1 is crucial for proper forebrain and pituitary development in both mice  is a homeobox transcriptional repressor that was initially isolated from murine embryonic stem cells (ESCs) over 25 years ago . Hesx1 ioth mice  and humaysplasia . It has man ESCs , and morman ESCs , suggestHesx1 expression in preimplantation embryos has not been reported (Hesx1 mRNA in wild-type (WT) embryos (C57BL/6J background) at 3.5 and 4.5 days post coitum (dpc) (Hesx1 from early (embryonic day 3.25 [E3.25]) to late blastocyst stages (E4.5) (Hesx1 expression was detected in the primitive endoderm (PrE) . Immunofluorescence staining against GFP, to detect YFP expression, revealed the presence of YFP+ blastomeres in one of nine 8-cell stage morulas, two of 18 3.5-dpc blastocysts and none of nine 4.5-dpc blastocysts derived from Hesx1Cre/+;R26YFP/YFP X Hesx1/−+ crosses  A. Analysum (dpc)  confirmes (E4.5) B. At lat crosses C. YFP exCre cDNA ).Figure Hesx1 mutants has not been previously described. However, the role of Hesx1 in diapause has not been investigated, despite the conservation of the core transcriptional circuitry operating in the preimplantation epiblast (Epi)  . To testst (Epi) D. A totan ratios , diapausn ratios D. These  matings E  and GATA6 (PrE), and the maximum fluorescence intensity (MFI) of the two markers was quantified. This analysis revealed a trend toward a reduction in the expression of both markers in Hesx1−/− blastocysts  data (Hesx1 promoter region. ChIP-qPCR on WT ESCs cultured in serum/leukemia inhibitory factor (LIF) revealed a significant enrichment in the amount of chromatin bound to SOX2 and NANOG and a non-significant increase in OCT3/4-bound chromatin  data   data A revealehromatin A. To asscontrols B. Simultcontrols B. SOX2 bpromoter , but theHesx1 mRNA levels compared with serum/LIF conditions  and glycogen synthase kinase 3 (CHIR), in addition to the cytokine LIF. These conditions stimulate the expression of factors involved in the maintenance of the naive state . Culturenditions C. Converand CHIR D. The upand Rex1 C and 2D.and Rex1 . To dissfter 5 h E. TogethHesx1 in ESCs may have molecular consequences, we generated Hesx1-deficient clones by direct derivation of ESC lines from Hesx1-deficient (Hesx1−/−) and Hesx1/++ control blastocysts. qRT-PCR detected the downregulation of pluripotency markers  and upregulation of lineage commitment markers  in Hesx1−/− relative to Hesx1/++ ESCs   D. We nexmulation E and 3F.mulation E and 3F,+/+ ESCs E and 3F.entation G. Howeve+/+ ESCs G. Responof Hesx1 H. TogethHesx1−/− ESCs may be prompted to transit from naive to Epi state. Hesx1/++ and Hesx1−/− ESCs were cultured in either serum/LIF or in the presence of activin and fibroblast growth factor 2 (FGF2) to induce Epi stem cell (EpiSC) differentiation . In addition, Hesx1−/− ESCs showed higher expression of Epi  and PrE  markers relative to control Hesx1/++ ESCs  revealed a significant enrichment of early developmental terms , whereas genes upregulated in Hesx1−/− ESCs belonged mostly to terms related to extraembryonic tissues and visceral organs  but around 5% in Hesx1/++ ESCs (5.14% ± 0.59%)  and upregulation of endodermal transcripts  in Hesx1−/− ESCs relative to Hesx1/++ control cells. In contrast, Hesx1over ESCs showed a complete lack of PrE markers expression at day 7  E. Hesx1 in 2iLIF E, which staining F. qRT-PCat day 7 G. These Hesx1 is a novel factor involved in the regulation of ESC fate , Hesx1Cre/+ . For ESC isolation, 3.5-dpc embryos were cultured on a layer of mitotically inactive mouse embryonic fibroblasts in N2B27 supplemented with 2iLIF.ckground ), Hesx1Cckground ), and HeChromatin immunoprecipitation was adapted from the Young lab . PrimersCells were lysed and analyzed for luciferase activity at 48 h post transfection. Total amount of transfected DNA was normalized by addition of an empty vector (pBluescript). Cells were harvested and assayed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega) and luminescence was measured using a BMG FLUOstar Optima multiplate reader (BMG Labtech). More details can be found in +/GATA6− or NANOG−/GATA6+) and measuring the MFI of NANOG or GATA6 across the line. For flow cytometry, cells were dissociated to a single-cell suspension and resuspended in FACS buffer . Cells were then stained with DAPI to eliminate dead cells and gated using SSC-H against SSC-W to exclude doublets. Samples were processed on a BD Fortessa (BD Bioscience) and data analyzed using FCS Express. A list of antibodies used can be found in See Gapdh via the ΔΔCt method. A list of primers used can be found in Total RNA from cells or embryos was extracted using RNeasy micro or mini kits (Qiagen). Reverse transcription was performed using the iScript cDNA synthesis kit. Real-time PCR was performed using an iTaq Universal SYBR Green Supermix (Bio-Rad). Gene expression was calculated relative to Hesx1/−+ pregnant females from Hesx1/−+ intercrosses were injected intraperitoneally with 10 μg of tamoxifen (Sigma) and 3 mg of Depo-Provera (Pfizer). Diapaused blastocysts were surgically transferred into the uteri of 2.5-dpc pseudo-pregnant CD1 recipient females and embryos collected at the specific developmental stages. All mouse experiments were performed under the umbrella of a UK Home Office Project Licence and by trained Personal Licence holders.For induction of diapause , 2.5-dpcUnless stated, all experiments were carried out on biological and experimental triplicates. All data represent mean ± SEM. Statistical significance was calculated with Student's t test.S.P. and J.P.M.-B. conceived and designed the study, S.P. carried out the majority of the experiments. S.B. performed experiments. J.A. performed the bioinformatics analysis. T.A.R. and J.M.B. helped with experimental design and data interpretation. S.P. and J.P.M.-B. wrote the manuscript.


Our group has developed the computationally optimized broadly reactive antigen (COBRA) technology to develop HA head-based strategies to elicit antibodies against H1, H3, and H5 influenza strains. These vaccines elicit broadly reactive antibody responses that neutralize not only historical and contemporary vaccine strains, but also co-circulating variants in mice. In this study, we used H1 and H3 HA antigens in a split, inactivated vaccine (IIV) formulation in combination with the AF03 squalene-in-water emulsion adjuvant in ferrets immunologically naïve to influenza virus. The H3 COBRA IIV vaccine T11 elicited antibodies with HAI activity against more H3N2 influenza strains compared to IIV expressing wild-type H3 HA antigens, except for IIV vaccines expressing the HA from A/Texas/50/2012 (Tx/12) virus. H1 COBRA IIV vaccines, P1 and X6, elicited antibodies that recognized a similar number of H1N1 viruses as those antibodies elicited by IIV expressing the A/California/07/2009 (CA/09) HA. Ferrets vaccinated with the P1 or X6 COBRA IIV were protected against CA/09 challege and cleared virus from the lungs of the ferrets, similar to ferrets vaccinated with the CA/09 IIV.Development of broadly reactive or universal influenza vaccines will be a paradigm shifting event for the influenza vaccine field. These next generation vaccines could replace the current standard of care with vaccines that elicit broadly cross-protective immune responses. However, a variety of  Influenza vaccination is the most cost-effective method to prevent influenza infections and spread within a community. Current influenza vaccination strategies primarily elicit antibodies that bind to influenza hemagglutinin (HA) and neuraminidase (NA) glycoproteins on the surface of the virus and block viral infection and spread from cell to cell , 2. Whilhttp://www.who.int/influenza/vaccines/virus/recommendations/en/). However, there is a need to develop influenza vaccine strategies that will elicit more broadly-reactive or more universal responses to recognize a larger number of circulating influenza variants within and across subtypes in any given season and over multiple seasons [Even though influenza vaccines have been used for ~50 years, several limitations still exist involving both their availability and their effectiveness . Antigen seasons , 2.To address the need for more broadly reactive influenza vaccines, our group has previously reported on the methodology of antigen design, termed computationally optimized broadly reactive antigen (COBRA), using multiple rounds of layered consensus building to generate influenza vaccine HA immunogens –21. COBRMustela putorius furo, female, 6 to 12 months of age), negative for antibodies to circulating influenza A  and influenza B viruses, were descented and purchased from Triple F Farms . Ferrets were pair housed in stainless steel cages  containing Sani-Chips laboratory animal bedding . Ferrets were provided with Teklad Global Ferret Diet  and fresh water ad libitum. Ferrets (n = 8) were vaccinated with inactivated split IIV vaccines expressing either one of 2 H3N2 COBRA IIV vaccines , one of 4 wild-type IIV vaccines , one of the 3 H1N1 COBRA , or the wild-type CA/2009 IIV vaccine. The split virion vaccine was inactivated with 0.1% Beta-propiolactone (BPL) for 24 h between 18-22C and 1% Triton X-100 in the presence of 500mg/L Tween 80 that was stirred for 1 h at 20C [Fitch ferrets , BEI Resources, the Centers for Disease Control (CDC), or provided by Virapur, LLC . Viruses were passaged once in the same growth conditions as they were received, in either embryonated chicken eggs or semi-confluent Madin-Darby Canine Kidney (MDCK) cell culture as per the instructions provided by the WHO . Virus lA panel of 13 co-circulating MDCK cell passaged reassortant (7:1) H3N2 variants from the period of 2010–2016 included: A/Alabma/05/2010 (AL/10), A/Netherlands/009/2010 (NL/10), A/Hessen/5/2010 (Hes/10), A/Norway/1330/2010 (Nor/10), A/Madagascar/0648/2011 (Mad/11), A/Utah/12/2011 (Utah/11), A/Norway/1186/2011 (Nor/11), A/Athens/112/2012 (Ath/12), A/Jordan/30502/2012 (Jor/12), A/Minnesota/10/2012 (MN/12), A/Denmark/96/2013 (Den/13), A/Hong Kong/12/2014 (HK/12/14), A/Stockholm/28/2016 (Stock/16). Reassortant viruses were generated by using an eight-plasmid reverse genetics system as described previously , 25.H1N1 egg passaged viruses used were A/Chile/1/1983 (Chile/83), A/Singapore/6/1986 (Sing/86), A/Texas/36/1991 (TX/91), A/Beijing/262/1995 (Bei/95), A/New Caledonia/20/1999 (NC/99) A/Solomon Islands/3/2006 (SI/06), A/Brisbane/59/2007 (Bris/07), A/California/07/2009 (CA/09), A/Michigan/45/2015 (Mich/15). The 9-member panel included viral antigens representing human viruses from 1983 to 2015. The P1, X6, and X3 HA rescued (7:1) viruses (PR8 core proteins and NA from CA/09) were detected by using a hemagglutination assay, and were fully sequenced to ensure the absence of unwanted mutations.4 PFU of the CA/09 (H1N1) or 1 × 107 PFU of the Wisc/05 (H3N2) virus in a volume of 1 ml. Ferrets were monitored daily for weight loss, disease signs, and death for 14 days after infection. Individual body weights and death were recorded for each group on each day post virus challenge. Experimental endpoints were defined as >20% weight loss. Nasal washes were performed by instilling 3 ml of PBS into the nares of anesthetized ferrets each day for 14 days after inoculation. Washes were collected and stored at −80°C until use. The University of Georgia Institutional Animal Care and Use Committee approved all experiments under the Animal Use Protocol #2016 02–011, which were conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals, The Animal Welfare Act, and the CDC/NIH’s Biosafety in Microbiological and Biomedical Laboratories guide.On day 56 post-vaccination, ferrets were challenged intranasally with 5 × 10The hemagglutination inhibition assay was used to assess functional antibodies, elicited by H3N2 vaccines, to the HA able to inhibit agglutination of guinea pig and as well as turkey erythrocytes. The protocols were adapted from the WHO laboratory influenza surveillance manual  and usesAtlanta Biologicals, GA, USA). All supplements except FBS were purchased from Thermo Fisher Scientific. For virus titering, a plaque assay was performed similarly to previously described protocols [5) in each well of a six-well plate. Samples were diluted  and overlaid onto the cells in 100μl of DMEM supplemented with penicillin- streptomycin and incubated for 1 h with intermittent shaking every 15 minutes. Samples were removed, cells were washed twice, and medium replaced with 2 ml of L15 medium plus 0.8% agarose  and incubated for 72 h at 37°C with 5% CO2. Agarose was then removed and discarded. The cells were fixed with 10% buffered formalin, and then stained with 1% crystal violet for 15 min. The plates were then thoroughly washed in distilled water (dH2O) to remove excess crystal violet, before being air-dried. Next the number of plaques were counted, and the PFU per milliliter was calculated for each virus.MDCK and MDCK-SIAT1 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with penicillin-streptomycin, bovine serum albumin fraction V 7.5% solution, 25 mM HEPES buffer and 10% heat-inactivated fetal bovine serum  were vaccinated with split inactivated virus (IIV) vaccines containing COBRA or wild-type HA proteins representing the H3 subtype. Previous studies have described the COBRA methodology using 6,340 human HA amino acid sequences to design H3 HA sequences to account for the evolution of H3N2 influenza viruses isolated in humans over the past 45 years , 21. Two05 virus . The Uru05 virus , with al05 virus . TX/12 I05 virus . Ferrets05 virus .In order to demonstrate the breadth of antibody activity against co-circularing H3N2 influezna strains between 2010 and 2016, HAI activity was assessed from the collected antisera following vaccination. Human H3 hemagglutinin sequences were identified to represent different H3N2 influenza phylogenetic clusters and thirteen representative drift viruses were used in a panel to assess the breadth of HAI activity elicited by the IIV vaccines as previously described . FerretsTo extend these observations, ferrets were vaccinated with IIV vaccines expressing H1 HA antigens, one IIV expressed as the HA from the wild-type (CA/09) influenza virus and three IIV vaccines expressed as H1 COBRA HA antigens  . These t7 PFU/ml) at week 8 post-vaccination. All ferrets lost less than 5% weight regardless if they were vaccinated or not (3 PFU/ml) were recovered from the nasal washes from the mock vaccinated ferrets infected with the H3N2 virus, and no virus was detectable in nasal washes from vaccinated ferrets. Ferrets vaccinated with H1 IIV vaccines were challenged with CA/09 virus (5×104 PFU/ml). Non-vaccinated ferrets (mock) had a rapid drop in weight, losing 15–20% of their body weight by day 7 post-infection  (4 pfu/ml) that rapidly declined to low or undetectable levels by day 5 post-infection  . In addinfection .In this study, reassortant viruses expressing H1 and H3 COBRA HA antigens were generated by combining the desired HA segment with the 7 segments from the A/Puerto Rico/8/1934 virus. These viruses were subsequently amplified and used to generate inactivated split influenza vaccines (IIV). The first goal was to evaluate these IIV vaccines in naïve ferrets for elicitation of antibodies. Previously, these COBRA HA antigens, delivered to the immune system of mice on the surface of a virus-like particle vaccines, which elicited high titer antibodies against a panel of historical influenza A strains , 21. We The two H3 and three H1 COBRA HA antigens were designed to cover multiple time periods and antigenic spaces based on their date of isolation, as previously described , 21. TheThe T7 COBRA IIV vaccine was more effective at eliciting antibodies with HAI activity against H3N2 viruses from the mid-2000s, and the T11 COBRA IIV vaccine was more effective at eliciting antibodies with HAI activity against H3N2 viruses from the late-2000s through 2016. These results were similar to those obtained from mice vaccinated with VLPs expressing these same H3 COBRA HA antigens . Ferretsin vitro and in vivo models will need to be used to assess vaccine formulations, and downselect the candidates to those with the most likely chance of success in human clinical trials.In order to address if the lower titers, and albeit reduced recognition of viruses in the panel by the vaccine elicited antibodies, was a result of expressing the COBRA HA antigens in an IIV formulation, naïve ferrets were also vaccinated with VLP vaccines. VLP vaccinated ferrets had statistically similar HAI titers against the same panel of H1 and H3 viruses as antibodies elicited in ferrets vaccinated with IIV versions of these same vaccines. In contrast to BALB/c mice, ferrets are outbred animals, and this may have contributed to the inconsistent rates of seroconversion and spread in the HAI titers within a group of ferrets vaccinated with the same vaccine. Recently, our group demonstrated that priming naive ferrets with historical seasonal influenza viruses, and then vaccinating these primed ferrets with broadly reactive H1 or H3 COBRA HA based VLP vaccines boosted pre-existing antibodies induced by the wild-type influenza virus infections. These COBRA HA antigens induced antibodies with HAI activity against multiple antigenically distinct H1N1 and H3N2 viral variants , 30. The


ABCB11), organic anion-transporting polypeptide 1A2 (OATP1A2), carbonic anhydrase II (CA2), Na+-HCO3− cotransporter (NBC1), and hydroxysteroid sulfotransferases (SULT2A1) play an important role in bile acids transport and sulfation in placentas during pregnancy. We also found the potential regulation role of ALDBSSCG0000000220 and XLOC_1301271 on placental SULT2A1. These findings have uncovered a previously unclear function and its genetic basis for bile acids metabolism in developing placentas and have important implications for exploring the potential physiological and pathological pathway to improve fetal outcomes.Placenta performs the function of several adult organs for the fetus during intrauterine life. Because of the dramatic physiological and metabolic changes during pregnancy and the strong association between maternal metabolism and placental function, the possibility that variation in gene expression patterns during pregnancy might be linked to fetal health warrants investigation. Here, next-generation RNA sequencing was used to investigate the expression profile, including mRNAs and long non-coding RNAs (lncRNAs) of placentas on day 60 of gestation (G60), day 90 of gestation (G90), and on the farrowing day (L0) in pregnant swine. Bioinformatics analysis of differentially expressed mRNAs and lncRNAs consistently showed dysregulation of bile acids transport and detoxification as pregnancy progress. We found the differentially expressed mRNAs, particularly bile salt export pump ( Placenta is a temporary organ supporting the normal growth and development of the fetus during pregnancy. During this period, placenta is not only responsible for exchange of oxygen, iron, and substrate transfer such as sugar and amino acids, but also participates in proteins and peptide hormones synthesis and placental immunology ,2. MoreoPregnancy is commonly associated with extensive physiological and metabolic changes to accommodate the growing needs of fetuses . A typicsus scrofa genome    and 274 lncRNA transcripts (170 up-regulated and 104 down-regulated) were differentially expressed in G90 placentas ( < 0.05) , whereas < 0.05) . In unsu < 0.05) C,D. Thes < 0.05) . The relp < 0.01, corrected p < 0.05)  .p < 0.01, corrected p < 0.05) altered in G90 placentas compared with G60 placentas , located in the apical membrane of human and rat hepatocytes and reported to export BA in ATP-dependent manner [SLCO1A2, also known as OATP-A), mediating hepatocellular uptake of organic anions, including conjugated BA and estrone-3-sulfate, accompanied by bicarbonate efflux [CA2), catalyzing the conversion of CO2 to the bicarbonate ion and protons [+-HCO3− cotransporter   C,D, whicSULT2A1) mediates sulfo-conjugation of BA [SULT2A1 was higher at G90 than at G60, indicating the improved detoxification function of placentas, but was significantly decreased at L0 compared with that at G60 or G90  in G90 placentas compared with that in G60 placentas  expression at G90 was twice that at G60, and the higher C5aR1 expression was maintained till L0 when maternal TBA levels restored to the levels at G60 , which promotes dissociation of the C3 convertase enzyme [C3 in G90 placentas. Expression of high levels of complement regulatory proteins including membrane cofactor decay accelerating factor  was reported to be critical for the trophoblast to prevent excessive complement activation in the placentas [CD55 at G90 than at G60 might be an adaptive response to activated complement system. In support of this notion, the gene F2, encoding for thrombin protein which is capable of generating the complement activation product C5a in the absence of C3 [C5aR1. Given that 80–90% of the complement proteins are produced by the liver [C3 and C5 expression in liver tissues and C5a concentrations in maternal peripheral serum and U-A, C-V, and U-C serum. No differences were observed in hepatic C3 and C5 expression and in serum C5a concentrations with the advance of gestation stage  was found since the number of cis-mRNAs was relatively small. There was also no significant enrichment (corrected p > 0.05) in GO analysis between L0 and G60, except “membrane-bound organelle”, which belongs to cellular component terms. We performed GO analysis on their trans-regulated target lncRNAs in two comparison groups. In GO terms, the significantly over-represented terms are shown in p < 0.001, corrected p < 0.05). Although the existence of many common GO terms, no significantly enriched KEGG pathway was found between L0 and G60  between G90 and G60 were olfactory transduction, bile secretion, staphylococcus aureus infection, and complement and coagulation cascades (corrected p = 0.08). The predicted function “bile secretion” and “complement and coagulation cascades” were same with mRNA, indicating the potential strong connection and interaction between lncRNAs and mRNAs. Remarkably, the up-regulated XLOC_1301271 and ALDBSSCG0000000220 were matched with SULT2A1 . A recent study indicates that BA are natural ligands of olfactory systems [Recent studies have demonstrated that lncRNAs can regulate the expression of nearby and remote protein-coding genes in cis and trans, respectively ,48. The  and G60 . We perf and G60 . The sig SULT2A1 B,C. The  systems . HoweverRaised maternal serum BA is one of the most common phenomena among humans, rodents, and pigs during pregnancy ,50,51,52MRP2, also known as ABCC2), transports BA [MRP3, also known as ABCC3), also transports BA in ATP-dependent manner [ABCC2 and ABCC3 in rat placenta [ABCC2 and ABCC3 from G60 to L0, but their expression were not significantly different among G60, G90 and L0 , including bovine CA and human CA1 and CA2, were potently inhibited by bile salts [2 diffuses across placenta predominantly in the molecular form rather than as bicarbonate ion [Studies in isolated membrane vesicles of human and rat placentas ,63 have e efflux ,29,30, me efflux . We furt OATP1A2 , was higed by BA . Interesl origin . Unexpecle salts  and showle salts ,66,67. Inate ion  and the nate ion , the decSULT2A1 was significantly increased from G60 to G90, whereas it decreased from G90 to L0. Maternal serum sulfated BA being higher at G90 than at G60, and not significantly different between G90 and L0, further verified the BA sulfation function of placental SULT2A1, especially when the expression of SULT2A1 in liver was not significantly different [lnc-HC and MEG3, participate in BA metabolism, through regulating cholesterol 7α-hydroxylase (CYP7A1) and small heterodimer partner (SHP) [ALDBSSCG0000000220 and XLOC_1301271 on placental SULT2A1.In addition, our data also revealed the regulated BA metabolism in developing placentas. In humans, the hydrosteroid SULT (SULT2) family is comprised of two subfamilies, SULT2A1 and SULT2B1. SULT2A1 mediates sulfo-conjugation of endogenous molecules, including androgens, estrogens, and glucocorticoids, as well as BA, and thus facilitating BA detoxification and elimination , whereasifferent . Furtherer (SHP) ,70. In tGpbar1)/ nuclear factor kappa B (NF-κB) [TNF, also known as TNFα) and FXR [Bile acids have also been reported to induce placental inflammation by activating G protein-coupled bile acid receptor 1( (NF-κB) , and pla and FXR ,72. The In summary, genes encoding proteins responsible for placental BA transport and detoxification were dysregulated as pregnancy advanced. The high consistency between mRNAs and predicted functions of dysregulated lncRNAs implies that lncRNAs may play an important role in regulating the expression of mRNAs during pregnancy. These findings have uncovered a previously unclear function and its genetic basis in developing placentas and have important implications for exploring the potential physiological and pathological pathway to improve fetal outcomes.n = 3, 3, and 4, respectively). Briefly, the placentas were collected 5 cm away from the umbilical cord and selected from fetuses with average body weight. The number of fetuses at G60, G90, and L0 is shown in A total of 10 placentas were sampled as described  from 10 The total BA (TBA) were measured by the enzymatic cycling method assay kits in 7020 automatic biochemical analyzer  as described.® Ultra™ Directional RNA Library Prep Kit for Illumina®  following manufacturer’s recommendations. Products were purified by AMPure XP system  and library quality was assessed on the Agilent Bioanalyzer 2100 system . The data were analyzed and annotated by a pipeline developed in-house. Briefly, high quality clean reads were aligned to the reference genome using TopHat v2.0.9 . The mapped read were assembled by both Scripture  [p-adjust < 0.05 were assigned as differentially expressed. GOseq R package  and KOBAS software  were used to perform the Gene Ontology (GO) and KEGG enrichment analysis of differential expression genes or lncRNAs target gene, respectively.Total RNAs were isolated and quality controlled. Ribosomal RNA (rRNA) and rRNA free residue were removed by Epicentre Ribo-zero™ rRNA Removal Kit  and ethanol precipitation, respectively. RNA-Seq was performed on an Illumina Hiseq 2000 platform and 150 bp paired-end reads were generated according to Illumina’s protocol. Sequencing libraries were generated using the rRNA-depleted RNA by NEBNextMA, USA)  and CuffMA, USA) . The canMA, USA) , with anTM 6 Flex Real-Time PCR system  to quantify mRNAs or lncRNA expression with a commercial SYBR Green kit . Specific primers were designed using the software of primer BLAST from NCBI  according to the manufacturer’s specifications. The complementary DNA (cDNA) was synthesized using a reverse transcription kit  and stored at 4 °C prior to real-time quantitative PCR (q PCR). Real-time PCR was performed on a QuanStudiorom NCBI . AmplifiT method ,80.The C5a level of uterine artery, umbilical cord vein, uterine vein, and maternal vein serum were measured using the Elisa assay kits  following the manufacturer’s instruction.p < 0.05 was considered statistically significant.All data were analyzed by using the SAS statistical package , except for the RNA-seq result. All values were expressed as means ± SE. Significant differences were analyzed by PROC GLM and least significant difference test or using the NPAR1WAY procedure of SAS 9.4 test for samples that were not normally distributed or homogeneous variances.


Ginseng products used as herb nutritional supplements are orally consumed and fermented to ginsenoside compounds by the intestinal microbes. In this study, we investigated antiviral protective effects of fermented ginseng extracts against different strains of influenza viruses in genetically diverse mouse models. Intranasal coinoculation of mice with fermented ginseng extract and influenza virus improved survival rates and conferred protection against H1N1, H3N2, H5N1, and H7N9 strains, with the efficacy dependent on the dose of ginseng samples. Antiviral protection by fermented ginseng extract was observed in different genetic backgrounds of mice and in the deficient conditions of key adaptive immune components . The mice that survived primary virus inoculation with fermented ginseng extract developed immunity against the secondary infection with homologous and heterosubtypic viruses. In vitro cell culture experiments showed moderate virus neutralizing activity by fermented ginseng extract, probably by inhibiting hemagglutination and neuraminidase activity. This study suggests that fermented ginseng extracts might provide a means to treat influenza disease regardless of virus strains. Influenza virus is a contagious pathogen that has been causing massive mortality for decades. Among the three major types  of influenza virus, type A is more prevalent due to diverse hosts. The Center for Disease Control and Prevention (CDC) estimates that influenza has resulted in between 12,000 and 56,000 deaths annually in the United States since 2010 ,4,5. AmoPanax ginseng has long been reported as one of the most common herbal medicines used in humans. Treatment of mice with ginseng extracts has been reported to reduce the production of inflammatory IL-6 and IL-8 cytokines and to increase antiviral cytokine interferon (IFN)-γ upon influenza virus infection ,12. GinsIn this study, we investigated antiviral effects of fermented ginseng extract samples on influenza viruses. Results in this study demonstrated that fermented red ginseng extracts had an antiviral protective activity against influenza viruses from different subtypes  in in vivo mouse models. In vitro cell culture studies suggested mechanistic insights into the antiviral activity of the fermented ginseng extracts. Fermented ginseng products showed higher in vivo antiviral effects against influenza viruses compared to nonfermented ginseng samples.Lactobacillus alimentarius M-2 (KCTC 11054 BP), Leuconostoc mesenteroides M-3 (KCTC 11055 BP), and pectolytic enzymes  for 7 days (Korean Patent No. KR-1008774890000). The fermentation for preparing fermented ginseng sample A (F.G.A) was processed for the extended time of 7 days for high levels of compound K production, whereas the fermentation time for fermented ginseng sample B (F.G.B) was shortened for 2 days so that the ginsenoside components (Rb1 + Rg1) would be retained above a level of 2.5 mg/g of ginseng sample. The condition of strains and enzymes was identical for preparation of F.G.A and F.G.B. The components of fermented ginseng extract samples A and B are presented in Panax ginseng were washed, steamed at 100 °C for 2–3 h and dried. The dried red ginseng roots were boiled in 4–5 volumes of water for 3 h, and the supernatant ginseng extracts were concentrated and manufactured for nonfermented ginseng extract products.Madin–Darby canine kidney (MDCK) cells purchased from ATCC  were cultured in Eagle’s Minimum Essential Medium (EMEM) and used for microneutralization tests and plaque assays. Subtypes A/Philippines/82 (H3N2), A/California/04/2009 (H1N1pdm), and A/WSN/1933 (H1N1) viruses were kindly provided by Dr. Huan Nguyen, Dr. Richard Webby, and Dr. Yumiko Matsuoka, respectively. The reassortant A/Vietnam/1203/2004 virus (A/VN1203), reverse genetic recombinant A/VN1203, rgH5N1 containing HA with polybasic residues removed and NA from A/VN1203, and six internal genes from A/PR/8/1934 (H1N1) was described in our previous study . The reatm1Knw/J, CD8 T cell-deficient B6.129S2-Cd8atm1Mak/J, B cell-deficient µMT B10.129S2(B6)-Ighmtm1Cgn/J, MHCII deficient (B6.129S2-H2dlAb1-Ea/J)) were purchased from the Jackson Laboratory . To determine in vivo antiviral effects, 25 µL of different strains of influenza viruses were mixed with 25 µL of fermented ginseng extract samples A or B and then incubated for 30 min at 37 °C. The amounts of ginseng samples in the mixtures were in the range of 50–1000 µg doses per mouse. Then, 50 µL of virus–ginseng mixtures were intranasally inoculated into naive mice [50) first. After 1 h postinfection, 1 mg dose of fermented ginseng sample A was intranasally inoculated into the infected mice every hour for five times. Wild-type mice (BALB/c and C57BL6) and mutant mice  of Georgia State University (approved protocol IACUC protocols A14025 on 20 October 2014 and A18001 on 18 September 2017).Mice were sacrificed at day 6 postinfection with A/Vietnam/1203/2004 (rgH5N1) and bronchoalveolar lavage fluids (BALF), and lung samples were collected from the individual mouse as previously described . Part of5) MDCK cells and cultivated for 18–22 h at 37 °C with 5% CO2. After discarding the supernatants, the mixtures with MDCK cells were fixed to the 96-well plate with 80% acetone for 10 min. For the ELISA OD value determination, the primary antibody  and the secondary antibody  were used. Virus control wells (VC: virus only) and cell control wells (CC: cells only) were set for OD value calculation: “X” = ((Average of VC wells) − (Average of CC wells))/2 + (Average of CC wells). All OD values below “X” were considered as a positive (virus negative) for the neutralizing activity. TCID50 (50% tissue culture infective dose) was used to measure the cell infection rate.Virus microneutralization assay using MDCK cells was performed as previously described . In brie2 and fixed with 4% formalin for 1 h after removing the agarose layer [For the plaque forming assays, A/California/2009 (H1N1) and A/Vietnam/1203/2004 (rgH5N1) were mixed with different ginseng samples at a final concentration of 10 mg/mL for 30 min at 37 °C. Then, the mixture was incubated with MDCK monolayer cells for 1 h at 37 °C. After the incubation for 1 h, the virus–ginseng mixture was discarded and the MDCK monolayer was covered with 1.5% agarose with 2× DMEM and trypsin. The whole MDCK monolayer with agarose was cultivated for 3–5 days at 37 °C with 5% COse layer . Crystalw/v) trypsin/0.53 mM EDTA. Viable cells were counted after staining with 0.4% trypan blue solution.For cell viability test, 2 mL of EMEM medium was mixed with different concentrations of F.G.A to make final concentrations of F.G.A to 5 mg and 10 mg/mL. The mixtures were incubated with MDCK monolayer for 6 h. The medium–ginseng mixture was then removed, and cells were washed out by 0.25%  and A/Vietnam/1203/2004 (H5N1) strains were determined using a standard method . FermentO-phenylenediamine dihydrochloride (OPD)  was used to check the color development, and plates were finally read in 450 nm after stopping the reaction.NA-specific inhibition activity by enzyme-linked lectin assays using virus substrates has been well reported in previous studies ,25,26,27t-test was used to determine whether the two different groups showed statistical significance. When p value was less than 0.05, it was considered as significant.A two-tailed student’s Orally taken ginseng supplements are digested and fermented in the intestines, producing active ingredients such as ginsenosides, including compound K. We tested whether fermented ginseng samples would have antiviral in vivo protection against a lethal dose of influenza virus infection. The red ginseng extracts were in vitro fermented with a mixture of bacteria and enzymes, and the product compounds were characterized as shown in 50) of rgH5N1 virus alone or rgH5N1 virus mixed with the fermented ginseng extracts of A or B (F.G.A or F.G.B) or nonfermented ginseng (non-F.G) extract. High and moderate doses  of F.G.A and high dose (500 µg) of F.G.B or non-F.G were first tested  were intranasally infected with a lethal dose , and naive mice that survived from a sublethal dose of rgH5N1 virus infection showed high levels of virus-specific antibody responses due to influenza viral replication. By contrast, the fermented ginseng sample A groups  showed no significant virus-specific antibodies, suggesting complete protection against viral replication C,D,E.50 of rgH5N1 to those groups that survived from the first infection (We then applied the secondary infection with 3 LDnfection B. Due ton = 5). As a positive control, a high dose (500 µg) of F.G.A was also included in this experiment. Moderate to low doses (100 and 50 µg) of F.G.A showed 100% protection without displaying weight loss, as observed in the high dose (500 µg) F.G.A group. However, the lowest dose (20 µg) of F.G.A did not show any antiviral protective effect against the high dose (10 LD50) of rgH5N1 virus  with a high dose of rgH5N1 virus were applied to the wild-type C57B/6 mice (50) did not induce virus-specific antibodies  to investigate whether CD8+ T cells were required for conferring protection virus by fermented ginsengs. The fermented ginseng sample A exhibited a similar pattern of protection against A/Philippines/82(H3N2) in CD8 KO mice. Both groups of CD8 KO mice with mixture of H3N2 virus and 500 µg and 250 of F.G.A were fully protected against body weight loss  with the mixture of fermented ginseng sample A and A/California/2009 (H1N1) resulted in complete protection as observed in wild-type mice. Similar results were observed in MHC-II knockout mice. As shown in the Induction of antibodies is known to be the most relevant immune correlate conferring protection against influenza virus. To test whether fermented ginseng sample A inoculation with H1N1 virus would have antiviral protective effects in a B cell-deficient mice, a µMT mouse model was used with a similar protocol as described in previous sections B. The in5 titers of egg infectious units). Treatment of F.G.A (1 mg) postinfection resulted in high lung viral titers, similar to the untreated naïve mice  in the lung C. Mice iInfluenza virus infection causes severe lung inflammation with high infiltrating cells in the airways and parenchymal tissues, as shown in our histopathology pictures  inoculation with fermented ginseng samples were exposed to the secondary H3N2 virus infection with an antigenically different subtype in the absence of additional ginseng treatment . The F.GSince fermented ginseng extract has in vivo antiviral protection against the influenza virus, we determined whether this ginseng sample has antiviral activity in vitro. The fermented ginseng sample B (10 mg/mL) only inhibited 10–15% of viral infection of H1N1, H3N2, and rgH5N1, as determined by the microneutralization method A. In comThe anti-influenza virus activities of fermented ginseng extract against H1N1 and rgH5N1 were further examined by a plaque assay. As shown in Different incubation time of H1N1 virus with sample A and B prior to infection was tested C. In theWe also investigated whether fermented ginseng extract would exhibit inhibitory effects on influenza virus plaque formation upon treatment after MDCK cells had been infected with influenza virus. Approximately 25% and 50% reduction in plaques were observed with 3 and 6 h incubation, respectively, of 10 mg/mL sample A with MDCK cells that were infected with H1N1 or rgH5N1 virus prior to ginseng sample treatment D. FermenNext, we determined the effects of fermented ginseng extract on hemagglutination and neuraminidase activity and on antibody binding to the virus. We found 10 mg/mL of F.G.A significantly decreased the capture ability of primary antibody of anti-H1 HA or anti-H5 HA to the coated H1N1 or H5N1 on 96-well plate, respectively E,F. MeanWe also tested the cytotoxicity of F.G.A in vitro and in vivo. Different concentrations of F.G.A were added to 2 mL of EMEM medium at final concentrations of F.G.A 5 mg and 10 mg, respectively. As shown in 3 EID50) was preincubated with equal volumes of ginseng extract, Rb1, or other ginsenosides (2 mg/mL) at 37 °C for 1 h. The virus and compound mixture were administered to naïve animals intranasally under anesthesia, reporting the antiviral activity of Rb1 ginsenoside. Rb1 was shown to interact with viral hemagglutinin protein, preventing viral attachment to the 2–3 sialic acid receptors on the target cells [This study presents data demonstrating that fermented ginseng extract with more saponin component ginsenosides  increased survival rates and protected the mice against body weight loss when coinoculated with influenza virus and ginseng samples. Fermented ginseng sample A (F.G.A) and sample B (F.G.B) presented different anti-influenza effects, possibly due to the different saponin components. Compared to F.G.B, F.G.A showed more potent antiviral activity against rgH5N1, H3N2, H1N1, and rgH7N9 influenza virus, not only in wild-type C57BL/6 and BALB/c mice but also in CD4 T cell-deficient mice, CD8 T cell-deficient mice, B cell-deficient mice and MHC-II-deficient mice with defects in CD4 and MHC II-antigen presentation. In F.G.A-treated group (250 µg or higher dose), there were no virus replication and inflammation in the lung, while the mice with F.G.B still had substantial levels of viral loads and inflammation due to the influenza virus infection. It is assumed that the dose  of F.G.A conferred in vivo protection, inhibiting viral replication to a low level and preventing disease (no weight loss). It also limited the IgG production but allowed low viral replication, priming cellular immunity such as cross-protective T cell responses. During the secondary infection with the homologous virus  or H3N2 et cells . Fermented ginseng samples appeared to exhibit more effective antiviral protection effects on influenza virus in vivo than in vitro. The data in this study suggest that direct contact between F.G.A and the virus resulted in antiviral effects in vivo, regardless of host adaptive immunity, such as CD4 and CD8 T cells. We did not observe protective effects of fermented ginseng extract when it was used to treat mice that had already received infection. Treatment of fermented ginseng extract before influenza infection via oral route  did not Moreover, antiviral activity of fermented ginseng was observed in a dose-dependent manner in vivo. High concentration of F.G.A (above 100 µg) related with full protection—no antibody response . NormallIt is of interest to investigate which ginseng saponin ginsenosides function as the real effective antiviral component. After fermentation, the fermented ginseng sample A and B were both found to contain higher levels of compound K and active ginseng saponin components ginsenosides . It is speculated that combined effects of multiple substances in fermented ginseng samples might have such antiviral effects against influenza viruses with diverse subtypes. High concentrations (>10 mg/mL) of the fermented ginseng extract samples in in vitro antiviral test assays would not be in a range of an efficacious therapeutic level. Administration of mixed ginseng samples and viruses would not have much clinical relevance. In other aspects, this study focused on in vivo protective effects on influenza virus by fermented ginseng samples after direct contact with the virus. Inoculation with mixed pathogenic virus and fermented ginseng samples at appropriate dose resulted in protection in naïve mice without displaying weight loss during primary infection. In addition, the mice that survived or were protected during primary infection could develop immunity against homologous and different strains during the secondary infection. This finding could be applied to develop a safer live vaccine formulation for high-risk populations. Further studies on the safety, immunogenicity, and efficacy aspects should be performed.Translating into clinical relevance, intranasal (spray) treatment with fermented ginseng samples at the site of virus entry during severe epidemic or pandemic seasons might have some protective benefits. Further studies remain to be done to uncover the anti-influenza virus mechanisms of fermented ginseng samples.In the present study, we found that fermented ginseng extracts containing ginsenosides  displayed more powerful antiviral effects against influenza viruses than nonfermented ginseng extracts. The antiviral protective effects were observed regardless of influenza virus strains, including various subtypes of H1N1, H3N2, H5N1, and H7N9. Mice that were inoculated with a moderate dose of fermented ginseng extract samples and a lethal dose of virus were protected against weight loss with 100% survival rates during primary infection as well as developed immunity against secondary viral infection.


Nε-heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and Nε-pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme’s binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS).  Escherichia coli leucyl-tRNA synthetase [Methanocaldococcus jannaschii tyrosyl-tRNA synthetase [The emergence of stop codon suppression to incorporate noncanonical amino acids (ncAAs) over the last fifteen years enabled the production of proteins that are site-specifically endowed with various chemical handles, mimics of natural posttranslational modifications (PTMs) and spectroscopic probes ,2,3,4. Inthetase , Methanonthetase  and pyrrNε-pentenoyllysine (Pek) and Nε-heptenoyllysine (Hek) (see We speculated that Hek) see A would bMethanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS) [Methanosarcina mazei PylRS (MmPylRS) with rationally enlarged amino acid binding pockets, as illustrated in Pek was recently identified as a low-affinity substrate for MbPylRS) . All PylMbPylRS) ,17. It hMbPylRS) ,19,20. TMmPylRS(Y384F), a well-known rational mutant which promotes the aminoacylation reaction without significantly altering the specificity [MmPylRS(Y384F). The mutation Y384F was kept in all subsequent PylRS tested. Next, we tested the variant MmPylRS(Y306A/Y384F), a well characterized unspecific synthetase accepting a broad spectrum of different amino acids [MmPylRS(C348A/Y384F) being the most efficient variant. The combination of Y306A and L309A reduced the protein yield drastically, the combination of L309A and C348A slightly. All variants tested are show in MmPylRS in different orientations, but an overly large cavity in the binding pocket allows for too much movement, thus reducing the efficiency of the reaction.Using enhanced green fluorescent protein (EGFP) with the internal stop codon N150amber as a model, we initially attempted to incorporate Pek and Hek with cificity . We founno acids ,19,20. WMmPylRSY84F, a weMmPylRS(Y306A/Y384F) with a yield of approx. 2 mg per liter of culture, EGFP(150Pek) could be expressed with MmPylRS(L309A/Y384F), MmPylRS(C348A/Y384F) or MmPylRS(L309A/C348A/Y384F) with a maximum yield of approx. 3 mg protein per liter of culture achieved with MmPylRS(C348A/Y384F) after purification  was expressed with tion see B. The prtion see C. It shoare slow . This ca1 and 2 readily reacted with allyl alcohol under aqueous conditions  with a TEV protease recognition site upstream of the C-terminal 6xHis tag used for purification, as we speculated that the presence of six histidine residues may interfere with our decoration chemistry see . As bothions see , we firs1Pek withIndeed, while olefin metathesis has been applied for protein decoration ,26, it aMmPylRS(Y306A/Y384F) and the first incorporation of Pek with acceptable yield using novel rational variants, in particular MmPylRS(C348A/Y384F). These amino acids allow posttranslational protein decoration and also for various tether lengths. The amino acid specificity of novel MmPylRS variants should be further assessed, as they are potentially promiscuous, enabling the incorporation of a wide array of novel noncanonical amino acids, also providing a basis for further engineering of MmPylRS.In conclusion, we present the first incorporation of Hek using the well characterized variant We used orthogonal protein expression and subsequent chemoselective thiol-ene conjugation of carbohydrates to produce biocompatible protein scaffolds potentially useful for noninvasive targeting in living systems. In particular, the site-specific conjugation of carbohydrate moieties on proteins decorated with olefins as biorthogonal tags offers new tools for the defined targeting and structural and functional investigation of various phenomena in the living cells. In this context, the significance of this work is threefold: We present a method of incorporating long-chain olefins into proteins in a site-specific manner and show the conjugation with a polysaccharide as a model reaction for protein surface decorations which mimics natural glycosylations and other relevant modifications. We speculate that access to protein conjugation chemistry occurring at greater tether lengths above the protein surface will increase the tolerance for the introduced conjugation partners. Our method would thus provide a useful tool to generate proteins with artificial glycosylations or other modifications for medicinal research and novel applications.MmPylRS(C348A/Y384F) presented in this study should be assessed for the incorporation of bulky ncAAs not currently accepted by known variants. The increase in binding pocket size could allow the activation of a large array of ncAA structures, and give rise to improved engineering strategies for second-generation PylRS variants, employing MmPylRS(C348A/Y384F) as starting sequence, as has previously been the case with MmPylRS/Y306A/Y384F) [In addition, the variant A/Y384F) .in vivo production of proteins containing metathesis-competent ncAAs, such as Pek and Hek, is especially attractive as metathesis is an important and versatile method of C-C bond formation that is highly chemoselective and should display a high level of bioorthogonality. Therefore, it is extraordinarily attractive to use the olefin metathesis for the chemical modification of proteins. Unfortunately, we could demonstrate that olefin metathesis is not a generally applicable method for protein decoration. The fact that a lipase known for organic solvent stability [Doubtless, the possibility for tability  precipittability .3) was activated with NHS (4) and the activated ester (5) subsequently substituted by Nα-Boc protected lysine (6). Nα-Boc-Nε-heptenoyllysine (7) was deprotected with TFA to give Hek (1) as TFA salt with a good global yield of 61% [6-Heptenoic acid (d of 61%  (see SchNα-Boc protected lysine (6) reacted with 4-pentenoylchloride (8), giving Nα-Boc-Nε-pentenoyllysine (9). Deprotection with HCl gave (2) as hydrochloride (see ride see .MmPylRS cloned to the suppression vector pJZ-Ptrp-MmPylTS [E. coli BL21(DE3) with pQE80L-EGFP(150amber)-6xHis for expression of the target protein (EGFP) with the mutation N150TAG. Suppression was tested by addition of 2 mM Hek or Pek and 1 mM inducing agent (IPTG) to cultures in mid-log phase (OD600 = 0.5–0.7) in shake flasks. Controls without added amino acid, with different amino acids and with wild-type EGFP were prepared in the same way. After expression overnight, samples were taken for expression analysis with western blot and suppressed proteins were purified via immobilized metal affinity chromatography using gravity-flow columns packed with nickel-NTA slurry . For storage and mass analysis, the buffer was exchanged to 50 mM Tris-Cl pH 7.5/50 mM NaCl.Mutations were introduced into -MmPylTS  with oveFor western blot analysis, lysed samples were separated by SDS-PAGE (same amount of lysate applied to each gel pocket), transferred to a PVDF membrane and stained using an antibody directed to the hexahistidin tag of full-length EGFP and a secondary antibody coupled to alkaline phosphatase  with BCIP/NBT.Purified proteins were analyzed with SDS-PAGE and HPLC-ESI mass spectrometry using a C5 column  on an Agilent 1260 for separation and a coupled Agilent QTOF 6530 or Bruker microTOF for mass analysis. Measured mass spectra were deconvoluted using software supplied with the instruments using the maximum entropy algorithm.For chemical decoration, TTL(221Pek) and TTL(221Hek) were expressed analogously. To prevent interference of 6xHis tag during subsequent decoration reactions, the expression construct contained a TEV protease cleavage site directly upstream of the C-terminal 6xHis tag. For tag removal, the proteins were incubated with 0.1 mg TEV protease per mg of TTL in 150 mM NaCl/50 mM Tris-Cl pH 7.5/2 mM DTT overnight and subsequently applied to a nickel-NTA column to retain uncleaved protein and the 6xHis-tagged TEV protease.−1 in 0.25 M PBS pH 6) were added MgCl2·6H2O , allylic alcohol  and 86 µL of a solution of 0.7 mg of HGII in tBuOH (386 µL). To 100 µL of TTL(Pek) (1.5 mg·mL−1 in 0.25 M PBS pH 6) were added MgCl2·6H2O , allylic alcohol  and 100 µL of a solution of 3.0 mg of HGII in tBuOH (500 µL). To 100 µL of TTL(Hek) (0.2 mg·mL−1 in 0.25 M PBS pH 6) were added MgCl2·6H2O , allylic alcohol  and 100 µL of a solution of 0.4 mg of HGII in tBuOH (500 µL). The mixtures were stirred at room temperature and the reaction was followed by mass spectroscopy. No desired product of the cross metathesis reaction was observed and the signal of the protein was no longer observed.To 200 µL of EGFP , synthesized in the group of J. Rademann , and the


Vaccines that elicit broadly cross-neutralizing antibodies, including antibodies that target the conserved stem of hemagglutinin (HA), are being developed as a strategy for next-generation influenza vaccines that protect against influenza across multiple years. However, efficient induction of cross-neutralizing antibodies remains a challenge, and potential escape mutations have not been well characterized. Here we elicited cross-neutralizing antibodies by immunizing animals with the hemagglutinins from H5 and H9 subtype influenza A viruses that are sensitive to neutralization by stem antibodies. We further isolated and characterized an HA stem monoclonal antibody 4C2 that broadly neutralizes group 1 influenza viruses and identified HA mutations that reduced sensitivity to stem antibodies. Our results offer insights for next-generation influenza vaccine strategies for inducing cross-neutralizing antibodies. Influenza continues to be a global infectious disease threat, causing seasonal epidemics and occasional pandemics due to the emergence of an influenza A virus containing a hemagglutinin (HA) subtype that has not recently circulated in humans. Humoral immune responses against the HA protein, the principal antigen in inactivated influenza vaccines, correlate with protection against influenza. Therefore, vaccination provides an important public health strategy.HA is comprised of the HA1 surface subunit, forming the globular head domain that mediates binding to cell surface sialic acid receptors, and the HA2 transmembrane subunit, forming the major part of the stem region that mediates membrane fusion between viral and endosomal membranes during endocytosis. HA1 and HA2 are disulfide linked and make additional non-covalent interactions between the N- and C-termini of HA1 and the ectodomain of HA2 in HA stem region. Most neutralizing antibodies elicited by influenza virus infection or vaccination target the receptor binding site and surrounding residues on the HA1 head domain , 4. Due Recently, broadly neutralizing antibodies targeting the HA stem have been discovered –14. The We and others previously showed that different virus strains, even those within the same subtype with identical stem epitopes, may have different susceptibilities to neutralization by stem antibodies and cross-neutralizing sera , 16, 17.Animals were housed at the Division of Veterinary Services in the FDA White Oak animal facility in Silver Spring, MD. Experiments were performed under protocols numbers 2009–28 and 2014–04, approved by the US Food and Drug Administration Institutional Animal Care and Use Committee.Human 293T cells and Madin-Darby Canine Kidney (MDCK) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, L-glutamine, Eagle′s minimum essential medium (MEM) non-essential amino acids, penicillin/streptomycin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and 10% fetal calf serum. Influenza viruses (A/Puerto Rico/8/1934) were generated by reverse genetics as described previously , 19. Bri6 MDCK cells with the virus-antibody mixtures. After a 1.5 h adsorption, virus inoculum was removed, and the cells were overlaid with serum-free media containing 2 μg/ml L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin and MAb at the desired concentration. Virus recovered at the highest concentration of MAb underwent five additional rounds of selection. Potential escape mutant viruses were tested for neutralization resistance. Consensus nucleotide sequences of viral HAs from resistant viruses were determined by direct DNA-sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products and compared with those of the parental, wild-type virus.To generate an escape mutant, H1N1 A/Puerto Rico/8/1934 viruses were incubated with the monoclonal antibody (MAb) over a range of concentrations from 60–1 μg/ml in a total volume of 0.6 ml for 1 h at room temperature, followed by infection of ~ 2.3 x 10Recombinant HAs of A/Singapore/1/1957 (H2), and A/chicken/Hong Kong/G9/1997 (H9) were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). HA of A/Vietnam/1203/2004 (H5) was from an inactivated H5N1 influenza virus vaccine (Sanofi Pasteur). Anti-A/Vietnam/1203/2004 HA head neutralizing MAb VN2.4 and anti-A/Vietnam/1203/2004 HA head non-neutralizing MAb VN2.5 (both IgG1) were provided by Jerry P. Weir (unpublished data). Anti-A/Puerto Rico/8/1934 HA head neutralizing MAb H17-L2 –23 was kFull-length HA ORFs from H1, H2, H3, H5, H7 and H9 viruses , and NA 6 RLU of luciferase activity were incubated with antibodies or sera for 1 h at 37°C. Pseudovirus and antibody mixtures (100 μl) were then inoculated onto 96-well plates that were seeded with 2.5 x 104 293T cells/well one day prior to infection. HA-pseudovirus infectivity was evaluated 48 h later in the luciferase assay. The antibody dilution causing a 95% reduction of RLU compared to control  was used as the neutralization endpoint titer [HA-pseudovirus neutralization was performed as described previously , 30, 31.nt titer . IC95 waAs described previously , plaque Two New Zealand white rabbits were primed subcutaneously with 75 μg of A/chicken/Hong Kong/G9/1997 (H9) HA (BEI Resources) mixed with Freund’s complete adjuvant , followed by two boosts of 75 μg of A/chicken/Hong Kong/G9/1997 (H9) HA mixed with Freund’s incomplete adjuvant  and two boosts of 45 μg of A/Vietnam/1203/2004 (H5) HA (Sanofi Pasteur) mixed with Freund’s incomplete adjuvant, at four-week intervals. Serum samples collected pre-immunization and 4 weeks after each immunization were heated to 56°C for 30 min to inactivate complement for the neutralization assay.Mouse hybridomas secreting HA-specific MAbs were prepared by Precision Antibody . Briefly, BALB/c mice were immunized intraperitoneally with 18 μg of A/chicken/Hong Kong/G9/1997 (H9) HA mixed with Freund’s complete adjuvant, followed by one boost of 18 μg of A/chicken/Hong Kong/G9/1997 (H9) HA mixed with Freund’s incomplete adjuvant, two boosts of 9 μg of A/Vietnam/1203/2004 (H5) HA mixed with Freund’s incomplete adjuvant, and one boost of 10 μg of A/chicken/Hong Kong/G9/1997 (H9) HA and 10 μg of A/Singapore/1/1957 (H2) HA (BEI Resources) mixed with Freund’s incomplete adjuvant, at two-week intervals. Serum samples collected pre-immunization and two weeks after each immunization were heated to 56°C for 30 min for neutralization assays. Splenocytes from immunized mice were fused with mouse myeloma cells Sp2/0, and the hybridomas were screened for H5, H2 and H9 HA-specific MAbs secretion by enzyme-linked immunosorbent assay (ELISA) using HA proteins. Positive hybridomas were subcloned to verify clonality. The heavy and light chain genes of MAbs were sequenced and cloned into expression plasmids by GenScript . Both IgG1 and IgG2a isotypes of MAb were expressed and purified by GenScript.2SO4 before recording OD450. The endpoint titer was defined as the highest dilution that gave an absorbance value greater than 0.050.To screen MAbs for binding to influenza virus HA, HA antigen (10 μg/ml) was coated onto ELISA plates. The coated plates were blocked and incubated with MAbs. The plates were then washed with PBS (pH 7.2) and incubated with peroxidase-conjugated secondary antibody . Unbound secondary antibody was washed off with PBS (pH 7.2), signal was developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate, and the reaction was stopped with 1M H450 was recorded.Competition ELISAs were used to determine whether the new cross-neutralizing MAbs overlapped other known neutralizing antibodies. H5 HA antigen (2 μg/ml) was coated onto ELISA plates. After blocking, the coated plates were incubated with mixtures of biotinylated MAb 4C2  and other antibodies at various concentrations. The plates were then washed with Tris-buffered saline (TBS)-0.05% Tween-20 and incubated with horseradish peroxidase-conjugated streptavidin . Signal was developed, and OD50) of A/Puerto Rico/8/1934 virus. Body weights were monitored daily and mice that reached a study endpoint of 25% loss in body weight were humanely euthanized using CO2. In passive MAb transfer experiments, mice were administered intraperitoneally with MAbs 6 h before challenge with 5 LD50 of A/Puerto Rico/8/1934 virus.Six week old BALB/c mice  were immunized with 9 μg of A/chicken/Hong Kong/G9/1997 (H9) HA mixed with TiterMax adjuvant , followed by 1 boost of 9 μg of A/Vietnam/1203/2004 (H5) HA mixed with TiterMax adjuvant, and 1 boost of 9 μg of A/Singapore/1/1957 (H2) HA mixed with TiterMax adjuvant by subcutaneous injection, at 2-week intervals. Two weeks after the final boost, mice were anesthetized and challenged intranasally with 5 50% lethal dose  using the Protein Data Bank (PDB) entries 1RU7, 3ZTN, 3GBN, 4HLZ, 3ZTJ, 3GBM.The interactions between escape mutation residues and their neighbor residues were modeled with the UCSF Chimera program (We selected HA immunogens from A/Singapore/1/1957 (H2), A/Vietnam/1203/2004 (H5), and A/chicken/Hong Kong/G9/1997 (H9) that we previously found to be sensitive to cross-neutralizing antibodies , 17. HigWe first immunized two rabbits with A/chicken/Hong Kong/G9/1997 (H9) HA three times, followed by two immunizations with A/Vietnam/1203/2004 (H5N1) vaccine. While the pre-immunization sera did not have neutralization titers to all tested H1, H2, H5, H7 and H9 HA-pseudoviruses , rabbitsTo further evaluate our immunization strategy and the protective ability of cross-neutralizing antibodies against heterosubtypic influenza viruses challenge, we immunized mice with recombinant HAs for A/chicken/Hong Kong/G9/1997 (H9) and A/Singapore/1/1957 (H2) and split, inactivated A/Vietnam/1203/2004 (H5) vaccine. We then challenged with a lethal dose of A/Puerto Rico/8/1934 (H1N1). Mice vaccinated with H9, H5, and H2 antigens generated not only strain-specific neutralizing antibodies against A/chicken/Hong Kong/G9/1997 (H9) HA, A/Vietnam/1203/2004 (H5) HA, and A/Japan/305/1957 (H2) HA, but also low-level cross-neutralizing antibodies against A/Puerto Rico/8/1934 (H1) HA in two of five mice . ImportaWe next tried to isolate MAbs that may have contributed to the cross-protection. We repeated our immunization strategy in mice and selected hybridomas that secreted MAbs against H9, H5, and H2 HAs. Among these hybridomas, clone 4C2 was found to produce a MAb that neutralized H9, H5, H2 and H1 HA-pseudoviruses, but not H3 and H7 HA-pseudoviruses . HoweverTo characterize MAb 4C2, we performed immunoprecipitation and competition ELISAs to detect MAb 4C2 binding to HA. Immunoprecipitation studies showed that MAb 4C2 binds to the neutral pH conformation of HA, but not to the low pH HA conformation . When MAin vivo protection of neutralizing stem antibody has been largely associated with Fc-mediated activation of humoral and cellular innate immune functions, and thus influenced by IgG isotypes [in vitro . As controls, PBS or an H5N1 HA MAb VN2.5 failed to protect mice from morbidity or mortality . The grein vivo. A second escape mutant isolate carried only the V444L substitution in HA. The control virus cultured without the presence of MAb 4C2 did not show any HA mutations.We next generated escape mutant viruses to MAb 4C2 by culturing A/Puerto Rico/8/1934 (H1N1) virus in MDCK cells in the presence of MAb 4C2. After a dozen attempts and extensive passaging, we obtained an escape mutant virus with a lysine to glutamic acid substitution at position 55 (K55E)  in HA1 and a valine to leucine substitution at position 444 (V444L) (H3 HA numbering or V115L in HA2 numbering) in HA2 that conferred low-level resistance to MAb 4C2. Additional resistance mutations would be needed for higher levels of resistance, and compensatory mutations might be needed to maintain fitness in vitro fitness cost for the escape mutant viruses. Since escape mutant viruses were selected by culturing virus in the presence of MAb 4C2 in MDCK cells, we further included the plaque reduction neutralization (PRN) assay that uses replicating viruses to evaluate the sensitivity of escape mutants to MAb 4C2. We found that PRN assay requires much higher concentrations of stem antibodies for neutralization compared to the HA-pseudovirus assay, consistent with prior reports [To determine whether these mutations conferred resistance to stem antibodies, we engineered viruses and HA-pseudoviruses with an HA gene containing single mutations and both mutations. Both wild type and the engineered escape mutant viruses replicate well in MDCK cells and have similar infectivity, suggesting no  reports . HoweverTo further understand the nature of these mutations, we modeled the changes using available high-resolution structures. Neither mutation identified in these escape viruses resides directly within the epitopes of well-characterized stem antibodies C179, CR6261, and FI6 . V444L . ModelinAltogether, these data and our modeling suggest that MAbs 4C2, C179, CR6261, and FI6 target similar regions in the HA stem, but mutations beyond the epitope may influence fusion peptide function and allosterically affect stem antibody interactions with their epitopes on HA.The induction of broadly neutralizing antibodies is a goal of many next-generation influenza vaccines that are being developed. Vaccination with or exposure to heterologous antigens as a general strategy for increasing breadth of immune responses to induce anti-HA stem antibodies has been proposed –46. Herein vivo [in vitro potency.Similar to the findings of others , 42, we in vivo , 42 sincThe stem antibody epitopes on HA are very conserved, which may reflect functional constraints on residues in this region and/or limited immune selection against these residues. Generation of escape mutant influenza viruses against MAb 4C2 required many passages, and others have reported that viruses resistant to stem antibodies are not easily generated , 47–49. The escape mutations that emerged in our studies have not been previously identified as contact residues for stem antibodies. Our structural modeling suggests that these residues may alter the fusion peptide, HA stem conformation, and flexibility in a way that affects stem epitope conformation and/or availability and thus influences stem antibody binding. Most reported neutralization escape mutations to stem antibodies are in and around the epitopes of stem antibodies. The major resistance mutations often directly reduce antibody binding affinity with the epitopes , 47, 55,Previously, we reported that conformational stability of HA is an important attribute of susceptibility to broadly neutralizing stem antibodies . HA stabS1 FigThe Amino acids sequences of MAb 4C2 heavy chain and light chain with color coded FR, CDR and Constant regions are shown.(EPS)Click here for additional data file.