Pasteurella multocida is a widespread respiratory pathogen in pigs, causing swine pneumonia and atrophic rhinitis, and the capsular serogroups A and D are the main epidemic serogroups in infected animals. This study investigated the protective effects of serogroup A and D bacterins against current circulating P. multocida strains, to better understand the immunity generated by bacterins.P. multocida strains were isolated, and used as inactivated whole cell antigen to prepare P. multocida bacterins. Mice were immunized with these bacterins at 21-day interval and intraperitoneally challenged with the homologous and heterologous P. multocida strains, respectively. The antibody titer levels and immunization protective efficacy of vaccines were evaluated.13 serogroup A (seven A: L3 and six A: L6 strains) and 13 serogroup D  All of the bacterins tested induced high titer levels of immunoglobulin G antibodies against the parental bacterial antigen in mice. Vaccination with the six A: L6 bacterins provided no protection against the parent strain, but some strains did provide heterologous protection against A: L3 strains. Vaccination with the seven A: L3 bacterins provided 50%–100% protection against the parent strain, but none gave heterologous protection against the A:L6 strains. Immunization with the thirteen D: L6 bacterins offered 60%–100% protection against the parent strain, and almost all D: L6 strains gave cross-protection.P. multocida vaccine development.This study found that the cross-protectivity of serogroup A strains was poor, while serogroup D strains was effective, which provided some insights for  Pasteurella multocida, a facultative anaerobic Gram-negative coccobacillus is named after Louis Pasteur, who first isolated and described this bacterium as the causative agent of fowl cholera in 1880  and live vaccine (serogroup B strain EO630), had no protective effect against the epidemic serogroup A and D strains  and serogroup D strains (D1–D13). Mice immunized with these bacterins were challenged with the parent strain and other serogroup A or D strains. Differences in the protective effects of these bacterins showed the cross-protection capacity between serogroup A strains and within serogroup D strains, providing reference results to guide the development of new multivalent P. multocida vaccines for pigs.orldwide  and capsorldwide , 17, whiorldwide . The preese pigs . Prior tese pigs . The dom B and A , 22, but B and A , 24. Ina B and A –27. Our  strains , drivingP. multocida strains of the capsule serogroups A (strains A1–A13) and D (strains D1–D13) were isolated from diseased swine in central and eastern China by the Veterinary and Biological Products Engineering Laboratory of Henan University of Science and Technology , between February 2011 and January 2019 (Twenty-six ary 2019  23). Ea. EaP. muad libitum, and acclimated for 3 days before commencing the experiments. The animals in this study were treated in accordance with the recommendations of the Guide for the Animal Care and Use Committee of Henan University of Science and Technology (No. 20220116002).Female Kunming (KM) mice aged 5–6 weeks were used for the vaccine study and 10–11 weeks mice were used for the virulence tests. The mice were purchased from the Animal Experiment Center of Zhengzhou University, China, and were housed in groups, provided with food and sterile water P. multocida strain A1, 25 female KM mice (10–11-weeks-old) were randomly divided into five groups. P. multocida strain A1 was grown to a bacterial peak in trypticase soy broth (TSB) with 4% healthy bovine serum and 0.1% whole blood with lysed blood cells, and serially diluted 10-fold in sterile phosphate-buffered saline (PBS) to obtain cultures of ~10–104 CFU/ml. The numbers of bacteria extracted in each dilution were checked using direct-plate viable counts. The first four groups were infected intraperitoneally with 200 μl aliquots of increasing dilutions, and the last group was injected with PBS alone as a control and showed no effects. Groups of mice challenged with the various culture dilutions were isolated and housed in a standard animal facility with ad libitum access to a normal rodent diet and water. The inoculated mice were observed for 14 d for signs of disease, and dead mice were dissected immediately to observe their gross pathology. The 50% lethal dose (LD50) was calculated on day 14 post-challenge using the Reed-Muench method , as previously described  and stored overnight at 4°C. The plates were then washed with ELISA washing buffer  and blocked using 15% (wt/vol) skimmed milk powder in PBS for 1 h at 37°C. The plates were rewashed three times. After the wells had been washed and blocked, the serum antibodies were added to the wells (100 μl/well) and incubated for 30 min at 37°C. The serum samples were then serially diluted two-fold from 1:20 to 1:81,920 in the plates using blocking buffer, and the serially diluted plates were then incubated for 30 min at 37°C. The antibody titers were detected using goat anti-mouse immunoglobulin G (IgG)  diluted in blocking buffer , and the sample-treated plates were washed four times and incubated with secondary antibodies for 30 min at 37°C. The plates were washed five times and incubated with TMB chromogenic solution (100 μl/well) for 10 min at room temperature in the dark, and then 100 μl of TMB reaction stop solution was added to each well. Finally, the optical density was measured using an ELISA reader at a wavelength of 450 nm.escribed . Blood sP < 0.05 (*), P < 0.01(**), or P < 0.001 (***).Data are expressed as mean ± standard deviation. Differences in the levels of antibodies among all groups of mice were analyzed using one-way analysis of variance (ANOVA). Comparisons of mouse survival rates were analyzed using Fisher's exact test, and survival times were analyzed using the Kaplan-Meier method. All statistical analyses were performed using the SPSS 20.0 software , and graphics were produced with the Prism 8.0.3 software . Comparisons were considered significantly different if 50 of serogroup A P. multocida strains A1–A13 and serogroup D strains D1–D13 were estimated using the Reed-Muench method  animals (P < 0.001) (P < 0.05). In addition, mice revaccinated with A7 bacterin produced a significantly greater serum IgG titer than the other three vaccines in test-2 (P < 0.001), the D11 bacterin group being significantly higher than the D10 and D13 bacterin groups after the booster immunization in test-6.We detected the production of IgG antibodies in mouse sera to evaluate the immune response of < 0.001) , and aftP. multocida serogroup A inactivated whole-cell vaccines against type A strains were investigated. KM mice were subcutaneously inoculated with A1–A13 bacterins on day 0, and day 21. On day-14 after the booster immunization, mice were intraperitoneally challenged with serogroup A strains of P. multocida. The test-1 result showed that vaccination with A1 or A2 bacterin provided the mice with 100% protection against challenge with 4 × the LD50 of the parent strain, and that vaccination with A3 bacterin provided 86% protection against a 4LD50 challenge with strain A1 (P < 0.001), and mice vaccinated with A2 bacterin showed significant differences in survival time compared with the A2 challenged controls  (P > 0.05)  , indicat > 0.05) . In addiP < 0.001) relative to the control group, all of which died within 4 days . Similarly, mice immunized with D1, D2, and D4 bacterins also showed significantly increased survival times (P < 0.05) when challenged with strain D3 , however, mice immunized with D7 showed longer survival times (P < 0.05) (P < 0.05)  . The D5  < 0.05) .P. multocida infection. The protective efficiency of bacterins against challenge strains and the survival curves showed a cross-protection effect between strains. Vaccination with the D11 bacterin resulted in 20% survival when challenged with strain D13, and the D13 bacterin resulted in 10% survival when challenged with strain D11 (P > 0.05) (P > 0.05)  , indicat > 0.05) , with no > 0.05) , showingP. multocida is a recognized primary pathogen causing economically significant diseases, with a serious impact on the health and economics of the pig-rearing industry worldwide  provided 86% protection against a challenge with strain A1 (A:L3), while A1 bacterin gave no protection against strain A3. A8 bacterin (A:L6) gave 67% or 17% protection against strains L3, A6, and A5, respectively, while A6 or A5 gave no protection against A8. These results indicated that even though the L6 type bacterin used in this study gave protection against the L3 type strain, the L3 type bacterin in turn was unable to protect mice against challenge with the L6 type strain. This may be related to the presence of multiple LPS structures generated by spontaneous mutations in the LPS biosynthesis genes in L3 or L6 type strains , and thain vitro and in vivo situations .L-jG and Z-qZ conceived and designed the study. L-jG and J-qY performed the experiments. L-jG, Q-yX, and Y-fF analyzed the data. L-jG, X-cZ, BT, and Z-qZ wrote and revised the manuscript. All authors read and approved the final version.


In this review, we summarize the current state of knowledge on the ATP-adenosine axis in ischemic stroke, discuss contradictory results, and point out potential pitfalls towards translating therapeutic approaches from rodent stroke models to human patients.In ischemic stroke, the primary neuronal injury caused by the disruption of energy supply is further exacerbated by secondary sterile inflammation. The inflammatory cascade is largely initiated by the purine adenosine triphosphate (ATP) which is extensively released to the interstitial space during brain ischemia and functions as an extracellular danger signaling molecule. By engaging P2 receptors, extracellular ATP activates microglia leading to cytokine and chemokine production and subsequent immune cell recruitment from the periphery which further amplifies post-stroke inflammation. The ectonucleotidases CD39 and CD73 shape and balance the inflammatory environment by stepwise degrading extracellular ATP to adenosine which itself has neuroprotective and anti-inflammatory signaling properties. The neuroprotective effects of adenosine are mainly mediated through AThe online version contains supplementary material available at 10.1007/s00281-023-00987-3. Stroke ranks among the leading causes of death and disability worldwide. With 7.6 million new cases in 2019, ischemic stroke accounted for 62% of all strokes, and 3.3 million patients died from this widespread disease [+) into the interstitial space in an activity-dependent and regulated manner. Release mechanisms include exocytosis, connexin or pannexin hemichannels, ATP-binding cassette (ABC) transporters, calcium homeostasis modulator (CALHM) channels, a macropore after P2X7 receptor overstimulation, maxi-anion channels (MACs), and volume-regulated ion channels (VRACs) as comprehensively reviewed by Giuliani et al. [Under physiological conditions, cells within a tissue, including the brain, release ATP and nicotinamide adenine dinucleotide . Three P2X receptor subunits form an ion channel that upon ATP-mediated gating conducts the rapid influx of calcium and sodium ions and efflux of potassium ions. The expression of P2X receptors has been ascribed to various brain-resident cells as well as to cells of the immune system  with P2X-P2X7. ThP2rx1 in mice diminished the activation and recruitment of neutrophils to inflamed arteriolar endothelial cells, accompanied by impaired platelet aggregation and fibrin generation [Of importance in the context of ischemic stroke, P2X1 is expressed on platelets. ATP (10 μM) increases platelet aggregation in vitro, which can be prevented by the P2X1 antagonist NF449 . P2X1 isneration . Therefoneration . Apart fneration . It has P2rx4 in myeloid cells using LysM-Cre mice, only female mice showed a reduction in infarct size, suggesting a sex difference in the P2X4 response after stroke [ATP released during cerebral ischemia triggers P2X4 opening on brain innate immune cells such as microglia or infiltrating monocytes/macrophages and sustained P2X4 activation contributes to the ischemic injury , 20. Amor stroke . Anotherr stroke . 5-BDBD r stroke . Anotherr stroke . In liner stroke . Given tr stroke . First Pr stroke . The affr stroke . Furtherr stroke . In prinr stroke . As anotr stroke . Therefor stroke .P2X7 is most likely the most extensively studied P2X receptor in brain ischemia. P2X7 activation is known as a potent trigger for the NLRP3 inflammasome formation in macrophages and microglia, leading to the release of pro-inflammatory interleukin-1β (IL-1β), a powerful driver of post-stroke inflammation –34. Yet,P2rx7-deficient and WT mice [First studies by Nancy Rothwell’s group did not show a difference in stroke size when comparing  WT mice , and i.c WT mice . However WT mice . P2X7 up WT mice . Other s WT mice . Studies WT mice  and late WT mice . The stu WT mice . Similar WT mice . In cont WT mice .P2rx7-deficient mice. As mentioned above, first stroke experiments with P2X7ko mice did not reveal differences in stroke size [As for P2X7 targeting compounds, contrasting results have also been reported in studies using oke size . Later ioke size . In contoke size . A recenoke size  exhibiteoke size  in mice oke size . This stoke size . Interesoke size . Since tWhen using antibody or nanobody-based therapeutics, one has to monitor for potential immune-related adverse events (irAE). Antibody-induced inflammation via complement activation or activation of natural killer (NK) cells and macrophages can be prevented by modifying the Fc-region of antibodies. Nanobody monomers lack Fc regions in principle but can be tailored as Fc-fusion proteins with desired or no Fc-related functions , 50. To P2rx7-deficient mice of different origin. For the latter aspect, differences between the GlaxoSmithKline (GSK) and the Pfizer P2X7ko mice have already been demonstrated [P2rx7 gene targeting. Indeed, our group could identify a P2rx7 passenger mutation in the commonly used P2X4ko mouse [P2rx4 and P2rx7 are direct neighboring genes, differentially expressed genes in congenic P2X4ko might also occur in congenic P2X7ko mice. If such passenger mutations also exist in the widely used Pfizer P2X7ko mouse [In general, though various studies suggest a role of P2X7 in stroke, many P2X7-related pathways and mechanisms that participate in post-stroke inflammation, thromboinflammation, BBB breakdown, neuroprotection and recovery remain to be revealed and connected. Contrasting results may be the consequence of (1) variations in the specificity of applied P2X7 antagonists, (2) different stroke models that do or do not allow reperfusion or that are based on photothrombosis, (3) differences related to administration routes or pre- versus post-treatment, or (4) the use of nstrated : GSK P2Xnstrated , whereasnstrated . GSK P2Xko mouse , 56. Sinko mouse  still neX. laevis oocytes or HEK293 cells by electrophysiology, the ATP concentration to induce the half-maximal response (K1/2) is about ten times lower for rat P2X1 (0.82 μM) than for rat P2X4 (11 μM). Rat P2X7, in turn, has a K1/2 of 130 μM and is accordingly about ten times less sensitive to ATP than P2X4 [Of note, the ATP-gated P2X receptors expressed in the brain differ greatly in their sensitivity to ATP. As measured in transfected han P2X4 . Thus, tP2Y receptors are G-protein-coupled receptors activated by ATP, ADP, and other naturally occurring nucleoside phosphates. The most abundant P2Y receptors in the brain are P2Y12 and P2Y13, expressed at high levels in microglial cells  which is further converted to AMP by ENPP1 [On the canonical pathway of adenosine production Fig. , ecto-nuby ENPP1 . On top by ENPP1 , 76.Fig.In mouse and human brain, CD39 expression is highest on microglia followed by endothelial cells –13, 76 , whereas vascular permeability in the brain was not affected [When interpreting and extrapolating results on CD73 from experimental stroke studies conducted in mice, one has to keep in mind that CD73 is absent on murine brain endothelial cells but highly expressed on peripheral endothelial cells and human brain endothelial cells , 77, 92 affected .+CD25+Foxp3+ Tregs co-express CD39 and CD73, and adenosine generated by their coordinated activity mediates both their immunosuppressive effects on activated T effector cells and their own expansion and functionality through A2AR signaling [Beyond brain-resident cells, infiltrating immune cells play a central role in secondary neuronal injury, and their CD73 expression can also influence stroke outcome. Murine CD4ignaling , 95. In ignaling . Analogoignaling . Given tignaling , modulat+ to ADPR on the alternative pathway of adenosine formation and controls NAD+ bioavailability and activity of NAD-dependent enzymes in the brain as reviewed by Guerreiro et al. [+ levels itself [+ depletion, on the other hand, leads to mitochondrial dysfunction and oxidative stress and is a common finding in neurodegenerative disorders [+ levels in healthy humans [The ectoenzyme CD38 catalyzes, inter alia, the conversion of NADo et al. . Besideso et al. , 101. Seo et al. . Regardio et al.  and reduo et al. . CD38 kno et al. . Benefics itself , 104 as s itself . NAD+ deisorders , 106. CDisorders  complemey humans . Spontany humans . Taken t2-dependent manner [The concentration of extracellular adenosine increases substantially during in vivo ischemia. By microdialysis technique, Melani et al. measured a basal concentration of 130 nmol/l in the rat striatum which increased 10-fold within the first hour after intraluminal pMCAO and then decreased again but remained elevated until the end of the observation period 4 h after stroke induction , 109. Tht manner . Consequt manner , 111. Unt manner , and adet manner –115. Giv1R [1, A2A, A2B, and A3 receptors are the four known AR subtypes, which all belong to the superfamily of G-protein-coupled receptors and differ regarding their affinity for adenosine and tissue distribution. Apart from inhibition or stimulation of adenylate cyclase, signaling occurs through phospholipase C (PLC), calcium, and mitogen-activated protein kinases (MAPK) [1 and A2A, but during ischemia, adenosine reaches concentrations in the low micromolar range sufficient to also activate low affinity A2B and A3R [1R shows an overall higher expression in mice than in humans with a quite even distribution across neurons in cortex, hippocampus, and cerebellum, oligodendrocytes, and astrocytes. At the cellular level, A2AR expression is highest on endothelial cells followed by astrocytes in both species, and at the spatial level, A2AR density is highest at post-synaptic neurons in the striatum. In the striatum, A2ARs mainly signal through activation of stimulatory Golf [2BR in the CNS, A3R expression at the cellular level is highest on microglia in both mice and humans and at the spatial level most dense on hippocampal neurons [Adenosine release during ischemia is considered an endogenous neuroprotective response as adenosine infusion through a microdialysis probe into the rat striatum reduced neurological deficits after tMCAO . Pre- an1R , therapes (MAPK)  Fig. 3)1R [1, A2 and A3R . A1R shoory Golf . While a neurons –13, 125  neurons . A revie neurons . In the 1R signaling in response to ischemia exerts protective effects by inhibiting excitatory synaptic transmission [1R agonists  was protective in a model of global cerebral ischemia in gerbils [1R upregulation and that detrimental effects of chronic A1R agonism are due to the phenomenon of A1R desensitization [1R signaling also plays a central role in mediating the beneficial effects of ischemic preconditioning in the brain [1R agonists for clinical application, mainly receptor desensitization and cardiovascular side effects such as bradycardia and hypotension, the neuroprotective potential of partial agonists  was recently investigated and proven in oxygen-glucose deprivation (OGD) as the most frequently used in vitro model of cerebral ischemia [Asmission –129, in smission –132. Acu gerbils –135, whe gerbils . Of note gerbils . It has tization  which hatization . A1R sighe brain , 139, a ischemia .2AR antagonism (CGS 15943) on ischemic injury were first demonstrated in 1994 by Gao and Phillis after global forebrain ischemia in gerbils [2AR antagonists—namely, CSC, CP 66,713, ZM 241385, SCH 58261—when being administered before ischemia [2AR exhibited smaller infarcts 2 h, 24 h, and 48 h after tMCAO [Adora2a encoding A2AR in cM1 correlated with improved recovery [2AR signaling are largely attributed to an increase of extracellular glutamate concentrations leading to excitotoxicity, thus counteracting the depression of excitatory transmission mediated by A1R [2AR stimulation: First, it increases neuronal glutamate release during ischemia in vitro [2AR deficiency was shown to reduce striatal glutamate outflow measured by microdialysis during tMCAO and reperfusion [2AR activation on astrocytes inhibits glutamate uptake [1 and A2AR can form heteromers on striatal glutamatergic nerve terminals in which A2AR activation reduces the affinity of the A1R for agonists shifting the balance towards A2AR effects in an adenosine-rich milieu such as ischemia [Protective effects of A gerbils . In the ischemia , 143 or ischemia –146. Theischemia . In lineer tMCAO , 149 ander tMCAO . Interesrecovery . Detrimed by A1R . Severalin vitro , 153, anerfusion . Second,e uptake –157. Thiischemia .2AR antagonists exert their protective effects through inhibition of phospho-p38 MAPK [2AR activation was shown to drive microglia process retraction into an amoeboid shape upon activation which represents a hallmark of neuroinflammation and modulates their phagocytotic capacities [2AR signaling on brain endothelial cells contributes to ischemic brain injury as endothelium-specific A2AR-deficient mice also showed reduced infarct volumes 24 h after stroke [Beyond the control of glutamatergic excitotoxicity, Ap38 MAPK  and by rp38 MAPK . Moreovepacities . A2AR sir stroke .2AR agonists  also conferred protection [2AR signaling. The substantial contribution of sterile inflammation to secondary neuronal injury is nowadays widely accepted and summarized elsewhere [2AR is abundantly expressed on innate and adaptive immune cells, and its activation serves as a strong anti-inflammatory signal [2ARs even increase in density and affinity on lymphocytes from ischemic stroke patients indicating the translational relevance of this pathway [2AR agonist CGS 21680 only conferred protection when being chronically administered, whereas delivery limited to the first 24 h after stroke did not reduce infarct volume [When given systemically and chronically 13 days before global brain ischemia or until day 7 after tMCAO, Aotection , 162. Thotection  and contotection  might aclsewhere , 165. A2y signal , 167. A2t volume  as it prt volume .2AR signaling in brain ischemia depending on the timepoint and the leading mechanism of injury: A2AR antagonists reduce ischemic injury early after stroke by reducing excitotoxicity, whereas A2AR agonists confer protection when being administered for several days post-ischemia by attenuating secondary sterile inflammation. From a translational perspective, therapeutic strategies with antagonists or agonists at the A2AR should therefore be carefully evaluated in terms of timing.Taken together, research over the last decades demonstrated a dual role of A2AR signaling, far less literature is available on the role of A2BR signaling in ischemic stroke. Though ubiquitously expressed [2BR for its endogenous ligand adenosine and the lack of potent and selective agonists and antagonists has hampered investigations into its specific function in health and disease for a long time. Moreover, the role of A2BR is often masked by co-expression of A2AR, which especially holds true for monocytes and macrophages, where A2AR remains the predominant adenosine receptor. Due to recent innovations regarding pharmacological tools and molecular approaches, A2BRs are increasingly recognized as orchestrators of immunity and inflammation as reviewed by Haskó and colleagues [2BR activation increases pro-inflammatory cytokine production by mast cells, whereas it shapes the function and cytokine profile of antigen-presenting cells such as DCs and macrophages towards an anti-inflammatory and tolerance-inducing phenotype [2BR knockout mice displayed a mild but significant increase in pro-inflammatory cytokines under basal conditions [Compared to Axpressed , the lowlleagues . In the henotype . A2BR knnditions .2BR signaling plays a central role in controlling barrier function. The protective effect is even potentiated under hypoxic conditions because of adenosine accumulation and A2BR upregulation [2BR knockout mice exhibited increased vascular leakage measured by Evans’s blue extravasation in multiple organs including the brain during hypoxia, whereas A1R, A2AR, and A3R deficiency did not accentuate hypoxia-induced vascular leakage compared to wild-type littermate controls [2BR knockout mice expressed higher levels of adhesion molecules at the peripheral vasculature resulting in higher numbers of leukocytes adhering at the endothelium under basal conditions [2BR signaling on hematopoietic cells mainly accounted for A2BR-mediated attenuation of hypoxia-associated neutrophil transmigration, while vascular A2BR conferred protection to fluid leakage [On endothelial cells, Agulation , 174. Cocontrols . A2BR knnditions  and incrnditions . Bone ma leakage , 175.2BR agonist BAY60-6583 after tMCAO diminished immune cell infiltration at day 2 and reduced infarct volume at day 7 after stroke [2BR signaling are contrasted by a study indicating a rather detrimental role of central A2BR signaling: In transient global brain ischemia induced by four-vessel occlusion in rats, the selective A2BR antagonist MRS1754 reduced the phosphorylation of p38 MAPK and subsequent activation of neutral sphingomyelinase 2 which otherwise leads to pro-inflammatory ceramide accumulation in hippocampal astrocytes [2BR signaling on astrocytes seems to exert a pro-inflammatory role, while A2BR activation on immune cells and endothelial cells dampens inflammation and vascular leakage.A recent in vivo study in rats demonstrated that systemic and chronic treatment with the Ar stroke . These ptrocytes . Taken t3R agonists: Pre-treatment with the A3R agonist CI-IB-MECA either once i.c.v. 15 min or twice i.v. 165 min and 15 min before transient MCA ligation reduced infarct volume at day 2 in rats and mice [3R knockout mice exhibited larger infarcts after tMCAO than WT controls [3R agonist LJ529 twice within the first seven hours after tMCAO reduced infarct volume at day 1 in rats. This effect was only reversed by application of the A3R antagonist MRS1523 but not by co-administration of an A2AR antagonist (SCH 58261) indicating that ischemic protection was indeed mediated by A3R [1/A3R agonist AST-004 via bolus and continuous infusion starting 2 h after tMCAO limited the lesion growth rate measured by repeated magnetic resonance imaging (MRI) scans and overall stroke volume at day 5 in macaques as an example of non-human primates [3R signaling on synaptic transmission and excitotoxicity during ischemia with both agonists and antagonists exerting ischemic protection under different experimental conditions [3R signaling depending on the severity of OGD in rat hippocampal slices: While A3R agonists had an acute A1R-like inhibitory effect on synaptic transmission during a short period of OGD (2–5 min), both antagonists  and prolonged exposure to agonists  prevented or delayed anoxic depolarization and were protective during severe OGD (7 min) [3R-mediated effects from beneficial to detrimental via sustained activation of PLC and protein kinase C through Gq proteins and subsequent calcium mobilization. The yet protective effects of chronic A3R agonist treatment are a consequence of receptor desensitization in an per se adenosine-rich microenvironment [3R [Several in vivo stroke studies demonstrated a protective effect of Aand mice . In the controls . With hid by A3R . Recentlprimates . In vitrnditions . In 2007 (7 min) . It is aironment , which hment [3R , 183.3R signaling in conditions of severe ischemia explains early findings by von Lubitz and colleagues, who showed that acute treatment with the A3R agonist IB-MECA right before global forebrain ischemia increased mortality until day 7 after ischemia in gerbils, whereas chronic pre-treatment over 10 days, probably leading to A3R desensitization, enhanced neuronal survival in the hippocampus [This noxious role of Apocampus .3R agonists are also mediated by immune cells as demonstrated by Choi et al. [3R agonist LJ529 when given twice at 2 h and 7 h after tMCAO reduced the migration of activated microglia and infiltration of monocytes into ischemic cortical and striatal lesions. In a microglial cell culture, LJ529 inhibited chemotaxis of microglia and monocytes, and co-treatment with A3 and A2AR antagonists indicated that this effect was selectively mediated by A3R [3R was found to be overexpressed in inflammatory tissue and on peripheral blood mononuclear cells from patients with different autoimmune inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn’s disease [3R agonists exerted anti-inflammatory effects across different experimental models of inflammation [3R agonist CF101 even proved effective and safe in a phase II clinical trial in rheumatoid arthritis [3R agonists in ischemic stroke mounts, CF101 might indeed represent an interesting therapeutic perspective.Beyond receptor desensitization, the protective effects of Ai et al. . The A3Rd by A3R . A3R was disease , 186. Vaammation  which isrthritis . If the 3R signaling takes on a noxious role in conditions of severe ischemia such as long OGD and in vivo stroke, and the yet protective effects of chronic A3R agonist treatment are mainly attributed to receptor desensitization.Taken together, A+ concentrations through ARTC2.2-mediated ADP ribosylation, as there is no human orthologue of ARTC2.2 [Mice and humans differ in their expression and regulation of P2 receptors, particularly with respect to P2X7. P2X7 is expressed at lower levels in humans (at least on circulating T cells) than in mice, as shown, for example, for Tregs, which have a very high P2X7 expression in mice, but low expression in humans , 190. Co ARTC2.2 , 193. P2 ARTC2.2 . OverallMajor differences between mice and humans also exist in the ectonucleotidases degrading ATP to adenosine. As mentioned earlier, CD73 is highly expressed on human, but not on murine brain endothelial cells , 77, 92.Due to these differences in purinergic signaling between mice and humans, results from mouse models need to be carefully evaluated and are unlikely to be directly applicable to humans.2AR), (iii) major cardiovascular side effects , (iv) gene expression differences between mice and humans , and (v) futility of pre-treatment strategies in human stroke. Especially when aiming at reducing post-stroke inflammation, e.g., through P2X7 blockade, the rapid onset of P2X7 signaling after stroke will probably leave a narrow time window for treatment. To our opinion, the engineering of P2X7-blocking nanobodies with improved ability to cross the BBB after i.v. injection could be a huge step towards translation. Further, the application of nanobodies via the intranasal route could be a promising therapeutic approach. A high dose of P2X7-blocking nanobodies could be given as nasal spray at the first signs of stroke. The successful distribution of nanobodies via the nasal route targeting, e.g., transthyretin in the CNS has already been demonstrated in mice [A large number of preclinical studies clearly demonstrated the involvement of the ATP-adenosine axis in stroke pathophysiology reaching from excitotoxicity over post-stroke inflammation to recovery. From today’s perspective, the translation of drugs targeting key players of purinergic signaling from experimental stroke research into clinical routine is mainly hampered by (i) the BBB that constitutes a major obstacle for the i.v. application of antibody-based therapeutic constructs, (ii) a potentially dual role of certain targets depending on the timepoint and leading mechanism of injury


Ir8a, Ir25a, or Ir76b, were severely impaired in attraction to human scent but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in “mosquito magnet” human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others provides insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.Some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. We tested mosquito attraction to human skin odor and identified people who are exceptionally attractive or unattractive to mosquitoes. These differences were stable over several years. Chemical analysis revealed that highly attractive people produce significantly more carboxylic acids in their skin emanations. Mutant mosquitoes lacking the chemosensory co-receptors  Chemical analysis of the skins of humans who are exceptionally attractive or unattractive over time to mosquitoes reveals higher levels carboxylic acids in the former. These compounds are sensed by mosquito chemosensory ionotropic receptors.  Aedes aegypti is a highly efficient vector of viruses including yellow fever, dengue, chikungunya, and Zika among human populations  and three IR co-receptors . In Aedes aegypti there are 116 ligand-selective ORs and 132 ligand-selective IRs  and the ionotropic receptors (IRs) . ORs andtive IRs . Togethetive IRs . Becausetive IRs . Neverthnditions . Mosquit overall . Finallyo humans . The reco humans .Orco co-receptor is not required for discriminating between them. Mutants lacking Ir8a, Ir76b, and Ir25a retained a preference for “mosquito magnets,” but showed an overall reduced attraction to human skin odor  was passed over each stimulus to convey volatile odors to mosquitoes downwind  two additional high attractors (Subjects 24 and 31), and two subjects with intermediate attractiveness (Subjects 30 and 32)  .Aedes aegypti  and tested the behavior of heterozygous animals of all 4 strains and the heteroallelic Ir76b32/61 and Ir25aBamHI/19 null mutants. Homozygous Ir76b and Ir25a mutants had difficulty blood-feeding and Ir25a homozygous mutants generally laid fewer eggs. Notably, Anopheles coluzzii Ir76b null mutants show normal attraction to human host cues but do not blood-feed or produce eggs  to identify compounds on human-worn nylon that were associated with mosquito attractiveness . BecauseUsing unbiased feature detection and data filtering we identified 204 molecular features that were enriched on subject nylons versus unworn nylons and method blanks, across all 4 experiments . High at10.5281/zenodo.5822538).To confirm our finding that highly mosquito-attracting humans produced more abundant carboxylic acids on their skin, we enrolled 56 new human subjects in a validation study  and usedWe again performed 4 replicate metabolomic experiments . We thenWe next performed a targeted re-analysis of carboxylic acids with 10–20 carbons in individual human subjects and control samples -H. The aTo test the hypothesis that high attractors have higher levels of mosquito attractant compounds on their skin, we performed a dose-response experiment, in which we competed different sized swatches of high attractor Subject 33 nylon against a standard 5.08 cm x 2.54 cm-sized swatch of low attractor worn nylon from Subject 19 or Subject 28 . We founIn this work, we establish that the differential attractiveness of individual humans to mosquitoes is a stable over many months and is associated with the abundance of skin-associated carboxylic acids. This finding is consistent with the discovery that mice at a specific stage of infection with malaria parasites were more attractive to mosquitoes, and that infected mice showed an overall increase in concentration of many emitted volatile compounds . Highly Our behavioral assay results corroborate anecdotal evidence that context matters for how attractive a person is to mosquitoes in a real-world setting, since mosquitoes feed opportunistically. If one human walks into a highly mosquito-infested environment alone, they may receive many bites, regardless of their overall attractiveness level because they are the only feeding option. Mosquito preferences matter more in group settings. The “mosquito-magnet” in the group may receive the most bites, leaving the less attractive humans largely untouched. This suggests that mosquitoes distinguish the scent of two human samples using cues that exist along a continuum. We propose that exceptionally high or low attractiveness to mosquitoes is a “fixed” trait, caused by factors that remain constant over a period of several years, even when environmental factors are not strictly controlled. It has been shown that identical twins are more similarly attractive to mosquitoes than fraternal twins , suggestIr8a, Ir76b, or Ir25a, or Orco was able to disrupt the ability of mosquitoes to discriminate between two subjects, we speculate that there is extensive redundancy in the detection of human-derived skin odors. This may be due to central olfactory coding mechanisms or to the recently described co-expression of ORs and IRs in the same olfactory sensory neuron .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Leslie B. Vosshall  and the Rockefeller University Institutional Animal Care and Use Committee (IACUC protocol 20068-H). Human subjects gave their written informed consent to participate in this study.All subjects who participated in our studies were selected solely based on their availability and willingness to participate in frequent nylon wearing experiments. The initial cohort of 8 subjects was recruited from scientists working in or nearby the Vosshall lab. Over several months, as we collected the nylon preference dataset shown in 10.5281/zenodo.5822538), including data for the 18 subjects whose data are plotted in 64 healthy human subjects participated in this study. Age at inception of study on 12/7/2017: mean 29.8, median 29, range 19–57 years. Self-identified gender: 37 female, 26 male, 1 non-binary. Due to sample size limitations, we intentionally did not subdivide these groups further to investigate the contribution of demographic factors such as sex, age, and ethnicity, or behavioral factors such as diet, personal care, or activity levels on mosquito attractiveness, which we determined empirically. The behavior experiments in Aedes aegypti wild-type laboratory strains (Orlando and Liverpool) were reared in an environmental room maintained at 70–80% relative humidity and 25–28°C, as previously described (escribed . All aniIr25a and Ir76b mutants were generated using methods described previously (http://crispor.tefor.net/)  using the following parameters: Genome, Aedes aegypti – yellow fever mosquito – NCBI GCF_002204515.2 (AaegL5.0); Protospacer Adjacent Motif (PAM), 20bp-NGG - Sp Cas9, SpCas9-HF1, eSpCas9 1.1. For each gene, two pairs of sgRNA with predicted MIT Specificity Scores ≥95 were selected for targeted double stranded break-induced mutagenesis, with each pair flanking roughly 250 base pairs within exon 2 for Ir76b and exons 2 and 3 for Ir25a. sgRNA DNA templates were prepared by annealing oligonucleotides as previously described using the following target sequences:eviously . sgRNA sIr25a-sgRNA1: GTTGAGCTACTAACCGTCGAIr25a-sgRNA2: TACTGACAGCAAAGGGCTGTIr25a-sgRNA3: CCTACGGTTTCCGCATCAACIr25a-sgRNA4: AAGAAGGCGACTTGAGGCAAIr76b-sgRNA1: GTTACACCGAACGTCAGAAIr76b-sgRNA2: TACTCTGGTCGGACGCGGTGIr76b-sgRNA3: CTCCTTTCAATCGGGACGTGIr76b-sgRNA4: CAACGGCCAGCAGCGATACCIn vitro transcription was performed using HiScribe Quick T7 kit (NEB E2050S) following the manufacturer’s protocol. Following in vitro transcription and DNAse treatment for 15 minutes at 37°C, sgRNA was purified using RNAse-free SPRI beads , and eluted in Ultrapure water . To further facilitate isolation of loss-of-function mutants, 200 bp single-stranded DNA oligodeoxynucleotide (ssODN) donors were designed as a template for homology-directed repair (Ir25a and Ir76b), flanking an insert with stop codons in all three frames of translation and a BamHI restriction site (IDT). Since homology arms contained sgRNA target sites for the outer most sgRNA target sites (sgRNA1 and sgRNA4 for both Ir25a and Ir76b), PAM motifs for the respective sgRNAs were mutated to avoid ssODN donor cleavage. The sequences for each ssODN follow below with left and right homology arms italicized, stop codons underlined, and BamHI restriction site highlighted in bold:d repair . The ssOIr25a-ssODN:TAATAAGGATCCATAACTAAGGAACCGGCCAAGAAGGCGACTTGAGGCAATAGCGATCTCTATCAAACGTAAAAAGCAACTATCTGTTGCAGGTTTGCTACAAAACTTTATTGCGCTGAACTATATAAGAAAGAACCCAAGCCTCGGACTTTCAGTTGAGCTACTAACCGTCGAATGAAACCGTACTGACAGCAAAGGGCIr76b-ssODN:TAATAAGGATCCATAACTAAGGGTGTGGATTTTGATTCTGGTATCGCTGCTGGCCGTTGGTCCAATCATCTACGGAATGCTGATTGTGCGGTACAAAATGACCAAAGACAATCTAATTGCATCGAACTCTCTTTCCCACGTTCAACAGGACTGGCCGCTGAGTTACACCGAACGTCAGAATAGTACTCTGGTCGGACGCGAedes aegypti (Liverpool LVP-IB12 strain) embryos were injected with a mixture containing recombinant Cas9 protein  at 300 ng/μl, 4 sgRNAs at 40 ng/μl each, and donor ssODN at 125 ng/μl. Embryos were injected by the Insect Transformation Facility at the University of Maryland Institute for Bioscience & Biotechnology Research. Embryos were hatched and G0 females were crossed to wild-type Liverpool males, and their G1 offspring were screened for germline mutation by PCR amplification and Sanger sequencing the regions flanking the sgRNA target sites. Two unique stable mutant lines, each resulting in an early stop codon due to a frameshift mutation, were isolated from each injection. For Ir25a, one isolated mutant line had a 160-bp deletion with an ssODN integration (Ir25aBamHI), and the other had a 19-bp deletion (Ir25a19). For Ir76b, one isolated mutant line had a 61-bp deletion (Ir76b61), and the other had a 32-bp deletion (Ir76b32). Virgin females from each mutant line were backcrossed to wild-type Liverpool males for 8 generations prior to establishment of stable homozygous lines. To control for potential CRISPR-Cas9 off-target effects on behavior, homozygous mutant lines were intercrossed to generate heteroallelic mutants that were tested in all behavior experiments alongside appropriate genetic controls. It was previously shown that although Ir76b mutant Anopheles coluzzii females show normal attraction to human host cues, they fail to blood feed and therefore produce no offspring  cage for 30 minutes at ambient temp/humidity. These cages contained a mixed population of males and females, which had been allowed to mate freely since eclosion. Mosquitoes had access to the entire hand and forearm of the subject. For context, normal strains feed to repletion on a human arm after 5–10 minutes, even when the arm is placed outside of the cage netting and not inside the cage. Feeding of these mutants was unsuccessful when the human arm was placed against the netting on the outside of the cage. About 50% of females were engorged at the end of a given 30-minute feeding session, while the rest were partially fed or unfed. Notably, a given female took much longer to probe the arm repeatedly before successfully feeding. No attempt was made to refeed females that did not feed during a given 30-minute feeding session. Four days after feeding, an oviposition cup was placed into the cage and as many eggs as possible were collected over a 4-day period. After this stage the entire process was repeated once or twice with the same cage to obtain enough eggs to propagate the strains and carry out experiments. It is our impression that Ir25a females but not Ir76b females laid fewer eggs than wild type, but we did not investigate this further in the course of this study. Unlike the observed difficulties with the homozygous mutants, heterozygous mutants fed normally and laid normal numbers of eggs.For each target gene, approximately 500 wild-type ffspring . We alsoGenotypes were confirmed using Phire Tissue Direct PCR Master Mix  followed by gel electrophoresis and Sanger sequencing (Genewiz) using the following primer combination for each mutant allele:BamHIIr25a, 19Ir25aForward: AATACTTGAGGAGTCGTTGAATReverse: GAAGCAATGCCTTGTACTTATG61Ir76bForward: AGCCGAATATGAAGGTCAAGCReverse: CAGCACCTGTTCCTTGTCTT32Ir76bForward: TGCATCGAACTCTCTTTCCCReverse: CGATAGCTAAGATGCCAGTACATIr25aBamHI allele was detected by either a 160 bp deletion or presence of an exogenous BamHI restriction site from the donor ssODN. BamHI restriction digest of the PCR product generated a single 764 bp fragment in wild-type animals and two fragments (275 bp and 329 bp) in the Ir25aBamHI mutant. For mutants with small deletions, the presence or absence of endogenous restriction enzyme target sites was used to distinguish between mutant and wild-type alleles. PCR products were generated and digested with the indicated enzyme, producing the indicated bands in mutant and wild type:The Ir25a19 with MspIIr25a19: 764 bpWild type: 502 and 262 bp; Ir76b61 with BstUIIr76b61: 764 bpWild type: 367 and 397 bp; Ir76b32 with BstNIIr76b61: 757 bpWild type: 392 and 365bp; All genotyping experiments were performed with a no DNA control as well as fragment size validation using 1Kb Plus DNA ladder . See Ir25a and Ir76b female homozygous mutants, it is difficult to maintain these as homozygous strains. For the benefit of scientists wishing to work with these new strains, we have devised a crossing scheme that uses heterozygous mutant females to propagate the mutant alleles. An important aspect of this approach is that Ir25a and Ir76b mutants do not carry a fluorescent marker at their respective gene loci. This strategy consists of first crossing homozygous Ir25a or Ir76b mutant males to heterozygous females from the corresponding Ir25a-QF2 and Ir76b-QF2 gene-sparing knock-in driver lines , so that the modified stocking was open on both ends. Subjects wore 2 nylon sleeves on each arm: a brown experimental nylon was worn next to the skin, and a black outer nylon was placed over the experimental nylon to minimize contamination of the inner nylon. Subjects wore nylons during the day and were allowed to perform typical daytime activities but were asked not to exercise or drink alcohol while wearing the nylons. After the 6-hour wearing period, nylons were deposited in Whirl-Pak bags and kept at −20°C for 1–10 days before behavioral and chemical analysis. For the “round-robin” two-choice assay experiment in 10.5281/zenodo.5822538). Air flow and CO2 conditions for the two-choice olfactometer assay were carried as described for the Quattroport . We asked subjects to remove both nylon sleeves they wore on each arm as a single “unit”  and to place these in a Whirl-Pak bag  in the freezer. This allowed us to determine which side of the brown experimental nylon had touched the subject’s skin (the side that was not touching the black outer nylon), and we marked this side with a fine point permanent marker. The side of the nylon that touched the subject’s skin was placed facing upwards in the stimulus box, to ensure consistency between trials. In all behavior experiments, nylon samples were de-identified before being cut and presented to mosquitoes in the behavioral assay (either the two-choice assay or the single stimulus olfactometer assay), such that experimenters were blinded to the Subject ID. Each piece of nylon was attached to a flexible plastic rectangle of the same size , using small binder clips . The color of the plastic rectangle indicated the de-identified label given to that nylon, corresponding to the source of the nylon. For instance, in a given experiment, Subject 33 was designated as Subject “A” by someone other than the experimenter, and then the experimenter cut the de-identified nylon pieces and attached all Subject “A” nylons to red plastic cards. The color of the card was chosen randomly for each experiment, and colors rotated between subjects. Each nylon piece was used for only one trial and then discarded. Binder clips and plastic cards were washed with soap and water as described above and set out to air dry at the end of each experiment.Statistical analyses were performed using R. Two-choice assay data were analyzed by comparing the percent of mosquitoes attracted to each of two stimuli, for several pairs of stimuli, using Wilcoxon rank-sum tests with Bonferroni correction -E. NonpaThe attraction score reported in Nylon sleeves that had been stored at −20°C for 1–10 days were thawed and cut into 5.08 cm x 2.54 cm pieces. Experimenters always handled nylon sleeves with gloves and cleaned scissors with 70% ethanol between samples. For each subject, 6 separate pieces (5.08 cm x 2.54 cm each) of the same nylon sleeve were analyzed in each of the 4 experiments shown in 100 μL of the liquid nylon extract was manually transferred using a pipet to a new glass autosampler vial, containing 100 μL of borate buffer . First 400 μL of 100 mM pentafluorobenzyl bromide , prepared in acetonitrile  and 400 μL cyclohexane  were added sequentially to the reaction vials. Samples were then sealed using crimp caps  and heated to 65°C with shaking for 1 hour to esterify carboxyl and hydroxyl groups . Samplesm/z 50–650. For all GC/QTOF-MS experiments, the sample injection order was randomized using the “RAND” function in Microsoft Excel. The same GC column was used for data acquisition shown in GC/QTOF-MS data was collected using an Agilent 7890B gas chromatograph (GC) and an Agilent 7200 quadrupole time-of-flight (QTOF) mass spectrometer (MS), fitted with a Gerstel MPS Robotic Autosampler. 1 μL of the sample volume was injected in splitless mode onto a DB5ms column . GC conditions were as follows: initial oven temperature 60°C (hold 1 minute), ramp at 25°C/minute to 300°C (hold 2.5 minutes), ramp at 120°C/minute to 60°C (hold 1 minute); the total run time was 16.1 minutes. The inlet, transfer line, chemical ionization (CI) source, and quadrupole temperatures were set to 280°C, 300°C, 150°C, and 150°C respectively. The emission current was set to 4.2 μA and data collection was in 2 GHz mode, mass range − ion, unless the peak was considered saturated, in which case the [(M+1)-H]− isotope was used consistently across all data files. For Experiments 1.1–1.4, we calculated the median abundance of each compound in each of 4 experiments for each of 2 high attractor subjects and 2 low attractor subjects    using auTo identify compounds of interest, we used the accurate mass of the molecular ion (M-H) to predict chemical formulas using Agilent Qualitative Analysis software (v10.0). Formula prediction was constrained to C, H, O, N atoms, and formulas within 100 ppm mass error of the measured mass were considered , step 9.All statistical analyses were performed using R version 3.6.3 . Details1Supplemental Figure S1 - Related to Figure 3Ir76b and Ir25a mutant strainsPCR genotyping (A-B) Genotyping schematics (left) and agarose gel electrophoresis images (right) of PCR fragments before (left) and after (right) restriction with the indicated restriction enzyme to genotype the indicated Ir76b (A) and Ir25a (B) mutants.2Supplemental Figure S2 - Related to Figure 5Additional description and validation of GC/QTOF-MS data(A) Mechanism of pentafluorobenzyl-bromide (PFB-Br) derivatization reaction.(B) Representative extracted ion chromatograms (EICs) for the indicated compounds.(C) Abundance of several carboxylic acids in low attractors  vs. high attractors . Each dot represents abundance of the indicated compound for one subject in one experiment (median of 4 replicate samples). Nonparametric linear mixed-effects model followed by Benjamini Hochberg FDR correction (p<0.1) was used. Violins labeled by different lowercase letters were significantly different. Among 9 compounds analyzed here, all were verified to be significantly more abundant in the high attractors than low attractors, except tridecanoic acid, which was not significantly different (p=0.126).(D) Representative extracted ion chromatograms (EICs) for three control compounds: two deuterated internal standards and one nylon-derived compound, from the indicated human subjects in Experiment 1.1.(E) Abundance of control compounds shown in (C). Each dot represents the abundance (median of 4 replicate samples) of the indicated compound for one subject in 1 of 4 experiments: Experiments 1.1–1.4).4Supplemental Figure S4 - Related to Figure 6Intra-individual stability of skin chemistryA) Timeline of behavior (green blocks) and GC/QTOF-MS experiments (purple blocks), relative to one another. 64 human volunteers participated in this study, and nylons from 18 of these were analyzed by GC/QTOF-MS. Nylons from four of these subjects  were repeatedly tested behaviorally over a three-year period and analyzed using GC/QTOF-MS in 2 sets of 4 replicate experiments  that were conducted 1 year apart.B) Heatmaps depicting quantified abundance of carboxylic acids with 10–20 carbons, averaged across 4 replicate samples per experiment, in 4 subjects. Each heatmap represents one of 8 independent experiments. Experiments 1.1–1.4 were conducted about a year before Experiments 2.1–2.4.(C) Heatmaps depicting quantified abundance of carboxylic acids with 10–20 carbons, averaged across 5 replicate samples per experiment, in 18 subjects from the validation cohort. Each heatmap represents one of 4 independent experiments, conducted about a week apart. Representative extracted ion chromatograms (EICs) for three control compounds: two deuterated internal standards and one nylon-derived compound, from the indicated human subjects in Experiment 2.1.(B) Abundance of control compounds shown in A. Each dot represents the abundance of the indicated compound for one subject in one experiment (median of 4 replicate samples).(C) Extracted ion chromatograms of three identified hit compounds, overlaid with extracted ion chromatograms of the same sample spiked with a known standard.


Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes. Influenza virus is a public health issue affecting millions of people globally each year. Commercial influenza vaccines are based on the hemagglutinin (HA) surface glycoprotein, which can change antigenically every year, demanding the manufacture of newly matched vaccines annually. HA stem epitopes have a higher degree of conservation than HA molecules contained in conventional vaccine formulations, and we demonstrate that we are able to design nanoparticles that display hundreds of HA stem fragments on nanoparticles. These designed nanoparticles are heat-stable, elicit antibodies to the HA stem, confer protection in mouse challenge models, and show cross-reactivity between HA subtypes. This technology provides promising opportunities to improve seasonal vaccines, develop pandemic preparedness vaccines, and facilitate the development of a universal influenza vaccine. Influenza virus infects millions of people worldwide every year and there is the ever-present threat of a new influenza pandemic causing worldwide deaths, exemplified by the 1918 influenza virus that killed millions –5 and elAll influenza HA subtypes have a similar protein structure  16,17].,17.16,17One global goal in public health is the development of a universal influenza vaccine ,25 that Nanoparticle vaccine designs for influenza have been previously attempted, including a construct targeting the H1 HA stem fragment. This fragment was grafted on to the small carrier B2 and displayed on the Flock House Virus (FHV) scaffold . HoweverIn this work, we designed a novel HA-stem nanoparticle platform using structure-guided techniques. Bioinformatics was used to identify a conserved peptide fragment of influenza HA, known as helix-A  for H1-H16 HA subtypes. The helix-A region, which contains 22 residues, was then isolated from the HA sequences. Helix-A consensus sequences representing HA subtypes were found to be conserved and sequence identity ranged from 50% to 100% between the HA subtypes  involve not only helix-A but other regions of HA2 and HA1 in the context of prefusion HA . Second,We created a library of 27 selected sequences to represent HA sequence diversity. The library contains at least one helix-A sequence from each HA (H1-H16) subtype . BioinfoWe designed nanoparticles that integrated sequences from our helix-A library into the exposed immunodominant loops of the HBV capsid scaffold . This scTo facilitate correct nanoparticle folding, we integrated flexible linkers flanking the helix-A insertions . HomologNanoparticles, one without any inserted antigen (scaffold) and one with helix-A from the H1N1 2009 pandemic influenza virus , were produced and purified. Purification of both scaffold and H1 nanoparticles by gradient centrifugation resulted in similar banding patterns consistent with the production of particles in excess of megadalton size . The purThe structures of the scaffold and H1 nanoparticle were found to be similar in size and shape . Cryo-elThe strong density of the core of the H1 nanoparticle observed by cryo-EM suggested that the nanoparticle may be a stable and robust platform for epitope display. To test the impact of temperature fluctuations on nanoparticle stability, purified H1 nanoparticles aliquots underwent different thermal conditions. Aliquots were either stored at 4°C, as would be done similar to a vaccine cold chain or heated to temperatures of 40°C or 90°C for one hour. All aliquots were then equilibrated back to room temperature. While there are some visual differences like levels of stain penetration  and someA mouse model was used to assess the immunogenicity of purified nanoparticles with and without heating . Enzyme-Sera from mice immunized with H1 nanoparticle kept at 4°C were analyzed against different H1 HA recombinant proteins by ELISA and western blot. ELISA analyses showed the mouse sera was able to bind trimeric full-length HA (HA0) and the trimeric HA ectodomain which lacks the transmembrane region of HA, but it did not bind to recombinant HA1 head protein (rHA1), which does not contain the HA stem region . The facTo probe stem binding, we used a group 1 stem (H1) construct of a headless stem trimer composed mostly of HA2 which has been previously used as a probe for stem antibodies in sera ,60. In wH1 nanoparticles were used to immunize mice in the presence and absence of adjuvant, Sigma Adjuvant System (SAS). Addition of SAS increased overall antibody levels . However, after boosting there is no statistical difference in the antibodies elicited to the nanoparticle with or without adjuvant, p = 0.1233  activity and microneutralization (MN) activity . Prior tEncouraged by the production, heat-stability, and immunogenicity of the helix-A H1 nanoparticle Figs  and 3, tTo further evaluate additional HA helix-A nanoparticles subtypes from the observed expressions , H7-nanoBecause certain monoclonal antibodies like FI6v3  can bindCross-reactivities of the H1 and H7 nanoparticle sera to H1 HA (group 1) and H7 HA (group 2) proteins were analyzed by ELISA. When binding to H7 HA was compared, both homosubtypic reactivity of H7 nanoparticle sera  with an HBV nanoparticle scaffold via a flexible linker region. We assessed the feasibility of design and production, analyzed the structure and stability, and investigated the immunogenicity and protective capacity of the engineered nanoparticles. Our results indicate that nanoparticles displaying the helix-A from H1 HA can be expressed and purified from a simple bacterial expression system, are heat-stable and are immunogenic for both group 1 and group 2 HA subtypes. Furthermore, this platform can be expanded to express helix-A nanoparticles for most HA subtypes, producing a library of vaccine candidates. Additionally, our nanoparticle displayed heat resistance immunogenicity and retained its particle structure with longevity. These attributes will aid in the development of vaccines with increased stability, having the potential to increase shelf-life.An emerging strategy in vaccine development is to use symmetrical nanoparticles to display a viral antigen with multiple copies displayed, multivalency, to the immune system ,32,61–63When choosing a nanoparticle scaffolding, the risk of eliciting antibodies that could be cross-reactive to human antigens via vaccine-induced autoimmunity should be minimized. Scaffolds of bacterial origin such as ferritin, encapsulin, and lumazine synthase maximize sequence divergence from similar proteins in humans to avoid this consequence ,62. EvenAccessibility of the antigen-presenting epitope is an important consideration in designing nanoparticle vaccine candidates. Compared to a previous study which grafted helix-A onto a scaffold of flock house virus which found 20% protection in mice , our nanHeat stability is another advantage of our helix-A nanoparticle design . CurrentConcern over vaccine and antigen stability is not unique to influenza vaccines. Trimeric viral glycoproteins like HIV Env were unstable and required the introduction of mutations and disulfide bonds for native-like trimer stabilization to produce SOSIP HIV Env trimers comparable to those displayed on the viral surface . Only afOne advantage of our helix-A nanoparticle design is that there is no need to mutate and then screen the viral glycoprotein sequences for trimer stabilization as the previous work by Yassine et al.  and ImpaOur H1 helix-A stem nanoparticle is immunogenic and protects from homosubtypic influenza virus challenge in mice. While the mechanism of protection is not fully elucidated, we found that the helix-A stem nanoparticle elicited antibodies that bound the rHA2 construct, indicating they were stem-directed antibodies . HemagglThe impact of adjuvant on the helix-A stem nanoparticle system was confounding as it increases immunogenicity of sera , but it The research and the use of nanoparticle-based vaccines are increasing. For example, COVID-19 vaccines based on lipid nanoparticles have focused attention on the potential applications of different types of nanoparticle vaccines ,89. In fOur study utilized the squalene-based adjuvant SAS which increased the total number of antibodies to the H1-nanoparticle, but these additional antibodies were not productive for conferring protection. While the mechanism of action of this adjuvant has not been extensively studied, MF59, a similar squalene-based adjuvant ,90 that The nanoparticle design concept described here in detail for H1, H5, and H7 can be applied more broadly to other influenza A HA subtypes, as we have demonstrated an expression library for the majority of influenza A HA subtypes . PresentSeveral approaches have been suggested to increase the breadth of the influenza vaccine response such as the addition of multiple HA subtype antigens to the vaccine mixture or formulations targeting cross-protective epitopes. A broadly efficacious vaccine could safeguard against antigenic drift of seasonal viruses as well as offer protection from emerging subtypes that currently do not circulate. In this study, we showed that the pandemic H1 and H7 helix-A nanoparticles elicited antibodies that could bind to phylogenetically distinct group 1 (H1) and group 2 (H7) HA proteins . When alWe have also not yet fully validated our mAbs that were isolated via helix-A nanoparticle immunizations to determine their breath of cross-reactivity and protection against additional HA subtypes, such as those with pandemic potential like H5 and H9. Additional, cryo-EM experiments would allow for the mapping of the binding epitope of our discovered mAbs which would give greater insight to how they confer protection. Once more is known about how these mAbs bind to the stem region they could possibly be used as prophylaxis with further development.In conclusion, our results indicate that helix-A sequences are conserved and can be displayed as antigenic epitopes on a nanoparticle scaffold. Using H1-nanoparticles as a proof of concept, we showed that the helix-A epitope is immunogenic and that the nanoparticles were protective in an H1N1 viral challenge model. Furthermore, the elicited antibodies exhibited homosubtypic and heterosubtypic binding between group 1 and group 2 HA proteins and isolated mAbs were cross-reactive to both H1 and H7 subtypes and could protect against H1N1 infection. We also established an expression library of helix-A nanoparticles for the majority of Influenza A HA subtypes. Due to the broad array of influenza subtypes that can be targeted and their robust properties, our results suggest that helix-A nanoparticle immunogens should be further explored to aid in the development of more efficacious and broadly protective influenza vaccines, which is a milestone on the path towards a universal influenza vaccine.All applicable international, national, and institutional guidelines for the care and use of animals were followed. All mouse experiments were performed under approved protocols. Approval was granted by the Animal Care and Use Committee (ACUC) at the National Institute of Allergy and Infectious Diseases (NIAID).fludb.org influenza hemagglutinin (HA) sequences was categorized. The database was divided into individual databases for H1 to H16, the amino acid sequence distributions of the helix-A regions were compiled for each HA subtype. The consensus sequence was determined per HA subtype. Consensus sequences were made by downloading sequences from the influenza research database (https://www.fludb.org), parsing the helix-A sequence and cataloging their respective subtypes by using Python macros, determining the consensus sequence of each subtype based on the helix-A sequences and by comparison of consensus sequences to produce a comparison matrix (Microsoft Excel macros). Further, the identity and occurrence of each variation from the consensus sequence was classified. Sequence identities were performed with EMBOSS-lite (formerly GCG-lite). Individual amino acid differences and total sequence identity variation between H1-H16 consensus sequences was determined using a matrix to compare sequence identifies between the helix-A consensus sequences (In order to better determine the antigenic variation in the helix-A stem region (A-helix), a time snapshot of the amino acid sequences for all equences .Helix-A nanoparticles were designed by making chimeric protein sequences by inserting two tandem copies of helix-A from HA stem regions into a HBV capsid monomer. Twenty-seven naturally occurring helix-A sequences were chosen from the influenza database to represent H1-H16 subtypes  These 27g for 30 min and resuspended in ice-cold PBS and then sonicated to release the crude nanoparticle. Cellular debris was removed by pelleting via centrifugation at 16,300xg for 10 min. The crude supernatant containing nanoparticles was then precipitated with ammonium sulfate at 45% saturation via incubation overnight at 4°C, and the precipitant was resuspended in PBS. This nanoparticle preparation was then layered on a gradient with 30%–50%–70% sucrose steps and centrifuged at 34,000 rpm using a SW55 Ti rotor for 2.5 hours. Sucrose fractions were collected, and the location of the nanoparticles were identified by SDS-PAGE. Fractions containing the nanoparticles were dialyzed overnight in PBS and then purified in three sequential CsCl gradients centrifuged in an SW55 Ti rotor at 38,000x rpm for 24 hours. The gradient band containing the nanoparticles were collected and dialyzed in PBS overnight. HBV capsid particles were purified in a similar fashion as H1 and H7 nanoparticles. Endotoxin was removed from the samples by adding 2.5% Triton X-114, mixing at 4°C for 1 hr, incubating at 37°C for 10 min, centrifuging at 16,500xg for 10 min at 25°C, and collecting the upper aqueous phase. Three cycles of Triton X-114 endotoxin removal were performed per purification. Endotoxin removal was confirmed by using the LAL chromogenic Endotoxin Quantitation Kit  according to the manufacturer’s instructions. Similar steps were done for the H5 nanoparticle purification.For production and purification of helix-A nanoparticle , the H1 and H7 constructs  were subcloned into pET21b expression vectors to create . These plasmids were transfected into Rosetta 2 cells and this system was used for expression according to manufacturer’s instructions (EMD Millipore) for expression. The E. coli Rosetta cells were grown in 2xYT liquid media at 37°C until the optical density (OD) at 600 nm was 0.8. Cells were then induced with 1 mM IPTG and expressed overnight at 25°C. Cells were then pelleted at 3300xNanoparticles were heat treated for stability and immunogenicity assessments. Aliquots of purified nanoparticles in PBS were maintained at 4°C, incubated at 40°C for one hour, or incubated at 90°C for one hour. Each of the three aliquots was then equilibrated to room temperature. One fraction of each aliquot was then used for negative-stain electron microscopy analysis and another fraction used for immunogenicity studies. To study long-term stability of nanoparticles, microliter aliquots of H1 and H7 nanoparticles that had been stored at 4°C for 3 years were used for negative-stain electron microscopy analysis.Antigen was applied to 96-well plates and incubated overnight at 4°C (1.25 μg/mL), following PBS washes and blocking , primary antibody was incubated for 2 hr at room temperature . Primary monoclonal antibodies were stocks at 1mg/ml and for ELISAs primary mAbs C179, FI6v3, and CR6261 was used at 0.5 μg/ml while discovered antibodies mAb-11, 49, 63, 67, 85 were tested at both 0.5 and 5 μg/ml as primary antibodies. Plates were washed and placed at 37°C for 1 hr with the addition of an HRP conjugated secondary antibody (goat anti-mouse IgG (H+L), Thermo Fisher Scientific). Colorimetric detection occurred for 15 min at room temperature and absorbance was read at 405 nm . Samples were run in quadruplicate. Endpoint titers levels were statistical defined per plate using saline sera to determine thresholds . StatistPurified recombinant full-length HA proteins were from Protein Sciences Corporation, Meriden, CT and HA ectodomains were from the International Reagent Resource and Sino Biological. Headless trimeric H1 stem protein was kindly provided by Jeffrey Taubenberger . HA protAll mouse experiments were performed under protocols approved by the Animal Care and Use Committee (ACUC) at the National Institute of Allergy and Infectious Diseases (NIAID). A combination of 30 female and 30 male BALB/c mice (Charles River Laboratories), aged 8–10 weeks were randomly assigned to antigen groups and underwent two immunizations (Day 0 and Day 21) and tail bleeds (Day 0 and Day 14) prior to a terminal bleed on Day 35. Mice were injected intramuscularly with 50 μL of antigen at a concentration of 0.5 mg/mL for a final experimental dose of 100 μg/mouse. 30 male mice were used for the heat-treatment immunogenicity studies following the same regimen. The H7-nanoparticle immunogenicity experiments were performed on 5 female BALB/c mice. Similar steps were done for the H5-nanoparticle immunogenicity. No sex differences were observed.50 ) of influenza H1N1 . Mice were observed twice daily for survival criteria  until Day 56, when all surviving mice were humanely euthanized. Differences in survival rates were compared using a Kaplan-Meier survival analysis (Graph Pad Prism).Experiments involving virus challenge were conducted using protocols approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee. 15 female BALB/c mice, aged 8–10 weeks, underwent the same inoculation protocol as the immunogenicity experiments’ (injections Day 0 and Day 21). On Day 35 mice were anesthetized and intranasal challenged with 10x LDTransformed E. coli cells containing plasmid expression vectors for 16 nanoparticle constructs representing H1-H16 HA subtypes were seeded into 4 mL of 2XYT liquid medium and induced with 1 mM IPTG to express overnight. Cells were subsequently isolated from medium via centrifugation (5000 rcf for 10 min) and lysed with B-PER (Thermo Fisher Scientific) for 15 min at room temperature; the content of the cell lysates were separated by centrifugation (16000 rcf for 7 min). Nanoparticle production was confirmed by Western blot as described above using an epitope tag antibody (10E11) to the scaffold (capsid) region (aa. 2–10) that is in all the constructs. Helix-A sequences in the nanoparticle constructs where from the following influenza viruses: A/California/4/2009 (H1N1), A/Japan/305/1957 (H2N2), A/Brisbane/10/2007 (H3N2), A/Bufflehead/California/Hkwf205/2007 (H4N8), A/Vietnam/1203/2004 (H5N1), A/Chicken/NewYork/14677-13/1998 (H6N2), A/Anhui/01/2013 (H7N9), A/Turkey/Ontario/6118/1968 (H8N4), A/HongKong/1073/1999 (H9N2), A/Americangreen-wingedteal/Alaska/44160-020/2006 (H10N7), A/Duck/Memphis/546/1974 (H11N9), A/Duck/Alberta/60/1976 (H12N5), A/Gull/Maryland/704/1977 (H13N6), A/Mallard/Gurjev/263/1982 (H14N5), A/Wedge-tailedshearwater/Westernaustralia/2576/1979 (H15N9), A/Shorebird/Delaware/172/2006 (H16N3). Note for the selected sequences H4 and H14 had the same helix-A sequences as well as H10 and H15.Spleens were isolated and used fresh for flow cytometry staining experiments. Spleen cells were washed with RPMI containing 10% fetal bovine serum and 1% Penicillin-streptomycin  and PBS and stained for flow cytometric sorting using the following antibodies and probes: F4/80 (Clone BM8), Gr-1 (Clone RB6-8C5), CD4 (Clone GK15), CD8a (Clone 53–67) BV510, B220 (Clone RA3-6B2) BV421, IgM (1B4B1) PECy7, IgG1 (Clone A85-1), IgG2a/b (Clone R2-40), IgG3 (Clone R40-82) FITC, H1 California09 PE, H7 Shanghai13 APC. LIVE/DEAD Fixable Aqua Dead Cell Stain  was included to identify non-viable cells. Following staining, cells were washed with PBS containing Fetal Bovine Serum (2%) and acquired on the BD FACS Aria sorter. H1 California09 and H7 Shanghai cross-reactive IgG memory B cells were identified as F4/80- Gr-1- CD4- CD8- B220+ IgM- IgG+ H1 California09+ and H7 Shanghai+ cells and sorted into 96-well plates (1 cell per well) for cDNA synthesis and immunoglobulin heavy and light chain amplification by PCR. Flow cytometry data analysis was done using FlowJo. The source of the H1 and H7 probes were similar as to previously described by Whittle et al. [Note: Additional materials and methods are presented in S1 Fig(A-C) Epitope footprints of three broadly-reactive stem antibodies to influenza HA were colored to illustrate the extent of the bound Fab footprint on HA. HA1 is colored red, HA2 is colored blue, and the A-helix is colored green. Overlaid on the HA structure is the footprint for antibodies (A) C179, (B) CR6261, and (C) FI6v3, with respective PDB codes 4HLZ, 3GBN, and 3ZTJ. Footprints were depicted as HA atoms within 5 Å of Fab atoms, hydrogen atoms excluded. Footprints for each broadly neutralizing antibody extend beyond the helix-A residues. C179 is a mouse HA stem antibody while CR6261 and FI6v3 are human stem antibodies. C179, CR6261, and FI6v3 epitope footprints not only involve helix-A residues but also regions outside of helix-A such as portions of HA2 and HA1.(TIFF)Click here for additional data file.S2 FigHemagglutinin sequences  (H1 to H16) were downloaded from the influenza sequence database. Sequences were grouped according to subtype and the helix-A region for each sequence was extracted. Consensus sequences for the helix-A for each HA subtype was derived by comparison of all sequences within a group. Pair-wise sequence identity comparisons between different HA subtypes (H1-H16) were then organized as a matrix. Identities ranged from 50% to 100% for comparison between two different subtypes. Consensus sequences were only used in sequence alignment analyses to compare the sequence identities between over fifty thousand helix-A sequences spanning different HA subtypes (H1-H16) by using a matrix comparison. For actual helix-A nanoparticle construct design, screening and purification, helix-A sequences in stem nanoparticles were direct HA sequences from each subtype (H1-H16 HA) based on a bioinformatic HA library covering HA sequences .(TIFF)Click here for additional data file.S3 Fig(A) Over 50,0000 sequences of HA from the influenza database were downloaded and curated into about 36,000 helix-A sequences for the 16 HA subtypes. A cDNA sequence library was created containing 27 HA helix-A nanoparticle sequences encompassing the HA subtypes. (B) Bioinformatic analysis using sequence identity threshold cut-offs when comparing the helix-A regions from the 27 HA-nanoparticle sequence library to the larger helix-A sequence database of over 36,000 HA sequences. Sequence identity threshold cut-offs are shown with each corresponding percent coverage of the larger helix-A sequence database.(TIFF)Click here for additional data file.S4 Fig(A) HBV capsid (scaffold) without helix-A insertions. The capsid sequence is represented by gray box regions. The immunodominant loop (c1 epitope) is denoted by an asterisk. (B) Construct for H1-nanoparticle (H1-nano) consists of two copies of helix-A of influenza H1 HA (CA09) inserted into the scaffold at the immunodominant loop region to flank the c1 sequence on both sides. Each copy of helix-A is represented by a green box. Each helix-A sequence is flanked by linker sequences (white boxes). The constructs contain an endogenous epitope tag for antibody 10E11 (black box) that recognizes residues 1–10 at the N-terminus of the capsid scaffold. (C) Diagram key to indicate the schematic representations for each region: HBV capsid scaffold, helix-A epitope insertion site into immunodominant lope (c1 epitope) of capsid scaffold, helix-A, linkers, and epitope tag. (D) Western blot to probe for expression of capsid scaffold (without helix-A) and H1-nanoparticle , respectively. Antibody 10E11 was used as primary antibody to detect the sequence tag. Molecular weight standards are denoted (std) and bands for scaffold and H1-nanoparticles are denoted with asterisks.(TIFF)Click here for additional data file.S5 Fig(A) Multiple molecular models of a dimeric unit of the H1-nanoparticle with the HBV capsid scaffold (gray) being fixed relative to the helix-A stem epitope having different conformations and orientations . The scaffold monomers (gray) each have two copies of HA helix-A inserted into the tip of the loop of the capsid protein. (B) A molecular model for the H1-nanoparticle with helix-A stem epitopes in different positions as indicated in panel A. For clarity all helix-A portions are green with the scaffold in gray.(TIFF)Click here for additional data file.S6 Fig(A) SDS-PAGE analysis of purified HBV capsid (scaffold) purification fractions. The first lane contains molecular weight standards (std) and subsequent lanes contain gradient fractions for increasing sucrose concentrations. A white arrow indicates the bands at about 20 kDa. (B) SDS-PAGE analysis of H1-nanoparticle construct purification fractions. The first lane contains molecular weight standards (std) and subsequent lanes contain gradient fractions for increasing sucrose concentrations. A white arrow indicates the H1-nanoparticle protein bands at about 25 kDa. (C) Negative-stain electron microscopy of purified HBV capsid (scaffold). Black arrows indicate capsid scaffold particles. Scale bar 50 nm. (D) Negative-stain electron microscopy of purified H1-nanoparticles. Black arrows indicate H1-nanoparticles. Scale bar 50 nm.(TIFF)Click here for additional data file.S7 Fig(A) Cryo-electron microscopy images of HBV capsid (scaffold) particles and (B) H1-nanoparticles. (C) 3D reconstruction cross-section for HBV capsid scaffold by cryo-electron microscopy. Scale bar is 5 nm. (D) 3D reconstruction cross-section for H1-nanoparticles by cryo-electron microscopy. Arrows denote the location of the insertion, where diffuse density is observed compared to the HBV capsid scaffold. Scale bar is 5 nm. H1-nanoparticle subunits that comprise the nanoparticle give the appearance of spikes on the surface. (E) Negative-stain electron microscopy of H1-nanoparticles incubated without Fabs and (F) incubated with Fabs derived from polyclonal sera from mice immunized with H1-nanoparticles. Scale bar is 100 nm. (G) Images of 2D class-averages from 591 PTA stained H1-nanoparticles classified into 4 classes. The best resolved class (upper-right) illustrates a protein shell, measures about 7 nm thick, and encapsulates a slightly darker core. H1-nanoparticle subunits that comprise the nanoparticle give the appearance of undulations of the particle surface when visualized by PTA stain. (H) Images of 2D class averages from 308 PTA stained H1-nanoparticles in complex with polyclonal Fabs were classified into 4 classes. The extra density surrounding the H1-nanoparticles, visible in all 4 averages, is consistent with a coat of bound polyclonal Fab. Scale bars are 10 nm. Note, for ease of comparison between HBV capsid (scaffold) structure and H1-nanoparticle, panel D is the same as main (TIFF)Click here for additional data file.S8 FigComparison of sera reactivity to (A) scaffold, (B) H1-nanoparticle (H1-Nano) and (C) full-length recombinant H1 HA protein from mice immunized with temperature-treated H1-nanoparticles (H1-Nano) via ELISA with serially diluted sera consisting of PBS (black) and H1-nanoparticle exposed to three temperatures .(TIFF)Click here for additional data file.S9 Fig(A) ELISA binding analysis of stem monoclonal antibodies  binding to scaffolding, H1 nanoparticle, and H1 rHA full-length protein. (B) ELISA used to probe the binding of monoclonals antibodies mAb_11 (black), mAb_49 (olive), mAb_63 (magenta), mAb_67 (gray) and mAb_85 (brown) at lower concentration to recombinant H1 and H7 proteins.(TIFF)Click here for additional data file.S10 Fig50) of H1N1 virus. (B) Time course of hemagglutination-inhibition activity (HAI) for mouse sera. (C) Time course of microneutralization (MN) activity for mouse sera. Greater than 10 HAI and >20 MN were chosen because these were above judged background levels of the assays. Since the sera were taken pre-challenge the denominators are 10 and for day 56. Only serum from challenge survivors were tested and thus there are lower denominators. Note, for ease of comparison S10A is a similar immunization figure as main (A) Schedule for mouse immunization and challenge. Mice were immunized on days 0 and 21 with H1-nanoparticle with and without adjuvant. Mice were challenged on day 42 with 10X Mouse Lethal Dose (MLD(TIFF)Click here for additional data file.S11 FigSaline sera tested for reactivity to different recombinant HA proteins: H1, H2, H5, H7, H10, and H15. Standards are denoted (std).(TIFF)Click here for additional data file.S12 Fig ELISA binding of sera from mice immunized with different nanoparticles  displayed in the main figures with the additional data point (red) depicting a respective HA polyclonal positive control at 5ug/ml, displayed at the highest concentration of sera. (A) INA414, Novus Biologicals, (B) PS6000, Protein Sciences, (C) PS6003, Protein Sciences, (D) PS6000, Protein Sciences, (E) PS6000, Protein Sciences. Anti-HA polyclonals were to those that reacted to their respective HA proteins .(TIFF)Click here for additional data file.S13 Fig(A) Probing for detectable ADCC activity for mouse monoclonal stem antibody C179 (positive-control), H1 nanoparticle mouse sera and control mouse sera. The reporter assay was a mouse FcγRIV ADCC Bioassay (Promega) with the target cells being A549 cells infected with influenza A/California/04/09 H1N1 and effector cells being a genetically engineered Jurkat T cell line that expresses mouse FcγRIV receptor and a luciferase reporter driven by an NFAT-response element (NFAT-RE). Co-culturing with a target cell and using an appropriate bridging antibody that can bind both antigen on target cells via Fab regions and can bind Fc receptors (a mouse FcγR) on effector cells via antibody Fc regions results in mouse FcγRIV signaling and NFAT-RE-mediated luciferase activity plotted as fold induction. C179 displayed ADCC activity while the H1 nanoparticle and control sera did not. . Assessment of antibody class and subclass identity in three samples: (B) C179 antibody, (C) H1 nanoparticle mouse sera, and (D) control mouse sera by the use of a mouse antibody isotyping cassette-based assay . Cassettes provide bands as a color-readout on the presence of antibody isotypes. The isotypes represented by the abbreviations on the cassettes are detailed below the cassettes. For example, G1 is IgG1, etc. (E) Samples and detected isotypes.  Note C179 is a stem monoclonal antibody that is IgG2a but purified from hybridoma from mouse ascitic fluid which may explain the presence of both IgG2a and IgG1 bands.  Both H1 nanoparticle and control sera samples displayed bands corresponding to multiple isotypes. For example, H1 nanoparticles sera had bands for IgG1, IgG2a, IgG2b, IgG3, IgA, IgM. Note: We confirm that we are the photographers for panels B, C, and D.(TIFF)Click here for additional data file.S14 Fig(A) SDS-PAGE of monoclonal antibody mAb-11 under reducing (R) and non-reducing conditions (NR). The molecular weights of the standards (M) are denoted. Similar SDS-PAGE analyses of monoclonal antibodies (B) mAb-49, (C) mAb-63, (D) mAb-67, (E) mAb-85. Samples under reducing conditions had dithiothreitol (DTT) added to the samples and non-reducing did not. Under reducing conditions bands appear consistent with heavy chains (~50kDa) and light chains (~25kDa). While under non-reducing conditions bands appears at ~150kDa which is consistent with a disulfide liked IgG consisting of with 2 heavy and 2 light chains (~150kDa).(TIFF)Click here for additional data file.S15 Fig(A) Structure of H1 CA09 in complex with Fabs of the broadly reactive stem human antibody FI6v3 (PBDID: 3ztn). HA1 is red and HA2 is blue with helix-A in forest green. (B) Binding to recombinant H1 HA CA09 via competition ELISA with increasing concentrations of antibody FI6v3 against mouse monoclonal antibody mAb-11. Increasing concentrations of FI6v3 deceases the binding of mAb-11 to H1 HA. FI6v3 and mAb-11 were primary antibodies with HRP conjugated secondary antibodies (goat anti-human IgG and goat anti-mouse IgG).(TIFF)Click here for additional data file.S16 FigMonoclonals mAb11, mAb85, mAb49, mAb63, mAb69 were from this study and like the stem antibody mAb FI6v3, the panel of discovered mAbs did not show detectable hemagglutination inhibition activity. Two negative controls were normal control goat serum  and monoclonal antibody FI6v3 . Three positive controls were a goat antiserum influenza A(H1N1) pdm09 , showing a HAI titer of 640, a pool of mouse monoclonal antibodies against influenza A(H1) pdm09 viruses (FR-572), showing a HAI titer of 80 and a pool of anti-influenza A mAbs (FR-1217), showing a HAI titer of 20. FR-reagents were from the International Reagent Resource. Back titrations are controls to make sure 4HAU/25ul are in each well.(TIFF)Click here for additional data file.S17 Fig50  of H1N1  virus on day 42. (B) Survival curves for mice immunized with saline control (black) or commercial vaccine (purple). (C) Weight-loss curves for challenged mice that were immunized with saline control (black) or commercial vaccine (purple). Trivalent Flublok contains recombinant H1 HA CA09, H3 HA, and influenza B HA (Victoria-lineage-like).(A) Schedule for mouse immunization with commercial vaccine Flublok (2016) containing rHA for H1, H3, and B influenza strains or saline control. Groups of mice (N = 5 per group) received intramuscular injections day 0 and 21. Mice were challenged with 10x MLD(TIFF)Click here for additional data file.S18 Fig(A) Probing for detectable ADCC activity for mouse monoclonal stem antibody C179 , mouse monoclonal antibodies  and control mouse sera (dashed black line). The reporter assay was a mouse FcγRIV ADCC Bioassay (Promega) with the target cells being A549 cells infected with influenza A/California/04/09 H1N1 and effector cells being a genetically engineered Jurkat T cell line that expresses mouse FcγRIV receptor and a luciferase reporter driven by an NFAT-response element (NFAT-RE). Co-culturing with a target cell and using an appropriate bridging antibody that can bind both antigen on target cells via Fab regions and can bind Fc receptors (a mouse FcγR) on effector cells via antibody Fc regions results in mouse FcγRIV signaling and NFAT-RE-mediated luciferase activity plotted as fold induction. C179, mAb_49, and mAb_11 displayed ADCC activity while mAb_63, mAb_67, mAb_85, and control sera did not.(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.