Multivalent and Sequential Heterologous Spike Protein Vaccinations Effectively Induce Protective Humoral Immunity against SARS-CoV-2 Variants.

The emergence of new SARS-CoV-2 variants continues to cause challenging problems for the effective control of COVID-19. In this study, we tested the hypothesis of whether a strategy of multivalent and sequential heterologous spike protein vaccinations would induce a broader range and higher levels of neutralizing antibodies against SARS-CoV-2 variants and more effective protection than homologous spike protein vaccination in a mouse model. We determined spike-specific IgG, receptor-binding inhibition titers, and protective efficacy in the groups of mice that were vaccinated with multivalent recombinant spike proteins (Wuhan, Delta, Omicron), sequentially with heterologous spike protein variants, or with homologous spike proteins. Trivalent (Wuhan + Delta + Omicron) and sequential heterologous spike protein vaccinations were more effective in inducing serum inhibition activities of receptor binding to spike variants and virus neutralizing antibody titers than homologous spike protein vaccination. The higher efficacy of protection was observed in mice with trivalent and sequential heterologous spike protein vaccination after a challenge with a mouse-adapted SARS-CoV-2 MA10 strain compared to homologous spike protein vaccination. This study provides evidence that a strategy of multivalent and sequential heterologous variant spike vaccination might provide more effective protection against emerging SARS-CoV-2 variants than homologous spike vaccination and significantly alleviate severe inflammation due to COVID-19.

Cross-protection against influenza viruses by chimeric M2e-H3 stalk protein or multi-subtype neuraminidase plus M2e virus-like particle vaccine in ferrets.

Current influenza vaccine is not effective in providing cross-protection against variants. We evaluated the immunogenicity and efficacy of multi-subtype neuraminidase (NA) and M2 ectodomain virus-like particle (m-cNA-M2e VLP) and chimeric M2e-H3 stalk protein vaccines (M2e-H3 stalk) in ferrets. Our results showed that ferrets with recombinant m-cNA-M2e VLP or M2e-H3 stalk vaccination induced multi-vaccine antigen specific IgG antibodies (M2e, H3 stalk, NA), NA inhibition, antibody-secreting cells, and IFN-γ secreting cell responses. Ferrets immunized with either m-cNA-M2e VLP or M2e-H3 stalk vaccine were protected from H1N1 and H3N2 influenza viruses by lowering viral titers in nasal washes, trachea, and lungs after challenge. Vaccinated ferret antisera conferred broad humoral immunity in naïve mice. Our findings provide evidence that immunity to M2e and HA-stalk or M2e plus multi-subtype NA proteins induces cross-protection in ferrets.

Supplementation of seasonal vaccine with multi-subtype neuraminidase and M2 ectodomain virus-like particle improves protection against homologous and heterologous influenza viruses in aged mice.

The conventional inactivated split seasonal influenza vaccine offers low efficacy, particularly in the elderly and against antigenic variants. Here, to improve the efficacy of seasonal vaccination for the elderly population, we tested whether supplementing seasonal bivalent (H1N1 + H3N2) split (S) vaccine with M2 ectodomain repeat and multi-subtype consensus neuraminidase (NA) proteins (N1 NA + N2 NA + flu B NA) on a virus-like particle (NA-M2e) would induce enhanced cross-protection against different influenza viruses in aged mice. Immunization with split vaccine plus NA-M2e (S + NA-M2e) increased vaccine-specific IgG antibodies towards T-helper type 1 responses and hemagglutination inhibition titers. Aged mice with NA-M2e supplemented vaccination were protected against homologous and heterologous viruses at higher efficacies, as evidenced by preventing weight loss, lowering lung viral loads, inducing broadly cross-protective humoral immunity, and IFN-γ+ CD4 and CD8 T cell responses than those with seasonal vaccine. Overall, this study supports a new strategy of NA-M2e supplemented vaccination to enhance protection against homologous and antigenically different viruses in the elderly.

Universal protection against influenza viruses by multi-subtype neuraminidase and M2 ectodomain virus-like particle.

Annual influenza vaccination is recommended to update the variable hemagglutinin antigens. Here, we first designed a virus-like particle (VLP) displaying consensus multi-neuraminidase (NA) subtypes (cN1, cN2, B cNA) and M2 ectodomain (M2e) tandem repeat (m-cNA-M2e VLP). Vaccination of mice with m-cNA-M2e VLP induced broad NA inhibition (NAI), and M2e antibodies as well as interferon-gamma secreting T cell responses. Mice vaccinated with m-cNA-M2e VLP were protected against influenza A (H1N1, H5N1, H3N2, H9N2, H7N9) and influenza B (Yamagata and Victoria lineage) viruses containing substantial antigenic variations. Protective immune contributors include cellular and humoral immunity as well as antibody-dependent cellular cytotoxicity. Furthermore, comparable cross protection by m-cNA-M2e VLP vaccination was induced in aged mice. This study supports a novel strategy of developing a universal vaccine against influenza A and B viruses potentially in both young and aged populations by inducing multi-NA subtype and M2e immunity with a single VLP entity.

Impact of hemagglutination activity and M2e immunity on conferring protection against influenza viruses.

To improve cross-protection of influenza vaccination, we tested conjugation of conserved M2e epitopes to the surface of inactivated influenza virus (iPR8-M2e*). Treatment of virus with chemical cross-linker led to diminished hemagglutination activity and failure to induce hemagglutination inhibiting antibodies. Conjugated iPR8-M2e* vaccine was less protective against homologous and heterosubtypic viruses, despite the induction of virus-specific binding IgG antibodies. In alternative approaches to enhance cross-protection, we developed a genetically linked chimeric protein (M2e-B stalk) vaccine with M2e of influenza A and hemagglutinin (HA) stalk of influenza B virus. Vaccination of mice with inactivated influenza A virus supplemented with M2e-B stalk effectively induced hemagglutination inhibiting antibodies, humoral and cellular M2e immune responses, and enhanced heterosubtypic protection. This study demonstrates the importance of HA functional integrity in influenza vaccine efficacy and that supplementation of influenza vaccines with M2e-B stalk protein could be a feasible strategy of improving cross-protection against influenza viruses.

Thermostable H1 hemagglutinin stem with M2e epitopes provides broad cross-protection against group1 and 2 influenza A viruses.

Hemagglutinin (HA) stem-based vaccines have limitations in providing broad and effective protection against cross-group influenza viruses, despite being a promising universal vaccine target. To overcome the limited cross-protection and low efficacy by HA stem vaccination, we genetically engineered a chimeric conjugate of thermostable H1 HA stem and highly conserved M2e repeat (M2e-H1stem), which was expressed at high yields in Escherichia coli. M2e-H1stem protein presented native-like epitopes reactive to antisera of live virus infection. M2e-H1stem protein vaccination of mice induced strong M2e- and HA stem-specific immune responses, conferring broadly effective cross-protection against both antigenically distinct group 1 (H1N1, H5N1, and H9N2 subtypes) and group 2 (H3N2 and H7N9 subtypes) seasonal and pandemic potential influenza viruses. M2e-H1stem vaccination generated CD4+ and CD8+ T cell responses and antibody-dependent cytotoxic cellular and humoral immunity, which contributed to enhancing cross-protection. Furthermore, comparable broad cross-group protection was observed in older aged mice after M2e-H1stem vaccination. This study provides evidence warranting further development of chimeric M2e-stem proteins as a promising universal influenza vaccine candidate in adult and aged populations.

A chimeric thermostable M2e and H3 stalk-based universal influenza A virus vaccine.

We developed a new chimeric M2e and H3 hemagglutinin (HA) stalk protein vaccine (M2e-H3 stalk) by genetic engineering of modified H3 stalk domain conjugated with conserved M2e epitopes to overcome the drawbacks of low efficacy by monomeric domain-based universal vaccines. M2e-H3 stalk protein expressed and purified from Escherichia coli was thermostable, displaying native-like antigenic epitopes recognized by antisera of different HA subtype proteins and influenza A virus infections. Adjuvanted M2e-H3 stalk vaccination induced M2e and stalk-specific IgG antibodies recognizing viral antigens on virus particles and on the infected cell surface, CD4+ and CD8+ T-cell responses, and antibody-dependent cytotoxic cell surrogate activity in mice. M2e-H3 stalk was found to confer protection against heterologous and heterosubtypic cross-group subtype viruses (H1N1, H5N1, H9N2, H3N2, H7N9) at similar levels in adult and aged mice. These results provide evidence that M2e-H3 stalk chimeric proteins can be developed as a universal influenza A virus vaccine candidate for young and aged populations.

Enhanced cross protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes.

Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.

An Update on mRNA-Based Viral Vaccines.

With the success of COVID-19 vaccines, newly created mRNA vaccines against other infectious diseases are beginning to emerge. Here, we review the structural elements required for designing mRNA vaccine constructs for effective in vitro synthetic transcription reactions. The unprecedently speedy development of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was enabled with previous innovations in nucleoside modifications during in vitro transcription and lipid nanoparticle delivery materials of mRNA. Recent updates are briefly described in the status of mRNA vaccines against SARS-CoV-2, influenza virus, and other viral pathogens. Unique features of mRNA vaccine platforms and future perspectives are discussed.

Towards Goals to Refine Prophylactic and Therapeutic Strategies Against COVID-19 Linked to Aging and Metabolic Syndrome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gave rise to the coronavirus disease 2019 (COVID-19) pandemic. A strong correlation has been demonstrated between worse COVID-19 outcomes, aging, and metabolic syndrome (MetS), which is primarily derived from obesity-induced systemic chronic low-grade inflammation with numerous complications, including type 2 diabetes mellitus (T2DM). The majority of COVID-19 deaths occurs in people over the age of 65. Individuals with MetS are inclined to manifest adverse disease consequences and mortality from COVID-19. In this review, we examine the prevalence and molecular mechanisms underlying enhanced risk of COVID-19 in elderly people and individuals with MetS. Subsequently, we discuss current progresses in treating COVID-19, including the development of new COVID-19 vaccines and antivirals, towards goals to elaborate prophylactic and therapeutic treatment options in this vulnerable population.

Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be expanding the pandemic disease across the globe. Although SARS-CoV-2 vaccines were rapidly developed and approved for emergency use of vaccination in humans, supply and production difficulties are slowing down the global vaccination program. The efficacy of many different versions of vaccine candidates and adjuvant effects remain unknown, particularly in the elderly. In this study, we compared the immunogenic properties of SARS-CoV-2 full-length spike (S) ectodomain in young adult and aged mice, S1 with receptor binding domain, and S2 with fusion domain. Full-length S was more immunogenic and effective in inducing IgG antibodies after low dose vaccination, compared to the S1 subunit. Old-aged mice induced SARS-CoV-2 spike-specific IgG antibodies with neutralizing activity after high dose S vaccination. With an increased vaccine dose, S1 was highly effective in inducing neutralizing and receptor-binding inhibiting antibodies, although both S1 and S2 subunit domain vaccines were similarly immunogenic. Adjuvant effects were significant for effective induction of IgG1 and IgG2a isotypes, neutralizing and receptor-binding inhibiting antibodies, and antibody-secreting B cell and interferon-γ secreting T cell immune responses. Results of this study provide information in designing SARS-CoV-2 spike vaccine antigens and effective vaccination in the elderly.

TBK1 regulates regeneration of pancreatic ß-cells.

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate ß-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in ß-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat ß-cells. Conversely, TBK1 overexpression decreased sensitivity of ß-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of ß-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of ß-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived ß-cells and human islets. TBK1 expression was increased in ß-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and ß-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional ß-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a ß-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional ß-cells.

Publisher Correction: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Essential Roles for the Non-Canonical IκB Kinases in Linking Inflammation to Cancer, Obesity, and Diabetes.

Non-canonical IκB kinases (IKKs) TBK1 and IKKε have essential roles as regulators of innate immunity and cancer. Recent work has also implicated these kinases in distinctively controlling glucose homeostasis and repressing adaptive thermogenic and mitochondrial biogenic response upon obesity-induced inflammation. Additionally, TBK1 and IKKε regulate pancreatic ß-cell regeneration. In this review, we summarize current data on the functions and molecular mechanisms of TBK1 and IKKε in orchestrating inflammation to cancer, obesity, and diabetes.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

ß-cell proliferation induction is a promising therapeutic strategy to restore ß-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of ß-cell regeneration. The most potent ß-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated ß-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of ß-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced ß-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1ß-cells and ß-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in ß-cell proliferation, ß-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional ß-cell mass.

Zebrafish aversive taste co-receptor is expressed in both chemo- and mechanosensory cells and plays a role in lateral line development.

Fishes rely on both chemical and tactile senses to orient themselves to avoid predators, and to detect and taste food. This is likely achieved by highly coordinated reception of signals by mechano- and chemosensory receptors in fish. A small co-receptor from zebrafish, receptor activity modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), was previously found to be involved in recognition of triterpene glycosides, a family of naturally occurring compounds that act as chemical defenses in various prey species. However, its localization, function, and how it impacts sensory organ development in vivo is not known. Here we show that RL-TGR is expressed in zebrafish in both i) apical microvilli of the chemosensory cells of taste buds including the epithelium of lips and olfactory epithelium, and ii) mechanosensory cells of neuromasts belonging to the lateral line system. Loss-of-function analyses of RL-TGR resulted in significantly decreased number of neuromasts in the posterior lateral line system and decreased body length, suggesting that RL-TGR is involved in deposition and migration of the neuromasts. Collectively, these results provide the first in vivo genetic evidence of sensory cell-specific expression of this unusual co-receptor and reveal its additional role in the lateral line development in zebrafish.

MicroXRF tomographic visualization of zinc and iron in the zebrafish embryo at the onset of the hatching period.

Transition metals such as zinc, copper, and iron play key roles in cellular proliferation, cell differentiation, growth, and development. Over the past decade, advances in synchrotron X-ray fluorescence instrumentation presented new opportunities for the three-dimensional mapping of trace metal distributions within intact specimens. Taking advantage of microXRF tomography, we visualized the 3D distribution of zinc and iron in a zebrafish embryo at the onset of the hatching period. The reconstructed volumetric data revealed distinct differences in the elemental distributions, with zinc predominantly localized to the yolk and yolk extension, and iron to various regions of the brain as well as the myotome extending along the dorsal side of the embryo. The data set complements an earlier tomographic study of an embryo at the pharyngula stage (24 hpf), thus offering new insights into the trace metal distribution at key stages of embryonic development.

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for ß-Cell Regeneration.

The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of ß-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in ß-cell regeneration.

Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model.

UNLABELLED: In chronic liver failure patients with sustained fibrosis, excessive accumulation of extracellular matrix proteins substantially dampens the regenerative capacity of the hepatocytes, resulting in poor prognosis and high mortality. Currently, the mechanisms and the strategies of inducing endogenous cellular sources such as hepatic progenitor cells (HPCs) to regenerate hepatocytes in various contexts of fibrogenic stimuli remain elusive. Here we aim to understand the molecular and cellular mechanisms that mediate the effects of sustained fibrosis on hepatocyte regeneration using the zebrafish as a model. In the ethanol-induced fibrotic zebrafish model, we identified a subset of HPCs, responsive to Notch signaling, that retains its capacity to regenerate as hepatocytes. Discrete levels of Notch signaling modulate distinct cellular outcomes of these Notch-responsive HPCs in hepatocyte regeneration. Lower levels of Notch signaling promote amplification and subsequent differentiation of these cells into hepatocytes, while high levels of Notch signaling suppress these processes. To identify small molecules facilitating hepatocyte regeneration in the fibrotic liver, we performed chemical screens and identified a number of Wnt agonists and Notch antagonists. Further analyses demonstrated that these Wnt agonists are capable of attenuating Notch signaling by inducing Numb, a membrane-associated protein that inhibits Notch signaling. This suggests that the antagonistic interplay between Wnt and Notch signaling crucially affects hepatocyte regeneration in the fibrotic liver. CONCLUSION: Our findings not only elucidate how signaling pathways and cell-cell communications direct the cellular response of HPCs to fibrogenic stimuli, but also identify novel potential therapeutic strategies for chronic liver disease.