, a novel coronavirus (nCoV or SARS-CoV-2) belonging to the betacoronavirus family emerged 1,2. All human betacoronaviruses are unique from one another, however, they do share a certain degree of genetic and structural homology. SARS-CoV-2 genome sequence homology with SARS-CoV and MERS-CoV is 77% and 50%, respectively 3. In contrast to the relatively smaller outbreaks of SARS-CoV in 2002 and MERS-CoV in 2012, SARS-CoV-2 is exhibiting an unprecedented scale of infection, resulting in a global pandemic declaration of Coronavirus Infectious Disease (COVID-19) on 11 March 2020 by the World Health Organization (WHO). On 1 June 2020, the World Health Organization reported >6 million confirmed cases and 371 thousand deaths globally. Of note, during the 1918 influenza pandemic, more death was observed in the second phase of outbreak 4. Similar to influenza, COVID-19 harbours the potential to become a seasonal disease 5. The high infection rate, long incubation period, along with mild-to-moderate symptoms experienced by many, make COVID-19 a troubling disease. A vaccine is crucial, in particular because data indicate asymptomatic transmission of COVID-19 6-8. More than 10 years ago, scientists predicted the pandemic potential of the coronaviruses 9. And for the past 30 years, a once-per-decade novel coronavirus has pushed our public health system to the limit, with SARS-CoV-2 being the most severe. Despite the repeated warnings and discussion, the world was not prepared for this pandemic. The rapid development, distribution and administration of a vaccine to the global population is the most effective approach to quell this pandemic and the only one that will lead to a complete lifting of restrictions. Challenges include the vaccine design itself, but also its manufacture and global distribution; cold chain requirements present logistical and fiscal barriers to the availability of important, life-saving vaccines in resource-poor areas of the world. Innovating vaccine delivery platforms and devices to break cold chain limitations are therefore an efficient solution to safeguard potent vaccination for both wealthy and lower-income countries.

Rat astrocytes, immunologically competent glial cells of the central nervous system (CNS), released a variety of cytokines after activation. Lipopolysaccharidestimulated astrocytes produced tumor necrosis factor (TNF) as demonstrated by Northern blot analysis using a mouse TNF probe and by functional assay. Biological activity of rat astrocyte-derived TNF was neutralized by rabbit antiserum against recombinant murine TNF. Stimulation of astrocytes by lipopolysaccharide also activated the interleukin 1 and interleukin 6 genes. We have also investigated whether a neurotropic paramyxovirus, Newcastle disease virus, triggers cytokine production by astrocytes. This virus induced astrocytes to produce TNF, lymphotoxin, interleukin 6, and a-and 13-interferons.Thus, stimulation by endotoxin and virus activated distinct, yet overlapping, sets of cytokine genes. We propose that astrocytes and the cytokines they produce may play a significant role in the pathogenesis of immunologically and/or virally mediated CNS disease, in CNS intercellular communication, and in the interactions between the nervous and immune systems.Astrocytes are macroglial cells of the central nervous system (CNS) that express a variety of immunological characteristics. Astrocytes stimulated with y-interferon (IFN-y) express class I and class II major histocompatibility complex (MHC) antigens in rodents (1-3). Astrocytes expressing class II antigens can present foreign antigen to T cells in a MHCrestricted fashion (3,4). Lipopolysaccharide (LPS) stimulates astrocytes to produce prostaglandins (5), complement components C3 and factor B (6), and cytokines with biological activities similar to interleukin 1 (IL-1) (5) and IL-3 (7). These observations indicate that astrocytes are immunologically competent cells that share many important functional characteristics with macrophages.Accumulating evidence has revealed that astrocytes, like macrophages (8) MATERIALS AND METHODSCell Cultures. Primary cultures of rat astrocytes were established as described (13) (13). WEHI 164 clone 13, a murine fibrosarcoma line (14), was used for TNF functional assays.RNA Preparation and Analysis. Total RNA was isolated from cells by the guanidinium isothiocyanate method (15). RNA was denatured by formaldehyde treatment, electrophoresed through a 0.8% agarose gel, and transferred to nitrocellulose as described (16,17). The transferred RNA blots were hybridized with probes of high specific activity. Membranes probed with 32P-labeled DNA fragments were hybridized for 2 days at 370C and washed at 550C, twice in 2X SSC (ix SSC = 0.15 M NaC1/0.015 M sodium citrate)/0.1% SDS and twice in 0.5x SSC/0.1% SDS. Membranes probed with 32P-labeled RNA were hybridized overnight at 650C and washed at 650C as described above.Probes. Mouse cytokine probes were used in all experiments. DNA probes for IL-la, IL-1f3, IL-3, and lymphotoxin were constructed by using an oligolabeling reaction kit (Pharmacia), and RNA probes for TNF and the type I IFN were prepared using an RNA probe vector s...

Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34 CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34 CDC2 -cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34 CDC2 -mediated phosphorylation and resulted in loss of p34 CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34 CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34 CDC2 .C5b-9, the membrane attack complex of complement, causes cell death by forming transmembrane pores (1). When the number of C5b-9 molecules is limited to a sublytic level, nucleated cells are able to escape cell death by eliminating membrane-inserted terminal complement complexes (TCC 1 ; C5b-7, C5b-8, and C5b-9) by endocytosis and/or membrane shedding (2-4). Among these complexes, C5b-9 is most potent in activating target cells. C5b-9 causes a Ca 2ϩ influx and generates intracellular second messengers, including phosphatidylinositol triphosphates, diacylglycerol, and ceramide (5-8). Membrane-inserted TCC activates the G i /G o family of G proteins (9). Activation of G i /G o by TCC is responsible for the G␤␥-mediated activation of cell cycle through activation of Ras, Raf-1, extracellular signal regulated kinase-1 (10), and activation of phosphatidylinositol 3-phosphate kinase (10, 11).Cell cycle activation by C5b-9 is associated with an increase in CDK4, CDK2, and p34 CDC2 activities, and this is followed by an increase in DNA synthesis and cell proliferation (11, 12, 24 -26). The C5b-9-induced DNA synthesis is abolished by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 and phosphatidylinositol 3-phosphate kinase (11). Cell cycle activation by C5b-9 in postmitotic cells, such as oligodendrocytes (OLG) and myotubes, is associated with expression of c-JUN and c-FOS protooncogenes and loss of differentiation (12)(13)(14).In an effort to find novel C5b-9-induced genes involved in cell cycle regulation, we cloned the rat Response Gene to Complement (RGC-32) using mRNA differential display PCR in OLG (15, 16). C5b-9 en...

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We showed previously that, after spinal cord injury (SCI), tumor necrosis factor-alpha (TNF-alpha) may serve as an external signal, initiating apoptosis in neurons and oligodendrocytes. To further characterize the apoptotic cascade initiated by TNF-alpha after SCI, we examined the expression of TNF-alpha, inducible nitric oxide (NO) synthase (iNOS), and the level of NO after SCI. Western blots and reverse transcription polymerase chain reactions showed an early upregulation of TNF-alpha after injury. A peak TNF-alpha expression was observed within 1 h of injury. By 4 h after injury, the expression of iNOS and the level of NO were markedly increased in the injured spinal cord. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive cells were also first observed in the lesioned area 4 h after SCI. The largest number of TUNEL-positive cells was observed between 24-48 h after SCI. Injecting a neutralizing antibody against TNF-alpha into the lesion site after injury significantly reduced the expression of iNOS, the level of NO and the number of TUNEL-positive cells in the injured spinal cord. Injecting the NOS inhibitors, N(G)-monomethyl-L-arginine monoacetate and S-methylisothiourea sulfate, or an NO scavenger, carboxy-PTIO, into the lesion site also significantly reduced the level of NO and the degree of DNA laddering in the injured spinal cord. These data suggest that after SCI, apoptosis induced by TNF-alpha may be mediated in part by NO via upregulation of iNOS, induced in response to TNF-alpha.

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a Giα-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qβ. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qβ VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

Assembly of C5b-9 on cell membranes results in transmembrane channels and causes cell death. When the number of C5b-9 molecules is limited, nucleated cells are able to escape cell death by endocytosis and by shedding of membranes bearing C5b-9. Sublytic CSb-9 induces proto-oncogenes, activates the cell cycle, and enhances cell survival. In addition, C5b-9 reverses the differentiated phenotype of postmitotic cells, such as oligodendrocytes and skeletal muscle cells. The signal transduction pathways responsible for cell cycle activation by CSb-9 include Gi-mediated activation of extracellular signal-regulated kinase 1 and phosphatidylinositol 3-kinase (PI3-K). Cell survival enhanced by C5b-9 is mediated by the PI3-K/Akt pathway, which inhibits apoptosis through regulation of BAD. These findings indicate that complement activation and membrane assembly of sublytic C5b-9 play an important role in inflammation by promoting cell proliferation and by rescuing apoptotic cells.