Can Plant Plastid Produce a Candidate Influenza Vaccine?

Recent global flu pandemic highlights the obvious need for a vaccine against the influenza virus that is both inexpensive to produce and can be produced fast enough for development on a large scale. The influenza virus coat protein hemagglutinin (HA) is a major surface antigen and has emerged as a good candidate antigen for subunit vaccine development. Previous attempts to express HA from the A/Sichuan/2/87 H3N2 influenza subtype in plastids achieved HA gene transcription but no protein accumulation, suggesting that the HA protein may be unstable in the plastid. The aim of this work is to investigate alternative strategies that might achieve HA antigen accumulation in plastids. Two series of gene constructs were made. The first series of constructs coded for the first 14 amino acids of GFP and RBCL N-terminally fused to the full length HA, a full length GFP N-terminally fused to HA and a full length GFP N-terminally fused to HA1. The second series of gene constructs coded for two candidate influenza virus hemagglutinin epitopes, HA91-108 and HA 307-319 fused singly as N and C terminal fusions and as double terminal fusions to full length GFP. A total of ten gene constructs were introduced into the chloroplast genome by biolistic bombardment. Transformants were generated and confirmed through molecular analyses. Transplastomic lines generated with the 14aaRBCL/HA and 14aaGFP/HA constructs accumulated a recombinant protein that was smaller than expected. Further analysis is required to confirm that these proteins are indeed truncated HA fusion proteins. No transformed plant lines were generated with the full length GFP/HA and GFP/HA1 constructs suggesting that the tobacco plastid is not a useful system for producing full-length flu virus HA and HA1 proteins suitable for vaccine development. Expression and stability of recombinant HA91-108/GFP and GFP/HA307-319 fusion proteins was clearly demonstrated showing that conserved influenza virus HA epitopes can be expressed in plant chloroplasts. No transgene transcripts could be detected in transplastomic lines generated with the GFP/HA91-108, HA307-319/GFP or HA307-319/GFP/HA91-108 gene constructs. No transformed lines were generated with the HA91-108/GFP/HA307-319 construct. Conditions for shoot regeneration and the parameters required for biolistic-mediated chloroplast transformation of lettuce were also assessed. However, despite numerous attempts, no plastid transformed lettuce shoots were obtained

Plant-Derived Polyphenols Modulate Human Dendritic Cell Metabolism and Immune Function via AMPK-Dependent Induction of Heme Oxygenase-1

Polyphenols are important immunonutrients which have been investigated in the context of inflammatory and autoimmune disease due to their significant immunosuppressive properties. However, the mechanism of action of many polyphenols is unclear, particularly in human immune cells. The emerging field of immunometabolism has highlighted the significance of metabolic function in the regulation of immune cell activity, yet the effects of polyphenols on immune cell metabolic signaling and function has not been explored. We have investigated the effects of two plant-derived polyphenols, carnosol and curcumin, on the metabolism of primary human dendritic cells (DC). We report that human DC display an increase in glycolysis and spare respiratory capacity in response to LPS stimulation, which was attenuated by both carnosol and curcumin treatment. The regulation of DC metabolism by these polyphenols appeared to be mediated by their activation of the cellular energy sensor, AMP-activated Protein Kinase (AMPK), which resulted in the inhibition of mTOR signaling in LPS-stimulated DC. Previously we have reported that both carnosol and curcumin can regulate the maturation and function of human DC through upregulation of the immunomodulatory enzyme, Heme Oxygenase-1 (HO-1). Here we also demonstrate that the induction of HO-1 by polyphenols in human DC is dependent on their activation of AMPK. Moreover, pharmacological inhibition of AMPK was found to reverse the observed reduction of DC maturation by carnosol and curcumin. This study therefore describes a novel relationship between metabolic signaling via AMPK and HO-1 induction by carnosol and curcumin in human DC, and characterizes the effects of these polyphenols on DC immunometabolism for the first time. These results expand our understanding of the mechanism of action of carnosol and curcumin in human immune cells, and suggest that polyphenol supplementation may be useful to regulate the metabolism and function of immune cells in inflammatory and metabolic disease

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Can Plant Plastid Produce a Candidate Influenza Vaccine?

Recent global flu pandemic highlights the obvious need for a vaccine against the influenza virus that is both inexpensive to produce and can be produced fast enough for development on a large scale. The influenza virus coat protein hemagglutinin (HA) is a major surface antigen and has emerged as a good candidate antigen for subunit vaccine development. Previous attempts to express HA from the A/Sichuan/2/87 H3N2 influenza subtype in plastids achieved HA gene transcription but no protein accumulation, suggesting that the HA protein may be unstable in the plastid. The aim of this work is to investigate alternative strategies that might achieve HA antigen accumulation in plastids. Two series of gene constructs were made. The first series of constructs coded for the first 14 amino acids of GFP and RBCL N-terminally fused to the full length HA, a full length GFP N-terminally fused to HA and a full length GFP N-terminally fused to HA1. The second series of gene constructs coded for two candidate influenza virus hemagglutinin epitopes, HA91-108 and HA 307-319 fused singly as N and C terminal fusions and as double terminal fusions to full length GFP. A total of ten gene constructs were introduced into the chloroplast genome by biolistic bombardment. Transformants were generated and confirmed through molecular analyses. Transplastomic lines generated with the 14aaRBCL/HA and 14aaGFP/HA constructs accumulated a recombinant protein that was smaller than expected. Further analysis is required to confirm that these proteins are indeed truncated HA fusion proteins. No transformed plant lines were generated with the full length GFP/HA and GFP/HA1 constructs suggesting that the tobacco plastid is not a useful system for producing full-length flu virus HA and HA1 proteins suitable for vaccine development. Expression and stability of recombinant HA91-108/GFP and GFP/HA307-319 fusion proteins was clearly demonstrated showing that conserved influenza virus HA epitopes can be expressed in plant chloroplasts. No transgene transcripts could be detected in transplastomic lines generated with the GFP/HA91-108, HA307-319/GFP or HA307-319/GFP/HA91-108 gene constructs. No transformed lines were generated with the HA91-108/GFP/HA307-319 construct. Conditions for shoot regeneration and the parameters required for biolistic-mediated chloroplast transformation of lettuce were also assessed. However, despite numerous attempts, no plastid transformed lettuce shoots were obtained

Can Plant Plastid Produce a Candidate Influenza Vaccine?

Recent global flu pandemic highlights the obvious need for a vaccine against the influenza virus that is both inexpensive to produce and can be produced fast enough for development on a large scale. The influenza virus coat protein hemagglutinin (HA) is a major surface antigen and has emerged as a good candidate antigen for subunit vaccine development. Previous attempts to express HA from the A/Sichuan/2/87 H3N2 influenza subtype in plastids achieved HA gene transcription but no protein accumulation, suggesting that the HA protein may be unstable in the plastid. The aim of this work is to investigate alternative strategies that might achieve HA antigen accumulation in plastids. Two series of gene constructs were made. The first series of constructs coded for the first 14 amino acids of GFP and RBCL N-terminally fused to the full length HA, a full length GFP N-terminally fused to HA and a full length GFP N-terminally fused to HA1. The second series of gene constructs coded for two candidate influenza virus hemagglutinin epitopes, HA91-108 and HA 307-319 fused singly as N and C terminal fusions and as double terminal fusions to full length GFP. A total of ten gene constructs were introduced into the chloroplast genome by biolistic bombardment. Transformants were generated and confirmed through molecular analyses. Transplastomic lines generated with the 14aaRBCL/HA and 14aaGFP/HA constructs accumulated a recombinant protein that was smaller than expected. Further analysis is required to confirm that these proteins are indeed truncated HA fusion proteins. No transformed plant lines were generated with the full length GFP/HA and GFP/HA1 constructs suggesting that the tobacco plastid is not a useful system for producing full-length flu virus HA and HA1 proteins suitable for vaccine development. Expression and stability of recombinant HA91-108/GFP and GFP/HA307-319 fusion proteins was clearly demonstrated showing that conserved influenza virus HA epitopes can be expressed in plant chloroplasts. No transgene transcripts could be detected in transplastomic lines generated with the GFP/HA91-108, HA307-319/GFP or HA307-319/GFP/HA91-108 gene constructs. No transformed lines were generated with the HA91-108/GFP/HA307-319 construct. Conditions for shoot regeneration and the parameters required for biolistic-mediated chloroplast transformation of lettuce were also assessed. However, despite numerous attempts, no plastid transformed lettuce shoots were obtained

Can Plant Plastid Produce a Candidate Influenza Vaccine?

Recent global flu pandemic highlights the obvious need for a vaccine against the influenza virus that is both inexpensive to produce and can be produced fast enough for development on a large scale. The influenza virus coat protein hemagglutinin (HA) is a major surface antigen and has emerged as a good candidate antigen for subunit vaccine development. Previous attempts to express HA from the A/Sichuan/2/87 H3N2 influenza subtype in plastids achieved HA gene transcription but no protein accumulation, suggesting that the HA protein may be unstable in the plastid. The aim of this work is to investigate alternative strategies that might achieve HA antigen accumulation in plastids. Two series of gene constructs were made. The first series of constructs coded for the first 14 amino acids of GFP and RBCL N-terminally fused to the full length HA, a full length GFP N-terminally fused to HA and a full length GFP N-terminally fused to HA1. The second series of gene constructs coded for two candidate influenza virus hemagglutinin epitopes, HA91-108 and HA 307-319 fused singly as N and C terminal fusions and as double terminal fusions to full length GFP. A total of ten gene constructs were introduced into the chloroplast genome by biolistic bombardment. Transformants were generated and confirmed through molecular analyses. Transplastomic lines generated with the 14aaRBCL/HA and 14aaGFP/HA constructs accumulated a recombinant protein that was smaller than expected. Further analysis is required to confirm that these proteins are indeed truncated HA fusion proteins. No transformed plant lines were generated with the full length GFP/HA and GFP/HA1 constructs suggesting that the tobacco plastid is not a useful system for producing full-length flu virus HA and HA1 proteins suitable for vaccine development. Expression and stability of recombinant HA91-108/GFP and GFP/HA307-319 fusion proteins was clearly demonstrated showing that conserved influenza virus HA epitopes can be expressed in plant chloroplasts. No transgene transcripts could be detected in transplastomic lines generated with the GFP/HA91-108, HA307-319/GFP or HA307-319/GFP/HA91-108 gene constructs. No transformed lines were generated with the HA91-108/GFP/HA307-319 construct. Conditions for shoot regeneration and the parameters required for biolistic-mediated chloroplast transformation of lettuce were also assessed. However, despite numerous attempts, no plastid transformed lettuce shoots were obtained

Studies on human biliverdin-IX alpha reductase and linear tetrapyrrole signaling

THESIS 6353Human Biliverdin-IXa reductase (BVR-A) has been cloned and overexpressed in E.coli as a GST- and Hexahistidine fusion protein. The full length cDNA encoding the enzyme has been amplified via PCR and hgated into the pGEX-KG and pTrcHis B expression vectors in order to produce the respective fusions. Induction of TGI cells transformed with the pGEX-BVR-A and pTrc-BVR-A constructs has culminated in the expression of a recombinant protein of the correct size and antigenicity in both cases. Purification of the GST-fusion protein on a glutathione affinity resin yields approximately 40 mg of fusion protein per litre of culture. The fusion protein has a molecular weight of 66 kDa, however, it is possible to remove the GST tag using the proteolytic enzyme, thrombin. The purified hBVR-A protein migrates on SDS-PAGE with a mobility corresponding to 40 kDa, however, mass spectroscopic analysis has confirmed the true relative molecular mass to be 34 kDa. A selenomethionine derivative of the recombinant hBVR-A protein has been prepared for use in Multiwavelength Anomalous Diffraction experiments and crystals diffracting at 3A have recently been obtained

Inflammasome activation: from inflammatory disease to infection

The recognition of pathogen-derived molecules by the innate immune system is mediated by a number of receptors, including members of the TLR (Toll-like receptor), RLH [RIG (retinoic acid-inducible gene)-like helicase] and the NLR (NOD-like receptor) families. NLRs in particular are also involved in the recognition of host-derived `danger?-associated molecules which are produced under conditions of cellular stress or injury. Activation of these receptors leads to the assembly of high-molecular-mass complexes called inflammasomes which in turn leads to the generation of active caspase 1 and to the production of mature IL-1? (interleukin 1?). The discovery that NLRP3 (NLR-related protein 3) can recognize host-derived particulate matter such as uric acid and cholesterol crystals has led to this inflammasome being implicated in a number of inflammatory diseases, including gout, atherosclerosis and Type 2 diabetes. In addition, aberrant NLRP3 activation has also been observed in a number of heritable disorders now referred to as cryopyrinopathies. On the other hand, a number of studies have reported that recognition of both viral and bacterial products by NLRs is required for effective pathogen clearance. The present review discusses both aspects of NLR activation and will highlight the role of additional inflammasome complexes in sensing infection

Immune modulation: IL-1, master mediator or initiator of inflammation

Interleukin-1 (IL-1) lies at the center of the inflammatory response. This proinflammatory cytokine was first pinned down for its ability to induce fever, and was later assigned additional biological functions including neutrophil recruitment, lymphocyte activation and induction of inflammatory mediators1. Excessive production of IL-1? drives the recurrent episodes of fever and systemic inflammation in certain autoinflammatory diseases, linked to mutations in genes regulating innate immunity; such conditions include familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS)2, as well as gout, a condition where IL-1? induced by monosodium urate crystals (MSU) causes inflammatory response in joints