Naïve CD4+ T Cell Activation in the Nasal-Associated Lymphoid Tissue following Intranasal Immunization with a Flagellin-Based Subunit Vaccine.

The nasal-associated lymphoid tissues (NALT) are generally accepted as an immune induction site, but the activation of naïve T-cells in that compartment has not been well-characterized. I wanted to determine if early events in naïve CD4+ T cell activation and the extent of antigen specific cell division are similar in NALT to that observed in other secondary lymphoid compartments. I performed antigen tracking experiments and analyzed the activation of naïve antigen-specific CD4+ T cells in the nasal-associated lymphoid tissues (NALT). I directly observed transepithelial transport of fluorescently labeled antigen from the lumen of the airway to the interior of the NALT two hours following immunization. One day following intranasal (i.n.) immunization with antigen and adjuvant, antigen-specific CD4+ T cells in the NALT associated as clusters, while antigen-specific CD4+ T cells in control mice immunized with adjuvant only remained dispersed. The antigen-specific CD4+ populations in the NALT and cranial deep cervical lymph nodes of immunized mice expanded significantly by day three following immunization. These findings are consistent with initial activation of naïve CD4+ T cells in the NALT and offer insight into adjuvant mechanism of flagellin in the upper respiratory compartment.

Rapid Transient Expression of Receptor-Binding Domain of SARS-CoV-2 and the Conserved M2e Peptide of Influenza A Virus Linked to Flagellin in Nicotiana benthamiana Plants Using Self-Replicating Viral Vector.

The development of recombinant vaccines against SARS-CoV-2 and influenza A is an important task. The combination of the conserved influenza A antigen, the extracellular domain of the transmembrane protein M2 (M2e), and the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (RBD) provides the opportunity to develop a bivalent vaccine against these infections. The fusion of antigens with bacterial flagellin, the ligand for Toll-like receptor 5 and potent mucosal adjuvant, may increase the immunogenicity of the candidate vaccines and enable intranasal immunization. In this study, we report the transient expression of RBD alone, RBD coupled with four copies of M2e, and fusions of RBD and RBD-4M2e with flagellin in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff. The yields of purified recombinant proteins per gram of fresh leaf tissue were about 20 µg for RBD, 50-60 µg for RBD-4M2e and the fusion of RBD with flagellin, and about 90 µg for RBD-4M2e fused to flagellin. Targeting to the endoplasmic reticulum enabled the production of glycosylated recombinant proteins comprising RBD. Our results show that plant-produced RBD and RBD-4M2e could be further used for the development of subunit vaccines against COVID-19 and a bivalent vaccine against COVID-19 and influenza A, while flagellin fusions could be used for the development of intranasal vaccines.

A triple-RBD-based mucosal vaccine provides broad protection against SARS-CoV-2 variants of concern.

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broad-spectrum protection against the initial infection and thereby curb the transmission potential. Here, we designed a chimeric triple-RBD immunogen, 3Ro-NC, harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold. 3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern. Notably, intranasal immunization with 3Ro-NC plus the mucosal adjuvant KFD (3Ro-NC + KFDi.n) elicits coordinated mucosal IgA and higher neutralizing antibody specificity (closer antigenic distance) against the Omicron variant. In Omicron-challenged human ACE2 transgenic mice, 3Ro-NC + KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung (85.7-fold) and the nasal turbinate (13.6-fold). Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies. Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection, pathology and transmission potential.

THE BEAST IN THE BEAUTY BOX

SESSION TITLE: Critical Care in Chest Infections Case Report Posters 2 SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Enterobacter species are notorious for causing nosocomial infection. They were found to be the third most common pathogen in the respiratory tract amongst isolates in the ICU. What makes the situation grim is the growing antibiotic resistance with regards to treating these infections. Such is the extent of this problem that in certain parts of the world the antibiotic sensitivity of Pluralibacter gergoviae is used as an indicator for the spreading antibiotic resistance in the environment. CASE PRESENTATION: A 73 year old female with past medical history significant for hypertension, atrial fibrillation, and Coronary artery disease s/p stent placement in 2019 presented to our facility with a 4 day history of fever, cough and chest discomfort. She had tested positive for COVID-19 two days prior to presentation and was initiated on remdesivir, tocilizumab, and dexamethasone. She was initially managed on the floors but in view of her increasing oxygen requirement she was transferred to the critical care where she was intubated due to respiratory failure. She continued to spike fevers and was persistently hypoxic. Initially this was attributed to COVID pneumonia and a trial of convalescent plasma was also given. After 3 weeks, she tested negative for COVID-19 while still intubated and precautions were taken off. However, she continued to spike fevers. Repeat chest X-ray was done and it showed multifocal airspace disease with increasing opacification in the left upper lobe. Her endotracheal aspirate grew carbapenemase producing Pluralibacter gergoviae sensitive for ciprofloxacin. Subsequently, she was started on IV levofloxacin and received it for a total of 21 days. Her treatment course was complicated by prolonged intubation requiring tracheostomy and development of Pneumatocele. After stopping the antibiotics, she did not have fever and her white blood cell count was within normal limits. DISCUSSION: P. gergoviae is a known contaminant in intravenous fluids, invasive medical devices, eye cream, children's shampoo, skin cream, hand cleaning paste, and cleansing wipes. Over the decades due to selective pressure especially in the cosmetic industry from preservatives it has gained antibiotic resistance via overexpression of detoxifying enzymes, flagellin, modification of membrane structure/function. Improving patient's oral hygiene, implementing infection control protocols strictly in the ICU, minimizing invasive medical devices/catheters and educating the stakeholders shall help in curbing these incidents. Once identified, early Infectious disease specialist involvement can help choose an apt antibiotic regimen on the basis of existing antibiograms. CONCLUSIONS: This case highlighted the importance of close microbiological surveillance, minimizing occurrence of nosocomial infection and treating atypical organisms. Reference #1: Enterobacter gergoviae adaptation to preservatives commonly used in cosmetic industry M. Périamé,J.-M. Pagès,A. Davin-Regli 14 May 2014 DISCLOSURES: No relevant relationships by Abinesh Sekar

High-Yield Production of Receptor Binding Domain of SARS-CoV-2 Linked to Bacterial Flagellin in Plants Using Self-Replicating Viral Vector pEff.

The development of recombinant vaccines against SARS-CoV-2 is required to eliminate the COVID-19 pandemic. We reported the expression of a recombinant protein Flg-RBD comprising receptor binding domain of SARS-CoV-2 spike glycoprotein (RBD) fused to flagellin of Salmonella typhimurium (Flg), known as mucosal adjuvant, in Nicotiana benthamiana plants. The fusion protein, targeted to the cytosol, was transiently expressed using the self-replicating vector pEff based on potato virus X genome. The recombinant protein Flg-RBD was expressed at the level of about 110-140 µg per gram of fresh leaf tissue and was found to be insoluble. The fusion protein was purified using metal affinity chromatography under denaturing conditions. To increase the yield of Flg-RBD, the flow-through fraction obtained after loading of the protein sample on the Ni-NTA resin was re-loaded on the sorbent. The yield of Flg-RBD after purification reached about 100 µg per gram of fresh leaf tissue and the purified protein remained soluble after dialysis. The control flagellin was expressed in a soluble form and its yield after purification was about 300 µg per gram of fresh leaf biomass. Plant-produced Flg-RBD protein could be further used for the development of intranasal recombinant mucosal vaccines against COVID-19.

A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge.

An ideal vaccine against SARS-CoV-2 is expected to elicit broad immunity to prevent viral infection and disease, with efficient viral clearance in the upper respiratory tract (URT). Here, the N protein and prefusion-full S protein (SFLmut) are combined with flagellin (KF) and cyclic GMP-AMP (cGAMP) to generate a candidate vaccine, and this vaccine elicits stronger systemic and mucosal humoral immunity than vaccines containing other forms of the S protein. Furthermore, the candidate vaccine administered via intranasal route can enhance local immune responses in the respiratory tract. Importantly, human ACE2 transgenic mice given the candidate vaccine are protected against lethal SARS-CoV-2 challenge, with superior protection in the URT compared with that in mice immunized with an inactivated vaccine. In summary, the developed vaccine can elicit a multifaceted immune response and induce robust viral clearance in the URT, which makes it a potential vaccine for preventing disease and infection of SARS-CoV-2.