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Background: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. Methods: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. Results: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. Conclusions: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

The Chandipura virus matrix protein plays a crucial role in virus assembly, budding, and the cytopathic effects observed in infected cells by interacting with several host proteins. The protocol presented here outlines the expression and purification of full‐length Chandipura virus matrix protein and two N‐terminally truncated constructs produced in Escherichia coli. This protocol results in high yields of monomeric matrix protein, which is suitable for structural studies. Additionally, GFP‐fused Chandipura virus matrix protein constructs can be expressed in mammalian cells for examination of intracellular localization. The Chandipura virus matrix protein model, generated using AlphaFold, features an intrinsically disordered N terminus and a structured C‐terminal core, similar to other Vesiculovirus matrix proteins. This research protocol offers a guide for the cloning, expression, and purification of the Chandipura virus matrix protein using E. coli. It also includes a step‐by‐step procedure for cloning, expressing, and conducting fluorescence imaging of GFP‐fused Chandipura virus matrix protein in mammalian cell lines. This study aims to facilitate interaction studies between the Chandipura virus matrix protein and human proteins.

The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.

•Modular capsomeres were prepared by non-chromatographic approaches.•Adjuvanted modular capsomeres induced high antibody titer after only a single shot.•“Goldilocks” level of contaminants provided a significant adjuvanting effect. Highly pathogenic avian influenza (HPAI) causes significant economic loss, reduced food security and poses an ongoing pandemic threat. Poultry vaccination significantly decreases these problems and recognizes that the health of humans, animals and ecosystems are connected. Low-cost manufacture of poultry vaccine matched quickly to the ever-changing circulating strain is needed for effective vaccination. Here, we re-engineered the process to manufacture bacterially synthesized modular capsomere comprising influenza M2e, previously shown to confer complete protection in challenged mice, for application in poultry. Modular capsomere was prepared using a simplified non-chromatographic salting-out precipitation method and its immunogenicity tested in vivo in poultry. Modular capsomere crudely purified by precipitation (pCapM2e) contained more contaminants than equivalent product purified by chromatography (cCapM2e). Unadjuvanted pCapM2e containing 80EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2EU per dose). However, addition of adjuvant to pCapM2e resulting in high immunogenicity after only a single dose of vaccination, yet without any local adverse reaction. This finding suggests a strong synergy between adjuvant, antigen and contaminants, and the possible existence of a “Goldilocks” level of contaminants, where high immunogenicity and low reactogenicity can be obtained in a single-shot vaccination. The simplified process offers potential cost and speed advantages to address the needs in influenza poultry vaccination in low-cost veterinary markets.

Nasopharyngeal carcinoma (NPC) is a rare disease in children with good prognosis and high cure rate. Nevertheless, certain patients have an unfavorable prognosis due to development of refractory NPC that is unresponsive to any therapeutic strategies. The current study studies a case of a 17 years-old female with non-keratinizing NPC type IIb (T2N0M0), who passed away as a consequence of resistance to chemo-, radio- and β-interferon therapy, and to an allogenic stem cell transplantation. In order to identify factors that lead to treatment failure and fatal outcome, immunohistochemical analyses of different tumor biomarkers and hierarchical cluster analysis were performed and compared with those of eight other patients with NPC who experienced complete remission following conventional therapy. Hierarchical cluster analysis of the immunohistochemical results clearly demonstrated that staining for immunological factors (CD4, CD8 and CD56) distinguished this patient from the others. To further investigate a potential role of the immune system, lymphocytic infiltration was assessed in tumor tissue by evaluation of hematoxylin and eosin-stained tumor sections. Indeed, no tumor infiltrating lymphocytes (TILs) were observed in this NPC case, while 7 out of 8 of the other NPC samples contained variable TIL amounts. The view that immunodeficiency of the patient may be a factor in the fatal outcome of treatment is supported by the fact that this patient with NPC was not positive for Epstein-Barr virus markers and also infected by several other viruses and fungi (herpes simplex virus, human herpes virus 6, Varicella zoster virus, and Candida). In conclusion, the investigation of rare NPC cases with poor prognosis may provide an improved understanding of the molecular mechanisms involved in refractory tumors and identification of novel potential therapeutic targets for NPC in the future.

Background & objectives: Primary central nervous system lymphomas (PCNSLs) are relatively uncommon, accounting for 2-3 per cent of primary brain tumours. Majority of these are diffuse large B cell lymphomas (DLBCL) occurring both in immunocompromised and immunocompetent patients. We undertook this study to classify PCNSL into germinal centre (GC) and non-germinal centre (NGC) type based on Hans classification and to find the role of Epstein-Barr virus (EBV) in pathogenesis both by conventional immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). Methods: The consecutive cases of PCNSL during a 10 years period were analysed by IHC for CD45, CD20, CD3, B-cell lymphoma 2 and 6 (Bcl-2 and Bcl-6), B-cell specific octamer binding protein-1 (BOB-1), multiple myeloma oncogene-1 (MUM-1), EBV latent-membrane protein 1 (LMP-1), cyclin-D1, CD10, CD5 and CD23, as well as by CISH for EBV. Results: During a period of 10 years, 65 PCNSL were diagnosed which comprised 0.69 per cent (65/9476) of all intracranial tumours. The mean age of presentation was 49 yr with sex ratio (M:F) of 1.4:1. Most common location was supratentorial region with predominant involvement of frontal lobe. Single lesions were seen in 38 (58.4%) and multifocal lesions in 27 (41.5%) patients. None of the patients were immunocompromised. All cases were B cell immunophenotype and were DLBCL except one case of follicular lymphoma. According to Hans classification, majority of them were NGC (n=51, 79.6%) and 13 (20.3%) were GC type. Bcl-2 expression was noted in 34 (52.3%) tumours. EBV was positive in three (4.6%) cases; two were detected both by IHC and CISH and one case by CISH only. Interpretation & conclusions: In Indian population, PCNSL occurs mainly in immunocompetent patients, and a decade earlier than in western population. Immunophenotyping revealed that all cases were DLBCL with predominance of NGC type. No prognostic difference was seen between GC and NGC DLBCL. Association of EBV was rare and this virus was possibly not involved in the pathogenesis of PCNSL in immunocompetent individuals. CISH was an easy, economical and less cumbersome method for detection of EBV in PCNSL.

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein and 5-lipoxygenase-activating protein (FLAP) are among a large number of membrane proteins that are poorly expressed when traditional expression systems and methods are employed. Therefore to efficiently express difficult membrane proteins, molecular biologists will have to develop novel or innovative expression systems. To this end, we have expressed the SARS-CoV M and FLAP proteins in Escherichia coli by utilizing a novel gene fusion expression system that takes advantage of the natural chaperoning properties of the SUMO (small ubiquitin-related modifier) tag. These chaperoning properties facilitate proper protein folding, which enhances the solubility and biological activity of the purified protein. In addition to these advantages, we found that SUMO Protease 1, can cleave the SUMO fusion high specificity to generate native protein. Herein, we demonstrate that the expression of FLAP and SARS-CoV membrane proteins are greatly enhanced by SUMO fusions in E. coli.

BACKGROUND: Regular reformulation of currently available vaccines is necessary due to the unpredictable variability of influenza viruses. Therefore, vaccine based on a highly conserved antigen with capability of induction of effective immune responses could be a potential solution. Influenza matrix protein-2 (M2) is highly conserved across influenza subtypes and a promising candidate for a broadly protective influenza vaccine. For the enhancement of broad protection, four tandem copies of consensus M2 gene containing extracellular (ED) and cytoplasmic (CD) without the trans-membrane domain (TM) reconstituted from H1N1, H5N1 and H9N2 influenza viruses were linked and named as 4sM2. The construct was effectively expressed in Escherichia coli, purified and proteins were used to immunize BALB/c mice. Humoral and cell-mediated immune responses were investigated following administration. RESULTS: Mice were intramuscularly immunized with 4sM2 protein 2 times at 2 weeks interval. Two weeks after the last immunization, first humoral and cell mediated immune response specific to sM2 protein were evaluated and the mice were challenged with a lethal dose (10MLD₅₀) of divergent subtypes A/EM/Korea/W149/06(H5N1), A/PR/8/34(H1N1), A/Aquatic bird/Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses. The efficacy of 4sM2 was evaluated by determining survival rates, body weights and residual lung viral titers. Our studies demonstrate that the survival of mice immunized with 4sM2 was significantly higher (80–100% survival) than that of unimmunized mice (0% survival). We also examined the long lasting protection against heterosubtype H5N2 virus and found that mice vaccinated with 4sM2 displayed 80% of protection even after 6 months of final vaccination. CONCLUSION: Taken together, these results suggest that prokaryotic expressed multimeric sM2 protein achieved cross protection against lethal infection of divergent influenza subtypes which are lasting for the long time.

Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.

The role of Epstein-Barr virus (EBV) in idiopathic pulmonary fibrosis (IPF) is uncertain. A study was undertaken to detect the virus in IPF as well as to clarify the influence of EBV on the clinical features of the disease. Twenty nine lung specimens were obtained from patients with IPF, as well as five specimens from patients with systemic sclerosis with pulmonary fibrosis (SSc) and 15 specimens from controls. EBV DNA and EBV latent membrane protein 1 (LMP1) were detected using the PCR method and immunohistochemical analysis, respectively. EBV DNA was detected in 24 of 25 patients with IPF (96%), in all five patients with SSc (100%), and in 10 of 14 controls (71%). The detection ratio was significantly higher in patients with IPF than in controls (p = 0.047, odds ratio (OR) = 9.60, 95% confidence interval (CI) 0.9 to 96.9). Immunohistochemical analysis revealed that cuboidal epithelial cells were positively stained with anti-LMP1 antibody in nine of the 29 lung specimens from IPF patients. In contrast, neither the patients with SSc nor the control subjects showed positive staining. In the follow up periods LMP1 positive patients with IPF died more frequently from respiratory failure than LMP1 negative patients (4/9 versus 1/20; p = 0.022, OR = 15.20, 95% CI 1. 3 to 168.0). EBV LMP1 positivity may be associated with more rapid disease progression in IPF.