Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

Oral administration of Euglena gracilis Z and its carbohydrate storage substance provides survival protection against influenza virus infection in mice.

Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has ß-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum. At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1ß, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection.

Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D. chilensis, continuous and long term feeding with E. gracilis cells favors immune response and reduces the damage caused by sewage pollution exposure on hemocytes. Nevertheless, diet and transplantation procedures may produce negative effects on the oxidative balance of gills and digestive gland and should be taken into account for bioremediation strategies.

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Immunomodulatory effects of dietary ß-1,3-glucan from Euglena gracilis in rainbow trout (Oncorhynchus mykiss) immersion vaccinated against Yersinia ruckeri.

Potential immunostimulatory effects of orally administered ß-glucan were investigated in combination with immersion vaccination against enteric redmouth disease caused by Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). A linear, unbranched and pure (purity ≥98%) ß-1,3-glucan (syn. paramylon) from the alga Euglena gracilis was applied at an inclusion level of 1% ß-glucan in feed administered at a rate of 1% biomass day(-1) for 84 consecutive days. Fish were vaccinated after two weeks of experimental feeding and bath challenged with live Y. ruckeri six weeks post-vaccination. Blood and head kidney were sampled at day 0, 13 (1 day pre-vaccination), 15, 55, 59 (day 3 post-challenge (p.c.)), 70 and 84. Vaccination induced significantly increased survival p.c., whereas the ß-glucan had no effect on survival in either unvaccinated or vaccinated fish. Expression in head kidney of genes related to the acute phase response, i.e. interleukin-1ß (IL-1ß), serum amyloid A (SAA), precerebellin, and hepcidin, was significantly different in vaccinated fish receiving ß-glucan compared to vaccinated controls at day 3 p.c., while no effect of ß-glucan was observed among unvaccinated fish. Significant interaction between ß-glucan and vaccination was found for the regulation of IL-1ß, tumour necrosis factor-α, interferon-γ, SAA, precerebellin and hepcidin p.c. For SAA, the significant effect of ß-glucan in vaccinated fish persisted at day 14 p.c. and 28 p.c. The difference in gene expression among vaccinated fish was mainly observed as down-regulations in vaccinated, ß-glucan fed fish compared to up-regulations or no regulation in vaccinated controls. Slightly increased levels of plasma lysozyme activity were found in fish (both unvaccinated and vaccinated) receiving ß-glucan at day 3 p.c. compared to control fed groups. This was associated with a faster clearance of Y. ruckeri in unvaccinated fish receiving ß-glucan. In contrast to the trend towards a beneficial effect of ß-glucan on plasma lysozyme activity, a trend towards suppression of plasma antibodies was seen in both unvaccinated and vaccinated fish receiving ß-glucan. However, the effects of ß-glucan were not reflected in the survival curves, and the differences seen in plasma lysozyme activity and antibody levels may have counteracted and set off each other as well as counteracted any potential effect represented by the differences in gene expression found.

Immunodetection of a protein related to mammalian arrestin in Euglena gracilis.

Six monoclonal antibodies directed against different epitopes of bovine retinal arrestin recognized a single polypeptide in extracts of achlorophyllous Euglena gracilis cells. This polypeptide had an apparent molecular weight slightly lower: 45kDa vs. 48 kDa, and a more basic isoelectric point compared to that of bovine visual arrestin. It was located in an insoluble cytoplasmic fraction. Immunofluorescence assays show a cytoplasmic punctuated pattern suggesting a cluster distribution or a linkage to some cell structure. The presence of arrestine-like molecules in achlorophyllous Euglena gracilis cells suggests that these proteins might be involved in a peculiar step of chemical signal transduction processes.

Presence of proteins recognized by mammalian cytochrome P-450 antibodies in Euglena gracilis.

We have attempted to probe three microsomal cytochrome P-450 isozymes in Euglena gracilis using immunochemical methods. They cross-react with anti-rat cytochrome P4502C11, cytochrome P4502E, and cytochrome P4502B. Activities of alkoxyphenoxazone dealkylation have been tested in living cells. In untreated cultures, the amount of proteins recognized by anti-cytochrome P4502C11 or anti-cytochrome P4502E is high. Phenobarbital treatment increased the levels of microsomal proteins recognized by antibody to cytochrome P4502B, as well as dealkylases of pentoxyresorufin, but decreased the level of proteins recognized by anti-cytochrome P450C11 or cytochrome P4502E. These results suggest that these unicellular algae may contain different isozymes of microsomal cytochromes P-450, comparable to those in mammalian liver. They are cytochrome P-450 equivalents of mammalian isoenzymes 2C, 2E and 2B. However, we could not demonstrate ethanol induction of cytochrome P-450 equivalent to isoenzyme 2E. Its role in xeno- or endobiotic metabolism remains to be elucidated.

Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody.

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.

[Immunofluorescent study of the basic chromosomal protein set of green algae and Euglena]. / Immunofluorestsentnoe issledovanie nabora osnovnykh khromosomnykh belkov nekotorykh zelenykh vodoroslei i évgleny.

Using antisera to fractions H1, H2a, H3 and H4 of the calf thymus histones, a comparative immunofluorescent investigation of these proteins in the nuclei of Chlamydomonas reinhardii, Haematococcus pluvialis, Dunaliella salina and Euglena gracilis was carried out. It has been shown that according to the immunofluorescent test, the nuclei of these algae contain proteins close to fractions H2a, H3 and H4 of the calf thymus histones. H1 fraction in these algae is either absent or can be considered as a protein immunochemically non-related to H1 fraction of the calf thymus histone. For quantitative evaluation (in units of the immunological distance) of the difference between histones of the algae and of the calf thymus in situ by indirect immunofluorescence, it was suggested to use the ultimate dilutions of antisera to histones. It was shown that the ultimate dilutions were correlated with titres of antisera in the reaction of microcomplement fixation. Such an approach and the data obtained are of interest for studying into the evolution of nucleosome histones in unicellular and multicellular eukaryotes.