Immunogenicity and efficacy of VLA2001 vaccine against SARS-CoV-2 infection in male cynomolgus macaques.

BACKGROUND: The fight against COVID-19 requires mass vaccination strategies, and vaccines inducing durable cross-protective responses are still needed. Inactivated vaccines have proven lasting efficacy against many pathogens and good safety records. They contain multiple protein antigens that may improve response breadth and can be easily adapted every year to maintain preparedness for future seasonally emerging variants. METHODS: The vaccine dose was determined using ELISA and pseudoviral particle-based neutralization assay in the mice. The immunogenicity was assessed in the non-human primates with multiplex ELISA, neutralization assays, ELISpot and intracellular staining. The efficacy was demonstrated by viral quantification in fluids using RT-qPCR and respiratory tissue lesions evaluation. RESULTS: Here we report the immunogenicity and efficacy of VLA2001 in animal models. VLA2001 formulated with alum and the TLR9 agonist CpG 1018™ adjuvant generate a Th1-biased immune response and serum neutralizing antibodies in female BALB/c mice. In male cynomolgus macaques, two injections of VLA2001 are sufficient to induce specific and polyfunctional CD4+ T cell responses, predominantly Th1-biased, and high levels of antibodies neutralizing SARS-CoV-2 infection in cell culture. These antibodies also inhibit the binding of the Spike protein to human ACE2 receptor of several variants of concern most resistant to neutralization. After exposure to a high dose of homologous SARS-CoV-2, vaccinated groups exhibit significant levels of protection from viral replication in the upper and lower respiratory tracts and from lung tissue inflammation. CONCLUSIONS: We demonstrate that the VLA2001 adjuvanted vaccine is immunogenic both in mouse and NHP models and prevent cynomolgus macaques from the viruses responsible of COVID-19.

Mass vaccination in response to the COVID-19 pandemic has substantially reduced the number of severe cases and hospitalizations. As the virus continues to evolve and give rise to new variants that cause local outbreaks, there is a need to develop new vaccine candidates capable of stopping the viral transmission. In this study, we explore the immune responses induced by the vaccine candidate VLA2001 in animal models. We highlight the vaccine's ability to induce an immune response capable of blocking the virus and eliminating infected cells. We show that it can protect the host from developing severe disease.

Nutrients, bioactive compounds, and minerals in the juices of 16 varieties of apple (Malus domestica) harvested in Austria: A four-year study investigating putative correlations with weather conditions during ripening.

This study was conducted to examine putative correlations between weather parameters during April-September and the amounts of nutrients, minerals and bioactive compounds in the juices of 16 apple varieties from four harvest years in Lower Austria. For most sugar-parameters, negative correlations were found with the total precipitation (r between -0.42 and -0.64). Conversely, positive correlations were observed with the mean air temperature (r between 0.32 and 0.66), the global radiation (r between 0.32 and 0.61) and the number of tropical days (r between 0.39 and 0.51). The sum of 14 polyphenols (HPLC quantitation) was positively correlated with the mean air temperature and global radiation (rs 0.44 and 0.42). Negative correlations were observed between the global radiation and potassium, magnesium and calcium contents (correlation coefficients -0.49, -0.68 and -0.69). We conclude that increased temperatures and global radiation can be correlated with enhanced sugar synthesis and polyphenol formation.

Influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated Japanese Encephalitis vaccine, IXIARO®.

Aluminum hydroxide is a critical raw material in the production of many vaccines. It is used as an adjuvant in the formulation of the final bulk vaccine, and for this it must meet the specifications of the European Pharmacopeia Monograph. We investigated whether vaccine stability was affected by the presence of trace amounts of elemental impurities in commercially available aluminum hydroxide. The content of residual elemental impurities in commercially available aluminum hydroxide was determined by selective and sensitive inductively coupled-plasma mass spectrometry and inductively coupled plasma atomic emission spectroscopy. We found significant differences between different suppliers, but also between different lots from the same supplier. Inactivated Japanese encephalitis vaccine, IXIARO(®), was used to study the effect of residual metals in aluminum hydroxide on antigen stability. We propose that antigen degradation occurred via a pathway involving the metal-catalyzed, auto-oxidation of a process-related impurity (sulfite). Thus, sulfite auto-oxidation resulted in antigen degradation when residual Cu was present at elevated concentrations in aluminum hydroxide.

Differences in pharmacokinetics of apple polyphenols after standardized oral consumption of unprocessed apple juice.

BACKGROUND: Polyphenols are chemical compounds of the secondary plant metabolism. High concentrations can be found in various fruits including apples, berries and grapes. Polyphenols are associated with numerous health beneficial effects including a reduced risk for cardiovascular disease or diabetes. The human body cannot synthesize or store polyphenols and relies on continuous replenishment by daily diet. Unfortunately, knowledge on absorption, metabolization and excretion is still limited. The aim of this study was to determine the pharmacokinetic fate of apple polyphenols in young healthy adults. METHODS: Volunteers consumed 500 mL of an unfiltered apple juice. Blood and urine samples were collected within a time period of ten hours and analyzed for their total phenolic content, concentration of selected individual polyphenolic compounds and antioxidant capacity. RESULTS: Large differences in apple polyphenol pharmacokinetics between single subjects were observed. Those could be divided into subgroups according to fast or slow rates of polyphenol metabolism. Some subjects showed no detectable metabolism within the study time frame at all. An increase in the total phenolic content over time did not correlate with an observed, highly elevated antioxidant capacity (AOC) in the blood plasma after apple juice consumption. The determined increase of the AOC was rather a result of a high fructose content of the apple juice. No differences in renal excretion were detected between female and male subjects. However, relative concentrations were slightly higher in male subjects. CONCLUSIONS: Apple derived polyphenols can be readily detected in human blood and urine after juice consumption. The existence of sub-populations with different pharmacokinetics suggests significant variations in the individual metabolism rates of polyphenolic substances with implications on bioavailability and potential health effects within the body. TRIAL REGISTRATION: O2413 (Ethics Commissions of Upper Austria) and 415-EP/73/233-2013 Salzburg (Ethics Commissions of Salzburg).

Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy.

BACKGROUND AND PURPOSE: Insulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin. EXPERIMENTAL APPROACH: Total internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein. KEY RESULTS: Using our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR. CONCLUSIONS AND IMPLICATIONS: Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.

Bioanalytical characterization of apple juice from 88 grafted and nongrafted apple varieties grown in Upper Austria.

The compositional characteristics of untreated pure juice prepared from 88 apple varieties grown in the region of Eferding/Upper Austria were determined. Many of the analyzed varieties are noncommercial, old varieties not present in the market. The aim of the study was to quantitate the mineral, phosphate, trace elements, and polyphenolic content in order to identify varieties that are of particular interest for a wider distribution. Great variations among the investigated varieties could be found. This holds especially true for the total polyphenolic content (TPC) ranging from 103.2 to 2,275.6 mg/L. A clear dependence of the antioxidant capacity on the TPC levels was detected. Bioinformatics was employed to find specific interrelationships, such as Mg²âº/Mn²âº and PO4³â»/K⁺, between the analyzed bio- and phytochemical parameters. Furthermore, special attention was drawn on putative effects of grafting on the phytochemical composition of apple varieties. By grafting 27 different apple varieties on two trees grown close to each other, it could be shown that the apple fruits remain their characteristic phytochemical composition. Finally, apple juice prepared from selected varieties was further characterized by additional biochemical analysis including cytotoxicity, epidermal growth factor receptor (EGFR) inhibition, and α-amylase activity tests. Cytotoxicity and inhibition of EGFR activation were found to be dependent on the TPC, while α-amylase activity was reduced by the apple juices independent of the presence of polyphenolic substances. Taken together selected apple varieties investigated within this study might serve as preferable sources for the development of apple-based food with a strong focus on health beneficial effects.

Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid.

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.

Conformation of receptor adopted upon interaction with virus revealed by site-specific fluorescence quenchers and FRET analysis.

Human rhinovirus serotype 2 (HRV2) specifically binds to very-low-density lipoprotein receptor (VLDLR). Among the eight extracellular repeats of VLDLR, the third module (V3) has the highest affinity for the virus, and 12 copies of the genetically engineered concatamer V33333-His(6) were found to bind per virus particle. In the present study, ring formation of V33333-His(6) about each of the 12 5-fold symmetry axes on HRV2 was demonstrated by fluorescence resonance energy transfer (FRET) between donor and acceptor on N- and C-terminus, respectively. In particular, the N-terminus of V33333-His(6) was labeled with fluorescein, and the C-terminus with a new quencher which was bound to the His(6) tag with nanomolar affinity (K(d) approximately 10(-8) M) in the presence of 2 microM NiCl(2).

Multiple receptors involved in human rhinovirus attachment to live cells.

Minor group human rhinoviruses (HRVs) attach to members of the low-density lipoprotein receptor family and are internalized via receptor-mediated endocytosis. The attachment of HRV2 to the cell surface, the first step in infection, was characterized at the single-molecule level by atomic force spectroscopy. Sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra, which suggests that multiple receptors bound to the virus in a timely manner. Unbinding forces required to detach the virus from the cell membrane increased within a time frame of several hundred milliseconds. The number of receptors involved in virus binding was determined, and estimates for on-rate, off-rate, and equilibrium binding constant of the interaction between HRV2 and plasma membrane-anchored receptors were obtained.

A new, simple method for linking of antibodies to atomic force microscopy tips.

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.

Mimicking early events of virus infection: capillary electrophoretic analysis of virus attachment to receptor-decorated liposomes.

The attachment of human rhinovirus serotype 2 to an artificial cell membrane was followed by capillary electrophoresis. The cell membrane was mimicked by liposomes (average diameter of about 190 nm) containing a lipid with a nitrilotriacetic acid (NTA) group. This group, in the presence of Ni(2+) ions, served as anchor for the his(6)-tags of recombinant derivatives of the very-low-density lipoprotein (VLDL) receptor comprising either modules 1, 2, and 3 (V123) or five tandem copies of module 3 (V33333). We demonstrate by capillary electrophoresis with laser induced fluorescence detection of the liposomes that the minor receptor group rhinovirus HRV2 binds specifically to the receptor-decorated vesicles; the major receptor group rhinovirus HRV14, which uses a different receptor for cell binding, does not attach to the liposomes.