Strategies to improve the immunogenicity of subunit vaccine candidates

Subunit vaccines contain highly defined macromolecular components of a pathogen that are capable of eliciting protective immunity. They possess several advantages over other vaccine types (e.g. live attenuated and inactivated) such as improved safety profiles, highly defined nature, ease of production, and potential for lower cost of goods. One critical limitation of subunit vaccines, however, is their weak immunogenicity owing to their inability to replicate, monovalent structures, and the absence of other immunostimulatory components. Common approaches to enhance the immunogenicity of subunit vaccines include polyvalent antigen display strategies, the use of adjuvants, etc. The polyvalent antigen display strategy requires the use of a scaffold, which can be protein-based or some other materials. Chapter 2 focuses on the biophysical properties of a potential scaffold for polyvalent antigen display-Bacillus anthracis lumazine synthase (LS), an icosahedral homo-oligomeric protein. LS in PBS buffer showed a minor thermal transition around 50 ᵒC, and a major one at 95 ᵒC. The minor transition arose from the dissociation of the LS/phosphate complex, which formed in PBS buffer at room temperature. The major transition corresponded to the dissociation of LS oligomers, thermal unfolding, and aggregation. In chapter 3, I describe an attempt to develop ricin vaccine candidates in which LS was used as a scaffold to achieve polyvalent display of a linear neutralizing epitope (designated PB10) from ricin. PB10 was genetically inserted onto the C terminus of LS, and the fusion protein (designated LS_PB10) was expressed in an E.coli system. LS_PB10 self-assembled into spherical particles. Fusion of the PB10 peptide did not affect the structure and stability of LS. LS_PB10 showed tight binding to a mAb targeting the PB10 epitope. Immunization of LS_PB10 in mice elicited a moderate level of anti-ricin serum titers, which, however, failed to offer protection during a challenge study using a 10x lethal dose of ricin. Such an unsatisfactory end result may be attributable to 1) limited efficacies of using the PB10 epitope alone, 2) loss of secondary structure of PB10 on LS; 3) an epitope suppression effect induced by the highly immunogenic nature of the LS scaffold. Studying antigen/adjuvant compatibility is critical for the development of adjuvanted vaccine formulations. Chapter 4 discusses the utilization of biophysical tools to understand effects of two emulsion-based adjuvants (designated as ME.0 and SE) on the structure and thermal stability of alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection. Both adjuvants are oil-in-water (O/W) emulsions using squalene as the oil phase. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a blue shift in Trp fluorescence emission spectra of AT. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. The goal of protein formulation development is to identify optimal conditions for long-term storage. Certain commercial conditions (e.g., high protein concentration or turbid adjuvanted samples) impart additional challenges to biophysical characterization. Formulation screening studies for such conditions are usually performed using a simplified format in which the target protein is studied at a low concentration in a clear solution. The failure of study conditions to model the actual formulation environment may cause a loss of ability to identify the optimal conditions for target proteins in their final commercial formulations. In chapter 5, we utilized a steady-state/lifetime fluorescence-based high-throughput platform to develop a general workflow for direct formulation optimization under analytically challenging but commercially relevant conditions. A high-concentration monoclonal antibody and an Alhydrogel-adjuvanted antigen were investigated. A large discrepancy in screening results was observed for both proteins under these two different conditions (simplified versus commercially relevant). This study demonstrates the feasibility of using a steady-state/lifetime fluorescence plate reader for direct optimization of challenging formulation conditions and highlights the importance of performing formulation optimization under commercially relevant conditions

Effects of MS-153 on chronic ethanol consumption and GLT1 modulation of glutamate levels in male alcohol-preferring rats

We have recently shown that upregulation of glutamate transporter 1 (GLT1) in the brain is associated in part with reduction in ethanol intake in alcohol-preferring (P) male rats. In this study, we investigated the effects of a synthetic compound, (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), known to activate GLT1 on ethanol consumption as well as GLT1 expression and certain signaling pathways in P rats. P rats were given 24-hour concurrent access to 15% and 30% ethanol, water and food for five weeks. On week 6, P rats received MS-153 at a dose of 50 mg/kg (i.p.) or a vehicle (i.p.) for five consecutive days. We also tested the effect of MS-153 on daily sucrose (10%) intake. Our studies revealed a significant decrease in ethanol intake at the dose of 50 mg/kg MS-153 from Day 1 through Day 14. In addition, MS-153 at dose of 50 mg/kg did not induce any significant effect on sucrose intake. Importantly, we found that MS-153 upregulated the GLT1 level in the nucleus accumbens (NAc) but not in the prefrontal cortex (PFC). In accordance, we found upregulation of nuclear NFkB-65 level in NAc in MS-153-treated group, however, IkB was downregulated in MS-153-treated group in NAc. We did not find any changes in NFkB-65 and IkB levels in PFC. Interestingly, we revealed that p-Akt was downregulated in ethanol vehicle treated groups in the NAc; this downregulation was reversed by MS-153 treatment. We did not observe any significant differences in glutamate aspartate transporter (GLAST) expression among all groups. These findings reveal MS-153 as a GLT1 modulator that may have potential as a therapeutic drug for the treatment of alcohol dependence

Nanoplanktonic diatom rapidly alters sinking velocity via regulating lipid content and composition in response to changing nutrient concentrations

Diatom sinking plays a crucial role in the global carbon cycle, accounting for approximately 40% of marine particulate organic carbon export. While oceanic models typically represent diatoms as microphytoplankton (> 20 μm), it is important to recognize that many diatoms fall into the categories of nanophytoplankton (2-20 μm) and picophytoplankton (< 2 μm). These smaller diatoms have also been found to significantly contribute to carbon export. However, our understanding of their sinking behavior and buoyancy regulation mechanisms remains limited. In this study, we investigate the sinking behavior of a nanoplanktonic diatom, Phaeodactylum tricornutum (P. tricornutum), which exhibits rapid changes in sinking behavior in response to varying nutrient concentrations. Our results demonstrate that a higher sinking rate is observed under phosphate limitation and depletion. Notably, in phosphate depletion, the sinking rate of P. tricornutum was 0.79 ± 0.03 m d-1, nearly three times that of the previously reported sinking rates for Skeletonema costatum, Ditylum brightwellii, and Chaetoceros gracile. Furthermore, during the first 6 h of phosphate spike, the sinking rate of P. tricornutum remained consistently high. After 12 h of phosphate spike, the sinking rate decreased to match that of the phosphate repletion phase, only to increase again over the next 12 hours due to phosphate depletion. This rapid sinking behavior contributes to carbon export and potentially allows diatoms to exploit nutrient-rich patches when encountering increased nutrient concentrations. We also observed a significant positive correlation (P< 0.001) between sinking rate and lipid content (R = 0.91) during the phosphate depletion and spike experiment. It appears that P. tricornutum regulates its sinking rate by increasing intracellular lipid content, particularly digalactosyldiacylglycerol, hexosyl ceramide, monogalactosyldiacylglycerol, and triglycerides. Additionally, P. tricornutum replaces phospholipids with more dense membrane sulfolipids, such as sulfoquinovosyldiacylglycerol under phosphate shortage. These findings shed light on the intricate relationship between nutrient availability, sinking behavior, and lipid composition in diatoms, providing insights into their adaptive strategies for carbon export and nutrient utilization

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

Three-dimensional modelling of blur property for conventional optical microscopes

Intensity diffusion caused by optical diffraction limits the imaging resolution of conventional optical microscopes, therefore modelling and measuring the intensity transmission and distribution property of the light sources is a significant research topic in system development and pattern recognition. However, the complicated wave propagation process in optical imaging makes it difficult to provide a direct, analytical and simple mathematical model to measure the relationship between the blur degree and various camera parameters. In this study, an improved intensity transmission and distribution calculation method for conventional optical microscopes was proposed; furthermore, a simple mathematical relation between the blur degree and camera parameters was achieved based on the proposed method. First, the light intensity distribution and propagation characteristics of a conventional optical microscope were modeled based on the property of the Fresnel circular hole diffraction combined with the practical optical parameters. Second, by analyzing the property of intensity distribution and blurring imaging, a quantitative simplified mathematical relationship between the blur degree and camera parameters in optical microscope imaging was obtained, and the three-dimensional (3D) blur property in the optical imaging process was analyzed under different conditions. Third, the connection between diffractive optics and geometric optics was obtained by summarizing and generalizing the 3D blur property curve of each monochromatic light source. Finally, the proposed method was verified through a series of simulations and experiments