Synthesis of Sporidesmin Based Antigens

<p>In a program aimed at developing a chemically derived vaccine against sporidesmin-A, the toxin which causes the pasture disease facial eczema, two haptens have been prepared, coupled to protein carriers, and tested for immunogenicity. The hapten, 2-amino-5-chloro-3,4-dimethoxybenzyl alcohol has been synthesised from vanillin and the general synthetic methods developed in the course of this work have been used to prepare a large number of related vanillin derivatives for use in cross-hapten studies. The carbon-13 nuclear magnetic resonance spectra of 16 vanillin derivatives have been obtained, and two independent methods of deducing vanillin substitution patterns have been developed. 5-chloro-6, 7-dimethoxy-N-methyl-lH-indole has been prepared by a new route and its 3-oxoacetic acid derivative has been synthesized for use as a hapten. The Vilsmeier-Haack intermediate, l-methyl-3-(N,N-dimethylimonio) methyl-lH-indole chloride was isolated and a number of possible synthetic routes from it to pyrroloindoles were explored. The vanillin and indole haptens have been coupled to protein carriers to form antigens which generated a low antibody response. However, the use of mycobacterium phlei as a carrier greatly increased the antibody response but the antibodies produced did not interact strongly with sporidesmin-A.</p>

Synthesis of Sporidesmin Based Antigens

<p>In a program aimed at developing a chemically derived vaccine against sporidesmin-A, the toxin which causes the pasture disease facial eczema, two haptens have been prepared, coupled to protein carriers, and tested for immunogenicity. The hapten, 2-amino-5-chloro-3,4-dimethoxybenzyl alcohol has been synthesised from vanillin and the general synthetic methods developed in the course of this work have been used to prepare a large number of related vanillin derivatives for use in cross-hapten studies. The carbon-13 nuclear magnetic resonance spectra of 16 vanillin derivatives have been obtained, and two independent methods of deducing vanillin substitution patterns have been developed. 5-chloro-6, 7-dimethoxy-N-methyl-lH-indole has been prepared by a new route and its 3-oxoacetic acid derivative has been synthesized for use as a hapten. The Vilsmeier-Haack intermediate, l-methyl-3-(N,N-dimethylimonio) methyl-lH-indole chloride was isolated and a number of possible synthetic routes from it to pyrroloindoles were explored. The vanillin and indole haptens have been coupled to protein carriers to form antigens which generated a low antibody response. However, the use of mycobacterium phlei as a carrier greatly increased the antibody response but the antibodies produced did not interact strongly with sporidesmin-A.</p>

In vivo imaging shows continued association of several IFT A, B and dynein complexes while IFT trains U-turn at the tip

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Two-color imaging of fluorescent protein-tagged IFT components indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B, and IFT dynein typically remain associated at the tip and anterograde trains convert directly into retrograde trains without disassembly but for possible splitting into strings of IFT complexes. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit preventing the build-up of IFT material in flagella.

Feasibility Study on the Use of Fly Maggots (Musca domestica) as Carriers to Inhibit Shrimp White Spot Syndrome

The shrimp aquaculture industry has encountered many diseases that have caused significant losses, with the most serious being white spot syndrome (WSS). Until now, no cures, vaccines, or drugs have been found to counteract the WSS virus (WSSV). The purpose of this study was to develop an oral delivery system to transport recombinant proteinaceous antigens into shrimp. To evaluate the feasibility of the oral delivery system, we used white shrimp as the test species and maggots as protein carriers. The results indicated that the target protein was successfully preserved in the maggot, and the protein was detected in the gastrointestinal tract of the shrimp, showing that this oral delivery system could deliver the target protein to the shrimp intestine, where it was absorbed. In addition, the maggots were found to increase the total haemocyte count and phenoloxidase activity of the shrimp, and feeding shrimp rVP24-fed maggots significantly induced the expression of penaeidins 2. In the WSSV challenge, the survival rate of rVP24-fed maggots was approximately 43%. This study showed that maggots can be used as effective oral delivery systems for aquatic products and may provide a new method for aquatic vaccine delivery systems.

pH-Sensitive Chitosan Nanoparticles for Salivary Protein Delivery

Salivary proteins such as histatins (HTNs) have demonstrated critical biological functions directly related to tooth homeostasis and prevention of dental caries. However, HTNs are susceptible to the high proteolytic activities in the oral environment. Therefore, pH-sensitive chitosan nanoparticles (CNs) have been proposed as potential carriers to protect proteins from enzymatic degradation at physiological salivary pH. Four different types of chitosan polymers were investigated and the optimal formulation had good batch to batch reproducibility, with an average hydrodynamic diameter of 144 ± 6 nm, a polydispersity index of 0.15 ± 0.04, and a zeta potential of 18 ± 4 mV at a final pH of 6.3. HTN3 encapsulation and release profiles were characterized by cationic polyacrylamide gel electrophoresis. The CNs successfully encapsulated HTN3 and selectively swelled at acidic pH to facilitate HTN3 release. Protection of HTN3 against enzymatic degradation was investigated in diluted whole saliva. HTN3 encapsulated in the CNs had a prolonged survival time compared to the free HTN3. CNs with and without HTN3 also successfully reduced biofilm weight and bacterial viability. The results of this study have demonstrated the suitability of CNs as potential protein carriers for oral applications, especially for complications occurring at acidic conditions.

Quantitative consequences of protein carriers in immunopeptidomics and tyrosine phosphorylation MS2 analyses

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20x protein carrier in pMHC analyses and a high (~100x) and low (~9x) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the control condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

Evaluation of Glycosylated FlpA and SodB as Subunit Vaccines Against Campylobacter jejuni Colonisation in Chickens

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and the handling or consumption of contaminated poultry meat is the key source of infection. C. jejuni proteins FlpA and SodB and glycoconjugates containing the C. jejuni N-glycan have been separately reported to be partially protective vaccines in chickens. In this study, two novel glycoproteins generated by protein glycan coupling technology—G-FlpA and G-SodB (with two and three N-glycosylation sites, respectively)—were evaluated for efficacy against intestinal colonisation of chickens by C. jejuni strain M1 relative to their unglycosylated variants. Two independent trials of the same design were performed with either a high challenge dose of 107 colony-forming units (CFU) or a minimum challenge dose of 102 CFU of C. jejuni M1. While antigen-specific serum IgY was detected in both trials, no reduction in caecal colonisation by C. jejuni M1 was observed and glycosylation of vaccine antigens had no effect on the outcome. Our data highlight inconsistencies in the outcome of C. jejuni vaccination trials that may reflect antigen-, challenge strain-, vaccine administration-, adjuvant- and chicken line-specific differences from previously published studies. Refinement of glycoconjugate vaccines by increasing glycosylation levels or using highly immunogenic protein carriers could improve their efficacy.