A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin from Megathura crenulata and used as an immunogen against Rhipicephalus microplus and Rhipicephalus sanguineus s.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate against Amblyomma mixtum and Ixodes ricinus species, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein. SDS-PAGE analysis of this conjugate demonstrated that it is highly heterogeneous in size, carrying from 1 to 18 molecules of pP0 per molecule of Bm86. Forty-nine out of the 54 lysine residues and the N-terminal end of Bm86 were found partially linked to pP0 by using LC-MS/MS analysis and the combination of four different softwares. Several post-translational modifications of Bm86 protein were also identified by mass spectrometry. High immunogenicity and efficacy were achieved when dogs and cattle were vaccinated with the pP0–Bm86 conjugate and challenged with R. sanguineus s.l. and R. microplus, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 × 10⁻⁸. These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

The protein composition of an Outer Membrane Vesicle (OMV) preparation that constitutes the active pharmaceutical ingredient of VA-MENGOC-BC®, an effective vaccine against Neisseria meningitidis serogroups B, and C is presented. This preparation has a high lipid content and five abundant membrane proteins (FetA, PorA, PorB, RmpM, and Opc), constituting approximately 70% of the total protein mass. The protein composition was determined by combining the use of the Hexapeptide Ligand Library and an orthogonal tandem fractionation of tryptic peptides by reverse-phase chromatography at alkaline and acid pH. This approach equalizes the concentration of tryptic peptides derived from low- and high-abundance proteins as well as considerably simplifying the number of peptides analyzed by LC-MS/MS, enhancing the possibility of identifying low-abundance species. Fifty-one percent of the proteins originally annotated as membrane proteins in the genome of the MC58 strain were identified. One hundred and sixty-eight low-abundance cytosolic proteins presumably occluded within OMV were also identified. Four (NadA, NUbp, GNA2091, and fHbp), out of the five antigens constituting the Bexsero® vaccine, were detected in this OMV preparation. In particular, fHbp is also the active principle of the Trumenba® vaccine developed by Pfizer. The HpuA and HpuB gene products (not annotated in the MC58 genome) were identified in the CU385 strain, a clinical isolate that is used to produce this OMV. Considering the proteins identified here and previous work done by our group, the protein catalogue of this OMV preparation was extended to 266 different protein species.