Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis

Abstract

A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys 1 pP0 and p64K-βAla 1 pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli , the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-Cys 1 pP0 showed a heterogeneous conjugate compared to p64K-βAla 1 pP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-Cys 1 pP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-βAla 1 pP0 this ratio was 5-7. Cys 1 pP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while βAla 1 pP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-Cys 1 pP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-Cys 1 pP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract

Differentiation of isobaric cross‐linked peptides prepared via maleimide chemistry using MALDI‐MS and MS/MS

“…The peptides were purified using a HP1100 HPLC system (Agilent Considering the integrity of structural variants (hydrolyzed and thiazine forms 17,18 ) of the linker moiety, which are relevant to the conjugation modes, four types of cross-linked peptides were synthesized (Table 1). P3-P4 and P1-P3 yielded only the hydrolyzed forms on incubation under alkaline conditions (see Section 2).…”

“… 10 These isomers are stable in vivo and are not subject to the retro‐Michael addition. This hydrolysis of the thiosuccinimide linker proceeds more rapidly at basic pH and also occurs during the sample preparation and proteolysis of conjugates regardless of the position of the cysteine residue that is involved in the thioether bond 16–18 . The thiosuccinamic acids, hydrolysis products derived from a thiosuccinimide, can be easily identified using the MS mode, based on the fact that the molecular masses of these species differ by 18 Da.…”

On the utility of the extracted ion chromatograms for assigning the conjugation sites in bioconjugates synthesized by the maleimide-thiol chemistry

“…The identification of the conjugation site by mass spectrometry is very important because it informs the linkage position(s) between the antigen/drug and the carrier protein and shed light on product homogeneity (8,9). The identification of the conjugation sites and side reactions in homogeneous and heterogeneous conjugate vaccines (10,11) can be achieved by the identification of type 2 peptides (12) with software (13)(14)(15)(16)(17) developed to study protein-protein interactions by combining chemical cross-linking and mass spectrometry (18). By definition, a type 2 peptide is composed by two proteolytic peptides covalently connected through a chemical linker as a consequence of a crosslinking reaction (12,13).…”

“…This aspect is determinant for veterinary conjugate vaccines characterized by their very low cost (28). We have been working on the development of a wide-coverage veterinary conjugate vaccine against different tick species (10,11,(29)(30)(31) based on the chemical crosslinking of two antigenic peptides Cys 1 pP0 (CAAGGGAAAAKPEESKKEEAK) and Ac-Cys 1 pP0 (CH3COhttps://doi.org/10.26434/chemrxiv-2023-3g9g6 ORCID: https://orcid.org/0000-0002-8875-3642 Content not peer-reviewed by ChemRxiv. License: CC BY-NC 4.0 CAAGGGAAAAKPEESKKEEAK) (29) to two carrier proteins, Bm86 (32) and p64k (33) using different chemistries, including the maleimide-thiol chemistry.…”

“…BMPS has also been used in the development of other human vaccines (5,6). BMPS also allowed the synthesis of several candidate anti-ticks conjugate vaccines (Bm86-Cys 1 pP0, Bm86-Ac-Cys 1 pP0, and p64k-Cys 1 pP0) in two steps (10,11).…”