Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs

Abstract

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

Identification of Immunogenic Linear B-Cell Epitopes in C. burnetii Outer Membrane Proteins Using Immunoinformatics Approaches Reveals Potential Targets of Persistent Infections

“…As previously shown in Table 4, between 20% and 33% of responders to the epitopes OMP-H (51)(52)(53)(54)(55)(56)(57)(58)(59) , OMP-H (91-106) , Com-1 (57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76) , Com-1 (191)(192)(193)(194)(195)(196)(197)(198)(199)(200)(201)(202)(203)(204)(205)(206) , and OMP-P1 (215-227) presented high levels of specific antibodies. Aiming to explore these data, we compared the clinical features of high responders (HR: RI > 2), low responders (LR: 1 > RI ≤ 2), and nonresponders (NR: RI ≤ 1) to each studied peptide.…”

“…Moreover, in the C. burnetii-reactive group, the frequencies of response against the epitopes OMP-H (51-59) (65%), OMP-H (91-106) (50%), Com-1 (57-76) (58%), Com-1 (191-206) (58%), and OMP-P1 (215-227) (15 responders; 58%) were higher than 50%. Moreover, the frequency of response to OMP-P1 (197-209) (23%) was statistically lower than the frequencies of response to OMP-H (51-59) (p = 0.0047), Com-1 (57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76) , Com-1 (191)(192)(193)(194)(195)(196)(197)(198)(199)(200)(201)(202)(203)(204)(205)(206) , and OMP-P1 (215-227) (p = 0.0227) (Figure 2b). Despite these differences, we 2c).…”

“…The antigenicity of predicted linear epitopes was evaluated using the VaxiJen algorithm. Based on this evaluation, eight predicted epitopes were considered antigenic, among which were the four sequences predicted as linear B-cell epitopes by the three used algorithms (OMP-H (51)(52)(53)(54)(55)(56)(57)(58)(59) , OMP-H (98)(99)(100)(101)(102)(103)(104)(105)(106) , Com-1 (57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76) , and Com-1 (191)(192)(193)(194)(195)(196)(197)(198)(199)(200)(201)(202)(203)(204)…”

“…Regarding the exposition of epitopes on protein quaternary structures, predicted epitopes OMP-H (51)(52)(53)(54)(55)(56)(57)(58)(59) and OMP-H (91-106) (Figure 1a,b), Com-1 (191-206) (Figure 1c,d), and OMP-P1 (197)(198)(199)(200)(201)(202)(203)(204)(205)(206)(207)(208)(209) and OMP-P1 (215-227) (Figure 1e,f) were exposed on the trimer surface, and did not interact with other chains in the oligomeric structure; meanwhile, the epitopes Com-1 (57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76) , Com-1 (26-34) (Figure 1d), and OMP-P1 (98-106) (Figure 1e) presented, 25%, 78%, and 56%, respectively, of their sequences interacting with other chains when the predicted OMP trimeric structure was analyzed. Therefore, predicted epitopes Com-1 (26-34) (Figure 1d…”

Insights into Leishmania Molecules and Their Potential Contribution to the Virulence of the Parasite

“…The host proteinases, such as matrix metalloproteinases, play a crucial role in the subversion of the host immune response. Other virulence factors include glycoinositolphospholipids (GIPLs) [ 21 , 22 , 23 ], lipophosphoglycan (LPG) [ 24 , 25 ], proteophosphoglycan (PPG) [ 26 ], A2 protein [ 23 , 27 ], cysteine proteinases [ 23 ], surface acid proteinase (Gp63Gp63) [ 23 , 27 ] and 11 kDa kinetoplastid membrane protein (KMP-11) [ 28 ]. These factors might modulate the interaction between Leishmania and host immune cells [ 17 , 19 , 29 ].…”