Identification of immunodominant Bartonella bacilliformis proteins: a combined in-silico and serology approach

Abstract

Background Bartonella bacilliformis is the aetiological agent of Carrión's disease, a biphasic and highly lethal illness formerly restricted to the South American Andes that is now spreading to adjacent areas. Reliable serodiagnostic approaches and vaccines are urgently needed. In this study, we aimed to identify immunodominant proteins of B bacilliformis and to establish novel and reliable serodiagnostic tools. MethodsWe used a reverse vaccinology approach in combination with an analysis of heterologous genomic expression libraries to identify immunodominant proteins, on the basis of the genome sequences of B bacilliformis strains KC583 and KC584. Antigens were screened with serum samples collected from Peruvian patients with B bacilliformis infections and from German healthy blood donors without history of travel to South America. We further analysed immunoreactive proteins of B bacilliformis with immunoblotting and line blots. We used selected target proteins to develop a diagnostic ELISA. To assess the performance of this ELISA, we did receiver operating characteristic analyses to assess the area under the curve, cutoff values, sensitivities, and specificities with 95% CIs. FindingsWe used serum samples obtained between Dec 23, 1990, and May 5, 2018, from 26 Peruvian patients with B bacilliformis infections and serum samples taken between Aug 28 and Aug 31, 2020, from 96 healthy German blood donors. 21 potentially immunodominant proteins were identified and recombinantly expressed, and their reactivity was assessed with immunoblotting and line blots. Of these 21 antigens, 14 were found to be immunoreactive. By using serum samples of Peruvian patients with Carrión's disease and of healthy German blood donors, we identified three antigens (porin B, autotransporter E, and hypothetical protein B) as suitable immunodominant antigens, and we applied them in a diagnostic ELISA using two different antigen combinations (porin B plus autotransporter E and porin B plus autotransporter E plus hypothetical protein B). For the combination of porin B and autotransporter E, with optical density measured at 450 nm (OD 450 ) cutoff value of 0•29, sensitivity was 80•8% (95% CI 60•7-93•5) and specificity was 94•8% (88•3-98•3) for all Peruvian patient samples. For a combination of porin B, autotransporter E, and hypothetical protein B, with an OD 450 cutoff of 0•34, sensitivity was 76•9% (56•4-91•0) and specificity was 93•8% (86•9-97•7) for all Peruvian patient samples. Interpretation This novel ELISA could represent a useful serodiagnostic tool for future epidemiological studies of B bacilliformis in endemic areas. Additionally, the immunodominant antigens we have identified could provide a first basis for future vaccine development to prevent the highly lethal Carrión's disease. Funding DRUID (Novel Drug Targets against Poverty-Related and Neglected Tropical Infectious Diseases) Initiative and Robert Koch Institute.

Bartonella henselae Recombinant Pap31 for the Diagnosis of Canine and Human Bartonelloses

“…berkhoffii genotype III, were also not reactive to these synthetic peptides, suggesting that three-dimensional epitope conformation is likely of critical importance for the documentation of Pap31 seroreactivity. These findings are consistent with the poor seroreactivity results of B. bacilliformis Pap31 linear epitopes reported in a previous study [35], where only 2 of the 26 Peruvian patients with B. bacilliformis infection were reactive to B. bacilliformis Pap31 linear epitopes in line blots. It is also possible that B. henselae Pap31 peptide epitopes represent a minimally immunogenic region of the B. henselae Pap31 protein, and hence were not reactive with sera from B. henselae IFA-positive dogs and humans in this current study.…”

“…Previously, while assessing B. bacilliformis recombinant Pap31 for the diagnosis of Oroya fever, the authors demonstrated that Pap31 antigens were highly induced in growing cultures of B. bacilliformis and were immunologically recognized dominant proteins in infected humans, supporting the application of Pap31 for the ELISA and WB diagnosis of human Bartonelloses [27]. Dichter et al (2021), using a reverse vaccinology approach in conjunction with an immunoproteomic approach, also highlighted the potential utility of Pap31 as an immunodominant target recognized by the serum samples obtained from Peruvian patients infected with B. bacilliformis [35]. In another study, B. bacilliformis recombinant pap31 was not reactive with sera from patients with Coxiella burnetti, Brucella spp., or B. henselae infection, supporting a lack of recombinant B. bacilliformis Pap31 cross-reactivity with these microorganisms [34].…”

In-silico identification of potential antigenic proteins in Bartonella bacilliformis for the serological diagnosis of Carrions’ disease

“…The antigenicity of Pap-31 was confirmed by Angkasekwinai et al (2014) [48] and Gomes et al (2016) [10]. Similarly, Pap-31 was detected in the study by Dichter et al (2021) [38](see section 4.3 on Known Proteins). Furthermore, Gomes et al (2021) [11] identified immunogenic peptides within Pap-31 and SCS-α, establishing their inter-and intrageneric discrimination capacity.…”

“…B. bacilliformis outer-membrane and extracellular proteins have been analyzed by the identification of epitopes in the design of an artificial multi-epitope protein with positive results in murine model [37]. In a recently published article, Dichter et al [38] tested the immunogenicity of 22 candidate B. bacilliformis proteins identified by a reverse vaccinology approach using Vaxign-1 software. The present study identified 19 exact or homologs of these proteins.…”

“…In agreement with these data, Pap-31 was identified in the present in-silico analysis. Furthermore, coinciding with Dichter et al (2021) [38], 3 other porins (A, B and C) were identified. Interestingly, these porins present one of the lowest percent similarities with other Bartonella species (48%-58%).…”