A Novel Pan-Proteome Array for High-Throughput Profiling of the Humoral Response toTreponema pallidumsubsp.pallidum: a Pre-Clinical Study
Authors: Campo Joseph J. , Romeis Emily R. , Oberai Amit , Pablo Jozelyn V. , Hung Christopher , Teng Andy A. , Shandling Adam D. , Phan Amber , Haynes Austin M. , Giacani Lorenzo 
Published: April 2024

ABSTRACTBackgroundGiven the resurgence of syphilis, research endeavors to improve current assays for serological diagnosis and management of this disease are a priority. A proteome-scale platform for high-throughput profiling of the humoral response toTreponema pallidum(T. pallidum) proteins during infection could identify antigens suitable to ameliorate the performance and capabilities of treponemal tests (TTs), which may require weeks to become positive following infection, cannot distinguish between active and previously treated infections, or assess treatment response. Additionally, because infection-induced immunity is partially protective, profiling the response toT. pallidumouter membrane proteins (OMPs) could help select vaccine candidates.MethodsWe developed a pan-proteome array (PPA) based on the Nichols and SS14 strain complete proteomes and used it to define the IgM and IgG humoral response to 1,009T. pallidumproteins in sera collected longitudinally from long-term infected rabbits, and from rabbits that were infected, treated, and re-infected.FindingsApproximately a third of the pathogen’s proteome was recognized in infected animals, with a marked IgG response detectable between day-10 and day-20 post-infection. We found early, gradual, and late IgG kinetic profiles, strain-dependent differences in humoral reactivity, and post-treatment fluctuation in reactivity for several antigens. Very few antigens elicited an IgM response. Several OMPs were significantly and differentially recognized, but few elicited a robust response.InterpretationThe PPA allowed the identification of antigens that could facilitate early diagnosis and of a core set of OMP that could explain protection upon re-infection. No antigen appeared suitable to monitor treatment response.FundingNIH SBIR-R43AI149804RESEARCH IN CONTEXTEvidence before this studyIn April 2024, we searched the PubMed database for articles on preclinical studies using high throughput proteome arrays containing at least 10% of the predictedT. pallidumproteome that aimed at identifying antibody reactivity toT. pallidumantigens during experimental syphilis infection. We could retrieve only one manuscript. In this work, an array containing theT. pallidumpartial proteome as annotated in the first sequenced Nichols strain genome (GCA_000008605.1) in 1998 was assembled using recombinant antigens expressed inEscherichia coli(E. coli). The resulting array was probed using pooled sera from three rabbits infected with the Nichols stain ofT. pallidum, attained from infected animals at five time points following intratesticular infection. The small number of reactive antigens (n = 106) identified in this early study was likely to be an incomplete set of all antigens recognized during infection because not all the predicted targets in theT. pallidumproteome were successfully expressed and tested. In retrospect, additional limitations of the study included an initial suboptimal annotation of the Nichols genome used to define the pathogen’s proteome, which has now changed with the availability of a re-sequenced Nichols strain genome devoid of sequencing errors that affected the initial annotation process, and the refinement of bioinformatic pipelines for the identification of open reading frames (ORFs). Furthermore (as acknowledged by the authors), the possible presence of amplification errors in their expression clones might have affected the sequence of some protein targets and antibody binding to the targets. As a result, some of theT. pallidumantigens known to elicit a robust humoral response during experimental infection were not detected in this antigenic screen. Lastly, employing only the Nichols strain in this early study did not consider that a significant portion of the circulating syphilis strains belong to the SS14 clade ofT. pallidum.Added value of this studyThis novel PPA, combined with a more robust experiential design than ever reported, allowed us to overcome most of the limitations associated with the study mentioned above, as we were able to a) use the most recent annotations for the selectedT. pallidumstrains based on accurate genome sequences, b) print the pathogen’s virtually complete proteome in the study array, c) analyze individual sera to account for rabbit-to-rabbit variability in the humoral response to infection rather than pooled sera, d) detect both IgM and IgG over 10 or 20 timepoints, depending on the experimental design, e) obtain information on how the humoral response evolved upon treatment and re-infection and, finally, f) evaluate all of the above in animals infected with twoT. pallidumstrains whose genetic background is representative of the two currently circulating clades of the syphilis agent.Implications of all the available evidenceOur study provides new and more comprehensive data on how humoral immunity for two classes of antibodies develops during infection and how it evolves in response to treatment and re-infection. The analysis of sera collected at tightly spaced time points post-inoculation and for an extensive period post-infection provides a wealth of information to improve the diagnostic performance of existing tests detecting treponemal antigens. The analysis of differential immunity specific to the pathogen’s putative OMPs provides a rationale for vaccine candidate selection.