Hypoxic response protein 1 as a potential immunogenic antigen against tuberculosis infection
Morelli María Paula1,2, Amiano Nicolas Oscar1,2, Peña Delfina1,2, Tateosian Nancy1,2, Pellegrini Joaquín1,2, Rolandelli Agustín1,2, Ciallella Lorena3, Levi Nicolás3, de Casado Graciela3, Palmero Domingo Juan3, García Verónica Edith1,2.

1 Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Universidad de Buenos Aires (UBA)-CONICET, Ciudad Autónoma de Buenos Aires (CABA), Argentina. 2 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, CABA, Argentina. 3 Hospital de Infecciosas “Dr. Francisco Javier Muñiz”, CABA, Argentina.

Hypoxic response protein 1 or Rv2626c, a protein encoded at the Dormancy Survival Regulator (DosR) regulon of Mycobacterium tuberculosis, is overexpressed in hypoxia. In this work, by using different experimental approaches, we analyzed the potential immunogenicity of Rv2626c to be used as a possible vaccine candidate. We previously demonstrated that stimulation of peripheral blood mononuclear cells (PBMCs) and whole blood with Rv2626c induced a specific IFNγ response from latently Mtb infected (LTBI) individuals1,2. Furthermore, LTBI subjects displayed significantly higher plasma levels of anti-Rv2626c IgG as compared to patients with active Mtb infection2. Here, we initially analyzed whether polyfunctional responses against Rv262c protein might be generated. Therefore, PBMCs from LTBI individuals, tuberculosis patients (TBP), and healthy donors (HD) were cultured with Rv2626c and the expression of IFNγ, IL-2, and TNF were analyzed by flow cytometry. Our results showed a significantly higher frequency of CD4+IFNγ+TNF+IL-2+ cells induced in LTBI subjects as compared to TBP and HD. Next, we searched for the immunodominant regions of Rv2626c to which only LTBI responded. For this, we employed overlapping peptides arranged in six pools (A-F) pools composed of six peptides each, covering the entire Rv2626c protein. We found that the pools B, D and F induced the highest IFN-γ levels, allowing to discriminate LTBI from HD. And TBP. In addition to our studies in humans and considering the relevance of multistage vaccines against tuberculosis, we initiated in vivo experiments in BALB/c mice. Animals were administered with three doses (separated by two weeks) of Rv2626c protein plus Ag85A protein and IL-12 DNA as an adjuvant. After 14 days of the last priming dose, strong IgG levels against both proteins were measured in sera from immunized mice. Moreover, when splenocytes from vaccinated mice were stimulated ex vivo with Rv2626c, Ag85A, or both antigens, a significantly increased secretion of IFNg by cells from mice that had received IL-12 DNA was observed. Furthermore, Rv2626c peptide pools also elicited IFNγ production from ex vivo-stimulated splenocytes of immunized mice. Surprisingly, similar immunodominant regions of Rv2626c protein were identified in both humans and mice. Thus, our findings indicate that the murine model might be useful in the development of new therapeutic or prophylactic tuberculosis vaccines including hypoxic response protein 1.

Reference:
1. Peña D, et al. A Mycobacterium tuberculosis Dormancy Antigen Differentiates Latently Infected Bacillus Calmette–Guérin-vaccinated Individuals. EBioMedicine. 2015.
2. Amiano NO, Morelli MP, et al. IFN-γ and IgG responses to Mycobacterium tuberculosis latency antigen Rv2626c differentiate remote from recent tuberculosis infection. Sci Rep. 2020

Funding: This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) [PICT2015-0611 to VEG and PICT2016-0022 START UP to NOA] and Universidad de Buenos Aires (UBACyT) [20020170100127BA to VEG].

Maria Paula Morelli MSc

University of Buenos Aires

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