Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

Abstract

Tuberculosis (TB) remains a major global health challenge and the development of a better vaccine takes center stage in fighting the disease. For this purpose, animal models that are capable of replicating the course of the disease and are suitable for the early-stage screening of vaccine candidates are needed. A Mycobacterium marinum infection in adult zebrafish resembles human TB. Here, we present a pre-clinical screen for a DNA-based tuberculosis vaccine in the adult zebrafish using an M. marinum infection model. We tested 15 antigens representing different types of mycobacterial proteins, including the Resuscitation Promoting factors (Rpf), PE/PPE protein family members, other membrane proteins and metabolic enzymes. The antigens were expressed as GFP fusion proteins, facilitating the validation of their expression in vivo. The efficiency of the antigens was tested against a low-dose intraperitoneal M. marinum infection (% 40 colony forming units), which mimics a primary M. tuberculosis infection. While none of the antigens was able to completely prevent a mycobacterial infection, four of them, namely RpfE, PE5_1, PE31 and cdh, led to significantly reduced bacterial burdens at four weeks post infection. Immunization with RpfE also improved the survival of the fish against a high-dose intraperitoneal injection with M. marinum (% 10.000 colony forming units), resembling the disseminated form of the disease. This study shows that the M. marinum infection model in adult zebrafish is suitable for the pre-clinical screening of tuberculosis vaccines and presents RpfE as a potential antigen candidate for further studies.

Animal Models of Tuberculosis Vaccine Research: An Important Component in the Fight against Tuberculosis

“…, and is widely used as an animal model in vaccine research owing to its advantages of small size, easy reproduction, and low cost [36]. A er infection by M. marinum, both adult zebrafish and larvae can form granulomas that are very similar to those observed in humans, and the innate and adaptive immune responses elicited against mycobacteria are composed of the same primary components found in humans [37][38][39]. In addition, the transparent characteristic of zebrafish larvae is also suitable for fluorescence imaging.…”

“…Although zebrafishes are very different to humans in genetic terms, the above characteristics of this model have helped to bridge the gap between fish and humans. Data obtained from zebrafish studies have already shown that BCG vaccination, as well as DNA vaccination, can protect adult zebrafish from M. marinum infection by reducing both the mortality and bacterial counts in a manner dependent on the adaptive immune response and enhanced production of IFN-γ [38,113]. In addition to its use for the preclinical screening of vaccines, the zebrafish model has been used in clarifying the mechanisms underlying granuloma formation [114].…”

Mycobacterium tuberculosis PE31 (Rv3477) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1

“…As previously, reported vaccine candidate (Myllymaki et al, 2017), the role of PE31 in host interactions and pathogenesis of M. tuberculosis has remained to be understood. M. tuberculosis survival within macrophages is necessitates effective neutralization of hostile environments such as acidification, oxygen radicals, and RNIs (Cossu et al, 2012;Li et al, 2017).…”

“…Previously, the PE31 interact with PPE18, and able to form a heterodimeric complex with TLR 2 (Mukhopadhyay and Balaji, 2011). While another report suggested that PE31 with PE51 are protective antigens (Myllymaki et al, 2017). However, the exact role of PE31 in pathogenicity, host-pathogen interaction and underlying mechanisms are unknown.…”

Zebrafish as an alternative animal model in human and animal vaccination research

“…An infection by Mycobacterium marinum in adult zebrafish resembles that of human tuberculosis, as demonstrated by Myllymäki et al [62]. Those authors proved that the M. marinum infection model in adult zebrafish was suitable for preclinical screening of tuberculosis immune's responses and vaccines.…”