Elsevier

Immunology Letters

Volume 231, March 2021, Pages 61-67
Immunology Letters

Oral co-administration of bivalent protein r-BL with U-Omp19 elicits mucosal immune responses and reduces S. Typhimurium shedding in BALB/c mice

https://doi.org/10.1016/j.imlet.2021.01.006Get rights and content

Highlights

  • A novel bivalent protein r-BL was produced by in-frame splicing of sseB and ompL genes of S.Typhimurium and was stably expressed in prokaryotic system.

  • Mucosal adjuvant U-Omp19 protected the recombinant vaccine molecule r-BL in the protease rich environment of the GI tract.

  • Mice immunized with r-BL+U-Omp19 demonstrated a significant rise in local mucosal immunity.

  • Oral immunization with r-BL+U-Omp19 effectively reduced the fecal shedding of S. Typhimurium upon oral challenge in mice model.

Abstract

The increase in international food trade and travel has dramatically increased the global incidences of Salmonellosis. In the light of widespread resistance to frontline antibiotics, oral vaccines remain the most reliable alternative. In this study, the fusion protein, r-BL was rationally constructed by splicing the Salmonella Typhimurium sseB and ompL genes through G4S linker by over-lap extension PCR. The oral coadministration of r-BL with B. abortus U-Omp19 protein with known protease inhibitor activity resulted in significant increase of mucosal IgA titres to antilog 4.5051 (p < 0.0001) and 4.806 (p < 0.0001) in the fecal samples and intestinal washes respectively. Antibody isotyping of the intestinal washes demonstrated increase in mucosal IgM, IgG1 and IgG2a isotypes also and demonstrated a significant reduction in fecal shedding of S. Typhimurium in challenge study. The r-BL + U-Omp19 treated mice demonstrated a complete termination of Salmonella fecal shedding by the 12th day of challenge as compared to other study groups. In summary, the bivalent protein r-BL when administered with the mucosal adjuvant U-Omp19 was successful in triggering mucosal arm of the immune system which forms the first line of defence in combating the infections caused by the enteric pathogen like Salmonella.

Introduction

Salmonellosis an enteric illness that manifests as non-typhoidal enterocolitis, non-typhoidal focal disease, or typhoid (enteric) fever is a major public health concern in both developed and developing nations contributing substantially to the global burden of illness and deaths caused by foodborne diseases [1]. The etiological agent, Salmonella belongs to the family Enterobacteriaceae and is further divided into two species S. bongori and S. enterica with around 2600 serotypes by Kauffman–White scheme [9]. Contaminated food and water are the main reservoirs of Salmonella and one out of four diarrheal infections worldwide is attributed to this etiological agent [23]. Infants, children and immunocompromised are at higher risk as compared to adults. S. enterica subsp. Enterica serovar Typhi exclusively infects humans whereas non-typhoidal Salmonella such as S. enterica subsp. Enterica serovar Typhimurium and S. enterica subsp. Enterica serovar Enteritis infects humans, poultry, and cattle [7]. Salmonellosis is extensively treated using broad-spectrum antibiotics like fluoroquinolones, ampicillin, chloramphenicol, and trimethoprim–sulfamethoxazole and has led to the emergence of drug resistance and fear of potential epidemics [9]. The first incidence of Salmonella resistance to chloramphenicol was reported in 1960 [9] followed by the isolation of first multi drug resistant (MDR) strain of S. Typhimurium DT104 in 1990 [13]. The statistical surveys have reported high numbers of MDR isolates of S. Typhi in the continents of Asia and Africa [9].

The global eradication of smallpox by 1980′s and of Polio by 1988 in 121 countries proves vaccination as a rational approach for controlling the incidences of salmonellosis [24]. The live attenuated Ty21a and subunit Vi polysaccharide (ViCPS) are the two licensed vaccines for typhoid fever with the modest protective efficacy of 50–60 % over 3 years while there is no licensed vaccine available for non-typhoidal salmonellosis [20]. The selection of immunization route for effective prophylaxis is largely influenced by the entry route of etiological agents inside the host. Salmonella enters the host through oral route, and mucosal secretory IgA antibodies along mucosal surfaces of GI tract elicited by mucosal vaccines can play an important role in effective bacterial neutralization before establishment of infection. Mucosal vaccines administered through oral route also facilitates large scale vaccine coverage, ease of immunization without risk of blood borne infections [24]. The secretory IgA antibodies in the GI tract comes with the inherent advantage of resistance to host proteases and inhibit the adhesion and invasion of the pathogens with a massive production of approximately 50 mg/kg body weight per 24 h in humans [14]. Although the major share of immune protection is credited to mucosal IgA antibodies, nevertheless locally produced IgG and IgM also contributes in protection [14,15].

The target antigen when efficiently delivered to the MALT system underlying mucosal epithelium rich in immune cells like macrophages, dendritic cells, B-cells, T-cells by an ideal mucosal adjuvant elicits stronger immune response and block the pathogen even before it establishes infection [8,22]. Susceptibility to protease rich environment of the gastrointestinal tract is a prominent shortcoming for majority of the mucosal adjuvants [17,29]. The un-lipidated version of outer membrane protein, U-Omp19 from Brucella abortus has demonstrated the protease inhibitor activity and used as mucosal vaccine adjuvant [17,25,29]. The TTSS proteins and outer membrane proteins (OMP) of Salmonella have remained the principal targets for vaccine development. SseB, a SPI-2 encoded TTSS protein is localized on the cell surface of Salmonella and forms the part of translocon. Its prime role is membrane pore formation in the mucosal epithelial cells making way for effector proteins to the cytoplasm [19,28,29]. OmpL is a transmembrane β-barrel protein with 12 transmembrane surface exposed loops. Being surface exposed, highly expressed and easily accessible to the immune cells makes the outer membrane proteins like OmpL yet another attractive candidate [10,33]. In the present study we aimed to uncover the mucosal immune protection developed after the oral co-administration of bivalent subunit vaccine r-BL derived from SseB and OmpL of S. Typhimurium along with the mucosal adjuvant U-Omp19 of Brucella abortus in mouse model.

Section snippets

Animals and Ethical Statement

In the present study, four to six-week-old BALB/c mice were used as per the guidelines and regulations by Institutional Animal Ethical Committee, Defence Food Research Laboratory, Mysuru (IAEC-2016/MB/15) completely accredited by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The mice were maintained in the standard animal care facilities and were fed with sterile food and water ad libitum. The period of one week was laid for acclimatisation to

Construction, expression and characterization of recombinant bl and U-Omp19

We employed overlap extension PCR for the in-frame splicing of sseB (579 bp) and ompL (648 bp) genes of S. Typhimurium ATCC 14,028 through polyglycine linker (G4S) encoded by 15 nucleotide bases. The splicing yielded the desired product length of 1458 bp (Fig. 1A). After cloning in pRSET A vector the integrity of recombinant plasmid pRSET A-r-BL was confirmed by PCR with T7 universal primers and sequencing. In case of the mucosal adjuvant; the PCR amplified Omp19 gene fragment from B. abortus

Discussion

Salmonella an enteric pathogen gains entry through oral route and initiates infection by invading the M cells in follicle associated epithelium of Peyer’s patches [12,16,26,27]. The host immune protection at these mucosal linings is by the secretory antibodies especially the IgA antibodies mediated immune exclusion [14,24]. Harnessing mucosal immune responses would help in neutralization of pathogens at the mucosal surfaces and prevent the onset of infection at very early stages. Both subunit

Declaration of Competing Interest

The authors declare no conflict of interest with the subject and its manuscript.

Acknowledgments

Authors are thankful to the Director, DFRL for providing necessary facilities to conduct the study. PN is funded by Senior Research Fellowship (SRF) from Defence Research and Development Organization, India.

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