development of an oral vaccine against candidiasis using
TRANSCRIPT
Seiji SHIBASAKI1, Mitsuyoshi UEDA2
1Hyogo University of Health Sciences, Kobe, Japan, 2Kyoto University, Kyoto, Japan
Introduction Candidiasis is one of serious infec.ous disease caused by the virulent fungus Candida albicans and other Candida species [1]. Pharmacotherapy of candidiasis o=en involves the administra.on of amphotericin B, caspofungin and micafungin. Unfortunately, several mutants of Candida with reduced suscep.bility to these drugs have emerged [2]. Preven.on of infec.on by vaccina.on against Candida species is important strategy in efforts to control candidiasis. Recently, we have developed the molecular display technology, which uses gene.cally engineered microorganisms to produce foreign proteins on their surface [3]. The use of molecular display to prepare target proteins is well established for use with the yeast Saccharomyces cerevisiae. We present that the an.gens displayed on yeast generated using molecular display technology offer a novel type of oral vaccine against candidiasis. The enolase 1 protein (Eno1p) from C. albicans was selected as the model an.genic protein to be displayed on the surfaces of S. cerevisiae cells [4, 5].
(1) Molecular display of Eno1p on yeast cell surface The fragment of the gene encoding ENO1 was inserted into pULD1 plasmid that had been digested with NotI and XhoI. Eno1p coding sequence was fused to 5’-‐end of the cell wall anchoring protein, α-‐agglu.nin, coding sequence in the plasmid.
(2) Oral administra.on of yeast displaying Eno1p Female C57BL/6 (7 w) mice were used for oral immuniza.on experiments. A suspension of S. cerevisiae cells displaying Eno1p on the surfaces (1.6 × 109 cells/400 ml) were administered to ten mice per dose at weeks 0, 1, and 3 and at week 7. The host strain of S. cerevisiae was used as a control. All inoculums were suspended in PBS (400 μL) and administered via an intragastric tube a=er 2 h of fas.ng, once per day for 5 days per week. Blood samples were taken at week 9 to determine the .ter of serum IgG. For survival studies, mice were infected with 1.1 × 105 cells of C. albicans resuspended in 100 μl PBS by tail-‐vein injec.on 2 weeks a=er the last immuniza.on.
Materials and Methods
Results
Conclusions
A vaccine produced using molecular display technology avoids the need for protein purifica.on. This oral vaccine offers a promising alterna.ve to the use of conven.onal and injectable vaccines for preven.ng a range of infec.ous diseases.
References [1] Nucci M & Marr KA (2005) Clin. Infect. Dis. 41: 521–526. [2] Rodloff C, Koch D & Schaumann R (2011) Eur. J. Med. Res. 16: 187–195. [3] Shibasaki S & Ueda M (2010) Recent Pat. Biotechnol. 4: 198-‐213. [4] Shibasaki S, Aoki W, Nomura T, Miyoshi A, Tafuku S, Sewaki T & Ueda M (2013) Pathog. Dis. 69: 262-‐268. [5] Shibasaki S, Aoki W & Ueda M (2013) Yakugaku Zasshi. 133: 1145-‐1151.
Development of an oral vaccine against candidiasis using molecular display technology
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Fig. 1. Fluorescence micrographs
(1) Construc.on of Eno1p-‐displayed yeast cell
Fig. 2. Evalua.on of surface display of Eno1p on yeast cells
Fig. 4. Survival ra.o of vaccinated mice a=er challenge of C. albicans
Fig. 3. An.body response in administra.on of yeast cells
(2) Oral administra.on of yeast
This study shows that the only availability of a DNA sequence-‐encoding the an.genic protein is needed to produce oral vaccine based on molecular display technology.
Eno1p-‐displaying yeast
Administra.on + C. albicans Examina.on of survival rate
+ pULD1 Yeast cell
C. albicans genome DNA
PCR
Gene.cally engineered cells were stained with IgG labeled with Alexa-‐488 (Fig.1). Fluorescent stained cells were analyzed to evaluate surface display quan.ta.vely (Fig.2).
An an.body response in oral administra.on was measured by ELISA (Fig.3). Survival rates were evaluated for vaccinated and control mice a=er challenge of C. albicans (Fig. 4).