Multiepitope fusion antigens and vaccines and their use in treatment of enterotoxigenic diarrhea

#The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 12, 2014, is named P12168-02_ST25.txt and is 30,350 bytes in size.

#Virulence heterogeneity among bacterial or viral strains or isolates remains as one major challenge in vaccine development. Like other infectious pathogens, enterotoxigenic Escherichia coli (ETEC) strains are genetically and immunologically heterogeneous. ETEC strains are the most common bacteria causing diarrhea, a disease continues to be the second leading cause of death to children younger than 5 years in developing countries and remains as a major threat to global health. These ETEC strains produce immunologically different colonization factor antigens (CFAs) to colonize host small intestines and enterotoxins to disrupt fluid and electrolyte homeostasis in small intestinal epithelial cells that leads to fluid hyper-secretion and diarrhea. There are 23 CFA or CS (coli surface antigen) adhesins and 2 very distinctive enterotoxins (heat-labile toxin, LT, and heat-stable toxin type Ib, STa) characterized from ETEC strains associated with human diarrhea. As ETEC strains expressing any one or two of these adhesins with either enterotoxin can cause diarrhea, developing vaccines to effectively protect against ETEC diarrhea continues to be challenging.

#It has been observed that immunity induced by individual antigens lacked in cross protection against ETEC, clearly due to immunological heterogeneity among CFAs and toxins. Early experimental vaccine studies showed that candidates carrying a single adhesin and/or toxin antigen induced immunity protecting against only ETEC strains expressing the homologous adhesin or toxin. The first ETEC vaccine candidate, killed ETEC prototype strain H10407 (O78:H11, LT+/STa+/CFAI+), induced anti-CFA/I and anti-LT immunity and protected against homologous challenge. Similarly to homologous protection from anti-CFA immunity, anti-LT antitoxin immunity protected against ETEC strains expressing the LT toxin but not against ETEC strains expressing STa toxin. Oral whole-cell ETEC vaccine candidates currently under development include rCTB-CF and ACE527. The rCTB-CF is a killed cocktail product of 5 strains expressing 6 CFA adhesins and recombinant B subunit of cholera toxin (CT) which is a homologue of LT. ACE527 carried 3 live attenuated E. coli strains that express 5 CFA adhesins, 1 CFA adhesin subunit, and LTB subunit. The killed rCTB-CF induced antibody responses protecting against 70% ETEC infection or against moderate to severe diarrhea to adults from developed countries travelling to ETEC endemic countries. This product, however, caused adverse effects and provided no significant protection against ETEC diarrhea when given to children especially very young children living in endemic areas, or failed to reduce overall diarrhea incidences among adult travelers. The live attenuated ACE527 initially showed some adverse effects, but these adverse effects were reduced or eliminated when a lower dose was used. ACE527 was shown to induce antibody responses to LTB, CFA/I, CS3, and CS6 among adult volunteers and to protect against severity of diarrhea outcome from homologous challenge.

#FIGS. 1A-1D. Construction and detection of an embodiment of the invention comprising ‘CFAI/II/IV-STatoxoid-LTtoxoid’ MEFA. (A) Construction of the CFAI/II/IV MEFA. The most antigenic epitopes of the CS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embedded into CFA/I major subunit by replacing the CfaB surface-exposed but less antigenic epitopes. (B) Construction of the 3×STaN12S-LTtoxoid fusion. Three copies of the STatoxoid STaN12S gene were genetically fused to the monomeric LTtoxoid (LTR192G/L211A) gene using SOE (splicing overlap extension) PCRs. (C) Construction of CFA-toxoid MEFA. A substitution of the first 150 amino acids of the 3×STaN12S-LTtoxoid (the N-terminal STaN12S and the first 131 amino acids of LTA subunit) with the CFA/I/II/IV MEFA created the CFA/I/II/IV-STaN12S-dmLT MEFA. Four linkers: LGA, GPVD (SEQ ID NO: 36), Gly-Pro linker GPGP (SEQ ID NO: 37), and L-linker (DPRVLSS, SEQ ID NO: 39) were used for the construction. (D) Western blot to detect the CFA/I/II/IV-STaN12S-LTtoxoid MEFA protein with anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant (1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300; Sigma) and anti-STa antiserum (1:3300; provided by Dr. D C Robertson). Extracted MEFA proteins separated in 12% PAGE gel were detected with each anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+) indicated the CFA/I/II/IV-STaN12S-LTtoxoid MEFA proteins, whereas lane (−) of extracted total proteins of E. coli BL21 host strain as the negative control. Lane M is the protein marker (in kilodaltons; Precision Plus Protein pre-stained standards; Bio-Rad).

#FIG. 2. Mouse serum anti-adhesin and antitoxin IgG antibody titers. Anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4/CS6 and anti-CS5/CS6, and anti-STa and anti-LT IgG antibodies in the serum of each mouse immunized with ‘CFA/I/II/IV-STaA14Q-LTtoxoid’ fusion antigen (solid circles) and the serum of each control mouse (blank circles) were titrated in ELISAs. CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6 adhesin heat-extracted from E. coli or ETEC strains in Table 1 (500 ng per well of a 2HB plate), STa-ovalbumin (10 ng per well of a Costar plate), or LT (List Biological Laboratories, Inc.; 100 ng per well of a 2HB plate) and HRP-conjugated goat-anti-mouse IgG (1:3300; the secondary antibodies) were used to titrate IgG antibodies specific to CFA/I, CS1, CS2, CS3, CS4/6, CS5/6 adhesins and to STa and LT toxins, respectively. The antibody titer was calculated from the highest dilution of a serum sample that produced an ELISA optical density of greater than 0.3 (above the background) and presented in a log10 scale. Each dot represented a mouse IgG titer, and the bars indicated the mean titer of the group.

#FIG. 3 depicts the titration of anti-CFA/I, -CS1, -CS2, -CS3, -CS4/CS6, -CS5/CS6, and anti-CS6 IgA antibodies in fecal suspension samples of the immunized mice (solid dots) and control mice (circles). Five hundred nanogram CFA adhesins heat-extracted from E. coli strains expressing CFA/I, CS1, CS2, CS3, CS4/CS6 or CS5/CS6 were coated to each well of an Immulon 2HB plate. Fecal suspension sample from each mouse (1:20 dilution), in triplicate, was added to each well. HRP-conjugated goat-anti-mouse IgA (1:1000) was used as the secondary antibodies. Optical densities were used to calculate anti-STa antibody titers (in log10).

#In accordance with one or more embodiments of the present invention, instead of randomly stacking individual epitopes from each adhesin with linkers to generate a multiepitope antigen, the CFA/I major subunit was used CfaB as the backbone, and antigenic prediction software was applied to embed epitopes of CS1-CS6 (by substituting surface-exposed exposed but less antigenic CfaB epitopes) so that the resultant multiepitope CFA protein would have antigenicity propensity similar to the CfaB subunit. This multiepitope CFA protein thereby has a relatively stable structure and a better presentation for the CS1-CS6 epitopes to induce host immune responses. When carried by the CFA/I Cfa operon, this multiepitope CFA protein can be expressed as a multiepitope CFA adhesin to be used for killed or live whole-cell vaccine development. This approach, described herein as an embodiment of the present invention, to construct multiepitope antigen against multiple virulence factors can be generally applied in developing multivalent vaccines against pathogens expressing heterogeneous virulence factors.

#Data from the embodiments of the present invention indicate that these constructed multiepitope CFA antigens elicited antibodies cross reactive to CAF/I, CFA/II and CFA/IV adhesins, and that anti-adhesin antibodies in the serum significantly inhibited adherence of ETEC or E. coli strains expressing these 7 adhesins to Caco-2 or T-84 cells, and shows its application in developing effective ETEC anti-adhesin vaccines. In conjunction to a toxoid fusion antigen that induces antibodies neutralizing against both LT and STa toxins, this multiepitope CFA antigens of the present invention can be used to develop broadly protective vaccines against ETEC diarrhea.

#In accordance with an embodiment, the present invention provides a polypeptide molecule comprising one or more of the colonization factor antigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6).