1. Novel chimeric vaccines against Clostridium difficile infection
Xingmin Sun, PhD
Assistant Professor
Department of Molecular Medicine Morsani College of Medicine University of South Florida

2. Clostridium difficile
Gram positive, toxin-producing, spore-forming anaerobic bacterium.
One of the three urgent antibiotic resistance threats in US.
Leading cause of nosocomial antibiotic-associated diarrhea (AAD) in developed countries.
The most common organism to cause healthcare-associated infections in US, leading to 14,000 deaths per year.
A continual rise in the incidence of severe C. difficile infection (CDI) has been observed worldwide.
Emergence of more virulent strains like NAP1/BI/027: increased toxin production and sporulation; altered antibiotic resistance pattern; secretion of additional toxin.
NAP1: North American Pulse-Field type 1
027: PCR ribotype 027
NAP1/027 strain: has increased toxin production, altered antibiotic resistance patterns (fluoroquilonone resistance), as well as increased sporulation capacity. It produces additional toxin i.e., binary toxin.

3. Major virulence factors
Toxin A (TcdA) and Toxin B (TcdB): potent cytotoxic enzymes that damage the human colonic mucosa.
Binary toxin (CDT): present ~6% of C. difficile isolates, but found in all hypervirulent strains.
TMD
RBD
CPD
GTD

4. Major colonization factors
Two C. difficile flagellar proteins FliC and FliD are involved in the attachment of the organism to host cells and mucus layer.
Serum antibody responses against both FliC and FliD are detected in patients with C. difficile infection.
C. difficile Cwp84 is a cysteine protease, and plays a critical role in the maturation of surface-layer proteins.
Immunization with Cwp84 provides significant protection in hamsters by delaying C. difficile colonization. Cwp84 is highly immunogenic and conserved.
More importantly, serum antibody responses against both FliC and FliD are generated in patients with C.difficile infection.

5. CDI: transmission through spores
Step 1- Ingestion of spores
Step 2- Germination into vegetative cells
Step 3 - Altered intestine flora allows proliferation of C. difficile in colon
Step 4 . Toxin production leads to colon damage +/- pseudomembrane
The ingested spores may geminate in the small intestine and vegetative cells colonize in the large intestine without causing any problem. When gut flora is disrupted by antibiotics or other means, C. diff are resistant to most antibiotics therefore thrive and then produce toxins. Two exotoxins TcdA and TcdB actually responsible for the disease. They disrupt…

6. CDI: Standard treatment and major challenges
Antibiotics: Standard treatment
Vancomycin (only FDA-approved treatment)
Metronidazole (most commonly used treatment)
Fidaxomicin (lower recurrence rates)
Recurrence is common
~20% after first CDI episode
~40% after first recurrence
~60% after 2 or more recurrences

7. No vaccine against CDI is currently licensed
“C. difficile vaccination could be cost-effective over a wide range of C. difficile risk, especially when being used post-CDI treatment to prevent recurrent disease.”
A recent computer simulation model shows that…
Lee et al., Vaccine, Jul 19;28(32):
The potential value of Clostridium difficile vaccine: An economic computer simulation model. 7

8. Novel vaccine candidate targeting both toxins: mTcd138
TcdA
543
769 N GT
CPD
TMD
RBD C
1848
2710
TcdB W
DXD N GT
CPD
TMD
RBD C 1
544
767
1851
2366
W102A
D288N GT
CPD N C
mTcd138
TcdB
TcdA
RBD
What is the rationale for making this construct?
The RBD is the immunodominant region of TcdA and has potent adjuvant activity.
The neutralizing epitopes in TcdB are primarily confined to the N terminus since preincubation of polysera from the atoxic TcdB-immunized mice with recombinant TcdB-RBD did not significantly reduce the serum neutralizing activity.
The N terminus in TcdB is more highly conserved between historical and hypervirulent strains than its RBD.
Neutralizing antibodies to the TcdA-RBD were shown to be cross-reactive with TcdB-RBD.
We hypothesized that fusion of the N terminus of TcdB with the receptor binding domain (RBD) of TcdA should be sufficient to protect against toxin mediated symptoms

9. Construction of mTcd138: fusion of the N terminus of TcdB with the receptor binding domain (RBD) of TcdA
The RBD is the immunodominant region of TcdA and has potent adjuvant activity.
The neutralizing epitopes in TcdB are primarily confined to the N terminus since preincubation of polysera from the atoxic TcdB-immunized mice with recombinant TcdB-RBD did not significantly reduce the serum neutralizing activity.
The N terminus in TcdB is more highly conserved between historical and hypervirulent strains than its RBD.
Neutralizing antibodies to the TcdA-RBD were shown to be cross-reactive with TcdB-RBD.
What are rationales for making this construct?
We hypothesized that fusion of the N terminus of TcdB with the receptor binding domain (RBD) of TcdA should be sufficient to protect against toxin mediated symptoms

10. Expression and purification of mTcd138
(B)
(C)
250 kDa
150 kDa
mTcd138
mTcd138
100 kDa
75 kDa
Fig. 2. Expression and purification of mTcd138
Analysis of purified 138 kDa fusion protein by SDS-PAGE (A) and Western blotting with anti-TcdA antibody (B) and anti-TcdB antibody (C).
Analysis of purified 138 kDa fusion protein by SDS-PAGE (A), and Western blot analysis with anti-TcdA antibody (B) and anti-TcdB antibody (C)

11. mTcd138 is atoxic (B) (A) Fig. 3. mTcd138 is atoxic
(A) Vero cells in a 96-well plate were exposed to TcdA, TcdB or mTcd138 at different concentrations for 72 h. MTT assays were performed, and cell viability was expressed as the percentage of surviving cells compared to cells without toxin exposure. (B) C57/BL6 mice were intraperitoneally challenged with 100 ng of TcdA or TcdB or 100 μg of mTcd138. Mouse survival was monitored.

12. mTcd138 immunization via intraperitoneal (i. p. ), intramuscular (i. m
mTcd138 immunization via intraperitoneal (i.p.), intramuscular (i.m.) or intradermal (i.d.) routes induces similar levels of antibody response
Mice were ip immunized 3 times at a 14-day interval with 10 ug of mTcd138 with alum as adjuvant, and serum IgG titers against both toxins show here. Immunization of mice with mTcd138 via i.p., i.m. or i.d. routes induced similar levels of IgG antibody responses against both TcdA and TcdB
We also included sera from previously cTxAB-immunized mice for comparison. As you can see mTcd138 immunization generated an IgG response comparable/similar to cTxAB.
Fig. 4. mTcd138 immunization via intraperitoneal (i.p.), intramuscular (i.m.) or intradermal (i.d.) route induces similar levels of antibody response
Groups of C57 BL/6 mice were immunized 3 times at 14-day intervals via i.p., i.m. or i.d. route with 10 µg of mTcd138 in the presence of alum (i.m. and i.p.) or double mutant E.coli heat labile toxin (dmLT) (i.d.) as an adjuvant. Sera were collected, and anti-TcdA (A) or anti-TcdB (B) IgG titers measured by standard ELISA.

13. mTcd138 immunization induced potent neutralizing antibodies against both toxins
mTcd138 immunization induced potent neutralizing antibodies against both toxins (Fig. 5) though neutralizing titers against TcdA were much higher than those against TcdB.
Fig. 5. Serum anti-toxin neutralizing titers of the mTcd138-immunized mice (i.p.)
Vero cells were used to assess in vitro neutralizing activities of serum samples. The neutralizing titer is defined as the maximum dilution of the samples that blocks cell rounding induced by toxin at a given concentration. This given concentration is two times the minimum dose of the toxin that causes all Vero cells to round after a 16 h exposure to the toxin, i.e., 2.5 and 0.1 ng/ml for TcdA and TcdB, respectively.

14. mTcd138 immunization protected mice against systemic toxin challenge
Then we challenged immunized and age-matched non-immunized mice by IP with lethal dose of toxins. The lethal dose for both toxins is 100 ng.
The non-immunized mice died rapidly when ip challenged with either 100 ng of TcdA or TcdB, whereas All mTcd138-immunized mice survived the intraperitoneal challenge of 200 ng of either toxin.
Fig 6. mTcd138 immunization protects mice against systemic toxin challenge
Kaplan-Meier survival plot of mTcd138-immunized (i.p.) or control mice challenged with lethal dose of TcdA or TcdB (i.p.).

15. mTcd138 immunization of mice provided full protection against infection with a hypervirulent C. difficile strain
(A)
(C)
(B)
We further evaluated the immunogen mTcd138 in a mouse model of CDI. After three immunizatins via i.p., i.m. or i.d. routes, mice were challenged with 106 spores of C. difficile UK6 (BI/NAP1/027). In all vehicle-immunized mice, significant disease symptoms, including weight loss (Fig. 7B), severe diarrhea (Fig. 7C), ruffled coat, and lethargy were evident by day 2 postinfection, and approximately 40% of the mice succumbed by day 4 postinfection (Fig. 7A). In contrast, mTcd138-immunized mice were fully protected and showed no signs of disease at any stage (Figs. 7B and 7C).
Fig 7. mTcd138 immunization of mice via i.p, i.m. or i.d. routes provides full protection against infection with a hypervirulent C. difficile strain.
Mice were challenged with C. difficile UK6 spores (106 /mouse) 14 days after the third immunization with mTcd138 or PBS. Kaplan-Meier survival plots (A) and means relative weight of all surviving mice (up to the day of death) (B) of different groups, and frequency of diarrhea (C) are illustrated. Data were presented as mean relative weight ± standard error.

16. Development of a vaccine which targets C
Development of a vaccine which targets C. difficile colonization and reduces C. difficile excretion
Two C. difficile flagellar proteins FliC and FliD are involved in the attachment of the organism to host cells and mucus layer.
Serum antibody responses against both FliC and FliD are detected in patients with C. difficile infection.
Since CDI is transmitted by spores, we proposed to develop…
Bacillus megaterium is a Gram-positive, aerobic spore-forming bacterium found in widely diverse habitats from soil to fish and dried food. It has been industrially employed for more than 50 years because of its high capacity for production of exoenzymes. It is also a desirable cloning host for production of recombinant proteins, given that the genetic tools are available with shuttle vectors carrying strong inducible promoters and affinity tags. There are several noticeable advantages of B. megaterium expression system over the E. coli system. These include the lack of alkaline proteases and stably maintaining plasmid vectors, lack of endotoxin LPS, and capability of secreting expressed heterologous protein into the medium

17. FliCFliD immunization of mice provides significant protection against infection with a hypervirulent C. difficile strain
After three IM immunizations with 20 µg of C. difficile flagellin proteins FliC plus FliD, mice (n=10) were challenged with 106 spores of C. difficile UK6. All vehicle-immunized mice developed diarrhea (100%; Fig 5C), significant weight loss (Fig. 5B) with a 40% survival rate (Fig 5A), while the mice immunized with FliC plus FliD developed much less severe disease symptoms including less weight loss (Fig 5B) and lower diarrhea rate (70%; Fig 5C) with a significantly higher survival rate (90%; Fig 5A).

18. FliCFliD immunization significantly reduces C
FliCFliD immunization significantly reduces C. difficile excretion from mice *
More interestingly, mice immunized with FliC plus FliD secreted about fold less C.difficile (Fig 5D).

19. Summary of vaccine development
We have constructed a novel vaccine targeting both C. difficile toxins.
Immunization with mTcd138 induced potent antibody and protective responses against both TcdA and TcdB.
We have generated a novel vaccine (FliCFliD) which can significantly reduce C. difficile colonization and excretion.

20. Acknowledgements Dr. Abraham L. Sonenshein Dr. Sun Lab
Yuanguo Wang
Ying Cai
Xianghong Ju
Previous members:
Keshan Zhang
Song Zhao
Yuankai Wang
Dr. Abraham L. Sonenshein
(Tufts University)
Laurent Bouillaut
Dr. Qiaobing Xu
Ming Wang
NIDDK (K01DK092352); NIAID (R21 AI113470) ; Tufts Collaborates! ; Tufts Institute Innovation Award