• History

• Work in progress

• Future questions/directions

Vaccine development for prevention ofMycobacterium ulcerans Disease

Homologous and heterologous immunity in infections ofMice with Mycobacterium ulcerans and Mycobacteriumbalnei (marinum)

Frank Fenner, 1956

Can M. ulcerans or BCG protect against infectionWith M. marinum?

The responses of mice previously inoculated with M. ulcerans,Dark circles), M. balnei (unfilled circles) orBCG (circle with dot I.v.) BCG (triangle footpad) X=controls toInoculation in the rt footpad with 106 M. ulcerans 5 wks laterIn.=106

Weeks after challenge inoculation

Can M. ulcerans, BCG or M. marinum protect againstM. ulcerans disease?

The response of mice previously inoculated withM. ulcerans, M. balnei or BCG inoculation in the rt footpadto challenge with a small dose (10,300) M. ulcerans5 wks later

Summary of Fenner studies:

• M. ulcerans provides poor protection against M. marinum

• BCG provides poor protection

• M. marinum and M. ulcerans show good protection against both low and high dose M. ulcerans; BCG provides some protection against low dose infection

• Protection against MM or MU is not provided by antibody transfer

Can exposure to virulent M. ulcerans or a mycolactone-M. ulcerans mutant confer protective immunity to subsequent challenge with virulent M. ulcerans?

Protocol: 13 Hartley guinea pigs; 5 were vaccinated with 105 wildtype M. ulcerans, 5 with 105 MU1615E, a ML- mutant, 3 with media

Inguinal injection of M. ulcerans 6 weeks Challenge by

M. ulcerans1615E (ML-) intradermal inoculation

with 107 WT M.ulcerans

Lesions harvested at 48 hrs, 14 days, 4 weeks, 6 weeks, and 6 months for culture and histology

GP Vacc. w/ 1615 or 1615E 6 weeks challenged with MU1615

Time post-challenge Vaccine Gross pathology CFU

48 hrs 1615 WT3mm erythema 3+1615E ML- 4mm erythema, indurated 3+

14 d Media 7mm hard, fibrous ulcer 3+ 1615 WT 12mm indurated ulcer 3+1615E ML- 15mm, pus-filled ulcer 1+

4 wk 1615 WT 20mm red, indurated ulcer -1615E ML- 40mm granuloma, pus -

6 wk Media 25mm ulcer, calcified,fibrous 2+1615 WT 30mm red, vascularized, ulcer -

pus present1615E ML- 5mm red, slightly thickened -

6 mo. Media 10mm healed scar, calcified -1615 WT 10mm hard, granulomatous -1615E ML- Slightly depressed scar -

Intradermal injection of WT MU 161521d PI

Extracellular bacteria: few inflammatorycells

7d post vaccination withMU 1615E

Significant cellular necrosisBacteria sequestered byImmune response

Vaccinated animal21d post inoculation

Widespread cellularNecrosis. Both intra and extracellularbacteria b

Summary

Vaccination with either wild type M. ulcerans or a ML- mutant may prevent Buruli ulcer in a guinea pig model of infection and lead resulting in clearage of M. ulcerans.

Vaccination with a ML- mutant resulted in formation of a sterile abcess.

An immune response to mycolactone may not be necessary for a protective immune response

Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer. Tanghe A, Content J, Van Vooren JP, Portaels F, Huygen K Infect Immun. 2001 Sep;69(9):5403-11.

Buruli ulcer, caused by Mycobacterium ulcerans, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine M. bovis BCG. To identify potential mechanisms for this cross-protection, we identified and characterized the M. ulcerans homologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in M. ulcerans, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of M. ulcerans- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads of M. ulcerans- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.

Mycobacterium bovis BCG vaccination as prophylaxis against Mycobacterium ulcerans osteomyelitis in Buruli ulcer disease. Portaels F, Aguiar J, Debacker M, Guedenon A, Steunou C, Zinsou C, Meyers WM. Infect Immun. 2004 Jan;72(1):62-5

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. Clinically, the disease presents in the skin as either nonulcerative or ulcerative forms and often invades bones either subjacent to the skin lesion (contiguous osteomyelitis) or remote from the skin lesion (metastatic osteomyelitis). Osteomyelitis represents a severe form of the disease that often requires numerous surgical interventions, even amputations. Surgery is accepted as the present definitive treatment for BU. In the absence of an effective drug treatment, the need for the development of preventive and control strategies becomes paramount. No specific vaccine, however, is presently available for BU. Of 372 consecutive patients in Benin presenting with BU (confirmed by microbiological and histopathological analyses) whose Mycobacterium bovis BCG scar statuses were known, 196 children (<15 years old) and 108 adults had neonatal BCG vaccination scars. Of 196 children with BCG scars, 17 (8.7%) had osteomyelitis, while 7 of 28 children without BCG scars (25.0%) had osteomyelitis. Of 108 adults with BCG scars, 17 (15.7%) had osteomyelitis, while 14 of 40 adults without BCG scars (35.0%) had osteomyelitis. Our results show that effective BCG vaccination at birth provides significant protection against the development of M. ulcerans osteomyelitis in children and adults. Therefore, health authorities should give attention to the enhancement of neonatal BCG vaccination coverage in all countries of Africa where BU is endemic. Protection against severe forms of BU and childhood tuberculosis would likewise be improved by this intervention.

The local immune response in ulcerative lesions of Buruli disease . E. Kiszewski*, E. Becerril*, L. D. Aguilar*, I. T. A. Kader, W. Myers, F. Portaels§ and R. Hernàndez Pando Clinical & Experimental Immunology Volume 143 Page 445 - March 2006

11 patients 45 days (6) 150 days (5)AFBs many fewGranulomas absent presentCytokine profile

inf-γ low highTGF-β NSD NSDTNF-α NSD NSDIl-10 high low

Conclusions: Local immunosuppression is a consistentfeature of early lesions“suggesting that strong suppressionof the protective immune response facilitates proliferation ofM. ulcerans”.

Work in Progress I

Dr. Audrey Tanghe, Pasteur Institute of Brussels in collaboration with K. Huygen, and P. Small

1. Testing BCG Ag85 M. bovisBCG Ag85 M. ulceransBCG given twice

Ag85 Mu recomb. DNA 2X, Mb 85 protein boostAg85 Mb recomb. DNA 3x, Mu 85 protein boost

These strategies resulted in a equal delay in footpadSwelling

2. Use of proteins from genes implicated in mycolactone production. Cloned proteins injected I.M. at 3x at 3 week intevals. TetR and Fur induced significant interferon-γ protection studies in progress.

3. May also look at protection from 6 mycolactone-negative mutantsprovided by Small lab

Work in Progress II

Dr. Ian Mckenzies lab in collaboration with Paul Johnson,Geoff Pieterz, P. Small, T. Stinear

1. Protective effect of anti-mycolactone antibodiesImmunization with an anti-Myl-KLH conjugate followed by challenge with M. ulcerans results in increased footpad swelling (Note; injection of mycolactone into mouse footpad results in increased pain). This may be due to Arthus reaction FcR-- mice do not show these results

2. Knock-out mice to study immune response to MU and mycolactone

3. Production of higher titer anti-mycolctone antibodies; monoclonals

4. Vaccine response to 25 recombinant peptides produced by Tim Stinear will be tested for protection

What can we learn from animal models for vaccine development? What criteria for protection?

Early Late

Mice Predom. intracellular AFBs intra + extra many inflam. cells mostly macsin necrotic areas painful?painful

Guinea pigs Predom.extracellular AFBs few AFBsfew �inflamm. cells in granulomasnecrotic areapainless painless

Humans Predom. extracellular AFBs few AFBsfew inflamm cells in granulomasnecrotic areapainless painless

Other models: grass cutter? Primates?

Host differences in innate immunity and susceptibility to pathogenic organisms: the importance of the local immune response and disease

Host specificity of Chlamydia trachomatis due to differences between INF-γ pathways in mice and humans mediated by IDO (indoleamine 2,3, dioxygenase). H. Caldwell, H. PNAS, 2005

Production of shigellosis in mice by reconstitution withHuman Il-8. P. Sansonetti, et al.Shigella species. J. Immuno. 2004

Animal models for vaccine developmentWhat are the limitations?What criteria for evaluation?

Is a vaccine for Buruli ulcer desirable?How and where would this be used?

What is the nature of the protective immune responseto Buruli ulcer?

What preliminary research is most important for evaluating the feasibility of a vaccine?

What are the most appropriate methods/models for determining the efficacy of proto-type vaccines