Laboratory of immunochemistry of

glycoconjugates

and

Laboratory of lignin,

Institute of Chemistry, SAS

Preparation and immunological characterization of glycoconjugates

based on cell-surface polysaccharide antigens from the yeasts of Candida

genus – potential vaccines

Yeasts of Candida genus represent serious social and medical problem. Our laboratory belongs to those few departments in the world where the potential vaccines against diseases caused by the Candida yeasts are being developed.

Our vaccine constructs are based on polysaccharide from the cell of yhe yeasts, which are responsible for the antigenicity of the yeasts.

Children < 2 yrs, elderly people and immunocompromised individuals can produce sufficient level of IgG antibodies only against T-cell dependent antigens (polysaccharides are T-cell independent). Conversion of polysaccharides to T-cell dependence antigens can be achieved via conjugation with protein carrier.

General strategy of glycoconjugate vaccine preparation:

Yeast cultivation (most frequently present human pathogens:

C. albicans, C. tropicalis, C. parapsilosis, C. glabrata), isolation and

purification of mannans

Mannan derivatization

Conjugation with protein

Immunization of animal model

Immunological characterization of the conjugate activity

Study of the structure and biological activity of fungal polysaccharides at the Laboratory of microbial

polymersBy means of acidic and alkaline extractions, cellular β-D-glucan was isolated from the cell walls of yeasts Saccharomyces cerevisiae and Candida albicans.

Using 13C-NMR spectroscopy and by observing the complex formation with Congo Red dye, it has been proven that β-D-glucans form helical structures depending on the pH value (single and triple helices).

Water-soluble derivatives of S. cerevisiae glucan – carboxymethyl- and sulfoethyl glucans were prepared by a chemical modification, and by means of the ultrasonic treatment, fraction precipitation, and liquid chromatography, their defined fractions with different degrees of derivatization and molecular weight values were obtained.

Water-soluble derivatives of S. cerevisiae glucan exterted protective effect in mice with experimental infection caused by bacteria Klebsiella pneumoniae.

Carboxymethyl glucan inhibited lipid peroxidation in liposomes and revealed antioxidant activity comparable to that of the established antioxidants such as D-mannitol, α-tocopherol, and ascorbic acid.

Carboxymethyl glucan and sulfoethyl glucan prevented growth and metastasis of Lewis lung carcinoma and lymphosarcoma tumors and showed synergistic antitumor effect with a known cytostatic drug cyclophosphamide, and moreover suppressed its toxic side-effects.

Water-soluble derivatives of S. cerevisiae glucan demonstrated antimutagenic and antigenotoxic effect and inhibited the oxidative degradation of DNA in lung cells.

The ability of carboxymethyl glucan to scavenge free radicals was for the first time directly proven by a spin-trap EPR spectroscopy.

Water-soluble derivatives of S. cerevisiae glucan revealed protective anti-arthritic activity in an experimental model of adjuvant arthritis in mice.

Antioxidant and antimutagenic properties were manifested also by other fungal polysaccharides: glucomannan from the yeast Candida utilis and glucan-chitin complex from the filamentous fungus Aspergillus niger.

- isolation of antigen (extraction of cell mass and LPS purification), separation or destruction of lipidic part Lipid A (whole LPS is responsible for septic shock)

- conjugate synthesis (conversion to T-cell dependent antigen). Conjugate consists of antigen covalently bound to protein carrier (different geometry, valency etc.)

- conjugate characterization and in vivo testing (animal model)

- disease caused by Gram negative bacteria Vibrio Cholerae, every year over

100 000 dead

- prevention: strict sanitation and vaccination

- cell surface of bacteria contains lipopolysaccharides (LPS). Immunity against these LPS prevents against disease.

LPS

Synthesis and characterization of neoglycoconjugates based on Vibrio Cholerae O1 LPS. Derivatization of LPS carboxylic groups using different

linkers and conjugation with proteins

CH

CH

CH2OH

CH

C

OH

OCH3

HC

OCH3

CH

CH

O

O

OCH3

O CH

OCH3

O

CH

H3CO

OH

O

OCH3

O

CH

CH

CH2

OCH3

OH

O

C OH

O

OCH3

H3CO

H3CO

HCOH

H3CO

CH

H3CO

OH OH

C O

O

H2COH

HOCH2 H3CO

HCOH

H2COH

O

H3CO

C

H

O

H2COH

HCOH

O

H2COH

C

O

H2COH

HCOH

H2COH

OH

HC

H2COH

H3CO

HCOH

H2COH

O

HC

HOCH2

HC

H2COH

HCOH

H3CO

HC

HOCH2H3CO

HCOH

HC

HC

H2C

HCOH

HC

HOH2C

HC

H3CO

H

1. application of lignin in polymer blends

2. preparation of novel lignin-based antimutagenic and anticarcinogenic compounds

- isolation and modification of various types of lignins

- characterisation of rheological and physico-mechanical properties of lignin-polyolefin blends

- examination of the stability of polyolefin blends during processing and life service

- influence of lignin concentration on vulcanization, rheological and mechanical properties of rubber composites

- determination of antioxidative effect of lignin samples on rubber blends

Lignin belongs among main biomass components - chemical Lignin belongs among main biomass components - chemical treatment of wood worldwide yields about 50x106 t/ year. treatment of wood worldwide yields about 50x106 t/ year.

In Lignin Laboratory under the supervising of professor Ing. B. Košíková DrSc. novel methods were developed for use of lignin biomass component:

0 – 20 % of tail DNA

20 – 40 % of tail DNA

40 – 60 % of tail DNA

60 – 80 % of tail DNA

80 –100 %of tail DNA

Cells non-pretreated Cells non-pretreated with ligninwith lignin

Cells pretreated Cells pretreated with ligninwith lignin

0 75

0 21

10 4

60 0

30 0

Reduction of oxidative damage of DNA by lignin preparations

was prooved on:

- cells on hamster cells V79

- human cells VH10

- human carcinoma cells Caco-2

- primary testicular cells and lymphocytes isolated from the rats in vitro

- lymphocytes and testicular cells isolated from the rats fed by diet containing lignin ex vivo