ANTIGEN

Molecules from a pathogen or foreign organism that provoke a specific immune response.

Microbes:

Capsules, cell walls, toxins, viral capsids, flagella, etc.

Nonmicrobes:

Pollen, egg white , red blood cell surface molecules, serum proteins, and surface molecules from transplanted

tissue.

Lipids and nucleic acids are only antigenic when combined with proteins or polysaccharides

IMMUNITY: “Free from burden”

Ability of an organism to recognize and defend itself against specific pathogens or antigens

IMMUNE RESPONSE:.

Involves production of antibodies and generation of specialized lymphocytes against specific antigens.

Vaccine

which usually follows natural infection but without causing disease

• To generate long-lasting immunity

• To interrupt spread of infection

Type acquired through passive immunization –

Natural maternal serum/milk

Artificial immune serum

Type acquired through active immunization –

Attenuated organisms (live)

inactivated organisms (dead)

Cloned genes of microbiological antigens

Purified microbial macromolecules

Synthetic peptides

DNA

Hepatitis B Virus Infection

• More than 350 million chronically infected worldwide

• Established cause of chronic hepatitis and cirrhosis

• Human carcinogen—cause of up to 80% of hepatocellular carcinomas

• More than 600,000 deaths worldwide in 2002

Hepatitis B vaccine (HBV)

• Originally based on the surface antigen purified from the blood of chronically infected individuals.

• Due to safety concerns, the HBV vaccine became the first to be produced using recombinant DNA tech-

nology (1986)

• Produced in bakers’ yeast (Saccharomyces cerevisiae)

Characterization of the structural modifications accompanying

the loss of HBsAg particle immunogenicity

Vanille J. Greiner, Catherine Manin, Eric Larquet, Nabila Ikhelef, Frédéric Gréco,Sophie Naville, Pierre-Emmanuel Milhiet, Frédéric Ronzon, Andrey Klymchenkoa,Yves Mély,∗

Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France

Centre de Biochimie Structurale CNRS UMR 5048—Inserm U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier Cedex, Francea r

AimTo study relationship between immunogencity and structure (HBsAG)

Objectives

Study on structure and properties of treated particle (3weeks at +60ºC ) with untreated particle.

EM and AFM of isolated particle at 20nm

IR spectroscopy and Circular dichroism of the particle

Steady state and Time resolved flurosence data of the particle

Preparation of vaccine samples

HBsAg virus-like particles were produced

using the recombinant yeast Hansenula polymorpha

(by Sanofi Pasteur )

Extraction

Purification.

Purified HBsAg particles (1.5 mg/mL) were kept at +4◦C in PBS buffer.

Loss of HBsAg immunogenicity

In vivo potency assay

Sample or reference vaccine was injected into 5 week-old female

Balb/c mice in parallel with placebo (12 mice per dose)

In vitro relative potency assay

Mice were bled 42 days

Individual sera were assessed antibodies against Hepatitis B (ELISA assay)

Hep-atitis B antigen assayed against anti-Hepatitis B IgM (primary antibody)

anti-Hepatitis B IgG monoclonal antibody(Secondary antibody)

Tetramethylbenzidine (substrate)

OD @450 and 630 nm

Surface plasmon resonance (SPR)

Antigenicity of untreated and heated HBsAg particles, their affinity toward HBsAg-specific mAb RF-1

( ImperialCollege London, UK) [26] was measured on a Biacore 3000TMinstru-ment (GE Healthcare).

Electron microscopy (EM)untreated and heated HBsAg

diluted in Tris 10 mM, NaCl 150 mM buffer, pH 7.4 to a con-centration of 20 g/mL

deposited on a 400 mesh full carbon-coated glow-dis-charged grid and stained with 2% uranyl acetate

Observation

Atomic force microscopy (AFM)Untreated and heated HBsAg samples were deposited on mica, treated, and observed, as previously described

Fourier transform infrared spectroscopy (FTIR) Untreated and heated HBsAg samples were concentrated usinga capped centrifuge device (Amicon, Millipore) with a 5 kDa cut-off up to 1.8 mg/mL in protein.

Circular dichroism (CD)Far-UV CD spectra of HBsAg particles (0.2 mg/mL) were recordedon a Jasco-810 spectropolarimeter

Fluorescence spectroscopy Fluorescence spectra were recorded at 20◦C on a Flu-oroMax spectrofluorometer at 400nm

Results and discussion

In-vivo and In-vitro Both assays revealed a strong decrease of the HBsAg potency

Changes in HBsAg morphology ( EM)

Untreated sample appeared as corrugated spherical particles and heated sample

observed as long chains of HBsAg particles, with aggregation.

HBsAg particles were further imaged using AFM.

Untreated particles appeared as round-shaped particles with a 25 nm diameter and

numerous protein protrusions at their surface

A highly different pattern with a much lower density of particles on the mica was observed with

heated particles (suggesting that the interactions between the particles and the mica were altered).

Changes in the secondary structure of HBsAg S proteins (FTIR )

Untreated HBsAg particles show intense amideI and II bands centered at 1655 and 1548 cm.

Curve fitting analysis of the FTIR spectra revealed that the secondary structure of S proteins in untreated

HBsAg particles is constituted by about 59% α-helix and negligible amount of β-sheet .

Heated samples show a 15% decrease in the α-helix content.

Changes in the secondary structure of HBsAg S proteins ( UV-CD spectra )

Untreated HBsAg particles, two strong negative bands at 208 and 222 nm

typical of protein α-helical conformation

Interestingly, CD spectra of heated particles show a significant change at 222 nm

and a more moderate change at 196 nm.

Changes in the secondary structure of HBsAg S proteins (Fluroscence spectroscopy)

Conclusion

• Using a combination of physical techniques, we characterized the structural modifications accompanying the loss of HBsAg

particle antigenicity and immunogenicity induced by 3 weeks incubation at +60◦C.

• Together with the appearance of long chains of aggregated particles, our data indicated that heated HBsAg particles exhibit

modifications in both their protein and lipid parts.

• The particle surface was particularly altered since the integrity of the epitopes, the surface topography, the polarity of surfac

Llipids, and the solvent-exposed Trp residues were affected.

• The decrease in the α-helix content of the S proteins and the chemical alteration (likely oxidation) of the Trp residues at the

protein surface together with the lipid modification at the particle surface can largely explain the loss of immunogenicity,

evidenced by potency assays and antibody recognition.

• Taken together, our data further evidenced the close relationship between the structure and integrity of the HBsAg particle

surface and its immunogenic properties.