5.vaccine 2018.pdf

VACCINE TECHNOLOGY

1

Definitions ◼Originally derived from the Latin word ‘‘vacca’’

meaning cow ◼Pharmaceutical suspension or solution of an immunogenic substance or compound(s) that is intended to induce active immunity ◼Preparations containing antigenic substances that have been shown to be capable of inducing a specific and active immunity in man

VACCINES • Immunization: a procedure designed to increase concentrations of antibodies and/or effector T-cells which are reactive against infection (or cancer). • Immunization procedure called vaccination and the immunizing agent called vaccine (or “serum” in historical references)

3

Principles of Vaccination Immunity

• • • •

Self vs. non-self Protection from infectious disease Usually indicated by the presence of antibody Very specific to a single antigen

Principles of Vaccination Active Immunity

• Protection produced by the person's own immune system • Usually permanent

Passive Immunity • Protection transferred from another person or animal as antibody

Principles of Vaccination inject or administer

Antigen • A live or inactivated substance (e.g., protein, polysaccharide) capable of producing an immune response

Antibody • Protein molecules (immunoglobulin) produced by B lymphocytes to help eliminate an antigen

Passive Immunity • Transfer of antibody from an exogenous source • Transplacental most important source in infancy • Temporary protection

Sources of Passive Immunity • Almost all blood or blood products • Homologous pooled human antibody (immune globulin) • Homologous human hyperimmune globulin • Heterologous hyperimmune serum (antitoxin)

Classification of Vaccines • Live attenuated – viral – bacterial

• Inactivated kill the virus -> test can we make the vaccine or not

Inactivated Vaccines Whole

we can prepare from:

• virus • bacteria

Fractional

a component that include in inactivated vaccines

• protein-based – subunit what is it? go home study it for exam – toxoid some of them help you prevent disease can cause immune response • polysaccharide-based some (hypersensitivity) -> need to purify carefully (phải high purity) – pure bc polysaccharide is very weak, easy to hydrolyze -> to conjugate -> need to protect polysaccharide in – conjugate have living system -> stable in the body

Live Attenuated Vaccines this vaccine required the microbes to replicate -> survive -> need to remove virulant

• Attenuated (weakened) form of the "wild" virus or bacteria remove the virulence -> replicate • Must replicate to be effective

replicate because for vaccine to maintain long life in body if not might can't live in body

can they give the co-stimulation: combine with your antibody but no activity or decrease the action

• Immune response similar to natural infection • Usually effective with one dose

because long half-life and can replicate

Live Attenuated Vaccines • Severe reactions possible

because replicate, they may become dangerous again (become virulant again)

• Interference from circulating antibody antibody will recognize and then they kill the microbe, therefore the activity of vaccine will slow down or even nothing.

• Unstable

Live Attenuated Vaccines • Viral

measles, mumps, rubella, vaccinia, varicella, yellow fever (oral polio) (rotavirus) (influenza)

• BacterialBCG, oral typhoid

Vaccines in (parenthesis) are not available in the United States.

Inactivated Vaccines • Cannot replicate because inactivated • Minimal interference from circulating antibody no antigen at all • Generally not as effective as live vaccines

• Generally require 3-5 doses • Immune response mostly humoral • Antibody titer falls over time

short halflife

Inactivated Vaccines Whole cell vaccines • Viral

• Bacterial

polio, hepatitis A, rabies (influenza) (pertussis) (typhoid) (cholera) (plague)

Vaccines in (parenthesis) are not available in the United States.

Inactivated Vaccines Fractional vaccines • Subunit hepatitis B, influenza, acellular pertussis, typhoid Vi (Lyme) • Toxoid

diphtheria, tetanus

Polysaccharide Vaccines have a lot of hydroxyl group, can conjugate

if no conjugate, then consider the quality of polysaccharide

Pure polysaccharide

• pneumococcal • meningococcal • Haemophilus influenzae type b

Conjugate polysaccharide • Haemophilus influenzae type b • pneumococcal

Pure Polysaccharide Vaccines • Not consistently immunogenic in children <2 years of age • No booster response

• Antibody with less functional activity • Immunogenicity improved by conjugation

VACCINES IN Immunization Passive versus active immunization: -> TYPE ACQUIRED THROUGH Passive Immunization – -> Natural maternal serum/milk -> Artificial immune serum

-> Type ACQURIED THROUGH Active Immunization – -> Natural infection -> Artificial infection*: Attenuated organisms (live) inactivated organisms (dead) Cloned genes of microbiological antigens Purified microbial macromolecules Synthetic peptides DNA *Artificial refers to steps involving human intervention

19

Mechanisms of VACCINES

Establish resistance to virus/pathological organism by evoking an immune response 1. Give host a foreign organism/protein in non-infectious form -> active immunization

2. Antibodies are generated Ab binds to surface proteins of organism -> passive immunization

Other vaccination components: •

let the product release slowly, prolong the effect

Adjuvant: chemicals in the vaccine solution that enhance the immune response – – –

Alum – Ag in the vaccine clumps with the alum such that the Ag is released slowly, like a time-release capsule gives more time for memory cells to form 20

Vaccine preparation • A vaccine renders the recipient resistant to infection.

• During vaccination a vaccine is injected or given orally. • The host produces antibodies for a particular pathogen.

• Upon further exposure the pathogen is inactivated by the antibodies and disease state prevented. • Generally to produce a vaccine the pathogen is grown in culture and inactivated or nonvirulent forms are used for vaccination.

21

Traditional preparation

– Grow in animals (vaccinia in calves for smallpox; rabbit brains for rabies) – Simple bacterial culture (Cholera vibrio) then inactivation – Grow in eggs (influenza, vaccinia) then inactivate

>100 million eggs used for influenza in the USA every year 22

Improved Preparation

• Recombinant DNA technology is being used to produce a new generation of vaccines.

 Virulence genes are deleted and organism is still able to stimulate an immune response.  Live nonpathogenic strains can carry antigenic determinants from pathogenic strains.  If the agent cannot be maintained in culture, genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e.g. E. coli.

23

Recombinant Vaccines

1. Subunit Vaccines peptide vaccines Genetic immunization 2. Attenuated Vaccines 3. Vector Vaccines 4.Antigen Delivery Systems

24

VACCINES Principle of Vaccination

25

VACCINES Subunit/Peptide Vaccines

•Do NOT use entire virus or bacteria (pathogenic agent) •Use components of pathogenic organism instead of whole organism •Advantage: no extraneous pathogenic particles i.e. DNA •Disadvantage: Is protein the same as in situ?

-> Cost?

26

VACCINES Subunit/Peptide Vaccines Development of Subunit vaccines based on the following observation: •

It has been showed that the capsid or envelope proteins are enough to cause an immune response:  Herpes simplex virus envelop glycoprotein O.  Foot and mouth disease virus capsid protein (VP1)  Extracellular proteins produced by Mycobacterium tuberculosis.

• •

Subunit Vaccines Antibodies usually bind to surface proteins of the pathogen or proteins generated after the disruption of the pathogen. Binding of antibodies to these proteins will stimulate an immune response. Therefore proteins can be use to stimulate an immune response.

• •

27

VACCINES Subunit/Peptide Vaccines Example for a subunit vaccine -> Tuberculosis • • • • • • • • •

Tuberculosis is caused by Mycobacterium tuberculosis. The bacterium forms lessions in the tissues and organs -> causing cell death. Often the lung is affected. About 2 billion people are infected and there are 3 million deaths/year. Currently tuberculosis is controlled by a vaccine called BCG (Bacillus CalmetteGuerin) which is a strain of M. bovis. M. bovis often responds to diagnostic test for M. tuberculosis. Six extracellular proteins of M. tuberculosis were purified. Separately and in combinations these proteins were used to immunized guinea pigs. These animals were then challenged with M. tuberculosis. After 9-10 weeks examination showed that some combinations of the purified proteins provided the same level of protection as the BCG vaccine.

28

VACCINES Subunit/Peptide Vaccines Selection & delivery of vaccine peptides: Use discrete portion (domain) of a surface protein as Vaccine These domains are ‘epitopes’ (antigenic determinants) -> are recognized by antibodies CARRIER PROTEINS Problem -> Small Peptides are often Digested -> no strong immune response -> Carrier Proteins Make more Stable + stronger immune response Make fusion protein of carrier + vaccine peptide -> inert carrier or highly immunogenic carrier (hepatitis B core protein) 29

VACCINES

need to remove virulence

Attenuated Vaccines • Attenuated vaccines often consists of a pathogenic strains in which the virulent genes are deleted or modified. • Live vaccines are more effective than a killed or subunit (protein) vaccines. Example -> Vaccine against Cholera •

cholera is caused by infection withVibrio cholerae and is transmitted through contaminated water. need to remove

•

V. cholerae produces a enterotoxin with an A1 subunit and 5 B subunits -> causes disease

•

Presently the cholera vaccine consist of a phenol-killed V. cholerae and it only last 3-6 months.

•

A live vaccine would be more effective.

30

VACCINES Attenuated Vaccines Example -> Vaccine against Cholera

550 bases deleted of A1 peptide

The final result is V. cholerae with a 550 bp of the A peptide deleted. -> Currently being tested.

31

VACCINES Vector Vaccines Virus as Antigen Gene Delivery System !!! Vaccinia good candidate for a live recombinant viral vaccine •benign virus •replicate in cytoplasm (viral replication genes) •easy to store -> The vaccinia virus is generally nonpathogenic. The procedure involves: • The DNA sequence for the specific antigen is inserted into a plasmid beside the vaccinia virus promoter in the middle of a non-essential gene e.g. thymidine kinase.

32

VACCINES Vector Vaccines The procedure involves: •

The plasmid is used to transform thymdine kinase negative cells which were previously infected with the vaccinia virus.

•

Recombination between the plasmid and vaccinia virus chromosomal DNA results in transfer of antigen gene from the recombinant plasmid to the vaccinia virus.

•

Thus virus can now be used as a vaccine for the specific antigen.

-> Recombinant Virus

33

VACCINES Vector Vaccines • A number of antigen genes have been inserted into the vaccinia virus genome e.g. ❖ Rabies virus G protein ❖ Hepatitis B surface antigen ❖ Influenza virus NP and HA proteins. • A recombinant vaccinia virus vaccine for rabies is able to elicit neutralizing antibodies in foxes which is a major carrier of the disease.

34

VACCINES Vector Vaccines Control of Viral Vaccines Post Innoculation

•Vaccinia virus is resistant to interferon -> due to presence of K3L protein •Use an interferon-sensitive strain of vaccinia virus -> delete K3L gene to create mutant

35

VACCINES Bacterial Antigen Delivery Systems

Antigen Gene

-> Use live nonpathogenic bacterium which contains antigen (Salmonella or epitope from cholera)

•Insert antigen gene into flagellin gene Bacterium

•Epitope is expressed on the flagellum surface

Antigen Proteins made on Bacterial cell -> Flagellin-engineered bacteria is VACCINE Advantage - Oral Administration Vaccinate Patient 36

VACCINES Vaccine Approval • Done by CBER (Center for Biologics Evaluation and Research), an arm of the FDA • Generally same clinical trial evaluation as other biologics and drugs

37

Vaccine Technology

Vaccine Technology

Vaccine Technology

Future Development scope of vaccines (a) Vaccine against Alzheimeir’s disease (b) Vaccine for Meningitis C (c) Super vaccine (d) Immunomodulators (e) Vaccination with Gas-Lighter (f) Vaccine against cervical-cancer (g) Vaccination without needles

VACCINE DESIGN

Polytope optimization •Successful immunization can be obtained only if the epitopes encoded by the polytope are correctly processed and presented. •The design of a polytope can be done in an effective way by modifying the sequential order of the different epitopes, and by inserting specific amino acids that will favor optimal cleavage as linkers between the epitopes.

Polytope starting configuration

Polytope optimization Algorithm • Optimization of four measures: 1. The number of poor C-terminal cleavage sites of epitopes (predicted cleavage < 0.9) 2. The number of internal cleavage sites (within epitope cleavages with a prediction larger than the predicted C-terminal cleavage) 3. The number of new epitopes (number of processed and presented epitopes in the fusing regions spanning the epitopes)

4. The length of the linker region inserted between epitopes.

Polytope final configuation

Prediction of antigens • Protective antigens • Functional definition (phenotype) • Which antigens will be protective (genotype)? • They must be recognized by the immune system • Predict epitopes (include processing) • CTL (MHC class I) • Helper (MHC class II) • Antibody

Function and conservation • Some of the epitopes must exist in the wild type • Conservation (http://www.ncbi.nlm.nih.gov/BLAST) • Function • When is it expressed? • Where is it trafficked to? • SecretomeP Non-classical and leaderless secretion of eukaryotic proteins. SignalP Signal peptide and cleavage sites in gram+, gramand eukaryotic amino acid sequences. TargetP Subcellular location of proteins: mitochondrial, chloroplastic, secretory pathway, or other. • Expression level?

Selection of antigens • Epitopes

• Polytope • Proteins • Helper epitopes

• Does it contain any • Can they be added • Hide epitopes • Immunodominant and variable ones

Examples of antigen selections

Clustering of HLA alleles

Inside out: 1. Position in RNA 2. Translated regions (blue) 3. Observed variable spots 4. Predicted proteasomal cleavage 5. Predicted A1 epitopes 6. Predicted A*0204 epitopes 7. Predicted A*1101 epitopes 8. Predicted A24 epitopes 9. Predicted B7 epitopes 10. Predicted B27 epitopes 11. Predicted B44 epitopes 12. Predicted B58 epitopes 13. Predicted B62 epitopes

Strategy for the quantitative ELISA assay C. Sylvester-Hvid, et al., Tissue antigens, 2002: 59:251

• Step I: Folding of MHC class I molecules in solution b2m Heavy chain peptide

Incubation

Peptide-MHC complex

• Step II: Detection of de novo folded MHC class I molecules by ELISA

Development

C Sylvester-Hvid et al., Tissue Antigens. 2002 59:251-8

Flow Chart of ELISPOT Assay PBMCs + Peptide

+ peptide

Culture in vitro for 10 days + peptide

- peptide Incubating in anti IFN- pre-coated plate for 18-20 h •Coating Ab: –Human IFN- MAb (ENDOGEN, Pierce Biotechnology, Inc) Washing off the cells •Detection Ab: –Human IFN- MAb, Biotin labeled Adding Biotin-anti IFN- –(ENDOGEN, Pierce Biotechnology, Inc) Adding Streptavidin-HRP after washing the plate

Adding a substrate Automatical counting

Selected pathogens Pathogen

HLA binding ELISPOT

Influenza

X

X

Variola major (smallpox) vaccine strain

X

X/VRC, NIH

Yersinia pestis

X

Francisella tularensis (tularemia)

X

LCM

X

Lassa Fever

X

Hantaan virus (Korean hemorrhagic fever virus)

X

Rift Valley Fever

X

Dengue

X

Ebola

X

Marburg

X

Multi-drug resistant TB (BCG vaccine)

X

X

Yellow fever

X

(X) T August

(X) A Sjostedt

(X) T August

Prediction of Class II epitopes

Prediction of MHC Class II binding • Virtual matrices – TEPITOPE: Hammer, J., Current Opinion in Immunology 7, 263-269, 1995, – PROPRED: Singh H, Raghava GP Bioinformatics 2001 Dec;17(12):1236-7

• Web interface http://www.imtech.res.in/raghava/propred • Prediction Results

Prediction of Antibody epitopes

• Linear

– Hydrophilicity scales (average in ~7 window) • Hoop and Woods (1981) • Kyte and Doolittle (1982) • Parker et al. (1986)

– Other scales & combinations • Pellequer and van Regenmortel • Alix

– New improved method (Pontoppidan et al. in preparation) • http://www.cbs.dtu.dk/services/BepiPred/

• Discontinuous – Protrusion (Novotny, Thornton, 1986)

Prediction of proteins structure

• Homology modeling • Secondary structure prediction