PEPTIDE AND SUBUNIT VACCINES

• Vaccines- Introduction • Vaccines- History• Immunological Adjuvants• Types

•DNA Vaccines

• Viral Vectors

•Mucosal Vaccines

•Peptide Vaccines

•Subunit Vaccines• Vaccine Manufacturing• Vaccine Safety Issues• Animal Testing of Vaccines• HPV Vaccine- A case study of Vaccine Development

COURSE CONTENTS

• Peptide Vaccine are formed by using a specific domain of an antigenic protein.

• Instead of encoding whole of the protein, only the epitopic region can be added in to the vaccine preparation.

PEPTIDE VACCINES

1. Epitope/ target peptide Identification

2. Linking

3. Delivery

BASIC STEPS IN PEPTIDE VACCINE PREPARATION

Antigen based or

Antibody based

1. EPITOPE IDENTIFICATION

• Synthetic peptides have been used as powerful tools to identify class II and class I-restricted T cell epitopes by systematic screening in functional assays

• MHC-bound peptides eluted directly from tumor material of patients were identified on the basis of their mass and partial sequence by highly sensitive mass spectrometry

• Pool sequencing of MHC-eluted peptides has revealed that peptides bind to MHC I via an allele-specific consensus motif of anchor residues

• MHC molecules therefore encoded by different gene loci bind their own distinct (sometimes overlapping) family of peptides

1. A. ANTIGEN BASED IDENTIFICATION

• Strategy based on peptides of the hemagglutinin (H) and fusion (F) MV neutralizing antibodies are directed against these glycoproteins

• Sequential epitopes for both the proteins have been identified that have the inherent property of stimulating either B or T cell epitopes

• Two sequential B cell epitopes have been identified on the H protein by screening a panel of neutralizing and protective antibodies against a complete set of overlapping synthetic peptides

MEASLES VIRUS- EPITOPE IDENTIFICATION

HA

• Cancer T-cell Epitopes (TCEs)• Cancer vaccines can be based on whole cancer cells*, Tumor

Associated Antigens (TAAs) or peptide fragments of TAAs.• Whole cells- difficult to attain• TAA- laborious, expensive and resemble self antigens causing a

problem in discrimination.• So, recombinant TAAs appear to be obvious antigens for tumor

vaccines. They are: •Most TAAs are not tumor specific•Peptides derived from TAAs are the true antigens recognized

by the T cells •Ease of expression as production of TAA peptides in large

quantities may serve as a tumor vaccine

EPITOPE IDENTIFICATION- CANCER CELLS

CANCER VACCINE-PREPARATION

Antigenic Determinants on Abs Fall in 3 Categories:

1. B. ANTIBODY/ PARATOPE BASED IDENTIFICATION

ISOTYPIC ALLOTYPIC IDIOTYPICConstant Region of Ab Even though same isotypes

within one species small differences (1-4 a.a) arise in different individuals (form of polymorphism)

Unique VH AND VL binds antigen but can also behave as antigenic determinant.

Anti-isotypic antibodies are generated in case injected in different species. No response in the same species.

If injected with such Ab you generate anti-allotype Ab e.g., blood transfusion during pregnancy

If you inject a monoclonal antibody into a genetically identical recipient then anti-idiotypic antibodies are generated. No anti-isotypic and no anti-allotypic Abs will be generated.

IDIOTYPIC NETWORK

• The idiotype (Id) i.e idiotypic determinant is associated with the hypervariable region of the antibody (Ab) molecule

• It represents the unique antigenic determinant of that Ab• An Ab-1 is defined as an Ab that recognizes a particular

antigen e.g a vaccine candidate• The Id on Ab-1 itself can act as an immunogen that can

elicit an immune response; the Abs that bind to the Id on Ab-1 are referred to as anti-idiotypic antibodies ( anti-Id) or Ab-2

• The paratope is the site on Ab-1 that binds to a particular antigen; thus the binding site of an anti-paratope antibody is a molecular mimic of the antigen.

1. B. ANTIBODY/ PARATOPE BASED IDENTIFICATION

ANTI-IDIOTYPIC ANTIBODIES TO CANCER CELLS- AN EXPERIMENT

• Organic stretches• Glycans and other sugars

2. LINKER

Peptides need to be linked to another molecule to prevent rapid degradation (HAPTEN).

1. Keyhole limpet hemocyanin (KLH)

•an inert carrier protein from a marine gastropos mollusk

2. Hepatitis B core protein (HBcAg)

•highly immunogenic carrier protein which self assembles into small particles

3. CARRIER MOLECULE

• Epitope must consist of a contiguous stretch of amino acids

• Not all peptides are effective in eliciting an immune response (may need two or more)

• Peptide must have the same conformation as in pathogen

• Amount of peptide required to elicit an immune response may be 1000X more than for inactivated pathogen

LIMITATIONS OF PEPTIDE VACCINES

Subunit vaccines consist of one or more antigens purified from the microorganism or produced by recombinant DNA

technology or chemical synthesis

SUBUNIT VACCINES

Development of subunit vaccines requires knowledge of:

•Protective antigen(s)

•Ability to produce and purify those antigen(s) on large scale

•Ability to prove their protective efficacy in appropriate animal models in vivo and/or in vitro assays

Identification of the potential candidates for development of subunit vaccines therefore has to be based on approach

combining study of:•Genetics

•Biochemistry

• Immunology

PROCESSING

• The identification of protective antigens is a complex problem and involves approaches that can differ for viruses, bacteria and parasites

• Viruses have generally small genomes encoding a few proteins that can easily be selected when compared with larger microorganisms

• Envelope proteins and glycoproteins are the primary candidates for the induction of neutralizing antibodies whereas core antigens are usually good candidates for CTL responses

• For bacteria and parasites there can be several hundred potential candidate antigens

PROTEIN BASED

• The first subunit vaccines developed have been the diphtheria and tetanus toxoids

• Semi-purified toxins are inactivated by chemical (formaldehyde) treatment and used as vaccines

• For these two vaccines the titers of serum antitoxin antibodies correlate well with the protection

• For encapsulated bacteria the capsular polysaccharide as an antigen was chosen for development of vaccine on the observation that mutants without capsule are non-pathogenic

• Meningococcal group specific immunity is mediated by serum antibodies directed against the group-specific capsular polysaccharide.

CARBOHYDRATE BASED

Purified capsular polysaccharides have been used to develop vaccines against

•MenC,

•N. meningitidis group A (MenA)

•N. meningititdis group Y (MenY)

•N. meningititdis group MenW135 (MenW135)

Using the polysaccharide, vaccines have been developed against

23 serotypes of S. pneumoniae

•Hib

•Salmonella typhi

EXAMPLES

All these vaccines have several drawbacks•Capsular polysaccharides are T-independent antigens and they induce transient antibody responses (mostly IgM and IgG2 isotypes) in individuals aged over 18 with no efficacy in infants

•Polysaccharides do not induce immunological memory•Repeated immunization not only fails to induce any increase in specific antibody titers but can also in some cases even induce tolerance (in adults)•To overcome these drawbacks the toxoids and the polysaccharides have been put together in the form of a conjugate vaccine

DRAWBACKS

CONJUGATION

• During 1920s it was demonstrated that the immunogenicity of saccharides was significantly increased when animals were immunized with the sugar covalently linked to protein which behaved as carrier molecule

• This process converts T-independent antigens into T-dependent antigens

• The only limitation of this technology is the overload of carrier protein that patients would receive in the situations where all conjugated vaccines use the same carrier

• Carriers not only help the production of anti-oligosaccharide antibodies but also induce specific antibodies against themselves

• Anti-carrier antibodies suppress the induction of anti-oligosaccharide antibodies in animals.

POSSIBLE SOLUTIONS

Hib• The first conjugate vaccine developed has been against

Hib:

• It has a high immunogenicity and efficacy

• Induces immunological memory

• Ig isotype switching

• Antibody affinity maturation in children aged less than 18 months

• Un-conjugated polysaccharide vaccine fails to induce any of the above responses

CONJUGATE VACCINES

• Identification of a relevant antigen can take several years• It is possible that antigens expressed during the infection

in vivo are not equally well expressed in vitro during cultivation

• Once a suitable antigen is identified most often it is expressed in genetically engineered prokaryotic or eukaryotic vectors

• Although this approach has been a success in the case of viruses it has failed up till now for complex pathogens like bacteria and parasites

RECOMBINANT DNA APPROACH FOR SUBUNIT

VACCINES

PARTICLES BASED APPROACH

• First application for Hepatitis B through expression of HBsAg gene in bakers yeast i.e S. cerevisae

• Notably expression of the HBsAg gene in E.coli gave rise to HBsAg polypeptides but not of HBsAg particles

• HBsAg is a virus like particle (VPL) in that its surface structure is similar to that of HBV virion

• Purified yeast derived HBsAg is adjuvanted with Aluminum salts for formulation as vaccines

• HBsAg has also been expressed in transgenic potato tubers (immunogenic and edible)

• HPV virion is a highly ordered structure with major protein L1

• Expression of L1 in eukaryotic cells results in the formation of VLPs that elicit HPV-neutralizing antibodies leading to protective immunity

PARTICLES

• Allergy• Oncology• Autoimmunity• Crude extracts from allergens such as ragweed can be used as

therapeutic vaccines• Therefore an allergen encoding gene is expressed in a hetrologous host

cell• DIABETES• Before the clinical symptoms of the disease develop, autoantibodies

become detectable to pancreatic beta cell autoantigens e.g insulin. Following which beta cells are destroyed by autoimmune attack.

• A range of recombinant autoantigens can prevent the development of type 1 diabetes in a mouse model

• Clinical trials show that subcutaneous injection of E.coli derived recombinant insulin into pre-diabetic patients resulted in a significant delay in development of clinical type 1 diabetes

VACCINES FOR NON-INFECTIOUS DISEASES

Bacteria• E.coli• Bordetella pertusis• Vibrio choleraeYeast• S. cerevisiae• Hansenula polymorphaMammalian cells• Chinese hamster ovary• African green monkey

kidney• Lymphoid cells

Mammals• Goat• Sheep• Cow

Plants• Tomato• Potato• Tobacco

HOSTS FOR THE EXPRESSION OF RECOMBINANT PROTEINS

Among the examples of failures of conventional subunit vaccines are three diseases:

•Acquired Immunodeficiency Syndrome

•Tuberculosis

•Malaria

AIDSApproaches to vaccine development have essentially focused onto the proteins of the HIV-1 envelope mainly gp120Problems:• Antigen variability• Poor ability of these vaccine constructs to induce antibodies that

are able to neutralize heterologous viral strains• Effective approach is to aim for inducing both antibody and

CTL responses

FAILURES IN THE DEVELOPMENT OF SUBUNIT

VACCINES

TuberculosisProblems:

Intracellular location of Mycobacterium tuberculosis, which needs the development of Th1-type and MHC class I-restricted CTL responses

Lack of ideal experimental animal models of infection

MalariaProblems

Immunity against malaria parasites is not only specie specific but also specific for each stage of development

The effector mechanisms required for protection are different for each developmental stage

Ideal malarial vaccine therefore should consist of a mixture of antigens from the different stages of development of the parasite

FAILURES IN THE DEVELOPMENT OF SUBUNIT

VACCINES

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