Advancement in vaccinology

Pooja Goswami

• What is vaccine and why it is important in our life

• Concept behind vaccine ( Similar to infection)

• Types of vaccine

• Generation of Vaccine

• Designing of vaccine

• Reverse vaccinology

• Difference between classical and new trend vaccine

• Edible Vaccine

Points to be discuss

3

Overview of the Immune Response

• When a microbe enters the body the immune system responds in an attempt to eliminate the infectious agent.– Innate immune system relies on immediate

recognition of antigenic structures common to many micro-organisms (pathogen associated molecular patterns /PAMPS)

– Adaptive immune response made up of T & B lymphocytes that have unique receptors specific to microbial antigens, take time to respond

Natural immunity: Infection

Artificially acquired immunity: vaccine

Herd Immunity

Vaccine and vaccination

• Vaccine immune the individual

• Vaccination in community lead to immunity in society

• Principle of Vaccine – The more similar a vaccine is to the natural

disease, the better the immune response to the vaccine.

• Concept of Vaccine– Generate memory cells

• To generate and sustain the number of antigen specific B & T cells against a particular pathogen / antigen sufficient to provide protection

– Trained immune system to face various existing disease agents (viruses & bacteria)

Concept vaccine

Vaccine: Vaccinous (Pertaining to cow)

First Generation Second Generation Third Generation

Live attenuated

Killed/Inactivated

Conjugated

Toxoid

Subunit

Recombinant Vector

DNA

Framework for vaccine designing • If a scientist get to know some infection spread

in glove. Scientists quickly determine that disease X is caused by a new species of toxic bacterium. They call it “bacterium X.” And X is resistant to antibiotics too

WHAT TO DO NOW

• Researchers would carefully study bacterium X.

HOW TO THINK ABOUT VACCINE

• What nutrients it requires, how it damages the tissue

• Analyze bacterium X’s genes.

• Explore how the immune system responds to bacterium X It should be able to induce Both T cell and B cell mediated response.

• Discover and study the toxin secreted by bacterium X.

This imaginary new disease is caused by a bacterium, but scientists would use approaches similar to those outlined below to develop a vaccine against a new virus

First Approach for live attenuated

• These vaccines contain living microbe should grow in suboptimal condition, they loose ability to cause disease but retain the capacity of transient growth.

• This weakening of the organism is called attenuation

• Because a live, attenuated vaccine is the closest thing to a natural infection, these vaccines are good “teachers” of the immune system & elicit strong cellular and antibody responses

• Major disadvantage reversion of virulent form– Ex. BCG), Sabin polio , measles, herpes

Live attenuated Vaccine

Second approach: Inactivated vaccine

• But if X, Live attenuated is good choice for virus due small no. of genes than bacteria

• Heat killed vaccine is predominant to only humoral antibody response and required booster dose

• An inactivated vaccine is more stable and safer than live vaccines &easily stored and transported in a freeze-dried form

• Ex. Salk polio,

Heat killed vaccine

Subunit or toxoid vaccine

• Scientists would certainly look into the possibility of a subunit vaccine for X. Instead of the entire microbe, subunit vaccines include only the antigens that best stimulate the immune system.

• Because our imaginary bacterium X secretes a toxin, or harmful chemical, a toxoid vaccine might work against it.

• Inactivate toxins by treating them with formalin, a solution of formaldehyde and sterilized water. Such “detoxified” toxins, called toxoids, are safe for use in vaccines.

• Ex Diphtheria and tetanus

Subunit / Toxoid vaccine

Polysaccharide and Conjugate approach• If bacterium X possessed an outer coating of sugar molecules

called polysaccharides with antiphagocytic property, researchers would try making a conjugate vaccine for X.– Ex. Streptococcus Pneumanae have 23 diff capsular polysaccharide

(Pneumovax 23), N. meningigitidis

• Limitation– Can only activate Bcells with IgM production

• So to overcome Scientist tried linked Polyacchride Ag to carrier protein to elicit Th response class switching and memory cell formation

• Ex Hib causing menigigtits develop polysacchride linked to protein carrier to tetanus toxoid

DNA vaccine

• After genetic analysis of X, scientists could attempt to create a DNA vaccine against it.

• Direct injection of naked DNA plasmid into muscles as vaccine

• DNA vaccine consist– origin of replication, strong promoter, cloning site

& antibiotic as selection marker

• So Ag can expressed directly cell of host similar to viral infection

• DNA vaccine inject via intramuscular or gene gun

Recombinant Vaccine

• Recombinant vector vaccines could be another possible strategy against bacterium X,

• These are similar to DNA vaccines, but they use an attenuated virus or bacterium to introduce microbial DNA to cells of the body.

• Vector refers to the virus or bacterium used as the carrier.

• First recombinant vaccine Hep B

Because of the rigorous research and testing each vaccine must go through, it would take years, possibly decades,

Recombinant DNA Technology

List of vaccine

Type of vaccine Disease

1 Live attenuated vaccine Measels, mumps, rubella, polio (sabin) yellow fever

2 Killed vaccine (Inactivated) Cholera, hep-A, Plague , Polio, (Salk), Rabies

3 Toxoid Diphtheria, Tetanus

4 Subunit Hepatisi-B, Pertusis, pneumania,

5 Conjugate HIb

Some human disease agents for which rDNA vaccines are being developed

Pathogenic agent Disease

Varicella-zoster virus Chicken pox

Hepatitis A and B viruses High fever, liver damage

Herpes simplex virus type 2 Genital ulcers

Influenza A and B viruses Acute respiratory disease

Rabies virus Encephalitis

Human immunodeficiency virus AIDS

Vibrio cholerae Cholera

Neisseria gonorrhoeae Gonorrhea

Mycobacterium tuberculosis Tuberculosis

Plasmodium spp. Malaria

Trypanosoma spp. Sleeping sickness

Reverse vaccinology

• Computational approach for identification of vaccine candidates against microorganisms from their genome sequences

Why reverse vaccinology is required• In conventional method, vaccines are basically of 2

type, either live or killed (subunit, toxoid, polysaccharide)

• So to identify we have, to grow, cultivates the mo’s then dissect into individual components and each fraction to be tested for its immune response

• Its allow only those Ag which are abundant protein to quantify so the less abundant protein can not be targeted

• it takes decades to identify and growing such target protein and still it get fail sometime after so much effort if it is not able to induce best immune response

• So to overcome this situation Reverse vaccinology is the best way to deal

Conventional Vaccinology

5-10 years

Reverse vaccinology

1-2 years

Rappuoli in 2000

Pan genome:• Pan genome:

– Set of all unique gene of all the strains of prokaryote species

– Due to species diversity, bigger than single genome sequence

Pan genome

variable genomeSet of gene in single strain

of a bacteria

Core genomeSet of gene by all strains of

same bacteria

How to predict Protein for vaccine

• NERVE, Veign, Veijen– Based on extracellular proteins ( adhesion mol.)

• Jenner Predict Server– Based on host pathogen interaction and functional

classes of protein involve in virulence i.e. invasion, colonization, porins, flagellin, binding protein

• Vacceed (First software for eukaryote) 2014– Based on extracellular proteins and host pathogen

interaction and functional classes of protein involve in virulence i.e. invasion, colonization, porins, flagellin, binding protein

Role of Epitope prediction in vaccinology• Humoral immunity may prevent infection, but cell mediated

immunity is required for clearance and control of hyper variable virus like HIV and HCV

• T-cell Epitope: T cell recognize Ag only when they presented by MHC-I&II with help of CD-4 &CD-8

• Epimatrix, Clusti matrix, blasti matrix used to predict T cell epitope

• B-cell Epitope defined specific surface region of antigenic protein

– Linear Epitope (short peptide), PEOPLE, PREDITOPE

– Conformational Epitope (AA foled in 3 dimensions) CEP and DiscoTope used as tool to predict

First time Success in Men B

• After decades of effort, Scientist almost lost hope to develop vaccine against Meningococcus B which cause 50 % meningitis to worldwide, due to similar capsular polysaccharide as human cell wall

• In literature till 1990 only 12 surface Ag on Meningococcus B was reported , and only 4 or 5 out of them bactericidal activity, but not enough to develop a vaccine

• Reverse vaccinology took charge

First vaccine :Reverse vaccinology (Men-B)

Reverse vaccinology

• For viral vaccine, we focus only Structural protein (envelop and core protein)• Spent 20 decades in every single form of HIV envelop

protein gp120,gp140,gp 160 and structural protein gp 55 & GAG inspite of having whole genome sequence

• After So many years we realized we have whole genome to study, so we can study, all proteins, those are not the part of envelop and available in very small amount.

• Tet, Naf, Pol and Reg showing promising result in HIV

Development of Vaccine• Pre-clinical development is research carried

out in lab assays and on animals.– Creation of the vaccine concept

– Evaluation of vaccine efficacy in test tubes and animals

– Manufacture of the vaccine to Good Manufacturing Practice standards

• Clinical development is when the vaccine is first tested in humans. It covers four stages over several years,

Different type development necessary to reach licensing

License

Phase-IVPost

marketing, safety

&efficacy of 1000 of subjects

for several years

Safety <10-50 ppl & <1 year

Safety &dose ranging50-100ppl & 1-3 year

Safety &efficacy1000 ppl& 3-5 year

Efficacy: ↓ in incidence of disease in vaccinated ppl

UIP (Universal immunization programme)

• WHO guidelines for 6 children disease i.e. Diptheria, pertussis, tetanus, polio, measels and tuberculosis

• UIP includes 6 vaccines: BCG, DPT, polio, mmr, DT and TT. These six vaccines are also known as the primary vaccines

• There are the so-called secondary vaccines for Selective Immunization (SI) against yellow fever, Japanese encephalitis, hepatitis B, typhoid, cholera, etc., required for specific risk groups or age groups or special situations across the whole country or selected states/regions.

Vaccine vial Monitor

How do you make a traditional vaccine?

What is Edible Vaccine ?

• Edible Vaccine involves introduction of selected desired genes into plant and then inducing these altered plants to manufacture the altered protein

• Edible vaccine mucosal immunity i.e. first line of defense

Edible vaccineBacterial or viral

Transformation via Agrobacterium tumifaciens

Electroporation

Gene gun method

Direct inoculation of plant virus

The Agrobacterium method

• Soil bacterium causing a root disease called crown gall– In the case of disease, A. tumefaciens invades the host plant and

transfers a piece of its own DNA to the host genome

– For transformation, A. tumefaciens has been engineered to carry and transfer transgenes and to not cause disease

– This transformation is common for docots plant

A. tumefaciens

bacterialchromosome plasmids

The Gene gun ( Biolistic method)• Gene shoots target gene into

plant

• DNA bound to tiny particle of gold or tungsten ( target gene ) which are shot into plant tissue

• Particle penetrates the cell wall and membrane of plant tissue

• DNA get separate from metal due to high pressure (He) and integrate into plant DNA inside the nucleus

Electroporation

• This method is usually used to enter the viruses or plasmids in the form of vectors or plasmids into the cell through pores in the cell membrane.

• Miniature pores can be created by using electrical current from an external source, the cell membrane becomes more permeable allowing foreign objects enter into it.

• If the whole experiment is well controlled then after sometime, the pores of the plasma membrane can reseal, but during that time the molecules or foreign objects can enter the cell's cytoplasm.

Which part of the plant to be induced

• New genes into the plants requires a promoter specific to the area where is to be gene expressed

• For ex. U wanna grow, genes only in rice grain not in leaves– Endosperm specific promoter to be used

RABIES VIRUS G PROTEIN IN TOMATO

• Gene linked to CaMV35S promoter

• Introduced to tomato plants by Agrobacterium- mediated transformation

• Expression of recombinant glycoprotein in leaves and fruits

•

(Mc Garvey et al.,1995)

Gene derived from an antigen

Modification of gene to facilitate transcription

Insertion of modified gene into plasmid containing plasmid promoter gene to increase protein expression

Chloroplast transformation

Screening of plant expressing antigen

Nuclear transformation

Purification of antigen

Vaccine

Plant vaccine

First plant vaccine

• The first vaccine in plants was produced by expressing the Streptococcus mutans surface protein antigen A (SpaA) in tobacco

• Potato based vaccine against Hep B showed promising result

• Banana based vaccine norovirus causing dirrhea showed good reslut

Tested edible vaccine

Name of the vaccine

Vector Pathological conditions

Etiological agent

Rabies Tobacco Rabies Rabies virus

Hepatitis B Potato, Tobacco

Hepatitis B HBs Ag

Gastroenteritis Maize Gastroenteritis Corona virus

HIV Tomato AIDS HIV virus

Cholera Potato Cholera Vibrio cholerae

j. inf. Disease, Vaccine 2000

Clinical Trial on

Norwalk Virus: 20 people fed with transgenic potato .

19 (95%)of them expressing Norwalk virus antigen showed seroconversion.

Hepatitis B: First human trials of potato-based vaccine against Hepatitis B have reported encouraging results.

The amount of HBsAg needed for one dose could be achieved in a single potato.

Advantages of edible vaccine

• Effective as delivery vehicle for immunization

• Edible vaccine can elicit mucosal immunity which is not observed in traditional vaccines

• Cost effective in storage, preparation, production and transportation

• Easily acceptable and do not require administration by injection

• Easy for mass production system by breeding as compared to an animal system.

Disadvantage

• Early ripening of fruits

• To overcome this, target Ag can grow in leave, so, we can dry them, grind them and placed in gelatin capsule

Hep-B antigen developed in tobacco plant

Int j. mol Sci.2013

The Hope

• 100 plants will yield a gram of purified vaccine (ie., 75,000 doses)

• Transient expression using “deconstructed virus” required 12 days from infection to harvest

Plague Vaccine Research

Step1 Generation of Ag

Step2 Isolation of Ag

Bacteria grown in bioreactor

Virus grown in cells of chicken or in cell lines

How to make a vaccine

Step3 Purification of Ag Chromatography

Step5

Step4 Formulation with adjuvant and preservatives, liposome,

Recombinant technology• Isolation and purification of DNA

• Both vector and target DNA molecules can be prepared

• Cleavage of DNA at particular sequences • The DNA fragment of interest is called insert DNA. DNA is usually

cleaved by nucleases and restriction endonucleases

• Ligation of DNA fragments• ligating the insert DNA to vector DNA (recombinant DNA or chimeric

DNA) by ligase

• Introduction of recombinant DNA into host cells• The direct uptake of recombinant DNA by a host cell is called genetic

transformation

• Replication and expression of recombinant DNA in host cells: Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell

• Identification of host cells that contain recombinant DNA of interest: Vectors usually contain easily scored genetic markers, or genes, that allow the selection of host cells that have taken up foreign DNA

How to make edible vaccine