Ch. 11. Vaccines
Subunit vaccines Attenuated vaccines Vector vaccines DNA vaccines
Subunit vaccines
Attenuated vaccines
Vector vaccines
DNA vaccines
Immunity
1. Innate Immunity (Skin barrier, acid pH of skin and stomach, lysozyme in tears, inflammation, phagocytosis, etc.) 2. Acquired Immunity Specificity --Antibody-antigen interactions Diversity --107 - 108 different antibodies Memory --Enhanced response to antigen after the initial exposure Recognition --Able to distinguish self from nonself
1. Innate Immunity (Skin barrier, acid pH of skin and stomach, lysozyme in tears, inflammation, phagocytosis, etc.)
2. Acquired Immunity
Specificity --Antibody-antigen interactions Diversity --107 - 108 different antibodies Memory --Enhanced response to antigen after the initial exposure Recognition --Able to distinguish self from nonself
Specificity --Antibody-antigen interactions
Diversity --107 - 108 different antibodies
Memory --Enhanced response to antigen after the initial exposure
Recognition --Able to distinguish self from nonself
The Immune Response i.e. Acquired
1. Humoral
Secretion of antibodies by activated B-cells
2. Cell-mediated
Cytotoxic T-cells (TC) kill infected and cancerous cells
Vaccines
Ex. -Diphtheria -Measles -Mumps -Smallpox -Polio -Tetanus -Whooping cough
Ex.
-Diphtheria -Measles -Mumps -Smallpox -Polio -Tetanus -Whooping cough
Needed:
New vaccines -Malaria. 1-2 million deaths/year -Diarrheal diseases. 4-5 million deaths/year -AIDS. ~40 million infected with HIV worldwide (12/04) -Cancer Existing vaccine improvements -Greater and longer lasting protection -Production methods Table 11.1
New vaccines
-Malaria. 1-2 million deaths/year -Diarrheal diseases. 4-5 million deaths/year -AIDS. ~40 million infected with HIV worldwide (12/04) -Cancer
-Malaria. 1-2 million deaths/year
-Diarrheal diseases. 4-5 million deaths/year
-AIDS. ~40 million infected with HIV worldwide (12/04)
-Cancer
Existing vaccine improvements
-Greater and longer lasting protection -Production methods Table 11.1
-Greater and longer lasting protection
-Production methods
Table 11.1
Traditional Vaccines --Most of the currently used and past vaccines 1. Killed pathogen
2. Live attenuated pathogen
1.) Not all pathogens can be cultured 2.) Growth of viruses requires cell culture -Slow -Expensive -Low yields 3.) Need for safety precautions during production 4.) Potential for spread of disease by vaccine -Incomplete killing of pathogen or -Reversion of attenuated strains to pathogenicity 5.) Inability to obtain vaccines for some diseases Ex. AIDS
1.) Not all pathogens can be cultured
2.) Growth of viruses requires cell culture
-Slow -Expensive -Low yields
-Slow
-Expensive
-Low yields
3.) Need for safety precautions during production
4.) Potential for spread of disease by vaccine
-Incomplete killing of pathogen or -Reversion of attenuated strains to pathogenicity
-Incomplete killing of pathogen or
-Reversion of attenuated strains to pathogenicity
5.) Inability to obtain vaccines for some diseases
Ex. AIDS
Molecular Approaches for Creating Vaccines
1. Live-attenuated vaccine -Delete virulence genes from pathogen 2. Subunit vaccine -Express cloned antigen genes, purify gene product and use as vaccine 3. Vector vaccine -Clone and express genes for antigens of pathogen in a nonpathogenic strain or vector Eliminates need to create an attenuated strain of the pathogen 4. DNA vaccine -Use a recombinant plasmid vector containing a gene for an antigen Gene is expressed by patient's cells that take up the DNA
1. Live-attenuated vaccine
-Delete virulence genes from pathogen
2. Subunit vaccine
-Express cloned antigen genes, purify gene product and use as vaccine
3. Vector vaccine
-Clone and express genes for antigens of pathogen in a nonpathogenic strain or vector Eliminates need to create an attenuated strain of the pathogen
-Clone and express genes for antigens of pathogen in a nonpathogenic strain or vector
Eliminates need to create an attenuated strain of the pathogen
4. DNA vaccine
-Use a recombinant plasmid vector containing a gene for an antigen Gene is expressed by patient's cells that take up the DNA
-Use a recombinant plasmid vector containing a gene for an antigen
Gene is expressed by patient's cells that take up the DNA
Live-Attenuated Vaccines
See Fig. 11.13
Ex. Cholera: caused by Vibrio cholerae
Secretes toxic protein in small intestine A nonpathogenic strain was created by deleting part of ctxA that coded for the A1 peptide of the toxin
Secretes toxic protein in small intestine
A nonpathogenic strain was created by deleting part of ctxA that coded for the A1 peptide of the toxin
Steps
1. Tetr gene was inserted into the chromosomal site of the V. cholera ctxA gene that encodes the A1 This disrupted the gene, making the strain nonpathogenic (and resistant to tetracycline) However, if the Tetr gene is excised, the strain would revert to a virulent strain Also, probably not a good idea to use antibiotic resistant organisms for vaccines So, 2. DNA encoding A1 was cloned using a plasmid vector 3. Most of the cloned DNA was deleted using restriction enzymes 4. The plasmid was transfered to the tetracycline resistant V. cholerae strain by conjugation 5. Homologous recombination exchanged the 5' and 3' ends of the A1 peptide DNA for the Tetr gene on the chromosome 6. Tet sensitive V. cholerae was isolated and used as a live-attenuated vaccine that could not revert to virulence
1. Tetr gene was inserted into the chromosomal site of the V. cholera ctxA gene that encodes the A1
This disrupted the gene, making the strain nonpathogenic (and resistant to tetracycline) However, if the Tetr gene is excised, the strain would revert to a virulent strain Also, probably not a good idea to use antibiotic resistant organisms for vaccines
This disrupted the gene, making the strain nonpathogenic (and resistant to tetracycline)
However, if the Tetr gene is excised, the strain would revert to a virulent strain
Also, probably not a good idea to use antibiotic resistant organisms for vaccines
So,
2. DNA encoding A1 was cloned using a plasmid vector
3. Most of the cloned DNA was deleted using restriction enzymes
4. The plasmid was transfered to the tetracycline resistant V. cholerae strain by conjugation
5. Homologous recombination exchanged the 5' and 3' ends of the A1 peptide DNA for the Tetr gene on the chromosome
6. Tet sensitive V. cholerae was isolated and used as a live-attenuated vaccine that could not revert to virulence
Subunit Vaccines
Vaccines derived from a component such as an antigenic protein of a pathogen rather than whole organism.
Ex. Purified outer membrane protein of an animal virus Fig. 11.1
Ex. Purified outer membrane protein of an animal virus
Fig. 11.1
Pluses and minuses
(+) May have lower side effects, not as many types of antigens (-) Costly to purify (-) Lower antigenicity, may not provide protection against pathogen Antibodies elicited against a pure antigen may not recognize it when present on the pathogen
(+) May have lower side effects, not as many types of antigens (-) Costly to purify (-) Lower antigenicity, may not provide protection against pathogen
Antibodies elicited against a pure antigen may not recognize it when present on the pathogen
Recombinant Subunit Vaccines
Ex. Hepatitis B virus (HBV)vaccine
~100,000 new cases/year in the U.S. More contagious than HIV Can't be grown in cell culture for vaccine production
~100,000 new cases/year in the U.S.
More contagious than HIV
Can't be grown in cell culture for vaccine production
Hepatitis B surface antigen (HBsAg) was developed into a subunit vaccine
-Forms 22 nm particles (protein aggregates) in infected individuals -Highly immunogenic
-Forms 22 nm particles (protein aggregates) in infected individuals
-Highly immunogenic
Cloned antigen gene not expressed at high levels in bacterial host
However, expression in yeast resulted in formation of antigenic particles
-Marketed by Merck as Recombivax
World's first genetically engineered vaccine
Vector Vaccines
Insert genes that encode a virus antigen into a harmless virus (the vector) that can infect humans
E.x. HBV core antigen/vaccinia virus vector Vaccinia virus causes cowpox Infects cows and humans producing only mild symptoms Previously used as safe vaccine for smallpox (now eradicated)
E.x. HBV core antigen/vaccinia virus vector
Vaccinia virus causes cowpox
Infects cows and humans producing only mild symptoms Previously used as safe vaccine for smallpox (now eradicated)
Infects cows and humans producing only mild symptoms
Previously used as safe vaccine for smallpox (now eradicated)
(See Fig. 11.16)
1. Antigen gene placed on a transfer vector, flanked by vaccinia virus DNA, and under the control of a vaccinia virus promoter
o Flanking DNA is from the thymidine kinase (TK) gene
2. Transfect vector into cultured animal cells infected with vaccinia virus
3. Antigen gene is inserted into vaccinia virus' TK gene by homologous recombination
o Recombination inactivates viral TK gene
4. Recombinant virus is selected for in cells that survive exposure to bromodeoxyuridine (nucleotide analog)
o Nonrecombinant virus has an intact TK gene and the enzyme phosphorylates bromodeoxyuridine which is incorporated into the cell's DNA, causing mutation and breaks in chromosomes that kill the cell
5. Vaccinate with recombinant vaccinia virus to elicit immune response that prevents infection upon exposure to the pathogen
Advantages
Resembles the natural infection process of the viral pathogen Immune system recognizes pathogen quickly Replication of viral vector amplifies amount of antigen after vaccination
Resembles the natural infection process of the viral pathogen
Immune system recognizes pathogen quickly
Replication of viral vector amplifies amount of antigen after vaccination
Disadvantage
Vector may cause serious infection in immunosuppressed individuals Ex. AIDS patients
Vector may cause serious infection in immunosuppressed individuals
Ex. AIDS patients
DNA Vaccines (Genetic Immunization)
1. Clone gene for an antigen of a pathogen Use E. coli plasmid vector with an animal virus promoter 2. Coat gold particles with plasmid DNA and inject into subject under high pressure (biolistics) 3. Expression of the antigen gene elicits formation of protective antibodies
1. Clone gene for an antigen of a pathogen
Use E. coli plasmid vector with an animal virus promoter
2. Coat gold particles with plasmid DNA and inject into subject under high pressure (biolistics)
3. Expression of the antigen gene elicits formation of protective antibodies
-Avoids time and expense of antigen production and purification
-Not yet approved for use in humans
Survival of mice vaccinated with influenza A DNA
See Fig. 11.8
Development of a DNA Vaccine for Malaria
Caused by Plasmodium falciparum -An intracellular parasite -An effective vaccine would elicit formation of specific cytotoxic T cells Would kill infected cells before a major infection could be established -Volunteers were vaccinated with plasmid DNA containing the gene encoding P. falciparium circumsporozoite protein (PfCSP) -Cytotoxic T cells were produced -Additional genes for other parasite antigens may be added to the plasmid to enhance the immune response
Caused by Plasmodium falciparum
-An intracellular parasite -An effective vaccine would elicit formation of specific cytotoxic T cells Would kill infected cells before a major infection could be established -Volunteers were vaccinated with plasmid DNA containing the gene encoding P. falciparium circumsporozoite protein (PfCSP)
-An intracellular parasite
-An effective vaccine would elicit formation of specific cytotoxic T cells
Would kill infected cells before a major infection could be established
-Volunteers were vaccinated with plasmid DNA containing the gene encoding P. falciparium circumsporozoite protein (PfCSP)
-Cytotoxic T cells were produced
-Additional genes for other parasite antigens may be added to the plasmid to enhance the immune response
This approach is also being tested using genes that encode HIV antigens to produce an AIDS vaccine
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