Immunological and Biological Products PDF

Immunological and Biological Products  Course Content Introduction to Biotechnology Introduction to genetic engineering Immunological products and biological products – General introduction – Different classes of immunological and biological products...

Immunological and Biological Products  Course Content Introduction to Biotechnology Introduction to genetic engineering Immunological products and biological products – General introduction – Different classes of immunological and biological products:  Immunological products: vaccines, antibodies,  Biological products: hormones, enzymes, growth factors, – Production, Formulation &Manufacturing, Handling and Dispensing of rDNA derived drugs 03/01/2025 2  Introduction to Biotechnology  At the end of this lesson you should be able to:- Defines the term biotechnology, Identify different applications of biotechnology Understand the impact of biotechnology in pharmaceutical care 03/01/2025 3  Introduction Biotechnology: is the technology that utilizes biological systems, living organisms or parts of this to develop or create different products. is defined as the application of the life sciences to chemical synthesis. It utilizes living cells and cellular materials to create pharmaceutical, diagnostic, agricultural, and other products to benefit society 03/01/2025 4  Introduction… According to the Convention on Biological Diversity (CBD), Biotechnology is defined as “Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use”. The living organisms or derivatives thereof most frequently used include micro-organisms, animals and plants (or their isolated cells) as well as enzymes. They can be utilized to process substances, usually other natural, renewable materials, or serve themselves as sources for valuable substances or goods. 03/01/2025 5  Introduction… Since the middle of the twentieth century biotechnology has rapidly progressed and expanded. In the mid-1940s, scale-up and commercial production of antibiotics such as penicillin occurred. The techniques used for this development were: – Isolation of an organism producing the chemical of interest using screening/ selection procedures, and – Improvement of production yields via mutagenesis of the organism or optimization of media and fermentation conditions. This type of biotechnology is limited to chemicals `occurring in nature 03/01/2025 6  Introduction…  Biotechnology and its applications in medicine, pharmaceutical and related industries represent one of the most influential developments in 21st century.  Several branches of industry rely on biotechnological tools for the production of  food,  beverages,  pharmaceuticals and biomedical products. 03/01/2025 7  Introduction… The definitions of biotechnology can be further divided into different areas known as red, green, blue and white. Red biotechnology: this area includes medical procedures such as utilizing organisms for the production of novel drugs or employing stem cells to replace/regenerate injured tissues and possibly regenerate whole organs. It could simply be called medical biotechnology. 03/01/2025 8  Introduction… Green biotechnology: Green biotechnology applies to agriculture and involves such processes as the development of pest-resistant grains and the accelerated evolution of disease- resistant animals. Blue biotechnology: Blue biotechnology encompasses processes in the marine and aquatic environments, such as controlling the proliferation of noxious water-borne organisms. White biotechnology: White (also called gray) biotechnology involves industrial processes such as the production of new chemicals or the development of new fuels for vehicles. 03/01/2025 9 Traditional application of biotechnology – Baking of bread – Fermentation of juices to alcoholic beverages – Production of spirits of wine (ethanol) – Vinegar manufacturing industry – Discovery of the fermentation properties of yeast – Description of the lactic acid fermentation by Pasteur – Detection of fermentation enzymes in yeast by Buchner – Discovery of penicillin by Fleming 03/01/2025 10  Pharmaceutical biotechnology Pharmaceutical biotechnology can simply be defined as the science that covers all techniques required for the production, manufacturing and registration of biological drugs. The aim of this pharmaceutical biotechnology is to design, produce drugs that are adapted to each persons genetic make up, which can give the maximum therapeutic effect. Biotechnolgy plays an important role in pharmaceutical science most especially in the pharmaceutical industries by creation of genetically modified organisms that can be used in industrial production. 03/01/2025 11  Pharmaceutical biotechnology… Pharmaceutical biotechnology… – is a relatively new and growing field in which the principles of biotechnology are applied to the development of drugs. – These products help treat and prevent diseases 03/01/2025 12 –  Applications of biotechnology Agriculture  Plant breeding to improve resistance to pests, diseases, drought and salt conditions  Bioinsecticide development modification for plants to improve nutritional and processing characteristics Chemical Industry  Production of bulk chemicals and solvents such as ethanol  Synthesis of enzymes and antibiotics 03/01/2025 13  Applications of biotechnology Medicine Development of novel therapeutic molecules for medical treatments Diagnostic tests Drug delivery systems Tissue engineering of replacement organs Gene therapy to cure genetic disease 03/01/2025 14  Applications of biotechnology Food Industry Production of bakers' yeast, cheese, yogurt and fermented foods such as vinegar Brewing and wine making Production of flavors and coloring agents Veterinary Practice Vaccine production Fertility control Livestock breeding 03/01/2025 15  Impact of biotechnology Positive impact  Accelerating the drug development process  Increase in Productivity Negative impact  Creation of genetically modified organisms can be potentially harmful to the environment and human health  E.g- environmental contamination 03/01/2025 16  Genetic Engineering At the end of this lesson, you should be able to:- – Defines the term recombinant DNA technology, – Identify different applications of genetic engineering, – know different techniques used in genetic engineering – Understand the application of different techniques used in recombinant DNA Technology 03/01/2025 17  Genetic Engineering  Genetic engineering  Is the process by which pieces of DNA are transferred from one organism to another.  is the process of using recombinant DNA (rDNA) technology to alter the genetic makeup of an organism.  is the direct manipulation of an organism’s genome using biotechnology.  It is combining DNA from two or more different organism to create recombinant DNA. 03/01/2025 18  Genetic Engineering  The principle of genetic engineering  is changing the genetic material of an organism by removing, changing or inserting individual gene.  E.g: Insertion of human insulin genes into bacteria to produce insulin  Amplification in rDNA technology is insertion of the target gene in a host. 03/01/2025 19  Genetic Engineering A strong foundation of genetic engineering and modern biotechnology was laid down by Cohen and Boyer in 1973 when they successfully introduced the desired genes of one organism into another, and clone of the new genes. In their experiments, they successfully recombined two plasmids (pSC 101 and pSC 102) and cloned the new plasmid in E.coli. Plasmid pSC 101 possesses a gene resistant to antibiotic tetracycline while plasmid pSC 102 contains a gene resistant to another antibiotic kanamycin. The newly developed recombined plasmid when incorporated into the bacteria exhibited resistance to both the antibiotics- tetracycline and kanamycin. 03/01/2025 20  Genetic Engineering… Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes. Genetic engineering primarily involves the manipulation of genetic material (DNA) to achieve the desired goal in a pre- determined way. Gene manipulation is defined as the formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation. 03/01/2025 21  Genetic Engineering  Recombinant DNA (rDNA)  is the main pillar of genetic engineering.  is a technique to alter genes of an organism or plant.  Recombinant DNA molecule is produced by joining of two or more DNA fragments originating from different organism  is a technology that uses enzymes to cut and paste together DNA sequences of interest  E.g.- production of human insulin, and third generation vaccines 03/01/2025 22  Genetic Engineering…  There are a key number of factors that make the field of biotechnology possible: Restriction enzymes Sequencing of DNA molecules PCR Southern and northern blotting Advancement of modern day computer 03/01/2025 23 Basic techniques of rDNA There are several techniques used in gene manipulation or recombinant DNA technology. The sum total of all genes in an organism makes up its genome. Genes are the segment of nucleic acids that code for a specific polypeptide. Genes are made up of nucleotide sequences where a combination of three nucleotides (codon) code for one amino acid. Genes are transcribed into mRNA that is then translated into polypeptide sequences. 03/01/2025 24  Techniques of rDNA… Chromosomal DNA is not the only genetic material, some bacteria posse‘s extra chromosomal genetic elements called plasmids. Plasmids are circular DNA molecules that can replicate independently. Plasmids contain the requisite genetic machinery, such as replication origin, which permits their autonomous propagation in a bacterial host or in yeast. A bacterial cell may possess single or multiple copies of the same plasmid. Some plasmids are present in one or a few copies per cell and replicate once per cell division as does the bacterial chromosome; their replication is said to be under stringent control. Since plasmids are small stretches of DNA sequences they are easy to handle in vitro and therefore are suitable vectors in genetic engineering. Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells. 03/01/2025 25  Techniques of rDNA… Genetic engineering is accomplished in three basic steps. – (1) The isolation of DNA fragments from a donor organism – (2) The insertion of an isolated donor DNA fragment into a vector genome and – (3) The growth of a recombinant vector in an appropriate host. 03/01/2025 26  Techniques of rDNA.. Isolation of the gene (DNA sequence)  Slice (cut) the desired DNA segment and introduce it into a vector (e.g., plasmid),  This is achieved using a specific bacterial enzyme called restriction enzymes or restriction endonucleases.  These enzymes are named with three letters based on the species where it was isolated. For example EcoRI is isolated from E. coli. 03/01/2025 27  Techniques of rDNA…  Each restriction enzyme cleaves DNA strand at a specific site called restriction site.  For example, Eco RI recognizes the sequence GAATTC and cleaves it between G and A (G↓A). 03/01/2025 28  Sometimes, the restriction site occurs on both Techniques of rDNA… the strands but in reverse direction. Such a segment of DNA with identical sequences but opposite in direction is called a palindrome. A palindrome site is a sequence of base pairs in double stranded DNA that reads the same backwards and forward across the double strand. When a restriction enzyme acts on palindrome, it cleaves both the strands of DNA molecule. While some enzymes cut the two strands 03/01/2025 29  Techniques of rDNA… Only those enzymes that cut the DNA asymmetrically are useful in rDNA technology. When such enzymes cleave DNA, they leave single stranded “sticky ends” on both strands. Same restriction enzymes are used to cleave the DNA molecule to be transferred and the vector. The circular structure of the plasmid is broken by the restriction enzyme; this process leaves a “sticky end” at either strand. The strand of DNA to be transferred must have two restriction sites; one on either side of the DNA segment of interest. When it is acted upon by restriction enzyme, it generates two sticky ends, one at either side of the segment. Since these sticky ends are generated by the same enzyme, they are complementary and hence are cohesive. 03/01/2025 30 03/01/2025 31  Techniques of rDNA… Vectors Bacterial plasmid is the most commonly used vector. Plasmids used in genetic engineering are said to be under relaxed control; their replication is totally independent of chromosomal replication. These plasmids may be present in copies of 10-700 per cell. Yeast artificial chromosome (YAC) is a specially constructed linear yeast chromosome that can incorporate DNA strands up to 1 million base pairs. 03/01/2025 32  Techniques of rDNA… The insertion of an isolated donor DNA fragment into a vector genome. The sticky ends of the cleaved DNA segment cohere with those of the vector, thus the cut DNA sequence can now be introduced into the plasmid. The cut ends are joined by DNA ligase enzyme and the introduced gene becomes a part of the plasmid. Ligase will join either "sticky" ends or "blunt" ends, but it is more efficient at closing sticky ends. The process of introducing foreign gene into a vector is called as cloning and the plasmid containing a cloned gene is called chimera. The DNA sequence that has been inserted into the vector is also called an ”insert”. 03/01/2025 33  Techniques of rDNA…  The chimera is then introduced into its host (e.g.,a bacterium) by various methods. Vectors carrying the genes must be incorporated into the living cells so that they can be expressed or replicated. The cells receiving the vector are called the host cell and once the vector is successfully incorporated into the host cell, the host cell is said to be “transformed”.  Foreign DNA cannot be readily sent across the membrane, following are few methods.  Heat shock: The chimera plasmids are placed in a solution containing cold calcium chloride and normal host bacteria. On heating suddenly to 42°C for 2-5 minutes the host bacterial membranes become permeable to plasmid chimeras, which pass into the cell.  Electroporation: The host cells are subjected to a high voltage pulse which temporarily disrupts the membrane and allows the vector to enter the cell.  Viruses: Since viruses have mechanism to infect susceptible cell and replicate themselves, a genetically engineered virus can deliver desired DNA sequence into 03/01/2025 the target host cell. 34  Techniques of rDNA… Gene gun: Gold particles coated with foreign DNA segments are fired into the host cell. Microinjection: A cell in held in place with a pipette under a microscope and foreign DNA is injected directly into the nucleus using fine needle. Liposome: Vectors can be enclosed in a liposome, which are small membrane bound vesicles. The liposome‘s fuse with the cell membrane (or nuclear membrane) and deliver the DNA into the cytoplasm/nucleus. 03/01/2025 35 03/01/2025 36  Nucleic acid blotting techniques  Nucleic acid Blotting: a technique used for locating a gene or sequence of interest from a complex mixture of DNA or RNA.  The most commonly used blotting techniques are:  Southern blotting for DNA  Northern blotting for RNA  Western blotting for protein  Dot blotting for DNA/RNA  Blotting refers to the process of immobilization of sample nucleic acids or solid support (nitrocellulose or nylon membrane)  Hybridization is pairing of complementary strands of nucleic acids. 03/01/2025 37  Nucleic acid blotting techniques… The first step of nucleic acid blotting is to separate a mixture of DNA or RNA on an agarose gel by molecular weight. The DNA or RNA is then transferred to a membrane usually made of nylon. Conventional capillary blotting, electro blotting or vacuum blotting (for fast and even transfer) are used the blotting technique. Use a specific probe directed against the sequence of interest to locate the presence and, in some cases, quantify the amount of target on your blot. This probe can be labeled with radioactivity, fluorescence or tagged with an enzyme that eventually generates a chemiluminescent signal when incubated with the appropriate substrate. Then the probe is incubated with the blot in hybridization buffer where it hybridizes to its complementary target on the membrane. 03/01/2025 38 Nucleic acid blotting techniques… Once the probes are hybridized on their complementary target, one proceeds to detection. Radioactive probes are detected directly with autoradiography or via phosphor imager screens and scanners. Chemiluminescent signal can get captured on both autoradiography and imaging systems, whereas fluorescent signal can only be captured on imaging systems. Then, the acquired signal may be converted to data via image analysis software for analysis or exportation to publication media. 03/01/2025 39 03/01/2025 40  Southern blotting Is a laboratory technique used to detect a specific DNA sequence in a blood or tissue sample. Southern blotting combines transfer of electrophoresis- separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. Is a technique that combines the use of restriction enzymes, electrophoresis, and DNA probes to generate, separate, and detect pieces of DNA. 03/01/2025 41  Southern blotting… Steps involved in southern blotting – DNA is digested with restriction endonucleases. High- molecular-weight DNA is digested into smaller fragments. – The DNA is then separated out by size by electrophoresis on an agarose gel. – A sheet of nylon or nitrocellulose membrane (purple) is placed on top of the agarose gel in a buffer solution. This is considered the blotting or transferring stage. Buffer transfer by capillary action is then used to move the DNA from the gel on to the membrane. 03/01/2025 42  Southern blotting… Steps involved in southern blotting… – Nitrocellulose membranes are baked by exposure to high temperatures (from 60 to 100 °C). Nylon membranes are exposed to UV radiation. These steps are used to ensure the permanent and covalent crosslink of the DNA present in the bands to the membranes. – The membrane is exposed to a radiolabeled probe. This probe is a single-stranded DNA fragment which has the sequence of interest that wanted to be detected. This probe is incubated with the membrane and allowed to hybridize with DNA on the membrane. Probes are usually radiolabeled so that they may be detected on film. – The pattern of hybridization is detected by visualization on X-ray film 03/01/2025 43 03/01/2025 44 Applications of Southern blotting technique – used for mapping the genes of an individual, – will deliver results where the genetic mutation lies, – used to develop a "DNA fingerprinting" of criminal suspects who leave behind their genetic information at crime scenes, – used for paternity testing, where DNA samples are usually plentiful 03/01/2025 45  Northern blotting  Northern blot  is a laboratory technique used to detect a specific RNA sequence in a blood or tissue sample.  It is analogous to Southern blotting except that the sample is RNA rather than DNA.  It involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to target sequence. 03/01/2025 46  Fig: Northern blotting technique 03/01/2025 47  Western  blotting Western blotting is a technique used to identify and locate proteins based on their ability to bind to specific antibodies.  Western blot analysis can detect your protein of interest from a mixture of a great number of proteins.  Western blotting can give you information about the size of your protein.  Western blotting uses gel electrophoresis ( Polyacrylamide) to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose), where they are probed (detected) using antibodies specific to the target protein.  Western blot is used in research to separate and identify proteins. 03/01/2025 48  Applications of Western blotting Western blot has various medical diagnostic applications – The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. – A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease’). – Western blot can also be used as a confirmatory test for Hepatitis B infection. 03/01/2025 49  Dot blotting  Dot blotting is a modification of a southern and northern blotting techniques.  In this technique, the nucleic acids (DNA/RNA) are directly spotted on to the filters (not subject to electrophoresis)  The hybridization procedure is the same as that of other blotting techniques.  It is useful in accumulating data for the evaluation of gene expression. 03/01/2025 50  Polymerase Chain Reaction (PCR)  A laboratory method used to make many copies of a specific piece of DNA from a sample that contains very tiny amounts of that DNA  For producing large quantities of a specified DNA  It is much faster and more sensitive than cell-based cloning  Sometimes called molecular photocopying  Gene cloning is the process of making an exact duplicate of the genetic material. 03/01/2025 51 Basic reaction:  To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA.  Next, an enzyme called "Tag polymerase" synthesizes two new strands of DNA, using the original strands as templates (guide).  This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA.  Then, each of these strands can be used to create two new copies, and so on.  The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.  The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermal cycler. 03/01/2025 52  Polymerase Chain Reaction (PCR)… Materials required i. Two primers, each about 20 bases long with sequence complementary to the sequence immediately adjacent to the DNA segment of interest ii. Target DNA fragment iii. DNA polymerase (e.g., Tag polymerase) which can sustain high temperature (> 60o C) iv. A large number of free 03/01/2025 deoxynucleotides (dNTPs) 53  Three stages of PCR  PCR is based on three simple steps required for any DNA synthesis reaction: 1. Denaturation  denaturation of the template into single strands  The double stranded DNA gets denatured and separation occurs on increasing the temperature to about 95° C for about 1 minute. 03/01/2025 54  Three stages… 2. Renaturation or annealing  annealing of primers to each original strand for new strand synthesis  the primers base join up with the complementary regions neighboring target DNA strands as temp. of the mixture is cooled to about 55°C. 3. Synthesis:  extension of the new DNA strands from the primers.  The initiation of DNA synthesis occurs 03/01/2025 at 3’-hydroxyl end of each primer. 55  DNA polymerase 03/01/2025 56 Application of Genetic Engineering  Study the arrangement, expression and regulation of genes,  Modification of genes to obtain a changed protein product,  Making multiple copies of a nucleic acid segment artificially,  Introduction of genes from one organism to another, thus creating a transgenic organism,  Creation of organism with desirable or altered characteristics 03/01/2025 57  Applications of Genetic Engineering in pharmacy  It is important for preparation of;  Human insulin,  human growth hormones,  follistim (FSH, for treating infertility),  human albumin,  monoclonal antibodies,  Recombinant vaccines, and many other drugs 03/01/2025 58  Different classes of Immunological products and biological products 03/01/2025 59 Introduction to Biopharmaceutical  Biopharmaceutical product is a pharmaceutical derived from biological sources and especially one produced by biotechnology. Biopharmaceuticals drugs structurally mimics compounds found within the body and are produced using biotechnologies. Biopharmaceuticals are representing a larger proportion of new drug around the world.  e.g. Extracted from living systems or produced by rDNA  The therapeutic target of a biologic is always a gene or a protein. 03/01/2025 60  Biopharmaceuticals… First-generation biopharmaceuticals are mainly copies of endogenous proteins or antibodies, produced by rDNA technology. – E.g. human growth hormone Second-generation biopharmaceuticals have been ‘engineered’ to improve the performance of the protein or antibody. – E.g. monoclonal antibodies Third generation biopharmaceuticals are original proteins – E.g. Etanercept is an excellent example of a third generation biopharmaceuticals. 03/01/2025 61  Biopharmaceuticals are distinct from chemical-based drugs  The primary difference is the method by which the drugs are produced:  Biopharmaceuticals are manufactured in living organisms such as bacteria, or yeast and much larger, more complex, and are composed of many more atoms with high molecular masses and structural complexity  Biopharmaceutical manufacturing methods, equipment, testing and the infrastructure required are much different, more complex and costly than for chemical 03/01/2025 62  Challenges in biopharmaceutical manufacturing  A challenge in biopharmaceutical manufacturing is both high complexity of proteins and the close similarity of variants to the desired proteins.  must be consistent in composition, and stable when stored correctly  Starting materials must be pure and standardized,  Equipment and facilities must be continuously checked,  Manufacturing processes must fall within defined ranges of temperature, pH, etc., and packaging, storage, and distribution must 63all 03/01/2025 IMMUNOLOGICAL PRODUCTS  Immunological Products  Immunotechnology deals with the practical aspects such as the production and engineering of antibodies, the application of antigens, the design of (recombinant) vaccines, strategies for immune intervention, etc. When was the world's first vaccination? – The smallpox vaccine was the first vaccine to be developed against a contagious disease in 1796, by the British doctor Edward Jenner. 03/01/2025 65  Immunological Products…  It is aimed at inducing active immunity in an individual, so that subsequent contact with microorganism following natural infection induces strong protective immune response.  Vaccine: a suspension of microorganisms (live or inactivated) that induce antibody production to protect against disease.  Immunity is the ability of the human body to tolerate the presence of 03/01/2025 66  Immunization vs Vaccination- what's the difference?  Vaccination is getting a vaccine to become protected against a disease.  Immunization is refers to the process of both getting the vaccine and becoming immune to the disease following vaccination.  How does immunization work?  All forms of immunisation work in the same way.  When someone is injected with a vaccine, their body produces an immune response in the same way it would 03/01/2025 67  Immunological Products… Properties of ideal vaccine: – Provide long lasting immunity. – Should induce both humoral (extracellular) and cellular (intracellular) immunity (efficacy). – Should not induce autoimmunity or hypersensitivity (safety). – Ease of administration (oral administration is the most preferred) – Ease to store (stability) – Should be inexpensive to produce (cost) 03/01/2025 68  Immunological Products… The vaccine vial may contain relevant:  Antigen….which generates the protective immune response.  Vaccine additives Excipients are all substances in the finished product, other than the active ingredients e.g. preservatives, diluents, stabilizers. Adjuvant: encourages a stronger immune response to the vaccine antigen. – E.g. Aluminium salts as helping to retain the antigen at the injection site long enough for an immune response to69 03/01/2025  Vaccines  Vaccines are substances administered to generate a protective immune response.  They can be live attenuated or killed.  Toxoids are inactivated bacterial toxins  Use of vaccines is now being extended to immunize against tumors or to block fertilization (contraceptive vaccines). 03/01/2025 70  Types of vaccines: A. Killed (Inactivated) Vaccines:  When it is unsafe to use live microorganisms to prepare vaccines, they are killed or inactivated.  These are preparations of the infectious, pathogenic microorganisms that have been rendered nonpathogenic,  usually by treatment with using  heat, formaldehyde or  gamma irradiation so that they cannot replicate at all.  Such killed vaccines vary greatly in their 03/01/2025 71  Examples of killed vaccines 03/01/2025 72  KILLED VACCINES… Advantages: – Safe to use and can be given to immunodeficient and pregnant individuals. – Cheaper than live attenuated vaccine – Storage not as critical as live vaccine or refrigerated storage not required Disadvantages: Weaker immune response than live vaccines; booster shots usually required, since the microorganisms cannot multiply Only humoral immunity can be induced. Most killed vaccines have to be injected. 03/01/2025 73 B. Live Attenuated Vaccine: These vaccines are composed of live, attenuated microorganisms that cause a limited infection in their hosts sufficient to induce an immune response, but insufficient to cause disease. To make an attenuated vaccine, the pathogen is grown in foreign host such as animals, embryonated eggs or tissue culture, under conditions that make it less virulent. The strains are altered to a non-pathogenic form These vaccines may be given by injection or by the oral route 03/01/2025 74  Examples of live vaccines 03/01/2025 75  Advantages of live vaccines  Strong immune response; often lifelong immunity with few doses Infectious microbes can stimulate generation of memory cellular as well as humoral immune responses. Since these can multiply in the host, fewer quantities must be injected to induce protection. A single administration of vaccine often has a high efficacy in producing long-lived immunity. – Multiple booster doses may not be required. 03/01/2025 76  Disadvantages of live vaccines  Requires refrigerated storage; may mutate to virulent form and cause disease. Live vaccines cannot be given safely to immune suppressed individuals. – Administration of live attenuated vaccines to people with impaired immune function can cause serious illness or death in the vaccine recipient. Since they are live and because their activity depends on their viability, proper storage is critical. 03/01/2025 77  Another vaccine classification SUBUNIT VACCINES: Subunit vaccines contain purified antigens instead of whole organisms. Such a preparation consists of only those antigens that elicit protective immunity. Subunit vaccines are composed of toxoids, subcellular fragments, or surface antigens. The effectiveness of subunit vaccines in increased by giving them with adjuvant. Adjuvants slow antigen release for a more sustained immune stimulation 03/01/2025 78  Examples of subunit vaccines 03/01/2025 79 Advantages subunit vaccine: They can safely be given to immune suppressed people They are less likely to induce side effects. Disadvantages: Antigens may not retain their native conformation, so that antibodies produced against the subunit may not recognize the same protein on the pathogen surface. Isolated protein does not stimulate the immune system as well as a whole organism vaccine. E.g. Haemophilus influenzae type b vaccine 03/01/2025 80  CONJUGATE VACCINES: are primarily developed against capsulated bacteria. they stimulate only humoral immunity. they generate short-lived immunity. Examples:  Haemophilus influenzae HiB polysaccharide is complexed with diphtheria toxoid.  Tetramune vaccine, which combines the tetanus and diphtheria toxoids, whole-cell pertussis vaccine, and H. influenzae type B conjugate vaccine. 03/01/2025 81  RECOMBINANT VACCINES: The vaccines are produced using recombinant DNA technology or genetic engineering. Recombinant vaccines are those in which genes for desired antigens of a microbe are inserted into a vector. Different strategies are: – Using the engineered vector (e.g. Vaccinia virus) that is expressing desired antigen as a vaccine – The engineered vector (e.g., yeast) is made to express the antigen, such is vector is grown and the antigen is purified and injected as a subunit vaccine. – Other expression vectors include the bacteria Escherichia coli, mutant Salmonella spp., and BCG. 03/01/2025 82 Advantages: – Those vectors that are not only safe but also easy to grow and store can be chosen. – Antigens which do not elicit protective immunity or which elicit damaging responses can be eliminated from the vaccine. – Example Cholera toxin A can be safely removed from cholera toxin. Disadvantages: – Since the genes for the desired antigens must be located, cloned, and expressed efficiently in the new vector, the cost of production is high. – When engineered vaccina virus is used to vaccinate, care must be taken to spare immunodeficient individuals. 03/01/2025 83  COVID-19 Vaccine A coronavirus disease 2019 (COVID-19) vaccine can prevent you from getting COVID-19 or from becoming seriously ill or dying due to COVID-19.  Messenger RNA (mRNA) vaccine.  This type of vaccine uses genetically engineered mRNA to give your cells instructions for how to make the S protein found on the surface of the COVID- 19 virus.  After vaccination, your muscle cells begin making the S protein pieces and displaying them on cell surfaces. 03/01/2025 84  COVID-19 Vaccine  This causes your body to create antibodies.  If you later become infected with the COVID- 19 virus, these antibodies will fight the virus.  Viral vector vaccine.  In this type of vaccine, genetic material from the COVID-19 virus is placed in a modified version of a different virus (viral vector).  When the viral vector gets into your cells, it delivers genetic material from the COVID- 19 virus that gives your cells instructions to make copies of the S protein 03/01/2025 85  COVID-19 Vaccine  Protein subunit vaccine.  Subunit vaccines include only the parts of a virus that best stimulate your immune system.  This type of COVID-19 vaccine contains harmless S proteins.  Once your immune system recognizes the S proteins, it creates antibodies and defensive white blood cells.  If you later become infected with the COVID-19 virus, the antibodies will fight the virus 03/01/2025 86  COVID-19 Vaccine  Covid-19 vaccine in Ethiopia; BioNTech, Pfizer vaccine Johnson & Johnson vaccine Oxford, AstraZeneca vaccine Sinopharm BBIBP vaccine inactivated No Vaccines in Clinical Trials in Ethiopia 03/01/2025 87  Components of vaccines and why are they present? 1. Active components The active component of a vaccine is known as the vaccine ‘antigen’. This is a modified or partial form of the virus, bacteria or the toxin that causes the disease against which the vaccine protects. The vaccine antigen is altered from its original form so it no longer causes disease but it can induce an immune response. 03/01/2025 88 2. Adjuvant Adjuvants are used to enhance the immune response to a vaccine. – Include various aluminium salts such as aluminium hydroxide, aluminium phosphate and potassium aluminium sulphate (alum). – One way adjuvants are thought to improve the immune response is by keeping the antigen(s) near the injection site so that they can be readily accessed by cells of the immune system. 03/01/2025 89 3. Diluents is a liquid provided separately and used to dilute a vaccine to the proper concentration prior to administration. This is usually sterile saline or sterile water. 4. Stabilizers Help maintain a vaccine’s effectiveness by keeping the antigen and other vaccine components stable during storage. Stabilizers prevent the vaccine components adhering to the side of the vaccine vial. Examples – lactose and sucrose (both sugars), – human or bovine (cow) serum albumin (both proteins). 03/01/2025 90 5. Preservatives Used to prevent fungal and/or bacterial contamination of vaccines, and are present in some but not all vaccines. Originally, preservatives were introduced to prevent bacterial contamination of multi-dose vials. – The preservatives used include thiomersal, phenoxyethanol and phenol. – Phenoxyethanol is an aromatic ether alcohol and is also used as a preservative in many cosmetics. – Phenol is an aromatic alcohol used as a preservative in very few vaccines 03/01/2025 91 6. Trace components used in the early stages of the production process of individual vaccines. Depending on the manufacturing process used this may include trace amounts of – cell culture fluids, – egg proteins, yeast, – antibiotics or inactivating agents. Usually, only minute traces of these substances are detected in the final vaccine product. 03/01/2025 92  Allergies to vaccines or vaccine components Vaccines rarely produce allergy or anaphylaxis (a rapid and serious form of allergic reaction). Overall, the total risk of anaphylaxis in children and adolescents after one vaccination has been reported as