Vaccines and Biotechnology-Based Diagnostics and Therapeutics PDF

Rubella and Smallpox – two vaccine-preventable diseases. Chapter 14 Vaccines and Biotechnology-Based Diagnostics and Therapeutics  History of Vaccination Variolation is an ancient Chinese medical practice in which dried scabs of smallpox lesion...

Rubella and Smallpox – two vaccine-preventable diseases. Chapter 14 Vaccines and Biotechnology-Based Diagnostics and Therapeutics  History of Vaccination Variolation is an ancient Chinese medical practice in which dried scabs of smallpox lesions were blown into a healthy individual’s nose In 1796, Edward Jenner produced the first vaccination; used the cowpox virus to protect individuals from smallpox – Smallpox is now eradicated In 1800s, Louis Pasteur developed rabies vaccine as well as anthrax vaccine for livestock At least 25 different infections are vaccine preventable Fears about vaccination developed in1998, after a paper published in The Lancet described a small study claiming a correlation between the MMR vaccine and autism – As a result of this now debunked study, there has been a resurgence in vaccine—preventable diseases For more on the vaccine debate: 2 https://www.pbs.org/video/frontline-the-vaccine-war/ Qualities of an Effective Vaccine 3 Vaccines Licensed for Use in the United States 4  Categories of Acquired Immunities Acquired Immunity Natural Immunity Artificial immunity acquired through the normal life experiences produced purposefully through not induced through medical means. medical procedures (also called immunization). Active Immunity Passive Immunity Active Immunity (same as vaccination) Passive Immunity is the consequence of a is the consequence of is the consequence of is the consequence of person developing his one person receiving a person developing his one person receiving own immune response preformed immunity own immune response preformed immunity to a microbe. made by another person. to a microbe. made by another person. © Photodisc/Getty Images RF © Photodisc/Getty Images RF © Photodisc/Getty Images RF © Photodisc/Getty Images RF 5  Herd Immunity Principle of vaccination is to stimulate a primary and secondary anamnestic response to prepare the immune system for future exposure to a virulent pathogen (at least 2 weeks following vaccination) Response to a future exposure will be immediate, powerful, and sustained Vaccines also can establish herd immunity (assuming that a certain percentage of the population is immunized, it is less likely that a non-immunized person will encounter the pathogen and thus even non-immunized individuals receive protective benefits) – The percentage of a population that must be vaccinated for herd immunity to be effective varies based on the pathogen – Highly infectious pathogens with airborne transmissionrequire higher vaccination rates 6 Recommended Immunization Schedule 7  Various Vaccine Preparations There are various vaccine preparations: 1. Killed whole cells or inactivated viruses Consist of whole inactivated pathogens, such as killed bacteria or inactivated viruses, or parts of pathogens Benefits: stable at room temperature, safe for immunocompromised individuals Drawbacks: Quickly cleared and boosters are often needed to achieve full immunity 2. Live, attenuated cells or viruses Contain pathogens that have been altered so that they do not cause disease, but are still infectious Benefits: Potent, long-lived, broader protection Drawbacks: Ability to cause disease in immunocompromised individuals 3. Subunit vaccines Include purified subunits, toxic vaccines, and conjugate vaccines Benefits: safe for immunocompromised individuals Drawbacks: require adjuvants (pharmacological additives) to stimulate a strong immune response 8  New Vaccines: DNA vaccines and Recombinant Vector Vaccines DNA vaccines use a pathogen’s DNA to stimulate an immune response To engineer a DNA vaccine, genes encoding highly immunogenic antigens must first be identified and then placed into a plasmid that is placed into a human host Recombinant vector vaccines place pathogen’s immunogenic genes into a harmless virus or bacterium which is then injected into the body 9  An Overview of Immunological Diagnostic Tests Immunological tests are rapid diagnostics that are used to identify a variety of cellular as well as viral pathogens and determining if a patient has been exposed to a particular pathogen or is immune to it Immunological tests rely on antibody-antigen interactions and are often a part of serology (study of patient serum) Serum is the liquid portion of blood that remains once all the formed elements (platelets and all blood cells) are removed by centrifuging (spinning) the sample at high speed 10  Agglutination Reaction Agglutination occurs when antibodies bind antigens in a clump Testing can identify either the antibody or the antigen in patient samples such as serum, urine, or cerebrospinal fluid Agglutination assays typically performed in a microtiter plate or on a slide Used for blood typing, to identify infections, and to diagnose noninfectious immune disorders such as autoimmune hemolytic anemia 11  Radial Immunodiffusion Test Immunodiffusion tests quantify the amount of a particular antigen (includes other antibodies) present in a sample Especially useful for quantifying the amount of a particular antibody isotype in a patient sample Both radial immunodiffusion test (Mancini method/single antigen detection) and double immunodiffusion tests (Ouchterlony method/multiple antigen detection) are used A known concentration of antibody is infused in a gel matrix and the solution being tested is added to a cut-out well in the gel The sample then diffuses from the well into the gel and create a precipitate at the zone of equivalence (an area where there is an equal or roughly equal amount of antigen and antibody molecules) The ring diameter is compared to a standard curve graph to determine antigen concentration – Larger ring = antigen is more concentrated in sample  Neutralization Reaction Plaque reduction neutralization test (PRNT) detects patient antibodies against a particular virus In this test, patient serum is extracted from a blood sample, the serum is serially diluted, and a purified preparation of the suspected virus is added Each serum/virus mixture is then added to separate petri plates of cultured cells and incubated for a few days – Similar level of plaques (compared to control) = Serum lacks/has low levels of virus-specific antibodies – Decreased plaques (compared to control) = Serum has high levels of virus-specific antibodies (neutralization occurred) Highly specific and allows for differentiation between related viruses Often preferred over other methods 13  Direct ELISA Allows for identification of an antigen in a sample A test solution containing antigens is added to microtiter plate wells and is then mixed with detection antibodies that are linked to a reporter enzyme – If the antigen of interest is in the wells, then the detection antibodies will directly bind it – Chemiluminescent or colorimetric detection is performed by adding a substrate to the wells that interacts with the antibody-linked reporter enzyme and then reading the signal via a microplate reader Downside: laborious and challenging to label diverse antibody isotypes 14  Indirect ELISA More common tool for determining if a patient has antibodies to a particular pathogen Method requires two antibodies: 1) antibody that recognizes antigen bound at the bottom of the plate wells and 2) antibody that is linked to an enzyme (detection antibody) Patient’s serum is added to plates that have wells that were pre-coated with known antigens and detection antibody is added – A substrate that interacts with the reporter enzymes is added and the resulting signal levels are then measured 15  Sandwich ELISA More sensitive than direct ELISA Requires two antibodies: 1) a capture antibody and 2) a detection antibody A sample containing antigens is added to plates that have well bottoms coated with capture antibody and a second antigen-specific reporter antibody is added and allowed to bind, thus “sandwiching” the antigen between two antibodies 16  Fluorescent-tagged antibodies Fluorescent-tagged antibodies are used in microscopy, immunofluorescence assays (IFAs), and in flow cytometry In florescence microscopy, florescent-tagged antibodies that recognize a specific antigen are incubated with tissue or cell samples, followed by observing the sample with a specialized microscope IFAs detect antigens or antibodies in a patient sample, but the detection antibody is linked to a fluorescent tag instead of an enzyme Flow cytometry can sort cells using a fluorescence-activated cell sorter (FACS) – Fluorescent-tagged antibodies are incubated with a patient blood sample. and the sample is loaded into the FACS machine, which counts tagged cells and sorts them from non-tagged cells 17  Interferon Gamma Release Assays Interferon gamma release assays (IGRA) provide a fast and relatively reliable way to detect TB in the early stages in vaccinated populations To perform the test, a patient’s blood is mixed with M. tuberculosis antigens and the level of INF-g released is measured T cells from a person who has TB (either a latent or an active infection) release more interferon gamma (INF-g) than T cells from someone who does not have TB The test can’t differentiate between an active versus latent TB infection 18  Western Blotting and Complement Fixation Western blotting is a protocol that detects specific proteins in a sample (including quantity of protein) – Western blot can detect the size of the protein (unlike ELISA) but is less sensitive than an ELISA – Protein is separated by gel electrophoresis, transferred to nitrocellulose membrane, and probed with antibodies Complement fixation assays detect patient antibodies in a serum Patient serum is mixed with antigen, complement proteins, sheep red blood cells, and an antibody to sheep red blood cells and hemolysis is observed 19  Polymerase Chain Reaction Polymerase chain reaction (PCR) is a very sensitive technique that allows for rapid replication of DNA, allowing for detection of small amounts of genetic material (i.e. a single viral particle) The starting material for PCR includes 1. Template DNA that contains the region that needs to be amplified 2. Oligonucleotide primers that are 15-20 synthetic nucleotides that are complementary to the ends of the target DNA 3. Deoxynucleoside triphosphates (dNTPs) that provide the precursors for DNA synthesis 4. Taq polymerase that is a heat-stable enzyme from the bacterium Thermus aquaticus that makes DNA PCR is performed in a thermocycler that cycles through: 1. Denaturation – Separation of double stranded DNA (95° C) 2. Annealing – Oligonucleotide primers bind to complementary nucleotides (50-65° C) 3. Extension – Taq polymerase synthesizes DNA (72° C) Other variations on PCR include real-time PCR (uses florescence imaging to monitor DNA as it is being made) and reverse transcription PCR (used to detect RNA within a sample) 20 Restriction Enzymes Restriction enzymes are made naturally by many species of bacteria as a protection from bacteriophages Hundreds are available commercially Restriction enzymes bind to specific DNA sequences (usually palindromic sequences) and cut the DNA into fragments with either “sticky ends” or “blunt ends” 21 Recombinant DNA Technologies Recombinant DNA (rDNA) refers to DNA that is generated or engineered by combining DNA from different organisms Recombinant DNA has many uses including drug development Steps in making rDNA: 1. Gene isolation and copying 2. Inserting gene into plasmid vector using restriction enzymes 3. Transforming the recombinant vector into host cells for gene expression 22  CRISPR-Cas9 Gene Editing https://www.cnn.com/videos/us/2015/10/30/orig-human-dna-genome-editing-crispr-cas9-wooly-mammoth-pioneers.cnn/vid eo/playlists/pioneers-orig / CRISPR-Cas9 is a gene-editing tool that is based on a prokaryotic immune response against bacteriophages 2 key components: 1. CRISPR - serves as a GPS for finding the desired genetic sequence that is to be cut out 2. Cas9 enzyme is the scalpel that cuts the DNA sequence once it is located Once cut, a vector containing the desired DNA can be inserted Various applications of the gene editing tool have been investigated but ethical concerns have arisen with human cell lines 23  Viral Vectors for Gene Delivery Gene therapy is the process of introducing genetic material into a human cell as a treatment Over 1,800 gene therapy clinical trials are currently in progress across 30 countries; most are aimed at treating cancer Engineered adenoviruses and retroviruses are the most common viral vectors used for gene delivery These viruses are genetically engineered to be nonpathogenic but infectious in order to deliver their genetic cargo to the host cell nucleus where it may or may not integrate into the host genome While gene delivery is efficient, the virus often elicits a host immune response Virus may also be used in conjunction with CRISP-Cas9 for gene editing 24  Genome Maps Requires documenting the position of every nucleotide along with the position of possible genes Many pathogens have had their genomes sequenced and mapped Genome maps can reveal how pathogens acquire virulence factors, the mechanism of pathogenesis, pathogenicity islands (regions in pathogen genomes that encode toxins, virulence factors, and resistance mechanisms), and genes needed for survival Information is useful for drug development and recombinant DNA technology 25  Gene Microarrays Useful tools for studying gene expression on a genome-wide scale Various uses including cancer genetics (microarrays help determine which genes may contribute to the development of cancers) and pathogen detection DNA probes (synthetic, single-stranded DNA oligonucleotides) are placed onto a DNA chip (a small piece of silicon glass) and allowed to bind with fluorescently- labeled cDNA extracted from various cell types (i.e. cancer cells and control cells) If cDNAs bind to a particular DNA sequence on the array, then a fluorescent signal will be detected with a specialized array reader (brighter “glow” = greater expression) – Cancer cell cDNA may be labeled green while the control is labeled red – Green glow = expression of gene is increased in cancer cell only – Red glow = expression of gene is increased in control cell only – Yellow glow = expression of gene is same in cancer and control cell  Q & A: Question 1 You receive a live, attenuated measles vaccine. The vaccine is an example of which of the following types of immunity? A) Natural, active B) Natural, passive C) Artificial, active D) Artificial, passive  Q & A: Question 1 Answer: C (Artificial, active)  Q & A: Question 2 Human Immunodeficiency Virus-1 (HIV-1) is an RNA virus with a long incubation period. Due to the long time needed for disease symptoms to develop, disease progression can alternatively be measured by detection of HIV genetic material in the bloodstream of infected patients. Which of the following molecular methods would be most appropriate for disease detection of HIV- 1 in patient’s bloodstream? A) Flow cytometry B) Reverse transcriptase polymerase chain reaction C) Complement fixation assay D) Western blotting  Q & A: Question 2 Answer: B (Reverse transcriptase polymerase chain reaction)  Q & A: Question 3 True or False, attenuated, live vaccines often require fewer boosters and doses than inactivated, whole agent vaccines. A)True B)False Q & A: Question 3 Answer: A (True)  Q & A: Question 4 Herd immunity describes: A) The process of immunizing animals so that people do not acquire zoonotic diseases B) The temporary immunity an infant receives from its mother during gestation and from breastfeeding C) The protection conferred to non-immunized people when a sizable portion of the rest of the population is immunized D) The process of spacing out vaccinations in infants to just one per doctor's visit  Q & A: Question 4 Answer: C (The protection conferred to non-immunized people when a sizable portion of the rest of the population is immunized)  Q & A: Question 5 Which of the following results would you expect from an interferon-gamma release assay from a patient suffering from an active tuberculosis infection? A) You would expect B lymphocytes to produce high titers of anti-tuberculosis IgM antibody IgD B) You would expect T lymphocytes to release high levels of interferon gamma C) You would expect B lymphocytes to release low levels of interferon gamma D) You would expect a large induration on the patient’s arm following injection of interferon gamma mixed with tuberculosis antigen  Q & A: Question 5 Answer: B (You would expect T lymphocytes to release high levels of interferon gamma )