Chapter 14: Vaccines, Diagnostics, Therapeutics, and Molecular Methods Flashcards
Chinese used variolation to combat smallbox 100 years ago…
powder made from dried scabs of smallpox; practitioner blew the powder into a healthy individuals nose; resulting small boz infections tended to be milder (only 1-2% mortality rates)
Edward Jenner
1796; milkmaids were not affected by small pox epidemics; most of them contracted cowpox; jenner suspected that a prior cowpox infection was protective against small poz; tested this by purposely inoculating a boy with cowpox pus, boy contracted it but recovered quickly he was then infected with small pox and did not show symptoms
Vaccination
derived from vacca the latin word for cow
Louis Pasteur developed…
late 1800s; early version of rabies vaccine to protect humans; vaccine to protect cattle against anthrax
How many different infections are currently vaccine preventable?
at least 25
The Lancet Paper
study of just 12 people; claimed a correlation between the measles, mumps, and rubella vaccine and the development of autism; resulted in many parents declining the MMR vaccine (as well as others); 2010 Lancet retracted the study and said it was bad science; additional studies have been done to prove it false
A drop in childhood vaccines has led to….
outbreaks of vaccine preventable disease; measles show the strongest re-emergence; resulted in states mandating vaccines
Herd Immunity
occurs when a sufficient percentage has been vaccinated (varies between pathogens); protects premature babies and immun-compromised patients; most ~85% vaccination but measles and whooping cough require ~95%
Live Attenuated Vaccines
contain altered pathogens that do not cause disease but are still infectious; developed multiple ways: cultivate pathogen in cell culture so it loses its pathogenicity (like flu strain in egg), genetic manipulation
Pros/Cons of Live Attenuated Vaccines
pro- stimulate potent immunological responses that are accompanied by long lived memory (bc it is a more natural imm response); cons- could cause disease in a immune compromised host, possible mutations to a infectious form, often need to be refrigerated
Inactivated Vaccines
include whole inactivated pathogens (the entire pathogen) or whole-agent and subunit vaccines ( a part)
Pros/Cons of Inactivated Vaccines
pros- safe for immune compromised patients and stable at room temp; cons- boosters required to achieve full immunity
Whole Agent Vaccines
contain the entire pathogen; pathogen is inactivated (not able to cause infection) by heat, chemicals or radiation
Subunit Vaccines
do not include whole pathogens; consist of purfied antigens (unable to cause disease) or parts of the infectious agent; require adjuvants; include purified subunit vaccines, toxoid vaccines, and conjugate vaccines
Adjuvants
pharmacological additives that enhance the bodys natural immune resposne to an antigen
Purified Subunit Vaccines
immunogenic portion of the pathogen; can be harvested from a natural pathogen or purified from a genetically engineered expression sys (recombinant subunit vaccines)
Toxoid Vaccines
purified and inactivated toxins; ex) tetanus and diptheria of DTap and Tdap
Conjugate (or polysaccharide) Vaccines
polysaccharide antigens cojugated to a more immunogenic protein antigen (bc immune sys no like recognizing sugars and respond better to proteins); ex) meningococcal vaccines, pneumococcal vaccines, and Hib vaccines
mRNA Vaccines
purified mRNA is encased in lipids chemically compatible with the cells plasma membrane; mRNA delivered to host cells; host cells translate the mRNA to build an antigenic protein that triggers an imm response; ex) moderna and pfizer vaccines for SARS-CoV-2
Recombinant Vector Vaccines
genetic material from the pathogen is packed inside a harmless virus or bacterium and inserted into the body; ex) johnson and john COVID vaccine
DNA Vaccines
genes encoding highly immunogenic antigens are identified; target genes are placed into a plasmid; plasmid is injected into a human host; human cells take up the plasmid and transcribe and translate the genes; cells become the antigen producers; results in a humoral (antibody) and a cellular immune response (T cells); focusing on HIV and cancer
Pros/COns of Biochemical Tests
biochemical tests detect pathways (like fermentation) and do not work with viruses; pros- useful for identifying bacteria that are responsible for an infection; cons- can take more than 24 hrs to perform, pathogen must be cultureable and cant identify noncellular pathogens
Antibodies
have 2 antigen binding sites; can attach to more than one antigen; can bind antigens into a clump (agglutination)
Agglutination Reactions can be seen when…
antibodies interact with cells that display multiple surface antigens or tiny synthetic beads coated with antigens
Treponema Pallidum Particle Agglutination Assay (TPPA) Test
tests for symphilis; detects patient antibodies against T. pallidum
Plaque Reduction Neutralization Test (PRNT)
patients serum is extracted and serially diluted; preparation of the suspected virus is added to the various tubes of diluted serum; each serum/virus mixture is added to petri plates of cultured cells and incubated
Negative Results of PRNT
no neutralizing antibodies present in the patient sample; patient sample and control culture exhibit same level of infection
Positive Results of PRNT
neutralizing antibodies present in the patient sample; antibodies bind to the added viruses and neutralizes them
Enzyme Linked Immunosorbent Assays
ELISAs; versatile and rapid diagnositc tests; rely on- antibody-antigen interactions, reporter enxyme attached to a monoclonal detecting antibody; chemically modifies an added substrate
Direct ELISA
allows for identification of antigens in a sample; solution possibly containing antigens is added to a microtiter plate wells; antigens stick to the bottom ofthe wells; detection antibodies are added and bind if the antigen is present; unbound antibodies are washed out of wells; substrate is added
Indirect ELISA
requires tow antibodies: first antibody (from patient) recognizes bound antigen and second antibody is enzme-linked
To Perform an Indirect ELISA
microtier plate is precoated with antigen; patients serum is added to plates; patient antibodies that recognize the antigens bind; enzyme linked detection antibody is added (binds to specific types of human antibodies); unbound detection antibodies are washed out; substrate is added; signal levels are measured
Performing the Sandwich ELISA
microtiter plate is coated with capture antibody; sample possibly containing antigen is added; antigens bind the capture antibody; detection antibody is added (sandwichs the antigen between two antibodies); unbound antibodies washed out; substrate is added and signal is measured
Immunofluorescence Microscopy
utilizes fluorescent tagged antibodies to recognize a specific antigen in a sample; requires a specialized fluorescent microscope
Immunofluorescence Assays (IFAs)
like ELISA, detects anitgens or antibodies in a patient sample; detection antibody is linked to a fluorescent tage instead of an enzyme; eliminates the need to add substrate for the detection step
Flow Cytometry
allows for enumeration of specific cells; requires fluorescence-activated cell sorter (FACS)
To Perform Flow Cytometry
fluorescent-tagged antibodies are incubated with a patient blood sample; unbound antibodies are removed; sample is loaded into the FACS machine; tagged cells are counted and sorted
Polymerase Chain Reaction (PCR)
sensitive enough to detect a single pathogen in a sample; creates billions of copies of a target gene in just a few hours; applications- facilitates gene sequencing for genetic disorders, diagnosing infections
Requirements to Perform PCR
thermocycler; reagents- template DNA to be copied, two single stranded DNA primers, Taq polymerase, deoxynucleotide triphosphates (dNTPs); a small tube with reagents mixed in the proper concentrations placed in the thermocycler
Thermocycler is programmed to cycle through a series of temperature changes…
melting step- high temp (95-99C) to separate double stranded DNA; annealing step- lower temp (50-65C) allows the primers to anneal with the template DNA; Extension step- optimal temp (65-75C) for the DNA polymersase to copy the target DNA
Real Time PCR
modified PCR that uses fluorescence imaging to visualize DNA copies as they are made; computer programs allow technicians to see the data immediately or in real time; sometimes called quantitative PCR (qPCR) (can measure how many copies of the target gene were initially present in the sample)
Reverse Transcription PCR
RT-PCR; useful for detecting RNA in a sample; requires reverse transcriptase (builds DNA that is complementary to target RNA molecules in a sample); ex) Covid 19 tests detects the genome of the virus
Genetic Information Nondiscrimination Act of 2008
GINA; attempt to protect pateints from discrimination based on genetic information; act is fairly weak; provides a number of loopholes for insureres and employers
Recombinant DNA Techniques
provide a way to insert a desired gene into an expression system; allows particular proteins to be produced in large amounts; DNA is generated or engineered by combining DNA from different organisms
Recombinant DNA Step 1:
Gene isolation and copying; building a DNA construct starts with isloating the gene that encodes the desired protein; PCR primers can be designed that flank the desired gene; PCR copies the desired gene through a series of thermocycles
Recombinant DNA Step 2:
inserting the desired gene into a plasmid; once the desired gene is copied, it is inserted into a cloning vector (cloning vectors are commercially available); desired gene copies and plasmid are cut with a restriction enzyme to generate sticky ends; resulting compatible sticky ends are joinged using DNA ligase; forms a completed recombinant vector
Recombinant DNA Step 3:
transforming the plasmid into cells for expression; newly made rDNA construct is inserted into host cells; prokaryotic cell lines are often used because they are usually cheap to culture and maintain; eukaryotic cells may be needed inc ertain cases due to certain protein modifications; host cells produce large quanities of the desired protein
CRISPR-Cas 9
gene editing tool; can locate a specific DNA sequence and cut it out with surgical precision; new DNA can be plugged into the cut site
CRISPR-Cas 9 Components
CRISPR- serves as a GPS for finding the desired genetic sequence that is to be cut out; Cas9 Enzyme- scalpel that cuts the DNA sequence once it is located
How CRISPR works…
guide RNA pairs with complementary target DNA; pairing signals the Cas9 enzyme to cut both strands of the DNA; generates a double strand break; new DNA can be added by delivering a desired sequence flanked by sequences in the area that have the double strand break; desired sequence is inserted
Gene Therapy
viruses used as gene delivery agents; adenoviruses and retrovirses are the most commonly used virsues; replacing mutated or missing genes with normal genes could help to control or even cure a disease
How Gene Therapy Works…
viruses are genetically engineered to be non pathogenic, but remain infectious; desired gene is then packaged into the virus; gene carrying virus is then introduced into the patient; virus enters a human cell; delivers the normal gene to host cell nucleus; normal gene is transcribed and translated
Pathogenicity Islands
regions of the pathogen genome that encode toxins, virulence factors and resistance mechanisms
Gene Microarrays
utilizes tools for investigating differences between healthy and diseased cells; utilizes complementary base pairing between nucleotides
