Microbiology: Basic and Clinical Principles Chapter 14 PDF

Microbiology: Basic and Clinical Principles Second Edition Chapter 14 Biomedical Applications: Vaccines, Diagnostics, Therapeutics, and...

Microbiology: Basic and Clinical Principles Second Edition Chapter 14 Biomedical Applications: Vaccines, Diagnostics, Therapeutics, and Molecular Methods Presented by Janet Dowding, Ph.D. St. Petersburg College Copyright © 2023 Pearson Education, Inc. All Rights Reserved Clinical Case Copyright © 2023 Pearson Education, Inc. All Rights Reserved A Brief History of Vaccines After reading this section, you should be able to: Review the early history of vaccines from variolation to Jenner’s advancements. Describe factors that contribute to the re-emergence of vaccine-preventable diseases. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (1 of 15) Hundreds of years ago Chinese used variolation to combat smallpox – Powder made from dried scabs of smallpox – Practitioner blew the powder into a healthy individual’s nose – Resulting smallpox infections tended to be milder (only 1–2% mortality rate) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (2 of 15) Edward Jenner (1796) – Milkmaids weren’t affected by smallpox epidemics – Most of them had contracted cowpox – Jenner suspected that a prior cowpox infection was protective against smallpox Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (3 of 15) Edward Jenner (1796) tested his hypothesis… – Purposely inoculated a boy with cowpox pus – Boy contracted cowpox, but quickly recovered – Jenner then infected him with smallpox, but he showed no symptoms Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (4 of 15) The word vaccination is derived from vacca, the Latin word for cow Smallpox vaccination was soon mandated for British soldiers and sought out by others Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (5 of 15) Louis Pasteur (late 1800s) developed: – Early version of the rabies vaccine to protect humans – Vaccine to protect cattle against anthrax As knowledge of pathogens increased, scientists developed more vaccines Currently, at least 25 different infections are vaccine preventable Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (6 of 15) Table 14.1 Vaccines Licensed for Use in the United States (1/6) Vaccine Administration Formulation Notes Adenovirus vaccine Oral administration; not Live attenuated Protects against routine for public adenoviruses 4 and 7 that cause colds Anthrax vaccine Intramuscular injection for Purified subunit Bacillus anthracis pre-exposure prophylaxis; bacterium; potential subcutaneous for post- biological weapon due to exposure prophylaxis; not the dangerous exotoxins it routine for public; adults only makes BCG (Bacille Intramuscular injection; not Live attenuated Mycobacterium Calmette–Guérin) routine for public tuberculosis; vaccine vaccine prevents severe forms of tuberculosis in children, but is variably effective in adults Cholera vaccine Oral; for people 18–64 years Live attenuated Protects against Vibrio old traveling to cholera- cholerae serotype O1 afflicted areas Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (7 of 15) Table 14.1 [continued] (2/6) Vaccine Administration Formulation Notes COVID-19 Injected; age for administration mRNA and vector Protect against COVID-19 disease vaccines* depends on vaccine brand formats available caused by SARS-Co-V-2 Dengue vaccine Subcutaneous injection; for use in Live attenuated Prevention of severe secondary children 9–16 years old who live in dengue disease endemic areas and who have had a laboratory-confirmed previous dengue Infection Diphtheria, Intramuscular injection; DTaP is Subunit/toxoid Protects against diphtheria caused tetanus, and routine pediatric vaccine; Tdap is combination by Corynebacterium diphtheria, acellular formulated with fewer diphtheria and tetanus caused by Clostridium pertussis (DTaP pertussis agents and is a booster tetani, and whooping cough and Tdap) vaccine caused by Bordetella pertussis vaccines Ebola Zaire Intramuscular injection; for use in Recombinant vector Prevents Ebola disease caused by vaccine people 18 years of age and older vaccine Zaire ebolavirus (first approved vector vaccine in U.S.) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (8 of 15) Table 14.1 [continued] (3/6) Vaccine Administration Formulation Notes Haemophilus B (Hib) Intramuscular injection; Conjugate Protects against Haemophilus vaccine routine pediatric vaccine influenzae type b bacteria Hepatitis A vaccine Intramuscular injection; Whole-agent Protects against hepatitis A virus routine pediatric vaccine inactivated Hepatitis B vaccine Intramuscular injection; Recombinant Protects against hepatitis B virus routine pediatric vaccine subunit Human papillomavirus Intramuscular injection; Recombinant Protects against the main strains of HPV (HPV) vaccines routine vaccine used in Subunit associated with genital warts and cancer pediatric and adult patients Influenza vaccines Intramuscular injection; Whole-agent Nasal mist made with live attenuated (many types routine vaccine used in inactivated and strains is no longer recommended in the available) pediatric and adult patients; purified subunit U.S. due to low efficacy annual vaccine available Japanese encephalitis Subcutaneous injection; Whole-agent Protects against Japanese encephalitis virus vaccine recommended for travelers to inactivated virus endemic areas Measles, mumps, and Subcutaneous injection; Live attenuated Protects against viruses that cause rubella (MMR) routine pediatric vaccine measles, mumps, and rubella vaccine Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (9 of 15) Table 14.1 [continued] (4/6) Vaccine Administration Formulation Notes Meningococcal Subcutaneous injection or intramuscular Recombinant Prevents meningococcal vaccine injection depending on the brand and subunit and disease caused by select manufacturer conjugate Neisseria meningitidis formulations serogroups available Pneumococcal Subcutaneous injection or intramuscular Conjugate Protects against the main vaccine (PCV) injection; PCV13 is routine pediatric serogroups of Streptococcus vaccine; PCV23 is a routine adult vaccine pneumoniae that cause and is also for pediatric patients over 2 pneumonia; PCV 13 protects years old who have an increased risk for against 13 serotypes; PCV23 pneumococcal disease protects against 23 serotypes Inactivated Intramuscular injection; routine pediatric Whole-agent Protects against poliovirus poliovirus vaccine inactivated vaccine (IPV) Rabies vaccine Intramuscular injection; only administered Whole-agent Pre-exposure and post- to at-risk groups inactivated exposure prophylaxis for rabies virus Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (10 of 15) Table 14.1 [continued] (5/6) Vaccine Administration Formulation Notes Rotavirus Oral; routine pediatric vaccine Live Protects against rotavirus gastroenteritis attenuated Smallpox Skin puncture with bifurcated needle Live Formulated using the vaccinia virus—a vaccine attenuated virus closely related to the cowpox virus; confers protection against variola virus (smallpox virus) Typhoid Oral for live attenuated or subcutaneous for Live Protects against typhoid fever caused vaccine conjugate formula; reserved for at-risk attenuated by the bacterium Salmonella typhi groups age 2 years and older and travelers and conjugate to endemic areas formulations available Varicella- Subcutaneous injection; routine pediatric Live Protects against chickenpox virus zoster virus vaccine attenuated vaccine Yellow Subcutaneous injection; reserved for at-risk Live Protects against mosquito-transmitted fever groups age 9 months and older and attenuated flavivirus vaccine travelers to endemic areas Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (11 of 15) Table 14.1 [continued] (6/6) Vaccine Administration Formulation Notes Zoster vaccine Subcutaneous Live attenuated Boosts immunity to injection; varicella zoster to reduce recommended for the risk of people herpes zoster (shingles) over age 50 Table based on 2021 Food and Drug Administration (FDA) recommendations *As of this text’s publication, some COVID-19 vaccines are being administered under FDA emergency use authorization. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (12 of 15) In addition to eradicating smallpox, global vaccination programs have saved millions of lives Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (13 of 15) It is estimated that between 2000 and 2030, routine vaccines against 10 pathogens (including measles, rotavirus, and hepatitis B) will prevent 69 million deaths in low- and middle-income countries Centuries of historical evidence and formal scientific research back the life-saving benefits of vaccines However, the practice has been a frequent source of controversy Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (14 of 15) In the 1800s: antivaccination societies formed in England to protest the growing use of vaccinations In early colonial India: many were offended that the smallpox vaccine was derived from cows (sacred to Hindus) In the 1900s: States enforced vaccination among citizens to prevent outbreaks of diseases – Spurred complaints over the loss of the individual right to choose Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccine History Includes Triumphs as Well as Controversies (15 of 15) In the 20th century: the Who helped bring vaccines to developing nations and served as an important partner in eradicating smallpox The next disease targeted for eradication is polio Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccination Fears Continue to Contribute to the Persistence or Re-Emergence of Preventable Diseases Fears about vaccination re-erupted in 1998 Paper published in The Lancet – Study of just 12 patients – Claimed a correlation between the measles, mumps, and rubella (MMR) vaccine and the development of autism – Shortly after publication, many parents in the United States and United Kingdom started to decline MMR and many other vaccinations for their children Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccination Fears and the Re-Emergence of Old Health Threats (1 of 4) In 2010 – The Lancet fully retracted the MMR/autism study as bad science – Study’s authors were funded by lawyers of parents with autistic children who were filing lawsuits against vaccine companies ▪ Major conflict of interest The main author has since lost his medical license amid charges of fraud and malpractice Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccination Fears and the Re-Emergence of Old Health Threats (2 of 4) Subsequent studies with 1,000s of participants found no link between vaccines and autism A 2015 study of 95,000 children showed no link between the MMR vaccine and autism Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccination Fears and the Re-Emergence of Old Health Threats (3 of 4) In both the United States and United Kingdom, a drop in childhood vaccination rates has led to outbreaks of vaccine-preventable diseases – Measles show the strongest re-emergence – CDC tracking data showed that the 2019 measles outbreak resulted in nearly 1,300 cases that spanned 31 states Copyright © 2023 Pearson Education, Inc. All Rights Reserved Vaccination Fears and the Re-Emergence of Old Health Threats (4 of 4) In response to increasing outbreaks, some areas changed school enrollment rules – No more opt out for personal reasons – California requires children receive at least 10 immunizations to attend school or daycare ▪ Only medical exemptions are allowed Copyright © 2023 Pearson Education, Inc. All Rights Reserved Overview of Vaccines After reading this section, you should be able to: Discuss the general immunology principles underlying vaccinations. Explain herd immunity and describe how it protects nonimmunized people. Describe the various types of vaccine formulations. State what adjuvants are and describe their purpose. Discuss how DNA vaccines and recombinant vector vaccines stimulate immunity. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (1 of 12) Immunity can be acquired by either natural or artificial means Vaccines are an important tools for stimulating artificially acquired active immunity To be effective, vaccines must stimulate immunological memory without causing the disease they aim to prevent. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (2 of 12) Vaccines do not provide immediate protection – It takes about two weeks for antibody levels to reach a peak Vaccines stimulate immunological memory – When the vaccinated individual encounters the real microbe at a later time, memory cells act quickly to prevent infection Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (3 of 12) Risks posed by routine vaccinations are extremely low for most people Some vaccines may not be recommended for certain patients (e.g., newborns, pregnant women or immune- compromised patients) Vaccinating a sufficient percentage leads to herd immunity for nonvaccinated individuals Protects premature babies and immune-compromised patients Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (4 of 12) Infected person The fewer disease-susceptible No immunity people in a community, the harder it is for a pathogen to be transmitted to a susceptible If no one is immunized... the disease spreads. host Immunized If just some are immunized... the disease still spreads. If most are immunized... the disease does not spread. Herd immunity protects those who cannot be immunized. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (5 of 12) Most pathogens require vaccination of ~85% of the population to have effective herd immunity Measles and whooping cough require ~ 95% vaccination rates for herd immunity Anyone who can be vaccinated should be vaccinated because herd immunity protects the most vulnerable people in the population Public health immunization initiatives aim to create herd immunity Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (6 of 12) In the United States, the CDC provides vaccination recommendations Routine childhood vaccines protect children against more than 15 different pathogens To stimulate optimal immunological memory, two or more subsequent boosters are often needed The doses are spaced apart to give the adaptive immune system time to respond and create more memory B and T cells against the vaccine agent Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (7 of 12) Some parents prefer to space out vaccinations – Can lead to a breakdown of herd immunity – Children who aren’t up to date with vaccines are not fully protected Keeping to the recommended vaccination schedule is most effective Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (8 of 12) Table 14.2 CDC’s Recommended Routine Pediatric Immunization Schedule for 2021 Vaccines Birth 1 2 mo s nth 4 6 9 12 15 mo s nth 18 19–23 4–6 11–12 16 mo nth mo s nth mo s nth mo s nth mo s nth mo s nth mo s nth y rs ea y rs ea y rs ea Blank Blank Blank Blank Blank Hepatitis B 1st 2nd 2nd 3rd 3rd 3rd 3rd dose 3rd dose dose dose dose dose dose dose Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Rotavirus* 1st 2nd dose dose Blank Blank Blank Blank Blank Blank Blank Diphtheria, 1st 2nd 3rd 4th dose 4th 5th tetanus, dose dose dose dose dose and pertussis (DTaP)** Blank Blank Blank Blank Blank Blank Blank Blank Blank Haemophil 1st 2nd 3rd 3rd dose us dose dose dose influenzae type b (H i b) caret (Hib ) ^ Blank Blank Blank Blank Blank Blank Blank Blank Pneumoco 1st 2nd 3rd 4th 4th dose ccal dose dose dose dose (PCV13) Blank Blank Blank Blank Blank Polio (IPV) 1st 2nd 3rd 3rd 3rd 3rd dose 3rd 4th dose dose dose dose dose dose dose Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (9 of 12) Table 14.2 [continued] Vaccine Birth 1 2 4 6 mo s nth 9 mo s nth 12 mo s nth 15 mo s nth 18 mo s nth 19–23 4–6 y rs ea 11–12 16 s mo nth mo nth mo nt mo s nth y rs ea y rs ea s s h Blank Blank Blank Blank Influenz Vaccine Vaccine Vaccine Vaccine Vaccine Vaccine Vaccine Vaccine Vacci a recomm recomm recomm recomme recomm recomm recomm recomme ne ended ended ended nded ended ended ended nded reco annually annually annually annually annually annually annually annually mme starting starting starting starting starting starting starting starting nded at 6 at 6 at 6 at 6 at 6 at 6 at 6 at 6 annu months months months months months months months months ally starti ng at 6 mont hs Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Measles, 1st dose 1st dose 2nd dose mumps, and rubella (MMR) Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Varicella 1st dose 1st dose 2nd dose (VAR) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (10 of 12) Table 14.2 [continued] Vaccines Birt 1 2 4 6 9 12 mo s nth 15 mo s nth 18 mo s nth 19–23 4–6 11–12 16 y rs ea h mo nth mo s nth mo s nth mo s nth mo s nth mo s nth y rs ea y rs ea Blank Blank Blank Blank Blank Blank Blank Blank Blank Hepatitis A 2 doses 2 doses 2 doses 2 doses 6 to 18 6 to 18 6 to 18 6 to 18 months months months months apart apart apart apart Meningococcal § Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Meningococcal, section sign. 1st 1st dose dose Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Diphtheria, Single tetanus, and dose pertussis booster (Tdap)** Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Human 2 doses papillomavirus (HPV ) ^^ (H P V) double caret Immunization doses are often given in a range of time. The ranges here represent the recommended time frames assuming the patient is on time for their vaccine dosing and does not have underlying conditions that require special adjustments. * Rotarix version shown; RotaTeq version requires a third dose at 6 months. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (11 of 12) ** DTaP is a combined vaccine against diphtheria, tetanus, and pertussis. Tdap protects against the same pathogens as the DTaP, but is formulated differently and is recommended as a single booster at age 11 (or older if not received at age 11) and in the third trimester of each pregnancy. ^ PedvaxHIB version; ActHIB, MenHibrix, Hiberix, or Pentacel consists of 4 doses given at ages 2, 4, 6, and 12–15 months. § Menactra / Menveo vaccine schedule shown; other versions may be recommended for certain high-risk groups and have a different dosing schedule. ^^11– 12 - year - olds get 2 doses that are at least 6 months apart; the 3-dose series is recommended for people with a weakened immune system and patients age 15 or older. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunization Programs Aim to Create Herd Immunity (12 of 12) The need for vaccines continues into late adolescence and adulthood – Bacterial meningitis vaccine is given to people 16 through 23 years of age – Pregnant women should get Tdap in the third trimester of each pregnancy – Everyone >6 months old should get an annual “flu shot” – Senior citizens should receive vaccines to prevent bacterial pneumonia and shingles Copyright © 2023 Pearson Education, Inc. All Rights Reserved  Vaccines Formulations Are Varied Vaccines can be injected, inhaled, or ingested and they come in diverse formulations Vaccines are categorized by how they are made Vaccine Categories Active Inactivated Attenuated Vector Whole-agent Parts of a pathogen vaccines vaccines vaccines Active virus or Genetically modified carrier Entire dead live bacterium rendered virus (vector) delivers genetic cellular pathogen or Subunit vaccines mRNA nonpathogenic information from the pathogen whole inactivated virus Portions of pathogen in vaccine vaccines Varicella-zoster vaccine Ebola vaccine (Erbevo) Polio vaccine currently mRNA that directs the used in U.S. (IPV) Purified subunit production of a specific Measles, mumps, and Certain COVID-19 vaccines, (natural and recombinant) protein found in or on rubella (MMR) vaccine including Johnson & Johnson/ Hepatitis A vaccine Hepatitis B vaccine the pathogen Rotavirus vaccine Janssen Certain influenza vaccines Certain influenza vaccines Certain COVID-19 Oral polio vaccine (OPV); vaccines, including not currently used in U.S. Toxoid (inactivated protein toxin) Moderna and Pfizer Diphtheria and tetanus components of DTaP and Tdap vaccines Conjugate (linked polysaccharides) Pneumococcal vaccine Haemophilus B (Hib) vaccine Copyright © 2023 Pearson Education, Inc. All Rights Reserved Live Attenuated Vaccines (1 of 2) Live attenuated vaccines – Contain altered pathogens that do not cause disease but are still infectious – Can be developed in a number of ways ▪ Cultivate the pathogen in cell culture so it loses its pathogenicity ▪ Genetic manipulation Copyright © 2023 Pearson Education, Inc. All Rights Reserved Live Attenuated Vaccines (2 of 2) Benefit – Simulate potent immunological responses that are accompanied by long-lived memory Drawbacks – Could cause disease in an immune-compromised host – Possible mutation to an infectious form – Often must be refrigerated Copyright © 2023 Pearson Education, Inc. All Rights Reserved Inactivated Vaccines: Whole-Agent and Subunit (1 of 5) Inactivated vaccines – Consists of whole inactivated pathogens – Includes whole-agent and subunit vaccines Benefits – Safe for immune-compromised patients – Stable at room temperature Drawbacks – Boosters required to achieve full immunity Copyright © 2023 Pearson Education, Inc. All Rights Reserved Inactivated Vaccines: Whole-Agent and Subunit (2 of 5) Whole-agent vaccines – Contain the entire pathogen – Inactivated by heat, chemicals, or radiation Copyright © 2023 Pearson Education, Inc. All Rights Reserved Inactivated Vaccines: Whole-Agent and Subunit (3 of 5) Subunit vaccines – Do not include whole pathogens – Consist of purified antigens or parts of the infectious agent – Require adjuvants ▪ pharmacological additives that enhance the body’s natural immune response to an antigen – Include purified subunit vaccines, toxoid vaccines, and conjugate vaccines Copyright © 2023 Pearson Education, Inc. All Rights Reserved Inactivated Vaccines: Whole-Agent and Subunit (4 of 5) Purified subunit vaccines – Immunogenic portion of the pathogen – Can be harvested from a natural pathogen or purified from a genetically engineered expression system (i.e., recombinant subunit vaccines) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Inactivated Vaccines: Whole-Agent and Subunit (5 of 5) Toxoid vaccines – Purified and inactivated toxins – Examples: tetanus and diphtheria of DTap and Tdap Conjugate (or polysaccharide) vaccines – Polysaccharide antigens conjugated to a more immunogenic protein antigen – Examples: meningococcal vaccines, pneumococcal vaccines, and Hib vaccines Copyright © 2023 Pearson Education, Inc. All Rights Reserved Inactivated Vaccines: mRNA Vaccines Purified mRNA is encased in lipids chemically compatible with the cell plasma membrane mRNA delivered to host cells Host cells translate the mRNA to build an antigenic protein that triggers an immune response – Examples: Moderna and Pfizer vaccines mRNA encodes a SARS-CoV-2 spike protein Copyright © 2023 Pearson Education, Inc. All Rights Reserved Recombinant Vector Vaccines Genetic material from the pathogen is packed inside a harmless virus or bacterium and inserted into the body Example: Johnson and Johnson’s Janssen COVID-19 vaccine uses adenovirus type 26 (Ad26) to deliver DNA that encodes a SARS-CoV- 2 spike protein When a person is vaccinated, Antigen target host cells are “infected” with the harmless vector virus and Gene for antigen will make the desired protein. This induces an immune response against the desired antigen. Pathogenic virus Recombinant Vaccine vector virus The virus cannot replicate and Vector virus therefore cannot cause disease or spread to other cells. Copyright © 2023 Pearson Education, Inc. All Rights Reserved New Vaccines Are in Development for Persistent and Emerging Diseases 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 and a cellular immune response ▪ Focusing on HIV or cancer Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunological Diagnostic Testing (1 of 2) After reading this section, you should be able to: Describe the advantages of immunological diagnostics over biochemical testing. Describe agglutination reactions and explain how they can be used to determine blood type. Describe how plaque reduction neutralization tests work and how they are clinically useful. Compare and contrast direct, indirect, and sandwich ELISA techniques. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunological Diagnostic Testing (2 of 2) After reading this section, you should be able to: Explain how fluorescent-tagged antibodies may be used in diagnostic testing. Discuss how interferon gamma release assays work and why they are an important development in the fight against tuberculosis. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunological Diagnostic Tests Often Rely on Antigen–Antibody Interactions (1 of 2) Benefit of biochemical tests – Useful for identifying bacteria that are responsible for an infection Drawbacks of biochemical tests – Can take more than 24 hours to perform – Pathogen must be culturable – Can’t identify noncellular pathogens Copyright © 2023 Pearson Education, Inc. All Rights Reserved Immunological Diagnostic Tests Often Rely on Antigen–Antibody Interactions (2 of 2) Immunological diagnostics are essential tools for identifying a variety of pathogens Immunological tests typically involve serology Often the goal is to determine if a patient has certain antigens and/or antibodies in their blood Copyright © 2023 Pearson Education, Inc. All Rights Reserved Agglutination Tests Reveal Antigen– Antibody Interactions (1 of 4) 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 – Tiny synthetic beads coated with antigens Copyright © 2023 Pearson Education, Inc. All Rights Reserved Agglutination Tests Reveal Antigen– Antibody Interactions (2 of 4) Agglutination reactions are usually used for: – Blood typing – Identify infections – Diagnose noninfectious immune disorders Testing can identify either antibody or antigen in patient samples (e.g., serum, urine, CSF) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Agglutination Tests Reveal Antigen– Antibody Interactions (3 of 4) Treponema pallidum particle agglutination assay (TPPA) test – Tests for syphilis – Detects patient antibodies against T. pallidum Negative result Positive result (no agglutination) (agglutination) Antibody Bead Antigen attached to bead Copyright © 2023 Pearson Education, Inc. All Rights Reserved Agglutination Tests Reveal Antigen– Antibody Interactions (4 of 4) Blood typing Agglutination occurs Surface Anti-B antigen B antibodies B or AB type blood Anti-B antibodies added No agglutination Surface Anti-A antigen B antibodies A or AB type Anti-A antibodies added blood Copyright © 2023 Pearson Education, Inc. All Rights Reserved Neutralization Reactions Are Useful for Detecting Immunity to Certain Viruses (1 of 2) Plaque reduction neutralization test (PRNT) – Patient’s 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Neutralization Reactions Are Useful for Detecting Immunity to Certain Viruses (2 of 2) Negative results: – No neutralizing antibodies present in patient sample – Patient sample and control culture exhibit same level of infection Positive results: – Neutralizing antibodies present in the patient sample – Antibodies bind to the added viruses and neutralize them Copyright © 2023 Pearson Education, Inc. All Rights Reserved Figure 14.8 Plaque Reduction Neutralization Test (PRNT) Virus Draw patient’s blood and isolate serum. Virus is added to a series of serum dilutions (one tube shown for simplicity). If antibodies against the virus are present, they coat the virus and limit its infectivity. Virus/serum mixture is added to cultured cells and incubated. Antibody-coated virus cannot infect cells. Observe incubated plate. Abundant plaques mean Reduced number of plaques patient lacks antibodies means patient has antibodies to virus. to virus. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Enzyme-linked Immunosorbent Assays (ELISAs) (1 of 2) Enzyme-linked immunosorbent assays (ELISAs) – Versatile and rapid diagnostic tests – Rely on: ▪ Antibody–antigen interactions ▪ Reporter enzyme attached to a monoclonal detecting antibody – Chemically modifies an added substrate Copyright © 2023 Pearson Education, Inc. All Rights Reserved Enzyme-linked Immunosorbent Assays (ELISAs) (2 of 2) Main formats for ELISA include: – Direct – Indirect – Sandwich Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct ELISA (1 of 3) Direct ELISA – Allows for identification of antigens in a sample – Solution possibly containing antigens is added to microtiter plate wells ▪ Antigens stick to the bottom of the wells – Detection antibodies are added and bind if the antigen is present – Unbound antibodies are washed out of wells – Substrate is added Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct ELISA (2 of 3) Direct ELISA – Substrate + antibody-linked reporter enzyme gives a colorimetric or chemiluminescent signal – Microtiter plate is inserted into a plate reader to measure the signal in each well Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct ELISA (3 of 3) Direct ELISA Enzyme-linked Signal develops when detection substrate interacts antibody with reporter enzyme. Antigen from test sample Signal analyzed by a plate Antigens from Add detection Excess detection reader sample are adhered antibody. antibody is rinsed away, to bottom of wells. and then substrate is added. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Indirect ELISA (1 of 3) Indirect ELISA – Requires two antibodies ▪ First antibody (from patient) recognizes bound antigen ▪ Second antibody is enzyme-linked Copyright © 2023 Pearson Education, Inc. All Rights Reserved Indirect ELISA (2 of 3) To perform the test: – Microtiter plate is precoated with antigen – Patient’s 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Indirect ELISA (3 of 3) Indirect ELISA Enzyme-linked Signal analyzed Patient detection by a plate reader antibodies antibody bind Antigen Plate comes with Patient serum Excess patient antibody Excess detection antibody antigen bound to added; patient is rinsed away before is rinsed away, and then bottom of wells. antibodies that adding detection substrate is added. recognize the antibody. antigen will bind to it. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Sandwich ELISA (1 of 2) To perform the test: – Microtiter plate is coated with capture antibody – Sample possibly containing antigen is added – Antigens bind the capture antibody – Detection antibody is added ▪ “Sandwiches” the antigen between two antibodies – Unbound antibodies are washed out – Substrate is added and signal is measured Copyright © 2023 Pearson Education, Inc. All Rights Reserved Sandwich ELISA (2 of 2) Sandwich ELISA – Requires two antibodies: ▪ Capture antibody and detection antibody Sandwich ELISA Enzyme-linked Signal analyzed Target detection by a plate reader antigen antibody Capture antibody Plate comes with Patient serum Captured antigen is Excess detection antibody capture antibody added; antigens that “sandwiched” between is rinsed away, and then bound to bottom can bind to the antibodies when detection substrate is added. of wells. capture antibody are antibody is added. retained in the wells after wells are rinsed. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Fluorescent-tagged Antibodies Can Detect Antigens or Antibodies in a Sample (1 of 4) Immunofluorescence microscopy – Utilizes fluorescent-tagged antibodies to recognize a specific antigen in a sample – Requires a specialized fluorescent microscope Copyright © 2023 Pearson Education, Inc. All Rights Reserved Fluorescent-tagged Antibodies Can Detect Antigens or Antibodies in a Sample (2 of 4) Immunofluorescence assays (IFAs) – Like ELISA, detects antigens or antibodies in a patient sample – Detection antibody is linked to a fluorescent tag instead of an enzyme – Eliminates the need to add substrate for the detection step Copyright © 2023 Pearson Education, Inc. All Rights Reserved Fluorescent-tagged Antibodies Can Detect Antigens or Antibodies in a Sample (3 of 4) Flow cytometry – Allows for enumeration of specific cells – Requires fluorescence-activated cell sorter (FACS) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Fluorescent-tagged Antibodies Can Detect Antigens or Antibodies in a Sample (4 of 4) 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Interferon Gamma Release Assays (IGRAs) Detect Tuberculosis Infections (1 of 2) Interferon gamma release assays (IGRAs) – Fast and reliable way to detect TB in the early stages in vaccinated populations – Measure patient’s T cells respond to Mycobacterium tuberculosis antigens – T cells from a person who has TB release more interferon gamma (INF - g ) than those from someone who does not have TB Copyright © 2023 Pearson Education, Inc. All Rights Reserved Interferon Gamma Release Assays (IGRAs) Detect Tuberculosis Infections (2 of 2) To perform the test: – Patient’s blood is mixed with M. tuberculosis antigens – Level of INF-g released is measured Copyright © 2023 Pearson Education, Inc. All Rights Reserved Selected Genetics Applications in Medicine (1 of 2) After reading this section, you should be able to: Describe the polymerase chain reaction (PCR), real-time PCR, and reverse transcription PCR(RT-PCR) and state the clinical applications of each. Describe the three general steps for producing a recombinant DNA(rDNA) vector, state how rDNA can be introduced into cells, and discuss the clinical applications of rDNA. Discuss the features and functions of restriction enzymes. Explain how the CRISPR-Cas9 protein system edits genetic material. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Selected Genetics Applications in Medicine (2 of 2) After reading this section, you should be able to: Describe the general process of gene therapy and state how viruses are used in this process. Explain what genome maps are and state why they are useful. Describe DNA microarrays and explain how they are applied to clinical diagnostics. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Polymerase Chain Reaction (PCR) Can Help Diagnose Infections and Genetic Disorders 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved PCR Technique (1 of 4) Requirements to perform PCR: – Thermocycler – Reagents: ▪ Template DNA to be copied ▪ Two single-stranded DN A primers ▪ Taq polymerase ▪ Deoxynucleotide triphosphates (dNTPs) A small tube with reagents mixed in the proper concentrations is placed in the thermocycler Copyright © 2023 Pearson Education, Inc. All Rights Reserved Figure 14.13 The Polymerase Chain Reaction Taq polymerase DNA sequence of interest Nucleotides Primers Cycle 1: Strands are separated and copied Yield: 2 copies Cycle 2: 4 copies Cycle 3: 8 copies Cycle 4: 16 copies Copyright © 2023 Pearson Education, Inc. All Rights Reserved PCR Technique (2 of 4) Thermocycler is programmed to cycle through a series of temperature changes: – Melting step: high temperature (95 - 99°C) to separate double-stranded DNA – Annealing step: lower temperature (usually between 50 - 65°C) allows the primers to anneal with the template DNA – Extension step: optimal temperature (usually between 65 - 75°C) for the DNA polymerase to copy the target DNA Copyright © 2023 Pearson Education, Inc. All Rights Reserved PCR Technique (3 of 4) During each series of heating and cooling cycles, new copies of the gene are made Each new copy becomes template for the next round of copies Number of copies of a desired gene doubles with each cycle Exhibits an exponential increase of 2n – n is the number of cycles Copyright © 2023 Pearson Education, Inc. All Rights Reserved PCR Technique (4 of 4) Amplified DNA can be analyzed using gel electrophoresis In gel electrophoresis, molecules are separated based on their size However, electrophoresis can take several hours Copyright © 2023 Pearson Education, Inc. All Rights Reserved Real-time PCR 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Reverse Transcription PCR (RT-PCR) 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 – Example: SARS-CoV-2 test detects the genome of the virus Copyright © 2023 Pearson Education, Inc. All Rights Reserved PCR for Genetic Testing (1 of 2) Screening for genetic disorders began in the early 1960s The sequencing of the human genome was completed in 2003 and advanced our understanding of genetics-based diseases Clinicians commonly order DNA-based tests – >2,000 genetics-based tests are available – Many are PCR-based Copyright © 2023 Pearson Education, Inc. All Rights Reserved PCR for Genetic Testing (2 of 2) Ethical considerations in genetics testing – Concerns have arisen regarding protection of patients’ genetic information Genetic Information Nondiscrimination Act of 2008 (GINA) – Attempt to protect patients from discrimination based on genetic information – Act is fairly weak ▪ Provides a number of loopholes for insurers and employers Copyright © 2023 Pearson Education, Inc. All Rights Reserved Drug Development Often Relies on Recombinant DNA Techniques Recombinant DNA techniques – Provide a way to insert a desired gene into an expression system – Allows particular proteins to be produced in large amounts – Recombinant DNA(rDNA) ▪ DNA is generated or engineered by combining DNA from different organisms Copyright © 2023 Pearson Education, Inc. All Rights Reserved Recombinant DNA Step 1: Gene Isolation and Copying Building a recombinant DNA construct starts with isolating 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Recombinant DNA Step 2: Inserting the Desired Gene Into a Plasmid (1 of 2) 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 joined using DNA ligase Forms a completed recombinant vector Copyright © 2023 Pearson Education, Inc. All Rights Reserved Recombinant DNA Step 2: Inserting the Desired Gene Into a Plasmid (2 of 2) Table 14.3 Common Restriction Enzymes Used in Molecular Cloning Restriction enzyme and DNA recognition sequence and cut point source EcoRI Eco R One Escherichia coli (generates sticky The figure illustrates the D N A recognition sequence and the cut point of Eco R 1. Eco R 1 from Escherichia coli (generates sticky ends) The recognition sequence is G A A T T C in 5 prime to 3 prime direction. The recognition sequence is palindromic. The cut point is between G and A on both the D N A strands. This generates 5 prime sticky ends on both the D N A strands. ends) BamHI Bam H One Bacillus The figure illustrates the D N A recognition sequence and the cut point of Bam H 1. Bam H 1 from Bacillus amyloliquifasciens The recognition sequence is G G A T C C in 5 prime to 3 prime direction. The recognition sequence is palindromic. The cut point is between G and G on both the D N A strands. This generates 5 prime sticky ends on both the D N A strands. amyloliquifasciens HindIII Hin d Three Haemophilus The figure illustrates the D N A recognition sequence and the cut point of Hind 3. Hind 3 from Haemophilus influenza The recognition sequence is A A G C T T in 5 prime to 3 prime direction. The recognition sequence is palindromic. The cut point is between A and A on both the D N A strands. This generates 5 prime sticky ends on both the D N A strands. influenza SmaI S m a One Serratia The figure illustrates the D N A recognition sequence and the cut point of Sma 1. Sma 1 from Serratia marcescens The recognition sequence is C C C G G G in 5 prime to 3 prime direction. The recognition sequence is palindromic. The cut point is between C and G on both the D N A strands. Since it cuts the D N A strands in the middle, it generates blunt ends with no overhangs of single-stranded D N A. marcescens Copyright © 2023 Pearson Education, Inc. All Rights Reserved 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 in certain cases due to certain protein modifications Host cells produce large quantities of the desired protein Copyright © 2023 Pearson Education, Inc. All Rights Reserved Figure 14.14 Recombinant DNA (rDNA) Copy viral surface protein gene using PCR. Gene for viral surface protein Treat the gene copies and the plasmid with restriction enzymes to generate sticky ends. Restriction enzyme cut sites Sticky ends of the gene segment and plasmid join by Purified plasmid treated complementary base pairing. with restriction enzymes Ligase binds them together. Plasmid Insert the plasmid into bacterial cells. The bacteria then express the gene to make viral proteins that can be purified and used in a vaccine. Recombinant vector Viral protein Vaccine Copyright © 2023 Pearson Education, Inc. All Rights Reserved CRISPR Can Edit Any Genetic Material (1 of 5) CRISPR-Cas9 – 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved CRISPR Can Edit Any Genetic Material (2 of 5) CRISPR-Cas9 components: – CRISPR ▪ Guides the Cas9 enzyme to the specific target sequence – Cas9 enzyme ▪ Scalpel that cuts the DNA sequence once it is located Copyright © 2023 Pearson Education, Inc. All Rights Reserved  CRISPR Can Edit Any Genetic Material (3 of 5) A guide RNA (the CRISPR part of the CRISPR-Cas9 system) A vector construct containing the correct version of pairs with the target DNA sequence. The Cas9 enzyme then the gene is introduced into the cells and is inserted cuts both strands of the target DNA at a specific location with into the cut DNA to repair the gene. respect to the guide RNA. Lung cell of cystic Cas9 enzyme Healthy fibrosis patient Guide Cut points in Desired gene from lung cell RNA both strands of donor construct the target DNA Mutation responsible for disease Target DNA sequence Repaired, corrected gene Corrected gene CRISPR-Cas9 system Copyright © 2023 Pearson Education, Inc. All Rights Reserved CRISPR Can Edit Any Genetic Material (4 of 5) How it 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved CRISPR Can Edit Any Genetic Material (5 of 5) The CRISPR-Cas9 system has been used in all types of cells (e.g., prokaryotic and eukaryotic) Even human cells can be manipulated with this system Possibilities for gene editing are endless Ethical concerns have spurred caution in the use of this system, particularly with human cell lines Copyright © 2023 Pearson Education, Inc. All Rights Reserved Viruses Can Deliver Genes to Human Cells (1 of 4) Replacing mutated or missing genes with normal genes could help to control or even cure a disease Gene therapy – Viruses used as gene delivery agents – Adenoviruses and retroviruses are the most commonly used viruses Copyright © 2023 Pearson Education, Inc. All Rights Reserved Viruses Can Deliver Genes to Human Cells (2 of 4) How it 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 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Viruses Can Deliver Genes to Human Cells (3 of 4) Harmless vector virus engineered to contain a human gene enters cell. Cell’s The virus delivers defective the gene to the copy of gene nucleus. Human cell The gene is expressed, producing the needed protein. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Viruses Can Deliver Genes to Human Cells (4 of 4) Over 1,800 gene therapy clinical trials are currently in progress across 30 countries – Most are aimed at treating cancer Copyright © 2023 Pearson Education, Inc. All Rights Reserved Genome Maps Reveal Valuable Information (1 of 2) Mapping an Base pairs 3,293-4,153 Base pair 86 (860 bp long) organism’s genome Gene for ampicillin requires documenting antibiotic resistance Base pairs 86-1,276 (1,190 bp long) the position of every Gene for tetracycline antibiotic resistance nucleotide Base pair 3,293 Name: pBR322 Length: 4,361 base pairs Many pathogens Base pairs 2,534-3,122 have had their (588 bp long) Origin of replication genomes sequenced Base pair 2,534 Base pair 1,915 and mapped Base pairs 1,915-2,106 (191 bp long) Gene that regulates how many copies of the plasmid will exist in the bacterial cell Copyright © 2023 Pearson Education, Inc. All Rights Reserved Genome Maps Reveal Valuable Information (2 of 2) Genome maps can reveal how microbes cause disease Pathogenicity islands – Regions of the pathogen genome that encode toxins, virulence factors, and resistance mechanisms Gene maps also show genes needed for normal cellular functions – Possible drug targets Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gene Microarray Technology Provides A Global View of Cellular Functions (1 of 2) Gene microarrays – Useful tools for investigating differences between healthy and diseased cells – Utilizes complementary base-pairing between nucleotides Copyright © 2023 Pearson Education, Inc. All Rights Reserved Gene Microarray Technology Provides A Global View of Cellular Functions (2 of 2) Tissue sample Lyse cells from tumor biopsy mRNA molecules cDNA is made by reverse transcriptase; all cDNA is fluorescently tagged. Labeled cDNA molecules The cDNA is applied to the microarray; cDNAs in the sample pair with complementary sequences on the array. DNA microarray Unbound cDNA is rinsed away; location and intensity of fluorescence for each spot on the array is analyzed to reveal which genes are expressed and their degree of expression. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Visual Summary: Biomedical Applications: Vaccines, Diagnostics, Therapeutics, and Molecular Methods Herd Immunity Agglutination- and Neutralization- Herd immunity protects those who cannot be vaccinated. If the vast majority are vaccinated, Based Diagnostics infectious disease will not spread through the Agglutination reactions: Neutralization reactions: population. Used in blood typing, to identify infections, andTests like the plaque reduction Immunized to diagnose noninfectious immune disorders. neutralization test detect patient Infected and antibodies against a specific virus. infectious Bead IgM Antibody-coated virus antibody Antigen cannot infect cells. attached to bead Vaccine Types Enzyme-Linked Immunosorbent Assays Attenuated Vaccines: Live (ELISAs) Active Agent weakened pathogen Sensitive rapid diagnostic tests that detect antigens or antibodies Vector in a sample; rely on a reporter enzyme linked to an antibody. Vaccines: Genetically modified Direct ELISA Indirect ELISA Sandwich ELISA carrier virus (vector) delivers genetic information from the pathogen Whole-Agent Vaccines: Inactivated pathogen Subunit Vaccines: Portion of pathogen is used to stimulate an Genetics Techniques for Diagnostics and immune response Purified Subunit Vaccines Therapeutics (Natural and Recombinant DNA techniques: CRISPR-Cas9 editing system: Recombinant) An expression system is engineered to make A gene-editing tool that locates a specific DNA sequence Inactivated Agent Includes natural or engineered parts of large quantities of a desired protein. and cuts it out so that a new sequence can be inserted. the pathogen Desired gene is isolate amplified by PCR, and cut Toxic Vaccines with restriction enzymes to generate sticky ends. Gene therapy: Inactivated protein toxin The process of using viruses to introduce genetic Conjugate (or Polysaccharide) Restriction enzyme cut sites material into human cells to treat disease. Vaccines Polysaccharides are conjugated or linked The virus to a component that The gene is delivers the enhances immunogenicity expressed, gene to the mRNA Vaccines Plasmid that gene will nucleus. be ligated into is also producing mRNA that directs the the needed Cell’s production of a specific protein cut with restriction defective found in or on the pathogen enzymes. protein. copy of Finalized construct put gene Polymerase chain reaction (PCR): into an expression system Copies genes for genetic engineering and Gene microarray (usually bacterial cells). to detect DNA in a sample; reverse transcription technology: PCR (RT-PCR) detects RNA in a sample; Provides a global view of cellular functions; useful both PCR and RT-PCR can be performed for detecting pathogens using real-time visualization methods. and for informing clinical Recombinant vector decisions for cancer therapies. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (1 of 4) Summary of the case: – Mariam, a school nurse, was examining 5-year-old Sam – He complained of a bad cough and felt extremely tired – During the examination he erupted into a coughing fit with loud wheezing sounds between coughs – Mariam suspected whooping cough – She called Sam’s parents and they arrived at the school – During consultation Sam’s mother said: ▪ He had a runny nose and felt warmer than usual a week earlier ▪ Two nights before, he broke into a coughing fit and then vomited, but he appeared to be fine the next morning, except for the cough Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (2 of 4) Summary of the case: – Mariam told the parents she suspected he had whooping cough caused by Bordetella pertussis in the lungs – Whooping cough is highly contagious, prolonged, severe, and preventable by a vaccine – The parents were unconvinced; they believed it was the flu – Mariam urged them to consult a doctor; they agreed and left – Afterward, Mariam checked his medical records and noticed he did not have any vaccinations due to a medical exemption – Mariam went to the principal’s office to discuss Sam’s condition and steps to be taken to warn parents if it was whooping cough Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (3 of 4) 1. Should Mariam be concerned for the other children who are in class with Sam? Why or why not? 2. Was Mariam being overly worried by telling the principal she suspected Sam had whooping cough? 3. If you were Sam’s pediatrician, what type of diagnostic tool would you most likely use in the clinic to quickly identify whether or not he has whooping cough? 4. If Sam does indeed have whooping cough, should the entire school body get vaccinated, or would another treatment be preferable? Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (4 of 4) 5. The parents seem convinced that their child contracted his respiratory infection, whatever it may be, at school. If the nurse is correct that Sam has whooping cough, would the school be a likely place for him to have contracted it? Be sure to explain. 6. If another child was exposed to Sam and did not have the pertussis vaccination for whooping cough, should the child be vaccinated immediately? Copyright © 2023 Pearson Education, Inc. All Rights Reserved Copyright This work is protected by United States copyright laws and is provided solely for the use of instructors in teaching their courses and assessing student learning. Dissemination or sale of any part of this work (including on the World Wide Web) will destroy the integrity of the work and is not permitted. The work and materials from it should never be made available to students except by instructors using the accompanying text in their classes. All recipients of this work are expected to abide by these restrictions and to honor the intended pedagogical purposes and the needs of other instructors who rely on these materials. Copyright © 2023 Pearson Education, Inc. All Rights Reserved