Microbiology Lecture Notes: Vaccines, Diagnostics, and Molecular Methods

Early History of Vaccines and Variolation

• Variolation Practice: Hundreds of years ago, the Chinese developed variolation to combat smallpox ($Variola$ virus).

  • Practitioners used a powder made from dried scabs of smallpox.

  • The powder was blown into the nose of a healthy individual.

  • This induced a milder case of smallpox with a mortality rate of only $1-2\%$.

• Edward Jenner (1796):

  • Observed that milkmaids were often spared from smallpox epidemics because they had previously contracted cowpox.

  • Hypothesis: Prior infection with cowpox provides protection against smallpox.

  • Experimental Test: Jenner inoculated a young boy with cowpox pus. The boy contracted cowpox and recovered quickly. Jenner then intentionally infected the boy with smallpox, but the boy remained asymptomatic.

  • Etymology: The term "vaccination" is derived from "vacca," the Latin word for cow.

• Mandates and Expansion:

  • Smallpox vaccination was eventually mandated for British soldiers.

  • In the late 1800s, Louis Pasteur developed early versions of the rabies vaccine (for humans) and an anthrax vaccine (to protect cattle).

  • Currently, at least 25 different infections are vaccine-preventable.

Licensed Vaccines in the United States (Table 14.1)

• Adenovirus Vaccine: Oral administration; protects against adenoviruses 4 and 7 (cause of colds); not routine for the general public; formulation is Live Attenuated.

• Anthrax Vaccine: Intramuscular (pre-exposure) or Subcutaneous (post-exposure); protects against $Bacillus\,anthracis$; adults only; formulation is Purified Subunit.

• BCG (Bacille Calmette-Guérin) Vaccine: Intramuscular; protects against $Mycobacterium\,tuberculosis$; prevents severe childhood TB; formulation is Live Attenuated.

• Cholera Vaccine: Oral; for ages 18-64 traveling to endemic areas; protects against $Vibrio\,cholerae$ serotype O1; formulation is Live Attenuated.

• COVID-19 Vaccines: Injected; mRNA and vector formats available; protects against SARS-CoV-2.

• Dengue Vaccine: Subcutaneous; for children 9-16 in endemic areas with laboratory-confirmed prior infection; prevents severe secondary disease; formulation is Live Attenuated.

• DTaP and Tdap (Diphtheria, Tetanus, Pertussis): Intramuscular; protects against $Corynebacterium\,diphtheria$, $Clostridium\,tetani$, and $Bordetella\,pertussis$; formulation is Subunit/Toxoid combination.

• Ebola Zaire Vaccine: Intramuscular; for adults 18+; prevents Zaire ebolavirus; first approved Recombinant Vector vaccine in the U.S.

• Haemophilus B (Hib) Vaccine: Intramuscular; routine pediatric; protects against $Haemophilus\,influenzae$ type b; formulation is Conjugated.

• Hepatitis A Vaccine: Intramuscular; routine pediatric; protects against Hepatitis A virus; formulation is Whole-agent Inactivated.

• Hepatitis B Vaccine: Intramuscular; routine pediatric; protects against Hepatitis B virus; formulation is Recombinant Subunit.

• Human Papillomavirus (HPV) Vaccines: Intramuscular; routine for pediatric and adult; protects against strains linked to warts and cancer; formulation is Recombinant Subunit.

• Influenza Vaccines: Intramuscular (most) or nasal mist (less common); annual; Whole-agent Inactivated or Purified Subunit.

• Japanese Encephalitis Virus Vaccine: Subcutaneous; for travelers; formulation is Whole-agent Inactivated.

• MMR (Measles, Mumps, Rubella): Subcutaneous; routine pediatric; formulation is Live Attenuated.

• Meningococcal Vaccine: Subcutaneous or Intramuscular; prevents $Neisseria\,meningitidis$ (select serogroups); Recombinant Subunit and Conjugate formulations.

• Pneumococcal Vaccine (PCV): Subcutaneous or Intramuscular; PCV13 is routine pediatric; PCV23 is for adults and high-risk pediatric; formulation is Conjugate.

• Inactivated Poliovirus Vaccine (IPV): Intramuscular; routine pediatric; formulation is Whole-agent Inactivated.

• Rabies Vaccine: Intramuscular; restricted to at-risk groups; Whole-agent Inactivated.

• Rotavirus Vaccine: Oral; routine pediatric; formulation is Live Attenuated.

• Smallpox Vaccine: Skin puncture with a bifurcated needle; uses vaccinia virus; formulation is Live Attenuated.

• Typhoid Vaccine: Oral (Live Attenuated) or Subcutaneous (Conjugate); for travelers and at-risk groups.

• Varicella-zoster (Chickenpox): Subcutaneous; routine pediatric; formulation is Live Attenuated.

• Yellow Fever Vaccine: Subcutaneous; for travelers/at-risk; formulation is Live Attenuated.

• Zoster (Shingles): Subcutaneous; for ages 50+; formulation is Live Attenuated.

Vaccine Impact and Controversies

• Successes:

  • Eradication of smallpox.

  • Global programs saved millions; projected to prevent 69 million deaths in low/middle-income countries between 2000 and 2030 (targets including measles, rotavirus, and Hepatitis B).

  • Polio is the next disease targeted for global eradication.

• Historical Resistance:

  • 1800s: Antivaccination societies in England.

  • Colonial India: Objection to smallpox vaccine derived from sacred cows (sacred to Hindus).

  • Individual Rights: Resistance to state-enforced vaccinations in the 1900s.

• The Lancet Controversy (1998):

  • A paper by Andrew Wakefield (study of 12 patients) claimed a link between the MMR vaccine and autism.

  • Consequences: MMR rates dropped in the U.S. and U.K.

  • Retraction (2010): The Lancet retracted the paper as "bad science."

  • Found massive conflict of interest: authors were funded by lawyers of parents suing vaccine companies.

  • Outcome: Wakefield lost his medical license due to fraud and malpractice. Subsequent massive studies (e.g., 95,000 children in 2015) found no link.

  • Re-emergence: 2019 U.S. measles outbreak reached nearly 1,300 cases across 31 states.

  • Policy Shifts: California removed "personal belief" opt-outs, requiring 10 immunizations for school entry (only medical exemptions allowed).

Immunology Principles and Herd Immunity

• Mechanism: Vaccines stimulate artificially acquired active immunity. They must trigger immunological memory without causing symptoms.

• Timeline: It takes approximately 2 weeks for antibody levels to reach peak levels after vaccination.

• Herd Immunity:

  • Protects the non-immunized (newborns, pregnant, immunocompromised).

  • Occurs when enough of the population is immune that the pathogen cannot find susceptible hosts to transmit.

  • Thresholds: Most pathogens require $85\%$ vaccination rate. Measles and whooping cough require $95\%$ vaccination to maintain herd immunity.

• CDC Pediatric Schedule: Often requires 2 or more subsequent boosters to optimize memory B and T cell populations.

Vaccine Formulations

Live Attenuated Vaccines

• Composition: Pathogens are altered to lose pathogenicity but remain infectious.

• Methods: Cultivation in cell culture or genetic manipulation.

• Pros: Strong immune response, long-lived memory.

• Cons: Risk to immunocompromised hosts, potential mutation back to infectious form, usually requires refrigeration.

Inactivated Vaccines

• Whole-Agent: Entire pathogen, inactivated by heat, chemicals, or radiation.

• Subunit: Purified antigens or parts of the agent. Requires adjuvants (pharmacological additives that enhance response).

  • Purified Subunit: Antigen harvested from natural source or expressed via recombinant system.

  • Toxoid: Inactivated toxins (e.g., tetanus, diphtheria).

  • Conjugate (Polysaccharide): Polysaccharide antigens linked to a protein to improve immunogenicity (e.g., Hib, pneumococcal).

Nucleic Acid & Vector Vaccines

• mRNA Vaccines: Purified mRNA is encased in lipids (chemically compatible with plasma membranes). Host cells translate the mRNA into an antigenic protein (e.g., SARS-CoV-2 spike protein).

• Recombinant Vector Vaccines: Genetic material is packed inside a harmless carrier (e.g., Johnson & Johnson uses Adenovirus type 26 to deliver DNA for SARS-CoV-2 spike protein).

• DNA Vaccines (Experimental): Target genes are placed in a plasmid, injected into the host, and host cells produce the antigen. Focused on HIV and cancer research.

Immunological Diagnostic Testing

• Biochemical Tests vs. Serology: Biochemical tests identify bacteria but take $>24\text{ hours}$ and require culturing. Serology (immunological testing) detects antigens or antibodies in serum, urine, or CSF.

• Agglutination Tests: Rely on antibodies binding to multiple antigens to form clumps.

  • Treponema pallidum particle agglutination (TPPA): Detects syphilis antibodies.

  • Used extensively for blood typing.

• Plaque Reduction Neutralization Test (PRNT):

  • Patient serum is serially diluted and mixed with a virus.

  • Mixture is added to cultured cells.

  • Positive: Reduced plaques (antibodies neutralized the virus).

  • Negative: Abundant plaques (patient lacks antibodies).

• Enzyme-linked Immunosorbent Assays (ELISAs):

  • Direct: Antigens adhere to wells; enzyme-linked detection antibody is added; substrate is added to generate a color/chemiluminescent signal.

  • Indirect: Precoated antigens in well → patient serum (primary antibody) → enzyme-linked detection (secondary) antibody which binds to human antibodies.

  • Sandwich: Capture antibody pre-applied → sample antigen added → detection antibody added. The antigen is "sandwiched" between two antibodies.

• Fluorescence-Based Tests:

  • Immunofluorescence Assays (IFA): Detection antibody is linked to a fluorescent tag instead of an enzyme.

  • Flow Cytometry: Uses Fluorescence-activated cell sorter (FACS) to count and sort specific cells (e.g., enumeration of CD4+ T cells).

• Interferon Gamma Release Assays (IGRAs): Used for TB detection. Measures interferon gamma release from T cells exposed to $M.\,tuberculosis$ antigens. High levels indicate infection.

Molecular Genetics Applications

Polymerase Chain Reaction (PCR)

• Components: Thermocycler, template DNA, two single-stranded primers, Taq polymerase, and dNTPs.

• Stages:

  1. Melting Step: High temperature to separate DNA strands.

  2. Annealing Step: Lower temperature ($50-65\,^\circ C$) so primers can bind.

  3. Extension Step: Optimal temperature ($65-75\,^\circ C$) for Taq polymerase to copy DNA.

• Growth Formula: Double with every cycle ($2^n$; where $n$ is the number of cycles).

• Variants:

  • Real-time PCR (qPCR): Uses fluorescence to visualize copies in "real time"/quantitatively.

  • Reverse Transcription PCR (RT-PCR): Uses reverse transcriptase to detect RNA (e.g., SARS-CoV-2 RNA genome).

Recombinant DNA (rDNA) Techniques

• Process:

  1. Isolation: PCR amplification of the target gene.

  2. Insertion: Use restriction enzymes to cut the gene and plasmid, generating "sticky ends." Join with DNA ligase.

  3. Transformation: Insert rDNA into host cells (often bacteria) for protein expression.

• Restriction Enzymes Examples:

  • Eco RI: From $E.\,coli$; generates sticky ends; recognition sequence $GAATTC$.

  • Bam H I: From $Bacillus\,amyloliquifasciens$; generates sticky ends.

  • Hin d III: From $Haemophilus\,influenza$; generates sticky ends.

  • Sma I: From $Serratia\,marcescens$; generates blunt ends; recognition sequence $CCCGGG$.

Gene Editing and Therapy

• CRISPR-Cas9:

  • Guide RNA (CRISPR): Acts as the GPS to find a DNA sequence.

  • Cas9 Enzyme: Acts as the scalpel to cut DNA.

  • Can be used in prokaryotic and eukaryotic (including human) cells.

• Gene Therapy: Uses non-pathogenic but infectious viruses (Adenoviruses, Retroviruses) to deliver healthy replacement genes into a patient's cell nucleus.

• DNA Microarrays: Compare healthy vs. diseased cells using nucleotide base-pairing for global views of cellular functions and clinical diagnostics.

Clinical Case: The Deadly Delay

• Patient: 5-year-old Sam.

• Symptoms: Bad cough, extreme fatigue, loud wheezing between coughing fits (whooping), history of runny nose/fever a week prior, vomiting after coughing.

• Suspected Pathogen: $Bordetella\,pertussis$.

• Factors: Sam had a medical exemption for vaccinations. Whooping cough is highly contagious and severe.

• Clinical Implications: Diagnostic tools like PCR or ELISA might be used. Decisions regarding herd immunity and school alerts are critical once identity is confirmed via lab tests.