Lecture 1: Vaccines

define vaccines

biological products that induce protective immunity without causing disease → one of the most effective public health tools

what effects have vaccines had on disease eradication and reduction?

• vaccination programs → reductions in incidence, basic reproductive number (R0, determines how likely illness is to spread or die out in a population), outbreak frequency

• global eradicationL smallpox (1980, first coordinated global vaxx program), wold poliovirus types 2&3, type 1 still in Pakistan and Afghanistan

what are the consequences of vaccines not being 100% effective and define what factors determine the severity

breakthrough infections can occur in individuals despite prior immunity

factors influencing severity of breakthrough infections:

• host factors : eg. elderly / immunocompromised

• vax type (durability of protection) and schedule

• hybrid immunity → natural infection + vax = lower severity

• waning immunity

• viral variants

• exposure/viral load

BUT… overall, vaccination lowers severity of breakthrough infections

how do vaccines lead to herd immunity?

vaccines cannot protect every individual in a population directly. if enough individuals are immune (eg. ~90-95% immunity for highly transmissible pathogens), transmission can be interrupted → provides indirect protection for those that cannot get vaccinated

briefly describe the history of vaccination from ancient practices, 18th century, 19th century, 20th century, and 21st century

• ancient practices: variolation in China, India, Ottoman Empire: deliberate exposure to dried small pox scabs. risky, but established principle that prior exposure to a pathogen confers protection

• 18th century: Edward Jenner used cowpox to protect against smallpox → concept of vaccination established → closely related but not identical virus still protected

• 19th century: Louis Pasteur developed vaccine for fowl cholera in chickens and rabies. Germ theory of disease (microbes vs miasma) strengthened vaccine science

• 20th century: YFV, influenza, polio, hepB, MMR vaccines developed

• mass immunization programs dramatically reduce infectious diseases

• 21st century: advances technologies: mRNA, viral vectors, recombinant vaccines

why is the innate immune system important in vaccination?

• early innate responses crucial for adaptive immune development

• PRRs and cytosolic sensors (eg. TLRs, cGAS-STING, RIG-I) on APCs are activated by PAMPs from viruses and adjuvants designed to mimic PAMPs or DAMPs

• APCs

what adaptive cells/processes are essential to vaccine-induced protection?

• T cell-dependent B cell activation: initiated by antigen recognition and CD4+ T cell-derived signals

• Germinal Centers: drive affinity maturation, isotype switching

• Short-Lived Plasma Cells: rapidly produce vaccine-specific Abs

• Long-Lived Plasma Cells: home to bone marrow, sustain Ab production

• Memory B Cells: mediate long-term immune memory

• Neutralizing Abs block viral entry; Fc-mediated functions enhance protection

• CD8+ Effector T Cells: eliminate infected cells and limit disease severity

• CD8+ Memory T Cells: expand rapidly upon re-exposure to eliminate infected cells

describe the differing pathways for adaptive immunity for vaccines presented on MHC I vs MHC II

• MHC class I: activates CD8+ T cell, leading to CD8+ effector and memory T cells

• MHC class II: activate CD4+ T cell which provides helper signals to B cells that encounter their soluble antigen → B cells proliferate and Ab response matures → memory B cell proliferation or plasma cell differentiation and antibody production

desribe the evidence of Abs as correlates of protection

• neutralizing Abs block viral entry and strongly correlate with protection against infection

• Ab deficiencies increase susceptibility to specific viral infections (eg. VZV), identifying key protective mechanisms

• Passive Ab transfer provides immediate protection against some infections → eg. maternal Abs across placenta, purifies neutralizing Abs from immune donors

vaccine protection relies on what?

immune memory

the ability of immune memory to defend against a future pathogen encounter depends on what?

1. incubation time of the infection

2. quality of the memory response

3. level of Abs induced by memory B cells

describe how incubation period impacts the ability of immune memory to defend against a future pathogen encounter

following the primary exposure to the pathogen, the level of protective Abs decline below the protective threshold. When the pathogen has a long incubation period (eg. hep B), upon secondary infection this allows enough time for antibody levels to get above the protective threshold. with a short incubation period (h. influenzae B) , there is insufficient time to raise protective antibody levels

• BUT in some cases Ab levels after primary vaccination remain above the protective threshold and can provide lifelong immunity

describe and provide examples of the classical vaccine platforms

1. Live-attenuated virus: whole, weakened viruses that can replicate but do not cause serious disease → eg. MMR vax

2. whole-inactivated virus: whole “killed” viruses that cannot replicate → eg. polio inactivated vax (IPV)

3. Protein Subunit: specific viral proteins or protein fragments → eg. hep B vax

4. virus-like particles (VLPs): viral proteins that mimic the virus but lack viral genetic material and cannot replicate → eg. HPV vax

describe live attenuated virus vaccines and how they can be produced

• whole, weakened viruses that can replicate minimally within host cells, mimicking natural infection

• often induce durable and broad immune responses

• produced by:

  • serial passage in non-human hosts or cells (eg. chicken embryo fibroblasts) → virus adapts to new host, loses virulence in humans

  • Cold adaptation → virus passaged at low temperatures (25-36C), loses ability to replicate efficiently at high temperatures

  • genetic engineering: targeted mutations or deletions in virulence genes

describe an example of a live-attenuated vaccine

Measles virus (MeV) adaptation to CD46 in cell culture:

• has several mutations that impact viral replication cycle

• after passaging, virus lost ability to bind to CD150/SLAM and PVRL4/nectin so can no longer replicate in immune cells → inhibited at level of cell attachment

• CD46/MCP found on all nucleated cells, CD150/SLAM found on immune cells (DCs, Mo), PCLR4/nectin found on epithelial cells and adenocarcinomas

describe the second example of a live-attenuated vax

cold-adapted influenza A virus → attenuated by affecting viral DNA/RNA synthesis

• impacts viral RNA-dep RNA pol (RdRp) → trimeric, composed of PB1, PA, PB2

• when passaged at lower temperatures, accumulated a few mutations in RdRp so cannot replicate at physiological temp → when given via nasal (has lower temp), can replicate but cannot replicate in the lungs

• possibility of reversion

describe whole-inactivated vaccines and how they can be inactivated

• whole viruses that cannot replicate within host cells → verification of inactivation is critical

• often need adjuvants, higher Ag doses, and booster doses to help compensate for weaker immunogenicity compared to live vax

• inactivated by chemical inactivation (more common) or physical inactivation

describe the methods for physical inactivation

• formalin (formaldehyde): crosslinks proteins And nucleic acids (eg inactivated polio vax)

• beta-propriolactone (BPL): alkylates nucleic acids (destroys genome), preserves protein structure better than formalin, used for flu, rabies, some COVID vax

describe the methods for physical inactivation

• heat: Denatures viral proteins (e.g., capsid proteins and envelope glycoproteins → loss of receptor binding or fusion capability), BUT can strongly alter antigenic structures

• Ultraviolet (UV) Inactivation : Direct photochemical damage to viral genomes, forms pyrimidine dimers in DNA or RNA, induces strand breaks, oxidative lesions, and cross-linking

• can do more damage to viral structure

describe an example showing the importance of verifying complete inactivation of whole-inactivated virus vaccines

The Cutter Incident (1955, USA):

• Two production pools of IPV made by Cutter Laboratories (accounting for 120,000

doses) were given to healthy children

• Inadequate virus purification during production led to the presence of cell debris in

the vaccine pools

• Cell debris blocked proper exposure of virions to formalin, preventing complete

inactivation

Outcomes:

• 40,000 cases of abortive poliomyelitis, 51 cases of permanent paralysis, 5 deaths

• Immediate tightening of federal regulations for vaccine production

how can complete inactivation be measured?

extended cell culture amplification by taking inactivated virus and “infecting” highly permissive cell lines → monitor for evidence of replication: CPE observation, genome amplification (PCR), antigen detection (immunoassay)

describe protein subunit vaccines

• Contain designated viral proteins rather than whole viruses → minimized risk since no infectious virus present

• Often require adjuvants - immune system may generate weak responses to single antigen proteins

• Often expressed in yeast, mammalian, or insect cell systems

describe an example of a protein subunit vaccine

Hepatitis B virus (HBV) vaccine:

• key proteinL HBV Surface Antigen HBsAg → S protein

• HBsAg → viral envelope protein composed of 3 related forms: Small (S), S protein only, Middle (M), S protein +PreS2 domain, and Large (L), S protein + PreS2+PreS1 domains

why is only the S protein used in recombinant HBV vaccines?

• major neutralizing epitopes: stimulates strong B and T cell responses

• highly conserved: broad protection against HBV strains

• established vaccine efficacy: induces protective anti-HB S antibodies after 3 doses

describe how the hepatitis protein subunit vaccine is produced using yeast

1. genetic material extracted from hepatitis virus

2. individual genes analyzed and identified

3. gene that directs production of surface protein is located

4. gene is removed from viral DNA and inserted into ‘plasmid’ → S protein moved to plasmid

5. plasmids inserted into yeast cells

6. yeast grown by fermentation

7. cells reproduce and generate more surface protein

8. result is large quantity of pure surface protein particles that provoke an immune response

9. surface proteins are combined with preserving agent (&adjuvant) and other ingredients to make vaccine

what are additional design and production considerations when making a protein subunit vaccine?

• Choice of antigen: must include protective epitope(s) that induce neutralizing Abs or effective T cell help

• Expression system: yeast (e.g., HBsAg), mammalian, or insect cells → affects glycosylation and folding; proper protein folding and post-translational modifications critical for immunogenicity

• Purification: removal of host contaminants while preserving native conformation

• Stability: formulation must maintain protein structure during storage and transport

• Dosing: multiple dosing and adjuvants often required for durable immunity

• Scalability: production must be compatible with large-scale manufacturing

describe virus-like particle (VLP) vaccines

• Viral structural proteins that self-assemble into particles that mimic that size, symmetry, and structure of native virions

• Genome-free → non-infectious and replication-incompetent

• Preserve conformational epitopes required for neutralizing antibodies

what effect do VLP vaccines have on B cells?

repetitive particle geometry of VLP-based vaccines optimally activated B cells:

• Strong B-cell activation requires at least 12–16 identical epitopes spaced ~5–10 nm apart (“immunons”) → VLPs naturally meet these criteria

• Enables efficient B cell receptor (BCR) cross-linking and recognition by IgM antibodies

• Promotes complement activation, plasma cell differentiation, and germinal center formation

• Induce durable antibody responses → especially valuable when target antigens are weakly immunogenic

describe an example of a VLP vax

Human Papillomavirus (HPV) vaccine:

• Licensed HPV vaccine antigen = L1 major capsid protein (immunodominant, highly immunogenic)

• Recombinant L1 self-assembles into icosahedral VLPs

• Displays native conformational neutralizing epitopes

• L2 minor capsid protein (highly conserved, less immunogenic) → considered a target for the development of a next-generation Pan-HPV vaccine

describe the key pros and cons of the live-attenuated, whole-inactivated, VLP, and protein subunit vaccines

describe some modern vaccine platforms

• mRNA and DNA vaccines: Use the host’s own protein synthesis machinery to produce immunogenic antigens → need to be delivered into cells for antigen expression

• recombinant viral vector vaccines:

describe the core design elements of mRNA vaccines

• produced by in vitro transcription (IVT) from DNA template
  

Conventional (non-replicating) mRNA:

• Synthetic mRNA is translated directly in the cytoplasm by host ribosomes

• Antigen output is dose-dependent

• Mimics the structure of endogenous mRNA with 5 sections: 5’ Cap, 5’ UTR, ORF, 3’ UTR, Poly-A Tail

Self-Amplifying mRNA (is replicating):

• Encodes antigen + viral replicase (RdRp); undergoes intracellular

RNA amplification

• Higher antigen expression at lower doses

what are the core design elements of DNA vaccines?

• Plasmid backbone: circular DNA molecule carrying the antigen gene; includes bacterial origin of replication and antibiotic resistance marker for propagation in bacteria

• Antigen coding sequence: gene encoding the target protein

• Eukaryotic expression elements: promoter, transcriptional terminator, and poly(A) signal to drive transgene expression in host cells

• Regulatory elements: sequences enhancing mRNA stability, nuclear localization, and efficient translation

• Codon optimization: adjusts gene sequence for efficient protein production in mammalian cells

what is a challenge with mRNA vaccines and how has this been overcome?

• mRNA is large (104-106 Da) & negatively charged → cannot pass through the anionic lipid bilayer of cell membranes

Techniques to deliver mRNA intracellularly:

• In vitro: electroporation, gene guns, ex vivo transfection

• In vivo: mRNA delivery vehicles (e.g., lipid nanoparticles)

lipid nanoparticles (LNPs)

• Encapsulate mRNA in their core → used in mRNA vaccine production

Consist of 4 Components:

• Ionizable lipids → help mRNA escape LNP

• Cholesterol or its variants → fusion to cell membrane

• Helper lipids → stabilize particle

• PEG-DMG → help extend circulation of LNP

what challenge do DNA vaccines have and how have we combatted this?

• DNA must cross plasma membrane and enter nucleus → nuclear localization signals often intrinsic to DNA viruses but can be engineered into plasmids

• variety of ways to transport DNA vax across plasma membrane → viral capsid, empty bacterial capsule, nanoparticle, liposome, gold beads

what are some current clinical applications of mRNA and DNA vaccines?

• infectious disease vaccines → encode 1-2 Ags, often cell attachment proteins

  • eg. SARS-CoV2, flu, RSV, Zika prM

• therapeutic cancer vaccines

• genetic adjuvants

describe an example of an mRNA vaccine

mRNA-1345: examples of structure-based vaxx design

• LNP-based mRNA vaccine candidate developed by Moderna for respiratory syncytial virus (RSV) infection

• Encodes for the RSV prefusion F (pre-F) glycoprotein → responsible for entry of the virus and cell-to-cell spread

• Recently authorized for adults > 60 years of age

why choose the pre-F glycoprotein as the antigen for an mRNA vaccine?

• metastable pre-F form exposes key neutralizing epitopes → epitopes lost in pre-F form

• strong correlation with protection

• high immunogenicity with lower Ag dose

• proven success across RSV platforms