m - Week 9 - L6 - Comprehensive Study Notes on Acellular, Subunit, Vector, and Nucleic Acid Vaccines

Overview of Acellular and Subunit Vaccines

• Acellular Vaccines: Contain only specific purified parts of pathogens capable of inducing a protective immune response, rather than whole killed or live-attenuated microbes.
• Subunit Vaccines: A subcategory of acellular vaccines that isolate one or more specific pathogen components.
  • Primary Application: Most effective when protection against disease requires exclusively a humoral (antibody-mediated) immune response against the targeted pathogen component.
  • General Characteristics:
    • Typically administered via parenteral injection.
    • Require multiple booster doses to establish long-term immunity.
    • Usually require an adjuvant to enhance immunogenicity (with limited exceptions).
    • Require cold chain storage.
    • Exhibit variable production costs depending on the manufacturing platform.
• Classification of Subunit Vaccine Types:
  1. Toxoid Vaccines (Requires culturing large pathogen quantities)
  2. Conjugate Vaccines (Requires culturing large pathogen quantities)
  3. Recombinant Antigen Vaccines (Does NOT require culturing large pathogen quantities)
  4. Synthetic Peptide Vaccines (Does NOT require culturing large pathogen quantities)
• Biosafety Selection Criteria: For dangerous pathogens requiring Physical Containment Level 3 ($\text{PC3}$) or Physical Containment Level 4 ($\text{PC4}$) containment, recombinant antigen or synthetic peptide platforms are selected to avoid culturing large volumes of hazardous agents.

Toxoid Vaccines

• Target Pathogens: Applicable exclusively to bacterial diseases because viruses do not produce toxins.
• Mechanism of Action:
  • Used when pathology is driven by a secreted bacterial toxin rather than cellular invasion by the microbe itself.
  • Induces neutralizing antibodies (termed antitoxins) that bind directly to the toxin, blocking its ability to interact with and destroy host cells.
• Manufacturing Process:
  • Pathogens are cultured in large-scale liquid fermenters.
  • The secreted toxin is purified from the culture supernatant.
  • The purified toxin undergoes chemical treatment (e.g., formaldehyde treatment) to alter its tertiary structure, neutralizing toxicity while retaining antigenicity. The inactivated form is called a toxoid.
• Adjuvant Requirement: Toxoid molecules are structurally small and weakly immunogenic on their own, requiring adjuvants such as alum (aluminum salts).
• Containment Limitations: Current standard protocols require culturing large volumes of pathogenic bacteria, making this approach unsuitable for high-containment ($\text{PC3}$ or $\text{PC4}$) pathogens unless recombinant expression systems are employed.
• Bacterial Toxin Examples and Pathologies:
  • Clostridium tetani: Produces tetanus toxin, causing rigid paralysis (tetanus).
  • Clostridium botulinum: Produces botulinum toxin, causing flaccid paralysis (botulism; toxin refined commercially as Botox).
  • Corynebacterium diphtheriae: Produces diphtheria toxin, causing severe tissue necrosis and a characteristic pseudomembrane over the tonsils and throat.
  • Bordetella pertussis: Produces pertussis toxin, causing whooping cough.
• Clinical Vaccine Formulations:
  • DTaP: Administered to infants and young children; protects against diphtheria, tetanus, and acellular pertussis.
  • Tdap: Formulated with reduced antigen concentrations for older children, adolescents, and adults.
  • Adjuvant Used: Alum (aluminum hydroxide/phosphate).

Conjugate Vaccines

• Target Pathogens: Used exclusively for capsulated bacteria, as viral pathogens lack polysaccharide capsules.
• Immunological Rationale:
  • Bacterial capsular polysaccharides mask cell surface proteins and evade host phagocytosis.
  • Capsular polysaccharides are T-independent antigens, making them weakly immunogenic (especially in infants).
  • Conjugation Strategy: Purified capsular polysaccharides are chemically linked (conjugated) to a strongly immunogenic carrier protein (often a toxoid protein). This converts a T-independent antigen into a T-dependent antigen, provoking a robust memory response.
• Carrier Proteins and Examples:
  • Salmonella enterica serovar Typhi (Typhoid vaccine): Uses Diphtheria toxoid carrier.
  • Neisseria meningitidis (Meningococcal meningitis vaccine): Uses Diphtheria toxoid carrier.
  • Streptococcus pneumoniae (Pneumococcal meningitis vaccine): Uses Diphtheria toxoid carrier.
  • Haemophilus influenzae type b (Hib vaccine): Uses Tetanus toxoid carrier.
• Outer Membrane Vesicles (OMVs) as Carriers:
  • Derived from Gram-negative bacterial outer membranes.
  • Highly immunogenic inherently; when OMVs serve as the carrier platform, chemical adjuvants are often redundant and unnecessary.
  • Clinical Trial Application: An intranasal COVID-19 vaccine trial conjugates the SARS-CoV-2 spike protein to Gram-negative OMVs.
• Adjuvants: Alum is standard when toxoids serve as carriers; omitted when using OMVs.

Recombinant Antigen Vaccines and Virus-Like Particles (VLPs)

• Methodology:
  1. Identify the protective antigen gene from a pathogen's known genome sequence.
  2. Clone the gene into an expression plasmid vector.
  3. Transform/transfect a safe expression host (e.g., Escherichia coli or Saccharomyces cerevisiae yeast).
  4. Express the antigen in large-scale bioreactors.
  5. Extract and purify the antigen, then formulate it with an adjuvant.
• Key Advantage: Completely bypasses the need to culture dangerous or unculturable pathogens, eliminating biosafety risks associated with $\text{PC3}$ and $\text{PC4}$ organisms.
• Hepatitis B Virus (HBV) Vaccine:
  • Developed in 1986 as the world's first licensed recombinant antigen vaccine.
  • Target: Hepatitis B surface antigen (HBsAg), which elicits neutralizing, protective antibodies in patient serum.
  • Production Pipeline: HBsAg gene isolated $\rightarrow$ cloned into a bacterial plasmid $\rightarrow$ transformed into yeast cells (S. cerevisiae) $\rightarrow$ mass-fermented in bioreactors $\rightarrow$ recombinant HBsAg extracted, purified, and adjuvanted.
  • Properties: Highly effective, fully non-infectious, and completely virus-free.
• Virus-Like Particles (VLPs):
  • Empty, non-infectious viral capsids assembled entirely without viral nucleic acids (DNA or RNA).
  • Self-assemble automatically upon recombinant expression of capsid structural proteins.
  • Chimeric VLPs: Created by cloning genes encoding target foreign antigens in-frame with the viral capsid protein gene, resulting in dense antigen presentation on the VLP surface.
  • Expression Host Options: Choice depends on structural complexity, post-translational modifications, and yield (determined via empirical trial and error):
    • Bacteria (E. coli): High yield, low cost, simple genetic manipulation.
    • Yeast system
    • Insect cells
    • Plant systems
    • Mammalian cells
  • Gardasil 9 HPV Vaccine:
    • Developed in Queensland, Australia.
    • Polyvalent recombinant VLP vaccine targeting 9 human papillomavirus (HPV) genotypes responsible for cervical cancer.
    • Cloned the L1 major capsid protein gene from each of the 9 HPV types into yeast expression vectors.
    • Purified individual VLP types are combined into a single 9-valent formulation.

Synthetic Peptide Vaccines and Epitope Identification

• Concept: Isolates and synthesizes only the specific minimal amino acid sequence (epitope) bound by the Fab region of a neutralizing antibody, rather than utilizing an entire protein subunit.
• Structure: Peptides are typically short, consisting of approximately $10$ to $20$ amino acids.
• Epitope Identification via B-Cell Cloning:
  1. A specific B cell binds its target antigen via its membrane-bound B-cell receptor (BCR).
  2. The B cell is activated, undergoes clonal expansion, and differentiates into antibody-secreting plasma cells.
  3. The single monoclonal antibody produced by the cloned B cell is isolated.
  4. Passive Immunization Note: Direct injection of these monoclonal antibodies provides immediate, short-term passive protection.
  5. Epitope Mapping: The Fab region of the antibody is sequenced and structurally modeled alongside the target antigen using computational matching algorithms to locate the precise structural fit.
• Peptide Synthesis & Formulation:
  • The identified amino acid sequence is programmed into an automated chemical peptide synthesizer.
  • May require structural modifications (e.g., conformational constraint or cyclization) to match native folding.
  • Delivered inside or presented on the surface of carrier structures: VLPs, OMVs, or liposomes.
  • Adjuvant Requirement: Essential due to extremely small peptide mass and low intrinsic immunogenicity.

Recombinant Viral Vector Vaccines

• Mechanism: Uses a live, non-pathogenic, or replication-defective virus to carry the gene encoding a target pathogen's antigen directly into host cells.
• Intrinsic Adjuvant Effect: Viral vector capsids naturally stimulate host Toll-like Receptors (TLRs), acting as built-in agonists that render external chemical adjuvants unnecessary.
• Immune Profile: Synthesizes antigens inside host cells, stimulating both humoral (antibody) and cell-mediated ($\text{CD8}^+$ T-cell) immune responses.
• Intracellular Processing:
  1. Viral vector enters the host cell (e.g., via intramuscular injection).
  2. Capsid uncoats, and the transgene migrates to the cell nucleus.
  3. Transgene is transcribed and translated by host cell machinery.
  4. Antigens are processed: degraded fragments are presented via MHC Class I molecules, or intact antigens are secreted to activate antigen-presenting cells (APCs).
• Viral Vector Platforms:
  • Replication-Defective Adenovirus: Gene-deleted to prevent human replication. Used in the Oxford-AstraZeneca COVID-19 vaccine; currently researched for HIV and Tuberculosis (TB).
  • Live-Attenuated Measles Virus Vector: Replication-competent, licensed, human-safe platform. Researched for COVID-19, HIV, and Ebola.
  • Canarypox Virus Vector: Avian poxvirus that cannot replicate in mammalian cells; enters human cells to express target proteins. Researched for HIV.
• Pre-existing Vector Immunity: Clinical studies demonstrate that pre-existing antibodies against the viral vector (such as prior measles vaccination) do not prevent a strong protective immune response against the encoded target antigen.

Live Bacterial Vector Vaccines

• Mechanism: Live, attenuated bacteria are transformed with a plasmid vector or chromosomally integrated gene encoding a foreign pathogen antigen.
• Features: Attenuated vector infects host tissue without causing disease; synthesizes foreign antigen in vivo, eliminating protein purification steps.
• Subcellular Localization of Target Antigen:
  • Intracellular (Cytoplasmic): Antigen released upon phagocytosis or lysis of the bacterial vector.
  • Cell Surface: Achieved by cloning a specific signal peptide sequence onto the antigen gene; enhances recognition by host immune cells.
  • Secreted: Antigen actively exported into host tissue by bacterial secretion systems.
• Bacterial Vector Platforms:
  • Live-Attenuated Salmonella typhi: Orally administered; targets mucosal tissue in the gastrointestinal tract to induce strong mucosal immunity. Researched for Helicobacter pylori (bacterial) and Influenza (viral) vaccines.
  • Live-Attenuated Salmonella enterica serovar Typhimurium: Orally administered. Licensed platform undergoing research for non-infectious target applications, including prostate cancer immunotherapy.
• Multivalent Advantage: Immunizes simultaneously against the carrier bacterium (e.g., Salmonella) and the engineered target antigen.

Nucleic Acid Vaccines: DNA vs. mRNA

• Fundamental Principle: Delivers genetic sequences (DNA plasmids or mRNA) directly into host tissue, forcing host cells to synthesize the viral or bacterial antigen in vivo.
• General Advantages: Fast design cycle, highly scalable, cost-effective, no whole-cell culturing, and rapidly adaptable to emerging pathogen variants (e.g., SARS-CoV-2 Alpha, Delta, and Omicron variants generated by viral RNA polymerase mutation rates).

Plasmid Design, Vaccine Mechanisms, and Administration Routes

• DNA Plasmid Vector Requirements:
  1. Origin of Replication (ori): Dual-functioning host origin recognized in both bacterial cells (for mass plasmid production) and eukaryotic host cells.
  2. Promoter Region: Strong eukaryotic promoter positioned upstream of the antigen gene, commonly derived from Cytomegalovirus (CMV promoter).
  3. Selectable Marker: Antibiotic resistance gene (e.g., Ampicillin resistance, $\text{Amp}^R$) for plasmid selection in bacterial cultures.
• Advanced DNA Physical Delivery Systems:
  • Gene Gun: Directs DNA-coated heavy-metal particles into epidermal host cells using high-pressure helium gas gas propulsion.
  • Electroporation: Intramuscular injection followed immediately by localized electrical pulses, opening transient pores in host cellular and nuclear membranes to facilitate nuclear uptake.
  • Cutaneous Suction-Based Transfection: Non-viral, painless intradermal injection method. Applies controlled, localized negative pressure (suction) over the injection bleb using a cup apparatus to drive plasmid uptake into cells without structural tissue damage.
• mRNA Administration Routes:
  • Intramuscular (IM): Primary clinical route (e.g., COVID-19 vaccines). High density of vascular structures and resident antigen-presenting cells (APCs).
  • Intradermal (ID): Targets dense dermal layers containing rich lymphatic network vessels, efficiently shuttling mRNA/APCs to regional lymph nodes to prime B and T lymphocytes.
  • Intravenous (IV): Direct systemic bloodstream delivery; supports large volume, but subject to shear forces from blood flow and systemic side effects.
  • Subcutaneous (SC): Administers large volume, but limited by low absorption rates, enzymatic degradation of mRNA, and sparse local immune cell populations.
  • Intranodal: Direct injection into peripheral lymph nodes; directly targets dense APC and lymphocyte populations.
  • Intranasal / Inhalation: Formulations under development for direct mucosal protection against airborne respiratory pathogens.