Epitope Selection, Filtering and Vaccine Construct Design for Subunit Vaccines

Welcome, future bioinformaticians! In the quest to develop effective vaccines, particularly subunit vaccines, two critical computational steps are the selection of the best possible antigenic components (epitopes) and the strategic design of how these components are assembled. This lesson will guide you through these processes, focusing on the rationale and methods involved.

Imagine you’re building a highly specialized toolkit to combat a specific disease. Epitopes are your custom-made tools, and the vaccine construct is the carefully organized toolbox that ensures these tools are delivered and function effectively.

1. Epitope Filtering and Selection: Choosing the Right Tools

Before we dive into filtering, let’s understand what we’re working with.

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What is an epitope?

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An epitope, or antigenic determinant, is the specific part of an antigen that is recognized by the immune system, such as by antibodies, B cells, or T cells. For vaccine design, we often focus on T-cell epitopes (recognized by T lymphocytes) and B-cell epitopes (recognized by B lymphocytes).

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Not all predicted epitopes are equally suitable for a vaccine. We need to filter them based on properties that suggest they will be effective, safe, and manufacturable. This is where physicochemical property assessment comes in.

1.1 Why Filter Epitopes?

Filtering predicted epitopes is crucial for several reasons:

• Enhanced Immunogenicity: Selecting epitopes that are more likely to provoke a strong and targeted immune response.
• Improved Solubility & Stability: Ensuring the epitopes (and the final vaccine construct) are soluble and stable, which is important for manufacturing, storage, and in vivo efficacy.
• Specificity: Avoiding epitopes that might cross-react with host proteins, which could lead to autoimmunity. (Though this is usually checked by other tools like allergenicity and autoimmunity checks, physicochemical properties indirectly contribute to overall suitability).
• Manufacturability: Properties like stability can influence how easily the vaccine can be produced, especially if it’s a recombinant protein.

1.2 The ExPASy ProtParam Tool

A popular tool for assessing various physicochemical properties of a protein or peptide sequence is ProtParam, available on the ExPASy server.

• Website: https://web.expasy.org/protparam/
• Function: It computes various physical and chemical parameters for a user-inputted protein sequence (either Swiss-Prot/TrEMBL accession number or raw sequence). These parameters include molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (GRAVY).

For epitope filtering, we are particularly interested in GRAVY, aliphatic index, estimated half-life, and the instability index.

1.3 Key Physicochemical Filtering Criteria

Let’s break down the criteria mentioned in the topic: “Epitopes with a grand average of hydropathicity (GRAVY) score less than 0 (i.e., hydrophilic), aliphatic index ≥ 70 (i.e., thermostable), estimated half-life in E. coli ≥ 10 hours, and classified as ‘stable’ were considered.”

1.3.1 Grand Average of Hydropathicity (GRAVY)

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What is GRAVY?

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The Grand Average of Hydropathicity (GRAVY) score is calculated as the sum of hydropathy values of all amino acids in a sequence, divided by the number of residues. It indicates the overall hydrophobicity or hydrophilicity of a peptide/protein.

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• Filtering Criterion: GRAVY score < 0
• Rationale (Why hydrophilic?):
  • Solubility: Hydrophilic peptides (negative GRAVY score) are generally more soluble in aqueous environments, like physiological fluids or buffers used in manufacturing. Poor solubility can lead to aggregation and reduced efficacy.
  • Surface Exposure: In a larger protein context, hydrophilic regions tend to be exposed on the surface, making them more accessible to immune cells and antibodies. While individual epitopes are small, this property is still generally favored.
  • Analogy: Think of oil (hydrophobic) and sugar (hydrophilic) in water. For our vaccine components, we prefer them to dissolve and interact well with the aqueous environment of the body, like sugar does.

1.3.2 Aliphatic Index

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What is the Aliphatic Index?

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The Aliphatic Index of a protein is defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine). It is regarded as a positive factor for the increase of thermostability of globular proteins.

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• Filtering Criterion: Aliphatic Index ≥ 70
• Rationale (Why thermostable?):
  • Stability: A higher aliphatic index often correlates with increased thermostability. This means the epitope (and the final vaccine construct) is more likely to maintain its correct three-dimensional structure at higher temperatures.
  • Manufacturability & Storage: Thermostability is beneficial during production processes (which might involve temperature fluctuations) and for the shelf-life of the vaccine, potentially reducing the need for strict cold-chain storage.
  • In Vivo Stability: A more stable peptide might resist denaturation in the host environment for longer, allowing more time for immune recognition.
  • Analogy: Imagine building with blocks. A high aliphatic index is like using very sturdy, heat-resistant blocks that won’t easily warp or melt, ensuring your structure (epitope) stays intact.

1.3.3 Estimated Half-life in E. coli

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What is Estimated Half-life?

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The estimated half-life is a prediction of how long a protein/peptide remains stable and functional before being degraded within a specific cellular environment or organism. ProtParam estimates this based on the N-end rule, which relates the N-terminal amino acid of a protein to its stability.

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• Filtering Criterion: Estimated half-life in E. coli ≥ 10 hours
• Rationale (Why long half-life, specifically in E. coli?):
  • Recombinant Production: E. coli is a common host system for producing recombinant proteins (including vaccine antigens). A longer half-life in E. coli means the protein is less likely to be degraded during expression and purification, potentially leading to higher yields.
  • General Stability Indicator: While this specific estimate is for E. coli, it can sometimes give a general indication of the peptide’s intrinsic stability against proteolytic degradation.
  • Analogy: This is like assessing how long a tool will last under typical workshop (E. coli expression system) conditions before it breaks down. A longer-lasting tool is more efficient to produce.

1.3.4 Instability Index (and ‘Stable’ Classification)

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What is the Instability Index?

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The Instability Index provides an estimate of the stability of a protein in a test tube. Proteins with an instability index smaller than 40 are predicted as stable, a value above 40 predicts that the protein may be unstable.

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• Filtering Criterion: Classified as ‘stable’ (which typically means Instability Index < 40).
• Rationale (Why ‘stable’?):
  • Overall Structural Integrity: This index considers dipeptides whose presence is statistically different in unstable versus stable proteins. A ‘stable’ classification suggests the peptide sequence is less prone to degradation or conformational changes in vitro.
  • Reliability: A stable epitope is more likely to maintain its intended structure, which is crucial for consistent immune recognition.
  • Analogy: This is like a quality check on your tool, ensuring it doesn’t have inherent weak points that would cause it to fall apart easily, even when handled carefully.

1.4 Step-by-Step Filtering Process (Hypothetical)

Let’s imagine you have a list of 5 potential epitopes predicted from a viral protein.

1. Obtain Epitope Sequences:

   • Epitope 1: SLFNTVATL
   • Epitope 2: KCYGVSPTK
   • Epitope 3: RPLPFFLLA
   • Epitope 4: TQIGCTLNF
   • Epitope 5: WEFVNTPPL

2. Analyze Each Epitope with ProtParam: For each sequence:

   • Go to ExPASy ProtParam.
   • Paste the epitope sequence into the sequence box.
   • Click “Compute parameters”.
   • Record the relevant values: GRAVY, Aliphatic Index, Estimated half-life (select “E. coli” if options are given, though ProtParam usually gives multiple), and Instability Index.

   Example for Epitope 1: SLFNTVATL (Hypothetical Values)

   • GRAVY: -0.250
   • Aliphatic Index: 95.56
   • Estimated half-life (E. coli): >10 hours
   • Instability Index: 30.00 (Stable)

   Example for Epitope 3: RPLPFFLLA (Hypothetical Values)

   • GRAVY: 1.500
   • Aliphatic Index: 120.00
   • Estimated half-life (E. coli): >10 hours
   • Instability Index: 25.00 (Stable)

3. Apply Filtering Criteria: Create a table to track this.

   Epitope	Sequence	GRAVY (< 0?)	Aliphatic Index (≥70?)	Half-life (E.coli ≥10h?)	Instability Index (< 40 / Stable?)	Pass All?
   Epitope 1	SLFNTVATL	-0.250 (Yes)	95.56 (Yes)	>10h (Yes)	30.00 (Yes)	Yes
   Epitope 2	KCYGVSPTK	-0.800 (Yes)	65.00 (No)	>10h (Yes)	45.00 (No)	No
   Epitope 3	RPLPFFLLA	1.500 (No)	120.00 (Yes)	>10h (Yes)	25.00 (Yes)	No
   Epitope 4	TQIGCTLNF	-0.100 (Yes)	80.00 (Yes)	5h (No)	35.00 (Yes)	No
   Epitope 5	WEFVNTPPL	-0.500 (Yes)	85.00 (Yes)	>10h (Yes)	38.00 (Yes)	Yes

4. Select Qualified Epitopes: Based on the table, Epitope 1 and Epitope 5 pass all criteria and would be selected for the next stage of vaccine design.

1.5 Interactive Checkpoint 1: Epitope Filtering

Let’s test your understanding of epitope filtering.

Place the following steps for filtering a *single* epitope using ProtParam in the correct order:

Click the items below in the order you think is correct.

• Record the GRAVY, Aliphatic Index, Estimated half-life, and Instability Index values.
• Go to the ExPASy ProtParam website.
• Compare the recorded values against the predefined filtering thresholds (e.g., GRAVY < 0).
• Paste the epitope's amino acid sequence into the input box and submit.
• Decide if the epitope passes all criteria.

Reset Check Order

Why is a GRAVY score of less than 0 generally preferred for vaccine epitopes?

• It indicates higher thermostability.
• It suggests the epitope is more hydrophobic and will integrate into cell membranes easily.
• It suggests the epitope is more hydrophilic, aiding solubility and potential surface exposure.
• It directly correlates with higher immunogenicity.

2. Vaccine Construct Design: Assembling the Toolbox

Once you have a set of promising epitopes, the next step is to design how they will be combined into a single molecule—a multi-epitope vaccine. This is typically done in silico (computationally).

The goal is to create a construct that:

• Presents multiple epitopes to the immune system.
• Includes an adjuvant to boost the immune response.
• Uses linkers to properly space epitopes and facilitate processing.

2.1 The Multi-Epitope Vaccine Approach

Why not just inject a mix of individual epitopes?

• Broader Coverage: A multi-epitope vaccine can elicit responses against various parts of a pathogen or even multiple strains.
• Enhanced Immunogenicity: Combining epitopes with an adjuvant in a single construct can be more potent.
• Overcoming HLA Restriction: Including multiple T-cell epitopes increases the chance that individuals with diverse HLA types (immune system genes) can mount an effective response.
• Simplified Production & Delivery: One molecule can be easier to produce, purify, and administer than a mixture of many small peptides.

This approach is often called a “string-of-beads” vaccine, where epitopes are the “beads” connected by “strings” (linkers), often with an adjuvant at one end.

2.2 Building Blocks of the Construct

A typical multi-epitope vaccine construct has three main types of components:

2.2.1 Adjuvants

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What is an Adjuvant?

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An adjuvant is a substance added to a vaccine to enhance the recipient's immune response to the supplied antigen(s). They help to stimulate a stronger, more durable, and more broadly protective immunity.

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• Purpose: Epitopes alone, especially small peptides, can be weakly immunogenic. Adjuvants act as a “wake-up call” for the immune system.
• Example: β-defensin.
  • β-defensins are antimicrobial peptides that also have immunomodulatory properties. They can recruit and activate antigen-presenting cells (APCs) like dendritic cells, which are crucial for initiating T-cell responses.
• Other Examples: Flagellin (a TLR5 agonist), MPLA (a TLR4 agonist), CpG ODN (a TLR9 agonist), Alum (aluminum salts).
• Placement: “at the N-terminus”
  • Rationale: The N-terminus is often chosen for adjuvants because:
    1. It can ensure the adjuvant is readily accessible to immune receptors.
    2. It might prevent interference with the folding or presentation of the downstream epitopes.
    3. Some adjuvants have structural requirements or functional domains that are best preserved when placed at an extremity.

2.2.2 Selected Epitopes

These are the high-quality epitopes you filtered in the previous step (e.g., Epitope 1 and Epitope 5 from our hypothetical example). The choice and order can be strategic:

• CTL and Th Epitopes: Often, constructs include both Cytotoxic T Lymphocyte (CTL) epitopes (for killing infected cells) and Helper T Lymphocyte (Th) epitopes (for helping activate CTLs and B cells).
• Order: The order might influence immune processing or the potential for creating unwanted “junctional epitopes” (new epitopes formed at the boundary of two linked components). Sometimes, Th epitopes are placed flanking CTL epitopes, or interspersed.

2.2.3 Linkers

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What are Linkers in a vaccine construct?

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Linkers are short amino acid sequences used to connect different components (like an adjuvant to an epitope, or one epitope to another) in a multi-epitope vaccine. They provide spacing and flexibility, and can aid in antigen processing and presentation.

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• Purpose:

  1. Separation: Prevent epitopes from interfering with each other’s folding or presentation.
  2. Flexibility/Rigidity: Provide appropriate conformational freedom or structural definition.
  3. Antigen Processing: Some linkers contain cleavage sites for proteasomes or other proteases, which helps in the generation of individual epitopes for presentation on MHC molecules.
  4. Preventing Neo-epitopes: Poorly designed junctions can inadvertently create new, unintended epitopes (junctional epitopes) that might be irrelevant or even harmful. Linkers can minimize this.

• Examples Given & Their Likely Rationale:

  • EAAAK: A rigid linker, known to promote α-helical structure. Provides good separation and defined conformation. Often used to separate domains in fusion proteins.
  • GPGPG: A flexible linker. The proline residues introduce kinks, and glycines provide rotational freedom. This flexibility can help epitopes fold independently and expose protease cleavage sites, aiding in antigen processing.
  • AYY, AK: These are very short linkers.
    • AYY (Alanine-Tyrosine-Tyrosine): Tyrosine is somewhat bulky. This might provide minimal, slightly rigid spacing.
    • AK (Alanine-Lysine): Lysine is charged. This could be used for minimal separation while maintaining solubility or providing a specific charge characteristic at the junction.
    • General Rationale for Short Linkers: Used when minimal separation is needed, or to simply break a sequence and potentially introduce a subtle structural turn or cleavage point.
  • KFERQ (often KFERQ for chaperone-mediated autophagy, or perhaps KFER as a short custom linker):
    • If KFERQ, it’s a motif recognized by Hsc70 for targeting proteins to chaperone-mediated autophagy, which could influence antigen processing pathways.
    • If just KFER, it’s a short tetrapeptide. Its specific properties would depend on context, but it’s likely chosen for specific spacing or to introduce particular amino acid characteristics. The context implies it’s used similarly to other linkers for epitope separation.

• Choosing Linkers: The choice of linker depends on the desired properties between connected components. For instance, a flexible linker like GPGPG might be used between epitopes to facilitate processing, while a more rigid one like EAAAK might be used to connect an adjuvant to the first epitope to maintain structural integrity.

2.3 Designing the Construct: A Conceptual Workflow

The design process is like assembling a custom piece of equipment from pre-validated parts.

1. Start with the Adjuvant: Place the chosen adjuvant (e.g., β-defensin sequence) at the N-terminus. Adjuvant -

2. Add the First Epitope with a Linker: Connect the adjuvant to the first selected epitope using an appropriate linker. The choice of linker here (e.g., EAAAK) might be to provide some structural separation from the adjuvant. Adjuvant - Linker1 - Epitope1 - Example: β-defensin - EAAAK - SLFNTVATL -

3. Add Subsequent Epitopes with Linkers: Connect the remaining selected epitopes one by one, each separated by a linker. The linkers between epitopes (e.g., GPGPG, AYY) are often chosen to facilitate antigen processing and prevent steric hindrance. Adjuvant - Linker1 - Epitope1 - Linker2 - Epitope2 - Linker3 - Epitope3 ... Example with two epitopes: β-defensin - EAAAK - SLFNTVATL - GPGPG - WEFVNTPPL

   Consideration for Linker Choice:

   • To promote cleavage and processing into individual epitopes, linkers like GPGPG or those containing known protease sites might be preferred between epitopes.
   • To avoid repetitive sequences if many GPGPG linkers are used, one might alternate with other short linkers like AYY or AK.

4. Optional: C-terminal Elements: Sometimes, additional sequences are added at the C-terminus, such as:

   • Purification Tags: e.g., a polyhistidine tag (His-tag) for easier purification of the recombinant protein.
   • End Sequences: Sequences to ensure proper termination of translation or enhance stability. (Though not explicitly mentioned in the topic, this is common practice).

Visualizing the Construct:

2.4 Interactive Checkpoint 2: Vaccine Construct Design

Let’s see what you’ve learned about assembling these constructs.

Complete the sentence about multi-epitope vaccine design:

A typical multi-epitope vaccine construct begins with an at the N-terminus to boost immunogenicity. This is followed by the selected , which are separated from each other by short amino acid sequences called . These separators, such as GPGPG or EAAAK, help in proper folding, prevent steric hindrance, and can facilitate antigen .

Check Answers

Linkers are primarily used in vaccine constructs to increase the overall molecular weight, making the vaccine more stable.

True False

Which of the following linkers is generally considered a 'rigid' linker, often promoting an α-helical structure?

• GPGPG
• AYY
• EAAAK
• KFER

3. Conclusion: From In Silico Design to Potential Vaccine

You’ve now learned the rationale and methods behind filtering epitopes based on their physicochemical properties and designing a multi-epitope vaccine construct in silico. These computational steps are vital for:

• Rational Vaccine Design: Making informed choices to maximize the potential efficacy and manufacturability of a vaccine candidate.
• Resource Optimization: Saving time and resources by computationally screening and designing before expensive and time-consuming laboratory experiments.

What’s Next? A computationally designed vaccine construct isn’t the end of the story. The sequence would then typically undergo further in silico validation (e.g., prediction of immunogenicity, allergenicity, population coverage, 3D structure modeling, docking with immune receptors) before it’s synthesized and tested in vitro (cell-based assays) and in vivo (animal models) to evaluate its actual safety and efficacy.

Mastering these bioinformatic techniques provides you with powerful tools to contribute to the exciting and impactful field of vaccine development!