HIV-1 gp140 Trimer Purification with Long Linker

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Solution Overview

Problem

Current methods for producing HIV-1 gp140 envelope protein trimers fail to generate authentic, stable forms that mimic native spikes, leading to challenges in eliciting neutralizing antibodies effective against diverse HIV strains.

Innovation Solution

A recombinant HIV-1 gp140 protein is engineered with a tag fused through a long linker at the C-terminus, allowing for efficient capture and purification using binding molecules, and optimized for maximal trimer production, ensuring proper cleavage and glycosylation to achieve native-like trimer structures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If current methods are used to produce HIV-1 gp140 envelope protein trimers, then production is simpler, but the trimers produced are not authentic and do not mimic native spikes

Engineering Contradiction:

Improvetrimer authenticityVSAvoidproduction method complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The gp160 precursor protein is segmented into gp120 and gp41 domains through controlled cleavage, allowing the production of authentic trimeric structures that mimic native spikes while maintaining manageable production complexity through stepwise assembly

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A furin cleavage site is introduced as an intermediary element that enables controlled processing of the gp160 precursor into gp120 and gp41, facilitating authentic trimer formation without requiring overly complex production methods

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If tags are fused directly to gp140 for purification, then purification is easier, but the tags interfere with trimer structure and stability

Engineering Contradiction:

Improvepurification easeVSAvoidtrimer stability

Core Design Contradiction:

Ease of manufactureVSStability of the object's composition

Solution Approach 1:

The purification tag is segmented and placed on a separate flexible linker rather than being directly fused to gp140, allowing easy purification while preventing interference with trimer structure and stability

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A flexible linker serves as an intermediary between the purification tag and gp140, enabling efficient capture and purification using binding molecules while maintaining proper cleavage and glycosylation for native-like trimer structures

Inventive Principle:

Principle #24Intermediary (Mediator)

3Shape

If short linkers are used to fuse tags to gp140, then the structure is more compact, but the tags are not accessible for binding during purification

Engineering Contradiction:

Improvestructural compactnessVSAvoidpurification accessibility

Core Design Contradiction:

ShapeVSEase of operation

Solution Approach 1:

The linker extends the tag into a different spatial dimension away from the compact trimer core, making the tag accessible for binding during purification while maintaining overall structural organization

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

Data Source

PatentUS10407470B2Method of inducing an immune response against human immunodeficiency virus comprising administering immunogenic compositions comprising authentic trimeric HIV-1 envelope glycoproteins containing a long linker and tag

Publication Date: 2019.09.10 CATHOLIC UNIV OF AMERICA

AI summary

Provided herein are HIV vaccines that encompasses recombinant trimers that mimic native HIV-1 envelope trimers. Also provided are methods of administering to a subject in need thereof an HIV vaccine provided herein to elicit antibodies against a recombinant trimer in the subject. A recombinant trimer is formed by a recombinant protein comprising a recombinant HIV-1 gp140 fused to a tag through a linker at C-terminus of the recombinant HIV-1 gp140, wherein the linker is sufficiently long so that the tag is accessible for binding by a binding molecule bound on a solid matrix during purification of the recombinant trimer.