Custom HSV-1 gB Protein Expression for Subunit Vaccine Research | AtaGenix

Study Context

Herpes simplex virus 1 (HSV-1) infects over 3.7 billion people globally, causing mucocutaneous lesions and, in rare cases, life-threatening encephalitis. Despite decades of research, no approved prophylactic vaccine exists. A key obstacle has been generating antigens that reliably induce the cellular immune responses needed to control latent HSV-1 infections.

A 2025 study published in Vaccine (DOI: 10.1016/j.vaccine.2025.127241) by Jingping Hu and colleagues investigated whether stabilizing HSV-1 fusion glycoprotein B in its prefusion conformation (H516P mutation) would improve immunogenicity in mRNA and subunit vaccine formats. The study found no significant difference between wild-type gB and gB H516P, but demonstrated that subunit vaccines adjuvanted with QS-21 and CpG ODNs induced robust T-cell responses — a promising finding for HSV-1 vaccine development.

The Challenge

The research team needed two variants of HSV-1 gB — wild-type and the H516P prefusion mutant — as coating antigens for ELISpot and ELISA assays. Both proteins had to meet stringent requirements: high purity for accurate immunological readouts, proper glycosylation to preserve conformational epitopes, and consistent quality between batches so that the wild-type vs. mutant comparison would not be confounded by production variability. Additionally, the H516P mutant introduced a structural constraint that risked affecting expression levels and protein stability.

AtaGenix's Approach

AtaGenix designed a parallel expression and purification workflow for both gB variants:

• Expression System: Chinese hamster ovary (CHO) cells were selected over HEK293 for this project — CHO provides more consistent glycosylation profiles across batches, which was critical for head-to-head comparison of two closely related protein variants.
• Construct Design: Both gB and gB H516P were expressed with C-terminal His-tags. Codon optimization for CHO maximized translational efficiency.
• Purification: Nickel column (Ni-NTA) affinity chromatography as the primary capture step, followed by polishing to achieve high purity. Both variants were processed under identical conditions to eliminate batch-to-batch variability.
• QC and Validation: SDS-PAGE confirmed molecular weight and purity. The research team subsequently validated both proteins in ELISpot (measuring IL-2 and IFN-γ production) and ELISA (measuring gB-specific IgG titers), confirming functional suitability as assay antigens.

Figure 1. Study design and immune response overview. The research compared mRNA and subunit vaccine formats using wild-type gB and prefusion-stabilized gB H516P, evaluating humoral and cellular immune responses in mouse models with QS-21/CpG ODN adjuvants.

Results and Impact

Both AtaGenix-produced proteins performed equivalently in immunological assays, which was itself a key finding — it demonstrated that the H516P prefusion lock did not improve immunogenicity over wild-type gB in these vaccine formats. More importantly for the vaccine field, the subunit formulations adjuvanted with QS-21 and CpG ODNs induced strong IFN-γ and IL-2 T-cell responses, suggesting that cellular immunity (critical for controlling latent herpesvirus) can be effectively elicited with properly formulated protein-based vaccines.

For the client, having access to well-matched, high-purity protein pairs allowed a clean head-to-head comparison that strengthened their publication and informed next-stage vaccine design decisions.

Figure 2. ELISpot validation using AtaGenix-produced gB proteins. Both wild-type gB and gB H516P showed comparable T-cell stimulation, confirming that the recombinant proteins retained native antigenicity and were functionally equivalent as assay reagents.

This case study is based on a published research collaboration. Results may vary depending on target protein, construct design, and project scope. All proprietary client information is subject to NDA.