Vaccination of Nonhuman Primates Elicits a Broadly Neutralizing Antibody Lineage Targeting a Quaternary Epitope on the HIV-1 Env Trimer

1. Research Background

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is the primary target of neutralizing antibodies, but its high variability poses challenges for vaccine development. Broadly neutralizing antibodies (bnAbs) are rare in natural infections and typically require years to emerge. Although Env trimer mimetics (e.g., BG505 SOSIP.664) can stably present native conformations, previous vaccine studies have mostly induced strain-specific neutralizing antibodies with limited coverage of globally prevalent HIV-1 subtypes. This study aimed to explore the induction mechanism of bnAbs targeting the conserved CD4 binding site (CD4bs) through a sequential immunization strategy using glycosylation-modified heterologous trimers.

2. Source of the Paper

• Research Team: A multinational collaboration led by Fabian-Alexander Schleich (Karolinska Institutet), Shridhar Bale, and Javier Guenaga (The Scripps Research Institute).
  
• Corresponding Authors: Gunilla B. Karlsson Hedestam (Karolinska Institutet) and Richard T. Wyatt (The Scripps Research Institute).
  
• Journal: Immunity (June 10, 2025, Volume 58), published by Elsevier.
  

3. Research Process and Results

1. Immunogen Design and Animal Experiments

Key Steps:

• Immunogen Engineering:
  
  • 16055 dg4 NFL trimer: Deletion of four N-glycosylation sites near CD4bs (N276/N301/N360/N463) to expose conserved epitopes.
    
  • Introduction of an interprotomer disulfide bond (CC3: A501C-A662C) to enhance trimer stability (Figure 1).
    
• Animal Model:
  
  • 12 rhesus macaques (non-human primates, NHPs) divided into two groups:
    
  • Group 1 (NHP1-6): Immunized with soluble trimers;
    
  • Group 2 (NHP7-12): Immunized with trimer-liposome conjugates.
    
  • Immunization Protocol:
    
  1. Pre-immunization: BG505/CH505 chimeric trimers + AMC016 trimer (targeting fusion peptide);
     
  2. Prime immunization: 16055 dg4 NFL trimer (focusing on CD4bs);
     
  3. Boosts: Heterologous fully glycosylated trimers (ZM233, CH119, JR-FL, etc.) at 12-week intervals (Figure 2a).
     

Experimental Results:

• Serum Neutralization Activity:
  
  • After two immunizations (post-2), NHP1 and NHP9 showed cross-neutralization against tier 2 viruses (e.g., JR-FL) (Figure 2c);
    
  • After six immunizations (post-6), neutralization breadth expanded to cover 70% of an 84-virus global panel (Figure 4).
    

2. Monoclonal Antibody Isolation and Characterization

Key Steps:

• Memory B Cell Sorting:
  
  • Antigen-specific B cells sorted via flow cytometry using biotinylated JR-FL NFL trimer probes (Figure S3a).
    
  • 185 paired heavy/light chain sequences isolated from NHP1, belonging to 49 clonal lineages.
    
• Antibody Properties:
  
  • LJF-0034 Lineage:
    
  • High somatic hypermutation (SHM: 12% heavy chain, 9% light chain);
    
  • Targets a quaternary epitope spanning CD4bs and V3 regions on adjacent protomers (Figure 5c);
    
  • Neutralization breadth: Covers 66% of 84 viruses (Figure 4), with mean IC50 of 2.14 μg/mL (LJF-0085).
    

Structural Insights:

• Cryo-EM Analysis:
  
  • Binding Mode: Light chain binds CD4bs (avoiding N276 glycan), heavy chain engages V3 (Figure 5d-e);
    
  • Epitope Features:
    
  • Salt bridges between light chain and conserved residues (D457/K282) (Figure 6a);
    
  • Heavy chain interacts with V3 residue R308 via HCDR2 (Figure 6c).
    

3. Natural Resistance Mechanisms and Epitope Optimization

Key Findings:

• Resistance Sites:
  
  • Neutralization resistance in AE recombinant strains (e.g., C1080.c3) linked to mutations in V3 (T305/T308/V316/R319) and CD4bs (G429/D474/R476) (Figure 7b);
    
  • Reversion mutations (e.g., T308R) restored neutralization sensitivity (Figure 7c).
    
• Glycan Impact:
  
  • Removal of N197 glycan significantly enhanced neutralization potency, whereas N276 glycan had no effect (Figure 7c).
    

4. Conclusions and Significance

1. Scientific Impact:

   • First demonstration that sequential heterologous trimer immunization can induce bnAbs targeting quaternary Env epitopes;
     
   • Reveals structural basis for cooperative CD4bs-V3 epitopes, informing vaccine design.
     

2. Translational Value:

   • Glycosylation engineering (e.g., N197 deletion) optimizes immunogen design;
     
   • LJF-0034 lineage antibodies are candidates for passive immunotherapy.
     

5. Highlights

• Innovative Methods:
  
  • Interprotomer disulfide (CC3) stabilizes trimer conformation;
    
  • Personalized Ig genotyping (IgDiscover algorithm) tracks antibody evolution.
    
• Key Discoveries:
  
  • Quaternary epitope bnAbs evade glycan barriers via bifurcated binding;
    
  • Natural resistance mechanisms guide vaccine strain optimization (e.g., V3 conservation).
    

6. Additional Information

• Data Availability:
  
  • Antibody sequences (GenBank), cryo-EM structures (EMDB/PDB) publicly accessible;
    
  • Code and algorithms on GitLab (IgDiscover).
    
• Clinical Translation:
  
  • 16055 dg4 NFL trimer in Phase I clinical trials (HVTN 313).