415 – Antigen Affinity Ceiling for Selection of Functional Mutations and HIV-1 Neutralization

Background

The mechanism by which B cells sense antigen affinity and initiate signaling that leads to full B activation and differentiation remains unclear. Understanding the relationship between antigen affinity/kinetic rates and selection of functional mutations is critical for HIV-1 Envelope (Env) protein immunogens designed to target broadly neutralizing antibody (bnAb) precursors. Previously, we had reported that B cell signaling strength is significantly associated with antigen binding kinetic on-rate and not affinity (K_D). Here, we performed immunization studies in a CD4 binding-site specific bnAb (CH235) precursor knock-in (KI) mouse model to test the hypothesis that a prime immunogen with the optimal K_D will favor germinal center (GC) selection of functional mutations and induction of autologous virus neutralization.  

Methods

Affinity (K_D) and kinetic rates of HIV Env (CH505M5 and CH505TF) trimers were measured by SPR. B cell activation was measured using calcium flux and phosphokinase signaling assays. Each group (6) of CH235UCA KI mice (expresses CH235 unmutated common ancestor BCRs) were immunized (4x) with each of 5 trimers of varying affinities (10.6nM-23.5µM). Sera antibody titer and the GC and memory B cells frequency were analyzed using ELISA and flow cytometric phenotyping, respectively. Neutralization titers by pseudovirus TZM-bl neutralization assay and NGS was done to identify frequencies of key functional mutations. 

Results

Increasing sera IgG binding titers against each of the trimer immunogen was observed. The weaker/moderate affinity (K_D=4.7/0.56µM) groups showed higher frequencies of Env-specific IgG+ GC B cells compared to the high affinity group (K_D=16.7nM). Both the weaker/moderate affinity immunogens induced significantly higher ID₅₀-neutralization titers when compared to either the high (K_D=10.6-16.7nM) or the weakest (K_D=23.5µM) affinity immunogens (p=0.0022). A significantly higher frequency of the key improbable mutation (K19T) was observed in the weaker affinity groups (p=0.0087; p=0.0043). Importantly, several previously defined key V_H  mutations (K19T+N52D+S57R) were significantly increased in the weaker affinity group (K_D=4.7µM) (p=0.0022). Thus, selection of key functional mutations was favored by weaker over higher affinity immunogens.

Conclusions

A lower and an upper affinity threshold set boundaries for selection of key functional mutations and the induction of higher titers of autologous HIV-1 neutralizing antibodies.  

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