Novel trimer-only

Background: Soluble immunogens that properly mimic the HIV-1 envelope glycoprotein (Env), such as SOSIP and native flexibly linked (NFL) trimers, are used as constituents for a number of HIV-1 vaccine approaches. However, most NFL and SOSIP constructs are not only expressed as trimers, but also produce undesired monomers and dimers that expose non-neutralizing epitopes. Usually, chromatography procedures are used to isolate the desired trimers for vaccination. However, this also implies that conventional SOSIP and NFL constructs might be less suitable for genetic vaccination. Here, we describe a novel HIV-1 Env construct that was designed to express as trimers only (TO). Methods: First, we combined the TO design with BG505 env to generate a BG505 TO-SOSIP construct as a proof-of-concept. Next, we used TO-SOSIP as a design template to generate a number of different HIV-1 Env immunogens: 1) TO-SOSIP in which immunodominant strain-specific glycan holes were hidden by glycan masking (GM) (TOSOSIP.GM). 2) TO-SOSIP containing germline-targeting (GT) mutations that enable binding by naive precursors of CD4bs broadly neutralizing antibodies (bNAbs) (TO-SOSIP.GT). Results: Transient transfection followed by lectin affinity chromatography showed that the BG505 TO-SOSIP construct expressed only as trimers, of which >85% displayed a native-like conformation, while BG505 SOSIP and NFL-SOSIP preparations contained significant amounts of dimers and monomers. Furthermore, TO-SOSIP interacted efficiently with quaternary dependent bNAbs, such as PGT145 and PGT151, while showing weak binding to most non-NAbs. Both glycan masked and germline targeting variants also expressed only trimers. TO-SOSIPGM showed negligible reactivity to glycan hole-targeting antibodies, while several TO-SOSIPGT variants showed efficient binding to inferred germline versions of several CD4bs bNAbs. Remarkably, the TO design approach also allowed us to generate influenza hemagglutinin (HA) immunogens that express only as trimers without heterologous trimerization domains. Conclusions: The TO design is a useful platform for generating Env and HA immunogens and is especially suitable for genetic vaccination purposes. In the context of DNA prime and protein boost vaccination strategies, the glycan masked and germline targeting TO-SOSIP constructs could prove essential for the early activation of potentially cross-neutralizing epitopes.