Stepwise Conformational Stabilization of a HIV-1 Clade C Consensus Envelope Trimer Immunogen Impacts the Profile of Vaccine-Induced Antibody Responses

Abstract

Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union’s H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development.

Systematic comparison of HIV-1 Envelope-specific IgG responses induced by different vaccination regimens: Can we steer IgG recognition towards regions of viral vulnerability?

“…Interestingly, shifting of Env-specific antibody responses away from V3 through concealment or elimination has been achieved, but was not accompanied by an increased production of Tier 2 neutralizing Abs (35,45). In line, studies into stepwise conformational stabilization of Env trimer immunogens demonstrated a decline of V3 reactivity with increasing trimer stabilization, yet also with declining overall Env immunogenicity and V2 responses 27 . These findings, in combination with our results, suggest that a strong V3 response does not negatively interfere with antibody responses to other Env regions and therefore the pursuit of inducing of bNAbs and binding antibodies against regions of putative viral vulnerability does not have to be mutually exclusive.…”

“…The peptide microarray used in our study, manufactured by JPT (Berlin, Germany), was designed to map IgG recognition of linear HIV-1 Env regions in preclinical and clinical vaccine studies and has been described previously ( 27 ). The array consists of 1034 15mer peptides with an 11 amino acid overlap to cover the whole gp160 extracellular domain of the HIV-1 Env.…”

“…Peptide microarrays were used according to the manufacturer’s instructions ( ) with minor modifications, as described before ( 25 , 27 ). The arrays were printed in a 4-well-system and each peptide was printed on the array in triplicates.…”

“…Envelope sequence analyses of breakthrough infections confirmed the selective pressure of V2-specific antibody responses in the RV144 trial ( 13 – 15 ) and further studies showed a parallel decline of vaccine efficacy and the level of anti-V2 IgG responses over time ( 12 , 16 – 18 ). During natural infection, antibodies against the highly variable V2 region are found in less than 50% of infected individuals ( 19 , 20 ) while anti-V3 antibodies can be found in almost all naturally HIV-1 infected individuals and are elicited by most vaccination regimen tested so far ( 18 , 21 – 27 ). The V3 sequence is the most conserved of all the variable Env regions ( 28 ) and important for the pathogenicity of the virus ( 29 ).…”

Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVCEnv) covalently complexed to two-domain CD4S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits

“…The CD4-bound conformation has not been widely explored, particularly in terms of immunogen development, as prefusion (ligand-free) states are known to favour neutralizing antibodies epitopes [34] , [35] , including conformational ones [9] , [36] while non-neutralizing antibodies sometimes target epitopes exposed upon CD4 binding such as the CD4-induced (CD4i) and the crown of the third hypervariable (V3) loop [37] , [38] epitopes. As such, immunogen development strategies have sought to maximize prevention of immunodominant (neutralization-hypersensitive) regions, targets of non-neutralizing antibodies, by engineering Envs with increasing mutations for structural stability of the trimer in its native state with much reduced CD4 affinity [39] , [40] . While it cannot be argued that the preferential antigenic properties of these improved trimers have been finely mapped and their improved immunogenicity in preclinical studies well demonstrated [11] , [41] , the limitations of focusing on the closed, ligand-free trimer as an immunogen have been highlighted here and in other studies [21] .…”