Hence, we performed Ni-NTA capture ELISA experiments with a comprehensive panel of bnAbs and non-nAbs, targeting all relevant sites in the Env protein (Physique 3). of ConCv5 KIKO as novel Env immunogens for use within the European Unions H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development. Keywords: HIV vaccine, envelope, stabilized trimer, clade C, consensus, centralized sequence, immunization, immunogen design, antibody response, peptide microarray 1. Introduction The HIV-1 Envelope surface glycoprotein (Env) is usually a trimer of heterodimers composed of non-covalently associated gp120 and gp41 subunits, which mature from a gp160 precursor unpon enzymatic cleavage by cellular proteases. Env mediates binding to the CD4 receptor on immune cells and, upon a series of conformational changes, facilitates contamination (as reviewed by [1]). Antibodies targeting Env in its native conformation can prevent binding to the CD4 receptor or the CCR5 or CXCR4 co-receptor on target cells, or interfere with the fusion process, and thereby prevent infection. However, the Rocuronium bromide induction of neutralizing antibodies (nAbs) is usually hampered by the dense glycan shield on Env, which covers almost the entire protein surface and thereby restricts accessibility for antibodies [2]. This, together with the structural flexibility and high degree of sequence variability of Env (up to 35% across all group M clades [3]) are regarded as the main impediments to the design of a successful vaccine capable of inducing antibodies that can neutralize a broad variety of HIV-1 strains (broadly neutralizing antibodies, bnAbs) (as reviewed by [4,5] as well as others). Therefore, Env immunogen candidates that are stabilized in a native, pre-fusion closed conformation are desirable, and centralized immunogen sequences [3,6] that represent circulating computer virus isolates as closely as possible would be additionally beneficial for the development of breadth. Over the past two decades, HIV-1 Env trimer-based immunogens have undergone fundamental improvements. The non-covalent association of the gp120 and gp41 subunits, which lead to gp120 dissociation and shedding of gp120, particularly on soluble forms of Env, was overcome by two strategies: (i) The introduction of a disulfide bridge between gp120 and gp41 (A501C/T605C) in combination with the I559P substitution in gp41 prevented shedding of gp120 and stabilized the trimer [7,8]. (ii) The replacement of the furin cleavage site by a flexible linker of suitable length allowed cleavage-independent trimers [9]. These strategies contributed to the generation of closed, native-like trimers with reduced exposure of epitopes that induce non-neutralizing antibodies [10]. This is of particular importance, as B-cell responses to off-target immunodominant regions such as V3 (as reviewed by [11]) might compete with other subdominant B-cells, recognizing weakly immunogenic neutralization-relevant regions Rocuronium bromide in the germinal centers [12]. Today, a variety of native-like trimers from different viral clades are available [13,14,15,16,17] and continuous optimization of stabilized Env trimers for favorable immunological traits is usually ongoing (as reviewed by [18]). These immunogens, however, were derived from distinct viral isolates and are therefore likely to induce strain-specific responses with low cross-reactivity. Induction of more broadly reactive antibody responses might be promoted by genetically and antigenically centralized immunogens such as consensus, ancestor, or mosaic proteins, which aim at representing the diversity of circulating viruses. Mosaic immunogens, being enriched for T-cell or B-cell epitopes, have demonstrated their ability to induce improved cellular immune responses Rocuronium bromide [19]. While ancestral sequence reconstruction infers phylogenetic associations, the generation of a consensus sequence utilizes the most frequently observed amino acids in each position of a multiple sequence alignment and therefore provides a sequence most closely related to all input sequences [20]. Notably, an ancestral clade C SOSIP gp140 trimer has proven successful to induce broadly neutralizing nanobodies against HIV-1 in camels [21]. Currently, only a small number of native-like Env trimer constructs derived from consensus sequences are Igf1r being explored. A soluble stabilized Env trimer was generated with a consensus sequence of clade C [22] and the underlying repair-and-stabilization concept (RnS) was successfully transferred to representative isolates from other clades [23]. Neither study, however, assessed the immunogenicity of these trimers. Further, the Env trimer ConM SOSIP, based on a consensus sequence covering the complete group M, induced neutralizing antibody responses in rabbits and macaques [24], and is currently being tested in a clinical trial in.