Background Current HIV-1 envelope glycoprotein (Env) vaccines are unable to induce cross-reactive neutralizing antibodies. coincided with CrNA development. These included a statistically shorter variable region 1 and a lower probability of glycosylation as implied by a high ratio of NXS NXT glycosylation motifs. Furthermore lesser probability of glycosylation at position 332 which is usually involved in the epitopes of many broadly reactive neutralizing antibodies was associated with the induction of CrNA. Finally Sequence Harmony recognized a number of amino acid changes associated with the development of CrNA. These residues mapped to numerous Env subdomains but in particular to the first and fourth variable region as well as the underlying α2 helix of the third constant region. Conclusions These findings imply that the development of CrNA might depend on specific characteristics of early Env. Env signatures that correlate with the induction of CrNA might be relevant for the design of effective HIV-1 vaccines. sequences from early HIV-1 clonal variants in clade B infected individuals that developed diverse levels of CrNA later on during contamination. We found that CrNA development correlated with early HIV-1 variants with shorter V1 regions lower probability of glycosylation and specific amino acid usage. These properties might open up avenues for vaccine design. Results Short V1 sequences correlate with the development of cross-reactive neutralizing activity To study Env determinants that may influence the induction of CrNA we retrospectively generated sequences from early HIV-1 variants in 31 individuals who at 2-4 years post-SC experienced diverse levels of CrNA in their serum (Physique?1) [35 79 We chose this experimental setup because contemporaneous viruses usually already have escaped from CrNA [25 34 and early viruses are proposed to be a major determinant for the induction of CrNA [80]. The individuals were matched for time between SC and CrNA measurement time between SC and clonal computer virus isolation CD4+ T cells/μl blood at set-point and viral weight at set-point (Additional file 1: Physique S1). Data on HIV-1 neutralizing activity in serum EGT1442 were available from previous studies (n = 292) [35 40 79 In short; sera were tested by Monogram Biosciences [81] for cross-reactive neutralizing activity in a pseudovirus assay including six tier two viruses with Envs from main isolates of HIV-1 subtypes A (94UG103) B (92BR020 and JRCSF) C (93IN905 and MGRM-C-026) and CRF01_AE (92TH021). This six viral panel covered 93% of the variance in neutralization of a larger pseudovirus panel (n = 15) [39]. It has been shown that classification of CrNA in sera as decided on this six computer virus panel was highly correlated with CrNA determination on a larger 23 computer virus panel [35]. The geometric mean IC50 titers in the sera of the selected individuals against a panel of 6 HIV-1 variants varied from 20 to 297; with an average of 98 (observe Table?1 for details). Phylogenetic analysis of all sequences using either neighbour-joining or maximum-likelihood methods revealed EGT1442 PPP2R1B clustering of sequences per individual excluding contamination but clustering of HIV-1 Env sequences from individuals EGT1442 with comparable geometric mean IC50 titer was not observed (Additional file 2: Physique S2). We observed that this geometric mean IC50 titer in serum correlated weakly with the mean length of V1 ((Physique?2B EGT1442 r = ?0.36; p = 0.049) but not with overall mean gp160 length (Determine?2A) nor with the total length of either the variable or the conserved regions (data not shown r = ?0.21; p = 0.25 and r = ?0.052; p = 0.80 respectively). Physique 1 Sampling of viruses EGT1442 and sera. Time bars showing the period in which viruses were isolated and the period in which sera were obtained for assessment of neutralizing activity. The white grey and dark grey bars represent the individuals with non-CrNA (n=9) … Table 1 Individual’s data summary Physique 2 Short V1 sequences and lower probability of overall glycosylation correlate with the development of cross-reactive neutralizing activity. Scatter plots of individual’s geometric mean IC50 titer across the 6 computer virus panel (x-axis) versus sequence … Lower.