Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate

Abstract

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

Figure 1

Analysis of the gp140_94UG018 protein. (A) The trimeric form of recombinant gp140 protein was verified and purified by a size-exclusion chromatography. (B) Fractions corresponding to the gp140 trimer were pooled and analyzed by Coomassie stained denaturing SDS PAGE to confirm the purity of the product.

Figure 2

Evaluation of IgG titers elicited in sera of immunized rabbits against the homologous trimeric gp140_94UG018. Five-fold dilutions of heat inactivated sera from pre-immunized (week 0) and immunized rabbits (week 12 and 14) were evaluated in ELISA for their reactivity with homologous gp140. Week 12 and 14 correspond to 4 and 6 weeks after the last antigen administration. The 50% binding is indicated for the week 12 and 14 sera.

Figure 3

Analysis of sequence homology. The gp120 amino acid sequence of the indicated HIV isolates were aligned with the 94UG018 clade A Ugandan isolate. Sequence homology between each isolate vs the 94UG018 isolate is indicated along the whole gp120 sequence.

Figure 4

Evaluation of IgG titers elicited in sera of immunized rabbits against heterologous HIV gp120. Five-fold dilutions of heat inactivated rabbits sera, collected at 4 weeks after the last antigen administration (week 12), were evaluated in ELISA for their reactivity with heterologous gp120s. The 50% binding is indicated.

Figure 5

Heatmap of binding to gp120 peptides’ pool. (A) Intensity of serum binding in ELISA to peptides’ pool covering the constant and variable regions of gp120 is shown as heatmap. Increasing O.D. value, indicating stronger binding, is represented as darker color. (B) Sequence divergence between peptides used as target in ELISA and the 94UG018 clade A Ugandan isolate is indicated along the whole gp120 sequence.

Figure 6

Heatmap of binding to individual gp120 peptides. Intensity of serum binding in ELISA to each overlapping peptide covering the indicated regions of gp120 is shown as heatmap. In each panel stronger binding is represented as darker color.

Figure 7

Neutralization activity of sera from immunized rabbits. (A) Neutralization activity in TZM-bl cells obtained with serial dilutions of IgG (expressed as μg/ml) purified from immunized sera is shown as heatmap for each virus tested. Neutralization activity >50% and <50% is indicated in red and orange, respectively, whereas absence of neutralization activity is indicated in grey. Pre-bleed sera showed a negligible neutralization activity (<10%). (B) Correlation matrix between neutralization activity and peptide binding to envelope gp120 regions. (C) Neutralization activity of TriMab.