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. 2015 May 26;10(5):e0126428.
doi: 10.1371/journal.pone.0126428. eCollection 2015.

Identification of Non-HIV Immunogens That Bind to Germline b12 Predecessors and Prime for Elicitation of Cross-clade Neutralizing HIV-1 Antibodies

Zheng Yang  1 , Jingjing Li  1 , Qingsheng Liu  1 , Tingting Yuan  1 , Yanyu Zhang  1 , Li-Qing Chen  2 , Qi Lou  2 , Zehua Sun  1 , Huazhong Ying  2 , Jianqing Xu  3 , Dimiter S Dimitrov  4 , Mei-Yun Zhang  5
Affiliations

Identification of Non-HIV Immunogens That Bind to Germline b12 Predecessors and Prime for Elicitation of Cross-clade Neutralizing HIV-1 Antibodies

Zheng Yang et al. PLoS One. .

Abstract

A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist

Figures

Fig 1
Fig 1. Identification of five (poly)peptides that bind to b12 germline and iAbs.
A: Flow cytometry of yeast-displayed P1-4 and P6 stained with biotinylated human (hub12ger) and macaque (Rhb12ger) b12 germline scFvs, and human (hub12iAbs) and macaque (Rhb12iAbs) b12 iAbs. Isotype control: human mAb unrelated to Env gp120. B: Binding of human and macaque b12 germline scFvs, iAbs and mature IgG1 b12 at 50 μg/ml to the isolated (poly)peptides by ELISA. IgG1s 2F5 and VRC01 were included as controls. The blank control contained no primary antibody. C: Sequences and origins of the isolated (poly)peptides. Underlined amino acids were added when constrained (poly)peptides were synthesized as conjugates to KLH.
Fig 2
Fig 2. Characterization of bleed 6 sera and IgGs for binding and neutralization activities.
A-B: Titration of bleed 6 sera for gp140SF162 trimer (A) and RSC3 (B). C-D: Competition of rabbit IgGs with mature human IgG1 b12 for binding to gp140SF162 (C) and RSC3 (D). Mature IgG1 b12 was included as control. For “blank”, no IgG was added. E: Neutralization breadth. Percent isolates neutralized by rabbit IgGs from each group (IC50 below 150 μg/mL) is shown. IgG1 b12 was tested at a maximum concentration of 20 μg/ml in the TZM-bl assay. IC50 > 20 μg/mL was defined as non-neutralizing. One-way ANOVA was used for statistical analyses using SPSS. Pre-immunization rabbit IgGs from each rabbit were also tested and all IC50s were > 150 μg/mL (not shown).
Fig 3
Fig 3. Profiling of bleed 4 and 6 rabbit IgGs from each group with the consensus clade B 15-mer peptides.
Profiles of bleed 4 and 6 rabbit IgGs from group 1 (A), 3 (B), 4 (C), 5 (D) and 6 (E) with the consensus clade B 15-mer peptides are shown. Locations of CD4bs peptides, b12 epitope (b12e) and VRC01 epitope (VRC01e), as well as Env variable loops and HR regions (HR1 and HR2) are indicated according to “Neutralizing Antibody Resources” (http://www.hiv.lanl.gov/content/immunology/neutralizing_ab_resources.html). Two serum samples from a same group were profiled separately, but the addition results of the two samples from the same group are shown. X axis: position of the peptides. Y axis: OD450nm.
Fig 4
Fig 4. Comparison of profiles of different bleed 6 rabbit IgGs with consensus clade B 15-mer peptides.
A-B: Profiles of rabbit IgGs from the immunization with or without (poly)peptide priming are compared. The profile of rabbit IgGs from immunization with P1-4 alone is included as a control. C: Profiles of rabbit IgGs from immunization with the same priming, but different order of the boosting immunogens are compared. D: Profiles of rabbit IgGs from two control groups without (poly)peptide priming and with different order of the boosting immunogens are compared. Locations of CD4bs peptides, b12 epitope (b12e) and VRC01 epitope (VRC01e), as well as Env variable loops and HR regions (HR1 and HR2) are indicated according to “Neutralizing Antibody Resources” (http://www.hiv.lanl.gov/content/immunology/neutralizing_ab_resources.html). Two serum samples from a same group were profiled separately, but the addition results of the two samples from the same group are shown. X axis: position of the peptides. Y axis: OD450nm.
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This work was supported by a General Research Fund (GRF) (#785112) from Hong Kong Research Grants Consuls (RGC) (http://www.ugc.edu.hk/eng/rgc/index.htm) and the China 12th 5-year Mega project for HIV/AIDS (#2012ZX10001006) (http://www.most.gov.cn/tztg/index.htm) to M-Y Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.