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. 2012 Jan;86(1):128-42.
doi: 10.1128/JVI.06363-11. Epub 2011 Oct 26.

Engineering, expression, purification, and characterization of stable clade A/B recombinant soluble heterotrimeric gp140 proteins

George Sellhorn  1 , Zane Kraft, Zachary Caldwell, Katharine Ellingson, Christine Mineart, Michael S Seaman, David C Montefiori, Eliza Lagerquist, Leonidas Stamatatos
Affiliations

Engineering, expression, purification, and characterization of stable clade A/B recombinant soluble heterotrimeric gp140 proteins

George Sellhorn et al. J Virol. 2012 Jan.

Abstract

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is composed of two noncovalently associated subunits: an extracellular subunit (gp120) and a transmembrane subunit (gp41). The functional unit of Env on the surface of infectious virions is a trimer of gp120/gp41 heterodimers. Env is the target of anti-HIV neutralizing antibodies. A considerable effort has been invested in the engineering of recombinant soluble forms of the virion-associated Env trimer as vaccine candidates to elicit anti-HIV neutralizing antibody responses. These soluble constructs contain three gp120 subunits and the extracellular segments of the corresponding gp41 subunits. The individual gp120/gp41 protomers on these soluble trimers are identical in amino acid sequence (homotrimers). Here, we engineered novel soluble trimeric gp140 proteins that are formed by the association of gp140 protomers that differ in amino acid sequence and glycosylation patterns (heterotrimers). Specifically, we engineered soluble heterotrimeric proteins composed of clade A and clade B Env protomers. The clade A gp140 protomers were derived from viruses isolated during acute infection (Q168a2, Q259d2.17, and Q461e2), whereas the clade B gp140 protomers were derived from a virus isolated during chronic infection (SF162). The amino acid sequence divergence between the clade A and the clade B Envs is approximately 24%. Neutralization epitopes in the CD4 binding sites and coreceptor binding sites, as well as the membrane-proximal external region (MPER), were differentially expressed on the heterotrimeric and homotrimeric proteins. The heterotrimeric gp140s elicited broader anti-tier 1 isolate neutralizing antibody responses than did the homotrimeric gp140s.

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Figures

Fig 1
Fig 1
Optimizing the coexpression of clade A and B gp140s. 293 cells were cotransfected with the indicated ratios of two plasmids, one expressing the His-tagged clade B gp140 and the other expressing the FLAG-tagged gp140. The relative expression of the clade A and clade B gp140s was determined with the use of an anti-FLAG MAb, specific for the FLAG-tagged clade A gp140, or a clade B-specific anti-V3 MAb, P3E1, as discussed in detail in Materials and Methods. Anti-HIV polyclonal sera (Env 2-3) were used to monitor the overall Env expression. Molecular weight standards are shown at the left of each panel.
Fig 2
Fig 2
Purification of heterotrimeric gp140s. (A) SDS-PAGE of the purified homotrimeric and heterotrimeric gp140 preparations. (B) Blue native PAGE of the same proteins as those in panel A. In both panels, the arrows indicate the presence of the 2 gp140s that migrate differently (heterotrimeric preparations) on the gel due to differences in molecular masses of the clade A and B gp140s (SDS-PAGE) or charge/mass ratios (native PAGE) (*, size differences between these clade A and clade B Envs determined by this method are negligible). Env visualization on both gels was done with Coomassie blue staining. (C) Detection of the His tag (clade B gp140) and/or FLAG tag (clade A gp140) by anti-His MAb or anti-FLAG MAbs of the indicated protein preparations. In the case of the heterotrimeric preparations, both the clade A and clade B gp140s are visualized by the use of both the anti-His MAb and anti-FLAG MAb. (D) Western blot assays of blue native gels of the indicated Env preparations. Envs were detected with either a purified pooled HIV+ IgG preparation (top left panel), an anti-FLAG MAb (top right panel), or an anti-His MAb (bottom left panel).
Fig 3
Fig 3
Relative epitope exposure on homotrimeric and heterotrimeric gp140s. The binding of the indicated MAbs to the indicated heterotrimeric and homotrimeric gp140s was determined as described in Materials and Methods. The statistical analysis compares the measured value for MAb binding to the heterotrimers to the theoretical value expected if there were no change in relative epitope exposure. Unpaired t test with Welch's correction. OD450, optical density at 450 nm.
Fig 4
Fig 4
Immunogenicities of the V1, V2, and V3 regions from clade A Envs. Animals were immunized with Q168/SF162L (A), Q461/SF162L (B), Q259/SF162NL (C), or Q461/SF162NL (D). Antibody reactivities to peptides derived from the V1, V2, and V3 regions of each clade A Env were determined by multiplex Luminex assay. Antibody reactivity to monomeric gp140 (m-gp140) was also determined. ■, sera from animals immunized with heterotrimeric gp140; ●, sera from animals immunized with mixtures of homotrimers. MFI, mean fluorescent intensity. V1, peptide spanning the V1 loop; V1V2 Junc, peptide spanning the N-terminal side of the V2 loop; V2 Crn, peptide spanning the crown portion of the V2 loop; V2 Ct, peptide spanning the carboxy-terminal site of the V2 loop; V3 Nt, peptide spanning the amino-terminal region of the V3 loop; V3 Ct, peptide spanning the carboxy-terminal region of the V3 loop (Table 1 shows peptide sequences). Statistical analyses were performed with the unpaired t test with Welch's correction analysis.
Fig 5
Fig 5
Immunogenicities of the V1, V2, and V3 regions from the clade B SF162 Env. The experiment was similar to that in Fig. 4, but the peptides were derived from the SF162 Env.
Fig 6
Fig 6
Immunogenicities of the 4E10 and 2F5 epitopes in the MPER of gp41. Sera were diluted at 1:100, and the antibody reactivities to the 4E10 (A) and 2F5 (B) peptides were determined (optical density at 450 nm [OD450]). Each point indicates an individual animal (3 animals per immunization group). The immunogens are indicated under the x axis. Heterotrimeric immunogens are indicated by a slash—for example, Q168/SF162L—while immunogens composed of the mixture of homotrimers are indicated by a plus sign—for example, Q168+SF162L. Statistical analyses were performed with the unpaired t test with Welch's correction analysis.
Fig 7
Fig 7
Immunogenicities of the His and FLAG tags. All clade A gp140s were tagged with FLAG, and the clade B gp140 was tagged with His. The anti-tag antibody responses in immune sera were determined at a 1:25 serum dilution, as described in Materials and Methods. Each bar indicates an individual animal. (A to D) Anti-His antibody responses. (E to H) Anti-FLAG antibody responses. (A and E) Sera from animals immunized with Q461 and SF162L; (B and F) sera from animals immunized with Q168 and SF162L; (C and G) sera from animals immunized with Q259/SF162NL; (D and H) sera from animals immunized with Q461/SF162NL.
Fig 8
Fig 8
Comparison of serum neutralizing activities in heterotrimeric and homotrimeric sera. (A) Each point indicates the log percent neutralization of a given serum against a given virus tested, irrespective of the immunogen. Only tier 1 viruses and one tier 2 virus (Q259) were included in this analysis. (B) The points represent the percentage of animals within each immunization group (n = 3) that neutralized a given virus. Red circles, sera from animals immunized with heterotrimeric gp140s; blue squares, sera from animals immunized with the mixture of homotrimeric gp140s. Statistical analyses were performed with Welch's t test.
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