Dataset Information

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

ABSTRACT: Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8?g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan "hole" naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

SUBMITTER: Bale S

PROVIDER: S-EPMC5976746 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

Bale Shridhar S Martiné Alexandra A Wilson Richard R Behrens Anna-Janina AJ Le Fourn Valérie V de Val Natalia N Sharma Shailendra K SK Tran Karen K Torres Jonathan L JL Girod Pierre-Alain PA Ward Andrew B AB Crispin Max M Wyatt Richard T RT

Frontiers in immunology 20180524

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a hi ...[more]

PMID: 29881382

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Project description:The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.

Project description:The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.

Project description:A vaccine regimen that elicits broadly neutralizing antibodies (bNAbs) is a major goal in HIV-1 vaccine research. In this study, we assessed the immunogenicity of the CAP256 superinfecting viral envelope (CAP256 SU) protein delivered by modified vaccinia virus Ankara (MVA) and DNA vaccines in different prime-boost combinations followed by a soluble protein (P) boost. The envelope protein (Env) contained a flexible glycine linker and I559P mutation. Trimer-specific bNAbs PGT145, PG16, and CAP256 VRC26_08 efficiently bound to the membrane-bound CAP256 envelope expressed on the surface of cells transfected or infected with the DNA and MVA vaccines. The vaccines were tested in two different vaccination regimens in rabbits. Both regimens elicited autologous tier 2 neutralizing antibodies (NAbs) and high-titer binding antibodies to the matching CAP256 Env and CAP256 V1V2 loop scaffold. The immunogenicity of DNA and MVA vaccines expressing membrane-bound Env alone was compared to that of Env stabilized in a more native-like conformation on the surface of Gag virus-like particles (VLPs). The inclusion of Gag in the DNA and MVA vaccines resulted in earlier development of tier 2 NAbs for both vaccination regimens. In addition, a higher proportion of the rabbits primed with DNA and MVA vaccines that included Gag developed tier 2 NAbs than did those primed with vaccine expressing Env alone. Previously, these DNA and MVA vaccines expressing subtype C mosaic HIV-1 Gag were shown to elicit strong T cell responses in mice. Here we show that when the CAP256 SU envelope protein is included, these vaccines elicit autologous tier 2 NAbs.IMPORTANCE A vaccine is urgently needed to combat HIV-1, particularly in sub-Saharan Africa, which remains disproportionately affected by the AIDS pandemic and accounts for the majority of new infections and AIDS-related deaths. In this study, two different vaccination regimens were compared. Rabbits that received two DNA primes followed by two modified vaccinia virus Ankara (MVA) and two protein inoculations developed better immune responses than those that received two MVA and three protein inoculations. In addition, DNA and MVA vaccines that expressed mosaic Gag VLPs presenting a stabilized Env antigen elicited better responses than Env alone, which supports the inclusion of Gag VLPs in an HIV-1 vaccine.

Dataset Information

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

Publications

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

Similar Datasets

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