Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C

Klasse, Per Johan; LaBranche, Celia C.; Ketas, Thomas J.; Ozorowski, Gabriel; Cupo, Albert; Pugach, Pavel; Ringe, Rajesh P.; Golabek, Michael; Gils, Marit J. van; Guttman, Miklós; Lee, Kelly K.; Wilson, Ian A.; Butera, Salvatore T.; Ward, Andrew B.; Montefiori, David C.; Sanders, Rogier W.; Moore, John P.

doi:10.1371/journal.ppat.1005864

Cited by 146 publications

(296 citation statements)

References 57 publications

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“…This trimer, located on the surface of virus particles, is therefore a focus of vaccine design programs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Of particular relevance is a subset of NAbs with activity, in vitro, against multiple circulating HIV-1 strains.…”

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confidence: 99%

“…These broadly neutralizing antibodies (bNAbs) target relatively conserved regions or motifs that span most of the trimer surface. Accordingly, immunogens that present the epitopes for multiple bNAbs are being developed and evaluated as components of strategies aimed at inducing similar antibodies in test animals and, eventually, humans (3,7,10,(12)(13)(14)(15). While the presence of a bNAb epitope on an immunogen does not guarantee that an appropriate immune response will be elicited, its absence means that such a response is highly unlikely.…”

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confidence: 99%

“…When used as immunogens in small animals, native-like (NL) SOSIP.664 trimers of several genotypes (for example, but not limited to, BG505 from clade A, B41 from clade B, and CZA97.012 from clade C) have elicited strong and consistent NAb responses to the autologous tier 2 (i.e., neutralization-resistant) viruses (3,7,33). This type of narrow-specificity response may serve as a necessary starting point for strategies intended to create neutralization breadth (7,(33)(34)(35)(36)(37)(38)(39).…”

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confidence: 99%

“…When SOSIP.664 changes were introduced into this env gene, the resulting Env proteins included dimers, pseudotrimers, and aggregates, together with a low yield of the desired NL trimers (22). The use of a PGT151 bNAb positive-selection column, however, allowed enough NL CZA97.012 SOSIP.664 trimers to be purified and tested as immunogens in rabbits, where they induced a strong and consistent autologous NAb response (3). To build on these observations, and also to learn more about trimer production in general, we have explored various ways to increase the yield of CZA97.012 trimers with an NL conformation.…”

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confidence: 99%

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Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

Ringe

Ozorowski

Yasmeen

et al. 2017

J Virol

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Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structureand antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes.IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.KEYWORDS HIV-1 vaccine, Env trimers

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

Ringe

Ozorowski

Yasmeen

et al. 2017

J Virol

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“…The prototype is the clade A trimer BG505 SOSIP.664, which is the basis of increasingly high-resolution structures (7)(8)(9)(10)(11) and which has induced autologous neutralizing MAbs against neutralization-resistant (classified as tier-2) viruses in animal experiments (12)(13)(14). Several other SOSIP.664 trimers have since been shown to have desirable antigenicity and immunogenicity properties (1,(12)(13)(14). A substantial body of antigenicity, structural, and immunogenicity data underpins why native-like trimers are now the basis of multiple vaccine design programs.…”

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confidence: 99%

High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers

Sullivan

Sulli

Nilo

et al. 2017

J Virol

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Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membranebound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

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cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV‐1 envelope glycoprotein vaccine candidate

Dey

Cupo

Ozorowski

et al. 2017

Biotech & Bioengineering

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We describe the properties of BG505 SOSIP.664 HIV‐1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native‐like trimers that are the basis for many structure‐guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV‐1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum‐free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size‐exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10. The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native‐like as judged by negative‐stain electron microscopy, and were stable over a multi‐month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native‐like Env glycoprotein trimers of various designs and genotypes.

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Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C

Cited by 146 publications

References 57 publications

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers

cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV‐1 envelope glycoprotein vaccine candidate

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