Making native-like HIV-1 Env trimers. The importance of both Env design and the purification method. John P. Moore Weill Cornell Medical College

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1 Making native-like HIV-1 Env trimers The importance of both Env design and the purification method John P. Moore Weill Cornell Medical College

2 Env proteins are usually not homogeneous Implications for purification Irrespective of the design (gp120 monomers, uncleaved gp140s, SOSIP.664 trimers), Env protein transfection supernatants contain multiple molecular species. Unwanted sub-populations can sometimes be eliminated on the basis of size (e.g., higher m.wt. forms by SEC). Other unwanted sub-populations have the same m.wt. as the desired product and cannot be size-fractionated. These problem proteins include non-native trimers and SSscrambled gp120 subunits. Hence attention must be given to purification methods.

3 Purifying Env proteins via a tag (e.g., a C-terminal His-tag) (similar issues can apply when using lectin columns) Input Mixture of native and non-native Env proteins, all with a tag All the input Env proteins bind to the column via the tag Elution Native Env with His-Tag The native and non-native Env proteins are all eluted from the column Non-native Env with His-Tag SEC can only remove non-native forms that differ in size

4 His-tags and lectin columns Both these strategies are best avoided for making nativelike SOSIP.664 trimers. Neither method is selective for native structures. Hence any non-native trimer subpopulations present will co-purify. For BG505 SOSIP.664 trimers, His-tag columns do work adequately, but these trimers are very forgiving (low % of non-native forms). Lectin columns can allow co-purification of antigenically inappropriate trimers (aberrant glycans?), as seen with the ZM197M clade C construct (Julien et al., submitted). There are also concerns about co-purifying cellular/serum glycoproteins that are hard to remove by SEC.

5 bnab columns for Env purification bnabs select for Env protein sub-populations that display the relevant epitope. In most cases, that means a properly folded Env protein can be purified. bnabs can be used to purify native-like trimers and the native fraction of gp120 monomers. It is highly unlikely that bnab columns can rescue uncleaved gp140s as the native structure content is too low. Multiple different bnabs are now known to a range of epitopes. Several are now being made under GMP conditions for clinical trials as therapeutics. bnab-env affinity is relevant: Too low, poor binding; Too high, poor recovery. In practice, most bnabs seem to be broadly in the right affinity range.

6 Purifying native trimers via bnab columms We strongly favor bnab affinity columns over any other technique, particularly C-terminal tags (e.g., His-tags). (Applies also to gp120 monomers). Standard method involves 2G12 columns followed by SEC. Positive selection columns using a quaternary-specific bnab (PGT145, PGT151) offer a simple way to select for native trimers when non-native forms are also present. For pre-clinical work, a PGT145/PGT151 column eliminates the need for an SEC column (i.e., one-step purification). Negative-selection methods (e.g., lectin/sec/f105) are complicated (3 columns) and offer no advantages. Also hard to see them as applicable to GMP production.

7 Epitopes for bnabs used to purify BG505 SOSIP.664 trimers Top View Side View Membrane 2G12 PGT145 PGT151 2G12 is not trimer-specific and will bind gp120 monomers. Although 2G12 has some selectivity for properly folded gp120 subunits, it will bind non-native forms of Env. PGT145 and PGT151 bind trimer-specific quaternary epitopes.

8 Input Mixture of native (epitope present) and non-native Env proteins (epitope absent) Purifying Env proteins via bnabs that select for a native conformation (trimer-specific or not) The native Env proteins bind to the bnab on the column Elution Examples 2G12 PGT145 PGT151 Native Env Non-Native Env The non-native Env proteins flow through the column and are discarded The native Env proteins are eluted and collected SEC still needed to purify trimer fraction unless quaternary bnab (PGT145, PGT151) used

9 2G12 columns for purifying SOSIP.664 trimers Most of our experience is with 2G12 (available as GMP reagent from Polymun, after clinical studies in 2008). Broadly reactive although less so with clade C Env. Key glycans can be knocked-in to restore epitope, even with clade C (e.g., ZM197M), although this does not always work (e.g., CZA97). Because 2G12 is not trimer-specific, SEC is needed to purify trimers away from monomers, dimers, etc. For the same reason, 2G12 columns are NOT suitable for SOSIP.664 trimers that contain non-native trimer subpopulations; the latter species co-purify and cannot then be separated by SEC.

10 PGT145 columns for purifying SOSIP.664 trimers PGT145 is being made as a GMP reagent as a Scripps CHAVI-ID supplement (provisionally). Very broadly reactive across clades, although some failures (e.g., CZA97, clade C). Completely trimer-specific, so SEC is NOT needed at preclinical level (i.e., one-step purification of native trimers). Can be used to eliminate V3-clipped clade B trimers (e.g., B41), but there are alternative ways to solve this problem. Elution can cause some closed SOSIP.664 trimers (e.g., BG505) to partially open up, as viewed by EM, but emerging antigenicity and immunogenicity data indicate that this is NOT a major concern.

11 If you don t have a native structure to start with, using a bnab column won t help Because the 2G12 epitope is not trimer-specific, 2G12 columns are NOT suitable for uncleaved gp140s that do not contain native trimers. A PGT145 column also won t work

12 Other bnab columns We use PGT151 columns to make at least one native-like SOSIP.664 trimer (CZA97, clade C). Like PGT145, PGT151 is fully trimer-specific (i.e., one-step purification). Max Crispin has used PG9 and b12 columns to purify BG505 SOSIP.664 trimers with comparable glycan content and EM appearance to 2G12- or PGT145-purified trimers. GMP-quality bnabs being made for therapy trials include 1074, 3BNC117, PGT121, VRC01. In principle some of them could be useful for Env protein purification.

13 The glycan profiles of Env proteins Influences of purification strategies Native-like SOSIP.664 trimers are homogeneous and have a high oligomannose content very similar to native (virionderived) trimers (60-70%). For BG505 SOSIP.664 trimers this is independent of the bnab used for purification. Uncleaved gp140s with non-native structures have a much lower (30-40%), non-native oligomannose content (i.e., their glycans are more highly processed). Gp120 monomers are heterogeneous; their glycan content can be influenced by the purification method, but is generally lower (~35-45%) than for SOSIP.664 trimers. Pritchard/Crispin et al. Cell Reports (in press)

14 Epitopes for bnabs used to purify BG505 SOSIP.664 trimers for glycan analysis 2G12 gp120-subunit epitope PGT145 Trimer-specific apex epitope PGT151 Trimer-specific gp120-gp41 interface epitope Top views Side views

15 BG505 SOSIP.664 trimers purified via different bnabs all have very similar high oligomannose (native-like) glycan profiles 2G12 PGT145 PGT T-cell expressed trimers, purified by bnab columns followed by SEC

16 In contrast, the mannose content of BaL gp120 monomers is influenced by how they are purified Ni 2+ /NTA-reactive (intermediate mannose) 2G12 non-reactive (low mannose) 2G12 reactive (high mannose) His-tagged BaL gp120 expressed in 293T cells and purified via Ni 2+ -NTA or 2G12 columns

17 Subtype B native-like B41 SOSIP.664 trimers (Pugach et al J Virol 89, 3380) Antigenically comparable to BG505, highly immunogenic (unpublished results). V3 clipping seen when expressed in 293T cells (common to most subtype B Env proteins of all designs). Clipping due to serum proteases. Prevented by expressing trimers in 293F cells without serum. Alternatively, purify trimers via a PGT145 column (clipped trimers don t bind). Purified, non-clipped B41 SOSIP.664 trimers are fully native-like when viewed by EM. Native-like, high-mannose glycan profile, as per BG505 SOSIP.664 trimers (Max Crispin unpublished results).

18 V3 clipping and how to prevent it Subtype B V3 contains scissile sites (rare in other subtypes) Thrombin-family proteases most important Clements/Moore ARHR 1991 Critical contributions to solving the problem from Bob Whalen s group: clipping proteases are in serum (e.g., FCS) Du et al Prot Exp Pur 59, 223 Pugach et al. J Virol T + serum, 2G12/SEC BG505 SOSIP kda 70 kda B41 SOSIP.664 Serum is the problem B41 SOSIP.664 trimers 293T CHO 293F 10% 1% 0% serum B41 293T + serum, different columns gp120 trimer trimer 2G12 PGT145

19 Env protein design and purification strategies both matter CZA97 and 92UG037 Env proteins a case history Uncleaved gp140-fd-his proteins based on the CZA97 (clade C) and 92UG037 (clade A) sequences have been extensively studied in recent years (*) and are or have been relevant to GMP/clinical trial programs. They are claimed to be native trimers after purification via Ni 2+ /NTA columns followed by SEC. We have made these proteins together with SOSIP.664-His and other versions, to study the influence of Env design and purification strategies on native structure. Nkolola et al J Virol 84, Kovacs et al PNAS 109, Nkolola et al Vaccine 32, Kovacs et al PNAS 111,

20 If you want to make native trimers you have to start with the right design Well, soorr, if I were going to Dublin I wouldn t be starting from here Is the road to Dublin, my good man? XXXXXXXXXXXXXXXXXXXX

21 My Env proteins bind a lot of bnabs (But what does bind mean?) Many/most of the claims that uncleaved gp140s have native conformations and bind conformationally sensitive bnabs are based on a misuse of SPR methods. A signal in certain assays, particularly SPR, is a not sufficient measure of trimer antigenicity. Suppose 5% of the Env proteins in a population are properly folded and express, say, the PGT145 epitope If the binding assay SELECTS for that 5% and ignores the 95%, you get a highly misleading answer about the properties of the bulk population

22 Stoichiometry matters the importance of having a sense of proportion Take Add Sample Mmmm, I can taste something sweet! Great for my breakfast!!!!

23 How not to analyze non-homogenous Env proteins by SPR Flow over chip Mixture of native (epitope present) and non-native Env proteins (epitope absent) bnab on chip Signals from bnab-bound native Env BUT The majority population of nonnative Env fails to bind, is discarded and is invisible in the assay Drain This commonly used assay format can detect the PRESENCE of an epitope among the total population of Env proteins in a heterogeneous mixture but says nothing about the PROPORTION of Env proteins that express it. What binds to the SPR chip is not representative of the bulk properties of the unfractionated immunogen.

24 A better way to use SPR to analyze Env proteins (Yasmeen/Klasse Retrovirology 11, 41) MAbs flow over chip Signals from MAb binding to native Env bnab-purified native Env with His-tag Chip-immobilized anti-his MAb The method measures the proportion of antigenic trimers as well as kinetic constants and thereby affinities; it is best used for comparative studies (e.g., native vs. non-native Env; NAbs vs. non-nabs).

25 But only if the cosmetic treatment involves improvements to BOTH the Env design AND the purification method

26 Summary Env design and the purification method are both important Native-like soluble trimers can be made by several methods that all involve stabilizing the gp120-gp41 and gp41-gp41 associations. The fully cleaved BG505 SOSIP.664 trimer is the present paradigm but many others are now being made. Traditional uncleaved gp140s cannot be rescued via purification. Uncleaved gp140s can adopt native-like structures but ONLY if a flexible linker is inserted between gp120 and gp41 AND additional stabilizing changes are added (at minimum I559P, better + SOS). BG505 SOSIP.664 trimers and several others (e.g., B41) can be purified by 2G12 or even via His-tags because they are ~100% native-like. For other SOSIP.664 constructs and stabilized uncleaved gp140s, bnab positive selection (e.g., PGT145) or, as used by other groups, negative selection (F105), is needed to isolate the native trimer fraction.

27 Immunogenicity of SOSIP.664 trimers vs. uncleaved gp140s (Sanders et al., Science, in press) Autologous Tier-2 NAb responses to various Env proteins in Tzm-bl assay

28 Acknowledgements WCMC AMC Scripps Al Cupo Rogier Sanders Gabe Ozorowski PJ Klasse Chris Cottrell Tom Ketas Andrew Ward Pavel Pugach Ian Wilson Rajesh Ringe Anila Yasmeen And all members of the WCMC/AMC/Scripps HIVRAD team Funding support: NIAID HIVRAD grant (Program Office Jim Bradac) XXXXX Trimers