Cleavage strongly influences whether soluble HIV-1envelope glycoprotein trimers adopt anative-like conformationRajesh P. Ringea, Rogier W. Sandersa,b, Anila Yasmeena, Helen J. Kimc, Jeong Hyun Leec, Albert Cupoa, Jacob Korzuna,Ronald Derkingb, Thijs van Montfortb, Jean-Philippe Julienc,d, Ian A. Wilsonc,d, Per Johan Klassea, Andrew B. Wardc,and John P. Moorea,1

aDepartment of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065; bDepartment of Medical Microbiology, AcademicMedical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands; and cDepartment of Integrative Structural and Computational Biology,International AIDS Vaccine Initiative Neutralizing Antibody Center and Center for HIV/AIDS Vaccine Immunology & Immunogen Discovery and dSkaggsInstitute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037

Edited by David Baker, University of Washington, Seattle, WA, and approved September 26, 2013 (received for review July 29, 2013)

We compare the antigenicity and conformation of soluble, cleavedvs. uncleaved envelope glycoprotein (Env gp)140 trimers from thesubtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence oftrimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bondand an I559P gp41 change, together designated SOSIP), wasassessed. Without SOSIP changes, cleaved trimers disintegrate intotheir gp120 and gp41-ectodomain (gp41ECTO) components; whenonly the disulfide bond is present, they dissociate into gp140monomers. Uncleaved gp140s remain trimeric whether SOSIP sub-stitutions are present or not. However, negative-stain electronmicroscopy reveals that only cleaved trimers form homogeneousstructures resembling native Env spikes on virus particles. In con-trast, uncleaved trimers are highly heterogeneous, adopting a va-riety of irregular shapes, many of which appear to be gp120subunits dangling from a central core that is presumably a trimericform of gp41ECTO. Antigenicity studies with neutralizing and non-neutralizing antibodies are consistent with the EM images;cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneu-tralizing gp120 and gp41ECTO epitopes that are occluded on cleavedtrimers. These findings have adverse implications for using solu-ble, uncleaved trimers for structural studies, and the rationale fortesting uncleaved trimers as vaccine candidates also needs tobe reevaluated.

Trimeric envelope glycoprotein (Env gp) spikes on the HIVtype 1 (HIV-1) surface mediate entry of the viral genome

into the target cell (1, 2). When spikes interact with their cell-surface receptors, a series of conformational changes within theEnv culminates in virus–cell membrane fusion. Neutralizingantibodies (NAbs) against various Env epitopes antagonize theseevents (2, 3). Hence, Env glycoproteins are a focus of vaccinedesign programs intended to induce NAbs and thereby preventHIV-1 transmission (3, 4). Env trimers are composed of threegp120 surface glycoprotein subunits and three gp41 transmem-brane glycoproteins, the six subunits all associated via non-covalent interactions (5, 6). A critical event in trimer assembly isproteolytic cleavage of the gp160 precursor into its gp120 andgp41 components, a process essential for HIV-1 entry not leastbecause it liberates the fusion peptide (FP) at the gp41 N ter-minus (5, 6).Trimer-based vaccine strategies involve expressing soluble,

recombinant versions of the virion-associated (i.e., native) spikes.To facilitate production and purification, the membrane-span-ning and cytoplasmic domains that anchor spikes to the virion,but that are not NAb targets, are eliminated (7–12). However, theresulting proteins, known as gp140s, are highly unstable and dis-integrate into their gp120 and gp41-ectodomain (gp41ECTO) com-ponents, making them useless as immunogens. Two fundamentally

different protein-engineering strategies have been used to creategp140s that can be produced and purified without falling apart(3, 4, 7–17). The most common method involves eliminating thecleavage site between gp120 and gp41ECTO, creating uncleavedgp140s (gp140UNC) where the two subunits remain covalentlylinked (7–12). Additional trimerization motifs are often added tothe gp41ECTO C terminus (10–12). Our alternative approach isbased on the premise that cleavage is a fundamental feature ofEnv structure and involves stabilizing fully cleaved gp140s. Thecritical changes are an appropriately positioned disulfide bond(referred to as “SOS”) to link gp120 to gp41ECTO covalently, andan Ile/Pro (IP) substitution at residue 559 to strengthen inter-gp41ECTO interactions (13–17). The resulting cleaved trimers aredesignated SOSIP gp140s (14). Additional modifications haveimproved their stability, homogeneity, and antigenicity (15–17).Our current design, based on the BG505 subtype A env gene,yields SOSIP.664 trimers that mimic native, virion-associatedEnv spikes antigenically and when viewed by negative-stain elec-tron microscopy (EM) (17–19).Here we show that cleavage is essential for producing stable,

soluble gp140 trimers that resemble native Env spikes. EMstudies reveal that purified, trimeric gp140UNC proteins areheterogeneous and that the irregularly shaped images rarelyresemble a native spike; we refer to them as “aberrant config-urations” (ACs). In contrast, cleaved SOSIP gp140 trimers arehomogeneous and mimic native spikes; we designate them na-tive-like (NL) trimers. The antigenic properties of the cleaved

Significance

Trimeric forms of HIV-1 envelope glycoproteins are being usedfor structural and vaccine studies. The most common way tomake these proteins is to eliminate the cleavage site betweenthe glycoprotein (gp)120 and gp41 subunits. We show thatdoing so creates trimers that adopt irregular, nonnative con-figurations. Cleaved, stabilized trimers, in contrast, resemblethe native spikes on the HIV-1 virus. Our findings will helpstructural and vaccine programs by showing how to makenative-like trimers. The rationale for vaccine trials based on theuse of uncleaved gp140 trimers should be reevaluated.

Author contributions: R.W.S., P.J.K., and J.P.M. designed research; R.P.R., A.Y., H.J.K.,J.H.L., A.C., J.K., R.D., T.v.M., and J.-P.J. performed research; R.P.R., R.W.S., I.A.W., P.J.K.,A.B.W., and J.P.M. analyzed data; and R.W.S., I.A.W., P.J.K., A.B.W., and J.P.M. wrote thepaper.

Conflict of interest statement: R.W.S., A.C., J.-P.J., I.A.W., P.J.K., A.B.W., and J.P.M. arelisted on a patent application relating to the design and use of soluble, cleaved envelopeglycoprotein trimers.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: jpm2003@med.cornell.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314351110/-/DCSupplemental.

18256–18261 | PNAS | November 5, 2013 | vol. 110 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1314351110

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

020

(NL) and uncleaved (AC) trimers, assessed by surface plasmonresonance (SPR) and enzyme-linked immunoabsorbance assays(ELISA), are consistent with the EM images. Nonneutralizinggp120 and gp41ECTO epitopes are exposed on gp140UNC trimersbut occluded on cleaved ones, whereas quaternary structure-dependent epitopes indicative of proper folding are present onlyon cleaved trimers. Our findings have substantial implications,because uncleaved trimers are being studied structurally anddeveloped as vaccine candidates (3, 9, 10, 12, 20).

ResultsCleavage and Trimer-Formation Properties of gp140 Mutants. Eightconstructs based on BG505 env were used to explore the in-fluence of cleavage and other stabilizing changes on trimer sta-bility and conformation. The constructs are grouped into twosubcategories: those that can (hexa-Arg or R6 cleavage site) orcannot (Ser-Glu-Lys-Ser or SEKS cleavage site) be cleaved.Mutant Env proteins also either contained or lacked the disulfidebond (SOS) between gp120 and gp41ECTO and/or the I559Pchange in gp41ECTO. In all eight constructs, gp41ECTO was ter-minated at residue 664, eliminating most of the membrane-proximal external region (MPER), the transmembrane domain,and the cytoplasmic domain (Fig. 1).The eight soluble gp140s were expressed transiently in

HEK293T cells; the Furin protease was coexpressed with thefour cleavable variants. The IP.R6 and WT.R6 variants werecleaved efficiently, but were too unstable to form gp140 trimers(Table 1) and were not studied further. The other six Env pro-teins were purified on a 2G12 mAb affinity column and analyzedby reducing SDS/PAGE (Fig. 2A). As expected, SDS/PAGEconfirmed that the two Env proteins containing the R6 site werefully (>95%) cleaved, whereas the four with the SEKS site werenot (<5%) (Fig. 2A). Low-level cleavage of SEKS variants maybe via a secondary-site eight residues upstream of the primaryFurin site (21). Nonreducing SDS/PAGE showed that, unlikeSOSIP.R6 and SOS.R6, the four gp140UNC preparations con-tained oligomers that survived heating with SDS (Fig. 2B) andthat are probably covalently linked via aberrant, intersubunitdisulfide bonds (10, 11).Blue native (BN)/PAGE analysis of 2G12-purified Env pro-

teins confirmed that the SOSIP.R6 construct produced trimersefficiently; the overall yield was ∼50–60% (Fig. 2C and Table 1).

The SOS.R6 variant produced some trimers (∼20%), but thepredominant Env species was monomeric gp140, that is, onegp120 linked to gp41ECTO via the SOS bond. As noted, the IP.R6and WT.R6 constructs formed trimers inefficiently (<5%). Thus,the only cleaved gp140 for which trimers predominated wasSOSIP.R6, consistent with previous reports that SOS and I559Pchanges are both needed to stabilize cleaved trimers (13, 14).However, the SOS bond alone (SOS.R6) was sufficient to allowsome cleaved trimers to be expressed from BG505 env. All four2G12-purified gp140UNC preparations contained trimers, butwere accompanied by Env dimers and aggregates (Fig. 2C).Nontrimeric Env species are commonly seen before trimer pu-rification on sizing columns (14, 22). Some monomeric gp120was also present in gp140UNC preparations, probably due to low-level cleavage at the secondary site (21). The presence or ab-sence of SOS and I559P changes had no effect on how gp140UNCproteins migrated, the lack of cleavage being sufficient fortrimer formation.The six 2G12-purified Env preparations were analyzed by size-

exclusion chromatography (SEC) (Fig. 3A), with the elutedfractions assessed by BN/PAGE (Fig. 3B). As previously found,the BG505 SOSIP.R6 construct formed trimers efficiently, withonly minor amounts of aggregates, dimers, and monomers visible(17). For SOS.R6, some trimers were produced but the greateramounts of aggregates and monomers eluted from the SECcolumn are consistent with the BN/PAGE study (Figs. 2C and3B). Significant amounts of SOS.R6 gp120 monomers were alsovisible. The contrast between the SEC profiles for SOS.R6 andSOSIP.R6 (gp120 monomers present vs. absent) suggests thatthe gp120–gp41ECTO disulfide bond forms more efficiently whenthe I559P substitution is present. All four uncleaved gp140syielded a major trimer peak as well as aggregates and gp120monomers.We conclude that only gp140 constructs with a covalent link

between gp120 and gp41ECTO form Env trimers efficiently. Thatlink can be either a peptide (between residues 511 and 512 forgp140UNC) or a disulfide bond (between residues 501 and 605 forcleaved SOS or SOSIP gp140).

Electron Microscopy of Cleaved and Uncleaved gp140 Trimers. Theabove six 2G12- and SEC column-purified trimers were viewedby negative-stain EM, and the reference-free 2D class averageswere inspected to determine their overall morphology. Thecleaved SOSIP.R6 trimers were regular and homogeneous (Fig.4 A and B), consistent with earlier reports of the same construct(there designated BG505 SOSIP.664 gp140) (17–19). A semi-quantitative analysis of the class averages showed that ∼100% ofthe SOSIP.R6 trimer images resembled virion-associated, nativetrimers from HIV-1 BaL (EMDB-5019) (Fig. 4B, Table 1, andFig. S1) (6). Hence, we refer to SOSIP.R6 gp140 trimers as“native-like.”About a third of the purified SOS.R6 trimers had an NL

configuration, the rest adopting a range of unusual shapes (Fig.4B, Table 1, and Fig. S1). Noting also the complete absence oftrimers from the WT.R6 and IP.R6 constructs, the gp120–gp41ECTO SOS bond is necessary and, to an extent, sufficient toyield NL gp140 trimers, at least from the BG505 gene. However,as most SOS.R6 trimers were of the irregular AC form, theI559P change is also important for making cleaved, NL gp140trimers quantitatively.UncleavedWT.SEKS gp140 trimers (i.e., the standard gp140UNC

design) were highly heterogeneous, with multiple different shapesvisible and no predominant conformation (Fig. 4B). These ACtrimers often resemble three gp120 subunits dangling off a centralcore, which is presumably a trimeric form of gp41ECTO. If so, thegp120 moieties must remain attached to gp41ECTO solely via theuncleaved link between the C5 region and the FP. Overall, onlya very small fraction (<2%) ofWT.SEKS gp140 trimers had the NLconfiguration (Table 1).The SOS.SEKS gp140UNC trimers, the standard gp140UNC

with an added SOS bond, were also mostly ACs, with <2% in NL

BG505 gp160

BG505 SOSIP.R6 gp140(=BG505 SOSIP.664 gp140)

<-gp120 gp41 ->

V1 V2 C2 V3 C3 V4 C4 V5 C5C1S-S T605C

R6 I559P

A501CT332NHR1 HR2

V1 V2 C2 V3 C3 V4 C4 V5 C5C1 HR1 HR2 TMM CT

complex glycan M - MPERoligomannose glycan unknown composi�on

T605CA501C

V1 V2 C2 V3 C3 V4 C4 V5 C5C1S-S

R6

T332NHR1

V1 V2 C2 V3 C3 V4 C4 V5 C5C1R6 I559P

T332N

HR1

T332NV1 V2 C2 V3 C3 V4 C4 V5 C5C1

R6HR1

V1 V2 C2 V3 C3 V4 C4 V5 C5C1

S-S T605C

SEKS

A501CT332N

V1 V2 C2 V3 C3 V4 C4 V5 C5C1

T332N

V1 V2 C2 V3 C3 V4 C4 V5 C5C1

T332N

V1 V2 C2 V3 C3 V4 C4 V5 C5C1T332N

BG505 SOS.R6 gp140

BG505 IP.R6 gp140

BG505 WT.R6 gp140

BG505 SOSIP.SEKS gp140

BG505 SOS.SEKS gp140

BG505 IP.SEKS gp140

BG505 WT.SEKS gp140

I559PHR1

HR2

HR2

HR2

HR2

S-S T605C

SEKS

A501C

HR1 HR2

SEKS I559PHR1 HR2

SEKSHR1 HR2

Fig. 1. Design of cleaved and uncleaved gp140s. Linear representation ofBG505 gp160 and the SOSIP.R6, SOS.R6, IP.R6, WT.R6, SOSIP.SEKS, SOS.SEKS,IP.SEKS, andWT.SEKS gp140 constructs. Modifications in red are described inResults and Materials and Methods. Env subdomains are as follows: fiveconserved domains (C1–C5); five variable domains (V1–V5); heptad repeats 1and 2 (HR1 and HR2); the MPER; transmembrane domain (TM); and cyto-plasmic tail (CT). Glycan assignments are as previously reported (17).

Ringe et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18257

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

020

form (Fig. 4B, Table 1, and Fig. S1). This low percentage was thesame as with WT.SEKS, so the subunit-linking SOS bond doesnot facilitate NL trimer formation when cleavage is prevented.The IP.SEKS trimers were also mostly ACs, although now

approximately a tenth of the images resembled NL trimers (Fig.4B, Table 1, and Fig. S1). The trimer-stabilizing I559P changedoes therefore allow a minor subset of gp140UNC trimers toadopt the NL configuration. However, the lack of cleavage stilldominates the outcome, as >90% of IP.SEKS trimers were of theAC type.Approximately two-thirds of the uncleaved SOSIP.SEKS tri-

mers that contained both the SOS and I559P changes adoptedthe NL configuration (Fig. 4B, Table 1, and Fig. S1). Thus,adding both the SOS and I559P changes partially rescues the NLphenotype in the absence of cleavage, albeit not completely. Wehave noted, however, that SOSIP.SEKS NL trimers seem quitefragile, in that the proportion in this form is much lower in somepreparations and may be dependent on incubation or storageconditions.

MPER Deletion Does Not Explain the Rarity of Native-Like gp140UNCTrimers. The above experiments used gp140s from which theMPER was deleted to increase trimer homogeneity (23, 24). Toassess whether the absence of the MPER influenced trimerconfiguration, we made SOSIP.R6-MPER, SOSIP.SEKS-MPER,and WT.SEKS-MPER constructs that are identical to the corre-sponding variants described above except for truncation at resi-due 681, not residue 664. The EM images show a large sphericalblob of density that masks the base of gp41ECTO in the MPER-

containing trimers (Fig. 4C and Fig. S1). This density is far largerthan can be accounted for by the additional 17 amino acids, butresembles a micelle that associated with similar KNH1144.681trimers when a small amount of detergent was added to preventaggregate formation (23, 24). Although there was some aggre-gation of the MPER-containing BG505 trimers, the extent wasmuch less than with the KNH1144 versions. The MPER-con-taining BG505 trimers may have acquired lipids or other cel-lular amphiphiles during production, creating a micelle aroundthe hydrophobic MPER that survives the purification process.Overall, the EM images of the three MPER-containing trimersare generally consistent with their MPER-deleted counterparts(Fig. 4C). Thus, a clear majority of 2D class averages from thevisible SOSIP.R6-MPER trimers had the NL configuration,whereas few if any WT.SEKS-MPER NL trimers were seen (Fig.4C, Table 1, and Fig. S1). We noted that, similar to SOSIP.SEKS,a substantial fraction of SOSIP.SEKS-MPER trimers adopted theNL form (Fig. 4C, Table 1, and Fig. S1), but again AC formswere also present. Finally, we studied two uncleaved, MPER-containing gp140 trimers based on other env genes: CN54(subtype C) and UG37 (subtype A) (12, 25). They were over-whelmingly in AC form, with almost no NL images observed(Fig. 4D, Table 1, and Fig. S2). We conclude that the presence orabsence of the MPER does not determine whether a solublegp140 trimer adopts the NL or AC configuration.

Antigenic Structure of Cleaved and Uncleaved gp140 Trimers.Cleaved and uncleaved, soluble or membrane-bound trimersexpress NAb and non-NAb epitopes very differently (13, 26–28).We therefore assessed the antigenicity of purified, D7324-taggedversions of SOSIP.R6, SOSIP.SEKS, and WT.SEKS gp140 trim-ers. An SPR study showed that NAb and non-NAb epitopes wereexpressed differently on the two versions of trimers (Fig. 5A).The cleaved, SOSIP.R6-D7324 NL trimers bound the quaternaryepitope-dependent NAbs PGT145 and PG16 strongly, whereasthe uncleaved WT.SEKS-D7324 AC trimers did so to a lesserextent (PG16) or not at all (PGT145). Note that PG16, but notPGT145, binds monomeric BG505 gp120 to a limited degree,which would account for PG16 reactivity with the AC trimers(17). In marked contrast, uncleaved WT.SEKS-D7324 AC trim-ers exposed the epitopes for non-NAbs F240 (gp41, cluster I)and b6 and b12 [gp120, CD4 binding site (CD4bs)] far more thancleaved, SOSIP.R6-D7324 NL trimers [NAb b12 is nonneutralizingfor HIV-1 BG505 (17)]. The two trimer forms bound broadlyneutralizing antibody (bNAb) 2G12 equivalently; this non-quaternary epitope is on the exposed surface of gp120. Theantigenic properties of the SOSIP.SEKS-D7324 trimers weregenerally intermediate between SOSIP.R6-D7324 and WT.SEKS-D7324 (Fig. 5A), which is consistent with the mixture ofNL and AC forms visible in the EM images of the SOSIP.SEKStrimers (Fig. 4B and Fig. S1).ELISA data were consistent with the SPR analysis. Cleaved

SOSIP.R6-D7324 trimers again bound bNAbs PG16 and PGT145far more strongly than uncleaved WT.SEKS-D7324 trimers,whereas the converse applied to non-NAbs F240, b6, b12, andF105 (also to the CD4bs). Like 2G12, the CD4bs bNAb VRC01(which neutralizes BG505) bound the two trimer forms compa-rably (Fig. 5B). The A32 epitope was not exposed on cleavedtrimers whether soluble CD4 (sCD4) was present or not, but thisnon-NAb bound well to uncleaved trimers. The 17b non-NAb

Table 1. Properties of cleaved and uncleaved gp140s

Mutant % cleavage* % trimers†Presence of

native-like trimers‡

SOSIP.R6 >95 ∼60 ++++SOS.R6 >95 ∼20 ++IP.R6 >95 <5 N/AWT.R6 >95 <5 N/ASOSIP.SEKS <5 ∼60 ++SOS.SEKS <5 ∼50 −IP.SEKS <5 ∼60 +WT.SEKS <5 ∼50 −SOSIP.R6-MPER >95 ∼60 ++++SOSIP.SEKS-MPER <5 ∼60 ++WT.SEKS-MPER <5 ∼50 −CN54 gp140 N/A N/A −UG37 gp140 N/A N/A −

N/A, not analyzed; but see data sheets at www.polymun.at.*gp140 and gp120 bands on reducing SDS/PAGE were quantified usingImageJ (National Institutes of Health). The percentage of total Env proteinthat was cleaved to gp120 and gp41ECTO was calculated.†The gp140 trimer, gp140 dimer, gp140 monomer, gp120 monomer, andEnv-aggregate bands on BN/PAGE of 2G12-purified Env proteins werequantified using ImageJ. The percentage of the total Env protein in theform of gp140 trimers was calculated.‡The formation of NL trimers was semiquantitatively assessed by negative-stain EM as described inMaterials and Methods, and categorized as follows: −,<2%; +, 2–10%; ++, 10–50%; +++, 50–100%; ++++, ∼100%.

Reducing SDS-PAGE

SOSIP.SEKS

38 kD49 kD

62 kD

98 kD

188 kD

SOSI

P.R

6

SOS.

R6

SOS.SEKS

IP.SEKS

WT.SEKS

AM

gp120gp140

Non-Reducing SDS-PAGE

M

gp140

B

669 kD440 kD

monomergp120

dimertrimeraggregate

C

BN-PAGE

MSOSIP.SEKS

SOSI

P.R

6

SOS.

R6

SOS.SEKS

IP.SEKS

WT.SEKS

SOSIP.SEKS

SOSI

P.R

6

SOS.

R6

SOS.SEKS

IP.SEKS

WT.SEKS

Fig. 2. Biochemical characterization of cleaved and uncleavedgp140s. Reducing SDS/PAGE (A), nonreducing SDS/PAGE (B),and BN/PAGE (C) analysis followed by Coomassie blue stainingof 2G12-purified Env proteins. Molecular weights of marker(M) proteins (thyroglobulin and ferritin) are indicated.

18258 | www.pnas.org/cgi/doi/10.1073/pnas.1314351110 Ringe et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

020

also bound more strongly to uncleaved trimers, but sCD4 in-duced its epitope on cleaved trimers (Fig. 5B).

Comparative Protease Sensitivity of Cleaved and Uncleaved Trimers.We assessed the sensitivity of SOSIP.R6 or WT.SEKS gp140trimers to trypsin, chymotrypsin, and proteinase K. The cleavedSOSIP.R6 trimers were completely resistant to trypsin and chy-motrypsin (1 μg/mL), whereas the uncleaved WT.SEKS trimerswere degraded at this concentration. Similarly, the uncleavedtrimers were markedly more sensitive to proteinase K (Fig. S3).

DiscussionOur principal conclusion is that Env cleavage is a critical de-terminant of the configuration of soluble gp140 trimers. Thus,cleaved, SOSIP-stabilized BG505 gp140 trimers [SOSIP.R6; re-ferred to elsewhere as “SOSIP.664” (17–19)] are highly homo-geneous and their configurations resemble native Env spikes, asjudged by negative-stain EM (17–19) (Fig. 4 A and B). The an-tigenicity of BG505 SOSIP.664 trimers correlates strongly withneutralization of the corresponding virus (17). In the context ofthe BG505 genotype, the gp120–gp41ECTO SOS bond is neces-sary, and to a limited extent sufficient, to produce these NLtrimers. Thus, the cleaved SOS.R6 construct (i.e., no I559Pchange) yielded some trimers (approximately one-third of thetotal) that appeared indistinguishable from SOSIP.R6. However,the overall SOS.R6 trimer yield was also reduced (by approxi-mately threefold), with gp140 monomers held together by theintersubunit disulfide bond predominating, and most SOS.R6trimers were of the AC form, that is, highly heterogeneous andnot at all like native spikes. The I559P substitution is, therefore,not only necessary to yield cleaved trimers efficiently (14) butalso helps keep them in the NL configuration. The cleaved gp140constructs lacking the SOS bond, IP.R6 and WT.R6, failed toform trimers, the gp120 components presumably dissociatingrapidly from a gp41ECTO monomer or trimer.We propose that the engineered SOS bond anchors the gp120

subunit to gp41ECTO, not by creating a new site of interaction butby supporting the weaker, noncovalent linkages that form natu-rally and which may be stabilized to some extent by the mem-brane-spanning domain. Membrane-associated, full-length (i.e.,gp160) SOS trimers are still fusion-competent, provided the

disulfide bond is reduced after receptor engagement; the SOSbond does not compromise trimer structure and function (29). Incontrast, the I559P change is incompatible with fusion, pre-sumably because it impedes the transition(s) from the prefusionform to the fusion intermediate and/or postfusion forms of gp41(14, 30). A loop-to-helix transition around residue 559 that isblocked by a helix-breaking proline may be involved in thoseconformational changes (14, 30).In marked contrast to cleaved SOSIP gp140 trimers, uncleaved

trimers adopted multiple, irregular conformations that only veryrarely (<2%) resembled native Env spikes. The simplest in-terpretation of the gp140UNC trimer EM images is that the threegp120 moieties dangle from a central gp41ECTO core (now in thepostfusion, six-helix bundle form), to which they remain tetheredby the still-intact linkage between the gp120 C5 domain and theFP region of gp41ECTO (Fig. 4E) (13, 31). The greater sensitivityof the WT.SEKS gp140UNC trimers to protease digestion, com-pared with the cleaved SOSIP.R6 versions, is consistent withuncleaved trimers adopting a splayed-out conformation that ismore accessible to the digesting enzymes (Fig. S3). The sameconclusions about the overall configuration of gp140UNC trimersof several genotypes were recently drawn from independent,biophysical analyses (31).We propose that, for gp140UNC trimers, the normal associa-

tion between gp120 and gp41ECTO either never forms or is toounstable to persist. Introducing the SOS bond into gp140UNC toform SOS.SEKS does not create NL trimers, whereas the I559Pchange is partially beneficial (5–10% in NL form). Introducingboth the SOS and I559P changes rescued a larger percentage ofSOSIP.SEKS gp140UNC NL trimers (approximately two-thirds),the rest being in AC form. The antigenicity properties of SOSIP.SEKS trimers are also consistent with their being a mixture of NLand AC forms. These NL trimers also appear to be less stable,and more sensitive to incubation/storage conditions, than SOSIP.R6. The stark contrast between gp140UNC proteins and cleavedSOSIP.R6 trimers (∼100% NL) clearly shows the importance ofcleavage for creating stable, soluble trimers that resemble Env

SOSIP.SEKS

M

monomergp120

dimertrimeraggregate

3 5 11 14 15 17 19 21 23

WT.SEKS

M

monomergp120

dimertrimeraggregate

5 7 8 10 12 15 22 25 27

SOSIP.R6

669 kD440 kD

M 6 8 10 12 14 15 16 19 23

SOS.R6

M 4 5 6 7 9 11 13 15 18

IP.SEKS

M 6 7 9 10 12 1 4 16 18 22

SOS.SEKS

M 3 4 5 6 7 9 10 12 16

669 kD440 kD

B

UV

Abs

orba

nce

Elution volume

IP.SEKS

SOSIP.SEKS

WT.SEKSSOS.SEKS

SOSIP.R6 SOS.R6

monomer

trimertrimer trimermonomer

monomer

monomer

trimer trimer

monomermonomer

aggregate

aggregate

aggregate

aggregate aggregate

A

trimer

aggregate

Fig. 3. SEC analysis of cleaved and uncleaved gp140s. (A) SEC profiles for2G12-purified Env proteins on a Superdex 200 26/60 column. (B) Selectedfractions were analyzed by BN/PAGE and Coomassie blue staining. Molecularweights of marker (M) proteins (thyroglobulin and ferritin) are indicated.

CN54

UG37

Na�ve-like trimers Aberrant trimers

Na�ve-like trimers Aberrant trimersTrimer

Micelle Micelle

SOSIP.R6

SOSIP.SEKS

SOS.R6

IP.SEKS

SOS.SEKS

WT.SEKS

B

D

E

A Side view Top view SOSIP.R6-MPER

WT.SEKS-MPER

C

SOSIP.SEKS-MPER

Na�ve-like trimer Aberrant trimer

Fig. 4. Negative-stain EM of cleaved and uncleaved trimers. (A) Comparisonof the reconstruction of the soluble BG505 SOSIP.R6 (NL) trimer (blue) (17)with the structure of the membrane-associated native Env spike of thesubtype B BaL strain (mesh) (6). (B) Reference-free 2D class averages forvarious 2G12/SEC-purified, MPER-deleted trimers. (C) Reference-free 2D classaverages for 2G12/SEC-purified SOSIP.R6-MPER, SOSIP.SEKS-MPER, and WT.SEKS-MPER trimers. (D) Reference-free 2D class averages for CN54 (subtypeC) and UG37 (subtype A) uncleaved gp140s. (E) Cartoon showing the NL andAC conformations of the Env trimer (see also refs. 13 and 31).

Ringe et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18259

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

020

spikes seen on virus particles. Although uncleaved gp140 trimerscontain an appropriate number of gp120 subunits (i.e., three),from the perspective of creating mimics of native Env spikes wesuggest they are trimers in name only (TINOs).Why do uncleaved gp140 trimers very seldom adopt NL

structures? Do such conformations rarely form, or are they toounstable to survive? Although both factors may be relevant, wethink that instability is the more important, namely that thenoncovalent bonds between gp120 and gp41ECTO are too weakfor soluble gp140 trimers to persist in the prefusion form for verylong after they are secreted. Without the engineered SOS bond,cleaved gp140 trimers are also highly unstable and disintegrate,and for many genotypes this also applies to cleaved trimerslacking the I559P change (14, 16). In that catastrophic process,the gp120 and/or gp41ECTO moieties physically separate but,when the same event takes place for their gp140UNC counter-parts, the dissociating subunits still remain attached via theuncleaved peptide link between C5 and the FP. The same con-clusion has recently been drawn independently (31). The re-sulting Env structures are “trimeric” in that three gp120 andgp41ECTO subunits are all physically associated, but as notedearlier, they are TINOs. These conclusions are not dependent onthe presence or absence of the MPER, as the EM images foruncleaved WT.SEKS and WT.SEKS-MPER trimers showed thatNL trimers were rarely seen in either case. MPER-containing,uncleaved gp140s of several genotypes also did not form NLtrimers when studied by EM (here) or using H/D-exchange massspectrometry, which is sensitive to local structural order (31).Why do the stabilizing SOS and I559P changes have different

quantitative and qualitative effects on cleaved and uncleavedtrimers, yielding SOSIP.R6 and SOSIP.SEKS NL structures at

approximate respective frequencies of 100% vs. 70% (less insome preparations and/or storage conditions)? We proposethat gp140 cleavage itself has a modest stabilizing effect on thetrimer. More specifically, we suggest that the normal gp120–gp41ECTO association is more likely to form, or to persist longer,after cleavage occurs, increasing the probability that an NLstructure will survive long enough to be further stabilized by theengineered SOS bond. One hypothesis is that cleavage-dependentrelease of the gp120 C5 region from the FP liberates gp120 and/orgp41ECTO residues necessary for formation of the complete inter-subunit binding site; the C5 region is certainly involved in gp120–gp41 association (32).The antigenicity data are consistent with how cleaved and

uncleaved trimers appear by EM. Both SPR and ELISA showthat cleaved SOSIP.R6 trimers express epitopes for the quater-nary structure-dependent NAbs PGT145 and PG16 far betterthan the WT.SEKS gp140UNC trimers. In contrast, CD4bs non-NAbs (for HIV-1 BG505) b12 and F105 bind uncleaved trimersfar more strongly than cleaved ones, and the same trend is seenwith b6. The VRC01 CD4bs bNAb, which does neutralizeBG505, binds cleaved and uncleaved trimers comparably inELISA. The A32 non-NAb bound only to the gp140UNC trimers.It is argued that this epitope is important for antibody-dependentcellular cytotoxicity (ADCC) activity (33), but we note that Envon the surface of virus-infected cells, the most likely ADCCtargets in vivo, is in the form of nascent virions on which Env ispredominantly cleaved (34). Cleaved SOSIP.R6 trimers alsoexpose the cluster I gp41ECTO epitope for the non-NAb F240to a far lesser extent than uncleaved WT.SEKS trimers. Thisgp41ECTO region is, therefore, inaccessible on the compact,cleaved trimers, buried under the properly sited gp120 compo-nents, but well-exposed on gp140UNC trimers, where the gp120subunits are physically separated from the six-helix bundle con-figuration of gp41ECTO (31).Here we have only studied how cleavage affects soluble gp140

trimers, but many studies show that cleaved and uncleaved,membrane-associated gp160 trimers differ antigenically in quitesubstantial ways. For example, multiple non-NAbs are muchmore reactive with various gp120 epitopes on uncleaved trimers,as are ones to the six-helix bundle form of gp41 (13, 26–28).Thus, we believe that the aberrant soluble gp140 structures thatwe have visualized by EM will have their counterparts on thesurface of cells expressing uncleaved Env proteins. Perhaps ad-ditional stability imparted by the transmembrane region will re-duce the extent to which membrane-associated trimers decayinto AC forms, but a wealth of antigenicity data strongly arguesthat such structures are commonplace on the surface of cellsexpressing uncleaved gp160s (13, 26–28).This study used a set of mutants based on the BG505 se-

quence, but the EM images of two other gp140UNC trimersof different genotypes are essentially identical to those based onBG505. Biophysical studies of gp140UNC proteins from severalgenotypes from various genetic subtypes yielded the same con-clusion about their nonnative conformation, and the addition ofnominally stabilizing changes such as a trimerizing foldon se-quence does not overcome such defects (31). Thus, the problemswe have identified with the heterogeneity and structural aber-rance of uncleaved BG505 gp140 trimers do appear generaliz-able, which could be verified experimentally. As gp140UNCtrimers are vaccine candidates, there are strategic implicationsfor the production of homogeneous antigens and how they be-have as immunogens (3, 9, 10, 12, 20). Although gp140UNCtrimers are modestly superior to the corresponding monomericgp120s at inducing NAbs in animals (10–12, 20), any such benefitnow seems very unlikely to be based on their mimicry of nativeEnv structures. One possibility is that the well-exposed gp41ECTOmoiety might confer an immunogenicity benefit, if any antibodiesto this subunit register as NAbs in assays such as the one basedon A3R5 cells (12). The greater size, and perhaps multivalency,of uncleaved trimers might also confer an immunological ad-vantage over a gp120 monomer, particularly if Env aggregates

100

80

60 PGT145

100

80

60 PG16400

600

2G12Diff

. (R

U)

A

2040

020

40

0

110

140140

110

b140

200

200

00

SOSIP.SEKS-D7324

SOSIP.R6-D7324

Res

p.

ff. (R

U)

0

20

60F240

0 300 600 900

80

50

20

b6

0 300 600 900 0

0 300 600 900

20

80 b12

0

SOSIP.SEKS D7324

WT.SEKS-D7324

Res

p. D

if

Time (s)( )

2G12

0.5

1.0

1.5

2.0 PGT145

0.5

1.0

1.5 PG16

0.5

1.0

1.5 b12

0.5

1.0

1.5B

OD

45

0

0.001 0.0 1 0.1 1 0.0

0.001 0.01 0.1 1 0.0 0.0

0.001 0.01 0.1 1 0.001 0.01 0.1 10.0

b6

1.0

1.5

2.0

0 5

1.0

1.5 F240 F105

0 5

1.0

1.5

1 01.5

2.02.5 VRC01

SOSIP.R6-D7324

0.001 0.01 0.1 1 0.0

0.5

0.001 0.01 0.1 1 0.0

0.5

0.0

0.5

0.001 0.01 0.1 1 0.001 0.01 0.1 1 0.00.51.0

A320.4

0.617b

0 60.81.0

WT.SEKS-D7324

WT.SEKS-D7324 + sCD4SOSIP.R6-D7324 + sCD4

Antibody (µg/ml)

0.001 0.01 0.1 1 0.0

0.2

0.001 0.01 0.1 1 0.00.20.40.6

Fig. 5. Antigenicity of cleaved and uncleaved trimers. (A) SPR sensorgrams(one of two or three similar replicates) for the indicated NAbs and non-NAbs,injected at 500 nM over purified, cleaved SOSIP.R6-D7324 and uncleavedWT.SEKS-D7324 or SOSIP.SEKS-D7324 gp140 trimers captured by D7324 ontoCM5 chips. Response units (RU) are given on the y axis as a function of time(s) after injection on the x axis (5-min association, 10-min dissociation). (B)ELISA binding curves (from two or three replicates) for the indicated NAbsand non-NAbs and purified, cleaved SOSIP.R6-D7324 and uncleaved WT.SEKS-D7324 gp140 trimers. The curves for A32 and 17b were derived withand without sCD4 (10 μg/mL), as indicated.

18260 | www.pnas.org/cgi/doi/10.1073/pnas.1314351110 Ringe et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

020

form during immunization. Whatever the case, hypotheses thatunderlie clinical trials of uncleaved trimers should be revisited.Moreover, the EM and biophysical methods described here andelsewhere (31) should be used to evaluate all Env trimers beingconsidered for clinical trials.We conclude that cleavage of the gp120–gp41ECTO junction is

essential for a trimer to adopt a stable native-like structure,probably because that event imparts additional robustness tointersubunit interactions. Uncleaved, soluble gp140 trimers haveaberrant, nonnative configurations when viewed by negative-stainEM, and their antigenicity profile is consistent with their appear-ance. Cleavage is not, however, sufficient for a cleaved, solubletrimer to be usefully stable, as these entities rapidly disintegrateunless they also include the stabilizing SOS and I559P changes. Theresulting soluble, cleaved, SOSIP-stabilized trimers are highly ho-mogeneous, resemble native Env spikes, and are useful for struc-tural (17–19) and immunogenicity studies. The cryo-EM and X-raydiffraction structures of the BG505 SOSIP.664 gp140 trimers at∼5 Å resolution show they adopt the prefusion form of Env (35, 36).

Materials and MethodsSoluble BG505 gp140s. The subtype A BG505 SOSIP.664 gp140 construct, heredesignated SOSIP.R6, is described elsewhere (17). It incorporates the fol-lowing sequence changes (HxB2 numbering): T332N in gp120 (restoration

of Asn332 glycan-dependent epitopes); A501C and T605C (SOS; gp120–gp41disulfide bond); I559P (IP; trimer-stabilizing); REKR to RRRRRR (R6) in gp120(cleavage enhancement); and the stop codon at residue 664 to delete theMPER (improved homogeneity and solubility). The following mutants weremade (Table 1): SOS.R6 lacks the I559P change; IP.R6 lacks the gp120–gp41ECTO SOS bond; WT.R6 lacks both I559P and SOS; WT.SEKS, the standardgp140UNC construct with the REKR cleavage site changed to noncleavableSEKS; SOS.SEKS, gp140UNC with the SOS bond added; IP.SEKS, gp140UNC withthe I559P change added; and SOSIP.SEKS, gp140UNC with both the SOS andI559P changes added. The four italicized constructs cannot be cleaved at theprimary site between gp120 and gp41ECTO. Variants bearing a D7324 epitopetag at the gp41ECTO C terminus were purified in the same way, and desig-nated by appending “-D7324” to the gp140 descriptor (e.g., SOSIP.R6-D7324, WT.SEKS-D7324). MPER-containing SOSIP.R6-MPER, WT.SEKS-MPER,and SOSIP.SEKS-MPER constructs were based on the SOSIP.681 gene (23, 24).See also SI Materials and Methods.

ACKNOWLEDGMENTS. We are grateful to D. R. Burton, J. Mascola, and P. D.Kwong for reagents. This work was supported by National Institutes ofHealth (NIH) Grants P01 AI82362, R37 AI36082, and R01 AI84817; Aids FondsNetherlands Grant 2011032; and the International AIDS Vaccine Initiative.J.-P.J. has a CIH Research Fellowship. R.W.S. has a Netherlands Organization forScientific Research Vidi Grant and a Starting Investigator Grant (ERC-StG-2011–280829-SHEV). EM data were collected at the National Resource forAutomated Molecular Microscopy at The Scripps Research Institute, sup-ported by NIH Grant P41 RR017573.

1. Melikyan GB (2008) Common principles and intermediates of viral protein-mediatedfusion: The HIV-1 paradigm. Retrovirology 5:111.

2. Klasse PJ (2012) The molecular basis of HIV entry. Cell Microbiol 14(8):1183–1192.3. Zwick MB, Burton DR (2007) HIV-1 neutralization: Mechanisms and relevance to

vaccine design. Curr HIV Res 5(6):608–624.4. Walker LM, Burton DR (2010) Rational antibody-based HIV-1 vaccine design: Current

approaches and future directions. Curr Opin Immunol 22(3):358–366.5. Checkley MA, Luttge BG, Freed EO (2011) HIV-1 envelope glycoprotein biosynthesis,

trafficking, and incorporation. J Mol Biol 410(4):582–608.6. Liu J, Bartesaghi A, Borgnia MJ, Sapiro G, Subramaniam S (2008) Molecular archi-

tecture of native HIV-1 gp120 trimers. Nature 455(7209):109–113.7. Earl PL, et al. (1994) Native oligomeric human immunodeficiency virus type 1 enve-

lope glycoprotein elicits diverse monoclonal antibody reactivities. J Virol 68(5):3015–3026.

8. Gao F, et al. (2005) Antigenicity and immunogenicity of a synthetic human immu-nodeficiency virus type 1 group M consensus envelope glycoprotein. J Virol 79(2):1154–1163.

9. Spearman P, et al.; HIV Vaccine Trials Network of NIAID (2011) A trimeric, V2-deletedHIV-1 envelope glycoprotein vaccine elicits potent neutralizing antibodies but limitedbreadth of neutralization in human volunteers. J Infect Dis 203(8):1165–1173.

10. Yang X, Wyatt R, Sodroski J (2001) Improved elicitation of neutralizing antibodiesagainst primary human immunodeficiency viruses by soluble stabilized envelopeglycoprotein trimers. J Virol 75(3):1165–1171.

11. Yang X, et al. (2002) Highly stable trimers formed by human immunodeficiency virustype 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fi-britin. J Virol 76(9):4634–4642.

12. Kovacs JM, et al. (2012) HIV-1 envelope trimer elicits more potent neutralizing anti-body responses than monomeric gp120. Proc Natl Acad Sci USA 109(30):12111–12116.

13. Binley JM, et al. (2000) A recombinant human immunodeficiency virus type 1 enve-lope glycoprotein complex stabilized by an intermolecular disulfide bond betweenthe gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associatedstructure. J Virol 74(2):627–643.

14. Sanders RW, et al. (2002) Stabilization of the soluble, cleaved, trimeric form of theenvelope glycoprotein complex of human immunodeficiency virus type 1. J Virol76(17):8875–8889.

15. Binley JM, et al. (2002) Enhancing the proteolytic maturation of human immunode-ficiency virus type 1 envelope glycoproteins. J Virol 76(6):2606–2616.

16. Beddows S, et al. (2007) A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virustype 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120. Virology360(2):329–340.

17. Sanders RW, et al. (2013) A next-generation cleaved, soluble HIV-1 Env trimer, BG505SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog 9(9):e1003618.

18. Julien JP, et al. (2013) Asymmetric recognition of the HIV-1 trimer by broadly neu-tralizing antibody PG9. Proc Natl Acad Sci USA 110(11):4351–4356.

19. Julien JP, et al. (2013) Broadly neutralizing antibody PGT121 allosterically modulatesCD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surroundingglycans. PLoS Pathog 9(5):e1003342.

20. Sanders RW (2011) Clinical evaluation of a soluble trimeric HIV-1 envelope glyco-protein vaccine. Expert Rev Vaccines 10(8):1117–1120.

21. Morikawa Y, Barsov E, Jones I (1993) Legitimate and illegitimate cleavage of humanimmunodeficiency virus glycoproteins by furin. J Virol 67(6):3601–3604.

22. Center RJ, Lebowitz J, Leapman RD, Moss B (2004) Promoting trimerization of solublehuman immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simianimmunodeficiency virus chimeras. J Virol 78(5):2265–2276.

23. Klasse PJ, et al. (2013) Influences on trimerization and aggregation of soluble, cleavedHIV-1 SOSIP envelope glycoprotein. J Virol 87(17):9873–9885.

24. Khayat R, et al. (2013) Structural characterization of cleaved, soluble HIV-1 envelopeglycoprotein trimers. J Virol 87(17):9865–9872.

25. Schiffner T, et al. (2013) Immune focusing and enhanced neutralization induced byHIV-1 gp140 chemical cross-linking. J Virol 87(18):10163–10172.

26. Si Z, Phan N, Kiprilov E, Sodroski J (2003) Effects of HIV type 1 envelope glycoproteinproteolytic processing on antigenicity. AIDS Res Hum Retroviruses 19(3):217–226.

27. Pancera M, Wyatt R (2005) Selective recognition of oligomeric HIV-1 primary isolateenvelope glycoproteins by potently neutralizing ligands requires efficient precursorcleavage. Virology 332(1):145–156.

28. Tong T, Osawa K, Robinson JE, Crooks ET, Binley JM (2013) Topological analysis ofHIV-1 glycoproteins expressed in situ on virus surfaces reveals tighter packing butgreater conformational flexibility than for soluble gp120. J Virol 87(16):9233–9249.

29. Binley JM, et al. (2003) Redox-triggered infection by disulfide-shackled human im-munodeficiency virus type 1 pseudovirions. J Virol 77(10):5678–5684.

30. Sanders RW, Busser E, Moore JP, Lu M, Berkhout B (2004) Evolutionary repair of HIVtype 1 gp41 with a kink in the N-terminal helix leads to restoration of the six-helixbundle structure. AIDS Res Hum Retroviruses 20(7):742–749.

31. Guttman M, Lee KK (August 21, 2013) A functional gp41-gp120 interaction is ob-served in monomeric but not oligomeric, uncleaved HIV-1 Env gp140. J Virol 87(21):11462–11475.

32. Helseth E, Olshevsky U, Furman C, Sodroski J (1991) Human immunodeficiency virustype 1 gp120 envelope glycoprotein regions important for association with the gp41transmembrane glycoprotein. J Virol 65(4):2119–2123.

33. Ferrari G, et al. (2011) An HIV-1 gp120 envelope human monoclonal antibody thatrecognizes a C1 conformational epitope mediates potent antibody-dependent cel-lular cytotoxicity (ADCC) activity and defines a common ADCC epitope in human HIV-1 serum. J Virol 85(14):7029–7036.

34. Parren PW, et al. (1998) Neutralization of human immunodeficiency virus type 1 byantibody to gp120 is determined primarily by occupancy of sites on the virion irre-spective of epitope specificity. J Virol 72(5):3512–3519.

35. Lyumkis D, et al. (2013) Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1Env trimer. Science, in press.

36. Julien JP, et al. (2013) Crystal structure of a soluble cleaved HIV-1 envelope trimer incomplex with a glycan-dependent broadly neutralizing antibody. Science, in press.

Ringe et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18261

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

020