Bacterial expression and characterization of nine polypeptides encoded by segments of the envelope gene of human immunodeficiency virus

Gene, 64 (1988) 121-134

121

Elsevier GEN 02339

Bacterial expression and characterization gene of human immunode~ciency virus (Recombi~lant

DNA;

therapeutics;

HIV/AIDS;

of nine polypeptides encoded by segments of the envelope

phage ;1 pL promoter;

antigenic/immunogenic

determinants;

diagnostics;

vaccine)

Kenneth P. Samuela*, Arun Sethb, Martin Zweig’ , Stephen D. Showalterd and Takis S. Papasa ” Laboratory of Molecular Oncology, NCI-Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) Tel. (301/698-1591,h Bionetics Research Facility, Basic Research Program, NCI-Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) Tet. (301)698-1587 and (.,”Program Resources, inc., NCI-Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) Tel. (301)698-1579, Ext. 1320 Received Revised

10 June 1987 15 September

1987

Accepted

23 December

Received

by publisher

1987 I8 January

1988

SUMMARY

Nine envelope {Env) polypeptides, encoding different regions of HIV gp120 and gp41 Env accounting for approx. 96% of the entire Env precursor glycoprotein complex (gp160) were ~‘~e~erie~~u coli at Ievels ranging from approx. 2 to 20”/, of total cellular protein. The recombinant were produced either as hybrid products fused to the cI1 gene fragment of the 3, vector or in an without interfering cI1 products. Partially purified protein fractions of each polypeptide were

proteins, and expressed in polypeptides unfused form characterized

serologically by Western-blot analysis against a panel of well characterized human immunodeficiency virus (HIV)-positive human reference sera. Most of the Env polypeptides were highly immunoreactive with anti-gp12O/gp41 antibodies present in the sera of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases, but the patterns of reactivity were different. These results demonstrate that some of the antigenic determinants residing on the viral gp160 complex are retained on the surfaces of the recombinant Env polypeptides, and suggest that these sites are differentially immunogenic. These results are therefore interpreted in the context of an ongoing process towards using bacterially expressed HIV Env ~~rre.~p~)~?~enl*e to: Dr. T.S. Papas, Oncology,

NCI-Frederick

Frederick,

MD 21701 (U.S.A.)

* Present

address:

Cancer

Research

Frederick.

Cancer

Program Facility,

Laboratory

Research

Bldg. 469.

Inc.,

base(s)

or

HIV, human 1000 bp;

deoxycholate;

Tel. (301)698-1576. Resources,

Bacto-yeast

NCI-Frederick

P.O. Box B, Bldg. 469, Room

MD 21701 (U.S.A.)

gene product;

of Molecular

Facility,

217,

extract

AIDS,

Ap. ampicillin;

ARC, AIDS-related

glucopyranoside:

bp, base pair(s);

from protein;

bovine

pancreas;

acquired

DTT,

immune

deficiency

complex; DNasel,

syndrome;

POGP,

p-octyl-

deoxyribonuclease

dithiothreitol;

Env,

etw, gene coding for Env; EtdBr, ethidium

I

envelope

bromide;

gp,

acid

PAGE,

phosphate-buffered Abbreviations:

Luria

(0.1 g), and

or orf, open

fluoride;

polymerase;

pal, retroviral

ment ofE. coli DNA polymerase

037X-1Il9:X8~$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

PMSF,

AND METHODS,

(IO g),

(0.04 g), for-

frame;

PA, poly-

PBS, Dulbecco’s and magnesium;

pL,

phenylmethylsulfonyl

PolIk, Klenow (large) frag-

I; R, resistance,

site; SDS. sodium

sodium

amine

MgCl, (0.94 g),

thymidine reading

calcium

promoter;

~~ATERIALS

NZ

PA gel electrophoresis; saline without

kb, kilo-

virus; Na-DOC,

NZYDT,

phage I major leftward

ribosome-binding

broth;

(5 g). NaCl (5 g), anhydrous

in g/l; ORF

acrylamide;

immunode~ciellcy

nt, nucleotide(s);

DL-diaminopimelic mulated

Tel. (301)698-1620.

LB,

dodecyl section h.

resistant; sulfate;

RBS,

TBS, see

122

polypeptides diagnostic

to help define biological and therapeutic

reagents

and structural

in the tight against

INTRODUCTION

epitopes

to aid in the development

of more sensitive

AIDS.

nant Env polypeptides

which represent

of the entire HIV gp160 Env precursor Human

immunodeficiency

genous infectious

that

most

agent linked to AIDS

patients

related to this syndrome,

protein.

virus (HIV) is the exoand AIDS-

related diseases (Barr6 Sinoussi et al., 1983; Popovic et al., 1984; Levy et al., 1984). Serologic studies show

approx. 96”,,

with AIDS

MATERIALS

AND

METHODS

or diseases

as well as a high proportion

of individuals who belong to the high-risk groups, have specific serum antibodies directed against HIVencoded proteins. The most prevalent and consistently identified antibodies are directed against the env

gene-encoded precursor glycoprotein (gp160) and its processed products, gpl20 and gp41 (Sarngadharan et al., 1984; 1985; Barin et al., 1985; Allan et al., 1985a). Seropositivity to the major viral core antigen, p24 (Kitchen et al., 1984; Steimer et al., 1986) and the pol/endonuclease (p64ip34) gene products (Allan et al., 1987; Laurence et al., 1987) may also correlate with the clinical state of infected individuals and could potentially serve as serologic markers of disease progression (Laurence et al., 1987; Pan et al., 1987). Serum antibodies to the other viral gene products, notably the 3’-orf p27 (Allan et al., 1985b; Franchini et al., 1987), Sor p23 (Kan et al., 1986), and pl4 tat111 (Barone et al., 1986; Aldovini et al., 1986) proteins have also been detected in infected individuals, but at a lesser frequency. Important aspects in the complex strategy against AIDS must involve the development of potent antiviral reagents designed to antagonize or abrogate viral replication and gene expression (reviewed in Mitsuya and Broder, 1987), as well as safe and effective vaccines to protect against viral infection. Along this strategy, and as part of our ongoing research program aimed at identifying and mapping structural and functional determinants located on the viral gpl60 glycoprotein complex, we have produced in quantity, recombinant HIV Env polypeptides for diagnostic, therapeutic, and vaccine applications. This report describes the bacterial expression, partial purification, and immunological characterization of nine fused and unfused recombi-

(a) Bacterial strains and HIV proviral clones E. coli strains N4830 (Gottesman et al., 1980), MZ-1, DC1148 and WPS-18 (gifts from Dr. Donald Court, NCI, Frederick) are 2 lysogens that harbor a mutant temperature-sensitive repressor coded by phage 1. gene ~1857, and are all derivatives of E. coli strain K-12 (Sisk et al., 1986). Strain DC1148 was constructed as strain WPSl8 except that it is Tn/O proc‘ ’ and contains an insertion of mini-ter ” in the

/or? gene

(Dr.

Thomas

Patterson,

NC],

Frederick. MD: personal communication). All strains Lverc gro\vn in LB or NZYDT broth at the permissive temperature (32’ C). Proviral HIV clones BH-X and BH-10 were derived from unintegrated linear DNA from H9 cells acutely infected M,ith HIV (Hahn et al.. 1984; Ratner et al., 1985) and \verc kindly provided by Dr. Robert Gallo. (b) Expression

vectors

The bacterial expression vector, pJL6 (Lautenberger et al., 1983) and its derivatives pJLA 16 (Lautenberger et al., 1984), pANH-1 (Seth et al., 1986), and pcIId3’-orf (K.P.S., unpublished), all contain the well regulated ,! p,. promoter and an N-terminal fragment of the i ~11 gene containing RBS and ATG start codon (Oppenheim et al., 1982). Detailed information on the construction of each expression vector is also provided in the references cited. Vector pcIId3’-orfcontains a 52-bp DruI-EcoRV fragment from the 3’-orf gene of HIV clone BH-8 (Ratner et al., 1985), which was fused to the 13 N-terminal codons of i, ~11 gene fragment of vector pJL6 at its blunted HirzdIII cloning site (K.P.S.. unpublished). Restriction enzyme cloning sites unique to each expression vector include

123

(i) HindIII, Hind111

CluI, and BamHI(pJL6); (pJLA16);

(iii) HpaI

(ii) NnrI and

(pANH-1);

and

or NZYDT culture)

broth (1: 100 dilution of a fresh overnight

containing

50 pg Ap/ml in 2-liter flasks. At

(iv) EcoRV (pcIId3’-orf).

cultures were brought quickly to A 590 N 0.5-0.7, 42’ C by rapid heating (< 30 s) with gentle shaking

(c) DNA manipulations

in a 90°C water-bath, continued

Retroviral prepared

and expression

from liter cultures

on CsCl-EtdBr

gradients

vector

plasmids

were

and purified by banding (Maniatis

et al.,

1982).

Recombinant plasmids were prepared from 3-ml cultures (Birnboim and Doly, 1979) for diagnostic screening

with two or more restriction

ases. For cloning in expression

plasmids,

all HIV env

elution through Elutip-d columns (Schleicher & Schuell, Keene, NH). DNA fragments were then resuspended in 10 mM Tris . HCl (pH 7.4)-O. 1 mM EDTA at concentrations of 0.5-1.0 mg/ml. of recombinant

ing at 42” C for time periods

production

then

with vigorous

shak-

ranging

from

2 to

60 min. Cells were collected by low-speed centrifugation, and pellets were washed once with cold PBS, drained,

and flash-frozen

for storage at -70” C, until

used.

endonucle-

gene fragments were isolated from 0.8% or 1% agarose gel slices by the phenol/freeze extraction method (Benson, 1984) and further purified by

(d) Construction

and protein

by further incubation

plasmids

Expression plasmids pJLA16, pANH-1 and pcIId3’-orf were linearized with Hind111 or NruI, HpaI, and EcoRV restriction endonucleases, respectively. An aliquot (0.1 pg) of the linearized vector DNA was treated with PolIk (BoehringerMannheim or New England BioLabs) or ligated directly to 1.0 pg of blunted env gene fragment in a 20- to 30-~1 reaction containing 50 mM Tris . HCl, pH 7.4, 10 mM MgCl,, 10 mM DTT, 0.4 mM ATP, and 5 units T4 DNA ligase (Boehringer-Mannheim) at 15’ C for 4 to 15 h. Following inactivation at 65’ C, l/4 vol. of each reaction was used to transform competent E. coli MZ-1, DC1 148, N4830, or WPS-18 cells (see section a, above) according to published protocols (Maniatis et al., 1982). Single ApK colonies were picked from LB or NZYDT agar plates containing 50 pg Ap/ml, grown at 32°C and plasmid DNAs then prepared from minipreps (Birnboim and Doly, 1979) and screened for insert orientation by diagnostic restriction digestion. (e) Protein production Recombinant env-containing plasmids in E. coli strains MZ-1, DC1148, N4830, or WPS-18, were grown with vigorous shaking at 32°C in 500 ml LB

(f) Partial purification

of recombinant

proteins

Cell pellets (1 to 3 g wet cell weight) from 500 ml induced cultures were lysed with 5 ml of 0.2 mg/ml of freshly prepared lysozyme in 50 ml Tris . HCl, pH 8.0, 2 mM EDTA, 1 mM DTT, 5% glycerol buffer (with or without 1 mM PMSF) at 4°C for 30 min. MgCl, was then added to 10 mM final concentration, followed by DNase I to 20 pg/ml, and further incubated at 4’ C for 30 min with occasional mixing. After centrifugation at 12 000 x g for 30 min, resulting pellets were sequentially extracted with 5 ml each of the same buffer containing detergent and chaotropes as described (Krippl et al., 1984; Samuel et al., 1985). Briefly, the resulting lysozyme pellets were each suspended in 5 ml of the same buffer containing 0.05% Na-DOC, vigorously vortexed for approx. 1 min, set on ice for 5 min, then made 1 M in final NaCl concentrations, and incubated with mixing at 4°C for approx. 3 h. Pellets were collected as before and extracted with 5 ml of buffer containing 1.5% j?OGP as before. The resulting pellets were then extracted with 2 M KSCN in buffer, and collected as before. The final KSCN pellet was solubilized in 1 to 2 ml of 8 M urea. All fractions were stored at -20°C until analyzed. (g) Source of human reference sera Sera from HIV-infected patients were previously characterized and determined to be positive for antibodies against HIV envelope glycoproteins gp120 and gp41, and were kindly provided for characterization of our recombinant Env polypeptides by Drs. L. Arthur (Frederick Cancer Research Facility), P. Levine (National Cancer Institute), and M.G. Sarngadharan (Bionetics Research, Inc.). The two

124

seronegative

human sera from uninfected

were provided

by M.Z.

individuals

Sera were not decoded

the basis of clinical histories of patients.

on

Each serum

was given an arbitrary identification number from 1 to 12. The antibody-positive HIV sera from AIDS and ARC patients were coded Nos. l-10, while antibody-negative HIV sera from clinically healthy individuals were coded Nos. 11 and 12. All sera were stored at -70°C

until used.

(b) SDS-PAGE

and Western-blot

negative ( - ) immunoreactivity

against the test area.

This is a subjective

based on the relative

intensity

evaluation

of the color reaction or the radioactive

band

on the autoradiogram.

RESULTS

(a) Construction

of recombinant plasmids

analyses

The complete nucleotide Protein samples (20 to 40 ,nl detergent or chaotrope fractions) were boiled for 3 min with an equal volume of 2 x SDS-sample buffer and fractionated on SDS-12.5% or 150/, PA gels using the discontinuous buffer system (Laemmli, 1970). After electrophoresis, gels were either directly stained with Coomassie brilliant blue 250 in acetic acid-isopropanol, or electroblotted onto nitrocellulose filters as described by Towbin et al. (1979). Initially, the membrane filters were incubated with BLOTTO (Johnson et al., 1984) for 1 h to block nonspecific binding sites, and then reacted with human sera at dilutions of 1: 100 in BLOTTO (3% Carnation dry milk in TBS) at 4°C for 12 h. After washing three times with BLOTTO, [ ‘251]protein A (NEN) was added to 1 x lo6 cpm/ml in BLOTTO, and the blots were incubated for 3 h at 37°C. Filters were then washed with BLOTTO, then in TBS (0.90,; NaCl, 10 mM Tris . HCl, pH 7.5), air-dried, and exposed to Kodak x-ray films at -70” C with intensifying screens. Alternatively, membrane filters were also reacted with a 1: 100 dilution of human sera in TBS-containing 0.5% Tween-20 and 0.5% bovine serum albumin overnight at room temperature. After washing three times with TBS, the filters were incubated with a 1: 1000 dilution of peroxidaseconjugated goat antibodies to human immunoglobulin G (Boehringer Mannheim Biochemicals) in TBS-2’:; horse serum at room temperature for 1 h. After another three washes with TBS, the filters were incubated with a 1: 1 solution of 4-chloro- 1-naphthol and hydrogen peroxide (Kirkegaard & Perry Laboratories) to visualize the stained immune complexes on the filters. The filters were then rinsed several times in deionized water and air-dried. Each polypeptide was graded as demonstrating either strong ( + + + ), good ( + + ), weak ( + ), uncertain ( f ), or

and predicted

amino acid

sequences of HIV proviral clones BH-8 and BH-10 envelope gene have been reported (Ratner et al., 1985). The enr gene, an ORF between nt coordinates 5781 and 8369, encodes a glycoprotein precursor (gp160) which is then proteolytically processed to generate the mature exterior glycoprotein (gp 120) and transmembrane glycoprotein (gp41) (Allan et al., 1985a; Veronese et al., 1985; Robey et al., 1985). Plasmid sub-clones of HIV isolates BH8 and BHlO, containing an approx. 2.7-kb KpnI fragment encoding most ofthe envgene, were starting materials from which all of the expression plasmids were constructed. Initial attempts at expressing gp160 by fusing the approx. 2.7-kb KpnI (sites at positions 5928 and 8596) insert containing the env gene of HIV clone BHlO into a unique KpnI cloning site of the expression vector, pANK-12 (Seth et al., 1986) were unsuccessful. Similar expression problems were encountered when most of the gp41 sequence was removed by truncation of the 2.7-kb KpnI env fragment

at its 3’-end

with the restriction

enzymes

BarnHI (site at nt position 8052) or Hind111 (site at nt position 7718). Although the specific reasons for lack of detectable protein expression were unknown, previous reports on problems of expression of foreign proteins in bacteria were attributed to a variety of factors, among which are message instability (Zabeau and Stanley, 1982) cell toxicity due to expressed proteins (Amann et al., 1984; Brosius, 1984) and metabolic instability of the protein itself (Bachmair et al., 1986). We also note that the recombinant plasmids harboring the HIV env gene grew slowly at the permissive temperature (32’ C), showing further restricted growth at the induction temperaturc (42 3C) (not shown). While there were initial reports on the successful expression of large HIV

125

Env proteins amount

in bacteria

of the protein

very low,

and

immunoblot

expressed

required

technique

To overcome

(Crow1 et al., 1985)

ATG start codon through a GTT (Val) codon at the

the

HpuI

was nevertheless

the use

of the

sensitive

for their identi~cation.

some of the aforementioned

because

of previous

successes

using

fragments

the env gene to generate

could

from regions

easily be fused

codons

of ,? ~11 gene at the H&zdIII or NnrI cloning

a tripartite

of the

I3 N-terminal

et al.. 1984); or (iii) as

fusion to the ORF of a 17-codon

portion

N-terminal codons of the /E ~11 gene fragment at a unique EcoRV site in the 3’-orf sequence of pcIId3’-

this

orf (K.P.S.,

specific

unpublished).

Briefly, the StuI restriction

of gp120 and gp41 which to the ORF

to the ORF

of the 3’-orf gene, which was then fused to the 13

approach for HTLV-I (Samuel et al., 1984; 1986). We took advantage of the presence of unique restriction sites within

(Seth et al., 1986); (ii) as a

fusion

sites of pJLA 16 (Lautenberger

prob-

lems in expression of larger env gene fragments, we chose smaller segments of the env gene for expression partly

site of pANH-1

hybrid

end of fragment

569

and HaeIII end of 566 (Fig. 1) were directly fused in-frame to the PolIk filled-in HindIII and NruI blunt

of A ~11 gene

ends, respectively, of vector pJLAl6. Both the StuI and HueIII env gene fragments originated from gp120 and gp41 regions, respectively, of HIV clone

fragment or to the GTT codon in the expression vectors. Each of the env gene fragments (identified by numbers and restriction end coordinates in Fig. 1) was linked at a unique cloning site in one of the bacterial vectors (see MATERIALS AND METHODS, section h) via either (if direct fusion to a synthetic

BH8. Similarly, the PvuII sites of fragments 3 18 and 1061, Sea1 site of fragment 719, and the Hind111 site of fragment 331 (previously made blunt with PolIk)

art

A

A Residue Number I

Kp~l

Bacterial Clones

Hind111

Y

486

\ Fig. 1. Diagrammatic mature

sca1 representation gpl20

and gp41 (expanded

clones (numbers

and scrvc only as illustration. regulatory protein); exons.

viral sequences sequence);

(Ratner

below each clone) harboring the LTR (long terminal

gag (coding for group specific antigens);

art (coding for anti-repression

transactivator);

et al., 1985). Hatched

fragment

for gpl60

of clone 503 represents

sequences

pal (polymerase);

em gene product translation

Diagrams non-coding

(gp160) is to generate

are also indicated.

bars below the expanded

gp120 and gp41 env gene fragments.

repeat

BamHI

and within the gp160 precursor

area). The ATG start and TAA stop codons

The open box in the AvctI-AmI

include

of the cw gene. The primary

(to remove a short leader sequence)

amino acid residues

Hind111 566

347

of HIV and subregions

at the N terminus

below the env gene represents

identify bacterial identi&ing

of the genome

cleaved rY” symbols)

Env products,

Numbering

503

Ill1

proteolytically

HaeIII

7ig HaeIII

ScaI

PVUII

gp160 complex

were not drawn to scale env sequences.

at the 5’- and 3’-ends); TAR (coding

Other

for transactivating

SOT(short orf); lat (coding for transactivating

and 3’-orf (3’ open reading frame). Both the tat and arf genes contain

regulatory two coding

126

were also fused by in-frame ligation to the HpaI blunt cloning site of pANH-1. The Asp718 (KpnI isoschizomer) PolIk,

site of fragment 486 was made blunt with

then fused in-frame

to the GTT

codon

of

pANH-1 via the HpaI site. Finally, the ScaI and AvaI blunt ends of fragments 347 and 503, respectively, were both fused by blunt-end EcoRV

site of pcIId3’-orf.

fragments BHlO.

originated

ligation

to the

All of these env gene

from the env gene of HIV isolate

Recombinant

plasmids

containing

env gene

temperature-sensitive temperature

slower growth characteristics

shown), thus mimicking characteristics fragments

production

Cultures were grown at 32°C until the (see MATERIALS AND METHODS, A 59,1N 0.5-0.7 section e), at which time the phage i cI857-coded

TABLE

expressed

previously

discussed

growth

larger env gene

section a). It was also dif-

(see RESULTS,

metabolically

(b) Protein

at 32’ C relative to the

for clones harboring

MZ-1 (Sisk et al., 1986), then transferred

in Table I.

by

production

other clones. The slow growth rates were even more evident at 42°C the induction temperature (not

ficult to identify

WPS18 (Sisk et al., 1986). All other recombinant plasmids carrying env gene segments were grown in either strains WPS-18 (clones 486, 719 and 1061) N4830 (clones 347 and 503), or DC1 148 (clones 3 18 and 33 1). A summary of the cloning data is presented

was inactivated

and protein

continued at 42’ C for the required time. Some of the transformants (e.g., clones 719 and 1061) exhibited

fragments 569 and 566 (herein referred to as clones 569 and 566) were initially cloned in E. coli strain into strain

repressor

shift to 42°C

any specific

new protein

bands

by either clone, even when cultures with [ 35S]methionine

labeled

were and/or

[ “Slcysteine. This lack of detectable protein expression by Coomassie blue staining of SDS-PA gels from analytical preparations of induced cells or by radio-labeling techniques, led us to employ the more sensitive immunoblot assay to identify the expressed products. Using this approach, we were able to identify the approx. 26-kDa and 39-kDa polypeptides expressed by clones 7 19 and 106 1, respectively (not shown). Time-course experiments further revealed that the approx. 26-kDa and approx. 39-kDa polypeptides were rapidly degraded following induction at 42°C (not shown), which may

I

Summary

of data on cloning

of HIV CWYgene fragments

Bacterial

Restriction

clone”

acid coordinates

and amino b

Expression

HIV

vector’

proviral

Type of fusion e

clones’i

486

KpnI-StuI (49-218)

pANH-1

BHIO

Direct

569

,%I-SCUI

pJLA16

BH8

Hybrid

318

PvuII-ScaI

(218-400)

PvuII-Hind111

1061 347

ScaI-AluI

719

ScaI-Hind111

566

HaeIII-Hue111

503

AvaI-AvaI

331

HindIII-BarnHI clones are designated

of each WY gene fragment vectors

d HIV proviral

supernatant)

in parentheses

T4

the enr gene fragment

by restriction

identify terminal

sites were as discussed

Direct

pANH- 1

BHlO

Direct

pcIId3’-orf

BHIO

Tripartite

pANH- 1

BHlO

Direct

pJLA16

BH8

Hybrid

pcIId3’-orf

BHIO

Tripartite

pANH-1

BHIO

Direct

end-points

in MATERIALS

et al., 1984) were derived

or (ii) hybrid

section a).

encoded

AND METHODS, iB (Hahn

following

the nomenclature

by each gene fragment.

by cloning SstI-restricted

section b.

unintegrated

proviral

DNAs (Hirt

et al., 1984).

ligation to either, (i) direct fusion to the ATG start codon via a single codon GTT, but fusion to the 13 codons

of the ~11 gene fragment;

was ligated to the ORF of a 52 bp 3’-orf gene fragment

of the vector (see RESULTS,

and amino acid coordinates,

amino acid residues

+ T-cell line, H9, into the phage vector igtWES.

were fused by in-frame

cl1 sequences;

BHIO

to the size (bp) of input env gene fragments.

is identified

BH8 and BHlO (Hahn

from the infected

interfering

(647-758)

and unique cloning

clones

C All env gene fragments without

(548-736)

according

et al. (1985). Numbers

‘ Expression

(405-647)

(732-863)

’ Location of Ratner

(294-647)

(405-523)

.I Bacterial

I

pANH-

(294-400)

previously

or (iii) a tripartite

fusion in which

fused to the 13 codons of the cI1 gene fragment

127

account for the low levels of production

of these Env

(Table II). Such anomaly

polypeptides.

polypeptide,

gels of bacterially

The

approx.

26-kDa

however, was more stably produced the 39-kDa

species, whose optimum

was about

3 min. To estimate

(5-10 min) than induction

visually

been observed

time

Significant

the expressed

levels of the other recombinant

Coomassie-blue-stained

intrinsic

to

proteins.

(c) Partial purification of recombinant polypeptides

Env

High-level accumulation of eukaryotic proteins in bacteria is known to result in formation of insoluble

protein

Moreover,

these

bands

resolved

smaller

inclusion

by

require

Env poly-

bodies (reviewed in Martson, strong denaturing

Consequently,

peptides were stably expressed to levels approximating 8-15 “/, of total bacterial protein, and for periods of up to at least 60 min at 42°C (Table II). The polypeptides produced by clones 486, 347 and 503 were also stably expressed to levels of > 5%. Clone No. 331 expressed a 15-kDa polypeptide to levels of approx. 5% (not shown). We also wish to note that the observed relative M,s (kDa units) of the expressed polypeptides may deviate from the size predicted on the basis of the amino acid coding capacity of the input env gene fragment, including any cII fusion peptide present

TABLE

proteins have previously et al., 1984; Samuel et al.,

blue (not shown).

polypeptides were also produced by clones 3 18, 569, and 566, as evidenced by the relative intensities of SDS-PAGE.

expressed

(Ferguson

on SDS-PA

1986) and may reflect certain properties

the relative

levels of each polypeptide expressed by clones 719 and 1061, preparative SDS-PA gels were stained with Coomassie

in mobility

1986), which

agents for solubilization.

purification

protocols

were designed

which have exploited this relative insolubility of fusion proteins, while maintaining most of the contaminating bacterial proteins in the soluble supernatant fractions (Krippl et al., 1984; Marston et al., 1986). We therefore utilized one of these protocols (Krippl et al., 1984) to obtain partially purified protein fractions which are enriched for the recombinant Env polypeptide of interest (see MATERIALS AND METHODS, section f). These soluble detergent or chaotrope fractions, and pellet fractions containing the inclusion bodies which were solubilized by 8 M urea, were analyzed by SDS-PAGE (Laemmli,

II

Summary

of data on synthesis

of HIV Env polypeptides

Bacterial

Region of

Prcdictcd

clone I*

Env’

.\I,

(X IO 7)’

Observed

Approximate

M, (x lo-s)d

protein

486

N-gpl20

18.0

16.5

569

M-gpl20

22.0

23.0

8.0

318

c-gpl20

12.0

17.0

15.0

amount

synthesized

of (%)”

5.0

341

c-gp 120

17.0

15.0

5.0

566

N-gp4 1

22.0

21.0

8.0

503

c-gp4 1

18.0

16.0

5.0

331

M-gp4 1

12.3

15.0

5.0

719

c-gp 120/

26.0

26.0

2.0

39.0

39.0

2.0

N-gp4 1 1061

c-gpl20/ N-gp4 1

.’ Bacterial

clones are as described

h C, C terminus;

N, N terminus;

in Table 1. and M, middle region, respectively,

’ The predicted M, is based on the calculated

coding capacity

of the gp120 and gp41 envelope

of each input gene fragment,

including

(see also Fig. 1).

contributions

due to cII and 3’-orf

gene fragments. ” The observed mobilities

M,, in most instances,

of recombinant

proteins

’ ‘,, values are rough estimates by visual observation

deviates expressed

of recombinant

of Coomassie-blue-stained

from the predicted in bacteria

polypeptides analytical

Mr. and is an intrinsic property

have previously expressed or preparative

been observed against

a background

SDS-PA

ofthe input gene sequence.

(Ferguson

et al., 1984; Samuel

of total bacterial

gels (see Fig. 2).

proteins,

Anomalous et al., 1986). determined

128

1970) under reducing

conditions

on a 150/, gel. The

gel was stained with Coomassie blue and the stained bands of recombinant polypeptides were identified (Fig. 2, arrowheads). 17 kDa produced

The polypeptides

of approx.:

by clone 318 (lane l), 22 kDa of

purification attained,

of the recombinant as evidenced

teins.

The higher the levels of recombinant

peptides

21 kDa of clone 566 (lane 6), remained insoluble throughout the extraction protocol, until the final

gent/chaotrope

2 M KSCN pellet fraction was solubilized

(K.P.S.,

extraction with 8 M urea. The approx. 15-kDa polypeptide of clone 331, which also remained with the KSCN with

pellet fraction 8 M urea.

(not shown),

The

other

Fig. 2. Detection

of expressed

Bacterially

proteins,

namely

the

by the sequential non-ionic

AND bands

HIV Env polypeptides,

extractions

detergents

of induced

and chaotropic

METHODS,

SDS-15”,,

gp120 and gp41 env-coded

expressed

detected

bacterial

agents

pellets with

(see MATERIALS

by staining

on

1970), and the resolved with Coomassie

25 1~1of the urea (the supernatant

fraction

poly-

enriched

section f), were fractionated

PA gel (Laemmli,

an

protein

blue. Aliquots obtained

of

after solu-

bilizing the KSCN pellet with 8 M urea) or 50 ~1 of the detergent fractions bands

of each identified

peptides 23.kDa

of approx.:

sample

I7-kDa

were analyzed.

fraction);

17.kDa

15-kDa

of clone

of clone 566 (lane 6, urea fraction).

peptide

of clone 331, which also extracted

include

lysozyme

r-chymotrypsinogen

17-kDa of clone 486 347 (lane 4, urea

503 (lane 5, /?OGP

21.kDa

was not included

the poly-

of clone 318 (lane I, urea fraction);

of clone

fraction.

The protein

in each lane include

of clone 569 (lane 2, urea fraction);

(lane 3, BOGP fraction);

protein

by arrowheads

fraction);

and

The 15.kDa

poly-

in the KSCN

in this study. Protein standards

(14.3-kDa), (25.7.kDa)

expressed,

final purification

poly-

the more efficient is the deter-

extraction

(Kripple

levels of 80-90%

et al., 1984), and can be attained

unpublished).

(d) Analysis of immunogenic

determinants

was solubilized

17-kDa polypeptides of clone 486 (lane 3) and clone 503 (lane 5) both extracted in the soluble /30GPdetergent fraction. In addition to extracting in the soluble BOGP fractions, about 50% of the 26-kDa and 39-kDa polypeptides produced by clones 719 and 1061, respectively (not shown), also extracted in the soluble Na-DOC fraction. Thus, varying levels of

peptides.

were

polypeptide fractions (Fig. 2, lanes 1 to 6) still contained varying levels of contaminating host pro-

clone 569 (lane 2) 15 kDa of clone 347 (lane 4) and

by further

polypeptides

by the fact that the crude

B-lactoglobulin

( 18.4.kDa),

(see left margin).

pellet (BRL) and

To further confirm the identity of the recombinant Env polypeptide bands visualized on the Coomassieblue-stained SDS-PA gel in Fig. 2, and to demonstrate the presence of immunogenic determinants residing on their surfaces, the partially purified protein fractions used in the experiment of Fig. 2 (see also RESULTS, section c) were resolved by fractionation on SDS-PAGE on either 15:; or 12.52, gels (Laemmli, 1970). The resolved polypeptides were electroblotted onto nitrocellulose sheets (Towbin et al., 1979) and the resulting filter strips cut from the sheets were reacted with a panel of well characterized human test sera as described in MATERIALS AND METHODS, section h. Results of the immunoblot analyses are shown in the data summarized in Table III. The results indicate that most of the recombinant Env polypeptides, with the exception of the 15-kDa and approx. 17-kDa polypeptides expressed by clones 33 1 and 486, respectively, were recognized by anti-gp 12O/gp4 1 antibodies present in the HIV-positive human reference sera (Table III, serum Nos. l-lo), but not by HIV-negative human control sera (serum Nos. 11 and 12). Immunoreactivity of the different polypeptides showed significant differences, and ranged from being strong (+ + + ), as demonstrated by the 21-kDa polypeptide of clone 566, to showing no detectable reactivity ( - ), as seen with the approx. 17-kDa and 15-kDa polypeptides of clones 486 and 33 1, respectively. These non-reactive polypeptides may not be immunogenic in patients with AIDS or AIDS-related diseases. Alternatively, such epitopes may not be reactive in immunoblot assays. This difference in the patterns of immunoreactivity detected with the various test sera suggests relative differences in the antigenic epitopes residing on the viral gp160 molecule, which may also reflect struc-

129

TABLE

III

lmmunoreactivity

of Env polypeptides:

Bacterial

Protein

clone”

fraction b

summary

Immunoreactivity

I

of Western-blot

against

data

serum number”

2

3

4

5

6

7

8

9

10

11

+

_ ND

12

318

XM mea

ND

+++

331

8M urea

ND

ND

+ _

++ _

++ _

++ _

+++ _

+ _

+ _

341

ND ND

+++ _

+ _

++ _

+++ _

+ _

_

_

_

486

8M urea /jOCP

_

_

_

f + _

503

BOGP

ND

+++

++

+

+++

+

+

++

+

++

ND

566

8M urea

ND

+++

+++

+++

+++

+++

+++

+++

+++

+++

_

569

8M urea

ND

+++

++

++

+

+

+++

+

+

+

ND _

719

Na-DOC/

+++

ND

++

++

+++

++

++

++

+

++

ND

-

+++

ND

+

++

+++

+

ND

ND

ND

ND

ND

_

_

+++

~

+++

_ _

BOGP 1061

Na-DOC/ fiOGP

,’ Bacterial h Protein

clones expressing fractions

enriched

Env polypeptides

are depicted

for the recombinant

diagrammatically

Env polypeptides

in Fig. 1 and summarized

were prepared

as described

in Table I.

in MATERIALS

AND METHODS,

section c.

’ Human test sera were given arbitrary Reactivity

of each serttm against

AND METHODS, immunoreactivity. against

identification

section h, and was graded ND indicates

the serum numbers

numbers

the Env polypeptides

from 1 to 12, as explained

was determined

as showing

that the Env polypeptides

either strong enriched

in MATERIALS

by the immunoblot

procedure

AND METHODS, as described

( + + + ), good ( + + ), weak ( + ), uncertain

in the protein

fractions

of the indicated

section g.

in MATERIALS

( k ), or no ( - )

clones were not analyzed

shown

tural constraints on antibody recognition of these epitopes. Further, some of these epitopes are highly immunogenic (polypeptides of clones 3 18, 503, 566, 569, 719). with the most strongly reacting and consistently immune sensitive Env polypeptide being the approx. 21-kDa polypeptide of clone 566 (Table III) which is encoded by the N-terminal region of gp41 (Fig. 1). The immunogenic epitopes recognized by the antigenic determinants residing on the various immunoreactive polypeptides display serologic profiles suggestive of a differential sensitivity and/or specificity to the various regions of the HIV Env complex. Because recombinant segments and synthetic peptides from gp41 have been the most widely utilized as diagnostic reagents for detection of HIV infections (Chang et al., 1985b; Cabradilla et al., 1986; Gnann ct al., 1987; Ho et al., 1987) it is ofparticular interest to identify and map all of the immunogenic and antigenic determinants residing on gp41. Of particular interest to us are epitopes recognized on the surfaces of the approx. 21-kDa clone 566 and 16-kDa clone 503 Env polypeptides. Although the 15-kDa polypeptide of clone 331 originated from a

region of gp4 1 that spans the C terminus of clone 566 polypeptide and the N terminus of clone 503 polypeptide (see Fig. l), this region was nevertheless not immunoreactive with any of the test sera used (Table III). Thus, gp41 immunoreactive epitopes do not appear to reside on clone 33 1 polypeptide, which may be due in part to the fact that this region of the gp41 molecule traverses the cell membrane (Kowalski et al., 1987). It is also reasonable to assume that since the Env polypeptides produced by our gp120- and gp41-expressing clones represent about 96% of the gp160 Env complex (Fig. l), at least 96% of the potential antigenic determinants residing within both conserved and variable regions of the HIV Env proteins (Modrow et al., 1986) were expressed. Although the exact nature of the strongly immunoreactive polypeptides of clones 347 and 486 with one of the two normal HIV-negative human reference sera (No. 11) used in this study is not yet known, such cross-reactivity may reflect the presence of comigrating bacterial proteins reactive with antibodies present in this serum sample. Antibodies to contaminating bacteria1 antigens are frequently

detected

in the sera of clinically

(Cabradilla

et al., 1986). Other

normal

individuals

strongly

immuno-

duced

by expression

in mammalian

cells (Chakra-

barti et al., 1986; Hu et al., 1986; Kieny et al., 1986).

reacting high-M, bacterial proteins were also recognized by this serum in the immunoblots (not shown).

Polyclonal antisera raised against such a bacterially expressed peptide to the C terminus of gp120 were

Since neither of these Env polypeptides

shown to neutralize HIV in a type-specific manner (Putney et al., 1986). But the nature and structure of

have shown

reactivity with any of the well characterized HIVpositive human sera nor with a second HIV-negative human

serum (No. 12) (Table III), it is unlikely that

the cross-reactive

epitopes

recognized

by serum

the neutralizing terized.

epitope have not been fully charac-

It remains

neutralizing

to be determined

epitope(s)

No. 11 were directed against the viral gp120. Homo-

as in clone 347 polypeptide)

geneous

in clone 3 18 polypeptide)

peptides,

preparations

of the recombinant

using a previously

developed

Env polypurification

protocol (Du Bois, 1986) are currently underway, and a more detailed analysis of the cross-reactive epitopes will be conducted against a larger and broader panel of human AIDS and normal sera. The potential diagnostic, prognostic or therapeutic values of these recombinant polypeptides and monoclonal antibodies currently being developed against them, will be evaluated.

DISCUSSION

We have constructed nine recombinant expression plasmids, each directing the synthesis of either a fused or unfused HIV Env polypeptide at production levels estimated to range from about 2’:” to about 15 “() of total cellular protein (Table II). The polypeptides, which range in size from approx. 15 kDa to 40 kDa, account for > 96% of the gp160 Env precursor complex. The results of the immunoblot analyses summarized in Table III indicate that the antigenic determinants residing along the HIV gp 160 Env complex were also retained on the surfaces of the recombinant Env polypeptides, and have remained immunogenic. The levels of immunoreactivity against a panel of well characterized HIV-positive human reference sera apparently reflect differences both in immunogenic and antigenic determinants, including their distribution over the entire HIV gp160 Env complex, since the different polypeptides reacted differently against the same panel of test sera. HIV Env proteins have also previously been expressed in bacteria (N.T. Chang et al., 1985; Crow1 et al., 1986; Putney et al., 1980) and even larger glycosylated Env polypeptides were also pro-

gp120 peptide (Putney

whether

reside at the C terminus or N terminus

the (e.g.,

(e.g., as

of the larger recombinant

et al., 1986). Other recombi-

nant polypeptides, as well as synthetic peptides encoding a highly immunogenic epitope located within the N-terminal region of gp41 (Chang et al., 1985b; Cabradilla et al., 1986; Ho et al., 1987; Gnann et al., 1987), are currently utilized as sensitive immunodiagnostic antigen reagents for detection of HIV infection. This region of gp41, including possible additional immunogenic determinants, also resides on the approx. 21-kDa polypeptide expressed in clone 566 of this report. Indeed, clone 566 polypeptide is the most immunoreactive of all the bacterially expressed Env polypeptides, reacting strongly against a selected panel of HIV-positive sera from AIDS patients (Table III). Further, the polypeptides produced by clones 3 18 and 569, expressing gp120 determinants, and 719 and 106 1, which encode both gp 120 and gp4 1 determinants, as well as the C-terminal gp41 encoding polypeptide of clone 503, all reacted well with most or all of the HIV-positive sera. But the patterns of immunoreactivity against the various HIV antibodypositive sera were distinctly different for each polypeptide. This difference in the relative antibody response to different regions of the HIV envelope may depend on the structure of the antigenic sites residing on gp120 and gp41 which is presented to the immune system of the infected individual. Thus it was observed that some of the representative HIVpositive sera, although demonstrating very strong immunoreactivity with the immunodominant epitape(s) on clone 566 polypeptide, reacted weakly or not at all with the other polypeptides (Table III). This pattern of immunoreactivity also suggests possible differential sensitivity and specificity of the various regions of the viral envelope for a limited number of immunogenic (conserved or non-conserved) epitopes recognized by HIV-infected indivi-

131

duals. In each experiment the same dilution The prediction

the blots were reacted with

(1: 100) of human of potential

antigenic

residing within both conserved of HIV gp 160 glycoprotein residing

this molecule,

on gp120 (Modrow

Since the recombinant

regions

complex shows that many

such sites exist throughout majority

determinants

and variable

polypeptides

with the

et al., 1986).

can account

et al., 1987) and syncytia formation 1987), virus-cell

test sera.

for

antibody-mediated

The development antibody

ticular reasons,

distributed

throughout

tially be identified

of the sites that

are

gp120 and gp41 could poten-

and their immunogenic

epitopes

mapped. Since the envelope glycoprotein coat is the first viral structure recognized by the immune system, and because gp120 and gp41 are the most immunogenic of the HIV antigens recognized in patients with AIDS and AIDS-related diseases (Barin et al., 1985; Allan et al., 1985), these studies could hold important implications for a HIV vaccine and the therapeutic effort. Furthermore, specific monoclonal antibodies directed against antigenic determinants within synthetic HIV Env peptides, recombinant Env polypeptides, or to disrupted or whole inactivated HIV virions (Chanh et al., 1986; Veronese et al., 1985; Gosting et al., 1987) have been used to identify the precursor gp160 or authentic HIV gp120 and gp41 glycoproteins from HIV infected cells. In addition, important virus neutralizing epitopes have been identified on polyvalent antibodies developed against HIV Env polypeptides expressed both in bacterial and mammalian systems (Lasky et al., 1986; Putney et al., 1986) on synthetic Env peptide antibodies (Chanh et al., 1986; Ho et al., 1987) as well as with monoclonal antibodies directed to gp 120 epitopes developed against inactivated whole HIV virions (Fung et al.. 1987). However, the structures and precise location of the specific neutralizing epitopes have so far not all been mapped. Thus, the availability of a more diverse group of recombinant and synthetic HIV antigens and anti-HIV immunological reagents may prove useful for the identification and mapping of important antigenic and immunogenic determinants located on the surface of the gp160 complex, including putative epitopes that are involved in the initial stages ofvirus-cell specific binding (McDougal et al., 1986; Maddon et al., 1986; Pert et al., 1986; Laskey

et al.,

neutralization

1987), (Lasky,

of more sensitive

diagnostic

antigen

and

assays for the early detection

of

HIV infection or as reagents for the prognostic evaluation of HIV-induced diseases, can be of par-

HIV gp160

many

virus

(Kowalski

fusion (Gallaher,

et al., 1986; Putney et al., 1986; Chanh et al., 1986; Robey et al., 1986) and immune suppression.

about 96% of the potential antigenic sites residing within both the conserved and variable regions of complex,

membrane

clinical

polyclonal

and

research

we are developing antibodies

Env polypeptides

For

these

both monoclonal

benefits.

and

directed

discussed

against

each of the

in this report.

In addi-

tion, purification of the polypeptides to homogeneity is currently in progress. The 17-kDa and 21-kDa polypeptides of clones 318 and 566, respectively, have been purified to near homogeneity (G.C. Du Bois and K.P.S., unpublished). The highly purified polypeptides will be evaluated against a larger and broader panel of well characterized sera from clinically healthy individuals, individuals from the various risk groups, and patients at different clinical stages of AIDS and from vastly different geographical regions, to further identify and map other structural and functional epitopes residing on the gp160 complex, and to evaluate their potential for use in diagnostic, prognostic, and therapeutic applications.

ACKNOWLEDGEMENTS

The authors

wish to thank Mr. Thomas

Pry and

Mrs. Sok L. Chen for their technical assistance, Mrs. Susan Toms for her excellent typing skills, Dr. James Lautenberger for helpful discussions, and Dr. Richard Ascione for reviewing the manuscript. This project has been funded at least in part with Federal funds from the Department of Health and Human Services under contract No. NOl-CO-74102. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

132

P.K., Chang,

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