AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 7, Number 10, 1991

Mary

Ann

Liebert, Inc.,

Publishers

Cross-Neutralizing Antibodies in Rabbits Immunized with HIV-1 gpl60 Purified from Simian Cells Infected with a Recombinant Vaccinia Virus

JAMES

KLANIECKI,1 TRACY DYKERS,1 BRUCE TRAVIS,1 ROBERT SCHMITT,1 MORGAN WAIN,1 ANDREW WATSON,1 PENNATHUR SRIDHAR,1 JAN McCLURE,1 BROR MOREIN,2 J. TERRY ULRICH,3 SHIU-LOK HU,1 and JAMES LEWIS1

ABSTRACT A recombinant vaccinia virus in which the transcription of the human immunodeficiency virus type 1 (BRU isolate) env gene is driven by the UK late vaccinia promoter yields about 10-fold higher amounts of gpl60 env protein upon infection of monkey cells than does a recombinant in which gpl60 is expressed using the 7.5K early-late promoter. The gpl60 was purified from detergent lysates of infected cells by lentil lectin affinity chromatography followed by immunoaffinity chromatography, and was obtained in yields of 1-2 mg/109 cells of material estimated to be about 70% pure. Pairs of rabbits were immunized with purified gpl60 using either one of five different adjuvants or an immunostimulating complex. In all cases a substantial humoral immune response was obtained after boosting, including an activity that neutralized the homologous (BRU) isolate of HIV-1. In some cases, this activity also neutralized two distantly related isolates, SF2 and MN.

INTRODUCTION

One

of

(between cysteine residues 301 and 336) might have been

important in obtaining protection.6 The rsgplóO used by Berman et al.4 did not include the transmembrane and cytoplasmic domains of gpl60 and thus might not be structurally and functionally representative of native gpl60. Complete gpl60 has, however, been purified '

candidate vaccine against acquired immune deficiency syndrome (AIDS) is that antibodies be elicited that will neutralize infection by a variety of isolates of human immunodeficiency virus type 1 (HIV-1). Neutralizing antibodies have been found in seropositive humans1 and these usually neutralize a variety of isolates of HIV-1. In contrast, neutralizing antibodies obtained by immunization of animals with a recombinant protein that includes most, but not all, of the external envelope glycoprotein (gpl20) neutralize only the homologous HIV isolate.2'3 In a more recent study, immunization with recombinant gpl20 protected chimpanzees against infection with the homologous strain of HIV-1, while recombinant truncated gpl60 (referred to as rsgplóO) did not.4 Protection of chimpanzees from HIV-1 infection following a complex scheme of immunization with diverse immunogens has also been reported.5 In both cases, the authors speculate that antibodies to the V3 type-specific dominant neutralizing region criterion for the success

a

from baculovirus infected-insect cells9" ' and from recombinant vaccinia virus-infected monkey cells.12 Immunization with gpl60 has been shown to elicit CD8+ cytotoxic lymphocytes (CTL) when administered to mice in the form of immunostimulating complexes (ISCOMs)10 and CD4+ CTL when adminis' ' tered to humans using alum as adjuvant. Type-specific, but not were elicited antibodies cross-isolate-reactive, neutralizing of with 160 under denaturimmunization goats gp purified upon ing conditions from insect cells, using complete Freund's adjuvant.9 Both type-specific and cross-neutralizing antibodies were elicited in goats immunized with gpl60 purified under native conditions from recombinant vaccinia-infected monkey cells, but only when complete Freund's was used as adjuvant,

'Bristol-Myers Squibb Pharmaceutical Research Institute Seattle, WA. department of Virology, National Veterinary Institute, Biomédical Center, S-751 23 Uppsala, Sweden. RIB1 Immunochem

Research, Inc., Hamilton,

MT.

791

792

KLANIECKI ET AL.

and then only after 6 boosts. Here we report that immunization of rabbits with gpl60 purified from recombinant vaccinia virus-infected cells produced antibodies that neutralized infectivity, not only of the homologous BRU isolate, but in some cases also of the heterologous SF2 and MN isolates of HIV-1. Neutralizing antibodies were present not only when complete Freund's adjuvant was used, but also when other adjuvants were used. Furthermore, the cross-neutralization reported here was seen after only 2 boosts, and some neutralizing activity was seen after only 1 boost.

METHODS Construction of the recombinant vaccinia virus vllK-ENVS expressing HIV-1 (BRU) gpl60 using the late 1 IK vaccinia promoter The plasmids pV-ENV5 and pV-ENV213 were used to prepare the gp 160 gene from the BRU isolate of HIV-1 for insertion into the shuttle vector that contains the late vaccinia UK promoter (pSCIO).14 To increase the efficiency of translation at late times during vaccinia infection,15 we removed from pVENV5 the HIV sequences upstream of the env initiation codon (96 base pairs from the residual of the BRU Avail site to the env ATG). The construction was done by standard methods.16 The method chosen to align env with the translation initiation codon provided in pSCIO introduced extra translated sequence 5' to env. Consequently the underlined 8 amino acid residues in the following sequence are expected to be added to the amino terminus of the env translation product: MNSRGSTMMRVKEK It is expected that these residues will be cleaved with the normal cleavage of the env signal sequence. vl lk-ENV5 differs from V-ENV5 not only in the use of the 1 Ik instead of the 7.5k vaccinia promoter, and in the fact that the gpl60 protein expressed by the former is expected to have an amino-terminally extended signal sequence, but also in that 91 base pairs of 5' untranslated env sequence is present in the latter, but not in the former. To determine whether the removal of this untranslated sequence in itself would affect gpl60 expression, a variant of V-ENV5 was constructed, termed V-ENV5 F6, in which an MboII site immediately upstream of the initiation codon for gpl60 was used to remove all 5' untranslated env sequences. This manipulation introduced an additional methionine codon immediately before the gpl60 initiation codon so that the signal sequence is expected to be amino-terminally extended by one methionine residue. The v-NY strain of vaccinia virus (New York City Board of Health strain of vaccinia virus purified from a commercial preparation of smallpox vaccine by Wyeth Laboratories, Marietta, PA) was used for the construction of the recombinant virus. Insertion of the HIV env gene from the shuttle vector into the vaccinia virus was accomplished as described by Mackett ...

etal.17

Infection of monkey cells

with vllK-ENV5

recombinant

African green

monkey kidney cells (BSC-40, a derivative of

BSC-1, ATCC No. CCL26)

were

cultured in 1

x

DMEM

(Gibco, Grand Island, NY), 5% fetal bovine serum (FBS) at 37°C, 5% C02. Typically forty 150 mm plates with a confluent

monolayer of cells were infected with vi 1K-ENV5 at 3 PFU (plaque-forming units) per cell. After 1 hour adsorption at 37°C, the inoculum was removed and 20 ml of media per plate was added. Following incubation for 48 h, the cells were scraped from the plates, pelleted, and washed with phosphate-buffered saline (PBS). Cells were disrupted by incubation for 1 hour at 4°C in 0.5 ml per plate of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 8.0), followed by sonicationon ice. The cell lysate was clarified by centrifugation at 1000 g for 10 min and the supernatant used for the purification of gpl60.

Expression of gpI60

in simian cells

recombinant vaccinia viruses

infected with

Confluent monolayers of BSC40 cells in 100 mm dishes were mock infected or infected with v-NY, V-ENV5, V-ENV5 F6, or vl 1K-ENV5 at a multiplicity of infection (MOI) of 10 pfu/cell. After 24 h at 37°C, the infected cells were washed twice with PBS and lysed in 0.75 ml of IX Laemmli sample buffer. After two cycles of boiling and freezing, 20 p.1 of each sample was loaded on a 7.5% sodium dodecyl sulfate poly aery lamide gel

(SDS-PAGE). After electrophoresis, proteins

were transferred nitrocellulose paper and probed with 125I-labelled monoclonal antibody as described by Linsley et al.18 The gpl60 proteins were visualized by autoradiography and quantitated by counting the excised bands using a gamma scintillation counter.

onto

Purification of gpl60 Two consecutive affinity chromatography steps were used to prepare gpl60 from the clarified cell lysate described above. Glycoproteins were first bound to a 25 ml bed volume column of lentil lectin Sepharose-4B (Sigma), equilibrated to 50 mM Tris-HCl, 150 mM NaCl, 0.25% Triton X-100, 0.1% aprotinin, pH 8.0 (Buffer A). The infected cell lysate (see above) was diluted 1:4, using buffer A without detergent to lower the detergent concentration to 0.25%, and slowly pumped over the lentil lectin column (< 0.5 ml/min). The column was then thoroughly washed with buffer A and the glycoproteins subsequently eluted with buffer A containing 1 M methyl-a-Dmannopyranoside. Initial elution was at < 0.5 ml/min for one void volume followed by incubation at zero flow rate for 30 to 60 minutes and then elution at < 1.0 ml/min. Fractions containing gpl60 were pooled and slowly (< 0.5 ml/min) passed over an immunoaffinity matrix made by using cyanogen bromideactivated Sepharose-4B (Pharmacia Fine Chemicals, Piscataway, NJ) to immobilize a mouse monoclonal antibody (110.1) directed to the carboxyterminus of gpl20 (Genetic Systems Corporation, Seattle, WA). Triton X-100 was removed by thorough washing with 50 mM Tris-HCl, 150 mM NaCl, 0.1% aprotinin, pH 8.0. Bound gpl60 was eluted with 1% triethylamine (v/v), 150 mM NaCl, pH 12, and immediately neutralized with 1 M Tris, pH 7.2. Fractions containing gpl60 were pooled and concentrated by ultrafiltration into PBS. Total protein concentrations were routinely determined using the bicinchoninic acid (BCA) assay from Pierce, using as standard bovine albumin fraction V. Some samples were further analyzed for total protein concentration and for composition by amino acid analysis after gas-phase hydrolysis of the samples

CROSS-NEUTRALIZING ANTISERA TO HIV

(110°C for 24 h in gaseous 6 N HC1 plus 1% phenol). A precolumn derivatization method using o-PA/FMOC chemistry followed by reverse-phase chromatography was implemented on a Hewlett-Packard Aminoquant system. AH amino acids were quantified except cysteine and tryptophan, and, in this particular case, proline residues. Immunization

of rabbits

For each of the five adjuvants and the immune-stimulating complex (ISCOM)19 tested, 2 New Zealand white rabbits were each immunized at weeks 0, 4, and 24 with 100 u.g of partially purified gpl60. The five adjuvants used were complete Freund's adjuvant (CFA) for the primary immunization followed by incomplete Freund's adjuvant (IFA) for the boosts, alum with 0.2% deoxycholate (Alum),12 and three oil-in-water emulsions from Ribi Immunochem Research, Inc. (Hamilton, MT) containing monophosphoryl lipid-A (MPL) mixed with either Mycobacterium phlei cell wall skeleton (CWS) or synthetic trehalose dicorynemycolate (S-TDCM) or both. The combination of MPL and CWS has been termed DETOX"', and has been used for immunotherapy in clinical trials.20

Assays of immune

response: ElA titers

of rabbit sera

to HIV-1

Rabbit anti-HIV titers were followed by EIA to 32 weeks postimmunization. Reagents and assay plates containing inactivated whole HIV virions were provided by Genetic Systems Corporation (Seattle, WA), and goat antirabbit immunoglobulin conjugated to horseradish peroxidase (HRP) was purchased from TAGO, Inc. (Burlingame, CA). Pools of paired serum samples were titered in duplicate, and the ratio calculated of the absorbance at 450 nm divided by the absorbance at 630 nm. The midpoint titer was determined as the inverse of the serum

dilution that gave

an

793

gpl60

and incubated for 45 minutes at room temperature, followed by washing three times in TNE. Color development used 1.0 ml/well of 50 p.g/ml 3-amino-9-ethyl-carbazoIe (Sigma Chemical Co., St. Louis, MO) in 50 mM sodium acetate-acetic acid buffer pH 5.0, plus 0.05% hydrogen peroxide diluted from a 30% solution. Development was stopped after 10 minutes by addition of distilled water. After drying, the plates were inspected using a microscope to identify infectious centers.

RESULTS

Expression of gpl60 by

vlIk-ENV5

gpl60

made in cells infected with vllkthe late vaccinia 1 IK promoter for the transcription of env, was compared with the amount made in cells infected by V-ENV5, which uses the constitutive early-late 7.5K promoter for the transcription of env. Lysates of infected cells were analyzed by Western immunoblot electrophoresis using radioiodinated monoclonal antibody 110.4.IX As reported previously,13 both gpl20 and gpl60 are present in the V-ENV5 lysate. Proportionately less gpl20 is present in the vl 1K-ENV5 lysate. The labeled band corresponding to gpl60 was excised and counted. The level of expression of gpl60 was tenfold higher with v 11 k-ENV5 (Fig. 1 ). A variant of v-ENV5 in which the 5' untranslated env sequences have been removed, V-ENV5 F6, gave the same level of gpl60 expression as did V-ENV5.

The amount of

ENV5, which

uses

absorbance ratio of 1.0.

Assays of immune response: Neutralizing antibodies The neutralizing titers of the rabbit sera were determined by an in vitro neutralization assay adapted from Chesebro and

HeLa CD4+ cells22 at 1.0 x 104 cells/ml plated into 24-well tissue culture dishes (2.0 x 104 cells/ well). One day later, the test sera were diluted into medium and mixed with an equal volume of diluted HIV-1 (either the BRU, MN, or SF2 strains) that had previously been titered to give at

KD

200-

Wehrly.2' Briefly, were

least 250 infectious centers per well in the absence of serum. Sera and virus were incubated for 45 minutes at 37°C. Medium was aspirated from the cells and 100 p.1 of DEAE-dextran at 250 u,g/ml was added. A 500 u.1 sample of the mixture of serum and virus was added per well and incubation at 37°C continued for 12-16 h. The inoculum was then aspirated, the cells washed with PBS, 2.5 ml of medium added, and incubation at 37°C continued for 3 to 4 days. The medium was then aspirated, the cells fixed in 100% methanol for 10 minutes at room temperature, and then washed three times in TNE ( 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 7.5, 1% FBS). HIV antibodypositive human sera (250 p.l/well at 1:100 dilution in TNE) was added and incubated for 45 minutes at room temperature. The cells were again washed three times in TNE. Goat-anti-humanHRP conjugate (250 u,l/well diluted 1:1000 in TNE) was added

9768-

43-

26FIG. 1. Expression of gp 160 in simian cells mock infected, or infected with v-NY, V-ENV5, V-ENV5 F6, or with vl IKEN V5.

KLANIECKI ET AL.

794

gplóO purification SDS-PAGE followed by staining with Coomassie Blue was used to analyze the purity of gpl60 (Fig. 2). After two affinity chromatography purification steps, the gpl60 is by far the major component present. The remaining contamination is due to small amounts of numerous species of molecular weight lower than that of gp 160. It became apparent during the course of this work that purified gpl60 bound substantially less Coomassie Blue than did the bovine serum albumin control. To estimate this difference, samples of purified gpl60 and of bovine serum albumin were subjected to amino acid analysis. An amount of each sample containing 10 p.g of amino acid residues in protein, as determined by amino acid analysis of an aliquot of each sample, was analyzed by electrophoresis followed by staining with Coomassie Blue, and the amount of stained material in each gel lane was quantitated by densitometry. As shown in Table 1, the sample of purified gpl60 binds Coomassie Blue far less efficiently than does an equal amount of peptide in a reference sample of bovine serum albumin. To estimate the difference in binding efficacy between BSA and gpl60, we have assumed that the contaminating proteins in the gpl60 preparation bind stain as well as does BSA so that the large difference in total amount of stain bound between the BSA and gpl60 samples is due entirely to lower binding capacity of gpl60. This seems a reasonable assumption since the gpl60 is the only single component present in significant amounts. Using

R-250,

12

3 4

5 6 7 '•""•tpi

-*-gp!60

Table 1. Densitometric Analysis of Protein Fractionated by Polyacrylamide Gel Electrophoresis and Stained with Coomassie Blue

Sample

(10 ßg ofprotein)

BSA

Sum of areas of all peaks Area of main peak Fraction of area in main peak

3.955 3.508 0.887

gp 160 eluted from

110. ¡column 1.825 0.747 0.409

Correcting" for estimated 3.8-fold lower binding of stain gpl60: 3.955 3.917 Sum of areas of all peaks 3.508 2.839 Area of main peak

to

Fraction of

area

in main

0.887

0.725

peak Areas of peaks are given in arbitrary densitometric units. aThe correction factor of 3.8 for the lower binding of Coomassie Blue to gpl60 was chosen by determining what factor was necessary to multiply the area of gpl 60 peak (0.747 units) such that the sum of the peak areas in the lane containing 10 ßg of protein in the gpl 60 sample would equal the sum of the peak areas in the lane containing 10 ßg of BSA sample (3.9 units). Thus (3.8 X 0.747) + ( 1.825 0.747) 3.917, where the second term represents the stain bound by the contaminants, which are assumed to bind stain as well as does BSA while the deficit in total stain bound by the gpl60 sample is assumed to be due to lower binding by gpl60. =

-

this normalization procedure, we estimate that gp 160 binds stain 3.8 times less than does BSA. Neglecting the difference in amount of stain bound by BSA and gp 160 gives an estimate from the densitometry of 41 % purity for gp 160. Allowing for the fact that gpl60 apparently is 3.8-fold less efficient at stain binding than an average protein, the gpl60 is 72% pure. Applying this method to the results shown in Figure 2, we estimate that gpl60 constitutes 18% of the clarified cell lysate, 46% of the material eluted from lentil lectin, and 72% of the material eluted from the

antibody (data not shown). Rabbit *

"*""

FIG. 2. Purification of gpl60 produced in cells infected with 11K-ENV5. Lanes 1 and 2 were loaded respectively with 10 or 25 u.g of total protein from crude detergent lysate; lanes 3 and 4 were loaded, respectively, with 10 or 25 u.g of total protein from the eluate from a lentil lectin affinity column; lanes 5, 6, and 7 were loaded respectively with 5, 10, or 25 u.g of total protein from the eluate from an immunoaffinity column. After electrophoresis, the gel was stained with Coomassie Blue, as described in Methods. v

serum

titers

against gpI60

Six pair of rabbits were immunized with gpl60 formulated using either one of five adjuvants or ISCOM. Sera from the two rabbits in each pair were pooled, and titer against gpl60 measured using EIA. In all cases (Fig. 3), titers rose rapidly within 6 to 8 weeks postimmunization (2-4 weeks after the first boost). The most dramatic responses were observed using CFA and MPL/CWS, with CFA giving titers 10- to 100-fold higher than the other adjuvants. During the course of these experiments it became clear that one of the two rabbits immunized using CFA was showing a very poor response for unknown reasons (see, e.g.. Table 2). Thus the EIA titer for the pooled CFA rabbits is likely to be the average of one very high and one very low titer. Antibody titers were either roughly constant or decreased no more than one log from week 8 through week 24, at which time the second boost was administered. Two weeks following the second boost, the titers obtained with all adjuvants except CFA were substantially increased to approach or equal the very high

795

CROSS-NEUTRALIZING ANTISERA TO HIV gpl60 Table 2. Neutralizing Antibodies

in

Rabbit Serum

Neutralizing titers Animal

Adjuvant formulation

number

186 187

ISCOMS

188 189 190 191

MPL/CWS

192

MPL/S-TDCM/CWS

193 194 195 196

MPL/S-TDCM

CFA ALUM/DOC

week 26

week 6 BRU

SF2

BRU

SF2

MN

50 100

50 50 50 50 0 25 0

25 200 50 100 100

NT

25 25 25 25 0 0 >400

0 100

0 50

50 0

0

0

100 800 (2) >400 400 >400 25 >400 (1) >400 (2) >400 200 (1) 800 (2) >400 0 (1) 200 (2) 200 0

197

(1)