Download Expression and immunogenicity of the entire human T cell

Journal of General Virology (1993), 74, 211-222.
211
Printed in Great Britain
Expression and immunogenicity of the entire human T cell leukaemia virus
type I envelope protein produced in a baculovirus system
J. Arp, 1 C. M . Ford, 1 T. J. P a l k e r , 2 E. E. King 1 and G. A. D e k a b a n ~*
1Immunology Group, The John P. Robarts Research Institute, London, Ontario, Canada N6A 5K8 and ~Duke
University Medical Center, Durham, North Carolina 27710, U.S.A.
The entire envelope gene of human T cell leukaemia
virus type I (HTLV-I) has been successfully expressed in
a baculovirus non-fusion vector system. The HTLV-I
envelope protein accumulated within the insect cells as
inclusion bodies which allowed efficient recovery of the
recombinant protein. In an attempt to study the role of
the HTLV-I envelope glycoprotein as an immunogenic
target, mice were immunized with the envelope protein
inclusion bodies (env-I.B.) in the presence or absence of
an adjuvant. Antibodies of broad specificity were
produced against the HTLV-I envelope protein in the
presence or absence of an adjuvant as detected by
Western blotting, radioimmunoprecipitation and peptide ELISA. Neutralizing antibody was detected when
env-I.B, immunizations were carried out in the presence
of high doses of a new adjuvant composed of a
mycobacterial cell wall extract. In a combined immunization regimen, env-I.B, were found to enhance and
broaden the antibody response to the HTLV-I envelope
glycoprotein, following priming with various recombinant vaccinia virus ( R W ) constructs expressing
either the entire native HTLV-I envelope (gp46 and
gp21) or just the surface envelope protein (gp46).
Increased titres of neutralizing antibodies were observed
following priming with the R W expressing gp46 only.
Results indicate that immunization regimes that involve
priming with RVV expressing HTLV-I envelope followed by boosting with recombinant baculoviral HTLVI envelope might be useful in eliciting protective immune
responses in vivo.
Introduction
infection have focused on the envelope glycoprotein.
Like those of other retroviruses, the HTLV-I envelope
protein appears to play a major role in the infection of
target cells (Dickson et al., 1982; Pique et al., 1990) and
elicitation of host antiviral immunity (Shida et al., 1987;
Nakamura et al., 1987). The HTLV-I-encoded envelope
is synthesized as a precursor gp63 protein which is
cleaved to form the mature gp46 glycoprotein and a
transmembrane protein, gp21. Recent animal protection
studies suggest that host immune effector functions
directed toward the HTLV-I envelope protein represent
an important mechanism for preventing infection and/or
the spread of the virus (Tanaka et al., 1991; Clapham et
al., 1984).
In an attempt to study the role of the HTLV-I
envelope glycoprotein as an immunogenic target and to
assess its vaccine potential against HTLV-I infection, we
have successfully expressed the entire HTLV-I envelope
protein using a baculovirus vector system. The accumulation of the HTLV-I envelope protein as inclusion
bodies in the insect cells simplified their isolation and
recovery. In this study, attempts were made to enhance
the immunogenicity of the HTLV-I envelope inclusion
bodies (env-I.B.) using various concentrations of adjuvant in the hope of generating a strong neutralizing
Human T cell leukaemia virus type I (HTLV-I) has been
firmly established by epidemiological and molecular
studies to be the aetiological agent of adult T cell
leukaemia (Yoshida et al., 1984; Robert-Guroff et al.,
1982) and is associated with a degenerative neurological
disorder known as HTLV-I-associated myelopathy/
tropical spastic paraparesis (HAM/TSP) (Osame et al.,
1986). The virus is principally transmitted in a cellassociated manner such that the major routes of
transmission involve sexual contact, mother-to-child
transmission, or contaminated blood either from transfusion of cellular blood components or sharing of needles
by intravenous drug abusers (Blattner et al., 1986;
Kinoshita et al., 1987; Satow et al., 1991). Public
education, discouraging breast feeding of infants by
HTLV-I-positive mothers and screening of blood products are the only methods currently available to prevent
transmission of this retrovirus. In developing countries
these methods of prevention are ineffective and impractical; thus there is a need for additional interventional strategies such as vaccines to control the
spread of HTLV-I infection.
Efforts to develop a subunit vaccine against HTLV-I
0001-1127 © 1993 SGM
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J. Arp and others
antibody response in mice. In addition, a combined
immunization regimen was also assessed. This involved
priming with one of three live recombinant vaccinia
viruses (RVV) each expressing a different version of the
HTLV-I envelope protein (Ford et al., 1992) followed by
boosting with env-I.B. The resultant sera have been
analysed using Western blotting, radioimmunoprecipitation, peptide ELISA and a syncytium inhibition
assay.
Methods
Cell lines. The Spodopterafrugiperda (Sf9) cells were obtained from
Dr P. Faulkner (Department of Microbiology and Immunology,
Queen's University, Kingston, Ontario, Canada) and grown in TC100
medium (Gibco-BRL) supplemented with 10% foetal bovine serum
(FBS; Gibco-BRL) and 100 gg/ml of gentamicin (Gibco-BRL). The
human T lymphocyte cell line M J-2 was obtained from Dr L. Arthur
(AIDS Vaccine Program, NCI-Frederick Cancer Research and Development Center). Two other T cell lines, HTLV-I-producing C91 PL
cells and non-infected indicator C8166 cells, were used in the syncytium
inhibition assay. All T cell lines were maintained in RPMI-1640
supplemented with 10% FBS and 100 units/ml of penicillin and
streptomycin.
Plasmids and viruses. The envelope fragment was derived from the
plasmid pMT-2 (provided by Dr R. C. Gallo, NCI/NIH; Ratner et al.,
1985). The baculovirus expression vector pVL1393 (provided by Dr M.
Summers, Texas A & M Agricultural Experimentation Station) was
chosen as the vehicle for insertion of the HTLV-I envelope glycoprotein
into the genome of Autographa ealifornica multiple capsid nuclear
polyhedrosis virus (AcNPV, provided by Dr P. Faulkner). The
procedures involving the production and isolation of the resultant
recombinant baculovirus were as defined by Summers & Smith (1987).
The RVV constructs RVV Els, RVV E3s, and the antisense construct
RVV Elas used in the combined immunization regimen (see below)
have been described in detail (Ford et al., 1991, 1992).
Indirect immunofluorescence of baculovirus-infected cells. Individual
monolayers of Sf9 cells were infected with wild-type bacnlovirus and
the two recombinant AcNPV clones (VHB5 and VHB6) at a
multiplicity of 0.5. Three days post-infection (p.i.), the cells were
harvested, washed and resuspended in PBS (pH 7.3) at a density of
5 x 106 cells/ml. Cell suspensions were spotted on tissue-grip (Fischer
Scientific) coated glass slides, allowed to air-dry and directly fixed in
cold acetone for 10 min. Slides were rinsed twice in PBS (pH 6.8) and
rinsed once in distilled water. Non-specific binding was blocked by
incubating the washed cells with a 3 % solution of BSA (Boehringer
Mannheim) in PBS for 1 h at room temperature. Cells were incubated
a further 30 min in this blocking solution (for second antibody
controls) or exposed to HTLV-I patient sera at a final dilution of 1 : 50
in blocking solution. The cells were rinsed in PBS and exposed to a goat
anti-human fluorescein isothiocyanate-conjugated antibody (GAMFITC; Jackson ImmunoResearch) at a final dilution of 1:100 in
blocking solution for 30 min at room temperature. Cells were rinsed
again before mounting with 2.5 % Dabco (Sigma) in glycerol-PBS (9: 1,
pH 8.7). Photography was with an Olympus BH-2 fluorescence
microscope with a fluorescein filter.
Detection o f secreted or cell-associated H T L V - I envelope protein.
Monolayers of 2 x 107 Sf9 cells were infected with either wild-type
AcNPV or recombinant baculovirus (infections of both clones VHB5
and VHB6 were tested) at a multiplicity of 0.2. Infected cells were
incubated in the presence of serum-flee medium (Excell 400, JRH
Bioscieuces). After 46 h, the infected cells and supernatants were
harvested by centrifugation at 3000 g for 15 min at 4 °C, in the presence
of the protease inhibitors 1 mM-PMSF and 1 mM-EDTA. The cell
pellets were stored at - 7 0 °C. To remove the majority of extracellular
virus from the supernatant, the supernatant was centrifuged at 14000 g
for 60 min at 4 °C. The supernatant was then dialysed against a buffer
of 0.1 mM-EDTA, ~ m~-Tri~HCl pH 7.4 for 2 days at 4°C. The
volume of the dialysed supernatant was then reduced to 200 ~tl by
lyophilization. Both the supernatants and their corresponding cell
pellets were resuspended in an equal volume of Laemmli buffer, boiled
for 5min and electophoresed on a 12% SDS-polyacrylamide gel
containing 6 M-urea (Hayden et al., 1986). Gels were then processed for
Western blot analysis.
Inclusion body isolation. Sf9 cells were infected with recombinant
baculovirus at a multiplicity of 10. Forty-six h p.i. the cells were chilled
for 10 rain and pelleted at 10000g for 20 min at 4 °C. The cells were
washed once in cell wash buffer consisting of 50 mM-Tri~HC1 pH 7.5,
1 mM-EDTA, l mM-DTT, l mM-PMSF (Nyunoya et al., 1990),
dispensed into 1 x 108 cell aliquots and the centrifugation was repeated.
The crude cell lysate was resuspended in TD buffer (Nyunoya et al.,
1990) and homogenized in a glass homogenizer in the presence of
DNase I (1 mg/ml) (Boehringer Mannheim). The suspension was
centrifuged for 20 rain at 10000 g. Pellets were sonicated in TD buffer
and incubated as described (Nyunoya et al., 1990). Future use of the
pellets decided the further processing of the env-I.B. (i) For mouse
immunizations, the env-I.B, pellets underwent three washes in PBS pH
7.2, interspersed by centrifugation at 10,000 g for 20 rain at 4 °C.
Pellets containing 10 gg of env-I.B, were stored at - 7 0 °C following
the final centrifugation and removal of the supernatants. (ii) For
SDS-PAGE, a pellet containing approximately 120 gg of env-I.B.
(protein equivalent of 5 x 107 cells) was resuspended by sonication, in
a Laemmli buffer containing 4 M-urea, 100 mM-DTT and 1 mM-PMSF.
This suspension was incubated for 1 h at 4 °C on a nutator, after which
it was divided into various required quantities for storage at - 7 0 °C.
Western blot assay. Inclusion body extract, solubilized in 4 M-urea
reducing buffer, was electrophoresed and transferred from a 12%
SDS/6M-urea polyacrylamide gel (Hayden et al., 1986) to an
Immobilon P membrane (Millipore). Blots were blocked in a solution
of 5 % skim milk powder (Johnson et al., 1984) at room temperature.
Blots were incubated in the appropriate primary antibody, diluted in
blocking buffer, overnight at 4 °C with rocking. Blots were then washed
in blocking buffer and exposed to goat anti-mouse, goat anti-rabbit or
goat anti-human IgG conjugated to alkaline phosphatase (Jackson
ImmunoResearch) at a final dilution of 1:5000, for 30 min at room
temperature. The blots were washed and exposed to substrate according
to the manufacturer's instruction (Blot Detection Kit: Amersham).
Each Western blot assay included three positive controls of (i) 1C 11, an
anti-gp46 mouse monoclonal antibody (MAb) (Palker et al., 1989), (ii)
anti-SP7 rabbit polyclonal serum (SP7 peptide sequence derived from
gp21; Palker et al., 1989) and (iii) human HTLV-I patient sera (TSP).
Immunizations. Three different inbred mouse strains, BALB/c
(Charles River), C57BL/6 (Charles River) and CFW/D (Ball &
McCarter, 1979) were immunized at 6 to 8 weeks of age by
intraperitoneal injection. Two different forms of HTLV-I envelope
protein immunogens were studied. For the adjuvant titration studies,
mice were injected with 10 gg of env-I.B, in the absence or presence of
various amounts of adjuvant formulated from a mycobacterial cell wall
extract (MCWE; Bioniche/Vetrepharm). MCWE is a purified and
deproteinized cell wall extract from a non-pathogenic species of
mycobacterium. The env-I.B, pellet was resuspended by sonication in
either 500 gl PBS (pH 7.2) or in 500 pl ofa 1:2 dilution of PBS-MCWE
emulsion. For the combined immunization regimen, mice were primed
with 4 x 106 p.f.u, of the appropriate purified RVV (RVV Els, RVV
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Immunogenicity of recombinant HTLV-I env protein
E3s or RVV Elas) diluted to 100lal with RPMI-1640. RVV Els
contains the entire unmodified HTLV-I envelope gene, whereas RVV
E3s contains only the portion of the HTLV-I envelope gene encoding
gp46. RVV Elas is identical to RVV Els except that the envelope gene
is in the antisense orientation. After 2 and 4 weeks, the primed mice
were boosted with either the same RVV preparation or with 10 gg of
env-I.B, suspended in 100 lal of 100 lag/ml MCWE adjuvant preparation. Terminal bleeds were recovered 2 weeks after the second
boost.
Immunoprecipitation of radiolabelled proteins. HTLV-I-infected human M J-2 cells were labelled with [35S]cysteine for immunoprecipitation of HTLV-I envelope proteins by serum of mice immunized
with HTLV-1 env-I.B. Cells (2x 107) were washed in cysteine-free
RPMI-1640 (Gibco-BRL Selectamine kit) with 1% dialysed FBS,
pelleted and incubated for 30 min at 37 °C with gentle mixing in
cysteine-free medium plus 1% FBS. The cells were pelleted and
resuspended in cysteine-free medium containing 0.5 mCi [35S]cysteine
(1000 Ci/mmol; Dupont, NEN) and incubated for 5 h at 37 °C with
mixing. Cell lysates were prepared as previously described (Dekaban et
al., 1984). The resultant cell lysate was precleared for 2 h at 4 °C by
incubating with Protein G Plus/A agarose (Oncogene Science), which
had been preincubated for 2 h at 4 °C with preimmune mouse serum.
The Protein G Plus/A agarose was pelleted and the resulting supernatant was divided into 5 x 106 cells equivalents. For the mouse test sera,
each aliquot of cell lysate was suspended in a total of 1 ml extraction
buffer containing 20 lal immune mouse serum and 30 lal of Protein G
Plus/A agarose. The positive control samples consisted of an aliquot of
precleared labelled cell lysate incubated with 30 lal of Protein G Plus/A
agarose and a mixture of rabbit polyclonal HTLV-I envelope
antipeptide sera raised against peptides SP-2, SP-4A, SP-6 and SP-7
(Palker et al., 1989). Immune complexes were allowed to form overnight
at 4 °C, washed with cold extraction buffer and then resuspended in an
equal volume of 2 x Laemmli buffer before loading onto a 12%
SDS-polyacrylamide gel. Gels were stained with Coomassie blue, fixed
for fluorography with Entensify Solution (Dupont, NEN), dried for 2 h
at 80 °C under vacuum and exposed for autoradiography at - 7 0 °C.
Peptide EL1SA. Binding of serum antibodies to HTLV-I envelope
synthetic peptides by ELISA was performed as previously described
(Palker et al., 1989) with the following exceptions: 2 lag of peptide per
microtitre well was used; and for efficient blocking, the reaction buffer
contained 2 % dried milk instead of 5 % BSA. The following synthetic
peptides containing hydrophilic sequences from HTLV-I gp46 or gp21
were chosen for the study: SP-2 [gp46, envelope amino acids (aa) 86 to
107], SP-4A (gp46, aa 190 to 209), SP-6 (gp46, aa 296 to 312) and SP7 (gp21, aa 374 to 392), all of which have been previously described
(Palker et al. 1989). The endpoint titre was defined as the serum
dilution at which the signal-to-noise ratio was >2.0; the mean
absorbance reading obtained with serum from a mouse injected with
100 lag MCWE alone was used to estimate background readings for
ELISA.
Syncytium inhibition assay. Neutralizing antibody titres were determined in a syncytium inhibition assay as previously described (Nagy
et al., 1983 ; Lal et al., 1991), by incubating 45 lal of HTLV-I-producing
C91 PL T cells with C8166 T cells (each cell line at 106 cells/ml in RPMI
with 10% FBS) overnight in a tissue culture incubator (5% CO v
37 °C) in the presence of 10 gl of serially diluted test serum (heatinactivated at 56 °C for 30 min). After 24 h, the presence of syncytia
was evaluated in an inverted microscope at 200-fold magnification and
the neutralizing titre was determined as the last serum dilution that
inhibited syncytium formation by greater than 90 %. Routinely, 100 to
200 syncytia could be obtained per microtitre well in the presence of
10 % normal mouse serum. All mouse sera were coded prior to testing
in the syncytium inhibition assay, and codes were broken only after
neutralizing titres had been measured. Neutralizing anti-HTLV-I
peptide antisera (Palker et al., 1992) and preimmune serum served as
positive and negative controls, respectively.
Results
Construction and characterization of recombinant
baculovirus
The entire HTLV-I envelope gene fragment was isolated
from the plasmid pMT-2 (Ratner et al., 1985) by a
BamHI-PstI partial digestion. This 1636 bp fragment
was inserted into the baculovirus transfer vector
pVL1393 downstream from the polyhedrin gene promoter as indicated in Fig. 1. In this pVLHTL construct,
the translation initiation codon of the envelope gene is
located 123 bp downstream from the non-functional
start codon of the polyhedrin gene and thus will result in
expression of the complete HTLV-I envelope protein in
the absence of additional polyhedrin protein sequences.
The pVLHTL plasmid was then transfected together
with AcNPV DNA into insect tissue culture cells (Sf9)
and virus was isolated from occlusion-negative plaques.
Southern blot analysis of digested recombinant viral
DNA generated the expected restriction fragments when
probed with an HTLV-I envelope-specific fragment.
(a)
A
Poly(A) s
P
i
~
g
~
n
a
Transcriptional
l
~
BamHI
HTLV-I env fragment
/
\
(1636 bp)
gp21
mrgp62/46
I
_--
r
(PstI)
Translational
stop
Cleavage
site
Ii uG)
)
Translational (BamHI)
start
J Transcription
(b)
gp46
SP-2
SP-4A
gp2 l
SP-6
SP-7
Fig. 1. (a) Gene transfer vector pVLHTL containing the entire HTLVI envelope glycoprotein gene used to generate the recombinant
baculovirus expressing the HTLV-I envelope glycoprotein. Horizontal
cross-hatch, 5' polyhedrin sequences; vertical cross-hatch, 3' multiple
cloning site sequences; dotted region, 3' polyhedrin sequences including
poly(A) addition signal. (b) Location of the synthetic peptides used in
the peptide ELISA to determine antibody reactivity to various regions
of the HTLV-I envelope proteins.
15-2
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J. Arp and others
(a)
(a)
(b)
12 3 4 5 6 7
(b)
1 2 3 4 5 6 7
1 2 3 4
(b)
1 2 3 4 5 6 7
o
42K
27K
(a)
5 6 7
-',63K
"q54K
"q43K
42K
27K
Fig. 2. Indirect immunofluorescence analysis with anti-HTLV-I
envelope MAb 1Cll. (a) Uninfected Sf9 cells; (b) wild-type
baculovirus-infected Sf9 cells; (c) recombinant VHB5 baculovirusinfected Sf9 cells; (d) recombinant VHB6 baculovirus-infectedSf9 cells.
Indirect immunofluorescence
To determine whether the envelope gene was expressed
by the recombinant baculovirus, indirect immunofluorescence using H T L V - I patients' sera was performed.
As illustrated in Fig. 2, normal Sf9 insect cells and those
infected with wild-type baculovirus failed to fluoresce,
whereas both recombinant baculovirus isolates (VHB5
and VHB6) revealed strong positive fluorescence. There
appeared to be aggregates of protein at the poles of
several envelope-expressing cells, and other recombinant
baculovirus-infected cells were stippled in appearance
suggesting that they may have been sequestering the
envelope protein within vacuoles.
Isolation and identification of the HTLV-I envelope
glycoprotein
In light of the indirect immunofluorescence observations,
it was important to determine whether any of the H T L V I envelope protein was being secreted by the Sf9 cells.
Western blots of the supernatant and corresponding cell
i!i!:¸¸
Fig. 3. Western blot analysis of supernatants (top two panels) and cell
pellets (lower two panels) following recombinant baculovirus infection. (a) Supernatant and cell pellet of wild-type baculovirus
infection. (b) Supernatant and cell pellet of recombinant baculovirus
infection. All blots were probed with the same series of primary
antibodies: lanes 1 and 2, normal mouse controls; lane 3, anti-gp46
1C11 mouse MAb; lanes 4 and 5, normal human controls; lanes 6 and
7, human HTLV-I patient sera (TSP patients).
pellet of a recombinant baculovirus infection (Fig. 3b)
revealed that the H T L V - I envelope glycoprotein was not
being secreted from the infected Sf9 cells but was
accumulating within the cells. The recombinant
baculovirus-infected cell pellet contained three major size
classes of H T L V - I envelope protein with M,. values
averaging 43K, 54K and 63K. All three size classes were
recognized by both the anti-gp46 1C11 M A b and h u m a n
H T L V - I patient sera. In addition, several minor protein
bands of lower Mr ranging in size from 30K to 39K were
also observed. N o specific immunoreactive proteins were
observed in the supernatant and cell pellet of a wild-type
baculovirus infection (Fig. 3a). The results of the
Western blots and the indirect immunofluorescence of
recombinant baculovirus-infected cells, combined with
the requirement for strong denaturing agents (4 M-urea
or 4 u-guanidinium hydrochloride) for solubilization of
the H T L V - I envelope protein, suggested that the recombinant baculovirus-infected cells stored the H T L V - I
envelope protein as inclusion bodies.
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Immunogenicity of recombinant H T L V - I env protein
(a)
1
2
94K- ....
67K-
....
a
43K- :
3
4
~:
::
"~ 63K
{
30K-;
{i{i{
20K(b)
A
B C D
94K-g~: N
67K-
"~ 63K
"~ 54K
-~ 43K
43K- !~
30K-
!i~i
20K:
Fig. 4. Analysis of HTLV-I envelope proteins produced as inclusion
bodies by recombinant baculovirus. (a) SDS-PAGE illustrating
expression and isolation of HTLV-I env-I.B. Lane 1, uninfected Sf9
cells; lane 2, wild-type baculovirus-infected Sfx) cells; lane 3,
recombinant baculovirus-infectedSf9 cells; lane 4, purifiedenv-I.B. (b)
Western blot analysis of env-I.B. Lane A, mouse anti-gp46 I C 11 MAb;
lane B, human HTLV-I patient serum {TSP patient); lane C, rabbit
anti-SP-7 peptide sera; lane D, competition assay with SP-7 peptide
and rabbit anti-SP-7 peptide sera.
The accumulation of the HTLV-I envelope protein as
inclusion bodies allowed its isolation from other cellular
and viral proteins to greater than 80% purity as
determined by SDS-PAGE. A modification of the
method developed by Nyunoya et al. (1990) allowed the
enrichment of these insoluble protein aggregates as
illustrated in Fig. 4(a). In our method, the addition of
DNase I was critical in obtaining maximum purification
of the env-I.B. Isolation of env-I.B, from an equivalent
amount of infected cells (Fig. 4a, compare lanes 3 and 4),
resulted in the recovery of the three major HTLV-I
envelope protein forms with minimal loss. No HTLV-I
envelope proteins were detected by Western blot analysis
in the cell lysate supernatants during the inclusion body
isolation (data not shown). The 43K, 54K and 63K
immunoreactive envelope proteins previously observed
in the total cell pellet (Fig. 3 b) were present in the same
relative amounts within the inclusion bodies (data not
shown).
When an equivalent amount of electrophoresed envI.B. (Fig. 4a, lane 4) was analysed by Western blotting,
the majority of extracted inclusion body material proved
to be of HTLV-I envelope origin, as shown in Fig. 4(b).
215
The three major proteins of 43K, 54K and 63K present
in the inclusion bodies proved to be immunoreactive
with both anti-gp46 1Cll MAb (Fig. 4b, lane A) and
anti-SP-7 peptide sera (Fig. 4b, lane B) and this suggested
that they represent different forms of the HTLV-I
envelope precursor protein. T o determine whether any of
these forms were the result of glycosylation, the effects
of tunicamycin were studied. Tunicamycin treatment
resulted in the disappearance of the 63K protein;
however, it had no effect on the production of the 43K
and 54K proteins. This suggested that the 63K protein
was the glycosylated precursor representing 5 to 10 % of
the total envelope protein, whereas the 43K and 54K
proteins were unglycosylated HTLV-I envelope precursor forms. Further confirmation of their precursor
origin came from competition Western blot assays in
which SP-7 peptide was found to inhibit binding of the
anti-SP-7 sera (SP-7 peptide sequence derived from
gp21) to the 43K and 63K proteins (Fig. 4b, compare
lanes C and D). The SP-7 peptide did not completely
inhibit the binding of the anti-SP-7 serum to the 54K
protein (Fig. 4b, lane D). The reason for this was not
clear. Control experiments using normal mouse and
rabbit sera, or sera raised against Sf9 cells infected with
unrelated recombinant baculovirus, did not possess
antibodies capable of binding to the 54K protein (data
not shown). Conversely, HTLV-I-specific sera did not
react with Western blots of unrelated recombinant
baculovirus cell pellets (data not shown).
Immunogenicity of inclusion bodies
Radioimmunoprecipitation and Western blot assays
revealed that injection of mice with env-I.B., in the
absence of adjuvant, could stimulate humoral responses
to the HTLV-I envelope protein. Serum from immunized
C57BL/6 mice possessed antibodies capable of
immunoprecipitating HTLV-I envelope proteins from
[aSS]cysteine metabolically labelled HTLV-I-infected M J2
cells (Fig. 5a). Normal C57BL/6 sera did not
immunoprecipitate these HTLV-I envelope proteins.
Western blot analysis confirmed the reactivity of the sera
from the immunized C57BL/6 mice to HTLV-I envelope
proteins (data not shown). Sera from B A L B / c and
C F W / D mice immunized with env-I.B, alone exhibited
similar humoral responses to the HTLV-I envelope
protein, as monitored by radioimmunoprecipitation and
Western blot assays (data not shown).
To stimulate an elevated humoral response to env-I.B.,
M C W E (Archambault, 1989) was employed as an
adjuvant. Since this was a new adjuvant, a titration
experiment was performed to determine the optimal dose
(0 to 500 gg) required to give the best antibody response.
From Western blot and radioimmunoprecipitation
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J. Arp and others
(a)
NMS
1
2
3
4
5
6
(b)
1
+ve
2
3
4
+ve
(c)
NMS
1
2
3
4
5
200K-- ..........
l16K97.4K-66.2K-54K
51K
46K
-,~54K
-*51K
.,t 46K
.,i 54K
-~51K
.,, 46K
42.7K. . . . . .
Fig. 5. Immunogenicity of the HTLV-I env-I.B, in the absence and presence of MCWE adjuvant preparation.
Radioimmunoprecipitationof HTLV-I envelopeproteins from HTLV-I-infectedMJ-2 cells with sera from individual C57BL/6 mice
(denoted 1 to 6 above the lanes) immunized with env-I.B, in the presence of (a) no adjuvant, (b) 50 gg dose of MCWE adjuvant
preparation or (c) 500 gg dose of MCWE adjuvant preparation. NMS, pooled sera from normal non-immunizedC57BL/6 mice; +ve,
rabbit polyclonal anti-peptide serum raised against envelopepeptides SP-2, SP-4A, SP-6 and SP-7.
assays, maximal seroconversion was observed in mice
immunized with 50 lag of M C W E adjuvant preparation
(Fig. 5b). Exceeding this dose resulted in a gradual decrease in mouse seroconversion with increasing amounts
of M C W E adjuvant preparation (Fig. 5c; 500gg
MCWE).
Characterization of the antibody response to env-LB.
In order to study further the effects of varying amounts
of M C W E adjuvant on the antibody response to envI.B., sera were assayed by ELISA for the ability to bind
to four synthetic peptides, SP-2, SP-4A, SP-6 and SP-7.
The locations of these peptides within the HTLV-I
envelope proteins are shown in Fig. 1 (b). The regions of
the envelope protein gp46, encompassed by the peptides
SP-2 and SP-4A, have been associated with virus
neutralization, whereas the SP-6 peptide region of gp46
has been shown to be immunogenic in humans (Palker et
al., 1989; Tanaka et al., 1991; Horal et al., 1991). The
SP-7 peptide spans another immunogenic region of the
HTLV-I envelope and has allowed us to monitor the
immune response to the transmembrane envelope protein, gp21. Mice injected with env-I.B, in the presence of
10 lag of M C W E adjuvant preparation produced sera
with the highest ELISA titres for all four synthetic
peptides (Fig. 6, group 4) and exhibited strong Western
blot reactivity (data not shown). As illustrated in Fig. 6,
inoculation of higher doses of M C W E resulted in a
corresponding decrease of serum reactivity with the
various peptides, with some mouse sera from these
high dose groups completely failing to recognize any of
the synthetic peptides. Those mice which failed to
generate antibody capable of recognizing the four
synthetic peptides also produced low levels of antienvelope antibody as detected by Western blotting and
radioimmunoprecipitation.
The peptide ELISA data helped to map the immunogenic regions of the recombinant HTLV-I envelope
protein present in the inclusion bodies. In all groups,
env-I.B, generated the highest ELISA titres to the
synthetic SP-6 peptide (Fig. 6). In fact, the level of SP-6binding antibodies stimulated by env-I.B, injection was
influenced only negligibly by the adjuvant dose received.
Intermediate antibody titres to the synthetic SP-4A and
SP-7 peptides were observed, with the lowest antibody
titres directed to the SP-2 peptide (Fig. 6). All env-I.B.immunized mouse sera were compared to sera from mice
injected with M C W E only (group 1) to determine
significant ELISA titres.
The various mouse sera were screened in a syncytium
inhibition assay to determine whether neutralizing
antibodies were generated. Neutralizing antibody titres
of 10 to 40 were observed in only a few mice receiving the
highest doses of M C W E (250 lag and 500 lag; data not
shown). These doses produced unwanted side-effects in
the mice, according to observation of their general
health.
Combined immunization regimens
Previous experiments (Ford et al., 1992) have shown that
RVV Els expressing the native HTLV-I envelope (gp46
and gp21), and RVV E3s expressing only the surface
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Immunogenicity of recombinant H T L V - I env protein
30000 -
I
I
I
I
I
I
I
I
I
SP-2
m
1000
m
300
m
100
m
30
m
10
m
3
30000 -
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
7
•
8
~'~
9
B
3000
1
I
SP-4A
10000
<
I
I
m
m
m
m
. . . . . .
I
SP-6
I
I
I
•
I
I
I
I
I
I
10000
3000
•
•
• •
A•••
. . . . . . .
•
I
m
m
m
m
m
m
I
SP-7
•
I
••
J ~ t
1000
m
AA
Ak
AA~
•
300
100
m
30
10
1
m
.....
1
2
I
3
I
4
5
I
6
I
7
. . . . . . . .
8
9
1
2
I
3
I
4
•
5
I
6
Group
Fig. 6. Peptide ELISA titres of sera from mice immunized with env-I.B, and various a m o u n t s of M C W E . All groups with the exception
of group 1 received 10 ~tg of env-I.B, in the presence of the appropriate a m o u n t of M C W E . G r o u p 1, 100 ~tg M C W E ; group 2, 0 ~g
M C W E ; group 3, 5 ~tg M C W E ; group 4, 10 ~tg M C W E ; group 5, 25 ~tg M C W E ; group 6, 50 ~tg M C W E ; group 7, 100 ~tg M C W E ;
group 8, 250 ~tg M C W E ; group 9, 500 ~tg M C W E . ELISA titre is the dilution that resulted in an absorbance equal to or greater than
twice background values obtained with control mouse sera injected with 100 ~tg of M C W E alone (group 1). Titres less than 50 1 were
assigned as values of 0.
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217
J. Arp and others
218
D a y 14 (+/-)
8
9
10
Day 37(+++)
Day 28 (+ +)
ll
7
12
8
9
10
7
11 12
8
9
10
~I .( q "i:iiiC ~ 4
11 12
3~!i K ~i!'41~34KK
; :
:N
?
,
Fig. 7. Monitoring anti-envelope seroconversion. Western blot reactivity of six C57BL/6 mouse sera (sera 7 to 12, denoted above the
lane) screened on days 14, 28 and 37 during the course of combined immunization with RVV E l s and env-I.B. (group B of Table 1).
Samples were taken prior to each env-I.B, boost and upon termination. Reactivity was graded: + , weak; + + , moderate; + + + ,
strong.
1. Characterization of the immune response induced in mice immunized with a combined R VV
and env-I.B, regime
Table
Group
A
B
C
D
E
F
G
Immunization regime
D a y serum
obtained
Western
blot assay*
Peptide ELISA (titre-l)] "
Immunogen
Day
SP2
RVV-Els
RVV-E 1s
RVV-Els
Termination
0
14
28
37
0
14
28
37
+ (2/4)
+(4/5)
+(4/4)
RVV-Els
env-I.B.
env-LB.
Termination
0
14
28
37
0
14
28
37
+(1/6)
+ +(6/6)
+ + +(6/6)
RVV-E3s
RVV-E3s
RVV-E3s
Termination
0
14
28
37
0
14
28
37
-(4/4)
+ (2/5)
+ +(5/5)
-
RVV-E3s
env-I.B.
env-I.B.
Termination
0
14
28
37
0
14
28
37
+(2/6)
+ +(6/6)
+ + +(6/6)
120(4/4)
RVV-Elas
env-I.B.
env-I.B.
Termination
0
14
28
37
0
14
28
37
--(4/4)
+(5/5)
+ + (3/3)
50(3/4)
1640 media
env-I.B.
env-l.B.
Termination
0
14
28
37
0
14
28
37
-(4/4)
+(4/4)
+ +(4/4)
-
RVV-Elas
RVV-EIas
RVV-Elas
Termination
0
14
28
37
0
14
28
37
-(3/3)
- (6/6)
-- (4/4)
.
-
SP4A
SP7
-
-
ND§
ND
ND
10-40 (4/4)
100(1/6)
383(6/6)
NO
ND
ND
--(5/5)
50(4/5)
-
ND
ND
ND
10 (1/6)
50(1/6)
230(4/4)
100(1/6)
540(4/4)
ND
ND
ND
10-20(3/4)
-
ND
ND
ND
-- (4/4)
-
100(6/6)
.
.
116(6/6)
.
460(4/5)
-
138(4/5)
650(4/5)
-
50(1/6)
430(5/6)
1610(5/6)
-
Neutralizing
antibody
(titre 1):~
-
ND
ND
ND
-(5/5)
ND
ND
ND
-- (4/4)
* Western blot reactivity was graded as the following: + , weak; + + , moderate; + + + , strong.
t Peptide ELISA titres depicted in the table are calculated m e a n s of positive samples only. Individual ELISA titres were
determined as the dilution that resulted in an absorbance equal to, or greater than, twice background values obtained with
control mouse sera (injected with 10 ixg of M C W E alone).
:~ Neutralization titre: the highest dilution that inhibited HTLV-I syncytium formation by greater than 9 0 % relative to
normal mouse serum controls.
§ ND, Not determined.
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Immunogenicity o f recombinant H T L V - I env protein
glycoprotein (gp46) were capable of inducing
neutralizing antibodies. In an effort to enhance
neutralizing antibody titres directed to the HTLV-I
envelope, env-I.B, was injected in combination with
RVV Els and RVV E3s. RVV Elas which contains the
antisense version of the native envelope gene was used as
a control. Following priming with either RVV Els or
RVV E3s, the mice were boosted twice with env-I.B, in
the presence of 10 tag of MCWE adjuvant. This dose of
adjuvant was chosen because it generated optimal
antibody titres to the biologically significant SP-2 and
SP-4A regions of gp46 (Fig. 6). The resulting sera were
characterized by Western blot assay, peptide ELISA and
syncytium inhibition assay (neutralization assay) and the
results are summarized in Table 1. Western blot reactivity
was recorded using a grading ( + , + +, + + +) system
as illustrated in Fig. 7.
Priming with RVV Els (Table 1, group B) and RVV
E3s (group D) when combined with boosts of env-I.B.
increased the overall antibody response to the HTLV-I
envelope protein as determined by the Western blot
assay, when compared to immunization with either RVV
Els or RVV E3s alone (groups A and C), or env-I.B.
alone (groups E and F). This did not translate into
increased ELISA antibody titres to the SP-2 and SP-4A
regions of gp46, when compared to the titres elicited by
env-I.B, alone (compare groups B and D with E and F).
We did not examine SP-6 since it is not associated with
virus neutralization. Priming with RVV E3s did enhance
neutralizing antibody titres when used in combination
with env-I.B. (compare groups C, D, E and F). As
previously demonstrated (Ford et al., 1992), RVV Els
(group A) efficiently induced neutralizing antibody in the
absence of significant antibody titres to the SP-2 and SP4A regions.
Discussion
In this study the entire HTLV-I envelope glycoprotein
was successfully expressed in a baculovirus system. It
was hoped that the presence of the complete envelope
sequence, in a non-fusion form, would permit the
presentation of both linear and conformational epitopes
to the immune system. Only truncated or fusion proteins
with smaller gene segments have been successfully
expressed in Escherichia coli (Kiyokawa et al., 1984;
Samuel et al., 1984; Chen et al., 1989). Expression of full
or nearly full length gp63 in E. coli has proven to be toxic
to the host cells. HTLV-I envelope protein expression in
the baculovirus system has been limited to recombinant
polyhedrin fusion proteins (Nyunoya et al., 1990).
Synthesis of the HTLV-I envelope glycoprotein by a
mammalian expression vector has also proven to be toxic
to the host murine cells (Vile et al., 1991). Indeed, only
219
very low levels of expression of the entire HTLV-I
envelope protein have been obtained in Saccharomyces
cerevisiae by Kuga et al. (1986).
Efficient expression of the HTLV-I envelope protein at
levels of up to 6 rag/1 of cell culture medium was observed
in our baculovirus system. The protein was not secreted
from the host cells but was stored intracellularly as
inclusion bodies. The envelope protein obtained from
purified inclusion bodies consisted of three major size
classes of protein averaging about 43K, 54K and 63K.
The 63K protein was equivalent in size to the expected
glycosylated envelope precursor gp63 of HTLV-I (Pique
et al., 1992). The glycosylated nature of the recombinant
63K protein was suggested by its disappearance upon
tunicamycin treatment. Western blot analysis and
tunicamycin treatment suggested that the 54K and 43K
proteins represented variant unglycosylated forms of the
envelope precursor. As predicted from the HTLV-I
nucleotide sequence (Ratner et al., 1985), the 54K
protein may represent the non-glycosylated envelope
precursor with its leader peptide still attached. Unfortunately, the precursor origin of the recombinant 54K
protein cannot be confirmed despite its ability to bind
with anti-SP-7 peptide serum (SP-7 peptide sequence is
derived from gp21), since SP-7 peptide could not
completely inhibit the binding of the anti-SP-7 serum to
the 54K protein. The reason for this lack of inhibition is
not known but it may be due to a heteroclitic response to
the SP-7 peptide. The identity of the non-glycosylated
43K protein is uncertain; however, we have
demonstrated that the protein contains the epitopes
recognized by the 1C11 MAb (specifically binds SP-4A
peptide) and anti-SP-7 polyclonal sera. Amino-terminal
sequencing of the 43K protein has been hindered by its
lack of solubility. The identities of the immunoreactive
low M r proteins of 30K to 39K observed on Western
blots have yet to be deduced. Similar low M r proteins
were seen upon expression of the human immunodeficiency virus (HIV) envelope glycoprotein in a similar
baculovirus system (Hu et al., 1987). They could
represent proteolytic degradation products of the envelope precursor protein or variant glycosylated forms of
the mature protein since some forms disappeared when
cells were treated with tunicamycin.
The accumulation of the HTLV-I envelope protein in
the form of inclusion bodies could be the result of several
related factors. The baculovirus expression system is
inefficient in the processing of viral glycoproteins such as
the haemagglutinin of influenza virus (Kuroda et al.,
1986) and the envelope glycoprotein of HIV (Hu et al.,
1987; Rusche et al., 1987), since these precursor proteins
failed to be processed into the mature form. It is likely
that the inefficient cleavage of the baculovirus-produced
HTLV-I envelope precursor protein may be due to
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220
J. A r p and others
incomplete or improper glycosylation. Sf9 cells are
capable of N-linked glycosylation but are unable to
perform the complex sugar linkages normally found on
the HTLV-I envelope proteins (Miller, 1988). This may
lead to the accumulation of the envelope precursor
protein since efficient proteolytic cleavage appears to
depend on proper glycosylation (Pique et al., 1992).
Another contributing factor may be that, like HIV-1
gp160/120, HTLV-I gp63/46 may have a sequence that
causes the retention of large amounts of the envelope
protein in the secretory pathway (Bonifacino et al., 1991 ;
Li et al., 1992). It may be that when Sf9 cells are made to
express large amounts of HTLV-I envelope precursor,
they retain more protein than is physiologically compatible, and hence the envelope proteins are stored in
inclusion bodies to maintain cell viability. Even in
mammalian expression systems, non-glycosylated and
partially glycosylated forms of the HTLV-I envelope
protein have been shown to accumulate within the cells
to significant levels (Pique et al., 1992).
The HTLV-I env-I.B, isolated from our baculovirus
system proved to be immunogenic in three strains of
mice. Efficient stimulation of envelope-specific antibodies
by env-I.B, was observed in animals immunized in the
absence of adjuvant. The significant anti-envelope
humoral response observed suggests that the packing of
the HTLV-I envelope protein into inclusion bodies may
mediate the slow release of the immunogen at the
injection site and cause a prolonged stimulation of the
animal's immune effector cells.
The injection of an emulsion of env-I.B, and MCWE
adjuvant preparation proved to elevate the HTLV-I
envelope-specific response significantly in immunized
mice. An MCWE dose of 10 gg produced maximum
antibody titres to the four regions of envelope
represented by the synthetic peptides used in the peptide
ELISA. However, 50 gg of MCWE produced the best
overall antibody response as determined by Western blot
and radioimmunoprecipitation assays. Exceeding the 10
to 50 gg dose range of MCWE adjuvant resulted in a
decrease in peptide ELISA titres and a drop in the
absolute number of mice that seroconverted.
These results suggest that MCWE can serve as an
effective adjuvant in immunization regimens as it
stimulated good antibody responses against important
regions of the HTLV-I envelope. Recent studies have
shown that the carboxy terminus of gp46 is highly
immunogenic in humans (Copeland et al., 1986; Palker
et al., 1989). Similar results are shown here, particularly
in the presence of MCWE. The carboxy-terminal SP-6
region elicited the highest antibody titres of the four
regions tested by peptide ELISA. Immune responses
against the SP-4A region are particularly significant
since this region encompasses a B cell epitope (Palker et
al., 1989), a T cell epitope (Kurata et al., 1989), a
cytotoxic T cell epitope (Jacobson et al., 1991) and a
virus-neutralizing epitope (Tanaka et al., 1991). In the
presence of MCWE, significant titres to the SP-4A region
were elicited. The SP-2 region has also been associated
with virus neutralization; however, this region within the
env-I.B, did not elicit as high an antibody response as did
other regions in the presence or absence of MCWE.
Neutralizing antibodies, as detected by the syncytium
inhibition assay, were elicited by env-I.B, only at high
adjuvant doses of MCWE; thus, there was no correlation
between the titre of antibody capable of binding to the
SP-4A and SP-2 peptides and the ability of the sera to
inhibit syncytium formation. This lack of correlation
suggests either that the HTLV-I envelope glycoprotein
possesses other epitope(s) capable of eliciting
neutralizing antibody, or that the generation of
neutralizing antibodies requires that the epitopes contained within SP-2 and SP-4A be presented in a specific
conformation that does not occur in the env-I.B, to a
significant extent.
Recently we have shown that RVV expressing different
versions of the HTLV-I envelope could induce
neutralizing antibody against HTLV-I (Ford et al.,
1992). In an effort to increase the neutralizing antibody
titres, a combined immunization regimen was devised
employing both env-I.B, and RVV. Priming with RVV
Els or E3s clearly enhanced the anti-envelope antibody
response as compared to RVV alone or env-I.B, alone.
Most notably, the combined immunization with RVV
E3s, expressing gp46, and env-I.B, generated higher
neutralizing antibody responses in comparison to mice
immunized with E3s alone. Interestingly, the RVV Els,
which expresses the native HTLV-I envelope of gp46 and
gp21, did not prime mice to induce higher levels of
neutralizing antibody. The reason for this is not clear
since RVV Els was capable of inducing neutralizing
antibody on its own. Perhaps the manner in which the
gp46 was presented to the immune system by RVV Els
and RVV E3s was different.
Previous studies (Ford et al., 1992) revealed that RVV
Els-infected human H9 T cells properly process and
express the native HTLV-I envelope proteins on the cell
surface to the same extent as HTLV-I-infected T cells, as
determined by fluorescence-activated cell sorting (FACS)
analysis. Thus we would anticipate that the majority of
the RVV Els-encoded envelope protein would be
presented to immune effector cells in association with
major histocompatibility complex (MHC) class I antigen
(Teyton et al., 1990). RVV E3s-infected H9 T cells do not
appear to retain HTLV-I gp46 envelope protein on their
surfaces as determined by FACS analysis, although RVV
E3s expresses higher levels of gp46 than RVV Els in
infected cells. This is in agreement with studies describing
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Immunogenicity o f recombinant H T L V - I env protein
the expression of HTLV-I gp46 (Pique et al., 1990) and
HIV gpl20 (Kieny et al., 1988) in the absence of their
respective transmembrane proteins, which demonstrated
that the majority of the HTLV-I gp46 and HIV gpl20 is
released from the cell surface. This suggests that the
exogenous gp46 secreted by RVV E3s-infected cells
would be processed and presented through the M H C
class II pathway (Teyton et al., 1990), the same pathway
expected to process and present the recombinant
baculovirus-produced env-I.B. An MHC class H presentation pathway shared by the RVV E3s- and
recombinant baculovirus-produced HTLV-I envelope
proteins suggests that similar envelope protein epitopes
would be presented to the immune system and thus may
explain why boosts of recombinant baculovirus envelope
protein (env-I.B.) enhanced the neutralizing response in
RVV E3s-primed mice.
In summary, we have successfully expressed the entire
HTLV-I envelope protein in the baculovirus expression
system. The envelope proteins were produced as inclusion bodies which could be efficiently recovered.
When env-I.B, were combined with an adjuvant, MCWE,
antibodies were raised to important regions of the
HTLV-I envelope, but this did not translate into
significant neutralizing antibody titres. The combined
immunization with RVV E3s, expressing gp46, and envI.B. did result in increased neutralization titres. It will be
interesting to determine whether this combined
immunization approach will prove effective in animal
challenge experiments.
We wish to thank Dr J. K. Ball for her initial advice and help with
the animal experiments. This work was financially supported by the
MRC of Canada, NIH/NCI CA40660 of the U.S. and the Leukemia
Society of America. J.A. is supported by an MRC of Canada
Studentship, G.D. is supported by an Ontario Ministry of Health
Career Scientist Award, and T. J. P. is supported by a Leukemia
Society of America Scholar Award.
References
ARCHAMBAULT,G. (1989). Effect of sodium diethyldithiocarbamate,
Corynebacterium parvum and mycobacterial cell wall extract on in
vitro hlastogenic responses of bovine blood lymphocytes. Cornell
Veterinarian 79, 11~4.
BALL, J. K. & MCCARTER,J. A. (1979). Biological characterization of
a leukemogenic virus isolated from the CFW mouse. Cancer Research
39, 3080-3088.
BLATTNER,W., NOMURA,A., CLARK, J., HO, G., NAKAO, Y., GALLO,
R. & ROBERT-GUROEE, M. (1986). Modes of transmission and
evidence for viral latency from studies of human T-cell
lymphotrophic virus type I in Japanese migrant populations in
Hawaii. Proceedings of the National Academy of Sciences, U.S.A. 83,
4895-4898.
BONIFACINO,J. S-, COSSON,P., SHAH,N. & KLAUSNER,R. D. (1991).
Role of potentially charged transmembrane residues in targeting
proteins for retention and degradation within the endoplasmic
reticulum. EMBO Journal 10, 2783 2793.
CHEN,Y. M. A., LEE,T. H., SAMUEL,K. P., OKAYAMA,A., TACHIBANA,
N., MIYOSHI,L, PAPAS,T. S. & ESSEX,M. (1989). Antibody reactivity
221
to different regions of human T-cell leukemia virus type I gp61 in
infected people. Journal of Virology 63, 4952-4957.
CLAPHAM,P., NAGY, K. & WEISS, R. A. (1984). Pseudotypes of human
T-cell leukemia virus type 1 and 2: neutralization by patients' sera.
Proceedings of the National Academy of Sciences', U.S.A. 81,
288~2889.
COPELAND, T. D., TSAI, W.-P., KIM, Y.D. & OROSZLAN, S. (1986).
Envelope proteins of human T-cell leukemia virus type I:
characterization of antisera to synthetic peptides and identification
of a natural epitope. Journal of Immunology 137, 2945-2951.
DEKABAN, G.A., BALL, J.K., ROBEY, W.G., ARTHUR, L.O. &
MCCARTER,J. A. (1984). Molecular biological characterization of a
highly leukaemogenic virus isolated from the mouse. IV. Viral
proteins. Journal of General Virology 65, 1791 1802.
DICKSON, C., E1SENMAN,R , FAN, F., HUNTER, E. & TEICH, N. (1982).
Protein biosynthesis and assembly. In RNA Tumor Viruses, pp.
513-646. Edited by R. A. Weiss, N. Teich, H. Varmus & J. Coffin.
New York: Cold Spring Harbor Laboratory.
FORD, C., ARP, J., KING, E., DEKABAN,G. A. & PALKER, T. (1991).
Expression and immunogenicity of HTLV-I envelope proteins by
recombinant vaccinia virus. In Vaccines 91: Modern Approaches to
New Vaccines, pp. 253-258. Edited by R.M. Chanock, H. S.
Ginsberg & F. Brown. New York: Cold Spring Harbor Laboratory.
FORD, C. M., ARP, J., PALKER,T. J., KING, E. E. & DEKABAN,G. A.
(1992). Characterization of the antibody response to three different
versions of the HTLV-1 envelope protein expressed by recombinant
vaccinia viruses : induction of neutralizing antibody. Virology" 191,
448-453.
HAYDEN, D. B., BAKER, N. R., PERCIVAL, M.P. & BECKWITH, P. B.
(1986). Modification of the photosystem II light-harvesting chlorophyll a/b protein complex in maize during chill-induced
photoinhibition. Biochimica et biophysica acta 851, 86~92.
HORAL, P., HALL, W. W., SVENNERHOLM,B., LYCKE, J., JEANSSON,S.,
RYMO, L., KAPLAN, M. H. 8z VAHLNE, A. (1991). Identification of
type-specific linear epitopes in the glycoproteins gp46 and gp21 of
human T-cell leukemia viruses type I and type II using synthetic
peptides. Proceedings of the National Academy of Sciences, U.S.A.
88, 5754-5758.
Hu, S. L., KOSOWSKI, S. G. & SCHAAF,K. F. (1987). Expression of
envelope glycoproteins of human immunodeficiency virus by an
insect virus vector. Journal of Virology 61, 3617-3620.
JACOBSON,S., REUBEN,J., STREILEIN,R. t~ PALKER,T. (1991). Induction
of CD4 ÷, HTLV-I specific cytotoxic T-lymphocytes from patients
with HAM-TSP: recognition of an immunogenic region of the gp46
envelope glycoprotein of HTLV-I. Journal of Immunology 146,
1155 1162.
JOHNSON, D.A., GAUTSCH, J.W., SPORTSMAN,J. R. & ELDER, J. U.
(1984). Improved technique utilizing nonfat dry milk for analysis of
proteins and nucleic acids transferred to nitrocellulose. Genetic
Analysis Techniques 1, 3 8.
KIENY, M., LATHE, R., RIVIERE,Y., DOTT, K , SCHMITT,D., GIRARD,
M., MONTAGNIER,L. & LECOCQ,J.-P. (1988). Improved antigenicity
of the HIV env protein by cleavage site removal. Protein Engineering
2, 219-225.
KINOSHITA, K., AMAGASAKI,T., HINO, S., DOl, H., YAMANOUCHI,K.,
BAN, N., MOMITA, S., IKEDA, S., KAMIHIRA, S., ICHIMARU, M.,
KATAMINE,S., MIYAMOTO,T., TSUJI, Y., ISH1MARU,W., YAMABE,T.,
ITO, M., KAMURA,S. 8z~TSUDA,T. (1987). Milk-borne transmission
of HTLV-I from carrier mothers to their children. Japanese Journal
of Cancer Research 78, 674-680.
KIYOKAWA,T., YOSHIKURA,H., HATTORI,S., SEIKI,M. & YOSHIDA,M.
(1984). Envelope proteins of human T-cell leukemia virus: expression
in Eseherichia coli and its application to studies of eriEgene functions.
Proceedings of the National Academy of Sciences, U.S.A. 81,
6202 6206.
KUGA, T., HATTORI, S., YOSHIDA, M. • TANIGUCHI, T. (1986).
Expression of human T-cell leukemia virus type I envelope protein in
Saccharomyces cerevisiae. Gene 44, 337-340.
KURATA, A., PALKER, T., STREILEIN,R., SCEARCE,R., HAYNES,B. t~;
BERZOFSKY, J. (1989). Immunodominant sites on human T-cell
leukemia virus type 1 envelope protein for murine T-cells. Journal of
Immunology 143, 2024-2030.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 04 May 2017 08:13:56
222
J. A r p and others
KURODA,K., HAUSER,C., ROTT, R., KLENK,H. & DOERFLER,W. (1986).
Expression of the influenza virus haemagglutinin in insect cells by a
baculovirus vector. EMBO Journal 5, 1359-1365.
LAL, R. B., RUDOLPH, D. L., PALKER,T. J., COLIGAN,J. E. 86 FOLKS,
T. M. (1991). A synthetic peptide elicits antibody reactive with the
external envelope glycoprotein of human T cell lymphotropic virus
type I. Journal of General Virology 72, 2321-2324.
LI, Y., Luo, L., RASSOL,N. & KANG, C. Y. (1992). Essence of human
immunodeficiency virus gpl20 folding for CD4 binding. Journal of
Virology (in press).
MILLER, L. K. (1988). Baculoviruses as gene expression vectors. Annual
Review of Microbiology 42, 177 199.
NAGY, K., CLAPHAM,P., CHEINGSONG-PoPov,R. & WEISSR. A. (1983).
Human leukemia virus type I : induction of syncytia and inhibition
by patients' sera. International Journal of Cancer 32, 321-328.
NAKAMURA,H., HAYAMI,M., OHTA,Y., ISHIKAWA,K., TSUIJIMOTO,H.,
KIYOKAWA, T., YOSHIDA,M., SASAGAWA,A. 86 HONJO, S. (1987).
Protection of cynomolgus monkeys against infection by human Tcell leukemia virus type-I by immunization with viral env gene
products produced in Escherichia coli. International Journal of
Cancer 40, 403-407.
NYUNOYA,H., OGURA,T., KIKUCHI,M., IWAMOTO,H., YAMASHITA,K.,
MAEKAWA, M., TAKEBE, Y., MIYAMURA, K., YAMAZAKI, S. &
SHIMOTOHNO,K. (1990). Expression of HTLV-I envelope protein to
hydrophobic amino-terminal peptide of baculovirus polyhedrin in
insect cells and its application for serological assays. AIDS Research
and Human Retroviruses 6, 1311-1321.
OSAME,M., USUKU,K., IZUMO,S., IJICHI,N., AMITANI,H., IGATA,A.,
MATSUMOTO, M. 86 TARA, M. (1986). HTLV-I-associated
myelopathy, a new clinical entity. Lancet i, 1031-1032.
PALKER, T., TANNER,M., SCEARCE,R., STREILEIN,R., CLARK, M. 86
HAYNES, B. (1989). Mapping of immunogenic regions of human Tcell leukemia type I (HTLV-I) gp46 and gp21 envelope glycoproteins
with env-encoded synthetic peptides and a monoclonal antibody to
gp46. Journal of Immunology 142, 971478.
PALKER, T. J., RIGGS, E. R., SPRAGION,D. E., MUIR, A. J., SCEARCE,
R.M., RANDALL, R.R., MCADAMS, M.W., McKNIGHT, A.,
CLAPHAM,P. R., WEISS,R. A. & HAYNES,B. F. (1992). Mapping of
homologous, amino-terminal neutralizing regions of human T-cell
lymphotropic virus type I and II gp46 envelope glycoproteins.
Journal of Virology 66, 5879-5889.
PIQUE,C., TURSZ,T. & DOKHELAR,M.-C. (1990). Mutations introduced
along the HTLV-I envelope gene result in a non-functional protein:
a basis for envelope conservation? EMBO Journal 9, 4243-4248.
PIQUE, C., PHAM, D., TURSZ,T. 86 DOKHELAR,M.-C. (1992). Human Tcell leukemia virus type I envelope protein maturation process:
requirements for syncytium formation. Journal of Virology 66,
906-913.
RATNER, L., JOSEPHS, S.F., STARCICH,B., HAHN, B., SHAW, G.M.,
GALLO, R.C. & WONG-STAAL, F. (1985). Nucleotide sequence
analysis of a variant human T-cell leukemia virus (HTLV-Ib)
provirus with a deletion in pX-l. Journal of Virology 54, 781 790.
ROBERT-GUROFF,M., NAKAO,Y., NOTAKE, K., ITO, Y., SLISKI,A. &
GALLO, R. C. (1982). Natural antibodies to human retrovirus HTLV
in a cluster of Japanese patients with adult T cell leukemia. Science
215, 975 978.
RUSCHE, J. R., LYNN, D.L., ROBERT-GUROFF,M., LANGLOIS,A.J.,
LYERLY, H. K., CARSON,H., KROHN, K., RANKI, A., GALLO, R. C.,
BOLOGNESI, D.P., PUTNEY, S.D. & MATTHEWS, T.J. (1987).
Humoral immune response to the entire human immunodeficiency
virus envelope glycoprotein made in insect cells. Proceedings of the
National Academy of Sciences, U.S.A. 84, 6924-6928.
SAMUEL, K.P., LAUTENBERGER,J.A., JORCYK, C.L., JOSEPHS, S.,
WONG-STAAL, F. & PAPAS, T. S. (1984). Diagnostic potential for
human malignancies of bacterially produced HTLV-I envelope
glycoprotein. Science 226, 1094-1097.
SATOW, Y., HASHIDO,M., ISHIKAWA,K., HONDS, H., MIZLrNO, M.,
KAWANA,T. 86 HAYAMI,M. (1991). Detection of HTLV-I antigen in
peripheral and cord blood lymphocytes from carrier mothers. Lancet
338, 915 916.
SHIDA,H., TOCHIKURA,T., SATO,T., KONNO,T., HIRAYOSHI,K., SEKI,
M., ITO, Y., HATANAKA, M., HINUMA, M., SUGIMOTO, M.,
TAKAHASHI-NISHIMAKI, F., MARUYAMA,T., MIKI, K., SUZUKI, K.,
MORITA, H., SASHIYAMA,H. 86 HAYAMI, M. (1987). Effect of
recombinant vaccinia viruses that express HTLV-I envelope gene on
HTLV-I infection. EMBO Journal 6, 3379-3384.
SUMMERS, M. D. & SMITH, G. E. (1987). A manual of methods for
baculovirus vectors and insect cell culture procedures. Texas
Agricultural Experiment Station Bulletin, no. 1555.
TANAKA,Y., ZENG, L., SHIRAKI,H., SHIDA,H. 86 TOZAWA,H. (1991).
Identification of a neutralization epitope on the envelope gp46
antigen of human T cell leukemia virus type I and induction of
neutralizing antibody by peptide immunization. Journal of Immunology 147, 354-360.
TEYTON,L., O'SULLIVAN,D., DICKSON,P. W., LOTTEAU,V., SETTE,m.,
FINK, P. & PETERSON, P.A. (1990). Invariant chain distinguishes
between the exogenous and endogenous antigen presentation
pathways. Nature, London 348, 39-44.
VILE, R. G., SCHULZ,T. F., DANOS,O. F., COLLINS,M. K. L. & WEISS,
R.A. (1991). A murine cell line producing HTLV-I pseudotype
virions carrying a selectable marker gene. Virology 180, 420~424.
YOSHIDA, M., SEIKI, M., YAMAGUCm, K. 86 TAKATSUKI,K. (1984).
Monoclonal integration of human T-cell leukemia provirus in all
primary tumors of adult T-cell leukemia suggests causative role of
human T-cell leukemia virus in the disease. Proceedings of the
National Academy of Sciences, U.S.A. 81, 2534-2537.
(Received 29 May 1992; Accepted 14 October 1992)
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