Humoral immune responses to T cell tropic
retrovirus simian T lymphotropic virus type III in
monkeys with experimentally induced acquired
immune deficiency-like syndrome.
M Kannagi, … , L M Waldron, N L Letvin
J Clin Invest.
1986;78(5):1229-1236. https://doi.org/10.1172/JCI112706.
The T cell tropic retrovirus of macaque monkeys simian T lymphotropic virus type III
(STLV-III) has morphologic, growth, and antigenic properties indicating that it is related to human T
cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the
etiologic agent of the acquired immune deficiency syndrome (AIDS) of humans. STLV-III
has recently been shown to induce an AIDS-like disease in macaque monkeys. In this study
the humoral immune responses of six experimentally infected monkeys have been
characterized to determine whether certain parameters of the antibody response to the virus
might be predictive of the clinical outcome of this infection. Two distinct patterns of antibody
responses were found. Four animals that died within 160 d of inoculation developed low
titer anti-STLV-III antibody responses that recognized only the viral envelope protein, and
progressive declines in total plasma IgG levels and absolute peripheral blood T4
lymphocyte numbers. The two animals that lived longer (one died at 352 d, the other
remains alive at 430 d) developed high titer anti-STLV-III antibody responses that
recognized both viral envelope and core proteins, increases in total plasma IgG, and a later
decrease in number of peripheral blood T4 lymphocytes. Interestingly, the single animal that
has remained clinically healthy after infection was the only one to develop detectable
STLV-III neutralizing antibodies.
Research Article
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Humoral Immune Responses
to
T
Cell Tropic Retrovirus Simian
T Lymphotropic
Virus Type
Illin
Monkeys with Experimentally
Induced
Acquired
Immune Deficiency-like Syndrome
Mari Kannagi, Masaya Kiyotaki, Ronald C. Desrosiers, Keith A. Reimann, Norval W. King, Linda M. Waldron, and Norman L.Letvin
Harvard Medical School, New England Regional Primate Research Center, Southborough, Massachusetts 01772
Abstract
The Tcell
tropic retrovirus
of macaquemonkeys simian
T lym-photropic virus type III(STLV-III)
hasmorphologic, growth, andantigenic properties
indicating
thatit
is related to human T celllymphotropic
virus typeIII/lymphadenopathy-associated
virus
(HTLV-III/LAV), the etiologic
agentof
theacquired
im-munedeficiency syndrome (AIDS) of humans. STLV-III
has recently been shown toinduce
anAIDS-like disease
in macaque monkeys. In thisstudy the humoral
immune
responsesof
sixexperimentally infected monkeys have been characterized
tode-termine whether
certain
parametersof the antibody
response to thevirus might be predictive of the clinical
outcomeof this
in-fection.
Twodistinct
patternsof
antibody
responses werefound.
Four
animals
that died within160
dof inoculation
developed lowtiteranti-STLV-III
antibody
responses thatrecognized
only theviral envelope
protein,
andprogressive declines
intotal
plasma IgG levels and absolute
peripheral
blood T4lymphocyte
numbers. The two
animals that lived
longer(one died
at352
d, theotherremains alive
at430
d) developed
hightiter
anti-STLV-IIIantibody responses that recognized both viral envelope and core
proteins, increases
in totalplasma
IgG,
and a later decrease innumber of peripheral blood
T4lymphocytes. Interestingly,
the
single animal
that hasremained
clinically healthy after
in-fection
wasthe
only
one todevelop detectable
STLV-III
neu-tralizing antibodies.
Introduction
Considerable
progresshas been
madein characterizing the
an-tibody
responsesofindividuals infected with
human T celllym-photropic virus
typeIII/lymphadenopathy-associated
virus(HTLV-III/LAV),'
the human acquired immune deficiencysyndrome
(AIDS)virus.
Themajor HTLV-III/LAV-encoded
proteins
recognized
by sera ofinfectedindividuals
(1,2) and theweak in
vitro neutralizing activity of
these sera (3, 4) have beendefined.
However, little
information is available
thatcorrelatesReceived
for publication
3April
1986andinrevisedform
9 June 1986. 1.Abbreviationsused inthispaper: AIDS, acquired immune deficiency syndrome; ELISA, enzyme-linked immunosorbent assay; gp,glycopro-tein;HTLV-III/LAV, humanTcell
lymphotropic
virus typeIII/lymph-adenopathy-associatedvirus; IL-2, interleukin 2; Mm, Macacamulatta;
RT, reverse transcriptase; STLV-III, simian T lymphotropic virus type III.
the nature ofthese antibody responses with clinical diseasecourse
ininfected individuals. Given the
large
number of individualsinfected
with HTLV-III/LAV, it
isimportant
to determinewhether
specific characteristicsof the
humoral immune responsetothe
virus might predict
the clinicaloutcomeofthose infections. Elucidation of suchpredictive
parameters would be mostreadily
accomplished in an animal model of AIDS in which experimentally infected animals could be regularly and inten-sively studied. Theonly
animal species shown to be infectable withHTLV-III/LAV is the chimpanzee. However,chimpanzees
havedeveloped no
signs
of disease following infection with the human virus (5-8).Thus,
whilethe chimpanzee will beimportant
for thedevelopment of an AIDS vaccine, it is not an ideal
ex-perimental animal in which to study the viral pathogenesis of AIDS.
ATcell tropic retrovirus with striking similarities to HTLV-III/LAV was recently isolated from macaque monkeys at the NewEngland Regional Primate Research Center (9). We have called this virus simian T
lymphotropic
virus type III (STLV-HI) of macaques. LikeHTLV-III/LAV,
STLV-HII can be isolated from Tlymphocytes
and growspreferentially
in T4+ rather thanT8+
lymphocytes (10). By
electronmicroscopy,
matureparticles have acylindrical nucleoid and the virus buds in a fashion typical of type C retroviruses(9).
Furthermore,
STLV-III- andHTLV-III/LAV-encoded proteins
areserologically crossreactive
(1 1). Inpreliminary studies with this agent, juvenile rhesusmonkeys
died with a
wasting
syndrome, opportunistic infections,
a pri-maryretroviral
encephalitis, and immunologic
abnormalities following STLV-III inoculation (12). Thus, STLV-III induces anAIDS-like disease in rhesusmonkeys.
In
this
study wehave
characterized the humoral
immune response of rhesus monkeysexperimentally
infected with STLV-III.This
wasdone
todetermine whether specific
parameters of theantibody
responseto STLV-IIImight
bepredictive
of the clinical outcome of this infection. We show that the pattern of theanti-STLV-IIIantibody
response of infectedmonkeys
does correlate with clinical disease course. These patterns ofvirus-specific
antibody
response also correlated withchanges
in levels of both the totalplasma
immunoglobulin
G(IgG)
and theab-solute
peripheral
blood T4 cells.Methods
Animal inoculations. Sixjuvenilerhesusmonkeyswereexperimentally
infectedaspreviously describedwith STLV-III thatwasisolated froma
rhesusmonkeythatdied withalymphoma (12). Each animalwas
in-oculatedwith1.7 mlof thesamecell-freesupernatant ofa
STLV-III-infectedinterleukin 2(IL-2)-dependenthumanperipheralblood
lym-phocyte culturecontaining 146,000 cpm/mlreverse
transcriptase
(RT) activity.Indirect
immunofluorescence.
An indirect immunofluorescencestainingmethodwasused fordetection of plasmaantibodies
against
J.Clin.Invest.©TheAmericanSocietyfor ClinicalInvestigation, Inc.
0021-9738/86/11/1229/08 $1.00
STLV-III. STLV-III-infectedanduninfectedH9 cells were used as
an-tigen-expressingand negativecontrol cells. Plasma from animals was inactivated at560Cfor 60 min. Smears of infected and uninfected H9 cellswere fixed with acetone at room temperature for 7
min,
dried, then incubated with plasma at 370C for 45 min. After three washes withphosphate-buffered saline (PBS), these smears were incubated with
flu-orescein-conjugatedgoat anti-serum to human polyvalent immunoglob-ulins(Meloy Laboratories Inc.,Springfield, VA) at370C for45 min,
washed three times with PBS, and examined under a fluorescence mi-croscope.
Enzyme-linked immunosorbentassay(ELISA). STLV-III was har-vested from producing Hut-78 cells -72 h after a 1 to 3 split. Cells were removed bycentrifugationat 500 g for 15 min and the supernatant was
filteredthrough disposable
0.2-itm
Nalgene filters to remove residual cells anddebris.Virus was thenpelletedat35,000g for 180 min in a Beckman type 19 rotor. The supernatant was removed and the virus was resuspendedovernightin TNEbuffer(20 mMTrisHC1, 100 mM NaCl, 1 mM EDTA, pH 7.5) at 40C. Afterfractionation by Sepharose 4B column chromatography,purifiedviruscontainedin thevoidvolume waspooled and pelleted again bycentrifugation (13).Purified virus was resuspended in 100,d
ofcalcium-magnesium-freePBScontaining 0.5%Triton X-100and0.6 M KC1foreach 1 literofculture. Lysed virus was stored at-70'Cbefore coatingwellsofELISAplateswithviralantigen.
Virus was diluted 1:1,000 withcalcium-magnesium-free PBSand 100
,ul
of thiswasaddedtoeach well of Nuncimmunoplates.
Afterincubationatroom temperatureovernight,the plates wererinsedwith water and unboundsiteswereblockedby 10 min incubation with 0.3% bovine serum albumin. The plates wererinsedagainwith water and stored at
4°Cin sealedbags beforeuse.
Plasmadilutedwith 10%heat-inactivated goat serum, and 0.05% Tween20in PBS wasincubated in antigen-coatedwells for 1 h at room temperature; wells were thenrinsedthreetimeswith 0.05% Tween 20 in PBS. 100Mlof alkaline phosphatase-conjugatedgoatantibody to hu-manIgG was added to each well and incubated for 1 h at room tem-perature. The wells were rinsedthree times again andp-nitrophenyl
phosphate substrate, prepared asdescribedby themanufacturer
(Kir-kegaardand PerryLaboratories, Inc.,Gaithersburg,MD), was added.
After30min, sodiumhydroxide was added to aconcentrationof 0.6 M and theabsorbancerecorded at 410 nmwithaDynatech600microplate reader. Plasma samples werediluted 1:20 followed by serial fourfold dilutions fortesting.Theantibody titer determinedby ELISA wasdefined
as thatdilution ofplasma that resulted in an opticaldensity reading
of<0.1.
Radioimmunoprecipitation. H9 orSTLV-III-infectedH9cells were
radiolabeled with
[35S]methionine
(150 uCi/ml,New England Nuclear, Boston,MA) for 4 h. The labeled cells were resuspended inlysis buffer (1% TritonX-100, 0.05 MTris-Hd
buffer,pH 7.2,0.15 M NaCl, 1 mMethylenediaminetetraacetic
acid, 1%aprotinin, and 1 mMphenylme-thylsulfonyl
fluoride) and cleared by centrifugation for1 h at 100,000g.Macaque plasma, 10-138 ul (the sample sizesbeingvaried tonormalize
for total IgGconcentrationin theplasma ofeachanimal),wereincubated withproteinA-sepharose CL-4B(Sigma Chemical Co.,St.Louis,MO)
and, after
washing,
antibody-coated beadswereincubated withthe cell lysate. Afterfurtherwashing,
immunoprecipitateswereelutedfromthe protein A-sepharose beads with sample buffer (5% 2-mercaptoethanol, 2%sodium dodecylsulfate (SDS), 0.08 M Tris-HC, pH 6.8, 10% glycerol, and0.001% bromophenol blue) by boilingat100°Cfor 3 min. Samples were analyzed by electrophoresis in 10% SDS-polyacrylamide gels with a 3% stacking gel according to the discontinuous buffer system of Laemmli (14).Neutralization. Cell-free STLV-IIIcontaining culture supernatants were aliquoted and
stored
inliquid nitrogen for subsequent use in the neutralization studies. Macaque plasma was heat-inactivated and 0.3 ml, diluted 1:2 with PBS, was mixedwith0.3 ml of various dilutions of supernatantcontainingvirus and incubated at37°Cfor 1 h. This mixture wasincubated with 2X106
human phytohemagglutinin-stimulatedpe-ripheralbloodlymphocytes(PBL)at
370C
for 2 h. Cells were then washed threetimes withPBS, resuspendedin 2.5 ml RPMI 1640mediumcon-taining10% fetal calfserumand0.1% recombinantinterleukin 2 (IL-2) (kindly provided by E.I.DuPont Nemours, Inc., Wilmington, DE). They were cultured in 24-well plates (NUNC, Boston, MA) at370Cin hu-midified air with 5%CO2. 1-mlaliquots of culture supernatants were harvested every 3 to 4 d and medium was changed at the same time.
Culturesupernatants werefiltered andstored at-70'C. RTactivities ofculture supernatants were measured as previously described (10).
Radioimmunoassay for total plasma IgGquantitation.IgGwas pu-rified from normal rhesus monkey serum by ammonium sulfate precip-itation and gel filtration through Sephacryl S-300 (Pharmacia Fine
Chemicals, Piscataway,NJ).Radioiodination ofmonkey IgG was
per-formed usingthemethodpreviously described (15).Monoclonal anti-human IgG antibody (kindly provided by Victor Raso, Dana-Farber CancerInstitute,Boston,MA) was adsorbedfor2 h at
40C
ontoflexible polyvinyl microtiter plates (Becton-Dickinson& Co., Oxnard, CA). Wells were then incubated with 1%bovine serum albumin in PBS for 1 h to blocknonspecific binding sitesontheplasticand washed three times with PBS. Equal volumes of appropriately diluted test plasma from in-oculated monkeys and'25I-labeled
monkey IgG were incubated in the wellsovernight.The wells were washed three times with PBS, cut from theplate,andcountedon anautomated gamma counter. Test plasma values were interpolated from a standard curve.Quantitation
of
circulating T4+ andT8+ lymphocytes. PBL were isolatedfrombloodsamples byFicoll/sodium diatrizoatedensity gradientcentrifugation, washed in Hanks' balanced saltsolution(HBSS), and
incubatedinaliquotsof1 X 106 cellsfor
29
min at40C
with monoclonalanti-T4andanti-T8antibodies(kindly providedby Stuart Schlossman, Dana-FarberCancerInstitute,Boston, MA). The cells were then washed in HBSS and incubated influorescein
isothiocyanate-conjugated
goatanti-mouseIg (Tago Inc., Burlingame,CA)for20 min at
40C.
After washing each sample in PBS and fixing with a 1% formalin solution,cytofluorographic analysis ofcellpopulationswasperformedona
flu-orescence-activated cell sorter (FACS-1; Becton-Dickinson & Co., Mountainview, CA).Theperipheralblood T4and T8 counts were
de-termined bythefollowing calculation: peripheralblood white cell count XpercentlymphocytesXpercent cells stained by that monoclonal
an-tibody.
Results
Clinical
courseof
disease. Six
juvenile
rhesusmonkeys
wereexperimentally
infected with STLV-III
(Table I) (12).
STLV-III
was
isolated from their plasma
at2
wkand
PBL at 17 wkafter
inoculation (12). Four died between 127 and 160 d after
inoc-ulation
(Macaca
mulatta
[Mm]
91-84,
Mm99-84,
Mm 101-84,
Mm106-83).
These four
monkeys
had diarrhea
by
1 moafter
infection
and
three
(Mm 91-84,
Mm101-84,
and
Mm106-83)
lost
13, 34, and 60%
of their body weight during
the
study.
At necropsy,the
thymuses of
the
four
monkeys
wereatrophic.
Three
(Mm 91-84,
Mm99-84,
and
Mm101-84)
had
evidence of
ex-tensive
necrotizing adenovirus
infections that involved
pancreas,lung, and
biliary
tract.All
four
died
with
aprimary
retroviral
encephalitis.
Perivascular
infiltrates oflarge, foamy macrophages
with occasional multinucleated
giant
cells
werescattered
throughout
the white
and grey matterof the brain(Fig.
1A).
Electron
microscopic
examination of these
lesions revealedSTLV-III
particles
in the
macrophages
(Fig.
1B).
A
fifth
animal (Mm
74-84) developed
transient
lymphade-nopathy
8moafter inoculation and died
4molater
having
lost
24%
of its body
weight.
Itwasnotedatnecropsytohavethymic
atrophy
andcryptosporidiosis.
Thefinal
monkey
(Mm
127-83)
developed transient
lymphadenopathy
1 yr after inoculation and hasotherwise remained
healthy.
Anti-STLV-III
antibody
responses. Plasma from bloodTableLClinicalHistoriesofSix STLV-III-infected Rhesus Monkeys
Day Lymphadenopathy posfinoculation
Animal syndrome of death Clinical features
Mm91-84 No 127 Weightloss;thymic
atrophy;adenovirus infectionof pancreas; encephalitis Mm101-84 No 142 Weight loss; thymic
atrophy;adenovirus
infectionof pancreas, biliary tract, and lung;encephalitis
Mm106-83 No 148 Weightloss; thymic
atrophy;encephalitis
Mm99-84 No 160 Thymic
atrophy,
adenovirus infection of pancreas;encephalitis
Mm74-84 Yes 352 Weightloss;thymic
atrophy;
cryptosporidiosis
Mm 127-83 Yes Healthy
assessed for anti-STLV-llI
antibody
titerby
indirect immuno-fluorescence. As shown in Fig. 2, two patterns of anti-STLV-III antibodyresponses
were seen. The four animals that died within 160 d of inoculation (Mm 91-84, Mm 99-84, Mm 101-84, and Mm 106-83)developed peak
antibody
titers ofonly
1: 16-1:64 by 17 d afterexperimental infection.
The titers fellto1:4 orbelow
in
theweeks before
their deaths. The
twomonkeys
that did not
die
within 160 d(Mm
74-84 and Mm127-83)
de-veloped peakanti-STLV-Ill antibody titers of 1:4,000-1:16,000
by 122 d after inoculation. Their high
antibody
titerspersisted.
The
titer ofanti-STLV-lI IgG antibody
in the six inoculatedmonkeys
wasalsomeasured
using
anELISA procedure.
Similar tothe
indirect immunofluorescence measurements,
Mm 74-84 and Mm127-83
developed
hightiter antibodies that
persisted
(Table
II). IgG
antibody appeared earlier
inthe
macaque thatis
stillalive (Mm
127-83)
then in the macaque that diedat352days (Mm
74-84).
The four animals that died 127-160 d pos-tinoculationdeveloped
little ornoantibody
detectableby
ELISA.Thus, the
titration of antibodies by ELISA
agreed
quite
well with theindirect immunofluorescence
measurements.Viralproteins recognized by
monkey
antibodies. Plasmasamples
from the six
inoculated animalswereanalyzed using
radioimmunoprecipitation
of labeledproteins
from infected cells followedby gel
electrophoresis
toassessthe presence ofantibodies thatrecognize the presumed
major
virus-encodedproteins
ofSTLV-Ill.
Asshown inFig.
3,
thefour
monkeys
that
died
within~ ~ *.e)
4"
I-0
. w**,MO
A4
.41
._
. _
4 ;604
0 ~ r
00
4 04
4
rl
A,
t;#R I
(3 -7 4 fs 1 O k
|RW1Ifs t 3~~~~~~~~4
ro
ty
0
t
SMI
"a
* 4 9
*
He'I,6
Figure 1.(A)Photomicrograph ofa
portion
ofthecerebralcortexin which thereare two(one large andonesmall)perivascular
infiltrates of macrophages.Thelarger infiltratealsocontainsamultinucleated,
syncytial
giantcell.The vesselsineachlesionaresectionedtangen-tially (hematoxylinand
eosin,
X350). (B)Electronmicrograph
ofaportion of thecytoplasm ofa
macrophage
fromaperivascular
infil-trateinthe brain. Therearemany
STLV-Ill
retrovirusparticles
con-tainedwithinmembrane-bound vacuoles
presumed
tobephagosomes.
Notethecylindrical-shaped
nucleoids in several of theparticles.
Nobudding particles
werefound in these cells.Antibody ResponsestoSimian
TLymphotropic
VirusType
III 1231-Y
16,384
Cr-.
H
.
> _-_j
U,_
0~
z po <.La
(L
<
2t
4096
-024.
256
64
16
4.
DAYS POSTINOCULATION
Figure 2. Plasmaanti-STLV-IIItitersofrhesusmonkeys determined
byindirect
immunofluorescence
afterexperimental
infectionwithSTLV-III.*Denotestimeof death (o,Mm
91-84;
.,
Mm99-84;
A, Mm101-84;*, Mm 106-84;a,Mm74-84;.,
Mm127-83).160 d
of
infection (Mm 91-84,
Mm99-84,
Mm101-84,
and Mm106-83) developed weak
transient antibody
responsesonly
to
the
160/120-kD viral envelope
glycoprotein
(gp). These weak
antibody
responsesdid
notsimply
reflect
low
plasma IgG levels,
but in fact
weredue
todecreased
specific antibody
responsessince the
amountof
plasma used for each
immunoprecipitation
wasvaried in
order tonormalize the
quantity
of total IgG in
each
sample.
In contrast,the
twoanimals that survived longer
(Mm 74-84 and
Mm127-83)
developed
strongantibody
re-sponses to gp
160/120.
Mm127-83 also
developed
adetectable
antibody
response to the
viral
core
proteins (p55,
p27) by day
32
(Fig.
3), while Mm 74-84 developed
ananti-core response
by day 235 (data not shown).
Comparative
studies of plasma
from
these
two animals, done without
varying
the
quantity of
plasma used
for
each
immunoprecipitation,
demonstrated
dif-ferences
in
their
antibody
responses.
Mm74-84, which died
-1
yr after experimental
infection,
developed
ananti-envelope
IgG
antibody
response
by
day
32
postinoculation,
which
persisted
until
death.
Antibodies
top55
and
p27
werenotdetected in the
plasma of Mm 74-84
until
day 235
postinoculation;
these
an-tibodies were no
longer detectable
by day
329
postinfection.
Mm127-83, the monkey that has remained healthy, developed
earlier
and more persistent antibodies to the envelope and core viralantigens. Antibody to gp 160/120 was detected by day 17postinfection and has persisted. Antibodies
top55 and p27
ap-peared by day 32,
werestill detectable
onday
329, but
were no longer present on day 367postinoculation.
Nosignificant
de-creasein
indirect immunofluorescence anti-STLV-III antibody
titer
ortotal plasma IgG levels
wasnoted in plasma samples
from
Mm 127-83during
thattime interval. The gp 160/120, p55,and
p27 werethe
only viral proteins that could be detected
using
plasma samples
from
these animals under these
experi-mentalconditions.
Plasma neutralization studies.
We assessed the STLV-IIIneutralizing activity of
plasma samples from the two monkeysthat
developed high titer antibodies specific for STLV-III
(Mm127-83
and Mm 74-84) (TableIII).
The paired plasmasamples
used in
this
studyshowed the
sameantibody titer
against STLV-III(1:46
by indirect immunofluorescence).
Thevirus isolate
uti-lized for
theneutralizing
assay wasthe
same as that used toinfect the six
monkeys.
Apreliminary
titration of the
virus-con-taining
supernatantshowed
that theminimum concentration
of
supernatant necessaryfor
aproductive infection of the
targetcells detectable by
RT assay wasbetween
adilution
of
1:32 and
1:64.
Whenthe virus-containing
supernatant wasadded
tothe assay at a greaterconcentration
than
a1:10 dilution, neither
plasma affected the generation of
RTby the
targetlymphocytes.
Ifthe virus containing
supernatant wasdiluted >1:36, its addition
to
the cultures did
notresult in
reproducible
infections. Viral
replication in the cells
infected
with the
mixture
of
virus-con-taining
supernatant diluted 1:16
or1:36 and plasma from Mm
127-83
wassignificantly
lower than in cells infected with
amix-ture
of virus and control
plasma.
Plasma from
Mm74-84
did
not
significantly inhibit
RTgeneration
in
this
assay.This
dif-ference
wasdemonstrated
consistently
in
four
separate experi-mentsusing paired
plasma from Mm 127-84 and
Mm74-84
drawn
between
days 123
and225
following experimental
infec-tion.
Total
plasma
IgG
levels.
While these results suggested
acor-relation
between the
natureof
the
antibody
responsegenerated
by the
STLV-III-infected monkeys
and their clinical
course,it
was not
clear
towhat
extentthese
patternsin
antibody
responses wereantigen
specific.
To
address this
issue,
total
IgG
levels
weredetermined
onplasma samples
taken
atvarious time
intervals
following
experimental
STLV-III
infection
of the
monkeys
(Fig.
4).
Table
II. Plasma Anti-STLV-IIIAntibody
Titers AsDetermined byELISA Experimentalyinfected monkeysDays
postinoculation Mm127-83 Mm74-84 Mm106-84 Mm101-84 Mm99-84 Mm91-84
15 <1:20 <1:20 <1:20 <1:20 <1:20 <1:20 30 1:320* <1:20 <1:20 <1:20 <1:20 <1:20 45 1:1,280 1:80 <1:20 <1:20 <1:20 <1:20 85 1:5,120 1:1,280 NTt NT NT NTT
120 1:20,480 1:5,120 <1:20 <1:20 <1:20 <1:20 240 1:20,480 1:20,480
300 1:20,480 1:20,480
330 NT 1:20,480
350 1:20,480
Day 32
Day
114
1
2 3
4 5 6
wUI~
1
2
3 4
5
6
Cto
~..*
_ .VAN
e eC'~~~~~~~~~~~~~~~:?.:.:,,. ,- Io
The twoanimals survivinglonger than 160 d (Mm 74-84
and Mm 127-83) showed significant increases intotal plasma
IgG after STLV-III infection. This increasehas been maintained inthe remaining live animal (Mm 127-83).In Mm 74-84, an
increasedplasmaIgG levelwasevidentatleast 250d postino-culation, but this had markedly declinedby the time of its death. All four animals that died by 160 dpostinfection showed
sig-nificant, progressive decreases in total plasma IgG levels after
virus inoculation.Nosignificantchangein total plasma protein
wasassociated with the decreases in total plasma IgG in the
animals(datanotshown). Thus,the changesintotal IgGin the
plasma of themonkeysdidnotreflectmerelygrosschanges in
plasma protein concentrations.
Table III. Neutralization ofIn Vitro STLV-IllInfection
by Plasma ofSTL
V-Ill-infected
Rhesus MonkeysVirus dilution Days of
Plasmasource* culture 1:10 1:16 1:36
Mm74-84 7 36,756t 19,003 2,215
11 31,680 18,554 15,005
Mm127-83 7 44,490 3,402 304
11 41,558 6,235 232 Uninfected control 7 44,791 23,232 4,711 monkey 11 37,972 22,078 16,364
* Plasmas from experimentally infected rhesus monkeys both showed
antibodytitersagainst STLV-IIIof1:46byindirect
immunofluores-cence.
tcpmRT
activity.
Figure 3. Detection ofantibodyresponses
toSTLV-III using radioimmunoprecipita-tion andgelelectrophoresis.
[35S]Methionineradiolabeled uninfected H9 (lane C)orH9-STLV-III-infected cell
ly-sateswere reacted with 10-138
gl
of ma-- caqueplasma
boundtoprotein
A-Sepha-roseCL-4B. (Sample sizes were variedto
normalizefor totalIgGconcentration in the plasma of eachanimal.) Lane 1,Mm74-84; lane 2,Mm91-84;lane3,Mm99-84;lane 4, Mm 101-84; lane 5,Mm106-83; lane6,
Mm 127-83;thesesampleswereall reacted withH9-STLV-III-infected lysates. LaneC,
Mm 127-83plasma was reacted withanH9 uninfected celllysate. The
immunoprecipi-tates were eluted andanalyzed in 10% SDS
polyacrylamidegel.
Circulating T4+ lymphocytes.
Finally,
wesought
todeter-mine ifthese patterns of clinical diseasecourse andhumoral
immune statuscorrelated with alterations in circulating T4+ lymphocytesinthe STLV-III-infectedanimals(Table IV).The number of T4+ PBLdecreased soonafter experimental
inoc-ulationwith STLV-III in the four monkeys that died
rapidly
andwhose totalplasma IgG levelsdecreased postinfection. Whilesomevariabilitywasnoted in their numbersof circulating T4+ lymphocytes, three of the four (Mm 91-84, Mm 101-84, and
Mm 106-83)demonstrated decreases in T4+PBLtobelowthe normal 1,000/mm3 (12, 16) by3 wk after STLV-IIIinoculation. The fourth monkey (Mm 99-84) demonstrated a decrease in
T4+PBLby 12 wk postinfection.Immediatelybefore death the
2,200
cu4 4800
<t o 1,400
1,000
200
o 200 300
DAYS POSTINOCULATION
Figure4.Totalplasma IgG levels determinedbyradioimmunoassay
afterexperimentalinfection of rhesusmonkeys with STLV-III.* De-notestimeofdeath (o, Mm 91-84; .,Mm99-84;A,Mm 101-84;A,
Mm106-84; 0,Mm74-84; .,Mm 127-83).
Antibody ResponsestoSimian TLymphotropicVirus Type III 1233
kD
200
-I I I
93
-
66-45
-U
Table IV. Changes in T4+ and T8+ Circulating Lymphocytes inRhesus Monkeys after Inoculation with STL V-III
Experimentally infected monkeys
Mm91-84 Mm101-84 Mm 106-83 Mm 99-84 Mm 74-84 Mm 127-83
Weeks
postinoculation T4* T8 T4 T8 T4 T8 T4 T8 T4 T8 T4 T8
Pre- 1,462 1,174 1,718 1,938 1,308 1,290 1,245 903 1,420 1,521 1,873 887 3 962 605 732 1,171 455 936 1,213 808 1,574 1,303 1,831 2,093 6 598 933 1,526 1,425 1,461 2,002 1,231 1,204 1,091 1,785 1,803 1,803 12 1,089 1,053 824 1,957 840 2,058 516 860 909 1,515 1,439 1,738
17 1,697 522 720 806 1,656 2,230 1,778 938
20 672 806 525 1,050 804 731
25 382 706 998 406
28 553 322 714 272
34 1,008 1,220 682 502
39 927 1,287 1,698 1,338
45 354 299 629 210
52 1,008 743
68 864 1,404
* The absolute T4+ or T8+ count was determined by the following calculation: peripheral blood white cell count times the percentage of lympho-cytes times the percentageofcellsreactivewith that monoclonalantibody. Resultsshow total numbers ofreactive cells per microliter.
T4+PBL
increased in
twoof
theanimals. In the two monkeysthat
survived longer and developed elevated plasma
IgG levels, adecline in
the numberof
T4+PBLdid
not occuruntilsignif-icantly later after STLV-III infection. This
decreasein
Mm 74-84 T4+ PBL occurred20
wkpostinoculation.
The T4:T8 ratio wasonly noted to be reversed in Mm 127-83 68 wk afterex-perimental
infection.
Discussion
Current estimates
suggestthat
in theUnited States
alonethere
arebetween
1and
2million individuals infected with
HTLV-III/LAV
(17). The clinical
outcomeof
this infection
has proven tobe
quite variable.
Some
infected individuals
manifest
no ap-parentdisease
oronly
transient
lymphadenopathy, while
othersdevelop
achronic
wasting syndrome
or afatal
immunodeficiency
syndrome punctuated
by
episodes
of
opportunistic
infections
and
tumors(18). The
presentstudies
wereperformed
in the
STLV-III/macaque model of AIDS
todetermine whether
any aspectsof the humoral immune
response tothis
HTLV-III/
LAV-related
retrovirus correlate with clinical
outcomeof
in-fection.
In
this prospective study of six
juvenile
rhesusmonkeys
in-oculated
with STLV-III,
twodistinct
patternsof
virus-specific
antibody
responses werefound.
Thefirst four
animals
todie
developed
lowtiter anti-STLV-III
antibody
responses thatrec-ognized only
the gp120/160 viral envelope
protein.
Thetwothatlived longer
developed
high
titeranti-STLV-III antibody
responses that
recognized
bothviral envelope
and core(p55,
p27) proteins.
Interestingly,
thesingle animal
that hasremained
clinically
healthy
wastheonly
one thatdeveloped
aneutralizing
antibody
response. While these resultssuggested
acorrelation
between the nature
of
theantibody
responsegenerated by the
STLV-III-infected monkeys
andtheir clinical
course, theseanti-STLV-III antibody
responses infact
also correlatedwith
totalplasma
IgG levels and the absolute number of peripheral blood T4+lymphocytes
in themonkeys.
Thefour
animals that diedby 160 d postinfection
showedimmediate and
progressive
de-clines
intotal
plasma IgG
levels and atrend toward
adecreasein
T4+ PBLpostinoculation, while the
twoanimals that survived
longer showed
significant increases in
totalplasma
IgG and noimmediate fall in
circulating
T4+ PBL.Thus, the natureof
theantibody
response toSTLV-III
inthe
six experimentally infected
monkeys
reflected
an even moregeneral
impairment of their
immune
function.
The present
studies
indicate
anumber
of parallels
betweenthe
natureof the
antibody
responsesof the rhesus
monkeys
toSTLV-III and that of humans to HTLV-III/LAV. The four
monkeys that died
shortly after
STLV-III
infection
developed
decreased
plasma
IgG
levels and
only
low
titer-specific antibody
responses
tothe
virus,
apatternof humoral
responseatypical
for human
AIDS.However,
theremaining
twoSTLV-III-in-fected monkeys
developed
increased
plasma IgG
levels
andalymphadenopathy syndrome,
just
asis
seenin humans
afterHTLV-III/LAV infection
(19).
Whilethese
twoanimals
devel-oped increased
plasma IgG levels,
suggesting
that
theregulation
of
their
Bcell function
wasabnormal, they
still
werecapable
ofgenerating
ahigh
titer
STLV-III-specific antibody
response. Theobservation
that the twomonkeys
which had a morechronicdisease
coursewerethe
only
inoculated
animals thatdeveloped
antibodies
tothe
coreviral
antigens
is consistent with
recentfindings
instudies of
HTLV-III/LAV-infected
humans. Thosestudies
suggestthat
AIDSpatients
areless
likely
tohave
anti-bodies
totheHTLV-III/LAV
coreantigen
thaninfected
indi-viduals with
less severemanifestations of disease
(20-24).
It
is also of
notethat,
aswith plasma of
HTLV-III/LAV-infected humans,
noobvious
correlation
could be made between theanti-STLV-III
antibody
titer
andantibody-neutralizing
ca-pacity
of plasma
from thesetwomonkeys.
Parallelplasma
sam-ples
from
Mm 127-83 andMm74-84,
whileexhibiting
differ-ences in
detectable anti-core
antibody
responses but thesameanti-STLV-III antibody
titers by indirect
immunofluorescence,
demonstrateddifferences
inneutralizing capacity.
Anumberof
result. The fine specificities of the anti-envelope antibody re-sponsesof Mm 127-83 and Mm 74-84 may be quite
different,
with only those of Mm 127-83 recognizing the particular deter-minant(s) of the virus envelope critical for infection oftarget
cells. Moreover, as has been shown in other lentivirus infections, invivo mutation of the infecting STLV-III virus may occur in these monkeys with a coincident drift in the specificity of the antibody response. Under such conditions, the
specificity
of the anti-envelope antibodies from these two monkeys for the original infecting virus might be very different. Finally, while little prec-edent exists for such aphenomenon,
the anti-core antibody may be capable ofplaying
acontributing
role inblocking
STLV-IIIreplication in vitro.This model provides a
unique
system forprobing
these and other questions relatedtothe pathogenesisof AIDS. Thestudy ofvirus-specific antibody responses in HTLV-III/LAV-infected humans iscomplicated
by anumber
ofimportant
variables. Even in geographically restricted human populations, various HTLV-III/LAVseropositive
individuals have been shown to beinfected
bystructurally distinct isolates of the AIDS virus
(25-28). Moreover, it is usuallyimpossible
todetermine
the precise time of infection of humans. It is also difficult toperform
pro-spective studies thatrequire
frequent,
invasiveinterventions.
Incontrast, in
working
with theSTLV-III/macaque
model forAIDS,
animals of like
ageand
environmental exposure
can beinoculated simultaneously with
asingle, known viral isolate
and studied prospectively with frequentdiagnostic
interventions. This model shouldfacilitatestudy
of theeventsin diseasepathogenesis
thatresult
in the extremevariation
inmanifestations
of AIDS. Itshould
also beuseful for vaccine development
andanti-viral
drug
testing
for AIDS.Acknowledgments
The authors thank Dr. D. Ho, Massachusetts GeneralHospital,Boston,
MAfor advice inestablishingtheneutralization assay; Drs. M. D. Daniel and R. D. Huntforhelpfulconversations duringthe courseof these
studies;B.Blakefor careful readingof thismanuscript;B.Roy for prep-arationof thismanuscript; and Dr. P.K. Sehgal for assistance in the
careof theexperimentalanimals.
This work was supported by National Institutes of Health grants
A120729,CA38205, CA34949, and CA40680, Division of Research
Re-sourcesgrantRR00168,andby contracts from the Massachusetts De-partment ofPublic Health and grant
NQI-CO-239
10 from the National CancerInstitute.Dr.Letvin isarecipientofanAmerican Cancer Society Junior Faculty Research Award.References
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