JOURNAL OF VIROLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Aug. 2001, p. 7470–7480 Vol. 75, No. 16
Postnatal Passive Immunization of Neonatal Macaques with a Triple
Combination of Human Monoclonal Antibodies against Oral
Simian-Human Immunodeficiency Virus Challenge
REGINA HOFMANN-LEHMANN,1,2JOSEF VLASAK,1ROBERT A. RASMUSSEN,1,2BEVERLY A. SMITH,1,2
TIMOTHY W. BABA,1,2,3VLADIMIR LISKA,1,2FLAVIA FERRANTELLI,1,2DAVID C. MONTEFIORI,4
HAROLD M. MCCLURE,5DANIEL C. ANDERSON,5BRUCE J. BERNACKY,6TAHIR A. RIZVI,6
RUSSELL SCHMIDT,6LORI R. HILL,6MICHALE E. KEELING,6HERMANN KATINGER,7
GABRIELA STIEGLER,7LISA A. CAVACINI,2,8MARSHALL R. POSNER,2,8
TING-CHAO CHOU,9JANET ANDERSEN,10AND RUTH M. RUPRECHT1,2*
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute,1Department of Medicine, Harvard Medical School,2
Beth Israel-Deaconess Medical Center,8and Harvard School of Public Health,10Boston, Massachusetts 02115; Division of
Newborn Medicine, Tufts University School of Medicine, Boston, Massachusetts 021113; Center for AIDS Research,
Department of Surgery, Duke University Medical Center, Durham, North Carolina 277104; Yerkes Regional
Primate Research Center, Emory University, Atlanta, Georgia 303225; University of Texas, M. D. Anderson
Cancer Center, Bastrop, Texas 786026; Institute of Applied Microbiology, University of Agriculture,
A-1190 Vienna, Austria7; and Molecular Pharmacology and Therapeutics Program,
Memorial Sloan-Kettering Cancer Center, New York, New York 100219
Received 20 February 2001/Accepted 24 May 2001
To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we
established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that
mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351–357,
1994). Using this model, neonates were protected from mucosal SHIV-vpu?challenge by pre- and postnatal
treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and
2F5 (Baba et al., Nat. Med. 6:200–206, 2000). In the present study, we used this MAb combination only
postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential
use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu?challenge,
while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used
as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination
against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably,
the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered
the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants
remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4?T-cell
decline. In contrast, all control animals had dramatic drops in their CD4?T cells by 2 weeks postexposure. We
conclude that our triple MAb combination partially protected against mucosal challenge with the highly
pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human
MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.
The role of neutralizing antibodies (NAbs) in protecting
against the human immunodeficiency virus (HIV) has recently
been investigated in macaques (3, 37, 38, 56) by using chimeric
simian/human immunodeficiency viruses (SHIV) (51, 52, 57).
These viruses contain a simian immunodeficiency virus (SIV)
isolate mac239 backbone and encode envelope glycoproteins
derived from HIV type 1 (HIV-1), which makes it possible to
test antibodies directed against HIV-1 envelope in rhesus ma-
Recently, passively infused antibodies were found to protect
against an intravenous (i.v.) SHIV challenge in macaques (3,
37, 56). We found complete protection in four adult rhesus
macaques challenged with SHIV-vpu?after an infusion of
F105, 2G12, and 2F5 (3). These human monoclonal antibodies
(MAbs) are directed against conserved epitopes on HIV-1.
F105 recognizes the CD4 binding site (CD4BS) on gp120 (49).
2G12 binds to a conformation-sensitive, glycosylation-depen-
dent, discontinuous epitope centered around the C3/V4 do-
main on gp120 of HIV (62), and 2F5 is directed against a
specific sequence, ELDKWA, within the external domain of
the gp41 (17, 45). We and others have also shown that infusion
of anti-HIV antibodies protected against mucosal transmission
of SHIV (3, 38). Mascola et al. (38) infused MAbs 2F5 and
2G12 with or without high-titer anti-HIV immunoglobulin into
adult rhesus monkeys 24 h prior to vaginal SHIV89.6PD chal-
lenge. The best protection was found in the cohort that re-
ceived the triple combination. Four of five animals were pro-
tected against infection, and the fifth monkey did not develop
CD4?T-cell depletion. In contrast, all control monkeys given
irrelevant control immunoglobulins developed high plasma
viremia and rapid CD4?T-cell decline.
* Corresponding author. Mailing address: Dana-Farber Cancer In-
stitute, 44 Binney St. JFB809, Boston, MA 02115-6084. Phone: (617)
632-3719. Fax: (617) 632-3112. E-mail: firstname.lastname@example.org
Our goal is to develop immune prophylaxis against mother-
to-child HIV-1 transmission. Previously, we established an
SIV/macaque model that mimics mucosal HIV-1 exposure of
neonates that can occur during delivery (2). Using this model,
we achieved complete protection of four neonatal rhesus ma-
caques with the human MAbs 2F5, F105, and 2G12 against
oral challenge with SHIV-vpu?, a chimeric virus that encodes
the env gene of the laboratory-adapted, T-cell-tropic HIV-1
IIIB (3). The neonates received transplacental MAbs before
birth, by passive therapy of the pregnant dams, as well as by
direct i.v. infusion after birth. Prenatal passive antibody ther-
apy of pregnant women requires large amounts of MAbs com-
pared to those needed to infuse only neonates. Thus, prenatal
immunoprophylaxis might be too costly and could render
large-scale use in humans impractical. In the present study, we
assessed the efficacy of immunoprophylaxis using only postna-
tal MAb treatment of infants. First, we conducted a pilot study
with two neonatal rhesus macaques that both received 2F5,
F105, and 2G12 prior to oral challenge with SHIV-vpu?.
In a second experiment, we determined whether human
MAbs administered postnatally would also inhibit mucosal in-
fection by a chimeric virus carrying the env gene of a primary,
and hence less neutralization-sensitive, HIV-1 isolate. Thus,
neonates were treated with MAbs after birth and challenged
orally with SHIV89.6P, an in vivo-passaged, pathogenic virus
which expresses envelope glycoproteins of a primary HIV-1
isolate (52, 53). The optimal combination of human neutraliz-
ing MAbs against SHIV89.6P was determined in vitro. Al-
though primary HIV-1 isolates are more resistant to neutral-
ization than laboratory-adapted strains (19, 44), MAbs 2G12,
2F5, and IgG1b12, a MAb which binds to a confirmation-
sensitive epitope that overlaps but is not precisely continuous
with the CD4BS (10), have broad neutralizing activity against
primary virus isolates (8, 10, 11, 17, 20, 61). In addition, we
detected strong synergism and complete neutralization of the
primary isolate, HIV89.6, in human peripheral blood mono-
nuclear cells (PBMC) with the triple combination F105, 2F5,
and 2G12 (3). Remarkably, although IgG1b12 alone did not
neutralize SHIV89.6P or SHIV-KB9, a fourth-passage molec-
ular clone of SHIV89.6P (18, 21, 27), the triple combination
2G12, IgG1b12, and 2F5 showed the strongest synergism.
Thus, we used this combination for the in vivo immunopro-
phylaxis studies with oral SHIV89.6P challenge.
MATERIALS AND METHODS
Human IgG1 MAbs. The human immunoglobulin G1 (IgG1) anti-HIV-1
MAbs used were 2F5 (17, 45), 2G12 (8, 62), IgG1b12 (10), and F105 (49). The
MAb preparations were of clinical-grade purity and endotoxin-free. For the in
vivo immunoprophylaxis studies, each of these MAbs was given at a dose of 10
MAb IgG1b12-secreting CHO cells (kindly provided by Dennis Burton,
Scripps Research Institute, La Jolla, Calif.) were expanded in endotoxin-free
Glasgow minimum essential medium (Glasgow MEM; Sigma, St. Louis, Mo.).
The medium was supplemented with 100 ?M MEM nonessential amino acids
(Gibco-BRL, Life Technologies, Grand Island, N.Y.), glutamic acid (60 ?g/ml),
asparagine (60 ?g/ml), 1 mM sodium pyruvate (Sigma), heat-inactivated fetal
calf serum (FCS; Gemini Bio-Products, Woodland, Calif.), penicillin (100 U/ml),
streptomycin (100 ?g/ml), 50 ?M methionine sulfoximine (Sigma), and the
nucleosides adenosine, guanosine, cytidine, uridine, and thymidine (Sigma), each
at 7 ?g/ml. The FCS concentration was stepwise reduced from initially 10% to
2%. Supernatants containing the MAb were collected on day 7 and clarified by
centrifugation. The antibody was purified by protein G chromatography (Amer-
sham Pharmacia Biotech Inc., Piscataway, N.J.), dialyzed against phosphate-
buffered saline under endotoxin-free conditions, tested for endotoxin, and stored
at 4°C until use. If necessary, endotoxin was removed by Detoxi-Gel (Pierce,
Rockford, Il.) according to the manufacturer’s instruction.
Virus stocks. We used two different chimeric SHIVs as challenge viruses.
SHIV-vpu?contains env from the laboratory-adapted, T-cell-tropic HIV-1 IIIB
on an SIVmac239 backbone (31, 36). SHIV89.6P encodes env of the primary,
dualtropic HIV-1 strain 89.6 originally isolated from PBMC of a 47-year-old man
with AIDS (16). The biological isolate was derived from SHIV89.6 after four
serial passages in rhesus macaques (51, 52) and is acutely pathogenic, causing
profound CD4?T-cell depletion within 2 weeks of virus exposure (52). Both
cell-free challenge virus stocks were propagated in mitogen-stimulated rhesus
macaque PBMC in the presence of human interleukin-2 (IL-2). Supernatants
were clarified by centrifugation, filtered, and stored in vapor-phase liquid nitro-
gen. Stocks were titrated by endpoint titration on CEMx174 cells as described
elsewhere (3). The SHIV-vpu?stock and the SHIV89.6P stock contained 2 ?
105and 2.6 ? 10450% tissue culture infectious doses per ml, respectively. To
determine the oral 50% animal infectious doses (AID50), seven neonatal rhesus
monkeys were exposed orally to serial dilutions of SHIV-vpu?; eight neonates
were used to titrate SHIV89.6P in a similar manner. The SHIV-vpu?stock
contained 3.79 oral AID50/ml (3); the SHIV89.6P stock yielded 30 oral AID50/ml.
In vitro SHIV89.6P neutralization assay. We adapted an MT-2 cell viability
assay (43) to measure neutralization of SHIV89.6P. MAbs were diluted serially
in RPMI 1640 (Gibco-BRL) supplemented with 15% heat-inactivated FCS,
penicillin (100 U/ml), streptomycin (100 ?g/ml), and 2 mM L-glutamine (Sigma).
Antibodies (50 ?l) were preincubated in triplicate with 50 ?l of SHIV89.6P
(diluted 1:5 in ice-cold medium; 50 ?l containing 4.2 pg of p27) in flat-bottom
96-well plates (Corning Costar Corporation, Boston, Mass.) at 37°C for 1 h.
Log-phase MT-2 cells (50 ?l of a 106-cell/ml suspension) were added to the
virus-antibody mixture and incubated at 37°C with 5% CO2. Virus-only controls
(containing SHIV89.6P and MT-2 cells only, without MAbs) and cells-only
controls were cultured in parallel. After 2 h, each well was supplemented with
100 ?l of medium and incubation was continued. Cell densities were reduced
after 3 days of incubation by replacing 100 ?l of each cell suspension with
medium. On day 5, cells were fed by exchanging 100 ?l of medium. Infection led
to extensive (?80%) syncytium formation after approximately 7 days in the
absence of MAb (virus-only wells). Cells were harvested, and viability was tested
by neutral red uptake (43). Cell suspensions (100 ?l) were transferred to 96-well
plates precoated with poly-L-lysine (Becton Dickinson, Franklin Lakes, N.J.). A
volume of 100 ?l of neutral red (1:300 solution; ICN Pharmaceuticals, Costa
Mesa, Calif.), diluted 1:10 in medium, was added to each well. The plates were
incubated for 1 h at 37°C with 5% CO2, followed by aspiration of the medium
and 2 gentle washes with phosphate-buffered saline. The neutral red absorbed by
viable cells was extracted with 1% acetic acid in 50% ethanol for 1 h at room
temperature under constant agitation. Optical density was read with a multiscan
Microplate Reader (Du Pont Instruments, Wilmington, Del.) at 540 nm. The
percentage of protection was defined as the difference in mean absorption
between test wells (cells plus virus plus antibody) and virus-only wells, divided by
the difference in mean absorption between cells-only wells and virus-only wells.
Determination of synergism (CI) and DRI. The complex interactions of MAb
combinations were assessed as outlined earlier (29, 30) by the Chou-Talalay
method (14, 15). Basically, this method yields two indices describing the inter-
actions among MAbs in a given combination: the combination index (CI) and the
dose reduction index (DRI). A CI of ?1 indicates synergism, a CI of 1 indicates
additive effects, and a CI of ?1 indicates antagonism. The DRI reflects the factor
by which the dose of each MAb in a combination may be reduced at a given
percent neutralization compared with the dose when each MAb is used alone
(12, 13). A high DRI correlates with a strong synergistic effect, and the amount
of MAb may be decreased accordingly in the combination.
Animals, animal care, and experimental design of the in vivo immunoprophy-
laxis study. We used 14 neonatal rhesus monkeys (Macaca mulatta) from a
retrovirus-free colony. The animals were kept according to National Institutes of
Health guidelines on the care and use of laboratory animals at the University of
Texas M. D. Anderson Cancer Center and at the Yerkes Regional Primate
Center, Emory University, Atlanta, Ga. These facilities are fully accredited by
the Association for Assessment and Accreditation of Laboratory Animal Care
International. Animal experiments were approved by the animal care and use
committees of these institutions and the Dana-Farber Cancer Institute. Monkeys
were anaesthetized intramuscularly with ketamine (5 mg/kg) before all proce-
dures that required removal from their cages. All animals described were found
to be negative for simian T-lymphotropic virus type 1 (32) and simian retrovirus
type D by PCR (34).
Two neonates, P1 and P2, were treated i.v. with the triple MAb combination
of F105, 2G12, and 2F5 on the day of birth (Fig. 1A). One hour after completion
VOL. 75, 2001 POSTNATAL PASSIVE IMMUNIZATION AGAINST SHIV CHALLENGE 7471
of the infusion, P1 and P2 were challenged orally in a nontraumatic manner (2)
with 10 oral AID50of SHIV-vpu?. A second MAb dose was given on day 8. Four
untreated control neonates (R9C, R10C, R11C, and R16C) were challenged
identically (Fig. 1B).
Four neonates (RCh-7, RFh-7, RIh-7, RWg-7) were treated i.v. with a com-
bination of three MAbs (IgG1b12, 2G12, and 2F5) within the first 3 days of
vaginal delivery (Fig. 1C). The treated animals were challenged orally with 15
oral AID50of SHIV89.6P 1 hour after completion of the MAb infusion. This
dose yielded a 99% probability of infection (58). A second MAb dose was given
on day 8. Four control group neonates (REh-7, RHh-7, RJh-7, and RUg-7) were
untreated prior to oral virus challenge (Fig. 1D).
Blood samples were collected from all animals on day 0 prior to MAb infusion
and virus inoculation (baseline collection) and on day 8, prior to the second MAb
infusion, to determine infection/protection of animals. Although desirable, no
additional samples could be collected after the MAb infusions to determine the
plasma titer of NAbs or to assess pharmacokinetics. The maximum blood volume
that can be sampled from approximately 500-g neonates (6 ml/kg of body weight/
month) did not allow it. MAb-treated animals were observed for 31 months after
SHIV-vpu?challenge and for 12 months after SHIV89.6P challenge, during
which time samples were collected regularly. When necessary, animals with
progressive disease were sacrificed via i.v. sodium pentobarbital injection.
Virus isolation; determination of proviral DNA and viral RNA loads. Methods
for PBMC coculture and DNA PCR have been described previously (2, 33). The
lower limit of detection of the DNA PCR was one copy per 150,000 cells (33).
Viral RNA loads were quantified by a one-tube fluorogenic probe-based real-
time reverse transcription (RT)-PCR (24). RNA was extracted from 140 ?l of
cell-free plasma with a QIAamp Viral RNA Mini kit (Qiagen, Valencia, Calif.).
The sensitivity of the RT-PCR is 50 copies per ml of undiluted plasma. Sodium
citrate-anticoagulated samples were not available from animal R16C for RT-
PCR or neutralization assays.
Serological assays. Plasma samples were analyzed as described elsewhere (2,
33) for the presence of specific antibodies, using commercially available Western
blot strips prepared from HIV-2 antigens which had been shown to cross-react
with SIV antisera. HIV-1 Western blot analyses were conducted as instructed by
the manufacturer (Epitope, Beaverton, Oreg.).
NAbs in saliva and plasma. Saliva was collected from the oral cavities of the
neonatal macaques using Weck-cel sponges (Xomed Surgical Products, Jackson-
ville, Fla.). Saliva samples were collected 1 h after the first MAb infusion and/or
prior to virus inoculation. After collection, sponges were saturated with 400 ?l of
medium (see above) for 15 min. Gentle squeezing with a sterile pipette eluted
the saliva, and the liquid (250 ?l) was sterile filtered. To measure neutralization,
serial dilutions of salivary filtrates were assayed in duplicate in an adapted MT-2
cell infection assay with SHIV89.6P as described above or with SHIV-vpu?as
described earlier (30). The salivary NAb titer is the reciprocal dilution which
protected 50% of MT-2 cells from virus-induced cytotoxicity. This corresponds to
90% reduction of the viral Gag synthesis (9). The assay’s lower limit of detection
titer was 10 for undiluted samples. The same assays were applied to quantify
NAbs in serially diluted, heat-inactivated (56°C, 1 h) plasma samples. Neutral-
izing activities against other SHIV variants, SHIV89.6 (51) and SHIV-KU2 (26),
and the heterologous T-cell-line-adapted HIV-1 strain MN (23) were measured
in an identical manner.
Quantification of CTL activity in the completely protected animal RCh-7. An
autologous B-lymphoblastic cell line (BLCL) was prepared by transformation of
PBMC from RCh-7 with herpesvirus papio (50). PBMC collected 46 weeks after
SHIV89.6P challenge were stimulated with paraformaldehyde-fixed, autologous
BLCL infected with SHIV antigen-encoding vaccinia virus constructs at 2 to 3
PFU per cell (Therion, Cambridge, Mass.) Recombinant human IL-2 (20 U/ml)
was added every 3 days. Cultures were tested on day 7 in a standard 5-h cytotoxic
T-lymphocyte (CTL) assay using autologous,51Cr-labeled BLCL target cells
infected with vaccinia virus constructs encoding SIV gag-pol, SIV nef, or HIV-1
IIIB env. Background cytotoxicity was measured using wild-type vaccinia virus-
infected control target cells. A PBMC culture from an animal with known CTL
activity was included as a positive control for the autologous stimulation and
cytotoxicity arms of the assay.
In vitro challenge of PBMC from the completely protected animal RCh-7.
PBMC were purified by Ficoll-Hypaque sedimentation from a sodium citrate-
anticoagulated blood sample collected from uninfected animal RCh-7 36 weeks
after SHIV89.6P challenge. Cells were washed and stimulated for 3 days with
concanavalin A (5 ?g/ml) in RPMI 1640 supplemented with 10% human type AB
serum, L-glutamine, and penicillin-streptomycin. Cells were washed, and CD8?
T cells were removed by negative isolation using anti-CD8 magnetic beads as
instructed by the manufacturer (Dynal, Lake Success, N.Y.). CD8-depleted cells
were exposed to SHIV89.6P (100 ng of p27) for 12 h at 37°C. The cells were
washed and then incubated for 17 days in the presence of recombinant human
IL-2 (40 U/ml). Supernatants were collected every 2 to 4 days and analyzed for
the presence of SIV p27 antigen, using a commercially available enzyme-linked
immunoassay kit specific for SIV (Beckman/Coulter, Hialeah, Fla.). PBMC from
FIG. 1. Experimental design of the in vivo oral challenge studies
with SHIV-vpu?(A and B) and SHIV89.6P (C and D). (A) Two
neonatal rhesus monkeys (P1 and P2) were infused twice i.v. with
human MAbs F105, 2G12, and 2F5 (10 mg/kg; solid black arrows). d0
and d8, days 0 and 8. (B) Four neonates (R9C, R10C, R11C, and
R16C) served as untreated control animals. All six animals were chal-
lenged orally with 10 oral AID50of SHIV-vpu?1 h after the first MAb
treatment on the day of birth (open arrows). Treated animals were
observed for 31 months, and serial blood samples were collected.
Control animals were sacrificed 6 to 12 months after virus challenge.
(C) Four neonatal rhesus monkeys (RCh-7, RFh-7, RIh-7, and
RWg-7) were infused i.v. twice with human MAbs IgG1b12, 2G12, and
2F5 (10 mg/kg; solid black arrows). (D) Four neonates (REh-7,
RHh-7, RJh-7, and RUg-7) served as untreated control animals. All
eight animals were challenged orally with SHIV89.6P (15 oral AID50)
1 h after the first MAb treatment or at the corresponding time point (3
days after birth) (open arrows). Animals were observed for 12 months,
and serial blood samples were collected. †, animals RWg-7, RHh-7,
RJh-7 were sacrificed 10, 7, and 9 weeks postexposure, respectively.
7472 HOFMANN-LEHMANN ET AL.J. VIROL.
an uninfected normal control animal were stimulated, CD8?T-cell depleted, and
infected in the same manner as a positive control.
Statistical analysis. For analysis, the viral loads were expressed as a log10
transformation based on the distribution of HIV RNA in human studies. Viral
loads below the limit of detection were set at half the limit of detection for
calculation. No imputation for missing or truncated data was employed. It is
noted that with four animals per group, there is low statistical power.
Synergistic neutralization of SHIV89.6P by a triple combi-
nation of human anti-HIV-1 MAbs in vitro. We previously
tested a large panel of human anti-HIV-1 MAbs for their
ability to neutralize SHIV-vpu?in vitro. MAbs IgG1b12, 2F5,
2G12, and F105 were synergistic when used in combinations (3,
29, 30). In addition, the triple combination of F105, 2G12, and
2F5 was used successfully to protect macaques from i.v. and
oral SHIV-vpu?challenge (3). In the present study, we ex-
plored the capacity of these four MAbs, used either alone or in
combination, to neutralize pathogenic SHIV89.6P. We also
compared the synergistic potency of F105 to that of IgG1b12 to
determine whether different MAbs directed against the
CD4BS were equally effective.
MAb IgG1b12 was inactive against SHIV89.6P; even high
IgG1b12 concentrations up to 100 ?g/ml did not significantly
neutralize SHIV89.6P (Table 1; Fig. 2). Nevertheless, IgG1b12
(included at a constant concentration of 50 ?g/ml) increased
the neutralization potency of other MAbs (2G12 and 2F5)
when used in combinations, as indicated by a leftward shift of
the curves (Fig. 2; Table 1). This allowed us to use a reduced
concentration of antibodies to achieve an equivalent level of
neutralization. For example, when used alone, 1.243 ?g of 2F5
per ml was required to obtain 50% SHIV89.6P neutralization
(Table 1); by adding IgG1b12, the dose of 2F5 necessary to
achieve the same result could be reduced 18.8 times (DRI) to
0.066 ?g/ml. The addition of IgG1b12 to the combination of
2F5 and 2G12 led to very strong synergism and to even greater
DRIs of 110 for 2F5 and 25.7 for 2G12 (Table 1). This effect
was stronger than that observed using F105. The addition of
F105 (also at a constant concentration of 50 ?g/ml) to a serial
dilution of 2F5 and 2G12 led to synergism at low concentra-
tions, whereas antagonism was seen at the desirable, high de-
TABLE 1. Synergistic neutralization of SHIV89.6P by human IgG1 MAbs in MT-2 cells
Single antibody (specificity) or antibody
Concn (?g/ml) for
Combinations of 2 MAbs
2G12 ? IgG1b12e
2F5 ? IgG1b12e
2F5 ? 2G12 (1:1)
3.8 Potentiation of 2G12 by IgG1b12d
Potentiation of 2F5 by IgG1b12d
Combinations of 3 MAbs
2F5 ? 2G12 (1:1) ? IgG1b12e
2F5 ? 2G12 (1:1) ? F105e
Very strong synergism
Antagonism at high degrees of
neutralization; synergism at low
degrees of neutralization
Combination of 4 MAbs
2F5 ? 2G12 (1:1) ? IgG1b12 ? F105e
0.265 0.540.80 11.32.2 Synergism
aThe neutralization dose for combinations of two and more MAbs was the sum of the dose of each MAb used in the combination regimen with the exception of
IgG1b12 and F105. See footnote e.
bCalculated by the Chou-Talalay method as described in Materials and Methods. CI ? 1, CI ? 1, and CI ? 1, indicate synergism, additive effects, and antagonism,
respectively. ED50and ED90, 50 and 90% effective doses.
cMeasured by comparing the doses required to reach 50% virus neutralization when the antibody was used alone and in combination with other antibodies.
dWhen one component in combination has no or negligible effect by itself (e.g., IgG1b12), no CI value can be calculated due to lack of dose-effect parameters. In
this case, the term “potentiation” is used instead of “synergism.”
eAlways used at a concentration of 50 ?g/ml.
FIG. 2. Human MAb neutralization of SHIV89.6P in MT-2 cells.
Neutralization was measured colorimetrically by the percentage of
viable cells after incubation with the virus-antibody mixture (y axis).
The antibody concentration (x axis) in combinations of two or more
MAbs is the sum of the concentrations of each MAb with the exception
of IgG1b12 and F105. When used in combinations, MAbs IgG1b12
and F105 were at 50 ?g/ml; as potentiators, these amounts are not
included in the total antibody concentration indicated on the x axis. In
addition, when MAbs 2G12 and 2F5 were tested alone or in combi-
nation with other antibodies, the concentration of each was identical.
Results are representative of two or three separate experiments for
VOL. 75, 2001POSTNATAL PASSIVE IMMUNIZATION AGAINST SHIV CHALLENGE 7473
7474HOFMANN-LEHMANN ET AL.J. VIROL.
grees of neutralization. Furthermore, the quadruple MAb
combination was less synergistic than the triple 2F5-2G12-
IgG1b12 combination, presumably because of competition be-
tween F105 and IgG1b12 for the CD4BS. Therefore, we se-
lected the triple combination of 2G12, IgG1b12, and 2F5 for
the in vivo SHIV89.6P studies.
Outcome of oral SHIV-vpu?challenge. Two neonatal rhesus
macaques were treated i.v. with a combination of 2G12, F105,
and 2F5 on the day of birth (day 0 [Fig. 1]) prior to oral
SHIV-vpu?challenge. Both animals received a second infu-
sion of this triple MAb combination 8 days later. Four un-
treated control animals were also challenged orally. These four
monkeys served as controls also for another study arm, re-
ported previously (3). All four untreated neonates, R9C,
R10C, R11C, and R16C, became infected. They were positive
for virus isolation and proviral DNA PCR, and all four animals
seroconverted (3). Plasma samples from R9C, R10C, and
R11C were tested by RT-PCR and yielded positive results
throughout the observation period (Fig. 3A).
In contrast, both MAb-treated animals were protected from
oral SHIV-vpu?challenge. No plasma viral RNA was detect-
able (Fig. 3B), virus isolation was persistently negative, and no
proviral DNA was detectable in PBMC samples obtained from
these animals between weeks 1 and 6 postexposure (data not
shown). Furthermore, no virus-specific antibodies were de-
tected by p27 enzyme-linked immunosorbent assay or by West-
ern blotting using HIV-2 strips (data not shown). Taken to-
gether, our results confirm that the triple combination of
neutralizing MAbs protects from oral SHIV-vpu?challenge
and demonstrate that protection is achieved with only postna-
Virological and clinical outcome of SHIV89.6P challenge.
Four neonatal rhesus macaques were treated i.v. with a com-
bination of 2G12, IgG1b12, and 2F5 within the first 3 days of
vaginal delivery (day 0 [Fig. 1]) and were challenged orally with
15 oral AID50of SHIV89.6P 1 h after completing the antibody
infusion. They received a second infusion of this triple MAb
combination 8 days later. Four untreated control animals were
All four untreated control animals (REh-7, RHh-7, RJh-7,
and RUg-7) became highly viremic (Fig. 3C and E). The peak
viral RNA levels in the three MAb-treated infants that devel-
oped viremia were 10 to 100 times lower than those in the two
control infants RHh-7 and RJh-7 (Fig. 3C and D), but because
of the low number of study animals, statistical significance
could not be reached. Without exception, the untreated ani-
mals developed rapid and profound CD4?T-cell depletion,
leading to nearly complete loss of peripheral CD4?T cells by
2 weeks after exposure (Fig. 3G), similar to SHIV89.6P infec-
tion of adult macaques (52, 53). The two untreated control
animals that had very high initial plasma viral RNA loads
(RJh-7 and RHh-7) had complete and persistent CD4?T-cell
depletion (Fig. 3C, E, and G). They subsequently developed
progressive disease (diarrhea, weakness, opportunistic infec-
tions, pneumonia, and lymphadenopathy) and succumbed to
AIDS 7 and 9 weeks postchallenge, respectively. Remarkably,
the other two untreated control animals (REh-7 and RUg-7)
recovered gradually from declines in CD4?T cells by week 8
postexposure, even though both had peak viral RNA loads
One of four MAb-treated neonates (RCh-7) was completely
protected from the oral virus challenge. No evidence of viral
replication was observed, as viral RNA remained undetectable
in the plasma by real-time RT-PCR (Fig. 3D) and PBMC
coculture was negative (Fig. 3F). Proviral DNA could not be
amplified from PBMC from this animal by DNA PCR (data
not shown). Furthermore, no evidence of virus-induced disease
was observed. The peripheral CD4?T-cell count from RCh-7
did not decrease after challenge (Fig. 3H). The remaining
three MAb-treated animals (RFh-7, RIh-7, and RWg-7) be-
came infected, as determined by virus isolation and RT-PCR
(Fig. 3D and F). Infant RIh-7 maintained its CD4?T-cell
counts above 955 cells/?l (Fig. 3H), in contrast to all four
controls, which suffered severe, acute drops of their CD4?T
cells to 0 to 170 cells/?l at week 2. The MAb-treated infant
RFh-7 showed a delay in an eventual decrease in CD4?T cells
(Fig. 3H). RFh-7 partially recovered and thereafter had slowly
declining CD4?T-cell counts. In contrast, RWg-7 had rapid
CD4?T-cell depletion and never recovered. This animal was
sacrificed 10 weeks after the SHIV89.6P challenge because of
disease that progressed to AIDS (diarrhea, weakness, oppor-
tunistic infections, severe thymic atrophy, and no detectable
Seroconversion and clinical correlation after SHIV89.6P
challenge. Assessment of Western blot-reactive antibodies and
NAb titers revealed three different patterns of humoral im-
mune responses among the animals in the SHIV89.6P study.
The protected neonate, RCh-7, neither seroconverted (Fig. 4)
nor developed a significant NAb titer (Table 2). This is con-
sistent with undetectable virus replication in this animal (Fig.
3D and F). Two untreated control animals, RHh-7 and RJh-7,
also had no detectable anti-SIV or anti-HIV-1 antibodies (Fig.
4 and data not shown) and no NAbs (Table 2). In these ani-
mals, however, rapid disease progression apparently precluded
the ability to mount any antiviral antibody responses. A second
pattern, observed in MAb-treated macaques RIh-7 and RFh-7
and untreated control animals REh-7 and RUg-7, was the
development of antibodies to SIV Gag (Fig. 4) and HIV Env
(data not shown), as well as significant titers (?100) of plasma
NAb (Table 2). In three of the four animals (RIh-7, REh-7,
and RUg-7), an increase in plasma NAbs preceded or coin-
cided with a decrease in plasma viral load and an increase in
FIG. 3. Plasma viral RNA load (A to D), virus isolation by coculture (E and F), and peripheral absolute CD4?T-cell counts (G and H) of
neonatal macaques challenged orally with SHIV-vpu?(A and B) or SHIV89.6P (C to H). P1 and P2 (B) received MAbs F105, 2G12, and 2F5.
No RT-PCR data were obtained for the fourth SHIV-vpu?control animal, R16C (not shown in panel A), because sodium citrate-anticoagulated
blood samples were not available. RCh-7, RFh-7, RIh-7, and RWg-7 (D, F, and H) received MAbs IgG1b12, 2G12, and 2F5. The sensitivity of the
RT-PCR assay is 50 copies/ml (A to D, dotted line). CD4?T-cell counts were not available for animal RCh-7 between weeks 9 and 43 (H). A
dotted line is drawn in panels G and H to indicate 750 CD4?T cells/?l, which defines severe T-cell depletion in human infants less than 12 months
of age. †, animals RWg-7, RHh-7, and RJh-7 were sacrificed 10, 7, and 9 weeks postexposure, respectively, due to progressed disease.
VOL. 75, 2001 POSTNATAL PASSIVE IMMUNIZATION AGAINST SHIV CHALLENGE 7475
CD4?T-cell counts (data not shown). A third pattern was
observed in the MAb-treated animal RWg-7, which transiently
developed low level anti-Gag antibodies (Fig. 4). Animal
RWg-7 progressed to disease and AIDS. In HIV-infected hu-
mans (7) as well as in SIV-infected macaques (2), selective loss
of anti-Gag antibodies is a grave prognostic sign heralding the
development of AIDS. The humoral response of RWg-7 fol-
lowed just such a pattern.
Potential mechanisms of the protection observed in three
MAb-treated animals. Neonates P1, P2, and RCh-7 were not
infected by all criteria tested and neither seroconverted nor
developed NAb responses (Fig. 4, Table 2, and data not
shown). P1, P2, and RCh-7 appeared to be protected from
mucosal virus challenge by well-characterized pure human IgG
MAbs. No mucosal NAbs of the IgA subtype were present in
our MAb combination. How could systemically administered
IgG MAbs protect against mucosal virus challenge? We tested
saliva samples collected from P1, P2, and RCh-7 1 h after the
first MAb infusion just prior to virus challenge in neutraliza-
tion assays. They had no more neutralizing activity than the
prechallenge samples collected from untreated control animals
(all titers ? 20). This observation was true for all MAb-treated
animals as well (all titers ? 20). Neutralizing titers, if present
at all in saliva at the time of virus challenge, were below the
lower detection limit of our assay. Thus, direct inactivation of
SHIV-vpu?and SHIV89.6P in saliva seems unlikely.
Given these results, it is possible that virus crossed the mu-
cosal barrier and went through an initial round of infection in
target cells of the local mucosal gastrointestinal tissue. The
potent neutralizing MAbs would have stopped further waves of
viral spread from these primary target cells. Nevertheless, lim-
ited local infection could have permitted the development of
virus-specific, cellular immune responses, which in turn could
have subsequently eliminated the small number of infected
cells. To test this possibility, we evaluated CTL activity of
PBMC obtained from RCh-7 46 weeks postchallenge; no sig-
nificant specific lysis was detected in51Cr release assays (data
not shown). However, measuring systemic CTL activity in pe-
ripheral blood may not reflect specific antiviral cellular immu-
nity at the level of the upper gastrointestinal mucosa itself.
To test for evidence of persistent mucosal immune protec-
tion, we rechallenged animal RCh-7 with 15 oral AID50
SHIV89.6P 54 weeks after the first challenge. We detected
viral RNA in plasma obtained 1 week after the rechallenge
(71,565 RNA copies/ml of plasma), and the CD4?T-cell
counts decreased from 1,516 to 684 cells/?l. Clearly, no pro-
tection was seen.
Potential mechanism that contributed to the recovery from
the acute pathogenic effects of SHIV89.6P. To gain more in-
sight into what immune mechanisms might have contributed to
the recovery of two of our four untreated controls (REh-7 and
RUg-7) from the acute pathogenic effects of SHIV89.6P, we
analyzed the humoral and cellular immune responses of these
animals. As mentioned above, both animals developed high
titers of NAbs against SHIV89.6P (Table 2). We evaluated the
breadth of NAbs by assessing their ability to neutralize other
SHIV variants and an HIV-1 strain as described earlier (18, 42,
43). We included plasma samples from REh-7 and RUg-7
collected 79 and 81 weeks postexposure, respectively. In addi-
tion, we assayed samples collected 35 and 135 weeks postex-
posure, respectively, from two other macaques, RDt-7 and
RGt-6, that had been inoculated orally with SHIV89.6P as
neonates and had recovered from the pathogenic manifesta-
tions of the infection. The plasma samples contained—in
FIG. 4. Western blot analysis of plasma collected from SHIV89.6P-exposed animals. Serial samples were analyzed using HIV-2 strips. The
number of weeks after challenge is indicated. Migration of HIV-2 proteins is shown on the left. The arrow on the right indicates migration of Gag
antigen. Plasma from SIV-free macaques or normal human serum and plasma from SIV-infected macaques or human anti-HIV-2 serum were
negative and positive controls, respectively (leftmost 4 lanes). †, sacrificed animal.
TABLE 2. SHIV89.6P plasma neutralization titers after passive
Plasma neutralization titera
Untreated control animals
aReciprocal dilution which protected 50% of MT-2 cells from virus-induced
cytotoxicity. This corresponds to 90% reduction of the viral Gag synthesis (9).
bWeeks after virus exposure. Animals RWg-7, RHh-7, and RJh-7 were sac-
rificed in weeks 10, 7, and 9, respectively. Therefore, samples obtained 7 weeks
postchallenge were analyzed.
7476 HOFMANN-LEHMANN ET AL.J. VIROL.
addition to anti-SHIV89.6P NAbs—variable and sometimes
potent neutralizing activity against the heterologous T-cell-
line-adapted HIV-1 strain MN (titers as high as 500) but no
activity against SHIV89.6 and SHIV-KU2 (all titers ? 20). We
also tested specific cellular immune responses in five of our
total six recovered macaques by using enzyme-linked immuno-
spot assays (28) and found a significant number of SIVmac239
Gag- and HIV89.6 Env-specific T lymphocytes in all five ani-
mals (data not shown).
Safety of human MAb administration to neonatal ma-
caques. Infusions of MAb combinations were tolerated well in
more than 15 neonatal macaques treated so far; no acute
reactions were reported after any of the more than 30 i.v.
infusions. One MAb-treated neonate (RCh-7) suffered a sei-
zure approximately 2 days after the first MAb infusion. RCh-7
had a low birth weight (420 g) but subsequently gained body
weight normally (1.68 kg at necropsy). No further seizures
were reported. As in all of the other animals, the MAb infusion
and the virus inoculation had been uneventful, suggesting that
a connection between these manipulations and the observed
seizure is unlikely. It is also improbable that it interfered with
the challenge protocol, since 2 days had elapsed between virus
challenge and the neurological events. Twenty-four days after
virus challenge, physical examination of RCh-7 revealed par-
tial paralysis of both lower legs (loss of reflexes); 7 days later,
contracture and atrophy of the affected musculature were re-
ported. Necropsy performed 55 weeks postchallenge revealed
that RCh-7 was in reasonably good condition with the excep-
tion of the previously observed pelvic and leg muscle atrophy.
In addition, focal cortical atrophy with sparing of the underly-
ing white matter and cavitation of the underlying tissue were
present in the left occipital-parietal junction, extending from
the parietal lobe into the premotor cortex. Hemosiderin-laden
macrophages were present at the borders of cavitated areas
consistent with previous cortical hemorrhage. No evidence of
vasculitis was detected. The unilateral lesion in the brain is not
the likely cause of motor dysfunction, given the sparing of the
motor areas and intactness of the cervical spinal cord. No
specific diagnosis for the neurologic deficits could be obtained;
however, the time course of events renders an etiologic asso-
ciation with the MAb treatment unlikely.
We infused triple combinations of human MAbs postnatally
into six neonatal rhesus macaques to evaluate the protective
potential against mucosal challenge with two different SHIV
strains. We achieved complete protection in two of two MAb-
treated neonates challenged with SHIV-vpu?and one of four
MAb-treated neonates exposed to SHIV89.6P, while all con-
trol animals exposed to either virus became viremic. An addi-
tional MAb-treated animal was protected from the rapid CD4?
T-cell depletion observed in all control animals after SHIV89.6P
challenge. Thus, the MAb combination was partially successful in
protecting animals from the pathogenic effects of the SHIV89.6P
infection. Furthermore, protection occurred without administra-
tion of large prenatal maternal MAb doses.
The triple MAb combination of F105, 2G12, and 2F5 pro-
tected neonates against oral SHIV-vpu?challenge after pre-
and postnatal immunoprophylaxis (3); it was therefore used in
the present study with SHIV-vpu?as well. For the SHIV89.6P
oral challenge study, F105 was replaced by IgG1b12 in the
triple combination because this regimen produced the stron-
gest in vitro neutralization against this virus. Interestingly, neu-
tralization of SHIV89.6P by 2G12 and 2F5 was potentiated by
the addition of IgG1b12. This was unexpected, since IgG1b12
alone did not inhibit SHIV89.6P replication in vitro. This result
supports earlier studies that used either SHIV89.6P or SHIV-
KB9, a molecular clone derived after the fourth passage of
SHIV89.6P (18, 21, 27). Neutralization resistance of viruses
might be due to a number of factors, including point mutations
leading to loss of the relevant epitope (41) or to global con-
formational changes that make the epitope inaccessible to the
MAb (47, 60, 64). The IgG1b12 epitope is present on the
envelope protein of SHIV-KB9; IgG1b12 binds soluble KB9
gp120 monomers with undiminished capacity (21). However,
the epitope seems less accessible for antibody binding in the
context of the naturally folded oligomeric envelope glycopro-
tein complex on the SHIV-KB9 virion surface (21). IgG1b12
binds other viral strains with equal or better avidity to the
oligomeric form of the envelope glycoprotein; this probably
accounts for its exceptional potency (22, 54). Resistance to
IgG1b12 has been mapped to the V1, V2, and V3 loops of the
HIV-1 envelope (21, 41, 54). Antibodies 2G12 and 2F5 recog-
nize epitopes unrelated to the V1, V2, or V3 loop or to the
CD4BS (8, 17, 45, 62). We postulate that binding of either
2G12 or 2F5 induces a conformational change in the oligo-
meric envelope glycoprotein complex of SHIV89.6P that al-
lows contact of IgG1b12 with its previously hidden cognate
The addition of a fourth antibody, F105, to the triple com-
bination of IgG1b12, 2G12, and 2F5 led to a decrease in
IgG1b12 seem to inhibit each other, although they use differ-
ent mechanisms to neutralize HIV. F105 inhibits the attach-
ment of the neutralized virus to the target cell (49), while
IgG1b12 inhibits the fusion entry process (39). Since they both
recognize epitopes that at least partially overlap with the
CD4BS, the diminished combined neutralization activity may
be due to steric hindrance and competition for the binding site.
We achieved complete protection against oral SHIV-vpu?
challenge in two neonatal rhesus macaques that received post-
natal treatment with a triple combination of human neutraliz-
ing MAbs. This chimeric virus encodes env of the laboratory-
adapted HIV-1 IIIB. To mimic HIV infection of human
newborns more closely, we then used a highly pathogenic chi-
meric virus that encodes env derived from the primary, dual-
tropic HIV89.6 and achieved partial protection. It is well
known that primary strains of HIV are more difficult to neu-
tralize than laboratory-adapted strains (19, 44). However, we
had optimized the MAb combination previously for high in
vitro neutralization of SHIV89.6P. Another factor that could
have contributed to the different outcome of the two immu-
noprophylaxis studies could also be the higher challenge dose
used of SHIV89.6P (15 AID50) than of SHIV-vpu?(10
Protection in the orally challenged neonates was achieved
with only postnatal treatment. We chose this approach because
the large amounts of each MAb required for treatment of the
mother during pregnancy may render passive immunoprophy-
VOL. 75, 2001 POSTNATAL PASSIVE IMMUNIZATION AGAINST SHIV CHALLENGE7477
laxis approaches to prevent maternal HIV transmission in clin-
ical trials too costly. Furthermore, we modeled our primate
study on the successful prevention of maternal transmission of
the hepatitis B virus, an enveloped virus that is transmitted
through the same routes as HIV. Anti-hepatitis B immuno-
globulin therapy is administered to newborn infants within 12 h
of birth; no prenatal treatment is given to infected mothers.
Passive immunization alone is 71% effective in preventing pas-
sage of hepatitis B virus to infants (5).
To our knowledge, the present study gives the first descrip-
tion of the course and outcome of SHIV89.6P infection of
neonatal rhesus macaques. In adult rhesus monkeys, i.v. inoc-
ulation of SHIV89.6P led to high initial viral peak RNA loads
and to profound, persistent depletion of CD4?T cells within 2
weeks after virus challenge (4, 25, 53, 59, 63). According to
most reports (42, 53, 63), SHIV89.6P infection leads to rapid
disease progression and to AIDS in adult macaques, leaving
the animals unable to seroconvert or mount any virus-specific
CTL responses. This course of infection was seen in only two
of our four naive neonates (RHh-7 and RJh-7). Interestingly,
the other two untreated control animals (REh-7 and RUg-7)
showed a rebound of CD4?T-cell counts 8 weeks after virus
exposure. These neonates seroconverted readily, developed
high titers of NAbs, and remained clinically well throughout
the 1-year observation period. A similar phenomenon was de-
scribed recently by Barouch and coworkers (4). Two of eight
rhesus monkeys that were challenged i.v. with 100 AID50of
SHIV89.6P did not show complete depletion of CD4?T cells;
one animal developed no significant disease, and only four of
the eight macaques died by day 140 postexposure (4). The
animals developed low virus-specific CD8?CTL responses.
However, in accordance with our observation in infant ma-
caques, two of the adult animals developed high NAb titers by
day 28 after virus exposure (4).
To further analyze the immune mechanisms that might have
contributed to the recovery from the acute SHIV89.6P patho-
genicity in our two untreated neonates, we assessed the
breadth of their NAbs using in vitro neutralization assays. We
included samples from REh-7, RUg-7, and two additional ma-
caques that had been inoculated orally with SHIV89.6P as
neonates and had recovered from the acute CD4?T-cell de-
pletion. Plasma samples from all four animals had NAb activity
against SHIV89.6P and the HIV-1 strain MN but not against
SHIV89.6 and SHIV-KU2. These results are similar to those
reported for plasma samples from SHIV89.6PD-infected ma-
caques (18, 42) and demonstrate the restricted nature of the
induced NAbs also in SHIV89.6P-infected animals. In addi-
tion, all four untreated, recovered macaques had strong virus-
specific cellular immune responses. In conclusion, these
SHIV89.6P-infected macaques that recovered from the severe
CD4?T-cell depletion and survived for a prolonged period
without significant disease demonstrated both strong NAb ac-
tivity and cellular immune responses. It remains to be deter-
mined what additional factors are responsible for the variable
outcomes observed among individual SHIV89.6P-infected
adult and neonatal macaques.
Virus replication was undetectable in the peripheral blood
of MAb-treated animals P1, P2, and RCh-7. The animals nei-
ther seroconverted nor developed NAb responses. Successful
in vitro infection of CD8?-depleted PBMC collected 36 weeks
after virus exposure from animal RCh-7 (data not shown)
excluded the possibility of natural resistance to SHIV89.6P due
to genetic predisposition, such as lack of coreceptors. There-
fore, the triple combination of human neutralizing MAbs must
have protected RCh-7 from systemic infection. It is not clear at
what level the infused IgG MAbs stopped the virus. As in our
previous study, in which neonates were completely protected
against an oral SHIV-vpu?challenge (3), we could not detect
any significant neutralizing activity in the saliva of P1, P2, and
RCh-7 at the time of challenge. While this does not rule out the
possibility that low amounts of MAbs interacted with the virus in
saliva, it is more likely that virus crossed the mucosal membrane
and underwent a single round of replication in target cells of the
local gastrointestinal tissues. Secondary waves of virus spread
might then have been prevented either through direct virus neu-
tralization by the parenterally infused IgG MAbs or via elimina-
tion of infected MAb-coated cells by antibody-dependent cell-
human anti-HIV MAb such as F105 were found to mediate
ADCC (48), and 2G12 has both ADCC- and complement-medi-
ated activity (62). Recently, ADCC or other cell-killing mecha-
nisms were also suggested to cause transient reductions of viral
load in SIVmac251-infected macaques infused with immuno-
globulins (6). Alternatively, the initial infection of a set of suscep-
tible target cells might have induced virus-specific cellular im-
mune responses. However, no direct evidence for cellular
immunity was seen—virus-specific CTL activity was not detected
in the peripheral blood, and there was no protection from oral
rechallenge 54 weeks after the first virus exposure.
The protection that we achieved in the four MAb-treated
neonatal rhesus macaques after oral challenge with pathogenic
SHIV89.6P may be compared to the level of protection re-
ported for antibody-treated, adult macaques challenged vagi-
nally with plasma-derived SHIV89.6PD (38). Similar doses (15
versus 10 to 50 AID50) and strains of virus (SHIV89.6P versus
SHIV89.6PD [35, 52]) were used in the two studies. However,
to achieve reproducible infection, the adult macaques were
treated with progesterone prior to vaginal challenge, resulting
in a thinning of the vaginal epithelium. This might have influ-
enced the MAb efficacy by allowing more antibodies to seep
across the mucosal barrier. In addition, we used a lower dose
of antibody (10 mg/kg) than used in the adult study (15 mg/kg),
and different combinations of antibodies were employed.
In conclusion, postnatal triple MAb combination overall
prevented infection in three of six treated infants. Among
SHIV89.6P-challenged animals, the MAb combination was
partially successful in preventing infection; half of the treated
infants were protected from the acute, severe T-cell depletion.
The failure to protect all MAb-treated animals from
SHIV89.6P infection did not seem to be related to neutraliza-
tion resistance. Viruses from MAb-treated animal RFh-7 and
untreated control animal REh-7, recovered 37 weeks postex-
posure, were still as sensitive to neutralization with the triple
MAb combination as the original SHIV89.6P inoculum (data
not shown). Even the partial protection that we report here is
encouraging since the macaque-adapted SHIV89.6P (52) is
more pathogenic in monkeys than HIV is in humans. To in-
crease the degree of protection in future primate studies, we
plan to include other synergistically acting MAbs in the com-
bination regimen and/or increase the neonatal MAb doses.
7478 HOFMANN-LEHMANN ET AL.J. VIROL.
Our approach is directly relevant for the development of a
new strategy against maternal-fetal HIV-1 transmission in hu-
mans, since the MAbs used are human antibodies directed
against HIV-1 glycoproteins. It may represent an important
addition to the antiretroviral therapy protocols established to
reduce mother-to-child HIV transmission. Being natural hu-
man proteins, human MAbs can be expected to have low tox-
icity. In symptomatic HIV-infected children, the prophylactic
use of i.v. immunoglobulin for the prevention of bacterial in-
fections was safe and was suggested as standard therapy (1, 55).
In addition, MAbs 2G12 and 2F5 have been infused safely into
HIV-infected adults in a phase I clinical trial (H. Katinger and
G. Stiegler, unpublished observations). The stability and long
half-lives of neutralizing human MAbs that we had noted in
our earlier study in neonates (3) may yield another clinical
benefit: the protection of infants against oral HIV transmission
through infected breast milk in the neonatal period. Protection
against this mode of maternal HIV transmission may be easier
to achieve than against our SHIV89.6P oral challenge, since
the infectivity of human breast milk is lower and does not yield
a 99% probability of infection (40, 46) as our SHIV89.6P
challenge dose did.
We thank Matthew Frosch (Department of Pathology, Brigham and
Women’s Hospital, Boston, Mass.) for review of the neuropathological
slides and helpful discussion. CHO cells producing MAb IgG1b12
were kindly provided by Dennis Burton (Department of Immunology
and Molecular Biology, Scripps Research Institute, La Jolla, Calif.).
We also thank Yulan Wang for technical assistance and C. Gallegos
and S. Sharp for preparation of the manuscript.
This work was supported in part by National Institutes of Health
grants RO1 AI34266, R21 AI46177, and PO1 AI48240 awarded to
R.M.R., RR00165 to H.M.M., AI26926 to M.R.P. and AI45320 to
L.A.C. It was also supported by the Pediatric AIDS Foundation grant
50864PG23 to R.M.R. and by Center for AIDS Research (CFAR) core
grant IP30 28691 awarded to the Dana-Farber Cancer Institute.
D.C.M. was supported by National Institutes of Health contract NO1
AI85343. R.H.-L. was supported by a grant from the Swiss National
Science Foundation (fellowship 823A-50315) and is the recipient of a
scholar award from the Friends of Switzerland, Inc. T.W.B. was a
recipient of National Institutes of Health Clinical Investigator Devel-
opment Award 30/35 KO8-AI01327.
1. Anonymous. 1991. Intravenous immune globulin for the prevention of bac-
terial infections in children with symptomatic human immunodeficiency virus
infection. The National Institute of Child Health and Human Developments
Intravenous Immunoglobulin Study Group. N. Engl. J. Med. 325:73–80.
2. Baba, T. W., J. Koch, E. S. Mittler, M. Greene, M. Wyand, D. Pennick, and
R. M. Ruprecht. 1994. Mucosal infection of neonatal rhesus monkeys with
cell-free SIV. AIDS Res. Hum. Retroviruses 10:351–357.
3. Baba, T. W., V. Liska, R. Hofmann-Lehmann, J. Vlasak, W. Xu, S. Ayehunie,
L. A. Cavacini, M. R. Posner, H. Katinger, G. Stiegler, B. J. Bernacky, T. A.
Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, Y. Lu, J. E. Wright, T. C. Chou,
and R. M. Ruprecht. 2000. Human neutralizing monoclonal antibodies of the
IgG1 subtype protect against mucosal simian-human immunodeficiency virus
infection. Nat. Med. 6:200–206.
4. Barouch, D. H., S. Santra, J. E. Schmitz, M. J. Kuroda, T. M. Fu, W.
Wagner, M. Bilska, A. Craiu, X. X. Zheng, G. R. Krivulka, K. Beaudry, M. A.
Lifton, C. E. Nickerson, W. L. Trigona, K. Punt, D. C. Freed, L. Guan, S.
Dubey, D. Casimiro, A. Simon, M. E. Davies, M. Chastain, T. B. Strom, R. S.
Gelman, D. C. Montefiori, and M. G. Lewis. 2000. Control of viremia and
prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA
vaccination. Science 290:486–492.
5. Beasley, R. P., L. Y. Hwang, G. C. Lee, C. C. Lan, C. H. Roan, F. Y. Huang,
and C. L. Chen. 1983. Prevention of perinatally transmitted hepatitis B virus
infections with hepatitis B immune globulin and hepatitis B vaccine. Lancet
6. Binley, J. M., B. Clas, A. Gettie, M. Vesanen, D. C. Montefiori, L. Sawyer, J.
Booth, M. Lewis, P. A. Marx, S. Bonhoeffer, and J. P. Moore. 2000. Passive
infusion of immune serum into simian immunodeficiency virus-infected rhe-
sus macaques undergoing a rapid disease course has minimal effect on
plasma viremia. Virology 270:237–249.
7. Binley, J. M., P. J. Klasse, Y. Cao, I. Jones, M. Markowitz, D. D. Ho, and
J. P. Moore. 1997. Differential regulation of the antibody responses to Gag and
Env proteins of human immunodeficiency virus type 1. J. Virol. 71:2799–2809.
8. Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M.
Purtscher, G. Gruber, C. Tauer, F. Steindl, A. Jungbauer, and H. Katinger.
1994. Generation of human monoclonal antibodies against HIV-1 proteins;
electrofusion and Epstein-Barr virus transformation for peripheral blood
lymphocyte immortalization. AIDS Res. Hum. Retroviruses 10:359–369.
9. Bures, R., A. Gaitan, T. Zhu, C. Graziosi, K. McGrath, J. Tartaglia, P.
Caudrelier, R. El Habib, M. Klein, A. Lazzarin, D. M. Stablein, L. Corey,
M. L. Greenberg, D. H. Schwartz, and D. C. Montefiori. 2000. Immunization
with recombinant canarypox vectors expressing membrane-anchored gp120
followed by soluble gp160 boosting fails to generate antibodies that neutral-
ize R5 primary isolates of human immunodeficiency virus type 1. AIDS Res.
Hum. Retroviruses 16:2019–2035.
10. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. Parren,
L. S. Sawyer, R. M. Hendry, N. Dunlop, P. L. Nata, M. Lamacchia, E.
Garratty, E. R. Stiehm, Y. J. Bryson, Y. Cao, J. P. Moore, D. D. Ho, and C. F.
Barbas. 1994. Efficient neutralization of primary isolates of HIV-1 by a
recombinant human monoclonal antibody. Science 266:1024–1027.
11. Capon, D. J., S. M. Chamow, J. Mordenti, S. A. Marsters, T. Gregory, H.
Mitsuya, R. A. Byrn, C. Lucas, F. M. Wurm, J. E. Groopman, et al. 1989.
Designing CD4 immunoadhesins for AIDS therapy. Nature 337:525–531.
12. Chou, T. C. 1991. The median-effect principle and the combination index for
quantitation of synergism and antagonism, p. 61–102. In T. C. Chou and
D. C. Rideout (ed.), Synergism and antagonism in chemotherapy. Academic
Press, San Diego, Calif.
13. Chou, T. C., and M. Hayball. 1996. CalcuSyn for Windows. Multi-drug
dose-effect analyzer and manual. Biosoft, Cambridge, United Kingdom.
14. Chou, T. C., and P. Talalay. 1981. Generalized equations for the analysis of
inhibitions of Michaelis-Menten and higher-order kinetic systems with two
or more mutually exclusive and nonexclusive inhibitors. Eur. J. Biochem.
15. Chou, T. C., and P. Talalay. 1984. Quantitative analysis of dose-effect rela-
tionships: the combined effects of multiple drugs or enzyme inhibitors. Adv.
Enzyme Regul. 22:27–55.
16. Collman, R., J. W. Balliet, S. A. Gregory, H. Friedman, D. L. Kolson, N.
Nathanson, and A. Srinivasan. 1992. An infectious molecular clone of an
unusual macrophage-tropic and highly cytopathic strain of human immuno-
deficiency virus type 1. J. Virol. 66:7517–7521.
17. Conley, A. J., J. A. Kessler, II, L. J. Boots, J. S. Tung, B. A. Arnold, P. M.
Keller, A. R. Shaw, and E. A. Emini. 1994. Neutralization of divergent
human immunodeficiency virus type 1 variants and primary isolates by IAM-
41-2F5, an anti-gp41 human monoclonal antibody. Proc. Natl. Acad. Sci.
18. Crawford, J. M., P. L. Earl, B. Moss, K. A. Reimann, M. S. Wyand, K. H.
Manson, M. Bilska, J. T. Zhou, C. D. Pauza, P. W. Parren, D. R. Burton,
J. G. Sodroski, N. L. Letvin, and D. C. Montefiori. 1999. Characterization of
primary isolate-like variants of simian-human immunodeficiency virus. J. Vi-
19. Daar, E. S., X. L. Li, T. Moudgil, and D. D. Ho. 1990. High concentrations
of recombinant soluble CD4 are required to neutralize primary human
immunodeficiency virus type 1 isolates. Proc. Natl. Acad. Sci. USA 87:6574–
20. D’Souza, M. P., D. Livnat, J. A. Bradac, and S. H. Bridges. 1997. Evaluation
of monoclonal antibodies to human immunodeficiency virus type 1 primary
isolates by neutralization assays: performance criteria for selecting candidate
antibodies for clinical trials. AIDS Clinical Trials Group Antibody Selection
Working Group. J. Infect. Dis. 175:1056–1062.
21. Etemad-Moghadam, B., Y. Sun, E. K. Nicholson, G. B. Karlsson, D.
Schenten, and J. Sodroski. 1999. Determinants of neutralization resistance
in the envelope glycoproteins of a simian-human immunodeficiency virus
passaged in vivo. J. Virol. 73:8873–8879.
22. Fouts, T. R., J. M. Binley, A. Trkola, J. E. Robinson, and J. P. Moore. 1997.
Neutralization of the human immunodeficiency virus type 1 primary isolate
JR-FL by human monoclonal antibodies correlates with antibody binding to
the oligomeric form of the envelope glycoprotein complex. J. Virol. 71:2779–
23. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F.
Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, et al. 1984. Frequent
detection and isolation of cytopathic retroviruses (HTLV-III) from patients
with AIDS and at risk for AIDS. Science 224:500–503.
24. Hofmann-Lehmann, R., R. K. Swenderten, V. Liska, C. M. Leutenegger, H.
Lutz, H. M. McClure, and R. M. Ruprecht. 2000. Sensitive and robust
one-tube real-time reverse transcriptase-polymerase chain reaction to quan-
tify SIV RNA load: Comparison of one- vs. two-enzyme systems. AIDS Res.
Hum. Retroviruses 16:1247–1257.
25. Iida, T., H. Ichimura, T. Shimada, K. Ibuki, M. Ui, K. Tamaru, T. Kuwata,
S. Yonehara, J. Imanishi, and M. Hayami. 2000. Role of apoptosis induction
VOL. 75, 2001POSTNATAL PASSIVE IMMUNIZATION AGAINST SHIV CHALLENGE7479
in both peripheral lymph nodes and thymus in progressive loss of CD4?cells Download full-text
in SHIV-infected macaques. AIDS Res. Hum. Retroviruses 16:9–18.
26. Joag, S. V., Z. Li, L. Foresman, D. M. Pinson, R. Raghavan, W. Zhuge, I.
Adany, C. Wang, F. Jia, D. Sheffer, J. Ranchalis, A. Watson, and O. Narayan.
1997. Characterization of the pathogenic KU-SHIV model of acquired im-
munodeficiency syndrome in macaques. AIDS Res. Hum. Retroviruses 13:
27. Karlsson, G. B., M. Halloran, J. Li, I. W. Park, R. Gomila, K. A. Reimann,
M. K. Axthelm, S. A. Iliff, N. L. Letvin, and J. Sodroski. 1997. Character-
ization of molecularly cloned simian-human immunodeficiency viruses causing
rapid CD4?lymphocyte depletion in rhesus monkeys. J. Virol. 71:4218–4225.
28. Larsson, M., X. Jin, B. Ramratnam, G. S. Ogg, J. Engelmayer, M. A.
Demoitie, A. J. McMichael, W. I. Cox, R. M. Steinman, D. Nixon, and N.
Bhardwaj. 1999. A recombinant vaccinia virus based ELISPOT assay detects
high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.
29. Li, A., T. W. Baba, J. Sodroski, S. Zolla-Pazner, M. K. Gorny, J. Robinson,
M. R. Posner, H. Katinger, C. F. Barbas, III, D. R. Burton, T. C. Chou, and
R. M. Ruprecht. 1997. Synergistic neutralization of a chimeric SIV/HIV type
1 virus with combinations of human anti-HIV type 1 envelope monoclonal
antibodies or hyperimmune globulins. AIDS Res. Hum. Retroviruses 13:
30. Li, A., H. Katinger, M. R. Posner, L. Cavacini, S. Zolla-Pazner, M. K. Gorny,
J. Sodroski, T. C. Chou, T. W. Baba, and R. M. Ruprecht. 1998. Synergistic
neutralization of simian-human immunodeficiency virus SHIV-vpu?by tri-
ple and quadruple combinations of human monoclonal antibodies and high-
titer anti-human immunodeficiency virus type 1 immunoglobulins. J. Virol.
31. Li, J. T., M. Halloran, C. I. Lord, A. Watson, J. Ranchalis, M. Fung, N. L.
Letvin, and J. G. Sodroski. 1995. Persistent infection of macaques with
simian-human immunodeficiency viruses. J. Virol. 69:7061–7067.
32. Liska, V., P. N. Fultz, L. Su, and R. M. Ruprecht. 1997. Detection of simian
T cell leukemia virus type I infection in seronegative macaques. AIDS Res.
Hum. Retroviruses 13:1147–1153.
33. Liska, V., A. H. Khimani, R. Hofmann-Lehmann, A. N. Fink, J. Vlasak, and
R. M. Ruprecht. 1999. Viremia and AIDS in rhesus macaques after intra-
muscular inoculation of plasmid DNA encoding full-length SIVmac239.
AIDS Res. Hum. Retroviruses 15:445–450.
34. Liska, V., N. W. Lerche, and R. M. Ruprecht. 1997. Simultaneous detection
of simian retrovirus type D serotypes 1, 2, and 3 by polymerase chain
reaction. AIDS Res. Hum. Retroviruses 13:433–437.
35. Lu, Y., C. D. Pauza, X. Lu, D. C. Montefiori, and C. J. Miller. 1998. Rhesus
macaques that become systemically infected with pathogenic SHIV 89.6-PD
after intravenous, rectal, or vaginal inoculation and fail to make an antiviral
antibody response rapidly develop AIDS, J. Acquir. Immune Defic. Syndr.
36. Lu, Y., M. S. Salvato, C. D. Pauza, J. Li, J. Sodroski, K. Manson, M. Wyand,
N. Letvin, S. Jenkins, N. Touzjian, C. Chutkowski, N. Kushner, M. LeFaile,
L. G. Payne, and B. Roberts. 1996. Utility of SHIV for testing HIV-1 vaccine
candidates in macaques. J. Acquir. Immune Defic. Syndr. 12:99–106.
37. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes,
M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb,
H. Katinger, and D. L. Birx. 1999. Protection of macaques against patho-
genic simian/human immunodeficiency virus 89.6PD by passive transfer of
neutralizing antibodies. J. Virol. 73:4009–4018.
38. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter,
C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, and M. G. Lewis.
2000. Protection of macaques against vaginal transmission of a pathogenic
HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat.
39. McInerney, T. L., L. McLain, S. J. Armstrong, and N. J. Dimmock. 1997. A
human IgG1 (b12) specific for the CD4 binding site of HIV-1 neutralizes by
inhibiting the virus fusion entry process, but b12 Fab neutralizes by inhibiting
a postfusion event. Virology 233:313–326.
40. Miotti, P. G., T. E. Taha, N. I. Kumwenda, R. Broadhead, L. A. Mtimavalye,
L. Van der Hoeven, J. D. Chiphangwi, G. Liomba, and R. J. Biggar. 1999. HIV
transmission through breastfeeding: a study in Malawi. JAMA 282:744–749.
41. Mo, H., L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. Parren, D. R. Burton,
J. P. Moore, and D. D. Ho. 1997. Human immunodeficiency virus type 1
mutants that escape neutralization by human monoclonal antibody IgG1b12.
J. Virol. 71:6869–6874.
42. Montefiori, D. C., K. A. Reimann, M. S. Wyand, K. Manson, M. G. Lewis,
R. G. Collman, J. G. Sodroski, D. P. Bolognesi, and N. L. Letvin. 1998.
Neutralizing antibodies in sera from macaques infected with chimeric simi-
an-human immunodeficiency virus containing the envelope glycoproteins of
either a laboratory-adapted variant or a primary isolate of human immuno-
deficiency virus type 1. J. Virol. 72:3427–3431.
43. Montefiori, D. C., W. B. Robinson, and S. S. Schuffman. 1988. Evaluation of
antiviral drugs and neutralizing antibodies to human immunodeficiency virus
44. Moore, J. P., Y. Cao, L. Qing, Q. J. Sattentau, J. Pyati, R. Koduri, J.
Robinson, C. F. Barbas III, D. R. Burton, and D. D. Ho. 1995. Primary
isolates of human immunodeficiency virus type 1 are relatively resistant to
neutralization by monoclonal antibodies to gp120, and their neutralization is
not predicted by studies with monomeric gp120. J. Virol. 69:101–109.
45. Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F.
Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of
human immunodeficiency virus type 1. J. Virol. 67:6642–6647.
46. Nduati, R., G. John, D. Mbori-Ngacha, B. Richardson, J. Overbaugh, A.
Mwatha, J. Ndinya-Achola, J. Bwayo, F. E. Onyango, J. Hughes, and J.
Kreiss. 2000. Effect of breastfeeding and formula feeding on transmission of
HIV-1: a randomized clinical trial. JAMA 283:1167–1174.
47. Parren, P. W., M. Wang, A. Trkola, J. M. Binley, M. Purtscher, H. Katinger,
J. P. Moore, and D. R. Burton. 1998. Antibody neutralization-resistant pri-
48. Posner, M. R., H. S. Elboim, T. Cannon, L. Cavacini, and T. Hideshima.
1992. Functional activity of an HIV-1 neutralizing IgG human monoclonal
antibody: ADCC and complement-mediated lysis. AIDS Res. Hum. Retro-
49. Posner, M. R., T. Hideshima, T. Cannon, M. Mukherjee, K. H. Mayer, and
R. A. Byrn. 1991. An IgG human monoclonal antibody that reacts with
HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection. J. Im-
50. Rabin, H., R. H. Neubauer, R. F. d. Hopkins, E. K. Dzhikidze, Z. V.
Shevtsova, and B. A. Lapin. 1977. Transforming activity and antigenicity of
an Epstein-Barr-like virus from lymphoblastoid cell lines of baboons with
lymphoid disease. Intervirology 8:240–249.
51. Reimann, K. A., J. T. Li, C. Lekutis, K. Tenner-Racz, P. Racz, W. Lin, D. C.
Montefiori, D. E. Lee-Paritz, Y. Lu, R. G. Collman, J. Sodroski, and N. L.
Letvin. 1996. An env gene derived from a primary human immunodeficiency
virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/
human immunodeficiency virus in rhesus monkeys. J. Virol. 70:3198–3206.
52. Reimann, K. A., J. T. Li, R. Veazey, M. Halloran, I. W. Park, G. B. Karlsson,
J. Sodroski, and N. L. Letvin. 1996. A chimeric simian/human immunode-
ficiency virus expressing a primary patient human immunodeficiency virus
type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus
monkeys. J. Virol. 70:6922–6928.
53. Reimann, K. A., A. Watson, P. J. Dailey, W. Lin, C. I. Lord, T. D. Steenbeke,
R. A. Parker, M. K. Axthelm, and G. B. Karlsson. 1999. Viral burden and
disease progression in rhesus monkeys infected with chimeric simian-human
immunodeficiency viruses. Virology 256:15–21.
54. Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas III, and D. R.
Burton. 1994. Recognition properties of a panel of human recombinant Fab
fragments to the CD4 binding site of gp120 that show differing abilities to
neutralize human immunodeficiency virus type 1. J. Virol. 68:4821–4828.
55. Schaad, U. B., A. Gianella-Borradori, B. Perret, P. Imbach, and A. Morell.
1988. Intravenous immune globulin in symptomatic paediatric human im-
munodeficiency virus infection. Eur. J. Pediatr. 147:300–303.
56. Shibata, R., T. Igarashi, N. Haigwood, A. Buckler-White, R. Ogert, W. Ross,
R. Willey, M. W. Cho, and M. A. Martin. 1999. Neutralizing antibody di-
rected against the HIV-1 envelope glycoprotein can completely block HIV-
1/SIV chimeric virus infections of macaque monkeys. Nat. Med. 5:204–210.
57. Shibata, R., F. Maldarelli, C. Siemon, T. Matano, M. Parta, G. Miller, T.
Fredrickson, and M. A. Martin. 1997. Infection and pathogenicity of chi-
meric simian-human immunodeficiency viruses in macaques: determinants
of high virus loads and CD4 cell killing. J. Infect. Dis. 176:362–373.
58. Spouge, J. L. 1992. Statistical analysis of sparse infection data and its impli-
cations for retroviral treatment trials in primates. Proc. Natl. Acad. Sci. USA
59. Ten Haaft, P., B. Verstrepen, K. Uberla, B. Rosenwirth, and J. Heeney. 1998.
A pathogenic threshold of virus load defined in simian immunodeficiency
virus- or simian-human immunodeficiency virus-infected macaques. J. Virol.
60. Thali, M., M. Charles, C. Furman, L. Cavacini, M. Posner, J. Robinson, and
J. Sodroski. 1994. Resistance to neutralization by broadly reactive antibodies
to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by
a gp41 amino acid change. J. Virol. 68:674–680.
61. Trkola, A., A. B. Pomales, H. Yuan, B. Korber, P. J. Maddon, G. P. Allaway,
H. Katinger, C. F. Barbas, D. R. Burton, D. D. Ho, and J. P. Moore. 1995.
Cross-clade neutralization of primary isolates of human immunodeficiency
virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J.
62. Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan,
K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger. 1996. Human
monoclonal antibody 2G12 defines a distinctive neutralization epitope on the
gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70:
63. Wyand, M. S., K. Manson, D. C. Montefiori, J. D. Lifson, R. P. Johnson, and
R. C. Desrosiers. 1999. Protection by live, attenuated simian immunodefi-
ciency virus against heterologous challenge. J. Virol. 73:8356–8363.
64. Wyatt, R., and J. Sodroski. 1998. The HIV-1 envelope glycoproteins: fuso-
gens, antigens, and immunogens. Science 280:1884–1888.
7480HOFMANN-LEHMANN ET AL.J. VIROL.