Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV
chimeric virus by passive infusion of neutralizing antibodies
John R. Mascola1, 2, Gabriela Stiegler3, Thomas C. VanCott1, Hermann Katinger3, Calvin B. Carpenter4, Chris E. Hanson4, Holly Beary1, Deborah Hayes1, Sarah S. Frankel1, Deborah L. Birx1
& Mark G. Lewis1
1 Division of Retrovirology, Walter Reed Army Institute
of Research and Henry M. Jackson Foundation, 1 Taft Court, Suite
250, Rockville, Maryland 20850,
USA
2 Department of Infectious Diseases, Naval Medical Research
Center, Forrest Glenn, Maryland 20910,
USA
3 Institute of Applied Microbiology, University of Agriculture
, Vienna, Austria
4 Division of Veterinary Medicine, Walter Reed Army Institute
of Research, Forrest Glenn, Maryland 20910
, USA
The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency
virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo
evaluation of anti-HIV-1 envelope glycoprotein immune responses1,
2,
3.
Using this model, others, and we have shown that passively infused antibody
can protect against an intravenous challenge4,
5. However, HIV-1
is most often transmitted across mucosal surfaces6,
7,
8,
9
and the intravenous challenge model may not accurately predict the role of
antibody in protection against mucosal exposure. After controlling the macaque
estrous cycle with progesterone10, anti-HIV-1 neutralizing monoclonal
antibodies 2F5 and 2G12, and HIV immune globulin were tested11,
12,
13.
Whereas all five control monkeys displayed high plasma viremia and rapid CD4
cell decline, 14 antibody-treated macaques were either completely protected
against infection or against pathogenic manifestations of SHIV-infection.
Infusion of all three antibodies together provided the greatest amount of
protection, but a single monoclonal antibody, with modest virus neutralizing
activity, was also protective. Compared with our previous intravenous challenge
study with the same virus and antibodies5, the data indicated
that greater protection was achieved after vaginal challenge. This study demonstrates
that antibodies can affect transmission and subsequent disease course after
vaginal SHIV-challenge; the data begin to define the type of antibody response
that could play a role in protection against mucosal transmission of HIV-1.
Because intravenous challenge allows the virus rapid access to CD4
+ target cells, it was not clear if similar high concentrations of
antibody would be required to protect against a mucosal challenge. Therefore,
using the HIV-1/SIV chimeric virus (SHIV) vaginal challenge model, single,
double, and triple antibody combinations were tested. Four groups of rhesus
macaques were infused with control intravenous immune globulin (IVIG,
n=5), monoclonal antibody 2G12 (n=4),
monoclonal antibodies 2G12 + 2F5 (n=5), or HIVIG +
2F5 + 2G12 (n=5). All five IVIG control monkeys developed
high plasma viremia and rapid CD4 cell decline following vaginal exposure
to SHIV89.6PD (Fig. 1). In contrast, no SHIV plasma
RNA was detected in 8 of 14 monkeys pretreated with antibody. Virus was also
not detected by co-culture of peripheral blood mononuclear cells (PBMC), or
by co-culture or polymerase chain reaction (PCR) for viral DNA from cells
of inguinal lymph nodes removed 3 weeks after SHIV challenge (data not shown).
In addition, these monkeys did not seroconvert to a SIV p27 gag antigen (Table 1). Therefore, we considered these eight monkeys to
be completely protected against infection. By these criteria, complete protection
was achieved in four of five monkeys in the HIVIG/2F5/2G12 group, two of five
in the 2F5/2G12 group, and two of four in the 2G12 group. These protected
monkeys also maintained normal CD4 cell counts. Six antibody-treated monkeys
became SHIV-infected and had detectable plasma RNA. Whereas IVIG control monkeys
maintained plasma RNA concentrations of 105 copies/ml
through 22 weeks after challenge, plasma viremia in these antibody-treated
monkeys declined to undetectable, or near undetectable, concentrations by
week 12. None of these six infected monkeys developed the profound loss of
CD4 cells that was seen in the five control monkeys.
Figure 1. Plasma SHIV RNA (a) and peripheral CD4% (b) for four
groups of monkeys after antibody infusion and vaginal SHIV89.6PD challenge.
The passively infused antibody is noted at the top of each graph. Percent
CD4 indicates percent of peripheral blood lymphocytes with CD4+
phenotype by flow cytometry. Open symbols signify monkeys completely protected
against infection.
Plasma monoclonal antibody concentration and neutralization titer were
measured 24 hours after antibody infusion (the day of SHIV89.6PD challenge).
Plasma monoclonal antibody concentrations ranged from 115 g/ml to 260 g/ml
(Table 2). Plasma 90% neutralization titers for the
monkeys in the double and triple antibody groups ranged from 1:29 to 1:88.
However, plasma from the monkeys that received monoclonal antibody 2G12 alone
were unable to achieve 90% virus neutralization at the highest dilution tested
(1:9). These data are consistent with the rather weak neutralization activity
of monoclonal antibody 2G12 against SHIV89.6PD5. Within treatment
groups, the plasma antibody concentrations and plasma neutralization titers
were generally similar and were not predictive of which monkeys were completely
protected against infection. Nonparametric statistical analysis using the
Mann-Whitney test did not show a significant correlation between plasma neutralization
titer and protection against infection. However, this analysis did not account
for possible local (mucosal) effects of antibody. Also, for those antibody-treated
monkeys that became SHIV-infected, infused antibody may have limited virus
replication and allowed adaptive immune responses to play a role in controlling
the infection.
Table 2. Plasma neutralization titers and monoclonal antibody concentrations
in plasma and mucosal compartments after passive antibody infusion
Monoclonal antibodies 2F5 and 2G12 could be detected in several mucosal
compartments after passive infusion (Table 2). Because
mucosal samples are subject to substantial dilution during collection, it
is difficult to estimate the actual concentrations of transudative monoclonal
antibodies present at the mucosal surface. Also, as a result of the variation
in mucosal monoclonal antibody concentration measured on sequential days of
collection for each animal, the values listed in Table 2
are an average from two or three mucosal collections during the first 6 days
after antibody infusion. This variation prevented a statistical comparison
of mucosal antibody concentrations in protected versus infected monkeys. Thus,
the average antibody concentration values are a qualitative indication that
transudative antibody was present at the mucosal surface. Virus neutralization
with mucosal samples has not yet been tested, but the concentration of monoclonal
antibodies 2F5 and 2G12 measured in the diluted vaginal washes is probably
not high enough to mediate virus neutralization in our PBMC neutralization
assay.
To study vaginal transmission, we used SHIV89.6PD that can be transmitted
to macaques across the vaginal mucosa14. Compared to our previous
experience with untreated female macaques, progesterone treatment facilitated
consistent SHIV89.6PD infection with a lower virus inoculum (M.G.L., manuscript
in preparation). Also, compared with our previous intravenous challenge study
with the same antibodies and virus, it seems that greater protection against
infection and disease was observed after vaginal challenge. Although this
observation is tempered by the fact that the intravenous and vaginal challenge
experiments were not done in parallel and the numbers of monkeys per arm was
small, a comparison of protection in these two experiments suggests important
differences. After vaginal challenge, complete protection was achieved in
four of five monkeys in the HIVIG/2F5/2G12 group, two of five in the 2F5/2G12
group, and two of four in the 2G12 group. In contrast, after intravenous challenge,
complete protection was achieved in three of six monkeys in the HIVIG/2F5/2G12
group, zero of three monkeys in the 2F5/2G12 group, and zero of three monkeys
in the 2G12 group. In addition, antibody-treated monkeys that became infected
after vaginal challenge displayed low or undetectable plasma RNA concentrations
and only modest declines in CD4 cell counts. This protection against SHIV-associated
disease was more pronounced in the vaginal challenge study. These data suggest
that antibody can more readily impact infection and disease when virus exposure
occurs across the vaginal mucosa, perhaps by interrupting initial events associated
with mucosal transmission and regional spread of HIV-1. Our previous in
vitro data showing that neutralizing antibodies can block both infection
of human dendritic cells and transmission to T cells are consistent with this
hypothesis15.
There are several limitations of these experiments and this animal model
of mucosal infection. To perform experiments resulting in consistent infection
of control monkeys, we treated female macaques with progesterone 30 days before
vaginal challenge. This treatment causes thinning of the vaginal epithelium10 and facilitates infection with SHIV89.6PD (our unpublished results).
We do not know how this alteration in the mucosal environment affected the
transudation of antibody across vaginal mucosa16, but it is
possible that this allowed more antibody to cross to the mucosal surface.
Also, the vaginal changes induced with a high intramuscular dose of progesterone
are probably more dramatic than changes that occur during the normal female
menstrual cycle. Although this vaginal challenge macaque model may allow general
conclusions about the role of antibody in protection, mucosal exposure, and
transmission of HIV-1 in humans is clearly a complex interaction that is affected
by variables not considered in these experiments.
In summary, this is the first study to directly evaluate the protective
effect of HIV-1 specific antibodies using the SHIV-macaque vaginal challenge
model. By controlling the macaque estrous cycle, we were able to conduct controlled
experiments of passive antibody transfer and vaginal challenge. Our data show
that antibodies can confer protection against vaginal exposure to a pathogenic
SHIV; if virus transmission occurs, their presence can ameliorate the subsequent
pathogenic manifestations of virus infection. Compared to our previous intravenous
challenge study with the same virus and antibodies, the data indicate that
greater protection was achieved after vaginal challenge. In both our intravenous
and vaginal challenge studies, a single monoclonal antibody, with modest virus
neutralizing activity, provided substantial protection. Because the greatest
amount of protection occurred when the most potent combination of neutralizing
antibodies were used, the data validate that in vitro neutralization
assays with PBMC target cells are one relevant measure of functional antibody
activity. Despite its limitations, this animal model probably provides relevant
data about the amount and character of antibody required to confer protection
against sexual transmission of HIV-1.
Methods Antibodies. HIVIG (manufactured as HIV-IG by NABI,
Boca Raton, Florida) is a preparation of purified polyclonal IgG derived from
the pooled plasma of several HIV-1 positive donors as described17.
The product is a 50 mg/ml solution that contains 98% monomeric IgG. Monoclonal
antibody 2F5 recognizes the gp41 sequence ELDKWA that is conserved among many
HIV-1 strains18. Monoclonal antibody 2G12 binds to a conformationally
sensitive epitope in the C3-V4 region of gp12019. Both human
monoclonal antibodies are subclass IgG1 and were produced by recombinant expression
in Chinese hamster ovary cells. Control IVIG, from HIV-1 seronegative individuals,
was provided by the manufacturer (Gammagard S/D, Baxter Healthcare Corp.,
Duarte, California).
Passive antibody transfer and virus challenge. Monkeys
used in this study were captive bred, adult female rhesus macaques and were
housed in a facility accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care in accordance with standards outlined
in the National Institute for Health (NIH) Guide for the Care and Use of Laboratory
Animals. All procedures were approved by the institutional animal care and
use committee. For antibody infusions, vaginal challenge, and blood and mucosal
collections, macaques were lightly anesthetized with ketamine HCl. A seed
stock of SHIV89.9PD was provided by Yichen Lu (AVANT Immunotherapeutics, Needham,
Massachusetts) and a challenge stock was grown and titrated in rhesus PBMC.
Detailed descriptions of the derivation of SHIV-89.6PD and its pathogenicity
have recently been published1,
14. Antibodies were infused intravenously
24 h prior to virus challenge. For all experiments, doses were IVIG, 400 mg/kg;
HIVIG, 400 mg/kg; 2F5, 15 mg/kg, and 2G12, 15 mg/kg. Vaginal SHIV challenge
was done by gently introducing 1 ml of a 1:5 dilution of virus stock (600
TCID50) into the vaginal canal of macaques using a 1-ml syringe.
Macaques were kept in a prone position for at least 15 min postchallenge.
Thirty days prior to vaginal challenge, macaques had received 30 mg of medroxyprogesterone
acetate (Depo-Provera, Upjohn, Kalamazoo, Michigan) by intramuscular injection.
Recent titration experiments in progesterone treated macaques demonstrated
that the monkeys were exposed to 10−50 animal infectious doses of SHIV89.6PD
(Lewis et al., manuscript in preparation). After vaginal challenge,
monkeys were followed clinically and by routine hematology, lymphocyte subset,
and blood chemistry measurements. Inguinal lymph node biopsies for viral co-culture
and PCR for viral DNA were done on all monkeys at 3 weeks post-virus challenge.
Assessment of SHIV-infection in macaques. SHIV-infection
was assessed by SHIV-specific antibody response, SHIV plasma RNA, virus isolation
from PBMC, and lymphoid tissues, and by PCR-based detection of SHIV gag DNA
in PBMC and lymphoid tissues. For viral cultures, monkey PBMC (or single-cell
tissue suspensions) were stimulated with phytohemagglutinin and Interleukin-2
for 3 d prior to the addition of PHA/IL2 stimulated human donor PBMC. Cultures
were followed for expression of p27-antigen by enzyme-linked immunosorbent
assay (ELISA). This method is highly sensitive for detection of virus infection20. Circulating concentrations of plasma viral RNA were determined
using an externally controlled reverse transcription PCR assay as previously
described20,
21. This assay has a lower limit of detection of
200 RNA copies/ml. A semiquantitative PCR assay was used to detect proviral
gag DNA in PBMC and tissue specimens20. The development of SHIV-specific
antibody responses were assessed by serial ELISA measurement of serum antibody
reactivity to p27-gag antigen. Monoclonal antibodies 2F5 and 2G12 do not bind
to SIV p27-gag antigen. HIVIG displays some reactivity in the p27-antigen
ELISA and therefore, for monkeys that received HIVIG, seroconversion to p27-antigen
was defined as a stable or rising titer of anti-p27 antibody by 12 weeks after
virus challenge. Assays for plasma-mediated virus neutralization were done
using PHA/IL2 stimulated human PBMC targets and a p27 gag-antigen ELISA read-out
as described13. Equal volumes of plasma and virus were mixed
30 min prior to the addition of PBMC. The plasma dilution was defined as the
final plasma dilution in the presence of virus and cells. Thus, for an initial
plasma dilution of 1:3, the final plasma dilution was defined as 1:9. Serial
dilution dose-response curves were fit using a quadratic function, and IC
50 and IC90 values were calculated by a least-squares regression
analysis.
Measurement of antibody concentrations in plasma and mucosal compartments.
Plasma and mucosal concentration of monoclonal antibodies 2F5
and 2G12 was determined by ELISA using standard curves generated from the
corresponding monoclonal antibody as described5. Plasma concentrations
of 2F5 and 2G12 were measured 15 min postinfusion, on days 1, 3, 6, and about
weekly for 4−6 weeks. Our previously published methods for mucosal collections
and specimen processing22 were modified for macaques. Vaginal
and rectal lavages were done using an 8-French pediatric feeding tube by flushing
3 ml of PBS into the vaginal canal or rectum and gently aspirating fluid back
into a 3-ml syringe. Nasal washes were done using a 6-French pediatric feeding
tube by instillation of 3 ml of PBS into each nostril and collecting draining
PBS into a 15-ml conical tube placed underneath the nostril. To collect oral
secretions, a total of six dacron, polyester-tipped swabs were brushed against
the mucosal surface of the oral cavity and antibody was eluted from swabs
with a total of 3 ml of PBS.
Received 7 November 1999; Accepted 1 December 1999
REFERENCES
Reimann, K.A. et al. A chimeric simian/human immunodeficiency 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 (1996). | PubMed | ISI | ChemPort |
Reimann, K.A. et al. 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 (1996). | PubMed | ISI | ChemPort |
Shibata, R. et al. Infection and pathogenicity of chimeric simian-human immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing. J. Infect. Dis.176, 362–373 (1997). | PubMed | ISI | ChemPort |
Shibata, R. et al. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nature Med.5, 204–210 (1999). | Article | PubMed | ISI | ChemPort |
Mascola, J.R. et al. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol.73, 4009–4018 (1999). | PubMed | ISI | ChemPort |
Piot, P. The science of AIDS: A tale of two worlds. Science280, 1844–1845 (1998). | Article | PubMed | ISI | ChemPort |
Miller, C.J. & McGhee, J.R. Progress towards a vaccine to prevent sexual transmission of HIV. Nature Med.2, 751–752 (1996). | Article | PubMed | ISI | ChemPort |
Spira, A.I. et al. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J. Exp. Med.183, 215–225 (1996). | Article | PubMed | ISI | ChemPort |
Frankel, S.S. et al. Replication of HIV-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid. Science272, 115–117 (1996). | PubMed | ISI | ChemPort |
Marx, P.A. et al. Progesterone implants enhance SIV vaginal transmission and early virus load. Nature Med.2, 1084–1089 (1996). | Article | PubMed | ISI | ChemPort |
Burton, D.R. & Montefiori, D.C. The antibody response in HIV-1 infection. AIDS11 Suppl. A, S87–S98 (1997). | PubMed | ISI |
Lambert, J.S. et al. Safety and pharmacokinetics of hyperimmune anti-human immunodeficiency virus (HIV) immunoglobulin administered to HIV-infected pregnant women and their newborns. J. Infect. Dis.175, 283–291 (1997). | PubMed | ISI | ChemPort |
Mascola, J.R. et al. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12. J. Virol.71, 7198–7206 (1997). | PubMed | ISI | ChemPort |
Lu, Y.C., Pauza, C.D., Lu, X.S., Montefiori, D.C., & Miller, C.J. 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. Hum. Retrovirol.19, 6–18 (1998). | PubMed | ChemPort |
Frankel, S.S. et al. Neutralizing monoclonal antibodies block human immunodeficiency virus type 1 infection of dendritic cells and transmission to T cells. J. Virol.72, 9788–9794 (1998). | PubMed | ISI | ChemPort |
Kutteh, W.H., Moldoveanu, Z., & Mestecky, J. Mucosal immunity in the female reproductive tract: correlation of immunoglobulins, cytokines, and reproductive hormones in human cervical mucus around the time of ovulation. AIDS Res. Hum. Retroviruses14 (Suppl. 1), 51–55 (1998). | PubMed |
Lambert, G. et al. Effect of maternal CD4+ cell count, acquired immunodeficiency syndrome, and viral load on disease progression in infants with perinatally acquired human immunodeficiency virus type 1 infection. J. Pediatr.130, 890–897 (1997). | PubMed | ISI | ChemPort |
Muster, T. et al. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol.67, 6642–6647 (1993). | PubMed | ISI | ChemPort |
Trkola, A. et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol.70, 1100–1108 (1996). | PubMed | ISI | ChemPort |
Lewis, M.G. et al. Limited protection from a pathogenic chimeric simian-human immunodeficiency virus challenge following immunization with attenuated simian immunodeficiency virus. J.Virol.73, 1262–1270 (1999). | PubMed | ChemPort |
Vahey, M.T. & Wong, M.T. in PCR Primer: A Laboratory Manual 313–338. (Cold Spring Harbor Press, Plainview, NY, 1995).
Artenstein, A.W. et al. Mucosal immune responses in four distinct compartments of women infected with human immunodeficiency virus type 1: a comparison by site and correlation with clinical information. J. Infect. Dis.175, 265–271 (1997). | PubMed | ISI | ChemPort |
Acknowledgments We thank Chris Sapan for supplying the HIVIG, Norman Letvin, Keith Reimann,
and Yichen Lu for the SHIV89.6PD, Robin Garner for statistical advice, Dawn
Harris for veterinary care and Francine McCutchan and Nelson Michael for manuscript
reviews. We appreciate the excellent technical assistance from Mark Louder,
Marcin Iwanicki, Jack Greenhouse, Mike Eller, and Jake Yalley-Ogunro. This
work was supported in part by grants from the National Heart Lung and Blood
Institute and by Cooperative Agreement between the U.S. Army Medical Research
and Materiel Command and the Henry M. Jackson Foundation for the Advancement
of Military Medicine.United States Department of Defense Disclaimer: The views
and opinions expressed herein are those of the authors and do not purport
to reflect the official policy or position of the U.S. Army, U.S. Navy or
the Department of Defense.
105 copies/ml
through 22 weeks after challenge, plasma viremia in these antibody-treated
monkeys declined to undetectable, or near undetectable, concentrations by
week 12. None of these six infected monkeys developed the profound loss of
CD4 cells that was seen in the five control monkeys.
g/ml to 260 
