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Journal of Virology, November 2000, p. 10514-10522, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effective Induction of Simian Immunodeficiency Virus-Specific
Systemic and Mucosal Immune Responses in Primates by
Vaccination with Proviral DNA Producing Intact but
Noninfectious Virions
Shainn-Wei
Wang,1
Pamela A.
Kozlowski,1
Gretchen
Schmelz,1
Kelledy
Manson,2
Michael S.
Wyand,2
Rhona
Glickman,3
David
Montefiori,4
Jeffrey D.
Lifson,5
R. Paul
Johnson,3,6
Marian R.
Neutra,1 and
Anna
Aldovini1,*
Department of Medicine, Children's Hospital, and
Department of Pediatrics, Harvard Medical School, Boston,
Massachusetts1; Primedica, Worcester,
Massachusetts2; Duke University, Durham,
North Carolina4; Retroviral Pathogenesis
Laboratory, AIDS Vaccine Program, SAIC Frederick, NCI-FCRDC,
Frederick, Maryland5; New England
Primate Research Center, Harvard Medical School, Southborough,
Massachusetts3; and Partners AIDS
Research Center and Infectious Disease Unit, Massachusetts General
Hospital, Charlestown, Massachusetts6
Received 28 April 2000/Accepted 15 August 2000
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ABSTRACT |
We report a pilot evaluation of a DNA vaccine producing genetically
inactivated simian immunodeficiency virus (SIV) particles in primates,
with a focus on eliciting mucosal immunity. Our results demonstrate
that DNA vaccines can be used to stimulate strong virus-specific
mucosal immune responses in primates. The levels of immunoglobulin A
(IgA) detected in rectal secretions of macaques that received the DNA
vaccine intradermally and at the rectal mucosa were the most
striking of all measured immune responses and were higher than usually
achieved through natural infection. However, cytotoxic T lymphocyte
responses were generally low and sporadically present in different
animals. Upon rectal challenge with cloned SIVmac239, resistance to
infection was observed, but some animals with high SIV-specific IgA
levels in rectal secretions became infected. Our results suggest that
high levels of IgA alone are not sufficient to prevent the
establishment of chronic infection, although mucosal IgA responses may
have a role in reducing the infectivity of the initial viral inoculum.
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INTRODUCTION |
Globally, more than 80% of the
transmission of human immunodeficiency virus (HIV) infection is via
mucosal routes. The ability of vaccines to induce mucosal immunity may
be required for protection against HIV infection or the
immunodeficiency syndrome that emerges after infection.
Stimulation of simian immunodeficiency virus (SIV)-specific mucosal
responses has been achieved with particulate antigens or with
microencapsulated killed virus (20, 21, 27, 29, 31-33, 37).
When the site of immunization targeted the iliac lymph node (TILN),
total protection from rectal challenge was achieved, while protection
from vaginal challenge was less consistent (33, 35). These
results suggest that mucosal responses might be a desirable feature of
an HIV vaccine (39). Since TILN vaccination or mucosal
administration of infected and fixed cells is unlikely to be employed
for large-scale immunization of humans, it is important to identify an
alternative vaccination strategy that engenders a similar protective
response but is more easily administered. Mucosal administration of DNA
vaccines may provide an alternative and safe strategy. DNA vaccines
have been successful in inducing antigen-specific mucosal responses in
mice, but little is known about the ability of DNA vaccines to
stimulate mucosal responses in primates. Furthermore, DNA vaccines have
been surprisingly less successful in stimulating antigen-specific
systemic immunoglobulin G (IgG) responses in primates than in
mice, so it is particularly important to determine whether
antigen-specific IgA production can be elicited in primates
through DNA vaccination.
DNA vaccines expressing HIV genes have been investigated in humans to
determine their safety and their ability to induce or boost
virus-specific immune responses (6). Several DNA vaccine constructs and vaccination protocols have been evaluated alone or
combined with other approaches for their ability to induce protection
against challenge with retroviruses (13-15, 19, 25, 34,
45). When challenged intravenously (i.v.), the animals sometimes
resisted the establishment of chronic infection and more frequently
achieved decreased viral load and had a more prolonged asymptomatic
state (13, 17, 45). These studies represent an interesting
first step in the study of SIV DNA vaccines, but they are limited by
several factors. In particular, these studies were not designed to
evaluate mucosal immunity, and the challenges did not involve mucosal exposure.
Viral genomes that produce noninfectious virus-like particles have
several features that make them attractive candidates for an AIDS
vaccine. They may be capable of engendering immunity similar to that
obtained with attenuated viral vaccines but do not establish the
persistent infection associated with attenuated viruses (8). Noninfectious virus-like particles are produced in host cells in the
same manner as a normal replicating virus and have protein components
whose conformational integrity is maintained. Ideally, an altered viral
genome would express proteins that assemble into noninfectious
particles which contain all of the immunogenic components of the virus
but which are unable to productively infect new cells.
We constructed a DNA vaccine with mutations in multiple structural
genes that produces SIV particles that are noninfectious and yet are
similar to normal SIV particles in protein content. We find that this
DNA vaccine candidate is immunogenic in rhesus macaques and can
stimulate significant levels of IgA antibodies in secretions when
administered at the rectal mucosa. Detailed analyses of the
immunological responses engendered by this vaccine and the ability of
vaccinated primates to resist a challenge with live SIV are presented.
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MATERIALS AND METHODS |
Vector construction.
All mutants of SIVmac239 were
constructed using the infectious clone pMA239 (14,110 bp)
(47), which carries a full copy of the molecular clone of
SIV mac239. Mutations were introduced in the SIV genome using
oligonucleotide-mediated site directed mutagenesis by overlapping
extension PCR (18) (Table 1).
The individual changes introduced in three SIV proteins are listed in
Table 1. In addition, the SIV 5' long terminal repeat (LTR) was
replaced by the cytomegalovirus (CMV) promoter or eukaryotic polypeptide chain elongation factor 1a (EF1a) promoter, and the SIV 3'
LTR was substituted with the polyadenylation site, poly(A), from pSG5
(Stratagene). The poly(A) fragment from the pSG5 vector replace
sequences 9505 to 10709 of SIVmac239 (44). A fragment containing the CMV promoter, derived by PCR from the pRL CMV vector, replaced the 5' SIV sequences up to the NarI site of pMA239.
The modified plasmid containing the CMV promoter, multiple mutations in
the HIV structural genes, and a replacement of the 3' LTR with a
poly(A) site was designated pVacc1. A similar strategy was used to
replace the 5' LTR with the EF1a promoter. The EF1a promoter DNA
fragment, obtained by PCR from pEBB (50), replaced the CMV promoter of pVacc1 to obtain the plasmid designated pVacc2. All mutated
viral sequences were confirmed by dideoxy sequencing. All DNA
manipulations were carried out according to previously published
procedures (3).
In vitro analysis of the SIV DNA vaccine candidates.
Transfection in 293T cells was carried out by the calcium phosphate
method. Quantitative reverse transcription-PCR (RT-PCR) assay was
carried out on cellular RNA extracted from 293T cells and DNase treated
to eliminate contaminating DNA according to a previously described
procedure (42). The protein and RNA contents of the viral
particles produced from the vectors were characterized biochemically by
Western blot and quantitative RT-PCR as previously described
(43). Electron microscopy was carried out on sections of
embedded particles according to standard procedures. Viral supernatants
derived from two independent transfections per construct were tested in
infectivity assays on CEMx174 cells as described elsewhere
(43). Cultures were maintained for 30 days after infection. Cleared supernatants were tested for virus content by SIV p27 enzyme-linked immunosorbent assay (ELISA). Nested PCR on cellular DNA
and RT-PCR on RNA from pelleted supernatants were also carried out on
cultures that scored negative in SIV p27 ELISA.
Vaccine formulation.
Plasmid DNA was purified by a CsCl
gradient, followed by passage through an endotoxin-free column
(Qiagen). For intradermal (i.d.) and intramuscular (i.m.)
administration of the DNA vaccine, saline solution (Sigma) was used to
resuspend the DNA, and the concentration was adjusted to 1 mg/ml. For
mucosal administration, the vaccine DNA was formulated in 20 mM
DOTAP-cholesterol liposomes (51) at a concentration of 0.5 mg/ml. For i.d. gene gun administration, the plasmid DNA was
precipitated onto 1.6-µm gold particles according to the manufacturer
protocol (Bio-Rad).
Macaque vaccination.
Nine rhesus macaques were vaccinated at
time zero and at 9 and 25 weeks with the SIV construct i.d. (group 1),
i.d. and at the rectal mucosa (i.d., R) (group 2), and i.d., at the
rectal mucosa, and i.m. (i.d., R, i.m.) (group 3). The animals in
groups 1, 2, and 3 received 0.5 mg of pVacc1 DNA i.d. (0.4 mg of DNA in
saline by needle injection and 0.1 mg DNA by gene gun) in the skin that
covers the gluteal area. Additionally, at all three vaccination time
points, 1 mg of pVacc1 DNA, mixed with liposomes according to the
protocol described above, was administered to the rectal mucosa of
animals in groups 2 and 3. Group 3 animals also received 1 mg of
vaccine pVacc2 DNA administered i.m. to the gluteal muscle. Control
animals were vaccinated like the animals in group 3, except that the
pUC19 plasmid replaced the SIV-related plasmids.
SIV-specific IgA analysis in rectal secretions.
Rectal
secretions were collected before and at intervals after immunization
with absorbent Weck-Cel sponges (Windsor BioMedical, Newton, N.H.)
using a modified wicking method that has been described in detail
elsewhere (27a). The volume of secretion eluted from each
sponge and dilution factors introduced by the premoistening saline and
elution buffer were calculated based on weights of fluid centrifuged
into 2-ml microcentrifuge lower-chamber tubes (Kozlowski et al.,
submitted). Blood contamination in secretions (assessed by measuring
hemoglobin using Boehringer-Mannheim ChemStrips 4) was found to be
negligible, representing only 0.01% of that in blood on average.
For detection of SIV and gp130-specific IgA and IgG in rectal
secretions, plates were coated with 250 ng of SIV viral lysate
or
purified native gp130 (both from Advanced Biotechnologies,
Rockville,
Md.) per well. Antibodies measured in these SIV ELISAs
likely do not
include those to gp130 since this envelope protein
could not be
detected on plates coated with viral lysate using
5 µg of anti-gp130
antibody (Advanced Biotechnologies) per ml.
Pooled serum from
SIV-infected monkeys was used to generate standard
curves in these
assays for interpolation of antibody concentrations
in samples. A
highly specific anti-monkey IgA mouse IgG monoclonal
antibody
(
52) was used as a secondary antibody, followed by
biotinylated goat anti-mouse IgG antibody (Southern Biotechnology
Associates, Birmingham, Ala.) from which antibodies cross-reactive
with
monkey IgG had been removed by passage through a column of
CNBr-activated Sepharose (Pharmacia) conjugated to monkey IgG.
In
assays for SIV-specific IgG in rectal secretions, plates were
developed
with a biotinylated (Pierce Sulfo-NHS-LC-Biotin EZ-link
kit) goat
anti-monkey IgG antibody (Accurate, Westbury, N.Y.).
To determine the
endpoint titers of antibody in secretions, the
last sample dilution
producing an absorbance value of greater
than or equal to the mean
absorbance ± 3 standard deviations in
eight control wells was
multiplied by the dilution factor introduced
into the secretion during
elution from
sponges.
To determine with accuracy whether rectal secretions contained
significant levels of SIV-specific antibodies and to facilitate
comparisons among animals in which total immunoglobulin concentrations
in secretions were highly variable, measured antibody concentrations
were divided by the total IgA or the total IgG concentration in
each
sample. Total IgA and IgG concentrations were quantitated
by ELISA
using plates coated with goat anti-monkey IgA or IgG
(Accurate), a
calibrated monkey serum standard provided by M.
W. Russell
(University of Alabama at Birmingham), and the above-described
secondary
reagents.
SIV-specific humoral responses in serum.
SIV-specific serum
IgG were measured by ELISA using plates coated with whole virus or
purified virus-derived gp130. Incubations were performed in duplicate,
and multiple double dilutions of each sample were evaluated. Bound
SIV-specific IgG were detected by incubation with an affinity-purified
donkey anti-human IgG-alkaline phosphatase conjugate (Jackson
Laboratories). Antibody-mediated neutralization of SIV was measured in
a CEMx174 cell-killing assay as described previously (30,
40). Neutralization was measured with two stocks of SIV: (i) a
laboratory-adapted stock of SIVmac251 produced in H9 cells and (ii)
molecularly cloned SIVmac239/nef-open produced in rhesus peripheral
blood mononuclear cells (PBMC) by using a vial of the original animal
challenge virus as seed stock. The former virus is highly sensitive to
neutralization, whereas the latter virus is extremely difficult to
neutralize in vitro (30, 40).
Cell-mediated immune responses.
Cytotoxic-T-lymphocyte (CTL)
assays were carried out according to previously described procedures
(23). Autologous herpesvirus papio-transformed B
lymphoblastoid cell lines (B-LCL) infected with a recombinant vaccinia
virus vectors expressing the SIV Gag, Pol, and Env were used as
stimulators and targets. Cytolytic activity was determined in a
standard 51Cr release assay. Lysis was generally examined
at effector/target (E/T) ratios of 40:1, 20:1, and 10:1. Based on
examination of SIV-specific CTL activity in over 20 negative controls
studied to date, SIV-specific CTL activity of 
5% at two different
E/T ratios was considered significant (23). In the subset of
vaccinated animals that express the Mamu-A*01 allele, the frequency of
CD3+ CD8+ cells specific for the SIV Gag 11C-M
epitope was determined using major histocompatibility complex (MHC) tetramers.
RT-PCR and PBMC limiting-dilution viral loads.
Plasma SIV
RNA levels were measured by a real-time RT-PCR assay, as described
elsewhere (49). The assay has a threshold sensitivity of 300 copy Eq/ml. Interassay variation is <25% (coefficient of variation).
Cell-associated virus loads were measured by limiting-dilution culture
of PBMC every month during the postchallenge time course as previously
described (53).
Flow cytometry.
Whole blood collected in EDTA was analyzed
for lymphocyte subset CD4 (OKT4a, Ortho, and/or Anti-Leu3a; Becton
Dickinson), CD8 (Anti-Leu2a; Becton Dickinson), and CDw29 (4B4; Coulter
Immunology) by a whole-blood lysis technique described previously
(53).
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RESULTS |
DNA constructs expressing genetically inactivated SIV
particles.
Our vaccine design strategy was to introduce a large
number of inactivating mutations into the viral genome, thereby
minimizing the probability of genetic reversion to an infectious virus
while retaining the ability to produce particles with protein content and immunogenic properties similar to wild-type virions. We have identified a combination of 22 mutations in three independent SIV genes
and substitution of the SIV LTRs that minimize the potential for
genetic reversion while retaining virus particle production, and we
have introduced these into a single construct. Knowledge of the
elements required for RNA packaging has led us to construct various SIV
and HIV-1 genomes that are capable of expression of particles which
lack genomic RNA (2, 43). To further improve the safety of
these particles, we selected residues that are crucial to the function
of reverse transcriptase and integrase as additional targets for viral
inactivation (9, 26). The collection of SIV mutated viruses
that have been produced is summarized in Table 1. All constructs
contain multiple CpG motifs, which are associated with improved
immunostimulatory properties of DNA vaccines (reference 28 and references therein).
Various promoters have been engineered to drive expression of these SIV
genes, as different promoters might produce different
levels of antigen
in the various cells targeted by DNA vaccination.
The different
promoter efficiencies were measured by evaluating
genomic viral RNA
accumulation by RT-PCR in 293T transfected cells
48 h after
transfection (Fig.
1A). The construct
pVacc2, containing
the EF1a promoter, produced higher levels of RNA
than construct
pVacc1, containing the cytomegalovirus (CMV) promoter,
or pMA22polyA,
containing the SIV LTR, and the increased RNA
accumulation correlated
with the increase in particle production from
the transfected
cells.
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FIG. 1.
Analysis of viral particle production by SIV mutated
constructs upon transfection in 293T. (A) RT-PCR analysis of total
viral RNA accumulated intracellularly after transfection with SIV
constructs. Nucleic acid amplification was carried out with SIV
Tat-related primers on total cellular RNA extracted from transfected
293T cells. Lanes show RT-PCR (a) and PCR (b) reactions carried out on
the total cellular RNA samples. The PCR control was a PCR reaction
carried out on pMA239 DNA (a) and in the absence of template DNA (b).
The mock control was a reaction carried out on RNA from supernatant
from mock-transfected 293T. Values reported in the panel reflect the
evaluation of the bands visualized in the figure. A value of 100 was
assigned to the band from pMA239. The intensity of the other bands was
calculated by dividing the intensity of each band by the intensity of
the pMA239 band. The average values from three independent experiments
performed in duplicate ± their standard errors were as follows:
pMA239, 100; pMA22polyA, 35.2 ± 1.73; pVacc1, 175.5 ± 22.4;
pVacc2, 292.2 ± 34.1. (B and C) Western blot analysis of pelleted
particles. In panel A the blot was probed with a macaque SIV polyclonal
serum that reacts predominantly with the SIV Env products. In panel B
the blot was probed with a macaque SIV polyclonal serum that reacts
predominantly with the SIV Gag products. The molecular weights of SIV
Env and Gag products are indicated in kilodaltons (kD). (D) RT-PCR
analysis of the genomic RNA content of SIV mutated particles. RNA
amplification was carried out with SIV Gag-related primers on viral RNA
extracted from pelleted virions. Lanes a and b show, respectively,
RT-PCR and PCR reactions carried out on the RNA samples. Standards are
derived from twofold dilutions of an RNA sample obtained from virions
produced by pMA239, which produces wild-type SIVmac239. (E)
Electron micrograph of noninfectious particles produced by pVacc1 after
transfection into 293T. Images were obtained at a magnification of
×90,000.
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The SIV vectors were tested for particle production and infectivity in
a tissue culture system. These vectors, containing
a total of 22 mutations affecting the function of three essential
genes of SIV,
efficiently produce particles containing all major
SIV proteins and no
detectable viral RNA (Fig.
1B and C). The
morphology of these mutant
particles was consistent with that
of immature particles lacking RNA
(
43). These particles were
determined to be noninfectious
when tested by a number of different
assays on CEMx174 (data not
shown).
Evaluation of immune responses to SIV antigens in macaques
vaccinated with a genetically inactivated SIV genome.
To evaluate
the induction of SIV-specific mucosal and systemic immunity in
primates, nine rhesus macaques were inoculated with SIV DNA (groups 1 to 3) and three rhesus macaques were inoculated with the control
plasmid pUC19 (group 4) according to the schedule and doses indicated
in Materials and Methods. Three different vaccination regimens were
used in order to investigate the ability of the DNA vaccine to prime
different immunological compartments. The rationale was to compare a
relatively simple regimen of immunization to more complex regimens.
Because hepatitis B virus is the only chronic virus for which a
protective vaccine is available and only one schedule could be
investigated with the limited number of animals available, we reasoned
that it might be appropriate to follow the hepatitis B virus
vaccination schedule.
Various samples were harvested during the course of the immunizations,
and the following immunological assays were performed:
SIV-specific IgA
and IgG in the rectal secretions, SIV-specific
IgG, IgA and
neutralizing activity in the serum, CTL activity
in PBMC, and tetramer
staining in Mamu A*01-positive macaques
(two of the nine that received
the vaccine). The results of the
immune response assays prior to live
virus challenge are briefly
summarized in Table
2. The most striking of all measured
immune
responses were the levels of virus-specific IgA detected in
rectal
secretions of animals in group 2, which received the i.d., R
regimen.
The data shown in Table
3 demonstrate
that the administration of a DNA vaccine at the rectal mucosa can
stimulate significant
SIV-specific IgA responses in primate rectal
secretions. Samples
from five of nine vaccinated animals were positive
at a secretion
dilution of 1:23 to 1:2,179. The absence of detectable
SIV-specific
serum IgA (data not shown) indicated that the IgA was
locally
produced. No virus-specific IgA was detected in serum samples
collected at the same time. The secretions from two animals were
also
SIV-IgG positive. Analysis of SIV-specific IgA content in
secretions
collected after the first and second vaccinations indicated
that three
rectal mucosal doses are necessary to induce significant
and consistent
SIV-specific IgA levels (data not shown). The magnitude
of the increase
in SIV-specific IgA content in most of the positive
rectal samples was
substantially higher than that seen thus far
in any other sample
analyzed in SIV-vaccinated animals or in animals
infected with SIV
(
29,
33,
37,
38; Kozlowski et al., submitted).
The i.m. administration of DNA together with rectal and
i.d. inoculations appeared to negatively affect the mucosal
responses
(compare fold increase in animals of groups 2 and 3 in Table
3).
This result provides preliminary evidence that simultaneous mucosal
and i.m. DNA vaccination cannot stimulate both the systemic and
the
mucosal arms of the immune system. However, the outcome might
be
different if simultaneous mucosal and systemic antigenic stimulation
is
provided by vaccines that are not DNA based or are administered
via
different
routes.
Humoral systemic virus-specific immunity was investigated by measuring
SIV-specific serum IgG in an ELISA assay. SIV-specific
IgG
responses were weak, ranging from 1:100 to 1:2,560 on the
day of
challenge (Table
4). Neutralization
assays carried out
with the same samples were negative when
SIVmac251 or the challenge
virus SIVmac239 was used in the
assay. Systemic cell-mediated
immunity was investigated by measuring
virus-specific CTL activity
in PBMC. CTL responses were sporadically
present at different
levels in different animals (Table
5). Animals with different
genetic
backgrounds respond differently to a vaccine. One animal
(animal 19775)
showed a very high level of CTL activity against
Env (25%) and Pol
(50%) when assayed 2 weeks after the third vaccination,
indicating
that this vaccine has the potential to stimulate significant
cellular
responses.
Evaluation of protection from the establishment of a persistent
SIV infection and/or simian AIDS after mucosal SIV challenge.
The
significant levels of IgA antibodies in rectal secretions elicited in
all three animals vaccinated i.d., R provided an opportunity for a
preliminary evaluation of the role of virus-specific IgA in prevention
of infection. Animals in all groups were challenged with live virus 2 weeks after the third immunization with 5,000 50% tissue culture
infectious doses of cloned SIVmac239, administered to the rectal
mucosa. This amount of virus was equivalent to 9 ng of p27 and is
estimated to be approximately 10 rectal macaque infectious doses
(AID50) (R. Desrosiers, personal communication). This challenge dose corresponds to 105
AID50 by i.v. titration. Because the small size of the
animal groups prevents a meaningful statistical analysis of the
challenge results, investigation of larger groups of animals immunized
via the mucosal route will be necessary to fully elucidate the role of
virus-specific mucosal immunity in the prevention of infection.
Anamnestic IgG responses were observed in all of the animals that were
previously SIV-IgG positive and became infected. Seroconversion
could
be documented in control animals 3 weeks after challenge
(Table
4).
Clear evidence of an anamnestic neutralizing antibody
response was
detected in serum from some animals 2 to 3 weeks
after challenge,
suggesting that priming for neutralization epitopes
was induced by the
DNA vaccine (Table
4). This anamnestic response
was
detected with SIVmac251 that is highly sensitive to
neutralization
but not with SIVmac239. This result was not
surprising as SIVmac239
is extremely difficult to neutralize
(
30,
40).
RT-PCR was carried out to detect RNA viral loads in serum samples from
the day of challenge to 25 weeks after challenge (Fig.
2). Cell-associated virus loads were also
measured in a limiting-dilution
cocultivation assay on a monthly basis
(data not shown). In the
infected animals, viral loads peaked 2 weeks
postchallenge and
subsequently decreased. Average viral loads were
lower for the
group vaccinated intradermally than for the control
group, with
differences of approximately 10-fold as measured by RT-PCR
(Fig.
2), possibly because on the day of challenge CTL responses, which
have been associated with viremia control (
22,
46), were
more
consistent and significant in the two i.d. vaccinated animals
that
subsequently became infected. Two of the nine vaccinated
animals
(animal 19796 of the i.d. group and animal 19821 of the
i.d., R group)
remained RT-PCR and PBMC cocultivation negative
postchallenge (the last
measurement was week 63 postchallenge).
PCR analysis of PBMC DNA
obtained from samples collected 2 weeks
after challenge, when viremia
peaked in all infected animals,
was also negative in the two animals
that resisted challenge (data
not shown). PBMC fluorescence-activated
cell sorter (FACS) analysis
was carried out for the T-cell
immunological markers CDw29, CD4,
and CD8 (Table
6). CDw29 measures a subpopulation of CD4
cells
(memory CD4 cells), and its decline has been observed as an early
indicator of the immunological decline that is correlated with
subsequent disease progression (
16). Two consecutive
measurements
of this marker that are <10% are considered an
indication of incipient
immunological decline. SIV-infected animals
vaccinated i.d. maintained
values of CDw29, CD4, and CD8 within the
normal range for a longer
period of time than the other infected
animals, while a decline
affecting in particular the CDw29 marker was
evident in most of
the other infected animals. Animals 19781 and 19784 in group 3
and animal 19816 in group 4 were diagnosed with an
AIDS-related
illness and euthanized 41 to 49 weeks after challenge.
These data
suggest that disease progression might be delayed in the
i.d.
vaccinated group compared to the other groups.
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FIG. 2.
SIV viral loads in macaques challenged rectally by
SIVmac239 (serum RT-PCR). Each time point represents the
average ± the standard error of the values of viral loads
detected in the three animals of one regimen group.
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DISCUSSION |
We have carried out a small pilot study to investigate whether
SIV-specific mucosal immunity can be induced by a DNA vaccine candidate
that produces noninfectious virus and whether virus-specific IgA
antibodies are a desirable component of a vaccine aimed toward the
prevention of mucosally transmitted AIDS. From this study, we can infer
the following. (i) A DNA construct that expresses all the SIV proteins
except Nef and produces a noninfectious virus due to mutations in three
structural genes is a safe and immunogenic reagent in macaques. (ii)
This DNA vaccine administered to macaques in a liposome formulation at
the rectal mucosa can stimulate significant levels of antigen-specific
IgA in mucosal secretions. These levels are higher than those achieved
through natural infection. (iii) Virus-specific IgA antibodies present
in rectal secretions may have a role in decreasing the infectivity of
the initial viral inoculum but alone are unlikely to be sufficient to
prevent infection. (iv) Simultaneous DNA vaccination via multiple
routes as described here did not result in efficient priming of various
immunological compartments.
There are a number of potential advantages to DNA vaccines that produce
virus particles compared to vaccines that produce individual antigens.
These noninfectious viral particles incorporate the Env proteins and
bind the viral receptor on target cells as well as the wild-type virus.
Binding of the receptor is thought to trigger conformational changes in
the Env protein that may reveal critical neutralizing epitopes on both
gp120 and gp41. Exposure of these epitopes may not occur with
Env-based vaccine preparations where the Env protein is not part of a
virus particle. It is also possible that DNA vaccines expressing
viral particles may be more efficient in priming CTLs. Although DNA
vaccines that produce virus particles and DNA vaccines that
produce individual antigens may prime equally well via the endogenous
antigen presentation pathway when targeted to hematopoietic cells, CTL
priming might occur more efficiently via the exogenous pathway with
particles, once they are taken up by antigen-presenting cells (APCs).
This may be particularly important if the cells targeted by the DNA vaccination are nonhematopoietic cells. When the DNA vaccine-derived antigen or the virus-associated antigen is expressed in a
nonhematopoietic cell, uptake of antigens by bone-marrow-derived APCs
is required to initiate antiviral CTL responses (10, 48).
The efficiency of this process might increase with particle-associated antigens.
The potential role of virus-specific mucosal immunity was not clarified
in this study. Antibodies in mucosal secretions alone seem
unlikely to protect from the establishment of chronic infection, since
animal 19786, who had the highest levels of IgA in secretions but
no detectable serum IgG, IgA, or CTLs on the day of challenge, became
infected. It has been suggested that secreted antibodies might provide
the first line of defense against the virus inoculum transmitted at the
time of exposure and that local interstitial antibodies and CTLs could
act as the second line of defense against virus that enters the mucosa
(4, 11-12, 14, 36). Indeed this combination has been
suggested as a key to resistance to HIV infection in studies of exposed
sex workers in Nairobi and Northern Thailand and discordant
heterosexual couples (5, 7, 41). Immunologically mediated
containment of the infection during its initial local phase might be
possible, and the presence of mucosal immunity could be critical to
achieve this goal. A future goal should be to devise practical
immunization regimens that are capable of stimulating consistent high
levels of both mucosal and systemic immunity.
Two animals in the experiment described here may have been protected
from infection by the immune responses induced through vaccination. It
is unlikely that the lack of chronic infection in these two
SIV-negative vaccinated animals resulted from poor infectivity of the
viral stock or natural resistance. The SIVmac239 utilized for the
rectal challenge in our experiments is a highly pathogenic virus that
quickly establishes high viremia and could easily infect PBMC from
these two animals in culture. Protection against this virus has been
difficult to achieve, and most successes have been in cases where
animals were vaccinated with attenuated viruses (8, 24). The
SIVmac239 virus stock used for the rectal challenge has been
administered rectally at the same concentration used in our experiment
to a total of 15 naive animals so far (Desrosiers, personal
communication). All of these animals have become persistently infected.
Despite multiple attempts, we were unable to demonstrate any direct or
indirect evidence of infection in the two animals that remained virus
negative after challenge. In addition, the postchallenge decline of
anti-SIV antibody titers in these animals implies a lack of further
antigenic exposure and is consistent with the animals being protected
from infection. No immunological correlates of protection could be
established in these two animals. Nevertheless, because some
immunological parameters potentially relevant to protection such as
levels of inhibitory chemokines (1) and SIV-specific mucosal
cellular responses were not evaluated in this study, it is possible
that immune responses induced by the vaccination may have played a role
in protecting these animals.
The small size of the groups does not lend statistical power to any
conclusions about protection. Nevertheless, it is intriguing that the
observed protection of one in three animals in two of the vaccine
groups is comparable to the rate of success obtained in other SIV DNA
vaccine studies (13, 45). A more thorough evaluation of the
role of mucosal immunity in protection from infection will require a
more extensive investigation with a larger number of animals.
Nevertheless, our ability to easily induce a high level of SIV-specific
IgA in rectal secretions by local DNA vaccination provides a simple
immunization strategy that could be easily transferred to the clinical
setting, if stimulation of mucosal immunity is a desirable feature in
an SIV and/or HIV vaccine.
| |
ACKNOWLEDGMENTS |
This study was funded by National Institutes of Health grants
AI41365, AI34757, and RR00168, contract AI85343, and, in part, with
federal funds from the National Cancer Institute, National Institutes
of Health, under contract NO1-CO-56000.
We thank John Altman (Emory University) for the gift of major
histocompatibility complex tetramers. We also thank M. Piatak and
L. Li for expert viral load analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Children's
Hospital, Enders 609, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 355-8426. Fax: (617) 355-8387. E-mail:
anna.aldovini{at}tch.harvard.edu.
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Abstract
Introduction
