AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 20, Number 4, 2004, pp. 425–434
© Mary Ann Liebert, Inc.
Protective Immunity to SIV Challenge Elicited by
Vaccination of Macaques with Multigenic DNA Vaccines
Producing Virus-Like Particles
SALLY P. MOSSMAN, 1,2,9 CHRISTOPHER C. PIERCE,1 ANDREW J. WATSON, 1
MICHAEL N. ROBERTSON,3 DAVID C. MONTEFIORI,4 LARENE KULLER,2
BARBRA A. RICHARDSON, 5 JEFFREY D. BRADSHAW, 1 ROBERT J. MUNN,6 SHIU-LOK HU,2
PHILIP D. GREENBERG,3 RAOUL E. BENVENISTE,7 and NANCY L. HAIGWOOD 1,8
ABSTRACT
We utilized SIVmne infection of Macaca fascicularis to assess the efficacy of DNA vaccination alone, and as a
priming agent in combination with subunit protein boosts. All SIVmne structural and regulatory genes were
expressed using the human cytomegalovirus Immediate Early-1 promoter in plasmids that directed the formation of virus-like particles in vitro. Macaques (n 5 4) were immunized intradermally and intramuscularly
four times over 36 weeks with 3 mg plasmid DNA. A second group (n 5 4) received two DNA priming inoculations followed by two intramuscular boosts consisting of 250 mg recombinant Env gp160 and 250 mg recombinant Gag-Pol particles in MF-59 adjuvant. These regimens elicited modest cellular immunity prior to
challenge. Humoral immune responses to Env gp160 were elicited and sustained by both vaccine protocols,
and as expected antibody titers were higher in the protein subunit-boosted animals. Neutralizing antibodies
prior to challenge were measurable in two of four subunit-boosted macaques. The two vaccine regimens elicited
comparable helper T cell responses at the time of challenge. Vaccinees and mock-immunized controls (n 5 4)
were challenged intrarectally at week 38 with uncloned SIVmne. Following challenge all macaques became infected, but both vaccine regimens resulted in reduced peak virus loads (p 5 0.07) and significantly improved
maintenance of peripheral CD41 T cell counts postchallenge (p 5 0.007, DNA alone and p 5 0.01, all vaccinees). There was no significant difference between the two vaccine groups in levels of plasma viremia or maintenance of CD4 1 T cell counts postchallenge.
INTRODUCTION
V
(HIV) are
needed to stem the worldwide epidemic. Despite promising results from immunizing mice with naked plasmid DNA
vaccines, results in primate models have been disappointing,
eliciting fully protective immune responses only to low pathogenicity challenge viruses.1,2 More recently responses induced
ACCINES FOR HUMAN IMMUNODEFICIENCY VIRUS
by naked DNA have been improved by the inclusion of adjuvants or cytokines3,4 or by using different methods of delivery.5,6 A number of groups have demonstrated the relative efficacy of priming responses using DNA vaccines, followed by
boosting with live virus vectors.4,7–11 We were interested in
comparing DNA alone with DNA plus subunit protein boosts
in the SIVmne challenge model of Macaca fascicularis. Boosting with protein in an appropriate adjuvant could potentially al-
1Seattle
Biomedical Research Institute, Seattle, Washington 98109.
Primate Research Center, University of Washington, Seattle, Washington 98195.
of Medicine, University of Washington, Seattle, Washington 98195.
4Duke University Medical Center, Durham, North Carolina 27710.
5Department of Biostatistics, University of Washington, Seattle, Washington 98195.
6Department of Pathology, University of California, Davis, California 95616.
7Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21702.
8Department of Pathobiology, University of Washington, Seattle, Washington 98195.
9Current address: Corixa Corporation, Seattle, Washington 98104.
2Regional
3Department
425
426
MOSSMAN ET AL.
low expansion of a CD4 and CD8 response primed by DNA.
Unlike viral vectors, which are often compromised by the elicitation of antivector responses, protein has the potential for delivering multiple subsequent boosting immunizations to maintain responses over time.
Primate lentivirus models for vaccine testing can have a
broad range of virus loads and highly variable time to CD41 T
cell decline.12,13 In the SIV mne model, the virus generally replicates to high virus loads in infected animals and leads to AIDSlike disease and CD41 T cell decline in 50–60% of macaques
within 3 years postinfection. The biological clone of SIVmne,
SIVmneE11S, shows reduced virus replication and slower CD41
T cell depletion relative to the parental uncloned stock.14 Priming with recombinant vaccinia viruses combined with subunit
protein boosts has shown some success in providing sterilizing
immunity against both intravenous and intrarectal challenge.15–17 These studies have indicated that complex multiple
antigen vaccines are more effective than Envelope-only vaccines.14 Protection against intravenous challenge was observed
in fewer animals than intrarectal challenge, leading to the conclusion that the mucosal barrier provides some protection, even
with the very high doses of virus used to ensure full infection
of all controls. The DNA vaccine described here was composed
of three plasmid constructs together encoding all of the structural and regulatory genes of SIVmneE11S , without the LTRs.18
Subunit boosts consisted of Gag-Pol particles mixed with
Envgp160, coformulated in MF-59 adjuvant. These antigens
were produced using recombinant vaccinia virus infection of
Vero cells. The choice of subunits in this experiment was governed by the desire to make direct comparisons with prior vaccinia virus priming and subunit boosting in this model. We
elected to challenge the macaques with SIVmne uncloned virus
stock by the intrarectal route, using the same viral stock used
in prior experimental work with vaccinia prime-boost. We present evidence that both of the vaccine strategies elicited protection from SIV mne -induced CD41 T cell decline.
MATERIALS AND METHODS
Expression plasmids
The structural and regulatory genes of SIV mneE11S , clone 8,19
were cloned into mammalian expression vectors under the control of the cytomegalovirus Immediate Early-1 promoter, as previously described.18 Three constructs were used in these experiments, pCI-env, pJW-gag-pol-env, and pcDNA3-nef.
Plasmids were prepared on a large scale using Qiagen EndoFree purification kits. The DNA vaccine for macaques comprised a pool of the three vectors in equal W:W ratios in endotoxin-free saline.
Animals and immunizations
Twelve juvenile cynomolgous macaques (Macaca fascicularis), 1 to 2 years of age and weighing 2–4 kg, were obtained
from and housed at The Washington Regional Primate Research
Center. All animals were negative for SIV and simian type D
retrovirus at the study onset. The DNA vaccine was administered at a dose of 3 mg DNA per animal in a volume of 1.5 ml.
This dose was divided in two and given intramuscularly into
the triceps and intradermally in the shoulder area. Recombinant
proteins were produced using vaccinia constructs as previously
described.15 Animals received a dose of 250 mg recombinant
gp160 (rgp160) plus 250 mg Gag-Pol particles.14 These were
coformulated in MF-59 adjuvant (Chiron Corporation) to a final volume of 1 ml per animal. The subunit boost was administered by the intramuscular route.
Challenge virus
SIVmne uncloned virus stock was grown in macaque PBMC
and titered in vivo in M. fascicularis. Macaques were challenged
with 20 macaque infectious doses (MID) by the intrarectal route.
Plasma virus load determination
Viral RNA in plasma was quantified using a quantitative
competitive-reverse transcriptase PCR (QC-RT-PCR) assay as
previously described.20 The limit of detection for this assay was
3000 copies/ml plasma.
Virus isolation
Cell-associated virus was detected postchallenge by coculture of peripheral blood mononuclear cells (PBMC) or lymph
node (LN) cells from macaques with CEMx174 cells. Virus in
supernatants from these cultures was detected by antigen capture ELISA. Cultures were maintained for 6 weeks before being discarded as negative.
Nested LTR-gag PCR assay
Nested set primers from the LTR/gag region of SIVmne were
used to amplify proviral DNA from 2 mg total DNA extracted
from PBMC or LN cells of macaques postchallenge. The first
round of PCR utilized a 1 mM concentration of the following
primers: 59-CCAGTGTGTGCTCCCATCTCTCCTAGTCGC
and 59-GTACTTTTTCTTCCCGCCGGGTCGTAGCC. The
reaction was carried out for 35 cycles, with an annealing temperature of 55°C. Of the first round reaction product 5 ml was
then amplified using a 1 mM concentration of an internal set of
primers: 59-CCTGACAAGACGGAGTTTCTCGCGCC and
59-CGATAATAAGAAGACCCTGGTCTG. This reaction was
performed for 35 cycles with an annealing temperature of 50°C.
The appropriately sized product was detected by agarose gel
electrophoresis and ethidium bromide staining.
Heteroduplex mobility assay
PCR products were tested for heteroduplexes using standard
procedures, essentially as described by Delwart et al.21 The SIV
clone 8 molecular clone env gene was used as a reference sequence by which to identify sequences identical to or divergent
from this clone in the V1–V2 regions.
Antibody assays
ELISA assays for measuring antibody titers to Envelope were
performed using plates coated with rgp160 at 2 mg/ml. Bound
antibodies in titrated sera were detected using goat antimonkey
IgG conjugated to horseradish peroxidase (Cappel). Western
blot analysis against vaccinia lysates was used to confirm that
antibody reactivity detected in this ELISA was gp160 specific
PROTECTION FROM SIV WITH MULTIGENIC DNA VACCINES
and not due to contaminating vaccinia proteins, which may have
been present in the subunit immunogens and in the protein used
to coat plates. Virus neutralizing antibodies against SIVmne uncloned stock were detected in CEMx174 cells as previously described.22
T cell proliferation assay
Ficoll-separated, cryopreserved PBMC were incubated in the
presence of 5 mg/ml rgp160 or 10 mg/ml phytohemagglutinin
in medium containing 10% heat-inactivated AB-positive human
serum for 3 days at 37°C. Cells were then pulsed overnight with
0.5 mCi of aqueous [methyl-3H]thymidine. Cells were harvested
the next day and counts incorporated measured on a scintillation counter. Proliferation was then expressed as the mean
counts per minute of triplicate wells in antigen minus counts in
medium alone (net CPM) and as a stimulation index.
Cytolytic T cell assay
Bulk cultures of PBMC from immunized and challenged macaques were stimulated twice in vitro with autologous adherent
PBMC infected with recombinant vaccinia virus expressing
Gag-Pol, Env, Nef, or LacZ, as an irrelevant control. T cells
were then assayed for lytic activity against autologous B-LCL
expressing SIV proteins using standard chromium release assay.23 For an assay to be significant, the specific target must be
killed at least 10% and it must exceed the killing of control targets by the mean plus 3 standard deviations of three replicas
for at least two different E:T ratios.
Statistical analyses
SPSS version 10.0 (SPSS, Inc., Chicago, IL) and S-Plus 2000
(Insightful, Inc., Seattle, WA) were used for all analyses. In all
cases, values below the limit of detection of an assay were transformed to be equal to the midpoint between zero and the lower
limit of detection of that assay. Differences between the experimental and control groups in median peak plasma viral load
at 2 weeks postchallenge and for levels of neutralizing antibodies postchallenge were tested using the Mann–Whitney U
test. Median area under the curve minus baseline values for
acute levels of plasma viral RNA between weeks 0 and 6
postchallenge were calculated for vaccinees and controls as previously described.24 Values were compared for statistically sig-
FIG. 1.
427
nificant differences using the Mann–Whitney U test. CD41 T
cell counts were defined as ,1000 only if the CD41 T cell
count at the subsequent time point was also ,1000. Differences
between the experimental and control groups for time to CD41
T cell count ,1000 were assessed using Kaplan–Meier survival
analysis and the log-rank test.
Detection of virus-like particles in vitro
pJW-gag-pol-env was cotransfected with plasmid pSV2neo
expressing the neomycin resistance gene (gift of Paul Luciw,
UC Davis) in COS cells using standard calcium chloride procedures. After 3 days transfected cells were selected by culturing in 400 mg/ml geneticin. Clones were expanded and examined for Gag and Env production by antigen-capture ELISA and
by radioimmune precipitation assays. Positive clones were fixed
in 2.5% glutaraldehyde in 0.1 M phosphate buffer and examined by electron microscopy. ELISA data estimated the yield
of virus-like particles (VLPs) at 120 ng/ml culture supernatant.
RESULTS
Prior to inoculation into macaques, all DNA vaccine constructs used in these studies were selected for optimal expression in vitro and immunogenicity in mice, as detailed elsewhere.18 Of particular interest from these preliminary studies
was the finding that the construct expressing Gag-Pol-Env was
capable of producing VLPs in transfected cells in vitro. COS
cells permanently transfected by the DNA construct were
screened for coexpression of Gag and Env by ELISA and radioimmunoprecipitation (data not shown). The highest expressing cell line was shown to produce VLPs by electron microscopy (Fig. 1).
Two groups of four cynomolgous macaques (M. fascicularis)
were immunized twice with 3 mg plasmid DNA expressing
multiple viral genes at weeks 0 and 8. The DNA alone group
then received a further two identical immunizations at weeks
16 and 36. The second group was boosted twice with 250 mg
rgp160 and 250 mg Gag-Pol particles formulated in MF-59 adjuvant. The four control animals received DNA immunizations
of pCI-neo plasmid, either four times (95013 and 95027) or
twice followed by two inoculations with MF-59 adjuvant alone
(95042 and 95057).
Electron micrograph to show virus-like particles budding from COS cells stably transfected with pJW-gag-pol-env.
428
MOSSMAN ET AL.
Humoral immune responses to Env gp160 (Fig. 2) and to
Gag (data not shown) were elicited by both vaccine protocols
and were sustained over the course of the immunization protocol. As expected, antibody titers were higher in sera from macaques boosted with recombinant protein, where geometric
mean end point titers to Env gp160 were 1/30,000 at time of
challenge, compared to 1/700 in animals receiving DNA as
a sole immunogen. Neutralizing antibody titers against
SIVmneE11S were detected in only two animals prior to challenge, and only after the fourth immunization (Fig. 3). Both animals were in the DNA plus subunit group (95022 and 95024).
Proliferative responses to rgp160 (Fig. 4) and to whole inactivated virus (data not shown) were induced equally well by
both vaccine regimens. Weak responses were detected as early
as postsecond immunization in three of the vaccinees (,2000
net cpm). Following the fourth immunization, at time of challenge, the number of responders (8 of 8) and the magnitude of
FIG. 3. Results of in vitro homologous virus neutralizing assay on pre- and postchallenge serum from macaques. Data are
expressed as titer at which 50% virus is neutralized. Titers for
animals vaccinated with DNA alone are shown in (A) 95010
(diamonds), 95011 (squares), 95045 (triangles), and 95047 (circles). Titers for animals vaccinated with DNA plus subunit
boost are shown in (B) 95014 (diamonds), 95018 (squares),
95022 (triangles), and 95024 (circles). Titers for control animals are shown in (C) those vaccinated with plasmid vector
alone are 95013 (diamonds) and 95027 (squares), and with vector plus adjuvant boost are 95042 (triangles) and 95057 (circles). Arrow represents time of challenge, and the horizontal
dotted line indicates the limit of detection of the assay.
FIG. 2. Pre- and postchallenge serum antibody titers to Env
gp160 by ELISA. Animals vaccinated with DNA alone are
shown in (A) 95010 (diamonds), 95011 (squares) 95045 (triangles), and 95047 (circles). Animals vaccinated with DNA
plus subunit boost are shown in (B) 95014 (diamonds), 95018
(squares), 95022 (triangles), and 95024 (circles). Control animals are shown in (C) those vaccinated with plasmid vector
alone are 95013 (diamonds) and 95027 (squares), and with vector plus adjuvant boost are 95042 (triangles) and 95057 (circles). Arrows represent times of immunization, and the vertical line at week 38 indicates time of challenge.
the responses had increased markedly, displayed as net counts
incorporated and converted to stimulation indices (Fig. 4).
We were unable to detect bulk SIV-specific cytolytic activity after in vitro stimulation of PBMC from any of the vaccinated animals prior to challenge, although CTL were readily
detected using this protocol in infected macaques postchallenge,
noted below. However, cytolytic T cell clones specific for Env
and Gag-Pol were detectable in two vaccinees following two
DNA immunizations.23
At 38 weeks postinitiation, 2 weeks postfinal immunization,
all macaques were challenged intrarectally with 20 MID of
SIVmne uncloned virus. Outcome postchallenge was monitored
by assessing viral burden over time, determining viral RNA in
plasma and proviral DNA in PBMC and lymph nodes, and also
by evaluation of CD41 T cell counts in peripheral blood. All
PROTECTION FROM SIV WITH MULTIGENIC DNA VACCINES
429
caques 95010 and 95011, immunized with DNA alone, by both
assays. Consistent with the QC-RT-PCR data, virus was undetectable in animal 95010 after the acute phase of viremia (week
8 postchallenge, week 46 postinitiation). Indeed, we were unable to detect proviral DNA in the lymph node of 95010, suggesting that this macaque may have been only transiently infected. Of the DNA plus subunit immunized macaques, provirus
was isolated from the PBMC only sporadically in 95022 relative to the other animals of the group, suggesting control of
virus load consistent with the quantitative plasma viral RNA
data. Although control animal 95013 had low levels of plasma
viremia, provirus was readily isolated from PBMC at most time
points tested.
To determine which species of virus was transmitted, we performed heteroduplex mobility analysis (HMA) of the V1–V2
FIG. 4. Lymphoproliferative responses to rgp160 in PBMC
from immunized macaques on day of challenge. Data are expressed as counts per minute of tritium incorporated, with background to medium alone subtracted (net CPM) and also as a
stimulation index.
animals became infected postchallenge, but protective immune
responses were elicited by both vaccine regimens.
Viral RNA in plasma was assessed by QC-RT PCR assay
over the course of 1 year postchallenge (Fig. 5). Three of four
macaques receiving DNA alone maintained low virus loads
(,5 3 104 copies/ml plasma) for the duration of the experiment. Two of these macaques, 95010 and 95011, had plasma
virus levels below the level of detection of the QC-RT-PCR assay at all time points, with the exception of a single time point
during the acute phase of viremia for animal 95010. One of the
subunit-boosted animals died at 8 months postchallenge due to
causes unrelated to SIV infection. Of the remaining three macaques, 95022 alone was able to control virus long term, with
plasma virus copy number being below the level of detection
for the assay at all but one time point following resolution of
the acute phase of infection. Macaque 95013 in the control
group was also able to control levels of plasma virus postchallenge, with the remaining three animals in this group showing
high acute peak virus loads and high viral set points out to 1
year postchallenge. There was a trend for a significant difference in peak viral load, measured at week 2 postchallenge, between DNA immunized animals compared to controls (p 5 0.1)
and between all vaccinees compared to controls (p 5 0.07). Furthermore, analysis of acute levels of viral RNA between weeks
0 and 6 postchallenge showed a similar trend (p 5 0.1 in each
case). There was no significant difference between the two vaccine groups in ability to control levels of plasma viremia postchallenge.
Detection of cell-associated proviral DNA by coculture assay and by PCR up to 20 weeks postchallenge (58 weeks postinitiation) is shown in Table 1. In general, detection of virus
in PBMC by culture in vitro was delayed in vaccinees relative
to controls. Provirus was only sporadically detectable in ma-
FIG. 5. Plasma virus load determinations postchallenge by
QC-RT-PCR assay. Number of viral copies per/ml plasma for
animals vaccinated with DNA alone are shown in (A) 95010
(diamonds), 95011 (squares), 95045 (triangles), and 95047 (circles). Results for animals vaccinated with DNA plus subunit
boost are shown in (B) 95014 (diamonds), 95018 (squares),
95022 (triangles), and 95024 (circles). Results for control animals are shown in (C) those vaccinated with plasmid vector
alone are 95013 (diamonds) and 95027 (squares), and with vector plus adjuvant boost are 95042 (triangles) and 95057 (circles). Arrow represents time of challenge, and the horizontal
dashed line indicates the limit of detection of the assay.
430
MOSSMAN ET AL.
TABLE 1. VIRAL COCULTURE
AND
PROVIRAL DNA
IN
PBMC
AND
LNMC POSTCHALLENGE a
Weeks postchallenge
Vaccine group
DNA alone
95010
95011
95045
95047
DNA plus Subunit
95014
95018
95022
95024
Controls
95013
95027
95042
95057
1
2
3
4
6
8
12
14 LN
16
20
2/2c
2/2
1/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/1
2/1
2/3
2/2
2/2
1/1
2/1
2/2
2/1
1/1
1/2
2/1
1/1
1/2
2/2
2/2
2/1
2/1
2/2
1/1
1/1
1/1
2/2
1/2
2/2
1/2
2/2
2/2
2/2
1/2
2/2
2/2
2/2
2/2
2/1
1/2
2/1
2/2
1/1
1/2
2/1
2/2
1/2
1/2
1/2
2/1
1/1
1/1
1/2
1/1
1/1
1/1
1/1
1/1
1/1
1/2
2/2
2/1
1/1
1/1
2/1
1/1
1/2
1/2
2/2
1/2
1/2
1/2
2/2
1/2
2/2
2/2
1/2
2/2
1/2
1/1
1/2
1/2
1/1
2/1
1/1
1/1
2/2
1/1
1/1
1/1
2/1
1/1
1/1
1/1
1/1
2/1
1/2
1/2
2/1
2/1
1/1
1/1
1/1
1/1
1/1
1/1
1/2
2/2
1/2
1/2
1/2
1/2
2/2
2/2
a Where
no result is indicated, assays were not performed due to lack of available sample.
PBMC were tested for all weeks except 14, where LNMC were tested. Results are shown as “1” or “2” at each time point
for provirus detection by viral coculture (left of slash)/nested LTR-gag PCR assay (right of slash).
env region of proviral DNA extracted from PBMC at 6 weeks
postchallenge. Amplified sequences from each animal were
mixed individually with the same region from molecular clone
8 of SIVmneE11S .19 The HMA of these mixtures showed no diversification, indicating that the predominant virus in the
SIVmne swarm (E11S) was transmitted to all of the animals,
vaccinees and controls (data not shown). This virus shares sequence homology with the Envelope, Gag-Pol, and Nef used
in the vaccine components, which were derived from the
SIVmneE11S clone 8.
CD41 T cell counts were monitored for 1 year postchallenge
as a further indication of the ability of macaques to control the
SIV challenge (Fig. 6). In the SIVmne infection and pathogenesis model in M. fascicularis, 50–60% of naive control macaques infected with either SIVmneE11S or uncloned SIVmne experienced relatively slow CD41 T cell decline and developed
AIDS-like diseases within 3 years after infection; however disease progression is more rapid with the uncloned virus.14 Within
the control group, only 95013 maintained normal CD41 T cell
counts by the termination of the study (94 weeks), but only
95027 declined to 500 cells/ml. We utilized 1000 cells/ml as a
cutoff in this study, based on published patterns of CD41 T cell
decline in the model, since this level is maintained in healthy
animals, and no recovery is seen once the CD4 cells drop below this level. Peripheral CD41 T cells were maintained above
1000 cells/ml blood in three of four macaques immunized with
DNA alone. The fourth animal in this group, 95045, dropped
below but close to this level after week 40 postchallenge (week
78 postinitiation). Of the three surviving DNA plus subunit
boost macaques, only 95022 had maintained CD41 T cell
counts above 1000 cells/ml blood by the end of the study at 1
year postchallenge. There was a statistically significant delay
in time to CD41 T cell decline to ,1000 cells/ml blood relative to controls for the DNA immunized macaques (p 5 0.007).
This statement is also true when all vaccinees were compared
with controls (p 5 0.01). As expected, the CD4 data show a
close inverse correlation with levels of plasma viremia. Consistent with plasma viremia data, there was no significant difference between the two vaccine groups in maintenance of CD4
counts following challenge.
Postchallenge cellular immunity was assessed in four vaccinees and two control macaques using chromium release cytolytic assays (Fig. 7). Bulk PBMC were tested without freezing and stimulated with recombinant vaccinia virus-expressed
SIV proteins. No CTL directed to Env or to Gag/Pol were detected in vaccinee 95018, which controlled viremia after the
acute phase (data not shown). When tested by stimulation with
Gag/Pol, Env, and Nef, PBMC from vaccinees with low or no
detectable viral replication postchallenge had borderline positive responses to Gag/Pol (95011) and Nef (95010) at 2 months
following challenge. DNA vaccinee 95045, with relatively poor
virus control, had strong Nef and weaker Gag/Pol responses at
5 months postchallenge. At 2 months postchallenge, bulk CTL
specific for Env (95027) and for Gag/Pol and Env (95057) were
seen at low levels in controls with poor postacute control of
viremia and ultimate disease progression (Fig. 7).
Anti-Envelope gp160 ELISA antibodies were boosted in
most macaques following exposure to challenge virus, and were
maintained at steady levels over the course of the study, out to
1 year postchallenge (Fig. 2). Interestingly, animal 95011 did
not exhibit an anamnestic boost in gp160 titers postchallenge,
further evidence of the low levels of viral replication in this animal, even at the acute phase, where viral DNA and RNA was
undetectable (Fig. 5 and Table 1). Neutralizing antibody titers
developed in most macaques by 16 weeks following challenge
(54 weeks postinitiation) (Fig. 3). In general, peak titers of neutralizing antibodies postchallenge were higher in vaccinees than
in control animals, suggesting priming of this response by the
PROTECTION FROM SIV WITH MULTIGENIC DNA VACCINES
FIG. 6. CD41 T cell counts in peripheral blood of vaccinated
macaques postchallenge. Number of CD41 cells per ml blood
for animals vaccinated with DNA alone are shown in (A) 95010
(diamonds), 95011 (squares), 95045 (triangles), and 95047 (circles). Results for animals vaccinated with DNA plus subunit
boost are shown in (B) 95014 (diamonds), 95018 (squares),
95022 (triangles), and 95024 (circles). Results for control animals are shown in (C) those vaccinated with plasmid vector
alone are 95013 (diamonds) and 95027 (squares), and with vector plus adjuvant boost are 95042 (triangles) and 95057 (circles). Arrow represents time of challenge.
vaccines. Indeed, there was a statistically significant trend for
higher neutralization titers in DNA plus subunit immunized animals compared to controls at 16 weeks postchallenge (week
54 postinitiation, p 5 0.1) and at 44 weeks postchallenge (week
82 postinitiation, p 5 0.07). In the DNA plus subunit group animal 95022 had the highest neutralizing titer prechallenge and
early postchallenge, and animals 95010 and 95047 had the highest postchallenge titers among the animals immunized with
DNA alone (Fig. 3). All three of these animals controlled viral
replication to some extent following challenge.
DISCUSSION
In this study we elected to challenge macaques by the mucosal (intrarectal) route with the uncloned stock of SIVmne. Heteroduplex mobility assay of proviral genomes from PMBC of
431
these animals postchallenge shows that the E11S variant alone
was detected in the blood. This variant, present in the complex
stock, appears to preferentially cross the mucosal barrier to establish infection, as has been reported previously.17 Sexual
transmission of a single viral species is also seen in infection
of humans with HIV-1.25,26 Thus, protection in this challenge
model may have additional relevance to the development of an
HIV vaccine that protects against mucosal transmission of the
virus. Failure to protect from infection by this variant, despite
its identity with the vaccine Envelope immunogen in all components (DNA and rgp160), suggests that improvements are
needed to elicit strong neutralizing antibodies at the site of infection.
Like others, we have demonstrated that naked DNA alone is
not sufficient to induce the levels of protection required by an
AIDS vaccine for induction of sterilizing immunity.27,28 However, the results presented here indicate that vectors engineered
to express multiple viral genes, to make VLPs in infected cells,
and additionally selected for optimal expression levels were
able to suppress viral replication and to significantly maintain
CD41 T cell counts above a threshold level following challenge
in three of four animals (p 5 0.007). Peak (week 2) viral loads
were lower in vaccinated macaques than in controls, trending
toward significance (p 5 0.07). Median peak viral RNA levels
were 3 logs lower in vaccinated animals than controls, but this
was not significantly different (p 5 0.1). The reduction in
viremia is less than that seen in SHIV89.6P challenge experiments reported recently.4 Whether this difference is due to the
immunogenicity of the specific vaccine constructs or to differences in control of viral replication between SHIV and SIV
models, or both, remains to be clarified. The SHIV89.6P model
has recently been the focus of questions due to its unusual
course of pathogenesis and CD41 T cell decline.29 In this study,
we had the choice of two challenge stocks of SIV that had been
titered in vivo and utilized extensively for vaccine studies: uncloned SIVmne and the biological clone SIV mneE11S . These models both have gradual rather than precipitous CD41 cell decline,
and depletion occurs more rapidly in some of the macaques infected with the uncloned virus than in those infected with
E11S.14 We chose to use the uncloned stock as a more robust
challenge that would provide us a greater opportunity to measure disease onset in a shorter period of time. A drop in CD41
T cells below 1000 in this species is uniformly followed by
gradual and irretrievable loss to very low levels with onset of
AIDS-like disease following. Because our study was less than
2 years in duration, we used a threshold of 1000 cells/ml below
which to predict disease progression in controls and in vaccinees. This level differs from that utilized in most published studies but was necessary due to the slow erosion of T cells seen
in the SIVmne disease course.
The mechanism of protective immunity induced by the DNA
vaccines is unclear from these studies. Immunization of macaques with DNA alone induced moderately high ELISA antibody titers to gp160. It is likely that the level of antibodies
achieved here is due to the production of VLPs in infected cells,
enhancing presentation of antigen to the immune system. Neutralizing antibodies were measured at higher titers and were
more sustained in the vaccinated groups compared with controls following challenge. Thus CD41 T cell help was effectively stimulated for the production of virus binding and neu-
432
MOSSMAN ET AL.
FIG. 7. Cytolytic T cell assays of bulk PBMC from SIV-infected macaques at various time points postchallenge. PBMC were
stimulated in vitro with recombinant vaccinia constructs expressing SIV antigens as noted in the individual graphs. These cultures were then tested for their ability to lyse autologous B-LCL target cells in an antigen-specific manner by chromium release
at three different effector to target (E:T) ratios. Specific targets were B-LCL infected with vaccinia viruses expressing GagPol,
Env, or Nef and uninfected B-LCL (Uninfected) and cells infected with vaccinia virus expressing b-galactosidase (Vacc-Lac)
were used as negative controls. PBMC from 95010, 95011, 95057, and 95027 were tested 9 weeks postchallenge; PBMC from
95045 were tested 5 months postchallenge.
tralizing antibodies. The presentation of the native Envelope
protein by expression of authentic Env gp160 in vivo after DNA
immunization likely facilitated this response. Although there
was not a universal correlation between the presence of neutralizing antibodies and control of plasma viremia in individual
macaques, it cannot be ruled out as a potential protective factor in certain animals. Those with the highest titers pre- and
early postchallenge controlled virus to some degree, and neu-
tralizing antibodies were higher in titer and sustained longer in
vaccinees relative to controls.
The production of particulate antigen by the gag-pol-envvector is likely to enhance the ability to induce a CD81 T cell response through phagocytosis by antigen-presentingcells and introduction of antigen into the cytosol.30 CTL responses in CD81
T cells were induced by the DNA, which would also utilize the
classic class I pathway in transfected cells, but these were of
PROTECTION FROM SIV WITH MULTIGENIC DNA VACCINES
insufficient frequency to be detectable in bulk cultures of
PBMC by chromium release assay prior to challenge. In macaques that were tested for the induction of CTL postchallenge,
bulk responses to Gag/Pol, Env, and Nef were readily detected.
Bulk CTL were more readily detected in those with detectable
postacute viremia. However, we did not obtain postchallenge
CTL data for all animals. More sensitive and quantitative methods for detection of antigen-specific CD81 T cells were not
used here due to technical and sample limitations, and thus the
role played by these effectors in vaccine induced protection is
not clear.
Surprisingly, boosting DNA-elicited responses with subunit
proteins in adjuvant did not further improve outcome postchallenge. Although humoral responses were increased relative to
DNA alone, antigen-specific T cell proliferation was equivalent between the two groups. The finding that DNA-subunit
prime-boost vaccines failed to induce sterilizing immunity after intrarectal challenge with SIVmne uncloned virus is in contrast to results in the same model with vaccinia virus priming
followed by subunit boosting.17 This observation underscores
the importance of elucidating differences between live virus and
DNA priming and of understanding the immunological correlates involved in protection.
We believe that the boosting immunogen could be improved
by using lower doses of protein for selection of T cells with
higher affinities,31,32 more potent adjuvants, and/or improved
methods for delivery of antigens to the MHC class I pathway
for presentation to CD81 T cells. For more optimal elicitation
of neutralizing antibodies, better presentation via the MHC class
II pathway may be needed, and this version of Envelope expressed in and extracted from vaccinia-virus-infected cells is
potentially compromised in key conformational determinants.
In addition, recent data suggest that the rational design of Envelope immunogens may be required for more effective induction of neutralizing antibody responses.3 Development in these
areas should improve the potential for using subunit proteins to
boost and maintain immune responses primed by gene-based
vaccines.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
ACKNOWLEDGMENTS
This work was supported by Public Health Service Grant AI26503 from the National Institute of Allergy and Infectious Diseases. The authors thank Chiron Corporation for the generous
provision of MF-59. We thank P.A. Luciw for a gift of pSV2neo
and for support of electron microscopy work; N. Doria-Rose,
V. Hirsch, and L. Stamatatos for critical review of the manuscript; and J. Oberembt for proofreading and formatting assistance.
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Seattle Biomedical Research Institute
307 Westlake Ave. N, Suite 500
Seattle, Washington 98109-5219
E-mail: nancy.haigwood@sbri.org
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