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Journal of Virology, May 1999, p. 4443-4446, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Immunization with a Live, Attenuated Simian Immunodeficiency
Virus Vaccine Leads to Restriction of Viral Diversity in Rhesus
Macaques Not Protected from Pathogenic Challenge
Donald L.
Sodora,1
Kristine E.
Sheridan,2
Preston A.
Marx,2,3 and
Ruth I.
Connor2,*
University of Texas Southwestern Medical
Center, Dallas, Texas 752351; Aaron
Diamond AIDS Research Center, The Rockefeller University, New York,
New York 100162; and The Tulane
Regional Primate Center, Covington, Louisiana
704333
Received 28 October 1998/Accepted 25 January 1999
 |
ABSTRACT |
Rhesus macaques immunized with simian immunodeficiency virus
SIVmac239
nef but not protected from SIVmac251 challenge were studied
to determine the genetic and biological characteristics of the
breakthrough viruses. Assessment of SIV genetic diversity (env V1-V2) revealed a reduction in the number of viral
species in the immunized, unprotected macaques, compared to the number in nonimmunized controls. However, no evidence for selection of a
specific V1-V2 genotype was observed, and biologically cloned isolates
from the animals with breakthrough virus were similar with respect to
replication kinetics and coreceptor use in vitro.
| |
TEXT |
The ability of live, attenuated
strains of simian immunodeficiency virus (SIV) to provide potent
protection in macaques against a pathogenic SIV challenge has been
documented by many groups (2, 3, 5, 6, 8, 14, 16, 18, 19, 23,
25-27). However, little is known about the genotype and
phenotype of viruses that emerge in immunized macaques that fail to be
protected, so-called "breakthrough viruses." To gain
insight into the mechanisms of protection, we studied cases where
immunization with SIVmac239nef failed to protect against SIVmac251
challenge. Phenotypic analyses were also undertaken to determine
patterns of coreceptor use and growth kinetics by using biologically
cloned isolates obtained from the unprotected animals.
PCR analysis of nef sequences in immunized, unprotected
animals.
Sixteen female rhesus macaques were immunized with a
live, attenuated SIV vaccine (SIVmac239nef) and challenged by the
intravenous route with a pathogenic, uncloned stock of SIVmac251 either
5, 10, 15, or 25 weeks after immunization, as described earlier
(6). To distinguish between the immunizing strain and the
challenge virus, DNA PCR was performed by using specific primers that
flank the deleted region of nef (Fig.
1) (6). By using one set of primers (Fig. 1A), sequences from both the wild-type (654-bp fragment) and nef (472-bp fragment) viruses were amplified. A second primer set (Fig. 1A) was used to increase detection of the wild-type nef product. One primer in this set hybridizes within the
deleted region of SIVmac239nef, resulting in amplification of only
wild-type nef sequences.
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FIG. 1.
DNA PCR of the nef gene. (A) SIVmac239nef
or SIVmac251 nef sequences were detected in PBMC samples
obtained at 100 days postchallenge by using two sets of primer pairs (1 and 2). (B) Products amplified by primer set 1 showing both the
nef fragment and the wild-type (wt) nef
fragment. (C) Products amplified by primer set 2 showing only wild-type
nef. Individual macaques are identified by the animal number
and a letter representing the challenge group: A, 5-week challenge; B,
10-week challenge; D, 25-week challenge. 1480-A and 1492-B represent
nonimmunized control animals in the 5- and 10-week challenge groups,
respectively.
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Of 16 immunized macaques, 7 were not protected from
challenge based on evidence of an increase in plasma viremia, a
decrease
in CD4
+ T cells, and the detection of SIVmac251 by
PCR (
6). To further
characterize viruses present in the
unprotected animals, DNA was
extracted from peripheral blood
mononuclear cell (PBMC) samples
obtained 100 days after challenge and
analyzed for the presence
of either SIVmac239
nef or SIVmac251
nef sequences. The results
of these experiments demonstrate
wild-type
nef in four of four
macaques challenged at 5 weeks
(macaques 1472, 1474, 1476, and
1478 [Fig.
1C]), two of four
challenged at 10 weeks (macaques
1482 and 1484 [Fig.
1C]), and
one of four challenged at 25 weeks
(macaque 1510 [Fig.
1C])
(
6). In addition, a smaller, 472-bp
PCR product
was observed in six of seven macaques with breakthrough
virus (macaques 1472, 1474, 1476, 1478, 1484, and 1510 [Fig.
1B]),
indicating the persistence of SIVmac239
nef. As expected, PCR
analysis of the 5- and 10-week control animals (macaques 1480
and 1492 [Fig.
1B and C, respectively]) revealed only wild-type
nef, consistent with SIVmac251
infection.
Genotypic analysis of the env V1-V2 region.
The
V1-V2 region of SIV env was used to assess the genetic
diversity of the breakthrough viruses. Preparation of viral RNA and DNA
for genotypic analysis, as well as nested PCR amplification of the
V1-V2 region, was performed as previously described (22). Three independently derived PCR products were pooled for analysis of
viral quasispecies, thereby increasing the number of viral species
within the PCR and reducing the possibility of sampling errors. Viral
load in plasma at the time of heteroduplex mobility assay (HMA)
sampling ranged from 1.5 × 104 to 5.4 × 105 RNA copies/ml on day 28 in the unprotected animals and
from 8.6 × 104 to 1.6 × 106 RNA
copies/ml on day 170, when lymph nodes were biopsied. In all cases, day
28 represented the earliest time at which SIVmac251 was detected in
PBMC after challenge and plasma SIV RNA increased.
The amplified products were analyzed by a modified version of the HMA
(
9-11,
22). A single-stranded radiolabeled probe derived
from the V1-V2 region of SIVmac239 was annealed to pooled,
nonradiolabeled
V1-V2 PCR product from each of the breakthrough
viruses, from
the SIVmac239
nef stock used for immunization, and from
the SIVmac251
challenge stock (Fig.
2).
When the radiolabeled V1-V2 probe from
SIVmac239 was hybridized
with nonradiolabeled PCR product from
SIVmac239, a single band
was seen on polyacrylamide gels, representing
a DNA homoduplex (Fig.
2A, lane 1). Similarly, a homoduplex was
observed when the radiolabeled
probe was annealed with V1-V2 PCR
product from SIVmac239
nef (Fig.
2A, lane 2), verifying that the
virus used for immunization contained
the same V1-V2 region as
SIVmac239.
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FIG. 2.
Single-stranded HMA of the env V1-V2 region.
Genetic diversity in env V1-V2 was assessed by HMA with DNA
extracted from infected PBMC (PB) (28 days postchallenge depicted for
all; 100 days postchallenge depicted for macaques 1474, 1482, 1484, 1488, 1490, and 1510) or from lymph node (LN) (170 days postchallenge)
or with plasma viral RNA (PL) (28 days postchallenge). (A) Animals
1472, 1474, 1476, and 1478 (5-week challenge group), animal 1480 (nonimmunized control), and animal 1510 (25-week challenge group); (B)
macaques 1482, 1484, 1488, and 1490 (10-week challenge group) and
macaque 1492 (nonimmunized control). S.S., single-stranded probe; H.D.,
homoduplex.
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Genotypes that differ from the SIVmac239 probe form heteroduplexes that
migrate with reduced mobility in polyacrylamide gels.
Many
distinct genotypes were observed with SIVmac251, representing
viral variants present in the uncloned challenge stock (Fig.
2B,
lane
17). Similarly, control macaques infected with uncloned SIVmac251,
without prior immunization, had several replicating genotypes
(macaques
1480 and 1492 [Fig.
2]). Although samples from 1480
showed fewer
viral species replicating in PBMC and plasma than
samples from 1492, multiple viral genotypes were observed in the
lymph nodes (Fig.
2A),
suggesting the presence of several replicating
variants. This is
consistent with previous results in experiments
with three rhesus
macaques inoculated with uncloned SIV (
22).
In each case,
macaques infected by the intravenous route had a
heterogenous
quasispecies population in either lymph nodes, plasma,
or PBMC, similar
to the infecting virus stock (
22).
HMA analysis of the immunized animals included assessment of the V1-V2
genotypes found in plasma, PBMC, and lymph nodes (Fig.
2). The results
of these experiments demonstrate a single band
corresponding to the
V1-V2 region of SIVmac239 in two macaques
(1488 and 1490), indicating
persistent infection with SIVmac239
nef
(Fig.
2B). The absence of
additional bands strongly suggests that
these animals were protected
from infection with SIVmac251. These
results are supported by the
absence of wild-type
nef sequences,
as determined by nested
DNA PCR (Fig.
1C). In contrast, breakthrough
virus was detected in
samples obtained from six other macaques
(1472, 1476, 1478, 1482, 1484, and 1510). Each of these animals
was also positive for wild-type
nef by nested PCR (Fig.
1C). One
other animal with
breakthrough virus, 1474, was positive for wild-type
nef by
nested PCR (Fig.
1C), but HMA analysis of plasma and PBMC
revealed only
SIVmac239
nef V1-V2 (Fig.
2A). Interestingly, only
one or two
genotypes were detected by HMA in the immunized, unprotected
macaques,
in contrast to the greater number of variants seen in
nonimmunized
controls (Fig.
2). This suggests that prior infection
with
SIVmac239
nef can restrict the number of replicating genotypes
in
immunized animals in the absence of complete protection. However,
the
heteroduplex mobilities of the breakthrough viruses were different
for
each macaque, arguing against selection for a common V1-V2
genotype.
Direct sequencing of the V1 region from three of the
unprotected
macaques (1472, 1476, and 1478) revealed no common
V1 sequence (Fig.
3). Additional sequence data obtained
from V2
(data not shown) and from biologically cloned viruses from two
nonimmunized control animals (1480 and 1492) confirmed these
observations
(Fig.
3).
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FIG. 3.
Sequence analysis of env V1 from macaques
with breakthrough virus and control macaques. (A) Nucleotide sequences
derived from PCR amplification and direct sequencing of the V1 region
of env from immunized, unprotected macaques (1472, 1476, and
1478) and nonimmunized controls (animals 1480 and 1492); (B) predicted
V1 amino acid sequences aligned to that of SIVmac239.
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Biological cloning, coreceptor use, and replication of breakthrough
viruses.
Biologically cloned isolates from the unprotected
macaques were obtained by limiting-dilution PBMC cocultures, as
previously described (7). Virus isolates were collected from
the culture supernatants, while DNA was extracted from the infected
cell pellets. The V1-V2 region was amplified by DNA PCR and analyzed by
HMA. Individual clones were selected in which the viral genotype of the
biological clone matched that of the breakthrough virus from the same
animal when analyzed on HMA gels (data not shown). Biological clones
representing the breakthrough viruses were obtained from four
immunized, unprotected animals (1472, 1476, 1478, and 1510) and from
two nonimmunized control animals (1480 and 1492). The isolates were
evaluated to determine coreceptor use by inoculation into GHOST.4 cells
expressing human CD4 and either rhesus CCR5 (4), rhesus
CXCR4 (4), human GPR15 (BOB [12, 13, 15]), or human STRL33 (Bonzo [12]). GHOST.4 cells contain
the gene for green fluorescent protein (GFP) under the control of the
human immunodeficiency virus type 2 long terminal repeat (kindly
provided by V. N. KewalRamani, New York University Medical
Center), and infection was assessed by measuring the levels of GFP
expression on day 3 after inoculation. The results of these experiments
demonstrate that virus isolates from each of the immunized, unprotected
and the nonimmunized control macaques use CCR5 for infection (Table 1). In addition, each of the biological
clones used GPR15 and to a lesser extent STRL33, but none was able to
infect cells expressing CXCR4; this is consistent with what has been
reported for SIVmac239 (4, 12, 21) and SIVmac251 (4,
21). No differences in the pattern of coreceptor use among
isolates from different animals were observed. In further experiments,
each of the isolates was used to infect activated rhesus PBMC and virus
replication was assessed by measuring SIV p27 in culture supernatants
on days 3, 7, and 9 (Fig. 4). The results
show that the isolates replicated to similar levels with approximately
a twofold difference in the peak values between viruses isolated from
immunized, unprotected macaques and from nonimmunized controls
(366 ± 158 and 707 ± 221 ng/ml, respectively).
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FIG. 4.
Replication of biologically cloned viruses. Isolates
from macaques with breakthrough virus (1472 [ ], 1476 [ ], 1478 [ ], 1482 [ ], and 1510 [ ]) and control macaques (1480 [ ] and 1492 [ ]) were obtained by limiting-dilution coculture
(7) and used to infect activated rhesus PBMC in vitro. Virus
replication was determined by measuring the levels of SIV p27 antigen
on designated days after infection.
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Discussion.
Earlier studies examined the temporal development
of protective responses in 16 rhesus macaques immunized with
SIVmac239nef (6). The results indicated that protection
develops between 10 and 15 weeks after immunization and that this
protection occurs in the absence of antibodies capable of neutralizing
the primary challenge virus in vitro (6). Of 16 rhesus
macaques immunized with SIVmac239nef, 7 were not protected from
SIVmac251 challenge. All but one of these macaques were challenged
after the peak of viremia (5 to 10 weeks), when plasma virus was low
and seroconversion had occurred (6). In the present study,
we examined the genotype and phenotype of viruses replicating in the
unprotected macaques. Because these animals were challenged with an
uncloned stock of SIVmac251, we were able to assess genetic diversity
within the V1-V2 region of env using a modified HMA
(9-11, 22). This region has been shown to be one of the
more variable regions of the SIV genome (1, 17, 20, 24). The
number of viral genotypes, as determined by HMA analysis, indicated
that fewer viral species from the challenge stock were replicating
within the immunized, unprotected macaques than in the nonimmunized
controls, suggesting that some selection had occurred as a result of
immunization. To address this in part, we evaluated genetic diversity
in other regions of the SIV genome (env V3 to V5,
gag, nef) by HMA and found no diversity within
these additional sites (data not shown). Alternatively, it is possible
that differences in viral diversity may have resulted from different
kinetics of viral evolution following infection of immunized and
control animals. However, we observed a significant reduction in viral
diversity by day 28 (the earliest day tested after challenge) in the
immunized, unprotected macaques, and further sampling of PBMC (day 100)
and lymph nodes (day 170) did not reveal any significant changes in the
patterns of diversity. Therefore, we favor the hypothesis that a
restriction in viral diversity occurred early after challenge in
unprotected macaques and may reflect preexisting antiviral immunity
induced by immunization with the live, attenuated SIV vaccine. Based on
HMA analysis and sequencing data, there was no evidence for selection
of a specific V1-V2 viral genotype among the animals with breakthrough
virus, and we found no evidence to suggest that the viruses replicating in these animals were distinct with respect to coreceptor use or growth
kinetics in vitro. Therefore, no common genotypic or phenotypic
property could be attributed to the breakthrough viruses among the
parameters studied here. Further experiments will be needed to identify
factors that are involved in reducing the number of replicating
variants in the immunized, unprotected animals and allowing penetration
and replication of the breakthrough strains.
Nucleotide sequence accession numbers.
The sequences of
1472-A, 1476-A, 1478-A, 1480-Acon, and 1492-Bcon have been deposited in
GenBank under accession no. AF129452, AF129453, AF129454,
AF129455, and AF129456, respectively.
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ACKNOWLEDGMENTS |
We thank Agegnehu Gettie for providing blood samples and Eric
Delwart for helpful discussions.
This work was supported by the NIH (AI28147 and AI36598), the Aaron
Diamond Foundation, and an Aaron Diamond Foundation Fellowship awarded
to D.L.S.
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FOOTNOTES |
*
Corresponding author. Mailing address: Aaron Diamond
AIDS Research Center, 455 First Ave., 7th Floor, New York, NY 10016. Phone: (212) 448-5040. Fax: (212) 725-1126. E-mail:
rconnor{at}adarc.org.
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Journal of Virology, May 1999, p. 4443-4446, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Fultz, P. N., Vance, P. J., Endres, M. J., Tao, B., Dvorin, J. D., Davis, I. C., Lifson, J. D., Montefiori, D. C., Marsh, M., Malim, M. H., Hoxie, J. A.
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Messmer, D., Ignatius, R., Santisteban, C., Steinman, R. M., Pope, M.
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