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Journal of Virology, October 2001, p. 9665-9670, Vol. 75, No. 20
Division of Vaccine Research, Institute of
Human Virology, University of Maryland Biotechnology Institute,
Baltimore, Maryland 21201
Received 1 March 2001/Accepted 27 July 2001
A prototype Shigella human immunodeficiency virus type
1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were
also vaccinated with a vaccinia virus-env
(vaccinia-env) vector or the gp120 DNA vaccine alone.
Enumeration of the CD8+-T-cell responses to gp120 after
vaccination using a gamma interferon enzyme-linked spot assay revealed
that a single intranasal dose of the Shigella HIV-1 gp120
DNA vaccine vector elicited a CD8+ T-cell response to
gp120, the magnitude of which was comparable to the sizes of the
analogous responses to gp120 that developed in mice vaccinated
intraperitoneally with the vaccinia-env vector or
intramuscularly with the gp120 DNA vaccine. In addition, a single dose
of the Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge.
Moreover, the number of vaccinia-env PFU recovered in mice
vaccinated intranasally with the Shigella vector was about
fivefold less than the number recovered from mice vaccinated
intramuscularly with the gp120 DNA vaccine. Since the
Shigella vector did not express detectable levels of gp120,
this report confirms that Shigella vectors are capable of
delivering passenger DNA vaccines to host cells and inducing robust
CD8+ T-cell responses to antigens expressed by the DNA
vaccines. Furthermore, to our knowledge, this is the first
documentation of antiviral protective immunity following vaccination
with a live Shigella DNA vaccine vector.
It is widely agreed that human
immunodeficiency virus type 1 (HIV-1)-specific effector
CD8+ T cells play a substantive role in controlling HIV-1
replication in infected individuals and are a prognostic determinant of
HIV-1 infection outcome (3, 18, 22, 31). Although the
mechanisms underlying the initiation and maintenance of effector
CD8+ T-cell responses during HIV-1 infection are still
unclear, vaccination strategies that are proficient at priming effector
CD8+ T-cell responses against HIV-1 antigens have been
developed (5, 11, 15). In this vein, a growing number of
macaque studies have reported compelling evidence showing an
association between CD8+ T-cell responses to HIV-1 antigens
and antiviral protection against the progression of simian/human
immunodeficiency virus (SHIV) or HIV-1 infections in nonhuman primates
(5, 15, 19, 25).
The central tenet of our HIV-1 vaccine development strategy is that the
induction of high-level antiviral protection against sexually
transmitted HIV-1 will be achieved only if the priming immunogen is
targeted to mucosal lymphoid tissues (8, 33). Indeed,
there is convincing evidence that mucosal immunity against HIV-1 will
play a crucial role in protection against sexually acquired HIV-1
(6, 19). However, while it is possible to boost mucosal
responses with parenteral immunogens in humans, the induction of strong
mucosal immune responses requires that the priming immunogen be given
mucosally (12). Thus, the aforementioned HIV-1 vaccines,
which are administered parenterally, do not induce strong local
cell-mediated immune responses in the mucosal lymphoid compartment
(5, 11, 15, 25). On the other hand, Wu et al.
(33) and Valentine et al. (28) have
demonstrated that live oral bacterial vectors deliver HIV-1 immunogens
to mucosal lymphoid tissues and induce mucosal immune responses against
HIV-1 antigens. Unfortunately, these first-generation bacterial HIV vectors did not induce measurable HIV-specific CD8+ T-cell
responses or antiviral immunity in laboratory animals (9, 27,
33).
More recently, an alternative bacterial vector modality that utilizes
attenuated derivatives of Shigella flexneri to deliver DNA
vaccines was reported. In this capacity, attenuated Shigella DNA vaccine vectors deliver passenger DNA vaccines to rodent
(26) and human (24) cells and stimulate
cytotoxic T-cell responses against DNA vaccine-encoded antigens in mice
(7). These observations suggest that attenuated
Shigella strains may serve as vectors for the delivery of
HIV-1 DNA vaccines.
The purpose of the studies described in this report, therefore, was to
determine whether a prototype Shigella HIV-1 DNA vaccine vector elicits CD8+ T-cell responses to HIV-1. Since
shigellae are host adapted, we made use of an experimental murine model
in which attenuated Shigella vectors are inoculated
intranasally (29) throughout the studies in this report.
Although infection by Shigella in intranasally inoculated
mice does not directly involve the gastrointestinal tract, as is the
case in human shigellosis, this model is useful in determining the
relative immunopotency of attenuated Shigella vector
strains. We show that vaccination of mice with the Shigella gp120 DNA vaccine induces robust CD8+ T-cell responses to
gp120 and significant levels of antiviral protective immunity against a
vaccinia virus-env (vaccinia-env) challenge.
Bacterial strains, plasmids, and media.
Attenuated
Shigella flexneri 2a
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9665-9670.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Vaccination with a Shigella DNA Vaccine
Vector Induces Antigen-Specific CD8+ T Cells and Antiviral
Protective Immunity
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ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
aroA iscA
strain CVD1203 has been described elsewhere (21). CVD1203
is capable of invading epithelial cells but undergoes minimal
intracellular proliferation (due to the aroA mutation) and
exhibits defective cell-to-cell spread (due to the ascA
mutation). (21). In a phase 1 volunteer trial, CVD1203 was
found to be attenuated and well tolerated at a dose of 106
bacilli (17). Escherichia coli strain
Stable2R was purchased from Life Technologies
(Gaithersburg, Md.). Eukaryotic expression vector
pcDNA3.1ZEO was purchased from Invitrogen Inc. (Carlsbad,
Calif.). Plasmid pEF1
syngp120MN, which served as
the source of DNA encoding HIV-1MN gp120, is described
elsewhere (2) and was kindly provided by Brian Seed,
Department of Molecular Biology, Massachusetts General Hospital,
Harvard Medical School, Boston, Mass. All bacterial strains were grown
on tryptic soy agar (Difco, Detroit, Mich.) or in tryptic soy broth
(Difco). Shigella strains H1016 (carries
pcDNA3.1ZEO) and H1012 (carries pOGL1) were cultured in
solid and liquid media supplemented with 100 µg of ampicillin (Sigma,
St. Louis, Mo.) per ml.
DNA vaccine construction.
gp120-encoding DNA was obtained by
PCR amplification of the synthetic HIV-1MN gp120 gene
(syngp120) in plasmid pEF1syngp120 (2) using forward
(5'-GGGGGGGGATCCATGCCCATGGGGTCTCTGCAACCGCTG) and reverse
(5'-GGGGGCGGCCGCTTATTAGGCGCGCTTCTCGCGCTGCACCACGCG) primers
specific for the 5' and 3' ends of syngp120, and standard PCR
procedures (4). The resultant PCR-generated DNA fragment was digested with restriction endonucleases BamHI and
NotI and annealed by ligation with T4 DNA ligase (New
England Biolabs Inc., Beverly, Mass.) to BamHI- and
NotI-digested pcDNA3.1ZEO DNA. Following ligation, the chimeric plasmid DNA was introduced into
transformation-competent E. coli strain Stable2R
and cultured overnight. Plasmid DNA was prepared from 2 ml of liquid
cultures of individual colonies and screened for the presence of
recombinant plasmids with the appropriate restriction endonuclease digestion pattern. One isolate, designated H1058, containing the modified pcDNA3.1ZEO with the
BamHI-NotI syngp120 fragment (designated pOGL1),
was stored at 
80°C. Additional characterization of the cloned
syngp120 DNA in pOGL1, including various restriction endonuclease digestions and dideoxynucleotide sequencing, was conducted to verify
that no significant alterations occurred during the construction of pOGL1.
Animal housing and handling. Specific-pathogen free, 18- to 20-g female BALB/cAnNCrlBR mice, purchased from the Charles River Laboratories (Wilmington, Mass.), were maintained in a specific-pathogen free, microisolator environment, and allowed to drink and eat ad lib. All murine studies were conducted in accordance with Institutional Animal Care and Use Committee-approved protocol no. 004-97 and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (20a).
Vaccination procedures. Shigella vector strains were cultured in 20 ml of tryptic soy broth at 37°C until the optical density at 600 nm reached 1.0 relative to sterile tryptic soy broth control. The bacterial suspensions were centrifuged at 5,000 × g for 15 min, and the bacterial pellets were washed twice in 20 ml of phosphate-buffered saline (PBS). Finally, the inocula were suspended in PBS at a density of 106 CFU per ml. These suspensions were used immediately to vaccinate groups of six mice intranasally as described previously (29).
In parallel, groups of six mice were vaccinated intraperitoneally with 107, 3 × 107, or 108 PFU of vaccinia-env vector vP1174, or 108 PFU of vaccinia-lacZ vector vSC8. The vaccinia vectors vP1174 and vSC8 were obtained from AIDS Research & Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, Md.). Inocula of vP1174 and vSC8 were prepared by propagating the vaccinia virus constructs in HeLa cells, which were cultured in CM (see above) at 37°C in 5% CO2 (23). Two additional groups of six mice were vaccinated intramuscularly with 20 µg of endotoxin-free (<0.5 endotoxin units [EU] per mg of DNA) pcDNA3.1ZEO or pOGL1 DNA suspended in normal saline (0.85% [wt/vol] NaCl). The gp120 DNA vaccine, pOGL1, and control DNA vaccine pcDNA3.1ZEO were formulated for intramuscular injection as described previously (32).Enumeration of IFN-
-secreting cells.
Single-cell
suspensions of splenocytes were prepared before and 3, 5, 9, 17, and 28 days after vaccination. Threefold dilutions of the splenocytes were
suspended in CM (see above) containing 10 IU of recombinant mouse
interleukin-2 (R&D Systems, Minneapolis, Minn.) either with or
without the immunodominant peptide of gp120, P18MN
(RIHIGPGRAFYTTKN) (10 µg/ml). The cell suspensions were used immediately (i.e., without in vitro expansion) in an
IFN--specific enzyme-linked immune spot (IFN--ELISPOT) assay to
enumerate the gp120-specific IFN--ELISPOTs by the method of
Versteegen et al. (30) as modified by Miyahira et al.
(20).
-ELISPOT assays.
Vaccinia-env challenge. The level of antiviral protection induced by each DNA vaccine modality was determined using a vaccinia-env challenge model as described previously (6). Briefly, inocula of vP1174 were prepared by culturing the recombinant vaccinia virus on BSC-1 cells until 90% of the cells were lysed. The lysed cells were removed from the culture supernatants by centrifugation at 4,000 × g for 10 min, and aliquots of the supernatants were stored in liquid nitrogen until used. The culture supernatants typically yielded about 5 × 109 vP1174 PFU/ml, as determined by a direct plaque assay on BSC-1 cells. Mice were inoculated with 108 PFU of vP1174 via intraperitoneal injection 28 days after vaccination. Six days after the challenge, the ovaries of the mice were harvested and homogenized with a mechanical tissue grinder. The homogenates were clarified by centrifugation at 4,000 × g for 10 min, and the number of vP1174 PFU in the resultant supernatants was enumerated by infecting BSC-1 cell monolayers with 10-fold serial dilutions of these fluids and counting plaques after 2 days in culture at 37°C in a 5% CO2 environment.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Induction of Env-specific CD8+ T cells.
The
antigen-specific IFN--ELISPOT assay is a rapid, reproducible, and
sensitive method for monitoring the immunogenicity of vaccine
modalities for CD8+ T-cell response (20, 30).
Therefore, this assay was utilized to assess the magnitude and kinetics
of the gp120-specific IFN--CD8+ T-cell responses that
transpired after vaccination of mice with prototype Shigella
gp120 DNA vaccine vector strain H1012.
-ELISPOTs were enumerated without in vitro
expansion (Fig. 1). Only a low number of
nonspecific IFN--ELISPOTs were detected 3 days after vaccination;
however, significant numbers of Env-specific IFN--ELISPOTs were
detected 5 days after vaccination with strain H1012 (Fig. 1).
|
) or
control strain H1016 (
). Env-specific IFN--ELISPOT response was due to the
presence of CD8+ T cells, whereas depletion of
CD4+ T cells only marginally altered the number of
gp120-specific IFN--ELISPOTs (Fig. 1, insert).
Immunoblot analysis showed that the Shigella gp120 DNA
vaccine vector did not express detectable levels of gp120 (i.e., <10 pg per 106 vector bacilli; data not shown). Thus, the
gp120-specific CD8+ T-cell responses that developed after
vaccination with the Shigella vector were most likely due to
expression of the gp120 DNA vaccine by host cells. Together, these data
confirm that Shigella vectors deliver passenger DNA vaccines
to host cells (7), resulting in the stimulation
CD8+ T-cell responses to the antigens expressed by host
cells harboring vector-delivered DNA vaccines.
Comparison of vaccine modalities.
In light of the above
results, we conducted a side-by-side comparison of strain H1012 with
gp120 DNA vaccine pOGL1 and vaccinia-env vector vP1174.
Parallel groups of mice were vaccinated as follows: intranasally with
104 CFU of strain H1012; intraperitoneally with
107, 3 × 107, or 108 PFU of
vaccinia-env vector vP1174; or intramuscularly with 20 µg
of endotoxin-free (<0.5 EU/mg) pOGL1 DNA. Negative-control mice were
vaccinated with Shigella strain H1016, control
vaccinia-lacZ vector vSC8, or pcDNA3.1ZEO DNA.
Nine days after vaccination, the gp120-specific IFN--ELISPOTs were
enumerated, revealing that the magnitude of the Env-specific
IFN--ELISPOT response elicited by H1012 was comparable to the
analogous responses elicited by vaccinia-env vector strain
vP1174 or gp120 DNA vaccine pOGL1 (Fig. 2). We concluded that these vaccination
modalities display similar capacities to prime gp120-specific
IFN--ELISPOT responses.
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Measurement of antiviral protection.
Recently, Belyakov et al.
(6) reported a vaccinia-env challenge model
that measures HIV-specific effector CD8+ T-cell responses
in mice. Therefore, we utilized this model to qualitatively assess the
gp120-specific CD8+ T-cell responses that developed in mice
vaccinated with the prototype Shigella gp120 DNA vaccine
vector. Thus, groups of BALB/c mice were vaccinated intranasally with a
single dose containing 104 CFU of strain H1012 or H1016.
For comparative purposes, two additional groups of mice were vaccinated
intramuscularly with 20 µg of pOGL1 or pcDNA3.1ZEO DNA.
Twenty-eight days after primary vaccination, the mice were challenged
with 108 CFU of vaccinia-env vector vP1174. Six
days after inoculation with the challenge virus, the numbers of vP1174
PFU in the vaccinated and control mice were enumerated. The results
showed that intranasal vaccination with the Shigella gp120
DNA vaccine vector afforded a significant level of protection against
the challenge virus (Fig. 3). Notably,
the number of vaccinia-env PFU recovered in mice vaccinated
intranasally with the Shigella vector was about fivefold
less than the number in mice vaccinated intramuscularly with the gp120
DNA vaccine alone (Fig. 3).
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Comparison of prime-boost vaccination protocols. The above findings demonstrated that Shigella HIV-1 DNA vaccine vectors have the potential to function as the priming component in a prime-boost HIV-1 vaccine, such as those currently being prepared for phase 1 volunteer trials (10, 15, 25). In this regard, strategies that incorporate a DNA vaccine as a priming vaccine and a viral vector as a boosting vaccine are believe to be highly effective at eliciting CD8+ T-cell responses to HIV-1 antigens (10, 15, 25) and in two instances have been shown to afford antiviral protection against HIV-1 (15) and SHIV (25) in nonhuman primates. For this reason, we compared the priming capacity of the Shigella DNA vaccine vector to that of the DNA vaccine alone in a prime-boost vaccination protocol that used a vaccinia-env vector as the boosting vaccine.
Accordingly, mice were vaccinated once intranasally with 104 CFU of strain H1016 or intramuscularly with of 20 µg of pOGL1 DNA. Control mice were vaccinated once intranasally with 104 CFU of strain H1012 or intramuscularly with 20 µg of pcDNA3.1ZEO DNA. Twenty-eight days after primary vaccination, the mice were given an intraperitoneal booster vaccination of 108 PFU of vP1174, and nine days after the boost, Env-specific IFN--ELISPOTs were enumerated (Fig.
4). Since mice vaccinated with H1016 or pcDNA3.1ZEO were not primed with gp120, the Env-specific
IFN--ELISPOTs that arose in these mice were solely due to the
vaccinia-env booster vaccine. Using this number as the
denominator, both DNA vaccine modalities were effective priming
vaccines, as the number of gp120-specific IFN--ELISPOTs in mice
primed with either H1012 or pOGL1 was significantly elevated than the
number in mice primed with the respective mock controls (Fig. 4).
Furthermore, the magnitude of the Env-specific IFN--ELISPOT response
in mice primed with Shigella gp120 DNA vaccine vector was
comparable to the analogous response in mice vaccinated with the gp120
DNA vaccine (Fig. 4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results in this report confirm that Shigella vectors are capable of delivering passenger DNA vaccines to host cells (24, 26) and inducing robust CD8+ T-cell responses to antigens expressed by the DNA vaccines (7). We extended these earlier findings by showing that vaccination of mice with a single dose of a prototype Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge. To our knowledge, this is the first documentation of antigen-specific antiviral protective immunity following vaccination with a live Shigella DNA vaccine vector. This finding has important ramifications, in light of mounting evidence indicating that CD8+ T-cell responses are capable of affording antiviral protection against HIV-1, as highlighted in recent studies that used parentally administered HIV-1 DNA vaccines to prime CD8+ T cells to HIV-1 antigens (5, 15, 25).
However, we believe that the live oral Shigella HIV-1 DNA vaccine vector system presents advantages over parentally administered HIV-1 DNA vaccines. In support of this notion, there is an emerging consensus that mucosal immunity to HIV-1 provides an important barrier against sexually acquired HIV-1 (6, 19). Moreover, attenuated oral Shigella strains retain the ability to enter colonic mucosal lymphoid tissues and induce both mucosal and systemic immune responses (13, 14, 16). Unfortunately, due to the restricted host specificity of Shigella, we could not directly assess the capacity of our prototype Shigella HIV-1 DNA vaccine vector to induce mucosal responses following oral administration. This question can be addressed only in nonhuman primates or in volunteers.
Nonetheless, the results presented in this report demonstrate that this vector has the capacity to function as the priming component of a prime-boost HIV-1 vaccine. We showed that the primary CD8+ T-cell response to gp120 following vaccination with the Shigella gp120 DNA vaccine vector was comparable to the magnitude of this response after vaccination with two commonly used vaccine modalities, a vaccinia-env vector and a gp120 DNA vaccine. In addition, the magnitudes of the secondary CD8+ T-cell responses to gp120 following a booster vaccination with the vaccinia-env vector were similar in mice primed with the Shigella gp120 DNA vaccine vector to those primed with the analogous gp120 DNA vaccine.
In summary, therefore, the data in this report indicate that Shigella vectors provide an effective and inexpensive method to deliver HIV-1 DNA vaccines to inductive lymphoid tissues. Given that the potency of the Shigella gp120 DNA vaccine vector was comparable to that of an analogous gp120 DNA vaccine, we believe this vector system holds promise for use as a public health tool in developed and developing countries.
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ACKNOWLEDGMENTS |
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We thank Robert Powel, who laid the essential groundwork to these studies, and Michael Boysun and Christine Obriecht for providing technical support throughout these studies. We also thank George Lewis for his guidance and encouragement throughout the studies in this report.
This work was supported in part by NIAID grants AI41914, AI42603, and AI43756.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Vaccine Research, Institute of Human Virology, 725 Lombard St., Baltimore, MD 21201. Phone: (410) 706-4685. Fax: (410) 706-4694. E-mail: hone{at}umbi.umd.edu.
Present address: Virology Laboratory, Lindsley F. Kimball Research
Institute, New York Blood Center, New York, NY 10021.
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Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, October 2001, p. 9665-9670, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9665-9670.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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