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*
Immunobiology Vaccine Center and Departments of Oral Biology and Microbiology, University of Alabama, Birmingham, AL 35294;
Department of Microbiology, National Institute of Public Health, Minato, Tokyo, Japan;
California Regional Primate Research Center, University of California, Davis, CA 95615;
§
AIDS Research Center, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan; and
¶
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 (Th1-type)- as well
as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover,
p55-specific CTL activity was demonstrated in lymphocytes from blood,
tonsils, and other lymphoid tissues. These results show that nasal
immunization with SIV p55 with cholera toxin elicits both Th1- and
selective Th2-type cytokine responses associated with the induction of
SIV-specific mucosal and serum Abs, and CTL activity. These results
offer a promise for the development of protective mucosal immunity to
SIV. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For development of an HIV vaccine, the rhesus macaque model of SIV infection is now widely used, since macaques infected with SIV through the reproductive tract develop an AIDS-like disease (8). Several studies have shown that SIV-specific immunity correlates with protection from SIV challenge. Intramuscular followed by intratracheal or oral immunization of microencapsulated SIV protected from intravaginal challenge with SIV (9). Recently, others have shown that targeted lymph node immunization with a combined vaccine containing gp120 plus p27 induced Ag-specific immunity in the rectum and protected from rectal challenge with SIV (10).
Numerous reports have shown that cholera toxin (CT)3 can support the induction of Ag-specific IgG and IgA Abs in both mucosal and systemic compartments (11, 12, 13, 14, 15, 16). For SIV vaccine development, our previous study has shown that oral immunization with SIV p55 plus CT induced Ag-specific IgG and IgA Abs in both serum and mucosal secretions, i.e., saliva and rectal washes, but failed to induce Ag-specific IgA Abs in vaginal washes (13). In the murine system, others have shown that nasal immunization with HIV peptide plus CT induced Ag-specific IgA Ab in vaginal washes (14). Our separate study also showed that nasal immunization of mice with vaccine proteins and CT induced mucosal and systemic Ab responses (15).
The major purpose of this study was to examine whether nasal immunization of nonhuman primates with SIV p55 plus CT as a mucosal adjuvant could elicit p55-specific Ab responses in mucosal effector sites, including the genital tract. Our findings showed that Ag-specific B cell and Th1 and Th2 cell subsets and CTL effector responses were induced in both mucosal and systemic compartments of rhesus macaques by nasal immunization. These results offer promise for the development of a mucosal vaccine with a simple immunization procedure to prevent sexually transmitted HIV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SIV gag gene encoding SIVmac251 p55 (p55) was derived from clone BMT 95102 (Quality Biologic, Gaithersburg, MD) and was produced under Contract N01-AI-05084 to the Vaccine Research and Development Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID) (13). CT was purchased from List Biologic Laboratories (Campbell, CA). B-subunit of CT (CT-B) was purified from the Escherichia coli strains containing the plasmids for rCT-B, as described previously (17).
Macaques
Eight female rhesus macaques (Macaca mulatta), which were captive bred, mature, and reproductively cycling (4.2–6.6 kg), were obtained from the California Regional Primate Research Center. They were confirmed negative for Abs to HIV-2, SIV, type D retrovirus, and STLV-1, and kept in accordance with American Association of Accreditation of Laboratory Animal Care standards (13).
Immunization method and schedule
Macaques were divided into three groups and nasally immunized with mucosal Ags containing: 1) 100 µg p55 alone (19145), 2) 100 µg p55 plus 10 µg CT (20119, 23681, 24941, and 25507), or 3) 100 µg p55 plus 100 µg CT (20976, 22511, and 25690). Macaques were anesthetized with ketamine and placed in dorsal recumbancy with head tilted back so that the nares were pointed upward. Vaccine solution (0.5 ml) was instilled dropwise into each nostril without inserting the syringe into the nasal cavity. Macaques were kept in that position for 10 min and then placed in lateral recumbancy until they recovered from anesthesia. Nasal immunizations were conducted on days 0, 7, 21, 35, and 49.
Collection of blood and external secretion samples
PBMCs were separated from heparinized blood using Lympholyte-Mammal (Cedarlane, Hornby, Canada). Serum, vaginal washes consisting of a mixture of cervical and vaginal secretions, rectal washes, and saliva were collected, as described previously (13).
Collection of tissue samples and isolation of lymphocytes
Macaques were sacrificed under anesthesia, and tissues of nasopharynx (NP), small intestinal lamina propria (LP), uterine cervix (UC), submandibular gland (SMG), tonsils, mesenteric lymph nodes (MLN), and spleen (SP) were collected. For the isolation of lymphocytes from different mucosal tissues, a modified enzymatic dissociation procedure was employed (18). NP, UC, and SMG were dissociated using collagenase type IV (0.5 mg/ml; Sigma, St. Louis, MO) in RPMI 1640 (Cellgro Mediatech, Washington, DC) for 30 min at 37°C. After removal of Peyer’s patches, the small intestine was treated with PBS containing 1 mM DTT, followed by 1 mM EDTA, and LP mononuclear cells were isolated by the same method used for NP. The lymphocytes from tissues were purified using a discontinuous 40 and 75% Percoll gradient (Pharmacia, Uppsala, Sweden) (18).
Ag-specific ELISA and ELISPOT assay
p55- and CT-B-specific IgG and IgA Abs in sera and secretions
were examined by ELISA, as described previously (13). Endpoint titers
were expressed as the last dilution giving an OD450 of
0.1 U above samples obtained from unimmunized controls. p55- and
CT-B-specific IgG and IgA Ab-forming cells (AFCs) were determined by
ELISPOT assay, as described before (11, 12, 13).
Cytokine-specific ELISA
PBMCs or purified lymphocytes from tissues were cultured at a
density of 1 x 106 cells/ml with or without 5 µg/ml
of p55 to detect Ag-specific T cell-derived cytokine production.
Culture supernatants were collected 3 days after incubation, and the
levels of Th1 and Th2 cytokines (IFN-, IL-4, IL-5, and IL-10) were
determined by ELISA (13). The concentration of cytokines was calculated
by the standard curves obtained using recombinant human cytokines.
p55-specific cytokine production was evaluated as the cytokine
concentration in culture supernatant of p55-stimulated cells minus that
without Ag.
Quantitative RT-PCR for cytokine-specific mRNA
For evaluation of cytokine-specific mRNA levels of p55-specific
CD4+ T cells, a quantitative RT-PCR was conducted (11, 12, 13).
Briefly, after 3-day culture of PBMCs or lymphocytes from the mucosal
tissues with Ag, as described above, the
CD4+CD8- T cells were purified by
FACStarPlus (Becton Dickinson, San Jose, CA). Total RNA was
isolated and subjected to reverse-transcriptase reaction using
oligo(dT) primer and Superscript II reverse transcriptase (Life
Technologies, Gaithersburg, MD), as described previously (13). The cDNA
from 1 ng of RNA was used for each cytokine-specific PCR with primers
specific for monkey IFN-, IL-2, IL-4, human ß-actin, IL-5, IL-6,
and IL-10 (Oligos Etc., Wilsonville, OR) (13). The PCR product was
quantitated by capillary electrophoresis with the laser fluorescence
detection system (LIF-P/ACE; Beckman Instruments, Fullerton, CA), as
described before (12), and cytokine-specific mRNA expression was
calculated against mRNA levels of ß-actin, which was considered to be
100. The relative peak area was determined as cytokine-specific mRNA
expression of p55-stimulated CD4+CD8- T cells
minus that of unstimulated CD4+CD8- T cells.
Detection of SIV-specific CTL activity
The details of culture and detection of bulk, secondary CTL
responses have been previously reported (19, 20, 21). Briefly, PBMC or
lymphocytes from tonsil and other lymphoid tissues of nasally immunized
macaques were stimulated with 10 µg/ml of Con A (Sigma, St. Louis,
MO) or with SIV-infected autologous CD4+ T cells and
cultured for 14 days in complete medium supplemented with 5% human
lymphocyte-conditioned medium (Hu IL-2; Hemagen Diagnostics, Waltham,
MA) and 20 U/ml of human rIL-2 (donated by Cetus, Emeryville, CA).
Autologous B cells were transformed by Herpes papio
(595S x 1055 producer cell line, provided by M. Sharp, Southwest
Foundation for Biomedical Research, San Antonio, TX), and infected
overnight with wild-type vaccinia virus (vvWR), or recombinant vv
expressing the p55gag (vvgag) or
gp160env (vvenv) of SIVmac239
(provided by L. Giavedoni and T. Yilma, University of California,
Davis, CA), and then labeled with 50 µCi of 51chromium
(Na2CrO4; Amersham Holdings, Arlington Heights,
IL) per 106 cells. Effector and target cells were added
together at multiple E:T ratios in a 4-h chromium-release assay, and
percentage of specific lysis was calculated from supernatant chromium
measured in a liquid scintillation counter (Microbeta 1450; Wallac
Biosystems, Gaithersburg, MD). Specific lysis was considered positive
if it was greater than twofold (3 SDs) above the lysis of vvWR targets
and if it was at least 10%. For some animals, a limiting dilution
assay for virus-specific CTL precursors was performed. The assay was
based on a previously described method (21), with the following
modifications. Briefly, isolated CD8+ lymphocytes were
diluted 11 times over the range of 5000 cells/well to 25 cells/well and
cultured in replicates of 24 wells. The cells were stimulated with Con
A (10 µg/ml; Sigma) and supplemented with human irradiated PBMC as
feeder cells at a concentration of 4 x 105/well. The
cultures were maintained in AIM-V medium (Life Technologies),
supplemented with 20% FCS and 5% human IL-2 (Hemagen Diagnostics,
Waltham, MA). The level of cytolytic activity was measured on day 14,
at which time the individual wells were split three ways and incubated
5 h with an autologous target cell infected with vvWR, vvgag, or
vvenv, as described above. Positive wells were identified as wells that
exceeded the mean chromium release from wells without effector cells by
3 SDs. Wells containing cells that lysed uninfected autologous targets
were eliminated from the calculations. The precursor frequency was
determined by 
2 analysis based on maximum likelihood
(22) by a computer program provided by Dr. R. Miller (University of
Michigan, Ann Arbor, MI).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum p55-specific Ab responses in macaques were determined.
p55-specific IgA and IgG Abs were induced in macaques, with highest
responses seen after the third immunization (Fig. 1
). Although the levels of p55-specific
IgG were similar in macaques given p55 only or p55 plus 10 µg CT,
those of p55-specific IgA Abs were generally higher in macaques given
p55 plus 10 µg CT (Fig. 1 and Table I).
In contrast, only one of three macaques given p55 plus 100 µg CT
produced p55-specific serum Abs (Table I). Although all macaques given
p55 only or p55 plus 10 µg CT exhibited AFC responses, the numbers of
the p55-specific IgA AFCs in PBMC were generally higher in macaques
given p55 plus 10 µg CT (Table I). Macaques given either 10 or 100
µg CT showed similar CT-B-specific Ab and AFC responses (data not
shown).
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Ag-specific Ab production was examined in saliva, rectal, and
vaginal washes. The peak titers of Abs occurred on days 42 to 49
(Table II). p55-specific IgG Abs were observed
in the secretions of all macaques given p55 plus CT. Although
p55-specific IgA Abs were observed in the secretions of all macaques
given p55 plus 10 µg CT and in one of three macaques given p55 plus
100 µg CT, levels of Ag-specific IgA Abs, especially in vaginal
washes, of macaques given p55 plus 10 µg CT were generally higher
than those of macaques given p55 plus 100 µg CT. The macaque given
p55 only did not show production of any Ag-specific Abs in external
secretions throughout the experiment. These data suggested that 10 µg
CT was the most effective nasal adjuvant for induction of
coadministered Ag (e.g., SIV
p55gag)-specific IgA Abs in external
secretions by nasal immunization.
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It was important to determine whether the Abs in external
secretions were mucosa associated or exudates from serum. Two macaques
(20119 and 23681) given p55 plus 10 µg CT were sacrificed to
determine Ag-specific AFCs in mucosal and systemic tissues.
p55-specific IgA AFCs were detected in all mucosal effector sites
examined, i.e., NP, LP, and UC, and in SP as a systemic compartment
(Fig. 2). These data confirmed that the
p55-specific IgA Abs in rectal and vaginal washes were indeed produced
in the mucosal sites. p55-specific IgG AFCs were also noted in NP, LP,
MLN, and SP (Fig. 2). A similar pattern of anti-CT-B AFC responses
was noted (data not shown).
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When lymphocytes from mucosal (e.g., MLN) and systemic (e.g., SP
and PBMC) tissues were assessed for proliferative responses,
p55-specific T cell responses were observed (data not shown). These
findings suggested that Ag-specific CD4+ T cells were also
induced by nasal immunization. To characterize Ag-specific
CD4+ T cells in mucosal and systemic tissues, Th1- and
Th2-specific cytokine production and mRNA expression were examined.
High levels of IFN- and IL-5 were detected in culture supernatants
of p55-specific CD4+ T cells from mucosal and systemic
tissues. In addition, IL-10 production was only associated with
intestinal LP (Fig. 3A). mRNA
expression for Th1-type cytokines, i.e., IFN- and IL-2, was abundant
in p55-stimulated CD4+ T cells isolated from both mucosal
and systemic tissues. In terms of Th2-type cytokines, mRNA for IL-5 and
IL-6 were expressed in high levels in CD4+ T cells obtained
from p55-activated cultures of MLN and UC, and IL-5, IL-6, and IL-10
mRNA were present in LP and SP CD4+ T cells (Fig. 3B). Neither IL-4 production nor IL-4 mRNA expression was
detected in p55-specific CD4+ T cells of mucosal or
systemic tissues (Fig. 3).
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Since p55-specific Th1-type T cell responses were also induced by
nasal immunization (Fig. 3
), it was important to examine whether
SIV-specific CTL responses were also generated. SIV
gag-specific CTL activity was detected in PBMC and tonsillar
lymphocytes from a macaque given p55 plus 10 µg of CT (Fig. 4). On the other hand, these lymphocytes
did not show cytolytic activity against target cells infected with vvWR
or vvenv. Altogether, three of five immunized monkeys tested at
necropsy had CTL responses in tonsillar lymphocytes, as detected by
bulk culture or by limiting dilution analysis (Table III). This CTL activity was weak and more
readily detected by precursor frequency analysis from limiting dilution
cultures. For example, monkey 25507 had no detectable CTL activity in
bulk cultures of PBMC or tonsil, but low CTL precursor frequencies of
74/106 CD8+ cells (95% confidence interval
29–119) in PBMC, and 29/106 CD8+ cells (95%
confidence interval 7–51) from tonsil. gag-specific CTL
responses were also detected in spleen or lymph node cells from some of
these animals. No CTL activity was detectable in the lymphocytes from
any tissue at necropsy of monkey 19145 that was immunized with p55
without CT (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we determined whether nasal immunization with p55 plus
CT as a mucosal adjuvant could induce p55-specific IgA and IgG Abs in
serum and in external secretions. According to the results obtained in
this study, there was a tendency that low dose of coadministered CT
supported high levels of Ag-specific immune responses after nasal
immunization. Thus, macaques nasally immunized with p55 plus 10 µg of
CT showed higher levels of p55-specific IgA Abs not only in serum but
also in secretions (Tables I and II). However, when a higher dose of CT
was employed, two of three macaques given p55 plus 100 µg of CT
showed only weak Ab responses against p55 in serum and secretions (Fig. 1 and Table I). A cytotoxic or inhibitory effect caused by a large dose
of CT could explain this result. Thus, it has been shown that CT can
inhibit T cell responses in vitro (28, 29). The new results obtained by
this study demonstrate that nasal immunization can induce SIV-specific
IgG and IgA immune responses in systemic sites (Fig. 1 and Table I) in
addition to mucosal compartments (Table II) in the presence of an
optimal dose of the mucosal adjuvant CT.
Recently, we have reported that oral immunization with p55 and CT
induced Ag-specific IgG and IgA Abs in serum and mucosal secretions,
i.e., saliva and rectal wash, but failed to induce Ag-specific IgA Abs
in vaginal washes (13). In the present study, it was clearly shown that
macaques nasally immunized with p55 plus 10 µg of CT developed
p55-specific IgA and IgG Abs in both digestive tract and genital tract
secretions. However, a macaque given p55 only failed to develop
p55-specific IgA and IgG Ab responses in secretions (Table II). These
findings further emphasize that appropriate mucosal delivery of vaccine
Ag together with mucosal adjuvant is important for the stimulation of
Ag-presenting and Th cells in inductive sites (e.g.,
nasopharyngeal-associated lymphoreticular tissues, NALT), which can
induce Ag-specific immune responses in distant effector sites such as
the reproductive tract.
In this study, we demonstrated that the mucosal tissues of macaques
nasally immunized with p55 plus 10 µg of CT contain Ag-specific AFCs
(Fig. 2). Moreover, analysis of mRNA expression and synthesis of
cytokines clearly showed that Ag-specific Th1- and Th2-type
CD4+ T cells producing IFN- (Th1) or IL-5, IL-6, and
IL-10 (Th2), respectively, were induced in both systemic and mucosal
tissues (Fig. 3). It is well known that IL-5, IL-6, and IL-10 support
the induction of IgA-producing cells (30, 31). According to the concept
of the common mucosal immune system (2, 26, 27), nasally administered
Ag would be processed by APCs and presented to B and T cells in NALT.
Ag-stimulated T and B cells leave NALT via efferent lymphatics and
reach the systemic circulation through the thoracic duct. These
lymphocytes then migrate into mucosal effector sites, e.g., nasal and
oral cavity, intestinal lamina propria, and the genitourinary tract.
Our findings suggested that nasal immunization resulted in the
induction of p55-specific Th1 and Th2 cells, especially IL-5, IL-6, and
IL-10, as well as IgA and IgG Abs in the mucosal effector compartments
via primary stimulation of immunocompetent cells in NALT.
Another effector mechanism to control HIV infection is HIV-specific
CD8+CTLs. CTLs appear to be temporally related to the
clearance of the virus during the acute phase of infection (32, 33). In
macaques intravaginally immunized with virulence-attenuated SHIV, the
presence of SIV-specific CTL was associated with protection from
vaginal challenge with pathogenic SIVmac (19). It is well
known that Th1-type cytokines (e.g., IFN-) are essential for the
induction of CTLs (34). On the other hand, the Th2 cytokine, IL-4, can
inhibit IFN- production (35). In this regard, our study showed that
macaques nasally immunized with p55 plus CT harbored elevated IFN-
production in addition to selected Th2 cytokine (IL-5, IL-6, IL-10)
synthesis. Thus, among the array of Th2 cytokines, IL-4 was not
detected in either mucosal or systemic compartments (Fig. 3). However,
our previous study, as well as those done by others, demonstrated that
IL-4 production by Ag-specific CD4+Th2-type cells was
increased in mice orally immunized with protein Ag and CT (12, 36).
Although several possibilities, including differences in species (e.g.,
mouse versus monkey), Ags (e.g., tetanus toxoid versus SIV p55), and
immunization protocol (e.g., nasal versus oral), are considered, we do
not have any specific explanation at this time. However, it is possible
that induction of IFN- may lead to the generation of selective Th2
cytokine-producing CD4+ T cells due to the inhibition of
IL-4 synthesis by IFN-. Recently, others have reported that nasal
immunization with HIV-1 peptide plus CT could induce peptide-specific
CTLs and protect against tumor development in mice (37). In our study,
SIV gag-specific CTL activity was detected in lymphocytes
from blood, tonsil and other lymphoid tissues (Fig. 4 and Table III).
Taken together, it is suggested that nasal immunization using CT as a
mucosal adjuvant has the potential to induce virus-specific Th1 and
CTLs in addition to mucosal and systemic Ab responses.
In this study, macaques nasally coadministered with CT did not exhibit clinical signs of toxicity (e.g., massive diarrhea). Nevertheless, it is well known that CT induces diarrhea in humans due to the toxic activity of the CT-A subunit (38). Recently, our group has developed mutant CTs (mCTs) in the ADP-ribosyltransferase cleft of CT-A subunit, which lost diarrhoeagenicity, but retained adjuvanticity when used systemically (17) and nasally (15). Thus, our current efforts are focused on determining whether mCTs can be used as mucosal adjuvants in macaques.
In summary, our studies demonstrate that nasal immunization using CT as a mucosal adjuvant is a simple and effective procedure for the induction of HIV/SIV-specific IgA Abs in mucosal compartments, including the genital tract. Nasal immunization with HIV-related Ag or attenuated-whole virus plus mCT as a mucosal adjuvant could be a practical regimen for the induction of protective humoral and cell-mediated immunity in both systemic and mucosal compartments, especially the genital tract, to prevent the sexual transmission of HIV.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan. E-mail address:
3 Abbreviations used in this paper: CT, cholera toxin; AFC, antibody-forming cell; CT-B, B subunit of cholera toxin; ELISPOT, emzyme-linked immunospot; LP, intestinal lamina propria; mCT, mutant cholera toxin; MLN, mesenteric lymph node; NALT, nasopharyngeal-associated lymphoreticular tissue; NP, nasopharynx; SMG, submandibular gland; SP, spleen; UC, uterine cervix; vv, vaccinia virus.
Received for publication April 23, 1998. Accepted for publication July 31, 1998.
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References |
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