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* Department of Mucosal Immunology, Research Institute for Microbial Diseases, and
First Department of Surgery, Osaka University, Osaka, Japan;
Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan;
AIDS Research Center, National Institute of Infectious Disease, Tokyo, Japan;
¶ Division of Mucosal Immunology, Department of Microbiology and Immunity, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
|| Immunobiology Vaccine Center, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-) and Th2 (IL-4-/-). In vitro
Ag-specific serum IgG Ab and vaginal wash samples possessing IgA and
IgG Abs that had been induced by nasal immunization with
gp160-HVJ-liposome were able to neutralize a clinically isolated strain
of HIV-MN strain isolated from Japanese hemophiliac patients.
Taken together, these results suggest that, for the prevention and
control of AIDS, nasally administered gp160-HVJ-liposome is a powerful
immunization tool that induces necessary Ag-specific immune responses
at different stages of HIV infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To effectively induce Ag-specific mucosal and systemic immune responses, in this study we used the viral-based hybrid Ag delivery system hemagglutinating virus of Japan (HVJ)4-liposome. The HVJ-liposome was developed by combining liposome with fusion proteins derived from the HVJ, also known as the Sendai virus, to enhance the efficiency of Ag delivery to targets (5, 6). In this system, vaccine Ags can be directly introduced into the cytoplasm by virus-cell fusion for the generation of Ag-specific CTLs (7). Moreover, this Ag delivery system has been shown to induce Ag-specific humoral immune responses (8). Thus, for the stimulation of two major arms of immune response including humoral (e.g., Ab) and cell mediated (e.g., CTL), HVJ-liposome is considered to be a useful vaccination Ag delivery vehicle. One interesting possibility entails the inclusion of HIV-surface glycoprotein gp160 into this HVJ-liposome. In a previous study, we demonstrated that, after nasal immunization, HVJ-liposome containing fluorescence-conjugated dextran is effectively taken up by epithelium-associated cells and APCs (8). Thus, HIV-liposome that contains gp160 could be effectively delivered into Ag-sampling M cells, epithelial cells, and APCs in nasopharyngeal-associated lymphoreticular tissue (NALT) and nasal passages (NP). Evidence that nasal immunization with HVJ-liposome containing OVA effectively induces both Ag-specific CTLs and IgA Ab responses (8) suggests that this novel mucosal Ag delivery vehicle could be implemented in the development of an HIV mucosal vaccine.
In this study, we examined whether nasal immunization with
HIV-gp160-encapsulated HVJ-liposome (gp160-HIV-liposome), in conditions
in which normal and Th cell functions are deficient, can induce
effective HIV-specific immunity in both the mucosal and systemic
compartments. To characterize the quality and quantity of HIV
Ag-specific immune responses, we tested mucosal gp160-HVJ-liposome with
both normal and Th1 (IFN--/-) and Th2
(IL-4-/-) immunodeficient mice. The
gp160-HVJ-liposome induced Ag-specific IgG and IgA Abs in both types of
mice. Postimmunization samples were also examined in vitro for isotype
and subclass distribution as well as neutralizing activity against a
clinically isolated strain of HIV. Furthermore, we also examined
Ag-specific CTL responses at both mucosal and systemic sites after
nasal immunization with gp160-HVJ-liposome. In short, the major aim of
the present study was to evaluate the potential for application of the
gp160-HVJ-liposome system in the development of a mucosal vaccine to
control of HIV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were obtained from SLC Japan (Hamamatsu, Japan) and
BALB/c strain mice with IFN- and IL-4 deficiency
(IFN--/- and
IL-4-/-) between 6 and 12 wk of age were
purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were
maintained in barrier-protected animal facilities under pathogen-free
conditions using the ventilated microisolator cage in the experimental
animal facility at the Research Institute for Microbial Diseases, Osaka
University (Osaka, Japan).
Preparation of HVJ-liposome
The preparation of HVJ-liposome has been described in detail
elsewhere (5, 6, 7, 8). Briefly, the lipid mixtures
(cholesterol, sphingomyelin, dioleoylphosphatidyle-thanolamine,
phosphatidylcholine, and phosphatidylserine) were deposited on the side
of the flask by removing the chloroform solvent in a rotary evaporator.
The dried lipid was hydrated in 200 µl of balanced salt solution (137
mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl; pH 7.5) containing HIV gp160 (100
µg; Protein Sciences, Meriden, CT). A liposome suspension was
prepared by vortexing, sonicating, and shaking to form liposome.
Purified HVJ (Z strain) was inactivated immediately before use by UV
radiation. The liposome-gp160 complex suspension was incubated with
10,000 hemagglutinating units of HVJ and collected by sucrose density
gradient ultracentrifugation. The preparation contained 
1 µg of
gp160/10 µl in the wet volume of HVJ-liposome.
Immunization and sampling schedule
Mice (n = 4 per group) were nasally immunized with gp160-HVJ-liposome (1 µg per 10 µl per mouse). Each group of mice was immunized once a week for four consecutive weeks. To monitor the induction of IgG and IgA anti-gp160 specific Abs, serum, saliva, fecal extract, and vaginal wash were collected at day 0 as preimmunization samples and, after the immunization, were collected five times at weekly intervals.
Detection of Ag-specific Ab production by ELISA
HIV-specific Ab titers in serum, saliva, fecal extract, and vaginal wash were determined by ELISA using modified methods as described previously (9). ELISA plates were coated with 100 µl of 2 µg/ml gp160 in 0.1 M carbonate buffer and incubated overnight at 4°C. The plates were then incubated with blocking solution (Block Ace; Dainippon Pharmaceuticals, Osaka, Japan) at 37°C for 2 h. Dilutions of all mucosal secretions starting at 1/4 and serum starting at 1/32 were made with blocking solution, and 50 µl of each dilution was added to duplicate wells of Ag-coated plates. After an incubation at 37°C for 2 h, the coated plates were washed with PBS-polyoxyethylene sorbitan monooleate (Tween 20; Wako Pure Chemical, Tokyo, Japan) and incubated with 100 µl of a 1/4000 PBS dilution of biotin-conjugated goat anti-mouse IgG and IgA detection Ab (Southern Biotechnology Associates, Birmingham, AL) at 37°C for 2 h. The plates were then washed with PBS-Tween 20 and incubated with 100 µl of a 1/2000 PBS-polyoxyethylene sorbitan monooleate (Tween 20; Wako Pure Chemical, Tokyo, Japan) dilution of streptavidin HRP (Life Technologies, Rockville, MD) for 1 h at room temperature. After incubation, color was developed with tetramethylbenzidine (Wako, Tokyo, Japan), stopped with 0.5 N HCl, and measured by OD450 on an ELISA reader (Lab System, Helsinki, Finland).
Isolation of mononuclear cells
Mononuclear cells from submandibular glands (SMG), NP, NALT, Peyer’s patch (PP), vaginal tissue (VT), and spleen (SP) were isolated as previously described (3, 8, 9, 10, 11, 12). In brief, mononuclear cells from NALT and SP were isolated by the mechanical dissociation method using gentle teasing through stainless steel screens. NP, SMG, VT, and PP mononuclear cells were isolated by an enzymatic dissociation procedure using collagenase type IV (Sigma-Aldrich, St. Louis, MO).
Detection of HIV-specific Ab-producing cells by ELISPOT assay
Mononuclear cells were analyzed for Ag-specific Ab production at
single cell level using Ag- and isotype-specific ELISPOT assay as
previously described (8, 9, 12, 13). Briefly, 96-well
filtration plates with a nitrocellulose base (Millititer HA; Millipore,
Bedford, MA) were coated with 2 µg of gp160 per well. Single cell
suspensions of mononuclear cells from different tissues were added at
varying concentrations and then incubated at 37°C for 4 h in air
with 5% CO2 and 95% humidity. After incubation
and washing, detection Abs consisting of 1 µg/ml of HRP-labeled goat
anti-mouse-µ, -, or -
(Southern Biotechnology Associates)
were added to the plate. The spots were developed by
3-amino-9-ethylcarbazole (Moss, Pasadena, MD) and counted under
a dissecting microscope.
Analysis of HIV-specific T cell responses
For analysis of gp160-specific T cell responses,
CD4+ T cells were isolated from NALT, NP, PP, VT,
and SP by a magnetic cell sorting system (MACS; Miltenyi Biotec,
Auburn, CA) and FACS (BD Biosciences, Mountain View, CA) as previously
described (14). Purified CD4+ T
cells (>98%) were suspended in complete medium and cultured at a
density of 1 x 105 cells/ml in the presence
of gp160 (5 µg/ml) and T cell-depleted and irradiated (3000 rad)
splenic feeder cells (1 x 106 cells/ml) in
flat-bottom 96-well microculture plates (Costar, Cambridge, MA)
(15). After 6 days of incubation, using cytokine-specific
murine cytokine ELISA kits (Amersham Life Science, Arlington Heights,
IL), culture supernatants of Ag-stimulated T cells were examined for
the presence of IFN-, IL-2, IL-4, IL-5, IL-6, and IL-10.
Ag-specific CTL assay
Lymphocytes isolated from nasally vaccinated mice were restimulated in vitro for 5 days with gp160 gene-transfected syngeneic BALB/c3T3 fibroblasts, termed 15-12, expressing an immunodominant epitope identified as a peptide composed of 15 aa (P18IIIB: RIQRGPGRAFVTIGK) (16). A long-term CTL line was also generated by repetitive stimulation of immune cells with mitomycin C-treated gp160 gene-transfected fibroblasts and 10% Con A supernatant condition medium according to a method described previously (17). After culturing for 5 days, the cytolytic activity of the Ag-restimulated cells was measured using a 6-h CTL assay with various 51Cr-labeled targets as previously described (16, 17, 18). For testing the peptide specificity of CTLs, effectors and 51Cr-labeled targets were mixed with various concentrations of peptide at the beginning of the assay. Percent-specific 51Cr release was calculated as follows: 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). Maximum release was determined by evaluating supernatants of cells that were lysed by the addition of 5% Triton X-100. Spontaneous release was determined by evaluating target cells incubated without added effector cells. SEMs of triplicate cultures were always within 5% of the mean. To confirm the surface phenotype and class I MHC restriction of the gp160-specific CTLs, the CTL effector cells were variously pretreated with anti-CD4 mAb (rat IgM, RL172.4 hybridoma) plus rabbit complement (Low-Tox Rabbit Complement; Cedarlane Laboratories, Hornby, Ontario, Canada), anti-CD8 mAb (rat IgM, 3.115 hybridoma) plus complement, or complement alone (16). These treated effector cell populations were then tested for killing activity on either fibroblasts transfected with gp160 gene (P18IIIB) or the control fibroblasts (18).
HIV neutralization activity assay
In vitro neutralizing assay of HIV was performed as previously described (19). Briefly, serum IgG Abs were purified from mice immunized with gp160-HVJ-liposome by using protein A-Sepharose (Amersham Biosciences, Little Chalfont, U.K.). Serum IgG was also purified from preimmunized and nonimmunized mice. In addition, vaginal wash was also tested for the presence of neutralizing activity. Appropriately, diluted serum and vaginal wash Abs were incubated with 100 medium tissue culture infective dose units of primary field isolates from Japanese hemophiliac patients (HIV-MNp) (19, 20) for 60 min at 37°C, and the mixture was shaken with 1 x 106 PHA-activated PBMCs for 60 min in a 37°C water bath (19). After washing, the cells were cultured in the presence of recombinant human IL-2 (40 U/ml; Shionogi, Osaka, Japan) for 7 days. After the incubation, culture supernatants were subjected to a p24 Ag ELISA (Dynabot, Tokyo, Japan) for the measurement of HIV. Data were expressed as percentage inhibition of p24 Ag production in the culture supernatants compared with results from cultures to which preimmunized or nonimmunized serum IgG or vaginal wash were added. Virus stocks were titrated on PHA-activated normal PBMCs, and the TCID50 of each virus was determined as described previously (19).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With high titers of gp160-specific serum IgG and IgA Abs
already detected between the second and third week after initial
immunization, gp160-specific Ab responses were clearly induced by nasal
immunization with gp160-HVJ-liposome (Fig. 1
). Furthermore, gp160-specific IgA Abs
were present in different mucosal secretions including fecal, salivary,
vaginal, and nasal (Fig. 2). Relevant to
sexual activity, relatively high titers of HIV gp160-specific IgA and
IgG Abs were present in the vaginal wash of mice nasally immunized with
gp160-HVJ-liposome. Taken together, these results indicate that nasal
vaccination with gp160-HVJ-liposome was an effective immunization
procedure for the induction of HIV gp160-specific systemic IgG and IgA,
as well as mucosal IgA and IgG, immune responses.
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The isolation of mononuclear cells from SP and different
mucosa-associated tissues (e.g., VT, intestinal lamina propria (i-LP),
NP, SMG, NALT, and PP) of mice nasally immunized with
gp160-HVJ-liposome provided further corroboration of the induction of
Ag-specific Ab responses at the cellular level. Isotype- and
Ag-specific ELISPOT assays revealed the presence of increased numbers
of gp160-specific IgG and IgA Ab-forming cells in the SP and other
parts of the systemic compartment (Fig. 3). In addition, Ag-specific IgG and IgA
Ab-producing cells were detected in all of the mucosa-associated
tissues, including VT, i-LP, NP, SMG, NALT, and PP (Fig. 3). As
anticipated, high numbers of gp160-specific IgA Ab-producing cells were
present in mucosal effector tissues, including VT, i-LP, SMG, and NP.
These results show that nasal immunization with gp160-HVJ-liposome
induced Ag-specific IgG- and IgA-producing cells in both the systemic
and mucosal compartments.
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To examine the potential for the application of nasal
gp160-HVJ-liposome in the development of a clinical therapeutic
vaccine, Th1 (IFN--/-) and Th2
(IL-4-/-) deficient mice were nasally immunized
with gp160-HVJ-liposome, after which increased levels of Ag-specific
IgG Ab in serum were induced in these immunodeficient mice (Fig. 4). However, the levels of
HIVgp160-specific IgG Abs induced in IFN--/-
and IL-4-/- mice were lower than in nasally
immunized normal mice (Figs. 1
and 4). More specifically, in serum and
vaginal wash, although the levels of HIVgp160-specific IgA Abs induced
in IL-4-/- mice were lower than in nasally
immunized normal and IFN--/- mice, IgG Abs
levels were similar (Figs. 1 and 4). These results suggest that nasal
vaccination with gp160-HVJ-liposome might be an effective way to induce
HIV Ag-specific immune responses even when the immunodeficient
condition of HIV-infected patients varies during the different stages
of progression of AIDS.
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We next investigated the IgG subclass of gp160-specific Abs in
serum isolated from immunodeficient (IFN--/-
and IL-4-/-) and wild-type mice nasally
immunized with gp160-HVJ-liposome. In wild-type mice,
gp160-specific IgG2a Ab level was highest, followed by Ab subclasses
IgG1, IgG2b, and IgG3 (Fig. 5). As
anticipated, Ag-specific IgG1, IgG2b, and IgG3 Abs, but not IgG2a Abs,
were induced in IFN--deficient mice (Fig. 5). In samples from
IL-4-/- mice, the highest titers of Ag-specific
subclass Ab were for IgG2a. Interestingly, high levels of
gp160-specific IgG3 Abs were consistently induced in these two
immunodeficient types and wild-type mice. The results obtained by the
analysis of wild-type mice suggest that the gp160-HVJ-liposome system
preferentially induced Th1-type responses based on the profile of the
Ag-specific IgG subclass distribution. It is worth emphasizing that the
HVJ-liposome system can overcome Th1- and Th2-deficient conditions and
induce selective Ag-specific IgG subclass Ab responses. These results
further indicated that nasal vaccination with gp160-HVJ-liposome holds
promise for inducing HIV Ag-specific Ab response in immunocompromised
patients.
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To characterize the nature of gp160-specific
CD4+ T cell responses, Th1-specific (e.g.,
IFN- and IL-2) and Th2-specific (e.g., IL-4, IL-5, and IL-6)
cytokine production was investigated. High levels of Th1-type cytokine
(e.g., IFN- and IL-2) were detected in the culture supernatant
harvested from in vitro gp160-stimulated CD4+ T
cells that were isolated from SP and mucosa-associated tissues
including VT, NP, NALT, and i-LP from normal mice nasally immunized
with gp160-HVJ-liposome (Fig. 6). The
findings reflect the results of analysis of Ag-specific IgG subclass
responses in wild-type mice nasally immunized with gp160-HVJ-liposome
(Fig. 5). A similar Th1 cytokine profile was also apparent in samples
from IL-4-/- mice nasally immunized with
gp160-HVJ-liposome. With IFN--/- mice, as
anticipated, the only Th1 cytokine detected was IL-2. It is also worth
noting that Th2-associated IgA-enhancing cytokines, such as IL-5 and
IL-6, were induced by gp160-specific CD4+ Th
cells isolated from systemic and mucosa-associated tissues of all of
the mice tested. Taken together, these findings suggested that nasal
immunization with gp160-HVJ-liposome induced both Th1- and Th2-type
CD4+ T cells in the mucosal and systemic
compartments.
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After confirming that Ag-specific Th1-type responses were elicited
by nasal immunization with gp160-HVJ-liposome (Fig. 6), we next tested
whether gp160-HVJ-liposome is able to induce MHC class I-restricted
gp160-specific CTL responses. At first, CTL activity against gp160 was
examined in splenic lymphocyte samples isolated from mice nasally
immunized with gp160-HVJ-liposome. After in vitro restimulation of the
immune splenic lymphocytes with gp160 gene-transfected 15-12 fibroblast
stimulator cells, we found that the presence of gp160-specific CTL
response was detected in mice nasally immunized with gp160-HVJ-liposome
(Fig. 7). When these splenic lymphocytes
were pretreated with mAb anti-CD4 or anti-CD8, the removal of
gp160-specific CTL activity was subsequently achieved by anti-CD8
mAb treatment (Fig. 7). These findings suggest that nasal
immunization with gp160-HVJ-liposome resulted in the priming of
Ag-specific CD8+ CTL responses.
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To better characterize virus neutralization activity, a
well-characterized, clinically isolated strain of HIV-MNp that
expresses IHIGPGRAFY at the core sequence of the HIV principal
neutralizing determinant (19, 21) was used as the vial
source. Serum IgG and vaginal secretory IgA (S-IgA) Ab samples
obtained from wild-type mice nasally immunized with gp160-HVJ-liposome
possessed neutralizing activity against HIV-MNp (Fig. 8). Similar neutralization activity was
observed with IgG immune serum purified from both IFN-- and
IL-4-deficient mice that had been nasally vaccinated with
gp160-HVJ-liposome (Fig. 8). Preimmunized IgG and vaginal wash samples
containing S-IgA, however, showed no neutralizing activity (data not
shown). These results indicated that nasal immunization with
gp160-HVJ-liposome was capable of strongly inducing gp160-specific IgG
and S-IgA Abs with virus-neutralizing activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nasal administration has been shown to be as effective at inducing Ag-specific immune responses, including Ab production and CTL activity, in both the mucosal and systemic compartments as oral immunization (15, 22, 23, 25, 26). Our own previous studies and work by other researchers has shown that coadministration of mucosal adjuvant is essential for the generation of Ag-specific mucosal and systemic immune responses via the respiratory and gastrointestinal immune system (3, 9, 13, 15). Thus, our previous report has shown that nasal vaccination with the fimbrial protein of Porphyromonas gingivalis and a well-known mucosal adjuvant cholera toxin (CT) induces protective immunity via the generation of Th2 cell-mediated Ag-specific mucosal IgA Ab responses (15). Nasal vaccine containing fimbrial protein alone, however, did not lead to the induction of Ag-specific Th and B cell responses (15). Furthermore, when female rhesus macaques were nasally immunized with p55 of SIV in the presence or absence of CT as a mucosal adjuvant (3), nasal vaccine containing both p55 and CT induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal, rectal, and salivary), but similar Ag-specific immune responses were not detected in primates nasally immunized solely with p55 (3).
In contrast with the previous investigations discussed above,
HVJ-liposome effectively induces Ag-specific Th1 and Th2 cells together
with the associated IgG and IgA Ab responses in both mucosal and
systemic sites without coadministration of mucosal adjuvant (e.g., CT
or Escherichia coli heat-labile enterotoxin). It is
pertinent to consider why HVJ-liposome induces mucosal and systemic
immune responses without mucosal adjuvant. One reason may be its
ability to directly deliver the Ag to cells associated with the mucosal
immune system. It was shown that HVJ-liposome directly attaches
to the cell membrane using envelope glycoproteins, the so-called fusion
proteins of the Sendai virus (27, 28, 29), which function
during natural infection via the respiratory epithelium (30, 31). Furthermore, in a previous study we found that HVJ-liposome
is able to preferentially attach to nasal epithelia and deliver
encapsulated Ag directly to the surface of epithelial cells, M cells,
and APCs (8). The immunoenhancing activity of envelope
glycoproteins composed with F and H proteins may bypass the need for
adjuvant. For example, it has been shown that virus-associated fusion
proteins possess immunoaugmentation or adjuvant activity (8, 29). Other reports have shown that the liposome inherently
possesses adjuvant activity (32, 33). Moreover, there is
evidence that, after Sendai virus infection of an epithelial cell line
(NIH 3T3), overexpression of NF-
B gene is induced, which leads to
the up-regulation of IFN-
mRNA induction (34).
Therefore, it is also possible that HIV-liposome is involved in the
activation of cytokine synthesis that enhances immune response. Taken
together, this evidence of the multiple immunoenhancing activities of
HIV-liposome is encouraging for the development of a novel and
effective Ag delivery vehicle that generates HIV Ag-specific mucosal
and systemic immune responses without administration of any additional
mucosal adjuvant.
HVJ-liposome, a novel viral-based hybrid consisting of liposome with Sendai virus fusion proteins, has proved effective as a delivery vehicle for introducing DNA into a host (5, 6, 7). Not only is HVJ-liposome capable of delivering the encapsulated oligodeoxynucleotide DNA into cell cytoplasm (5, 6, 7, 8), this delivery also results in presentation of MHC class I-mediated peptide that induces cell-mediated immune responses (29). Nasal immunization with HVJ-liposome also induces Ag-specific CTL responses at systemic lymphoid tissues in an MHC class I-dependent manner (8). Direct intracellular delivery of Ag via the fusion process demonstrated an encapsulated Ag to the MHC class I pathway (8, 29). In addition to these MHC class I-mediated CTL responses, nasal immunization with gp160-HVJ-liposome induced high levels of gp160-specific CD4+ Th1 and Th2 cell-mediated Ab responses in both systemic and mucosal tissues. This result suggests that mucosally administered HIV-liposome can activate MHC class II-mediated CD4+ T cell responses for the subsequent induction of gp160-specific IgG and IgA Abs. To this end, our previous study demonstrated that HVJ-liposome is capable of presenting peptide Ag together with MHC class II molecules of epithelial cells and macrophages (8). Other evidence from previous studies has also shown that mucosal epithelial cells are able to present Ags (35, 36). Thus, nasally administered HIV-liposome appears to use the mucosa-associated Ag presentation pathway for the maximum induction of gp160-specific IgA immune response. These different findings, taken together, provide evidence that gp160-HVJ-liposome effectively enhances gp160-specific MHC class I-mediated CD8+ CTL and class II-regulated CD4+ Th1/Th2 cell activities.
The established cytokine profile of Ag-specific Th1 and Th2 cells
predicts the presence of IFN-- and IL-4-dependent IgG subclass Ab
responses in mice nasally immunized with gp160-HIV-liposome, and our
findings show a profile of Th1 and Th2 cytokines in line with
anticipated IgG subclass Ab responses, which indicates that nasally
administered HVJ-liposome causes simultaneous induction of Ag-specific
serum IgG1 and IgG2a Ab responses. It was long ago shown that IFN-
is the key cytokine for generation of IgG2a Abs and that IL-4 is the
key cytokine for IgG1 Abs (37). Our current results
corroborate our previous finding that nasal immunization with
OVA-fusogenic liposomes results in the generation of both the Th1 and
Th2 cytokine-mediated IgG2a and IgG1 responses (8). Even
so, our previous and current investigations of the induction of
Ag-specific IgG3 subclass Ab responses differ. Whereas in the previous
study Ag-specific IgG3 Abs were not induced in wild-type mice nasally
immunized with HIV-liposome containing OVA (8), in the
current study, using an identical Ag delivery vehicle, HIV Ag-specific
IgG3 Ab responses were in evidence. We do not have any conclusive
explanation, other than that the discrepancy may be due to the
differing nature of the antigenicity of OVA and gp160.
One of the most significant findings of this study is that nasal
immunization with gp160-HVJ-liposome is capable of inducing high levels
of gp160-specific serum IgG and mixed S-IgA and IgG in vaginal wash
samples isolated from both in immunodeficient Th1 type (IFN-) or Th2
type (IL-4) mice and in wild-type mice (Fig. 4). In human AIDS, it has
been debated whether Th1- and Th2-type CD4+ T
cells are predominantly depleted in HIV-infected individuals. It has
been reported that, during the development of HIV infection, there is a
change from Th1 to Th2 responses (38). However, another
study suggested that a switch from Th1 to Th2 responses does not occur
during the progression of HIV infection (39). Meanwhile,
it has been shown that HIV preferentially replicates within
CD4+ T cells with phenotypical Th0 and Th2
cytokine synthesis (40). Although the exact state of
CD4+ T cell deficiency is unknown, we are making
provisions for this in the development of a mucosal vaccine that can
offer induction of effective Ag-specific immune responses in
HIV-infected patients at different stages of CD4+
T cell deficiency. At the same time, we are concerned with developing a
therapeutic vaccine system that is able to effectively counteract
different Th-type cell deficiency profiles in AIDS. Because nasal
administration of the gp160-HVJ-liposome vaccine results in the
induction of Ag-specific mucosal IgA and IgG as well as serum IgG Abs
in both Th1- and Th2-type immunodeficient mice, our current findings
show promising prospects for the application of gp160-HVJ-liposome in
the development of a therapeutic mucosal vaccine (Figs. 4 and 6).
This study has also shown that nasal vaccination with
gp160-HVJ-liposome is capable of inducing high levels of Ag-specific
neutralizing Abs in both serum and mucosal secretions in both wild-type
mice and Th1- and Th2-type immunodeficient mice (Fig. 8). The rapid
clearance of HIV from the peripheral blood after primary HIV infection
is generally thought to be the result of the development of
neutralizing Abs or the formation of Ag-Ab complexes (41).
The majority of neutralizing Abs detected in HIV-infected individuals
react with epitopes that are present on HIV gp120 and gp41
(42). These Abs have been shown to neutralize either a
single particular isolate (type specific) or several isolates with
varying degrees of sequence homology (group specific). One recent
report has in fact demonstrated in vitro that the vaginal wash
obtained from mice vaginally immunized with inactivated HIV-1 captured
Con A-immobilized polystyrene nanospheres contained an in vitro
neutralizing activity against the strain used for the immunization
(43). Furthermore, other reports suggest that nasal
immunization of mice with gp160 protein is capable of inducing systemic
and mucosal HIV-1-neutralizing Abs (25, 44, 45). These
results have shown that, after mucosal immunization with Ags and
adjuvant, vaginal wash secretions are capable of neutralizing HIV.
However, in fact, mucosal Ag-specific Abs were not induced by
systemic immunization with different forms of HIV Ags, including gp160
and gp41 (44, 45). The current study found that nasal
immunization with gp160-HVJ-liposome in the absence of coadministered
adjuvant induces, in vaginal secretions, neutralizing Abs against a
clinically isolated strain of HIV-MNp. Because vaginal wash samples
contained both S-IgA and IgG Abs, it would be interesting to know which
isotype of Ag-specific Abs preferentially provides protective immunity
at the reproductive epithelium. To directly address this issue, it is
important to purify individual isotypes of Ag-specific Abs from vaginal
washes. We were unable to do this in the current study, because the
amount of sample was limited. A future task remains: the elucidation of
whether a particular isotype or both have proneutralizing activity in
the reproductive compartment of mice nasally immunized with
gp160-HIV-liposome.
In summary, for the prevention and control of HIV infection, our present investigation has demonstrated the potential prophylactic and therapeutic usefulness of a nasal vaccine based on gp160-HVJ-liposome. It is especially significant that, as well as systemic CTL activity, gp160-HVJ-liposome can induce high levels of gp160-specific mucosal IgA or IgG and systemic IgG Abs with neutralizing activity.
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Acknowledgments |
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Footnotes |
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2 G.S. and T.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka, Japan 565-0871. E-mail address: kiyono{at}biken.osaka-u.ac.jp
4 Abbreviations used in this paper: HVJ, hemagglutinating virus of Japan; NALT, nasopharyngeal-associated lymphoreticular tissue; NP, nasal passage; SMG, submandibular gland; PP, Peyer’s patch; VT, vaginal tissue; SP, spleen; MNp, MN strain isolated from Japanese hemophiliac patients; i-LP, intestinal lamina propria; S-IgA, secretory IgA; CT, cholera toxin.
Received for publication July 15, 2002. Accepted for publication November 4, 2002.
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) (1 µg/mouse of gp160) or HVJ-liposome alone
(
) once per week for four consecutive weeks. Serum was collected
weekly and analyzed by ELISA for gp160-specific IgG and IgA Abs
responses. It should be noted that nasal immunization with gp160 alone
did not lead to the induction of Ag-specific IgG response. The levels
of gp160-specific Abs are expressed as reciprocal end-point titers. The
levels of total serum IgG and IgA Abs were also measured at 5 wk after
the initial immunization with gp160-HVJ-liposome (IgG, 850 ± 450
µg/ml; and IgA, 360 ± 130 µg/ml). The levels of total serum IgG
and IgA Abs were very similar between mice nasally immunized with
gp160-HVJ-liposome and HVJ-liposome alone. Results represent the values
(mean ± SEM) for four mice per group from three different
experiments.
or 
). CTL activity was measured by
51Cr-releasing assay (A). B,
To confirm the surface phenotype and MHC class I restriction of the
gp160-specific CTL, we performed specific subset deletion CTL assays.
Complement treatment along with the relevant mAbs was conducted to
disrupt CD8 T cells (•) and CD4 T cells (
) and
immune-deficient mice (IFN-