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Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
Department of Otolaryngology and Head and Neck Surgery, Wakayama Medical University School of Medicine, Wakayama, Japan; and
AIDS Research Center, National Institute of Infectious Disease, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-)- or
Th2 (IL-4-/-)-immunodeficient mice. Further, IgG3 was
highest among V3 peptide-specific IgG subclass Ab responses
in these immunodeficient mice as well as in wild-type mice. In
addition, this Ag-specific serum IgG Abs induced by nasal immunization
with rBCG-V3J1 possessed the ability to neutralize clinical isolate of
HIV in vitro. These results suggested that the nasal rBCG-V3J1 system
might be used as a therapeutic vaccine in addition to a prophylaxis
vaccine for the control of AIDS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite intensive debate about whether CTL and/or neutralizing Abs are important for the control of HIV infection, virus-neutralizing Abs are still considered one of the major effector mechanisms against HIV (2). One goal of vaccination should therefore be to induce cross-reactive Abs that can neutralize different strains of the virus for the reduction of the initial virus load while a type-specific host immune response is being activated. A principal neutralizing determinant has been identified in the V3 loop of gp120 (3). Of course, cell-mediated immunity including the induction of CTL and Th1-type responses is also effective against HIV, and the other important goal of vaccination should be to derive cross-reactive CTL, which can rapidly remove virus-infected cells.
The tuberculosis vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG)4 is a wildly used vaccine with a low rate of serious complications, especially the Tokyo strain (4). This vaccine has been shown to possess a strong systemic and mucosal adjuvant activity, which can induce both humoral and cell-mediated immune responses (5, 6, 7, 8). In previous study a recombinant BCG (rBCG) vector-based vaccine that contained the V3 principal neutralizing, but not a classical, CTL epitope of HIV induced Ag-specific immune responses to the epitope and prevented the viral infection following systemic immunization (5). By using for targeting the sequence of HIV-1 with which most Japanese researchers concur, a chimeric V3 loop protein secretion vector was constructed by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant-peptide secretion system in BCG Tokyo strain (rBCG-expressing V3J1; rBCG-V3J1) (5). When guinea pigs were systemically immunized with rBCG-V3J1, a principal neutralizing determinant-specific delayed-type hypersensitivity skin reaction was induced (5). Further, this rBCG-V3J1 immunization induced HIV-specific cytotoxic T lymphocytes in mice (5). The immune serum of IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a PBMC-based virus neutralization assay (5). These findings suggested that the rBCG system could be considered an effective Ag delivery vehicle for the development of HIV vaccine.
In this study we examined a potential of rBCG-V3J1 for mucosal
immunization to induce effective V3J1-specific immunity. To accomplish
this goal, an rBCG-based mucosal vaccine was tested in normal as well
as Th1 (IFN--/-)- and Th2
(IL-4-/-)-type immunodeficient mice to
characterize the quality and quantity of virus-specific immune
responses. Further, Abs obtained from these immunized mice were
examined for neutralizing activity against HIV, including clinical
isolate in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J mice and mice of the C57BL/6J background with IFN-
and IL-4 deficiency (IFN--/- and
IL-4-/-) between 8 and 10 wk of age were used
in the experiments and purchased from The Jackson Laboratory (Bar
Harbor, ME). Mice were maintained in barrier-protected animal
facilities under pathogen-free conditions using ventilated
microisolator cage in the experimental animal facility at the Research
Institute for Microbial Diseases, Osaka University (Osaka, Japan).
Immunization and sampling schedule
Several groups (n = 4–8/group) of mice were
immunized with rBCG-V3J1 by the nasal (10 µg live rBCG; 
1 x
106 bacilli), oral (100 µg), or systemic (s.c.;
100 µg) route. Each group of mice was immunized once per week for 3
consecutive wk, since our preliminary experiments demonstrated that one
immunization did not lead to the induction of an Ag-specific Ab
response. Serum, saliva, fecal extract, and vaginal wash were collected
at weekly intervals for 6–12 mo and monitored for IgG and IgA
anti-V3J1-specific Abs.
Detection of Ag-specific Ab production by ELISA
HIV Ag-specific 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 2 µg/ml V3J1-conjugated keyhole limpet hemocyanin (KLH) protein (V3J1-KLH) or KLH alone resuspended in 0.1 M carbonate buffer. The plates were then incubated with blocking solution (Block Ace, Dainihon, Osaka, Japan). Dilutions of saliva and fecal extract starting at 1/4 and of 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 incubation at 37°C for 2 h, the coated plates were washed with PBS-Tween and incubated with 100 µl of a 1/4000 PBS dilution of peroxidase-conjugated goat anti-mouse IgG and IgA detection Abs (Southern Biotechnology Associates, Birmingham, AL). After incubation for 2 h, plates were washed with PBS-Tween and incubated with 100 µl of a 1/2000 PBS-Tween dilution of streptavidin-HRP (Life Technologies, Gaithersburg, MD). After incubation for 1 h, color was developed with TMB (Wako, Tokyo, Japan), stopped with 0.5 N HCl, and measured by absorbance at 450 nm on an ELISA reader (Lab System, Helsinki, Finland).
Isolation of mononuclear cells
Mononuclear cells from submandibular glands (SMG), nasal passage (NP), and nasopharyngeal-associated lymphoreticular tissue (NALT), Peyer’s patch (PP), intestinal lamina propria (i-LP), and spleen (SP) were isolated as previously described (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, i-LP, and PP mononuclear cells were isolated by the enzymatic dissociation procedure with collagenase type IV (Sigma, St. Louis, MO).
Detection of V3 peptide-specific Ab-producing cells by ELISPOT assay
Mononuclear cells were analyzed for Ag-specific Ab production at
the single-cell level using Ag and isotype-specific ELISPOT assay as
previously described (13). Briefly, 96-well filtration
plates with a nitrocellulose base (Millititer HA, Millipore, Bedford,
MA) were coated with 2 µg V3J1-KLH/well. Single-cell suspensions of
mononuclear cells from different tissues were added at varying
concentrations. After incubation and washing, detection Abs consisting
of 1 µg/ml HRP-labeled goat anti-mouse 
or anti-mouse
(Southern Biotechnology Associates) were then added to the plate. The
spots were developed by 3-amino-9-ethylcarbazole (Moss, Pasadena, MD)
and counted under a dissecting microscope.
Analysis of cytokine production by V3 peptide-specific T cells
For analyzing V3J1-specific T cell responses,
CD4+ T cells were isolated from NALT, NP, SMG,
PP, and SP as previously described by FACS and MACS (10).
Purified CD4+ T cells (>98%) were suspended in
complete medium, cultured at a density of 1 x
106 cells/ml in the presence of V3J1-KLH (10
µg/ml) along with 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)
(14). Following 3-day incubation, culture supernatants of
Ag-stimulated T cells were examined for the production of IFN-,
IL-2, IL-4, IL-5, IL-6, and IL-10 by cytokine-specific ELISA.
Ag-specific CD4+ T cell-derived Th1 and Th2
cytokines were measured by murine cytokine ELISA kits (Amersham,
Arlington Heights, IL).
In vitro HIV neutralization assay
An in vitro neutralizing assay of HIV was performed as previously described (5, 15). Briefly, serum IgG Abs were purified from mice nasally immunized with rBCG-V3J1 using protein A-Sepharose (Pharmacia, Poole, U.K.). Serum IgG was also purified from pre- and nonimmunized mice. The diluted serum Abs were incubated with 100 medium tissue culture infective dose units of HIV-MN (H9/HTLV-IIIMN, AIDS Research and Reference Reagent Program, National Institutes of Health, Bethesda, MD) or HIV-MNp (primary field isolates from Japanese hemophiliac patients; AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan) for 60 min at 37°C, and the mixture was shaken with 1 x 106 cocultured PHA-activated human PBMCs for 60 min in a 37°C water bath (5, 15). After washing, the cells were cultured in the presence of recombinant human IL-2 (40 U/ml; Shionogi, Osaka, Japan) for 7 days. Following the incubation, culture supernatants were subjected to a p24 Ag ELISA (Dinabot, Tokyo, Japan) for the measurement of HIV. Data were expressed as the percent inhibition of p24 Ag production in the culture supernatants compared with that in the cultures to which preimmune or nonimmunized serum IgG was added. Virus stocks were titrated on the PHA-activated normal PBMCs, and the 50% tissue culture infective dose of each virus was evaluated as described previously (5, 15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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High titer values for V3 peptide-specific Abs responses were
induced by nasal and oral immunization of rBCG-V3J1 (Fig. 1
). The levels of mucosally induced
Ag-specific IgG responses were comparable to those induced by the
systemic route (Fig. 1). High titers of V3J1-specific IgG Abs were
detected 4 wk after initial immunization by all tested administration
routes (Fig. 1). It is important to note that high levels of V3
peptide-specific Abs responses were maintained >1 yr (e.g., 54 wk)
after initial immunization via nasal and systemic routes compared with
oral administration. Further, a lower dose (10 µg) of rBCG-V3J1 was
required for the induction of an Ag-specific immune response in nasally
immunized mice compared with orally immunized mice (Fig. 1). However,
V3J1-specific IgA Ab was not detected during the entire period of Ab
monitoring in either systemic (e.g., serum) or mucosal secreted (e.g.,
saliva, fecal extract, and vaginal wash) samples (data not shown).
Taken together, these results indicated that nasal vaccination with
rBCG-V3J1 was an effective mucosal immunization route for the induction
of an Ag-specific systemic immune response. Nasal immunization with
rBCG-V3J1 can induce and maintain high levels of HIV-specific IgG Abs
responses for >1 yr. Further, histological analysis revealed no
evidences for the development of local pathological change in mice
mucosally immunized with rBCG-V3J1. Thus, the detailed analysis of
quality and quantity of V3 peptide-specific immune responses was
focused on nasal vaccination with rBCG-V3J1 in the rest of this
study.
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Mononuclear cells were isolated from SP and different
mucosa-associated tissues (e.g., NALT, NP, SMG, PP, and i-LP) of mice
nasally immunized with rBCG-V3J1 to confirm the induction of
Ag-specific Ab-forming cells. The isotype and Ag-specific ELISPOT assay
revealed the presence of increased numbers of V3 peptide-specific IgG
Ab-forming cells in the systemic compartment (e.g., SP; Fig. 2). In addition, Ag-specific IgG
Ab-producing cells were detected in several mucosa-associated tissues,
including NALT, PP, NP, and i-LP (Fig. 2). To this end, some
V3J1-specific IgG Abs were detected in mucosal secretions (e.g., log2
titer of 4.7 ± 0.6 in fecal extract at 7 wk after immunization).
As indicated by the ELISA data (data not shown), however, V3J1-specific
IgA Ab-producing cells were not seen in all isolated tissues (Fig. 2).
These results showed that nasal immunization with rBCG-V3J1 was capable
of inducing Ag-specific IgG-producing cells in both SP and
mucosa-associated tissues, including NP, NALT, PP, and i-LP.
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Inasmuch as high levels of V3J1-specific IgG responses were
induced in the systemic immune compartment by nasal vaccination with
rBCG-V3J1, it was important to examine the nature of V3J1-specific Th
cell responses (e.g., Th1 and Th2 types) induced in these nasally
immunized mice. To characterize Ag-specific Th1- and Th2-type
responses, CD4+ T cells were isolated from SP and
mucosa-associated tissues of mice given nasal vaccine and restimulated
with V3J1-KLH in vitro. Culture supernatants from V3J1-stimulated
CD4+ T cells were then examined for the presence
of Th1 and Th2 cytokines by ELISA. High levels of Th1 (e.g., IFN-
and IL-2) cytokines were detected in the culture supernatant harvested
from in vitro V3J1-stimulated CD4+ T cells
isolated from SP and mucosa-associated tissues, such as NP, NALT, and
PP (Fig. 3). In contrast, Th2-type
cytokines were generally not detected. Taken together, these findings
suggested that nasal immunization with rBCG-V3J1 preferentially induced
Ag-specific IFN-- and IL-2-producing Th1-type cells in both systemic
and mucosa-associated sites.
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To seek a potential applicability of nasal rBCG-V3J1 for the
development of therapeutic vaccine, Th1
(IFN--/-)- and Th2
(IL-4-/-)-deficient mice were nasally immunized
with rBCG-V3J1. Increased levels of Ag-specific IgG Abs were induced in
these immunodeficient mice (Fig. 4). At
levels similar to nasally vaccinated wild-type mice, these high levels
of V3 peptide-specific IgG Abs responses were also maintained for at
least 27 wk in both IFN-- and IL-4-deficient mice (Fig. 4). When the
kinetics of these Ag-specific IgG Ab responses was compared, the
induction of high levels of V3 peptide-specific IgG Abs was delayed a
few weeks in IFN--/- mice compared with
IL-4-/- and wild-type mice (Fig. 4). These
results raised the interesting possibility that an rBCG-V3J1 nasal
vaccine might be an effective immunization tool to induce V3
peptide-specific immune responses under the different immunodeficient
conditions of HIV-infected patients through several progression stages
of AIDS.
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We next examined the IgG subclass of V3 peptide-specific Abs
induced by nasal vaccination in these immunodeficient and normal mice.
In wild-type mice, the levels of V3J1-specific IgG2a- and IgG3-isotype
Abs responses were highest, followed by IgG1 and IgG2b (Fig. 5). As one might expect, Ag-specific IgG1
and IgG2a Abs were not detected in IL-4- and IFN--deficient mice,
respectively (Fig. 5). However, high levels of V3J1-specific IgG3 Abs
were induced in these two immunodeficient mice. These results indicated
that high levels of V3 peptide-specific serum IgG3 responses were
induced and maintained by rBCG-V3J1 nasal vaccine for at least
0.5–1 yr despite different immunological conditions.
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For the characterization of virus neutralization activity, a
well-characterized laboratory strain of HIV-MN and one primary clinical
isolate HIV-MNp, which expressed IHIGPGRAFY at the core sequence of the
HIV principal neutralizing determinant (3, 5), were used
as the virus source. Nasal rBCG-V3J1-induced serum IgG Abs possessed
some neutralizing activity against HIV-MN in wild-type mice (Fig. 6). Further, HIV-MNp was neutralized by
the IgG immune serum (Fig. 6). A similar neutralization activity was
noted by the IgG immune serum purified from both IFN-- and
IL-4-deficient mice nasally vaccinated with rBCG-V3J1 (Fig. 6).
However, preimmune IgG showed no neutralizing activity (Fig. 6). These
results indicate that rBCG-V3J1 nasal vaccine is capable of inducing
high V3 peptide-specific IgG Abs with some virus-neutralizing activity.
Furthermore, it should be noted that high levels of V3 peptide-specific
IgG Abs with a detectable level of neutralizing activity were
maintained for a long time (e.g., 7–27 wk) after the nasal
immunization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-)- and Th2
(IL-4-/-)-deficient mice.
An important finding of the present study is a demonstration of the
induction of prolonged V3 peptide-specific IgG Ab responses with
neutralizing activity by nasal immunization with rBCG-V3J1. To this
end, a high titer of Ag-specific IgG Abs was induced and maintained
over 1 yr following three doses of nasal immunization without any
booster vaccination (Fig. 1
). Further, the original BCG vaccination has
been shown to induce prolix immunity after minimum doses of
immunization (24). Our finding suggested that rBCG
carrying the V3J1 gene may intracellularly coincide in selected tissue
of nasally immunized mice for continuous delivery of the peptide to
immunocompetent cells. In our separate study the current effort is
focused on the analysis of a dissemination pathway and determining the
source of inhabiting cells and tissues. Our preliminary results
suggested that specific message for BCG can be found in NALT and SP 1
wk after final nasal immunization (data not shown). However, we could
not yet recover actual BCG from these tissues of mice nasally immunized
with rBCG-V3J1. Based on the immunological evidence obtained by the
present study, it is plausible to speculate that intracellular rBCG
produces V3J1, which continuously stimulates a Th1-type response that,
in turn, induces neutralizing IgG Ab responses in nasally
immunized mice.
When a subclass of V3 peptide-specific IgG Abs was examined in normal
mice nasally immunized with rBCG-V3J1, Ag-specific Ab was found in all
subclasses (Fig. 5). However, higher levels of Ag-specific IgG2a and
IgG3 Abs were noted compared with IgG1 and IgG2b. According to the
profile of V3J1-specific IgG subclass distribution, one would predict
that the balance between Th1- and Th2-type responses is shifted toward
the former response. Analysis of Th cytokine synthesis by
V3J1-stimulated CD4+ T cells from different
mucosal and systemic tissues of nasally vaccinated mice further
reinforces this view, since a predominant production of Th1 cytokines
(e.g., IFN- and IL-2) was observed in CD4+ T
cells isolated from these tissues (Fig. 3). It has been also shown that
intracellular micro-organisms such as Mycobacterium,
Listeria, and Salmonella, have been shown to
preferentially induce Th1-type responses (25, 26, 27).
It was interesting to note that increased titers of V3 peptide-specific
IgG3 Abs were also induced in addition to Th1-regulated IgG2a and
Th2-dependent IgG1 responses (Fig. 5). The latter two subclasses were
known to be involved in anti-viral and anti-protein responses,
respectively (28). Thus, this Ag-specific IgG subclass Ab
could be a key player in the HIV-neutralizing activity induced by nasal
immunization with rBCG-V3J1. In the case of IgG3, this subclass was
shown to involve carbohydrate-specific Ab responses (29).
At this time we do not know of any explanation or mechanism for the
induction of this subclass of Ab by nasally administered rBCG
expressing the peptide. Further, the exact contribution of V3
peptide-specific IgG3 in virus-specific immunity is still not
known.
It is well known that BCG immunization generally induces a strong
Th1-type immune response, including those of high IFN- synthesis
(30, 31). Thus, it was important to examine whether
V3J1-specific Th1-type immune responses were induced by nasal
vaccination with rBCG-V3J1. As one might expect, high levels of
Ag-specific Th1-type CD4+ Th cell-induced IFN-
and IL-2 cytokines were noted (Fig. 3). To this end, high levels of Th1
cell-regulated IgG subclass responses were also induced in these
nasally immunized mice (Fig. 5). However, V3J1-specific IgA Ab was not
detected through the period of Abs monitoring not only in mucosal
secreted samples (saliva, fecal extract, and vaginal wash), but also in
serum (data not shown). Because of the preferential induction of
Ag-specific Th1-type responses by nasal immunization with rBCG-V3J1,
Th2-type immune responses were totally inhibited. Inasmuch as Th2
cell-derived cytokines such as IL-5 and IL-6 have been shown to be
essential for the induction of IgA (32), the deficiency of
the Th2-type response could be a major contributing factor for the lack
of Ag-specific IgA in mice nasally immunized with rBCG-V3J1.
Another important aspect of this study is that nasal vaccination with
rBCG-V3J1 is capable of inducing high levels of prolonged Ag-specific
neutralizing IgG responses in Th1- or Th2-type immunodeficient mice in
addition to wild-type mice for at least 0.5–1 yr (Figs. 1, 4, and 6).
In human AIDS, a dichotomy of Th1 and Th2 predominance in HIV-infected
individuals has became a central issue of debate. It was originally
demonstrated that a switch from Th1- to Th2-type responses occurs
during the development of HIV infection (33). However,
other studies suggest that a shift from Th1 to Th2 responses does not
occur during the progression of HIV infection (34).
Furthermore, it was demonstrated that HIV preferentially replicates
within CD4+ T cells with the phenotype of Th0 and
Th2 cytokine synthesis (35). When we consider the
development of a mucosal vaccine, especially for the purpose of a
therapeutic vaccine, we must also account for these dysregulated Th1
and Th2 cell profiles in AIDS. Our present finding has provided a
potential application of rBCG-V3J1 for the therapeutic mucosal vaccine,
since nasal administration of the vaccine construct resulted in the
induction of V3J1-specific IgG Abs with neutralizing activity in Th1-
and Th2-type immunodeficient mice (
Figs. 4–6).
In summary, our findings have presented a new possibility that the
mucosal rBCG-V3J1 vaccination system is a useful immunization regimen
for not only the prevention of HIV infection but also for the
development of therapeutic vaccine. Further, rBCG-V3J1 can induce
prolonged V3 peptide-specific IgG Abs with neutralizing activity for
more than 0.5–1 yr in both normal and immunodeficient (e.g.,
IFN--/- and IL-4-/-)
mice. Since rBCG is confirmed for biological safety, and the original
Tokyo strain has been already used for a human tuberculosis vaccine,
the concept of a nasal rBCG-V3J1 vaccine can be considered for the
development of the next generation of AIDS vaccine. However, we have to
emphasize that the safety and effectiveness of mucosal rBCG-based HIV
vaccine must be continuously and carefully examined both in in vivo and
in vitro systems before possible application to humans.
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Acknowledgments |
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Footnotes |
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2 T.H. and H.G. 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 565-0871, Japan. E-mail address: kiyono{at}biken.osaka-u.ac.jp
4 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; CT, cholera toxin; i-LP, intestinal lamina propria; KLH, keyhole limpet hemocyanin; NALT, nasopharyngeal-associated lymphoreticular tissue; NP, nasal passage; PP, Peyer’s patch; rBCG-V3J1, recombinant BCG vector-based vaccine that secretes the V3J1 principal neutralizing epitope of HIV; SMG, submandibular glands; SP, spleen.
Received for publication March 12, 2001. Accepted for publication September 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing T cells, T cells receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues. J. Immunol. 148:2030.[Abstract]
, and MIP-1
as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811. in the generation of protective T cells against Mycobacterium bovis BCG in mice. Immunology 91:529.[Medline]
in response to bacillus Calmette-Guérin. J. Immunol. 163:4246.This article has been cited by other articles:
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  J.-S. Yu, J. W. Peacock, W. R. Jacobs Jr., R. Frothingham, N. L. Letvin, H.-X. Liao, and B. F. Haynes Recombinant Mycobacterium bovis Bacillus Calmette-Guerin Elicits Human Immunodeficiency Virus Type 1 Envelope-Specific T Lymphocytes at Mucosal Sites Clin. Vaccine Immunol., July 1, 2007; 14(7): 886 - 893. [Abstract] [Full Text] [PDF]
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J.-S. Yu, J. W. Peacock, S. Vanleeuwen, T. Hsu, W. R. Jacobs Jr., M. J. Cayabyab, N. L. Letvin, R. Frothingham, H. F. Staats, H.-X. Liao, et al. Generation of Mucosal Anti-Human Immunodeficiency Virus Type 1 T-Cell Responses by Recombinant Mycobacterium smegmatis{triangledown} Clin. Vaccine Immunol., November 1, 2006; 13(11): 1204 - 1211. [Abstract] [Full Text] [PDF]
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 M. Kanekiyo, K. Matsuo, M. Hamatake, T. Hamano, T. Ohsu, S. Matsumoto, T. Yamada, S. Yamazaki, A. Hasegawa, N. Yamamoto, et al. Mycobacterial Codon Optimization Enhances Antigen Expression and Virus-Specific Immune Responses in Recombinant Mycobacterium bovis Bacille Calmette-Guerin Expressing Human Immunodeficiency Virus Type 1 Gag J. Virol., July 15, 2005; 79(14): 8716 - 8723. [Abstract] [Full Text] [PDF]
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K. Someya, D. Cecilia, Y. Ami, T. Nakasone, K. Matsuo, S. Burda, H. Yamamoto, N. Yoshino, M. Kaizu, S. Ando, et al. Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guerin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif J. Virol., February 1, 2005; 79(3): 1452 - 1462. [Abstract] [Full Text] [PDF]
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) and 27 wk (
)
and from immune-deficient mice (IFN-
) and
IL-4-/- (
)) 6 mo after the initial nasal immunization
with 10 µg rBCG-V3J1. These immune sera were analyzed for the
presence of neutralizing Ab against a clinically isolated HIV-1 strain
(HIV-MNp) or a laboratory strain (HIV-MN). As a control, serum samples
from preimmunized mice were tested and did not show any Ag-specific
neutralization activity (
). Results are the values (mean ±
SEM) for four mice in each experimental group.