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Journal of Virology, April 2000, p. 3427-3429, Vol. 74, No. 7
Department of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania1; Henry M. Jackson
Foundation, Rockville, Maryland2;
Primedica Mason Laboratories, Worcester,
Massachusetts3; and Department of
Medical Microbiology and Immunology, University of South Florida,
Tampa, Florida4
Received 11 October 1999/Accepted 22 December 1999
An important limitation of DNA immunization in nonhuman primates is
the difficulty in generating high levels of antigen-specific antibody
responses; strategies to enhance the level of immune responses to DNA
immunization may be important in the further development of this
vaccine strategy for humans. We approached this issue by testing the
ability of molecular adjuvants to enhance the levels of immune
responses generated by multicomponent DNA vaccines in rhesus macaques.
Rhesus macaques were coimmunized intramuscularly with expression
plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma
interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs
encoding human immunodeficiency virus Env and Rev and simian
immunodeficiency virus Gag and Pol proteins. We observed that the
cytokine gene adjuvants (especially IL-2 and IL-4) significantly
enhanced antigen-specific humoral immune responses in the rhesus
macaque model. These results support the assumption that
antigen-specific responses can be engineered to a higher and presumably
more desirable level in rhesus macaques by genetic adjuvants.
Numerous vaccines that stimulate the
production of protective antibodies have proven successful for
combating diseases such as hepatitis A and B, measles, and
poliomyelitis. As a novel and important vaccination technique, nucleic
acid or DNA immunization delivers DNA constructs encoding specific
immunogens directly into the host (10-12, 14, 15). These
expression cassettes transfect host cells, which become the in vivo
protein source for the production of antigen. This antigen then is the
focus of the resulting humoral and cellular immune responses. Nucleic
acid immunization is being explored as an immunization strategy against
a variety of infectious diseases (10-12, 14, 15).
To support the ultimate use of this vaccine technology in humans, it
may be important to translate the results originally observed in
small-animal systems to similar levels in primate model systems
(4). The nonhuman primates represent an important and
relevant model for vaccine evaluation (2, 7). These animals
are the closest species to humans, and there are numerous challenge
models for various infectious agents.
On the other hand, it has been reported that primates may have a
limited ability to produce DNA vaccine-encoded proteins through direct
genetic inoculation into muscle (3). An exact mechanism for
producing such proteins is unclear, and a major challenge of
investigating DNA immunization in nonhuman primates is the difficulty
in eliciting potent immune responses. For instance, DNA immunizations
alone in primates were not sufficient to generate high levels of
antigen-specific antibody responses (6). Intramuscular immunization of a human immunodeficiency virus type 1 (HIV-1) gp120 DNA
vaccine construct using a large dose (2 mg of DNA given eight times at
4-week intervals) in rhesus macaques elicited only a low level of
antigen-specific binding and no detectable neutralizing antibodies
(6). These observations of reduced humoral immunogenicity of
DNA vaccines in nonhuman primates suggest the need for higher doses in
humans. Thus, strategies to enhance the level of immune responses to
DNA immunization may be important in the further development of this
vaccine strategy for humans.
Several groups, including ours, have been investigating the use of
molecular adjuvants as a method of enhancing and modulating immune
responses induced by DNA immunogens. Codelivery of these molecular
adjuvants consisting of an expression plasmid bearing genes coding for
immunologically relevant molecules, including costimulatory molecules,
cytokines, and chemokines, with DNA vaccine constructs led to
modulation of the magnitude and direction (humoral or cellular) of the
immune responses induced in mice (1a, 2a, 5, 9, 16). It has
been reported recently that the modulation of immune responses through
this approach may modulate disease progression in several mouse
challenge models (9, 16). These results support the idea
that disease can be modulated by the use of cytokine adjuvants, at
least in mice; however, the effects of this strategy in nonhuman
primates have not been extensively reported.
In this study, we examined the use of cytokine cDNAs to enhance the
level of humoral immune responses generated by DNA vaccines in rhesus
macaques. We coimmunized rhesus macaques with expression plasmids
bearing genes encoding either Th1 (interleukin 2 [IL-2] and gamma
interferon [IFN- Five groups of two rhesus macaques each were immunized with specific
DNA vaccine constructs. The first group was immunized with constructs
coding for HIV-1 MN Env and Rev (pCEnv) and Rev-independent SIV Gag and
Pol (pCSGag/pol) antigens along with a control vector, pCDNA3. The
second group was immunized with pCEnv plus pCSGag/pol plus IL-2
constructs. The third and fourth groups were immunized with pCEnv plus
pCSGag/pol plus IL-4 and pCEnv plus pCSGag/pol plus IFN- Both pre- and postimmunization serum samples from the immunized
macaques were collected, and binding reactivities to recombinant HIV-1
envelop and SIV Gag proteins were determined by enzyme-linked immunosorbent assay. As shown in Fig. 1,
macaques immunized with pCEnv plus pCSGag/pol plus pCDNA3 had minimal
levels of anti-envelope or anti-Gag antibody responses following
immunization. On the other hand, a significant enhancement of the
levels of anti-envelope or anti-Gag antibodies was observed in the
animals immunized with pCEnv plus pCSGag/pol plus IL-2. In fact, the
magnitude of antibody response enhancement with IL-2 codelivery in
macaques was even greater than that observed in mice: a 4-fold increase
in endpoint titer was seen in mice compared to an over 100-fold
increase in titer in macaques against envelope and Gag proteins (Fig.
1) (5). Similarly, IL-4 coimmunization also positively
modulated the antigen-specific antibody responses. The group immunized
with pCEnv plus pCSGag/pol plus IL-4 developed a significant level of
anti-envelope-specific antibodies and a high antibody response against
Gag. Macaques immunized with pCEnv plus pCSGag/pol plus IFN-
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Modulation of Antigen-Specific Humoral Responses in Rhesus
Macaques by Using Cytokine cDNAs as DNA Vaccine Adjuvants
ABSTRACT
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])- or Th2 (IL-4)-type cytokines and DNA vaccine
constructs encoding HIV-1 MN Env and Rev (pCEnv) and simian
immunodeficiency virus (SIV) mac239 Gag and Pol (pCSGag/pol) proteins.
We observed that antigen-specific humoral immune responses could be
modulated positively in the macaque models using this approach.
,
respectively. The last control group was immunized with a control
vector, pCDNA3. These macaques were immunized with 200 µg of each DNA
at weeks 0, 6, and 12 and boosted with 500 µg of each DNA at week 28. These constructs were formulated and mixed prior to injection into the
quadriceps muscle (1).
had a
more moderate response against both envelop and Gag proteins. These
results demonstrate that antigen-specific antibody responses can be
driven to a higher and presumably more desirable level through the use of cytokine genetic adjuvants in rhesus macaques.
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FIG. 1.
Modulation of antibody responses in rhesus macaques.
Five groups of two rhesus macaques were immunized with 200 µg of each
DNA vaccine construct at weeks 0, 6, and 12 and boosted with 500 µg
of each DNA at week 28. Serum samples were collected from the immunized
macaques at weeks 0, 18, and 36. Binding reactivities to recombinant
HIV-1 gp120 envelope and SIV p27 Gag proteins (ImmunoDiagnostics, Inc.,
Bedford, Mass.) were determined by enzyme-linked immunosorbent assay as
previously described (1). Specific binding (absorbance at
450 nm) was calculated by subtracting A450
values from serum samples bound to bovine serum albumin (control) from
A450 values from serum samples bound to gp120,
that is, the A450s of experimental wells minus
the A450s of control wells. The endpoint
antibody titers for immunized rhesus macaques were determined as
previously described (1).
We also examined the ability of the antibodies from immunized macaques
to neutralize homologous HIV-1 MN or heterologous HIV-1 IIIB (Table
1). Although IL-2 or IL-4 coimmunization
resulted in enhanced levels of serum antibody responses, these animals showed only a minimal level at best of neutralizing antibodies against
the homologous HIV-1 MN isolate. On the other hand, similar to what
occurs with protein vaccines, none of the serum antibodies were able to
neutralize HIV IIIB, a divergent virus. Although these neutralizing
titers were still low overall, they illustrate the potential of this
approach. Higher dosages and frequencies of injections are expected to
further enhance the observed levels of neutralizing antibodies.
Combinations of envelopes might broaden the observed responses.
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These results indicate that the cytokine gene adjuvants can positively enhance antibody responses in rhesus macaques by intramuscular injections of DNA vaccines. This simple strategy has important implications for vaccines and immunotherapy approaches using the DNA platform. The use of molecular adjuvants (especially IL-2 or IL-4) to enhance antibody responses might be important in disease models such as hepatitis B, where the generation of antibodies is sufficient to provide protective immunity.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants from the NIH to D.B.W. and from NHLBI/NIH to K.E.U.
We thank R. Ciccarelli from WLV for thoughtful discussion and providing material for this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104. Phone: (215) 349-8365. Fax: (215) 573-9436.
Present address: Merck Research Laboratories, West Point, PA 19486.
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Journal of Virology, April 2000, p. 3427-3429, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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