Gene Ther Mol Biol Vol 3,
157-165. August 1999.
Rational
vaccine design through the use of molecular adjuvants
Jong J. Kim1,
Liesl K. Nottingham2, Jim Oh1, Daniel Lee1,
Ken Wang1, Mera Choi3, Tzvete Dentchev1,
Darren Wilson, Devin M. Cunning2, Ara A. Chalian2, Jean
Boyer, Jeong I. Sin1, and David B. Weiner1
1Department of Pathology and Laboratory Medicine; 2Department
of Otolaryngology/Head and Neck Surgery, University of Pennsylvania,
Philadelphia, PA 19104, 3Bryn Mawr College
_________________________________________________________________________________________________
Corresponding Author: Jong J. Kim, Ph.D., Department of Pathology and Laboratory Medicine,
University of Pennsylvania, 505 Stellar-Chance, 422 Curie Blvd., Philadelphia, PA
19104. Tel: (215) 662-2352; Fax:
(215) 573-9436; E-mail: jonger@seas.upenn.edu
Received: 18 September 1998;
accepted: 25 September 1998
Nucleic acid
immunization is an important vaccination strategy which delivers DNA constructs
encoding for a specific immunogen into the host. These expression cassettes
transfect the host cells, which become the in
vivo protein source for the production of antigen. This antigen then is the
focus of the resulting immune response. This vaccination technique is being
explored as an immunization strategy against a variety of infectious diseases
as well as cancer. The first generation DNA immunization experiments have shown
that the DNA vaccinesÕ ability to elicit humoral and cellular responses in vivo in a safe and well-tolerated
manner in various model systems, including humans. As we explore the next
generation of DNA vaccines, our goal is to refine the current strategy to
elicit more clinically efficacious immune responses. A more clinically
effective vaccine may need to elicit a more specific immune response against
the targeted pathogen. It would be a distinct advantage to design immunization
strategies which can be ÒfocusedÓ according to the correlates of protection
known for the particular pathogen.
In order to focus the immune responses induced from DNA immunization, we have investigated the co-delivery of genes for immunologically important molecules, such as costimulatory molecules and cytokines which play critical regulatory and signaling roles in immunity. We and others have shown that the use of these molecular adjuvants could enhance and modulate immune responses induced by DNA immunogens. Co-administration of costimulatory molecules (CD80 and CD86), proinflammatory cytokines (IL-1a, TNF-a, and TNF-b), Th1 cytokines (IL-2, IL-12, IL-15, and IL-18), Th2 cytokine (IL-4, IL-5 and IL-10), and GM-CSF with DNA vaccine constructs led to modulation of the magnitude and direction (humoral or cellular) of the immune responses. These studies demonstrate the potential utility of molecular adjuvant strategy as an important tool for the development of more rationally designed vaccines.
Although
the injection of DNA into tissues was originally reported in the 1950s, the
technology has gained more attention in recent years as a safe means of
mimicking in vivo protein production
normally associated with natural infection (Stasney et al, 1955; Paschkis et al, 1955; Ito,
1960). Nucleic acid or DNA inoculation is an important vaccination technique
which delivers DNA constructs encoding specific immunogens directly into the
host (Wolff et al, 1990; Tang et al, 1992; Wang et al,
1993; Ulmer et al 1993; Kim et al, 1997a; Agadjanyan et al, 1997, Tascon et al,
1996, Conry et al, 1996). This injection results in the subsequent expression of the foreign
gene in that host and the presentation of the specific encoded proteins to the
immune system. DNA vaccine constructs are produced as small circular vehicles
or plasmids. These plasmids are constructed with a promoter site which starts
the transcription process, an antigenic DNA sequence and a messenger RNA stop
site containing the poly A tract necessary for conversion of the messenger RNA
sequence into the antigen protein by the ribosomal protein manufacturing
machinery (Figure 1). This antigen
then is the focus of the resulting immune response. This vaccination technique
is being explored as an immunization strategy against cancer as well as a
variety of infectious diseases including AIDS.
II. Potential advantages of DNA vaccines
Nucleic acid
immunization may afford several potential advantages over traditional
vaccination strategies such as whole killed or live attenuated virus and
recombinant protein-based vaccines (Kim
and Weiner, 1997; Chattergoon et al, 1997). Since DNA vaccines are non replicating and the vaccine components are
produced within the host cells, they can be constructed to function safely with
the specificity of a subunit vaccine. However, DNA vaccine cassettes produce
immunological responses that are more similar to live vaccine preparations. By
directly introducing DNA into the host cell, the host cell is essentially
directed to produce the antigenic protein, mimicking viral replication or tumor
cell marker presentation in the host. This process has been reported to
generate both antibody and cell mediated, particularly cytotoxic T
cell-mediated, immunity (Figure 2).
Unlike a live attenuated vaccine, conceptually there is little risk from
reversion to a disease-causing pathogen from the injected DNA, and there is no
risk for secondary infection as the material injected is not-living and
not-infectious. In addition, genes which lead to undesired immunologic
inhibition or cross-reactivity (autoimmunity) may be either altered or deleted
altogether. Finally, DNA vaccines can be manipulated to present a particular
genome of the pathogen or display specific tumor antigens in non-replicating
vectors (Figure 1).
Figure 1. Potential immunologic targets for DNA
vaccination against HIV-1. These
targets include env, gag, and pol genes as well as the four accessory
genes.
Figure 2. Induction of antigen-specific humoral and cellular immune responses.
III. Molecular adjuvants as a immune modulation strategy
The
overall objective of any immunization strategy is to induce specific immune
responses which protect the immunized individual from a given pathogen over his
or her lifetime. One major challenge in meeting this goal is that the
correlates of protection from an individual pathogen vary from one infectious
agent to the next. The first generation DNA immunization experiments have shown
that the DNA vaccinesÕ ability to elicit humoral and cellular responses in vivo in a safe and well-tolerated
manner in various model systems, including humans. As we explore the next
generation of DNA vaccines, our goal is to refine the current strategy to
elicit more clinically efficacious immune responses. A more clinically effective
vaccine may need to elicit a more specific immune response against the targeted
pathogen. It would be a distinct advantage to design immunization strategies
which can be targeted according to the correlates of protection known for the
particular pathogen (Figure 3). Such
refinement could be accomplished by co-delivering genes for immunologically
important molecules, such as costimulatory molecules and cytokines which play
critical regulatory and signaling roles in immunity (Kim
and Weiner, 1997). These molecular adjuvant constructs could be co-administered along
with immunogen constructs to modulate the magnitude and direction (humoral or
cellular) of the immune responses induced (Figure
4).
There has been several reports of immune modulation by
protein delivered cytokines. However, the results in general appeared marginal.
More recently, we and others have focused on analyzing immune responses induced
to such gene delivery. Raz et al. observed that intramuscular injections of
plasmids encoding IL-2, IL-4, or TGF-b1 modestly modulated immune responses to transferrin protein delivered
at a separate site (Raz et al, 1993). IL-2 immunization resulted in an enhancement of antibody and T helper
proliferative responses while TGF-b1 immunization reduced anti-transferrin responses.
Figure 3. The potential utility of the molecular adjuvant network.
Tailoring the induction of specific immune responses by vaccination programs
against viral, bacterial, or parasitic diseases could be beneficial.
Figure 4. Cytokines as immune
response regulators. Cytokines play critical roles in the immune and
inflammatory responses. Based upon their specific function in the immune system
these cytokines could be further grouped as proinflammatory, Th1, and Th2
cytokines. Along with costimulatory molecules, these cytokines also play
important roles in the activation and proliferation of T and B cells.
IV. Modulation of immune responses using cytokine molecular adjuvants
In order to
focus the immune responses induced from DNA immunization, we have investigated
the co-delivery of molecular adjuvants. We first reported that co-immunization
of GM-CSF genes with DNA vaccine constructs increases antigen-specific antibody
and T helper cell proliferation responses while co-immunization with IL-12
genes results in weaker antibody responses and enhanced T helper cell
proliferation (Kim et al, 1997b,c). In addition, IL-12 co-immunization resulted in a significant
enhancement of CTL responses. Importantly, we observed a significant
enhancement of CTL response in vivo with the co-administration of murine IL-12
genes with four different HIV-1 DNA immunogens (gag/pol, envelope, vif, and nef)
which were CD8+ T cell- and MHC class I-restricted. In contrast,
almost no effect on CTL induction was observed with the genes for GM-CSF in
these studies. Moreover, Iwasaki et al. (1997) reported that GM-CSF and IL-12
co-delivery with DNA immunogen encoding for influenza NP resulted in enhanced
cellular immune responses. Moreover, Agadjanyan et al. (1997) reported that
co-administration of IL-12 genes with HIV-2 DNA immunogen resulted in a
dramatic enhancement of both Th and CTL responses. Furthermore,
co-administration of IL-12 genes with DNA immunogens strongly directed the
antigen specific immune response towards a Th1 type immunity and induced
delayed type hypersensitivity (DTH) to contact allergens as an in vivo model of
the Th1 response (Kim et al, 1998a). In addition to these reports, Chow et al. reported that either
injection of plasmid co-expressing hepatitis B surface antigen (HBsAg) and IL-2
or co-injection of IL-2 genes with plasmid expressing HBsAg resulted in the
enhancement of both antibody and T helper cell responses (Chow
et al, 1997).
More recently, we investigated the induction and
regulation of immune responses from the co-delivery of proinflammatory
cytokines (IL-1a, TNF-a, and TNF-b), Th1 cytokines (IL-2, IL-15, and IL-18), and Th2
cytokines (IL-4, IL-5 and IL-10) (Figure
5) (Kim et al, 1998b). We observed enhancement of antigen-specific humoral response with the
co-delivery of Th2 cytokines IL-4, IL-5, and IL-10 as well as that
of IL-2 and IL-18. A dramatic increase in antigen-specific T helper cell
proliferation was seen with IL-2 and TNF-a co-injections. In addition, we observed a significant enhancement of
the cytotoxic response with the co-administration of TNF-a and IL-15 genes with HIV-1 DNA immunogens. These
increases in CTL response were both MHC class I-restricted and CD8+
T cell-dependent. We also investigated whether the Th1 or Th2-type immune
responses are more important for protection from HSV-2 infection (Sin
et al, 1998). We co-delivered DNA expression construct encoding for HSV-2 gD
protein with the gene plasmids encoding for Th1-type (IL-2, 12, 15, 18) and
Th2-type (IL-4, IL-10) cytokines in an effort to drive immunity induced by
vaccination. We then analyzed the vaccine modulatory effects on resulting
immune phenotype and on the mortality and the morbidity of the immunized
animals following HSV lethal challenge. We observed Th1 cytokine gene
co-administration not only enhanced survival rate, but also reduced the
frequency and severity of herpetic lesions following intravaginal HSV challenge
(Figure 6). On the other hand,
co-injection with Th2 cytokine genes increased the rate of mortality and
morbidity of the challenged mice. Again, among the Th1 type cytokine genes
tested IL-12 was particularly a potent adjuvant for the gD DNA vaccination.
V. Modulation of immune responses using costimulatory molecule adjuvants
The
generation of the T cell immune response is a complex process that requires the
engagement of T cells with professional APCs such as dendritic cells,
macrophages, and B cells. These professional APCs possess large surface areas
for interaction with T cells. They also express high levels of MHC class I and
II molecules, adhesion molecules, and costimulatory molecules which are critical
for efficient antigen presentation and T cell activation. Professional APCs
initiate T cell activation by binding antigenic peptide-MHC complexes to T cell
receptor molecules. In addition, the APCs provide secondary signals through the
ligation of costimulatory molecules with their receptors (CD28/CTLA-4) present
on T cells. These costimulatory signals are required for the clonal expansion
and differentiation of T cells. The blocking of this additional costimulatory
signal leads to T cell anergy (Schwartz
et al, 1992). Among different costimulatory molecules, CD80 and CD86 have been
observed to provide potent immune signals (
Lanier et al, 1995, Linsley et al, 1990).
The CD80 and CD86 molecules are surface glycoproteins
and members of immunoglobulin superfamily which are expressed only on
professional APCs (Lanier et al, 1995, Linsley et al, 1990, June et
al,1994). Although both CD80 and
CD86 molecules interact with either CD28 or CTLA-4 molecules on T cells, CD80
and CD86 expression seem to be differentially regulated. CD86 is constitutively expressed by the
APCs whereas CD80 is expressed only after activation of these cells (Freeman
et al, 1989; Azuma et al, 1993; Freedman et al, 1991). Thus, CD86 may be important in the early interactions between APCs
and T cells during the induction phase of the immune response.
Figure 5. Each cytokine gene was cloned into expression plasmids under the
control of a CMV promoter.
Figure 6. Protection from lethal
HSV-2 challenge. Each group of mice (n=10) was immunized with gD DNA vaccines
(60 mg), and/or cytokine genes (40 mg) at 0 and 2 weeks. Three
weeks after the second immunization, mice (n=8) were challenged i.v. with 200 x
LD50 of HSV-2 strain 186 (7 x 105 pfu).
We recently reported that CD86 molecules play a
prominent role in the antigen-specific induction of CD8+ cytotoxic T
lymphocytes when delivered as vaccine adjuvants (Figure 7) (Kim et al, 1997a). Co-administration of CD86 cDNA along with DNA encoding HIV-1 antigens
intramuscularly dramatically increased antigen-specific T-cell responses
without a significant change to the level of the humoral response. This
enhancement of cytotoxic T lymphocyte (CTL) response was both major
histocompatibility complex (MHC) class I-restricted and CD8+ T
cell-dependent. Similar results have been obtained by other investigators who
also found that CD86, not CD80 co-expression results in the enhancement of T
cell-mediated immune responses (Tsuji
et al, 1997; Iwasaki et al, 1997). Accordingly, we speculate that engineering of non-professional APCs
such as muscle cells to express CD86 costimulatory molecules could empower them
to prime CTL precursors. On the other hand, the enhancement effect of CD86
co-delivery could also have been mediated through the direct transfection of a
small number of professional APCs residing within the muscle tissue.
Subsequently, these cells could have greater expression of costimulatory
molecules and could in theory become more potent.
As
summarized in Figure 8, we observed
that significant modulation was possible using molecular adjuvants. This
cytokine gene adjuvant network underscores an important level of control in the
induction of specific immune responses to tailor vaccination programs more
closely to the correlates of protection which vary from disease to disease.
This type of fine control of vaccine and immune therapies was previously very
difficult to obtain. Controlling the magnitude and direction of the immune
response could be advantageous in a wide variety of vaccine strategies. For
instance, in a case where T cell mediated response is paramount, but the
humoral response may not be needed or even be harmful, IL-12 genes could be
chosen as the immune modulator to be co-delivered with a specific DNA
immunogen. On the other hand, for building vaccines to target extracellular
bacteria, for example, IL-4, IL-5 or IL-10 genes could be co-injected. Furthermore,
in cases where both CD4+ T helper cells and antibodies play more
important roles in protection, GM-CSF as well as IL-2 could be co-delivered.
Lastly, in cases where all three arms of immune responses are critical, TNF-a could be co-injected to give a combined enhancement
of antibody, T helper cell, and CTL responses. In this regard it will be
important to examine combination delivery in the presence or the absence of
costimulatory genes to further control the immune responses. Furthermore, additional
molecular adjuvant candidates, such as chemokines, should be further developed
and tested. Cumulatively, these studies demonstrate the potential utility of
molecular adjuvant strategy as an important tool for the development of more
rationally designed vaccines.
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Red-labeled (red) a-gp120 antibodies. (A) A slide from
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