I. Introduction
Advances in molecular medicine
are based on the possibility to efficiently deliver genes into specialized
tissues. Two essentially
independent disciplines are predicated on this technological approach, gene
therapy (Mulligan, 1993) and DNA vaccination (Cohen, 1993; Donnelly et al.,
1997). In both instances success
is determined by a series of factors all of which depend on the efficiency of
gene delivery and gene expression in vivo. Strategies have been
developed to realize gene delivery via receptor mediated pathways (Ferkol et al., 1995; Ferkol et al.,
1996; Wu et al., 1989) exploiting specific structures on somatic cells and
their mechanisms to internalize and transport macromolecules. Targeted delivery
of DNA also needs to be gauged through the specificity of regulatory elements,
i.e., promoters and enhancers, which allow the transgene to be transcribed and
translated only in selected tissues.
The efficiency of these processes constitutes the rate limiting factor
for in vivo efficacy of both gene
therapy and DNA vaccination.
The use of polynucleic acid for
vaccination is complicated by the necessity to achieve sufficient secretion of
the transgene product in an immunogenic form. An ideal DNA
vaccine should yield molecules with sufficient conformational resemblance to
the native antigen to elicit immunity with the greatest cross-reactive
potential. In addition, the
process of DNA vaccination should also be tailored to include in the process
the participation of antigen-presenting cells to heighten both humoral and
cellular immune responses.
It is based on this reasoning
that we developed a new, rational method of immunization using DNA molecules,
somatic transgene immunization (STI), to combine selective targeting of B lymphocytes and their antigen-presenting
capacity with expression of conformationally constrained antigen molecules
(Gerloni et al., 1997). We
directed our considerations to immunoglobulin (Ig) genes as
“immunogens” in DNA vaccination. In this chapter we will review what known about the
principles of immunogenicity by DNA
and subsequently describe our findings on somatic transgenesis as they
relate to in vivo targeting of B
lymphocytes.
II. DNA and
immunogenicity
DNA itself is scarcely
immunogenic as it has proven extremely difficult to induce a response against
DNA (Madaio et al., 1984) even
though there exist clinical situations in which autoantibodies to double and
single stranded DNA are produced (Koffler et al., 1969; Pincus et al., 1969;
Tan et al., 1966). The immunogenicity
of DNA per se depends on its origin, i.e., eukaryotic or prokaryotic. For instance it was shown that mice
immunized with Escherichia coli
DNA complexed with methylated BSA in adjuvant produce significantly greater
amounts of antibodies than mice similarly immunized with calf thymus DNA
(Gilkeson et al., 1989; Gilkeson et al., 1989). This indicated that DNA molecules differ in their
immunogenic potential, a characteristic likely due to unique sequences or
structures present in bacterial DNA but rarely in mammalian DNA (Gilkeson et
al., 1989; Gilkeson et al., 1989).
Bacterial DNA possess immunostimulatory properties (Tokunaga et al., 1984), is mitogenic for B cells
and induces polyclonal antibody production (Messina et al., 1993), and enhances
the lytic activity of natural killer cells with production of IFN-g (Tokunaga et al.,
1984; Yamamoto et al., 1992). This
stimulatory properties are linked to a six-base nucleotide motif consisting of
an unmethylated CpG dinucleotide (Krieg et al., 1995) expressed nearly twenty
times more frequently in bacterial than in vertebrate DNA (Cardon et al.,
1994).
In 1992 Tang and coworkers
reported that inoculation of plasmid DNA induces specific immunity in vivo (Tang et al., 1992). There is a fundamental difference between Tang’s
accomplishment and previous attempts to immunize against DNA as it showed that
it was possible to use functional genes to generate immunity against a specific gene product. Implicit in this discovery was the fact
that factors regulating gene expression would also regulate immunogenicity
in vivo. Many reports have appeared
since demonstrating the use of plasmid DNA in eliciting immunity against
viruses (Davis et al., 1993; Raz et al., 1994; Ulmer et al., 1993; Wang et al.,
1993), bacteria (Huygen et al., 1996; Tascon et al., 1996), parasites (Doolan et
al., 1996; Sedegah et al., 1994; Xu and Liew, 1995), tumor antigens (Conry et
al., 1994), self antigens (Gilkeson et al., 1996; Waisman et al., 1996) and allergens (Hsu et al., 1996; Raz et
al., 1996). The rapidly
raising hope to develop simple and cost effective methods of vaccination was
followed by the demonstration that plasmid DNA could be used to induce antibody
responses (Cox et al., 1993; Davis et al., 1994; Fynan et al., 1993; Raz et
al., 1994; Robinson et al., 1993; Sedegah et al., 1994; Ulmer et al., 1993;
Wang et al., 1993) as well as cell-mediated responses of helper (Xiang et al., 1994) or cytotoxic T-cell type (Raz
et al., 1994; Sedegah et al., 1994; Ulmer et al., 1993; Wang et al., 1993).
III. The development
of somatic transgene immunization
Immunization via DNA
inoculation relies on in vivo
transfection, production and possibly secretion of the transgene product, and
antigen presentation by specialized cells. However, in most studies neither the
in vivo transfected cells nor the
antigen presenting cells involved in the process have been identified. Most
experiments use foreign DNA under the control of viral promoters which have
limited tissue specificity.
Therefore, no tissue-specific
control of expression is possible other than the site of DNA
inoculation.
Since the quality of nucleic
acids and gene expression are intimately connected with the vaccination process
we began studies considering together three factors that, alone or in
combination, could affect the success
of DNA immunization: (i) the
efficiency of in vivo transfection
including DNA uptake by the host cells, (ii) the efficiency with which transfected cells can
utilize the DNA and synthesize the transgene product, and (iii) the ability of in vivo transfected cells to serve as antigen-presenting
cells (APCs). Most reports still
indicate that expression of the transgene, synthesis of the corresponding gene
product, its presentation to immunocompetent cells and induction of immunity
occur after repeated inoculations of plasmid DNA (Whalen and Davis, 1995).
The genetic organization and
the molecular events leading to expression of immunoglobulin genes are well,
albeit not completely, understood
and consist in a cascade of tissue-specific genetic events occurring during
B-cell differentiation and resulting in the synthesis of immunoglobulin
molecules (Alt et al., 1987; Sleckman et al., 1996). Studies in vitro
have shown that transfection of B cell lymphomas with rearranged Ig gene is followed by a prompt utilization of the transgene and by secretion of
immunoglobulins encoded by the transgene (Morrison, 1985). A variety of rearranged functional Ig
genes have also been introduced in the germline to create mice with homogeneous
antigen receptor expression on B cells and secretion of immunoglobulins with
identical chemical and immunologic
characteristics (Storb, 1987). It
is then obvious that DNA
immunization with Ig genes, e.g.,
heavy (H) chain genes under the
control of their own tissue-specific promoter and enhancer elements (Banerji et
al., 1983; Gillies et al., 1983; Grosschedl and Baltimore, 1985; Mason et al.,
1985) could be a way to target B lymphocytes and satisfy basic requirement for
immunogenicity such as synthesis and secretion of the transgene product, and
antigen presentation.
In our first experiments we
inoculated adult C57Bl/6 mice with a plasmid DNA containing a chimeric
(mouse/human) immunoglobulin H chain gene with tissue-specific promoter and
enhancer elements. We immediately observed
that a single intraspleen inoculation was followed by (i) uptake and persistence of the transgene in B
lymphocytes for 3-4 months, (ii)
secretion of transgenic immunoglobulins (transgene H chain + endogenous L
chain) in amounts ranging between 15 and 30 ng/ml, and (iii) production of IgM anti-Ig antibodies. The circulating H chain transgene
product was found to be associated prevalently with k light (L) chains a fact per se not surprising since k L chains represent 95% of the pool of genes utilized in vivo in the mouse.
A booster immunization with Ig coded by the same transgene at various
times after priming with DNA showed the generation of a typical secondary
immune response with IgG1 and IgG2b antibodies (Gerloni et al., 1997). Thus, a single inoculation of an Ig H chain gene targeted to
spleen lymphocytes was sufficient to initiate immunity and establish
immunologic memory. We termed this
process somatic transgene immunization (STI) to reflect the fact that a foreign
gene is transported inside somatic cells
and initiates immunity (Gerloni et al., 1997). It became evident that from this point on the
physiological machinery provided by STI could be exploited to program
one’s individual immune response in a rational way.
IV.
Investigations on the transgene
Intraspleen inoculation of the
H chain transgene resulted in immunity and established immunologic memory. To explore the basis for this
phenomenon we undertook a systematic analysis of (i) the tissue
Figure 1. Schematic
representation of plasmid g1NANP.
The plasmid contains the Ig H chain construction which is the product of
the fusion between the productively rearranged murine VH62 gene and the human g1
C region gene present in genomic configuration in plasmid vector pNeog1
(Sollazzo et al., 1989). The VH gene was modified by insertion in the CDR3 of
36 bp heterologous sequence coding for three repeats of the amino acid sequence
Asn-Ala-Asn-Pro (NANP). The plasmid DNA carries the regulatory elements,
promoter (Pr) and enhancer (En) needed for tissue-specific expression. Neor
= neomycin and Ampr =
ampicillin, are the resistance
genes. CDR =
complementarity-determining region.
FR = framework region.
distribution of the transgene, (ii) its fate in vivo over time, (iii) the cell type involved in targeting, and (iv) the potential for somatic mutation of the transgene
in vivo. These studies were based on an Ig H chain gene
purposely engineered in the CDR3 of the VH domain to have a 36 base-pair
exogenic molecular marker (Figure 1).
A. Tissue distribution
Genomic DNA extracted from
various lymphoid (i.e., spleen,
lymph-nodes and bone-marrow) and non-lymphoid (i.e., liver, kidney, lung and muscle) tissues explanted at
different times, was analyzed for specific amplification of the transgene VDJ by
PCR and Southern blot hybridization.
An amplification product was readily visible in splenic genomic
DNA. No specific amplification
occurred in any of the other tissues (Table 1). This did not vary at any of the time
points analyzed. To control for specificity
and increase the sensitivity of the reaction, two additional PCR assays were
performed using primers designed to anneal sites within the VDJ region. One set of primers (pSE/pNAD)
specifically amplified the molecular maker; another (inner primers: pNEL/pNED)
served for nested PCR (Figure 2). The results confirmed those obtained
with VDJ amplification. Southern
blot analysis using a probe
specific for the molecular marker further confirmed the PCR results (Table 1). Thus,
after intraspleen inoculation the transgene H chain persists in vivo for a period of 3 months in the organ in which it was
inoculated.
B. Fate of the transgene
PCR and Southern blot
hybridization were also used to monitor the kinetics of the presence of the
transgene in vivo in mice analyzed at various times after
DNA inoculation (Xiong et al., 1997).
Amplification of the transgene VDJ region was visible up to 12 weeks
after a single DNA inoculation. No
amplification was seen at subsequent time points (16, 24, 36 and 52 weeks) (Table 1).
Southern blot hybridization with the marker-specific probe further confirmed
the PCR results (Table 1).
C. The transgene is harbored in B lymphocytes
As discussed above mice
undergoing STI produce transgene Ig
(15-30 ng/ml) for a protracted period of time in vivo. Coupled with the demonstration that the transgene
could only be found in the spleen (the organ of inoculation) and
consistent with the use of a gene
under control of promoter and enhancer elements specific for B lymphoid
cells, it became obvious that B lymphocytes could be the cell population
accounting for the initiation of
STI (i.e., transgene uptake, the
persistence of the transgene, its transcription and secretion of transgenic
Ig).
the Ig H chain gene used lacks a transmembrane domain,
rendering cell surface anchoring unlikely, experiments were performed to assess
accumulation of mutations as a result of protracted in vivo persistence in integrated form. The transgene VDJ
region was amplified from splenic genomic DNA, subcloned and sequenced by the
dideoxy termination method. No
evidence of hypermutation was found in the VDJ region of the transgene in
vivo even after 3 months (Table 2).
V.
Concluding remarks
The studies presented in this chapter indicate that
the biological phenomenon of
somatic transgenesis relies on series of cellular events which we begin to
understand and which seem to fulfill most of the conditions posed as requirements
for a new, rational approach to DNA-based immunization. For sake of brevity our comments
will concentrate on four points essential in our opinion to understand the
phenomenon and the future potential of our work (Figure 6).
First, is targeting of B lymphocytes
in vivo. As mentioned above two main considerations guided our
experiments in this direction: One
consideration is that B lymphocytes have receptors with specificity for DNA. This makes it possible that DNA, which sticks to the surface of a cell
due to its negative charge, is subsequently internalized by receptor-mediated endocytosis. As
pointed out both Ig and non-Ig receptors for DNA exist on normal B lymphocytes (Bennett et al., 1985; Glotz et al.,
1988; Holmberg et al., 1986). Presumably, after internalization only a fraction
of the DNA resists degradation in the endosomes and reaches the cytosol. For instance, 24 hours after in vivo receptor mediated gene transfer there exists only 1
copy of DNA per cell compared to 100 copies at four hours (Wilson et al.,
1992). To analyze conditions of binding and internalization new experiments are
in progress to determine whether the same effect can be obtained in vitro. The
other consideration is that tissue specificity is provided by the Ig gene regulatory
elements (see below).
Second, is integration. Studies in mice transgenic for Ig genes
have sufficiently shown that transgenes are generally integrated in multiple
tandem copies at one or a few sites in the host genome. Importantly, integration does not
occur into the homologous H or L locus (Storb, 1987). Similarly, somatic cell
hybridomas and non-secreting B-cell lymphomas transfected with Ig genes both
harbor integrated foreign gene(s) (Morrison, 1985) randomly. On the other hand, site specific
integration can be achieved using suitably modified expression vectors such as
replacement (Kardinal et al., 1996) or integration (Lang and Mocikat, 1994)
vectors. From the foregoing, it does not surprise that during somatic
transgenesis integration occurs randomly. It is quite likely that the transgene enters the
nucleus during cell division (Figure 6).
An aspect intimately connected
with integration of the transgene and its expression in B cells is its relation
with the endogenous Ig gene since, as a rule, a single B cell expresses only
one H chain together with one L chain, allelic exclusion (Pernis et al.,
1965). Although at this stage we
have no data in favor or against allelic exclusion during somatic transgenesis,
it is of note that germ-line transgenic mice have variably shown some leakage
and that the endogenous Ig gene is expressed together with the transgene
product (Storb, 1995). Apart from
the cell biological relevance, even partial lack of allelic exclusion in
somatic transgenic cells would lead to secretion of mixed antibody
molecules. Further studies will
need to address this point and their potential implication for immunization.
Third, is the process of
transcription and translation.
Possibly this is the step that confers the highest degree of selectivity
to the entire process. It is well known that Ig gene expression is restricted to lymphoid cells and among
them to cells of the B-cell lineage (Banerji et al., 1983; Gillies et al., 1983;
Grosschedl and Baltimore, 1985; Mason et al., 1985). Therefore, the use of B cell specific promoter and enhancer
elements introduces a stringent control mechanism on tissue
specificity and utilization of the transgene in vivo. One
essential aspect to a full understanding of the events starting somatic
transgenesis is “How does the first B cell become activated and how does this B cell begin to
produce transgenic Ig molecules?”
A plausible explanation is that bacterial DNA of the plasmid backbone
possesses immunostimulatory properties for B cells (Messina et al.,
1993; Tokunaga et al., 1984), an activity
mediated by a six-base, unmethylated CpG dinucleotide (Krieg et al.,
1995). Thus, activation of B cells
by plasmid DNA may be crucial to initiate transcription and translation, and
ultimately to set in motion the immunogenic process.
Fourth, is somatic
mutation. It was appreciated
early on that Ig genes isolated from myelomas and hybridomas are mutated in the
V region compared with the corresponding germ-line (Crews et al., 1981). Hypermutation occurs frequently in the
VDJ region, mainly in the CDRs.
This is commonly explained with hypermutation taking place during
antigen selection and affinity maturation of the antibody response, and is an
important means to increase antibody diversity (Griffiths et al., 1984).
Hypermutation arises through one of two mechanisms: antigen selection or
intrinsic mutational bias independent of selection. In the first case it is required that the Ig is expressed at the surface of B
lymphocytes for antigen to exert selective pressure. As indicated in Figure 1 the
transgene used in our studies lacks a transmembrane domain. This renders cell surface anchoring
unlikely with no possibility for somatic mutation to occur via this mechanism. Another consideration is that we used a H chain transgene coding for an already rearranged V region segment, hence ruling out the possibility of
mutations introduced during rearrangement. Others have shown that transgenic mice engineered with
already rearranged k chain genes do not mutate unless hyperimmunization is
in place (O'Brien et al., 1987).
In the second case, i.e.,
transcriptional error or transcription-driven hypermutation, there appears to
be a dependence on the physical distance between the exon and the promoter
element (Peters and Storb, 1996).
This does not seem to apply to our model of somatic transgenesis as no
mutation was found among the clones examined, albeit only a limited number of
clones at selected time points
were sequenced (Table 2). Understanding lack of
transcription-driven hypermutation during somatic transgenesis will need to be
addressed with further studies.
In conclusion, we have shown and discussed the use of
H chain Ig genes in somatic transgenesis as an in vivo step of targeted transgene expression which
preceding the phase of
immunogenicity which is central to our attempts to develop a new rational
approach to immunization, somatic transgene immunization. Others have used a similar rationale to
target B lymphocytes in vivo using retroviral vectors for stable
transgene expression (Sutkowski et al., 1994). The considerations made in this paper are relevant to better
understand the nature of somatic transgenesis and to its future applications
for DNA vaccination and gene therapy.
Acknowledgments
This work was supported by NIH
grant AI36467. During the
performance of the experiments reported in this article S.X. was on leave of
absence from Shanghai Medical University of the People’s Republic of China.
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