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of General Virology (1998), 79, 2965–2970. Printed in Great Britain
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Compatibility of plasmids expressing different antigens in a
single DNA vaccine formulation
R. Braun, L. A. Babiuk and S. van Drunen Littel-van den Hurk
Veterinary Infectious Disease Organization, 120 Veterinary Rd, University of Saskatchewan, Saskatoon, SK Canada S7N 5E3
One anticipated advantage of DNA immunization is
the potential to create multivalent vaccines. We
have examined the effects of mixing plasmids into
single formulations using plasmids expressing four
different membrane bound glycoproteins from
bovine herpesvirus-1 (BHV-1), bovine parainfluenzavirus-3 (bPI3) and human influenza virus
(HINF). Plasmids were delivered by intradermal
injection into the tails of mice and the types of
responses generated were clearly affected by the
expressed antigen. Plasmids expressing glycoproteins B and D of BHV-1, and the haemagglutinin/
neuraminidase of bPI3, generated responses with a
predominance of IgG1, suggestive of a Th2 type of
Introduction
Immunization with plasmid DNA has been shown to be an
effective method for generating protective immune responses
to a number of pathogens (Hassett & Whitton, 1996 ; Manickan
et al., 1997 ; Robinson & Torres, 1997). DNA immunization
involves the expression in vivo of antigens encoded by
plasmids that are delivered typically either by intramuscular or
by intradermal injection, or by a gene gun into the skin. DNA
vaccines hold many possible advantages when compared with
traditional vaccines (Hassett & Whitton, 1996 ; Whalen &
Davis, 1995). Immunization with DNA offers a safer vaccine
than live or attenuated vaccines, as well as an ability to
generate cell mediated responses, which subunit vaccines
typically cannot. Other benefits of DNA vaccines include the
potential for increased duration of immunity, an ability to
immunize neonates, as well as low cost, high stability and ease
of preparation.
One presumed advantage of DNA immunization is the
potential for multivalent vaccines, in which several plasmids
are mixed together in a single formulation. Unlike protein or
viral vaccines that may require specific solubilizing conditions,
possibly affecting other components of a multivalent vaccine,
Author for correspondence : S. van Drunen Littel-van den Hurk.
Fax ­1 306 9667478. e-mail vandenhurk!sask.usask.ca
0001-5634 # 1998 SGM
response. In contrast, the plasmid expressing HINF
haemagglutinin induced an antibody response
biased towards IgG2a, indicating a Th1 type of
response. In most instances the mixing of plasmids
had only slight effects on the magnitude or bias of
the responses to the individual components. However, under certain conditions we found that
addition of a second plasmid converted an IgG2a
biased response to a response with primarily IgG1
antibody. The reverse situation (i.e. an IgG1 conversion to IgG2a), however, was not found. These
findings have important implications for the
development of multivalent vaccines.
there should be no physical incompatibilities between plasmids.
However, interference at either the level of antigen production,
or the response to antigen, is possible. Production of
multivalent vaccines would be of great benefit in situations
where repeated vaccination is too costly or cumbersome, such
as with veterinary vaccines or vaccines for third world
countries (Babiuk et al., 1998). Several papers have described
immunization with more than one type of plasmid (Gerdts
et al., 1997 ; McClements et al., 1996) ; however, no detailed
examination has been carried out on how the responses to
individual components are affected by the mixing of plasmids.
One recent report has shown that responses to individual
plasmid encoded antigens may be altered by the administration
of a second plasmid (Cardoso et al., 1998). In this study we
have used plasmids encoding four different viral membrane
glycoproteins to examine, in detail, the compatibility of
plasmids mixed for DNA immunization.
Methods
+ Plasmids. Genes for glycoproteins B and D from bovine
herpesvirus-1 (BHV-1 gB and gD ; Tikoo et al., 1990 ; Whitbeck et al.,
1988), and the haemagglutinin}neuraminidase from bovine parainfluenzavirus-3 (bPI3-HN ; Suzu et al., 1987) were inserted into the
pSLIA vector (Braun et al., 1997) creating plasmids pSLIAgB, pSLIAgD
and pSLIAHN respectively. These plasmids have backbone sequences
derived from pSL301 (Invitrogen) and the promoter from human
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R. Braun and others
cytomegalovirus (HCMV) including the intron A, and a 3« region of the
bovine growth hormone (BGH) gene. The haemagglutinin from human
influenza virus A}PR}8}34 (HINF-HA ; Young et al., 1983) was expressed
from plasmid V1J-HA, which was a gift from D. Montgomery (Merck,
West Point, PA, USA) and also uses the HCMV}intron A promoter and
the BGH 3« region, but has a backbone sequence derived from pUC19
(Montgomery et al., 1993). Plasmids pV1J-gD (BHV-1 gD in pUC19
background) and pSLIAHA (HINF-HA in pSL301) were created by
exchanging the transcriptional units (promoter, gene and part of the BGH
3« region) from VIJ-HA and pSLIAgD.
Plasmids were grown in E. coli DH5α and purified by anion exchange
resin (Qiagen). Concentration and purity were assessed spectrophotometrically, and constructs were confirmed by restriction enzyme
digestion followed by agarose gel electrophoresis.
+ Animals. Plasmids in saline were injected in a single dose
intradermally into two to four sites in the tails of C57Bl}6 mice (Charles
River). Mice were bled at 2 weeks and 4 weeks, and then at monthly
intervals for up to 20 weeks. All animal experiments were approved by
the University Committee on Animal Care and Supply at the University
of Saskatchewan.
+ ELISA. Antibody titres (either total or IgG subclass) were determined
by ELISAs as described previously (Braun et al., 1997) and are represented
as the optical density obtained from a given serum dilution. Appropriate
dilutions were chosen so that the optical density values fell within the
linear region of the titration curve. Endpoint titres of the responses were
typically between 640–2560. For gB and gD specific ELISAs, in vitro
synthesized recombinant forms of BHV-1 gB and gD (Kowalski et al.,
1993 ; Li et al., 1996) were used to coat the plates. For the HN and HA
specific ELISAs, virus purified on sucrose gradients was used. bPI3 virus
was grown in Vero cells (Breker-Klassen et al., 1995) and influenza virus
A}PR}8}34 was grown in fertile eggs (Hoskins, 1967).
Statistical analyses of ELISA data were done using programs in the
Sigmaplot software package (Jandel Scientific). An independent, two
tailed t-test (P ! 0±05) was used to determine significant differences
between the means of experimental groups.
+ Transfections. COS-7 cells were transfected using lipofectamine
(Gibco-BRL) as described by the manufacturer. Surface antigen was
detected by incubating unfixed, transfected cells with antiserum
generated against the viral glycoproteins by DNA immunization, at
37 °C for 1 h. The cells were washed with PBS, pH 7±2, and incubated
with an FITC-conjugated goat anti-mouse antiserum (Zymed) for a
further 1 h at 37 °C. After washing the cells with PBS, the cells were
examined under fluorescent light and labelling was compared with that of
cells stained in a similar manner, but transfected with the null plasmid.
Results
Responses to individual plasmids
Initial experiments catalogued the magnitude and character
of the serum antibody response to antigens expressed by the
four different plasmids. Cell surface labelling of transfected
COS-7 cells with specific antiserum confirmed that all of the
plasmids produced proteins that were expressed on the cell
surface (data not shown). The time-course of antibody
production after injection of plasmids (Fig. 1) demonstrated
that by 2–4 weeks the responses to pSLIAgD, pSLIAgB and
pSLIAHN were all close to maximal with only slight increases
occurring after this time. The response to V1J-HA, however,
differed slightly with virtually no antibody present at 2 weeks
CJGG
Fig. 1. Time-course of total serum antibody production in mice given
single plasmid formulations. Antigen specific ELISAs were carried out with
pooled serum from seven mice taken at various times after immunization.
Values have been converted to % maximal response to harmonize the
scale between the different ELISAs. x, pSLIAgB injected mice ; y,
pSLIAHN injected mice ; E, pSLIAgD injected mice ; *, V1J-HA injected
mice. Mice were given a single dose of DNA into two sites with either
10 µg of pSLIAgB, pSLIAgD, V1J-HA, or 15 µg pSLIAHN. The total amount
of DNA for each injection was made up to 30 µg with pSLIA. Serum
dilutions used for ELISAs were 1/640.
Fig. 2. Serum IgG subclass titres generated by injection of individual
plasmids. Antigen specific ELISAs were carried out on serum collected 16
weeks after immunization and are reported as the average³SEM for
seven individual mice at a serum dilution of 1/160. Injected plasmid is
noted on the graphs. (A) Mice injected with pSLIAgB, pSLIAgD and V1JgD plasmids. (B) Mice injected with pSLIAHN, V1J-HA, and pSLIAHA
plasmids. Shaded bars represent levels of IgG1, open bars represent levels
of IgG2a. Total amount of DNA given was 30 µg divided between two
sites with the following doses for each plasmid : 10 µg for pSLIAgB,
pSLIAgD, V1J-gD, V1J-HA and pSLIAHA, and 15 µg for pSLIAHN.
Formulations with less than 30 µg were made up to 30 µg with pSLIA.
and only approximately 20 % of maximum at 4 weeks. All
responses remained high for the duration of the experiment (20
weeks) with no decrease in titres found.
Two distinct types of responses were found in the mice
injected with single types of plasmid DNA. BHV-1 gB and gD,
and bPI3 HN antigens when expressed by plasmids generated
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Fig. 3. Effect of mixing plasmids on total
serum antibody levels. Antigen specific
total serum antibody levels were
determined by ELISA in mice given
various plasmid mixtures. Values are the
averages³SEM for seven individual mice
in serum (1/640 dilution) taken 16
weeks after immunization. Antigens used
to test sera are indicated on the graphs.
Bars are labelled with the antigens
expressed by the other plasmids within
the mixtures. Doses of each plasmid are
the same as in Fig. 1. * Groups in which
the mean differs significantly from the
single plasmid group.
responses with high levels of IgG1 and low levels of IgG2a
(Fig. 2). The ratio of IgG1 to IgG2a suggests a Th2 biased
response. In contrast, antibodies generated against the HINF
HA protein had high levels of IgG2a and low levels of IgG1,
suggesting a Th1 type of response. Although serum antibody
IgG1}IgG2a ratios are only indicators of a Th1 or Th2 type of
response, in this experiment they clearly demonstrate a
difference in the response to the HA antigen. Over the course
of the experiment, IgG subclass biases did not change.
Backbone sequences have been found to affect immune
responses under certain conditions. One known nucleotide
sequence with immunomodulatory activity, AACGTT (Sato et
al., 1996), occurs three times in the pSLIA vector and twice in
V1J-HA. All the AACGTT sequences are in the ampicillin
resistance genes and none occur within the different viral
genes. Because the V1J-HA plasmid has a different backbone
sequence, the genes from V1J-HA and pSLIAgD were
exchanged, to ensure that plasmid backbone sequences (either
AACGTT or other unknown sequences) were not responsible
for the effects seen above. After a single intradermal injection
of pV1J-gD and pSLIAHA plasmids, serum antibody levels in
mice showed no significant differences in the magnitude, or the
bias, of the response in comparison to the original plasmids
(Fig. 2). Thus, although effects of backbone sequences on
immune responses have been documented (Roman et al., 1997 ;
Sato et al., 1996), they do not appear to be responsible for the
differences in the IgG subclass distributions reported in this
study.
Responses to plasmid mixtures
The effect of mixing plasmids, within a single vaccine, was
then determined by examining the magnitudes and the IgG
subclass of the antibody responses generated to the individual
components of two-component or three-component vaccines.
A single submaximal dose (5–20 µg) was used to ensure that
any effects due to mixing were not hidden by an excess of
injected DNA. Importantly, in all experiments null plasmid
(pSLIA) was included, so that all injections contained equivalent
amounts of total DNA. Total serum antibody responses
measured by ELISAs are presented in Fig. 3. Overall, only
slight effects of mixing were found on the total antibody
responses, with the trend suggesting slightly lower levels of
antibody when plasmids were mixed. Any inhibition measured
was typically equivalent to one fourfold endpoint titre, and is
thus not excessively strong. The use of submaximal doses
enhances variability between animals such that most apparent
inhibition seen in Fig. 3 is not statistically significant. However,
the consistency of the inhibition we have found over several
experiments leads us to believe it is real, with the plasmid
pSLIAgD showing the strongest inhibitory effect. For example,
in all four separate experiments where pSLIAgD and pSLIAHN
were mixed, HN-specific serum antibody levels were lower
compared to immunization with a single plasmid, although
twice the differences were not statistically significant (data not
shown).
IgG subclass ratios also showed little effect from the mixing
of plasmids (Table 1). Since three of the four plasmids
individually generated a IgG1 type response, it was not
surprising that mixing of these plasmids did not alter the IgG
bias. However V1J-HA, which generated a Th1 type response,
maintained its IgG2a bias when mixed with the other plasmids,
which themselves maintained their Th2 type of response.
One three-component formulation containing the
pSLIAgD, pSLIAgB and V1J-HA plasmids was also tested.
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R. Braun and others
Table 1. IgG1/IgG2a ratios in serum from mice immunized with different plasmid
mixtures
Values are the ratios of the OD values measures by ELISAs using subclass specific secondary antibodies at a
1}160 serum dilution. Primary plasmid denotes plasmid that expresses the antigen measured in ELISA. Mixed
plasmids are the other components in the vaccine. The ‘ three-plasmid ’ mix contains pSLIAgB, pSLIAgD and
V1J-HA.
Mixed plasmids
Primary plasmid
pSLIAgD
pSLIAgB
pSLIAHN
V1J-HA
pSLIA
pSLIAgD
pSLIAgB
pSLIAHN
V1J-HA
Three
plasmids
8±3
2±9
6±0
0±22
–
2±2
8±7
0±71
8±2
–
5±7
0±3
7±8
4±3
–
0±1
10
3±2
2±5
–
6±2
4±2
–
0±32
Fig. 4. Effect of dose and plasmid
expressed antigen on IgG subclass bias in
mixed vaccines. Antigen specific IgG
subclass titres were determined by ELISA.
Values are reported as the average³SEM
for six individual mice of serum taken 12
weeks after immunization. Serum
dilutions used were 1/160 for the HA
specific ELISA and 1/640 for the gD
specific ELISA. Shaded bars represent
IgG1 antibody, and open bars indicate
IgG2a antibody. (A) V1J-HA plasmid
(5 µg). Amounts of pSLIAgD mixed in are
indicated on the x-axis. (B) pSLIAgD
plasmid (5 µg). Amounts of V1J-HA
mixed in are indicated on the x-axis. Total
amount of DNA injected was 40 µg into
four sites, with pSLIA added to bring all
combinations to 40 µg. Ratios of
individual animals expressing a
predominantly IgG1 type antibody
response are shown above each group.
Total antibody responses (Fig. 3) and IgG subclass ratios (Table
1) suggest that the extra plasmid had no additional effect on
the antibody responses.
In the previous experiment, mixing of pSLIAgD and VIJHA seemed to show more pronounced effects on total
antibody levels and the IgG1}IgG2a ratio than other mixtures
(Fig. 3, Table 1). To extend these results, and test the limits of
mixing plasmids, an experiment was carried out by injecting
mice with combinations of high (20 µg) and low (5 µg) doses of
pSLIAgD and VIJ-HA. With a 5 µg dose of VIJ-HA, addition
of 5 µg of pSLIAgD to the mixture caused an increase in the
relative amounts of IgG1 specific for HA (Fig. 4 A). More
significantly, 20 µg of pSLIAgD in the mixture caused a
complete conversion in the ratio of serum IgG subclasses to a
very strong IgG1 response. Serum from mice immunized with
pSLIAgD alone did not bind influenza virus in the ELISA : thus
the IgG1 antibodies are HA specific. With a 20 µg dose of VIJ-
CJGI
HA the same pattern was found where 5 µg of pSLIAgD
enhanced the IgG1 ratio to approximately 1 : 1, and the 20 µg
dose of pSLIAgD produced a ratio of 10 : 1 (data not shown).
These results are consistent with the previous experiment in
which pSLIAgD does appear to cause an alteration of the
response to HA. In the final experiment the effect of pSLIAgD
seems to be stronger, which may be due to experimental
variations or because more total DNA was injected (40 µg in
comparison to 30 µg) in the final experiment. Because the
effects appear to be related to the dose of pSLIAgD, not the
relative ratios of the two plasmids, it suggests that the strength
of the IgG1 biased response may influence the conversion of
the response. In mice given V1J-HA and 5 µg of pSLIAgD, the
IgG1}IgG2a ratio of approximately 1 : 1 found for HA specific
antibodies is a result of averaging values from individual mice.
Individual mice, however, have typically either a predominant
IgG1 or IgG2a subclass, not a balanced ratio. The results in Fig.
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Mixed DNA vaccines
4 are therefore affected by the number of responders, and the
number of mice that responded with a predominant IgG1
response are shown on the figure.
The reverse condition, i.e. the addition of V1J-HA to
pSLIAgD, showed no effect on the IgG subclass ratios of the
gD specific response, with all groups having a strong bias to a
Th2 type of IgG subclass ratio (Fig. 4 B).
Discussion
Multivalent vaccines represent a cost effective and simple
method to generate protective immunity against multiple
pathogens. Unfortunately, many vaccines cannot be mixed
because of physical incompatibilities. DNA vaccines are
thought to be ideal for multivalent vaccines because of their
similarity of form. Experiments described in this paper
represent the most comprehensive examination to date of the
effects that may occur by mixing different antigen expressing
plasmids.
One interesting feature of the antibody responses to the
different antigens expressed alone was the difference in the
IgG bias. The bias of the response to HA is consistent with
previous work where a Th1 type of response was found with
saline injection of V1J-HA (Feltquate et al., 1997). As well, the
Th2 type response to BHV-1 gD has been described (Braun et
al., 1997) following intradermal injection of pSLIAgD. The
method of plasmid delivery and the cellular localization of the
expressed antigens are known to affect the bias of a response,
presumably by altering the types of antigen presenting cells
(APCs) that take up and process antigen (Feltquate et al., 1997).
In the present study, plasmid delivery and antigen localization
were the same for the different antigens, suggesting that the
antigen itself may influence the type of response. Although the
proteins used in these experiments are membrane bound, they
will transfer from the cells that have taken up the plasmid to
APCs (Doe et al., 1996). Because the antigens used in this study
are viral membrane glycoproteins that are known to interact
with extracellular receptors, the possibility thus exists that
these viral antigens, if released from the cell, may target to
different cell populations, creating the differences in the
responses.
When plasmids were mixed there appeared to be a slight
interference with the total antibody response in many
instances. We believe that the inhibition may be a general low
level of interference resulting from either reduced antigen
production or antigen competition. As the amount of antigen
expressed from cells increased, as would occur if two or more
plasmids were mixed, the added burden on cellular resources,
or potential problems of antigen toxicity, may reduce the
efficiency of synthesis, and thus lower antigen load. As well,
the increase of antigen produced by a multivalent vaccine
could also cause increased competition for APCs. We have
previously found that the relative efficacy of DNA
immunization decreased with increasing amounts of DNA
added (Braun et al., 1997), which is consistent with these
results. Because the cells responsible for the induction and
perpetuation of the immune response to intradermally injected
DNA are unknown, we are unable at this time to identify
which factors may be responsible for the slight inhibition of the
immune responses due to mixing plasmids.
Mixing of plasmids generally showed little effect on the
antibody bias which is interesting, because the cytokine
microenvironment during the initiation of the immune response in the lymph node is expected to dictate the type of
response (Paul & Seder, 1994 ; Mosmann & Sad, 1996). These
data suggest a segregation of the antigens at some point,
possibly as mentioned previously, because of targeting of
antigens to different APCs. Although physical separation
could be involved, a temporal difference may also be possible.
In the response to V1J-HA, IgG2a antibody tended to be
delayed in comparison to the IgG1 antibody generated to the
other plasmids (Fig. 1, unpublished observation). This raises
the possibility that the antigen responses segregate in time, not
space. Regardless of the exact mechanisms involved, the
evidence suggests that DNA plasmids with different types of
responses can be mixed and have little or no effect on the
vaccine partners.
In contrast to examples described above, pSLIAgD,
especially at higher doses, was found to cause a strong
alteration in the response to V1J-HA. Presumably in this case
the increased strength of the response to gD increased either
the spatial or temporal distribution of Th2 type cytokines
enough to influence the cytokine environment for the response
to the HA protein. Cardoso et al. (1998) found a similar effect
using plasmids encoding the HA and NP antigens from
measles virus. They specifically showed that a Th2 biased
response could alter a Th1 response. This is in contrast to
several experiments in which DNA vaccines override the Th2
type of response to protein, and generate a Th1 response (Raz
et al., 1996). These experiments were different, however,
because plasmid DNA was mixed with protein and large
amounts of DNA were used, so that backbone immunostimulatory sequences may have had an effect. In our
experiments, and those of Cardoso et al. (1998), plasmids were
mixed and less DNA was used, such that the mechanisms for
immune deviation likely differ from those in which plasmid and
protein are mixed. Why a Th2 type of response dominates
when plasmids are mixed is unknown, but it is of interest that
the Th1 responses have little effect on the Th2 response. This
result could give more credence to the possibility that there
may be a temporal difference in the Th1 versus Th2 response.
Thus, even if higher levels of V1J-HA plasmid are present, the
effect of Th2 cytokines on IgG class switching during the
response to pSLIAgD may precede the effect of Th1 cytokines.
In summary, our results indicate that there may be some
effects of mixing plasmids, but this is dependent on the dose
and on the specific antigen. In some situations it may be
possible to mix plasmids and have little interference among the
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R. Braun and others
components. However, under certain conditions responses
may be altered by the other components of the vaccine. If
protective immunity to a particular pathogen requires a specific
type of response, induction of an inappropriate response may
be detrimental. Conversely, mixing of plasmids could be used
as a simple way to direct responses to certain components. It
would appear, however, that Th2 type responses may be
dominant. Data described in this paper indicate that multicomponent vaccines must be thoroughly tested to ensure
appropriate responses. Because there are many factors that
may influence a particular response including route, antigen,
antigen form, dose and animal species, it is important to test
multivalent vaccine candidates in the target species, ideally
with a challenge model, such that the efficacy can be truly
evaluated.
We would like to thank members of the animal care group at VIDO
for the handling of animals, and Dr D. Montgomery for the plasmid V1JHA. This work was supported by grants from the National Science and
Engineering Council of Canada, Medical Research Council of Canada,
Alberta Agriculture Research Institute, Alberta Cattle Commission, the
World Health Organization, Beef Industry Development Fund and the
Agricultural Development Fund.
Published as VIDO journal series no. 237.
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Received 17 April 1998 ; Accepted 18 August 1998
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