,
therapeutic
vaccines,
adjuvants
and RNA vaccines)
Table of contents :
)
can be used as vectors for DNA vaccines as they free the carried plasmid
after cell lysis.
(e.g. VP22)
at sites of injection :
activating plasmacytoid
dendritic cells (PDCs)
eliciting Th1
polarization. Methylation of the palindromic CpG sequences of bacterial
expression vectors abolished their ability to induce an antibody response
to the transgene product (a highly immunogenic viral glycoprotein) in mice.
The antibody response could be rescued by concomitant injection of oligonucleotides
carrying immunostimulatory sequences. The B cell response to 2 plasmid
vectors, both expressing the same viral glycoprotein but containing a different
content of the highly stimulatory AACGTT motif, was compared. Comparable
B cell responses were induced to the 2 constructs given at an optimal vaccine
dose while the vector containing additional palindromic sequences resulted
in higher antibody titers at a suboptimal dose. These data indicate that
deletion of CpG motifs or methylation of such sequences in plasmid DNA
can abrogate the immune response to the vector encoded antigen and might
thus enhance their usefulness as gene therapy vehiclesref.
may be an obstacle for effective induction of immune responses by gene
vaccines, and should be corrected by codon optimizationref
can modulate immune responses induced by DNA vaccines as they form specific
complexes with DNA and this formulation serves to increase transfectant
DNA protection as well as local deliveryref,
but stimulated only modest levels of overall antibody production, with
relatively low level of IgG2a
exhibits strongest Th1
stimulatory activity whereas Tween 80 showed weakest Th1 activity,
as determined by IgG2a production, and saline formulation induced
weak T cell proliferation and DTH, in animals inoculated with a DNA construct
expressing the FMDV capsule protein VP1. Furthermore, co-inoculation of
levamisole increases the production of IFN-g
by > 100-fold as compared to that by DNA inoculation formulated in salineref
occursref,
but not alwaysref
and is usually transient
: current good manufacturing practice (cGMP) conditions for obtaining clinical-grade
plasmid DNA
and adaptive immunity
would certainly diminish the probability of such a phenomenon, because
the recombinant tumour antigen would be the only one being repetitively
boosted.)
engineered to secrete antigens will load the MHC class II processing pathway
during the phagolysosomal phase of its life cycle and the MHC class I pathway
during the cytoplasmic phase.
delivery of DNA drives a strong
Th2
response. In an effort to counter this, the Th1
cytokines, IL-12
and IL-23,
were genetically fused to the hemagglutinin (HA) gene from influenza APR/8/34,
and delivered these DNA constructs to Balb/c mice. Gene gun delivery of
the HA gene was able to induce antibody production by all vaccinated mice.
Linking of IL-12 caused almost complete suppression of immune responses
whereas mice vaccinated with IL-23HA showed long-lived IgG1 antibody
levels. Splenocytes from IL-23HA vaccinated mice also tended to produce
more IL-5 and IFN-g after restimulation in
vitro than splenocytes from HA vaccinated mice. While codelivery of
IL-23 did not change the type of immune response it may increase its longevity
following vaccinationref.
A comparative study of domestic pig, minipig and mouse in regard to local
tolerance and antigen expression of HIV immunotherapeutic using PMED. Pig/minipig
is considered a good model for the safety assessment of DNA vaccines due
to the similarity to human skin. Local reactions were evaluated at 10min,
4, 24 and 48h. Histology of administration sites revealed epidermal necrosis
with associated dermal inflammation at 10min and 4h, and subsequent regeneration
with repair at 24 and 48h. The degree and extent of these changes varied
according to species. Domestic pig and minipig showed superficial epidermal
necrosis and complete repair, while the mouse showed full-thickness epidermal
necrosis and partial repair. Expression of HIV antigen was confirmed using
immunohistochemistry in all three species at 4, 24 and 48h. The results
showed that PMED is an effective system for DNA vaccine delivery as demonstrated
by the antigen expression seen as early as 4href,
a molecule expressed on activated CD4 T lymphocytes and critical for T
cell helper function, can elicit significant titers of antigen-specific
immunoglobulins in serum and Tc1 CD8 T cell responses in CD4-deficient
mice. To investigate whether this approach leads to CD4 T cell-independent
vaccine protection against a prototypic AIDS-defining infection, Pneumocystis
carinii pneumonia, serum from mice vaccinated with PC-pulsed, CD40L-modifed
DCs was used to immunoprecipitate PC antigens. Kexin, a PC antigen identified
by this approach, was used in a similar DNA vaccine strategy with or without
CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L
showed significantly higher anti-PC IgG titers as well as opsonic killing
of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted,
Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge
model. Adoptive transfer of CD19 cells or IgG to SCID mice conferred protection
against PC challenge, indicating a role of humoral immunity in the protection.
The results of these studies show promise for CD4-independent vaccination
against HIV-related or other opportunistic pathogensref
for DNA vaccines :
: to assess GM-CSF immune accessory effects in tumor-bearing mice, an animal
tumor model was established by inoculating SP2/0 myeloma cells s.c. into
the flank of Balb/c mice and 14 days later, injecting either 400 mg
recombinant pcDNA3.1/mGM-CSF or a blank plasmid s.c. or i.m. into the tumor
four times. The tumor weight, the activities of CTL and NK, the serum levels
of IFN-g, IL-2 and lymphocytes infiltrating
in tumor tissue were analysed 8 weeks later with MTT, ELISA and pathological
section methods. The tumor lump was reduced in mice injected s.c. (0.880
+/- 0.405 g) or i.m. (0.378 +/- 0.411 g) with pcDNA3.1/mGM-CSF compared
with control mice injected s.c. (1.548 +/- 0.221g, P < 0.01)or i.m.
(1.554 +/- 0.249g, P < 0.001) with a blank vector. Lymphocyte infiltration
in tumor tissues was very apparent in mice injected i.m. with pcDNA3.1/mGM-CSF.
In contrast, there was no lymphocyte infiltration in tumor tissues of control
mice. In addition, the serum concentrations of IFN-g,
IL-2 and the activities of CTL and NK cells were significantly increased
in mice injected with pcDNA3.1/mGM-CSF compared with a control mice (P
< 0.01)ref
adaptor molecule such as the TRIF or MyD88 were constructed and administered
to mice to determine if these molecules can act as an adjuvant. A DNA vaccine
incorporated with the MyD88 genetic adjuvant enhanced antigen-specific
humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced
cellular immune responses. Incorporating the TRIF genetic adjuvant in a
DNA vaccine targeting the influenza HA antigen or the tumor-associated
antigen E7 conferred superior protectionref.
: to determine if the properties of IL-18 could be optimized for use as
a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to
identify variants of human IL-18 that were predicted to improve receptor
interactions and potentially bioactivity. The linkage of mature IL-18 to
a secretion signal sequence provided improved protein expression from mammalian
cells and signal peptidase cleavage of this protein produced the authentic
N-terminus. The IL-18 variant proteins secreted this way were bioactive,
as demonstrated by their ability to induce IFN-g
expression by human peripheral blood mononuclear cells (PBMCs) and to bind
to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were
inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of
IL-18, as measured by neutralization of the IFN-g
response in PBMCs. One variant, V11I/T63A, demonstrated increases both
in bioactivity and mammalian cell expression as compared to native IL-18,
indicating that this molecule may be particularly well suited for use as
a DNA-encoded vaccine adjuvantref.
and a gene expression enhancer in in vitro DNA viral infection,
including HSV-1 and CMV. A study sought to determine if increasing antigen
expression of DNA vaccines through inhibition of HDAC-induced chromatin
silencing using TSA would enhance DNA vaccine efficacy in vivo.
A luciferase assay was used to detect effects of TSA on different promoters
in
vitro. The effects of TSA on DNA vaccination of mice were determined
by neutralization assays for antibody production and interleukin staining
for detection of specific T cell responses. Mice receiving TSA in combination
with a DNA vaccine exhibited higher antibody responses to the vaccine than
mice not given TSA. Co-administration of TSA also enhanced specific CD8
T cells response. Drugs such as TSA that reduce initial gene silencing
by preventing histone deacetylation can increase immune responses to DNA
vaccinationref
has been shown to increase immune responses not only by promoting coclustering
of T cells and DCs in the lymph nodes and spleen but also by regulating
their immunogenic potential for the induction of T cell responses. Using
HPV 16 E7 as a model antigen, a chemotactic-antigen plasmid DNA vaccine
(pSLC-E7-Fc) was constructed by linking SLC and Fc gene sequences to each
end of E7 and evaluated its potency of eliciting specific immune response.
Immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte
proliferative and CTL responses than control DNA. All the mice receiving
pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous
inoculation of TC-1 cells, while those given control DNA all developed
tumors. These tumor-free mice were also protected against TC-1 rechallenge.
Complete tumor regression with long-term survival occurred in 72% of mice
given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis
model wherein TC-1 cells were intravenously injected, therapeutic vaccination
with pSLC-E7-Fc significantly reduced the number of tumor nodules in the
lung. In vivo depletion with antibodies against CD4+
or
CD8+ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced
immunotherapeutic effect. These data indicate that the DNA vaccine constructed
by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is
an effective approach to induce potent anti-tumor immune response via both
CD4+ and CD8+ T cells dependent pathwaysref.
DNA vaccine (see also protein
subunit vaccine
and therapeutic vaccines):,
which acts as a vector to carry HIV genetic material into host cells. AVX101
is the first vaccine to be tested that has been designed specifically to
target HIV-1 subtype C, which is most prevalent in South Africa, rather
than subtype B, which is predominant in the developed world and for which
other vaccine trials are underway
encoding HIV gag and pro genes and a portion of the reverse transcriptase
gene. Unlike many vaccines currently in clinical trials, tgAAC09 potentially
could be a single-shot vaccine, which would be useful in developing countries.
In addition, animal trials have shown that the vaccine candidate can stimulate
both an antibody and a cell-mediated response to HIV. The vaccine focuses
on HIV subtype C, which is the most common strain of the virus in India,
so India has begun its first-ever phase I clinical trial of an HIV vaccine,
which is being conducted by the Indian
Council of Medical Research and the International
AIDS Vaccine Initiative, is testing the vaccine candidate called The
Indian trial is part of a multicountry Phase I trail of tgAAC09, which
already has started in Europe with German and Belgian researchers testing
the vaccine in partnership with IAVI. IAVI beginning in March 2001 provided
$3 million over 18 months to the Indian government to fund HIV vaccine
development efforts. The trial, which is expected to take about 15 months
to complete, will test the vaccine on 30 male and female volunteers ages
18 to 45 who are HIV-negative and do not have any other significant illnesses.
The trial will take place at the National
AIDS Research Institute in Pune, India. ICMR Director N.K. The candidate
vaccine might stimulate an immune system response that could protect volunteers
from potential HIV infection. The volunteers have been made aware of the
risks involved, they have been counseled and their health status will be
monitored all the time. The European trial -- conducted at the Centre
Hospitalier Universitaire Saint-Pierre in Brussels, Belgium -- is testing
the safety and immune response of the vaccine in 50 male and female volunteers.
If the vaccine is shown to be safe, it might advance to larger clinical
trials, which could involve thousands of people at high risk of contracting
HIV, such as commercial sex workers. Targeted Genetics expects to report
data from the European trial in the first half of 2005. Web
resources :
Henry J. Kaiser
Family Foundation : kaisernetwork.org.
and CD40L.
The cDNA for macaque IL-12 and CD40L were cloned into DNA vectors. Groups
of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag
alone or in combination with either IL-12 or CD40L. CD40L did not appear
to enhance the cellular immune response to SIVgag antigen. However, more
robust results were observed in animals co-injected with the IL-12 molecular
adjuvant. The IL-12 expanded antigen-specific IFN-g+
effector cells as well as granzyme B production. The vaccine immune responses
contained both a CD8 component as well a CD4 component. The adjuvanted
DNA vaccines illustrate that IL-12 enhances a CD8 vaccine immune response,
however, different cellular profileref
delivered alone or in combination with the IL-15
gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein
induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6)
vector expressing a modified Env glycoprotein with C-terminal mutations
intended to mimic a fusion intermediate, in which the most divergent region
encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus
was replaced with the dual-tropic 89.6 viral strain. Using a recombinant
vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge
model, we observed that IL-21 plasmid produced sustained resistance to
viral transmission when injected 5 days after DNA vaccination. Moreover,
IL-21 in a synergistic manner with IL-15 expression vector augmented the
vaccine-induced recall responses to the vBD3 challenge compared with those
elicited by immunization in the presence of either cytokine alone. The
synergistic combination of IL-21 and IL-15 plasmids promoted expansion
of CD8+CD127+ memory T cell pools specific for a
subdominant HLA-A2-restricted Env121-129 epitope (KLTPLCVTL).
Coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced
CD8+ T cell function that was partially independent of CD4+
T cell help in mediating protection against vBD3 challenge. Furthermore,
the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular
cytotoxicity and complement-dependent lysis of Env-expressing target cells
through augmentation of Env-specific IgG Ab levels. These data indicate
that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of
the response to DNA vaccinesref
DNA vaccine (see also protein
subunit vaccine)
:-based
DNA vaccination to induce multi-specific CTL responses to HBV DNA vaccination
in normal mice and marked immune responses to multivalent HBV DNA immunization
in larger animal speciesref.
In rhesus macaques intramuscular vaccination with electroporation using
the plasmid encoding for HBV preS(2)-S antigen and an adjuvant plasmid
encoding for hIL-2 and hIFN-g can significantly
enhance the immunogenicity of the DNA vaccines, resulting in greatly improved
antibody responses as well as peptide-stimulated IFN-g
producing T cell responses. In addition, different antibody response behaviors
resulted from different electro-pulse parametersref
as an adjuvant improves antibody responses to HBV S antigen. Higher seroconversion
rates and higher antibody titers were also observed. Prothymosin a
appears to increase the number and affinity of HBsAg-specific, IFN-g-secreting
T cells and to enhance cellular immune response to the preS2S DNA vaccine.
Interestingly, administering the DNA separately from the prothymosin a
plasmid abrogated the enhancement of DNA vaccine potency. The results suggest
that prothymosin a may be a promising adjuvant
for DNA vaccinesref
DNA vaccine :
DNA vaccine : HDV DNA vaccine can produce Th1 and CTL immune
responses but only a low anti-HDV antibody titer is generated with a large
hepatitis D antigen (L-HDAg) construct. In contrast, DNA vaccine expressing
small hepatitis D antigen (S-HDAg) can generate a high titer of anti-HDV
antibodies. Whether the low humoral immunity of L-HDAg DNA vaccine is due
to inadequate dosage or can be ameliorated by other modes of immunization
needs further evaluation. Plasmid (p25L) encoding L-HDAg and plasmid (pS/p25L)
coexpressing HBsAg and L-HDAg were used in this study. We compared the
humoral response generated in mice using different plasmid DNA dosages
and modes of immunization, including gene gun and in vivo electroporation
(EP). I.m. injection with a high dose of plasmid DNA (10 mg/kg) produced
strong antibodies to HBsAg earlier than the usual dose did, but did not
augment the anti-HDV response. Gene gun DNA immunization could not provide
a better humoral immune response to HDV. EP DNA immunization had a higher
anti-HDV seroconversion rate of 80%, but the anti-HDV antibody responses
were generally weak (titer < or = 400:1). The low humoral immunogenicity
of DNA vaccine with L-HDAg cannot be ameliorated by different dosage, gene
gun immunization, or in vivo EP intramuscular injection. DNA vaccine
with a L-HDAg construct may not be a candidate HDV vaccine to generate
anti-HDV humoral immunityref
DNA vaccine : gene fragments encoding p166 and p179, which contain
the neutralization antigenic epitopes of a Chinese strain of HEV genotype
IV, were cloned into 2 different eukaryotic expression vectors (pTR421
and pCDNA3.1), respectively. The in vitro expression level of p166
and p179 in HepG2 cells transfected by each of the recombinant plasmids
with lipofectamine2000 was examined by means of immunofluorescence and
Western blot. Meanwhile, the in vivo expression level in muscles
of mice was examined with immunohistochemistry staining. 4 recombinant
plasmids, pTR421-166, pTR421-179, pCDNA3.1-166 and pCDNA3.1-179, were constructed
successfully and confirmed correct with restriction endonuclease analysis
and nucleotide sequencing. The antigen expression was only detected in
HepG2 cells transfected by pTR421-179 and in myocytes of the mice injected
with pTR421-179. Neither in vitro nor in vivo antigen expression
was detected with pTR421-166 although p166 was only 13 amino acids shorter
than p179 at N terminus. Neither pCDNA3.1-166 nor pCDNA3.1-179 was expressed
in
vitro and in vivoref.
To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and
ORF3 chimeric gene and to assess its ability to elicit specific immunologic
response in mice, the gene encoding the structural protein of HEV ORF2
fragment and full-length ORF3 was amplified by PCR. The PCR products were
cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid
pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly
thrice at a dose of 100 or 200 mug. Mice injected with empty pcDNA3 DNA
or saline served as control and then specific immune responses in the mice
were detected.RESULTS:After 2-3 times of inoculation, all mice injected
with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte
proliferation. The lymphocyte stimulation index in the group immunized
with pcHEV23 (3.1+/-0.49) was higher than that in the control group (0.787+/-0.12,
P<0.01). None in the control group had a detectable level of anti-HEV
IgG. DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully
induce specific humoral and cellular immune response in miceref.
DNA vaccine (see also protein
subunit vaccine,
killed
vaccines
and attenuated
vaccine):
intranasal (i.n.) vaccination represents an attractive non-invasive alternative
to needle-based injection and provides superior protection at mucosal surfaces.
However, new formulations are needed to improve efficacy and reduce the
refrigerated storage and distribution requirements associated with standard
liquid vaccines. Powder offer improved sterility and stability, as well
as ease of storage and transportation, enabling cost effective worldwide
vaccine delivery. A dry powder formulation of a reporter gene-encoding
plasmid DNA was IN administered in rats and reportr gene expression measured
in nasal and lung tissues. The results demonstrate elevated gene expression
in the nasal cavities, without spill-over into the lung tissues.
Using a model plasmid DNA vaccine for influenza, IN dry powder delivery
has been demonstrated to induce potent immune responses. The magnitude
of the serum antibody response was stronger than the corresponding titers
achieved via intramuscular injection or IN administration of a liquid formulation.
at the cell surface. In vitro, cell lines expressing FliC stimulated
production of proinflammatory cytokines and the up-regulation of costimulatory
molecules on monocytes. Mice given the FliC expression vector intradermally
exhibited site-specific inflammation and, in combination with vectors expressing
Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly,
mice also developed strong Ag-specific MHC class I-restricted cellular
immunity. To determine whether vaccination using FliC vectors could elicit
protective immunity to an infectious agent, mice were given dermal injections
of FliC expression vector together with a vector encoding the influenza
A virus nucleoprotein. This vaccination strategy elicited protective
immunity to lethal influenza A virus infectionref.
3 groups of 12 healthy adult subjects received a single dose on day 0 of
either 1, 2 or 4 mg of DNA vaccine, delivered
as 1, 2 or 4 PMED administrations. The PMED influenza DNA vaccine elicited
serum hemagglutination-inhibition (HAI) antibody responses at all three
dose levels, with the highest and most consistent responses in subjects
vaccinated with the highest dose level. Antibody responses were greatest
at the last time point tested, day 56. Treatment-related reactions were
mild to moderate, and included skin reactions at the vaccine site. These
results provide a preliminary indication of the safety and immunogenicity
of a prototype epidermal DNA vaccine for influenzaref
DNA
vaccine
: the small rodent model for ZEBOV - important for
the development of antiviral agents and vaccines, was established in 1998
and the minigenome-based reverse-genetic system was developed inn 1999.
Protection of non-human primates against a lethal challenge with ZEBOV
was first achieved in 2000 and in 2001 an infectious clone for ZEBOV was
created.
|
|
|
|
GP |
vaccinia virus |
guinea pig |
3/5 |
cynomolgus macaque |
0/3ref |
||
VEEV replicon |
guinea pig |
8/10ref |
|
mouse |
18/20ref |
||
cynomolgus macaque |
0/3ref |
||
DNA |
guinea pig |
13/15ref1, ref2 |
|
mouse |
50-100% (dependent on the dose of DNA administered)ref |
||
sGP |
DNA |
guinea pig |
8/11ref |
GP and NP |
VEEV replicon |
cynomolgus macaque |
0/3ref |
DNA |
guinea pig |
8/8ref |
|
DNA and adenovirus |
cynomolgus macaque |
4/4ref |
|
NP |
VEEV replicon |
mouse |
20/20ref1, ref2 |
cynomolgus macaque |
0/3ref |
||
DNA |
guinea pig |
5/8ref |
|
mouse |
70-80% (dependent on the dose of DNA administered)ref |
||
VP24 |
VEEV replicon |
mouse |
37/60 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)ref |
VP30 |
VEEV replicon |
mouse |
30/60 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)ref |
VP35 |
VEEV replicon |
mouse |
23/59 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)ref |
VP40 |
VEEV replicon |
mouse |
32/60 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)ref |
-based
vaccination vector. Vaccine-production will utilise Crucell's human designer
cell line PER.C6ref.
(that also encodes the replicase function and is capable of efficient intracellular
self-amplification) incorporated into virus-like replicon particles (VLP)ref
DNA vaccine : attenuated recombinant replication-competent VSV vectors
expressing either the EBOV glycoprotein or MARV glycoprotein. A single
intramuscular injection of the EBOV or MARV vaccine elicited completely
protective immune responses in nonhuman primates against lethal EBOV or
MARV challenges. Notably, vaccine vector shedding was not detectable in
the monkeys and none of the animals developed fever or other symptoms of
illness associated with vaccination. The EBOV vaccine induced humoral and
apparent cellular immune responses in all vaccinated monkeys, whereas the
MARV vaccine induced a stronger humoral than cellular immune response.
No evidence of EBOV or MARV replication was detected in any of the protected
animals after challenge. These data suggest that these vaccine candidates
are safe and highly efficacious in a relevant animal model. After being
rendered harmless through gene shuffling, VSV vectors were covered with
distinctive proteins that the human body uses to recognize and marshal
defences against either Marburg or Ebola. 6 macaques were given a jab against
Marburg disease; 4 were then infected with Marburg virus and 2 were infected
with Ebola. The 4 infected with Marburg survived; the others died within
10 days. The same procedure was used for jabs against Ebola disease; again,
the 4 that got the right shots survived; the other 2 died. There is concern
that VSV will revert to a dangerous form or hop over into cattle. Both
teams hope their vaccines will be on the market in three to seven years.
The jabs could be used earlier than that, however, if they prove useful
in preliminary studies and an outbreak demands their use on compassionate
groundsref.
Replication-competent vaccine based on attenuated recombinant vesicular
stomatitis virus (rVSV), as a postexposure treatment for MARV haemorrhagic
fever. A rhesus macaque model of MARV haemorrhagic fever that produced
100% lethality was used. rVSV vectors expressing the MARV Musoke strain
glycoprotein were administerede to 5 macaques 20–30 min after a high-dose
lethal injection of homologous MARV. Three animals were MARV-positive controls
and received non-specific rVSV vectors. We tested for viraemia, undertook
analyses for haematology and serum biochemistry, and measured humoral and
cellular immune responses. All 5 rhesus monkeys that were treated with
the rVSV MARV vectors as a postexposure treatment survived a high-dose
lethal challenge of MARV for at least 80 days. None of these 5 animals
developed clinical symptoms consistent with MARV haemorrhagic fever. All
the control animals developed fulminant disease and succumbed to the MARV
challenge by day 12. MARV disease in the controls was indicated by: high
titres of MARV (103–105 plaque-forming units per mL); development of leucocytosis
with concurrent neutrophilia at end-stage disease; and possible damage
to the liver, kidney, and pancreas. Postexposure protection against MARV
in non-human primates provides a paradigm for the treatment of MARV haemorrhagic
fever. Indeed, these data suggest that rVSV-based filoviral vaccines might
not only have potential as preventive vaccines, but also could be equally
useful for postexposure treatment of filoviral infectionsref
DNA vaccine : DNA vaccines encoding structural proteins of VEEV can
elicit immune responses and protection against VEEV though this protection
is incomplete against airborne VEEV. Particle-mediated epidermal delivery
of a DNA vaccine encoding the E2 glycoprotein of VEEV can be boosted with
a mucosally-delivered Ad-based vaccine encoding the same E2 glycoprotein.
This results in an improved Th2-type IgG response, an increase
in neutralising antibody and a significant increase in protection against
airborne VEEV. This indicates that prime-boost may be a suitable immunisation
regimen for providing protection against airborne VEEVref.DNA
vaccine
:
nucleoprotein or glycoprotein gene expressed from recombinant replicon
RNAs derived from an attenuated strain of VEEV
(that also encodes the replicase function and is capable of efficient intracellular
self-amplification) incorporated into virus-like replicon particles (VLP).
A single intramuscular vaccination of a replication-competent vaccine against
Lassa virus based on attenuated recombinant vesicular stomatitis virus
vectors expressing the Lassa viral glycoprotein elicited a protective immune
response in 4 nonhuman primates against a lethal Lassa virus challenge.
Vaccine shedding was not detected in the monkeys, and none of the animals
developed fever or other symptoms of illness associated with vaccination.
The Lassa vaccine induced strong humoral and cellular immune responses
in the four vaccinated and challenged monkeys. Despite a transient Lassa
viremia in vaccinated animals 7 d after challenge, the vaccinated animals
showed no evidence of clinical disease. In contrast, the 2 control animals
developed severe symptoms including rashes, facial edema, and elevated
liver enzymes, and ultimately succumbed to the Lassa infectionref
DNA
vaccines (see also attenuated
vaccine)
are currently in preclinical trials :
with WNV coat proteins (source : Acambis) is in phase I clinical trialDNAvaccine
: NIAID Vaccine Research Center
(VRC) produced a DNA vaccine encoding the spike (S) glycoprotein of
the SARS-CoV induces CD4 and CD8 T cell and neutralizing antibody responses,
as well as protective immunity, in a mouse model. Viral replication was
reduced by more than six orders of magnitude in the lungs of mice vaccinated
with these S plasmid DNA expression vectors, and protection was mediated
by a humoral but not a T-cell-dependent immune mechanismref.
Clinical trials began in December 2004. A secreted recombinant expression
plasmid, pVAX-S1, encoding the partial S protein with a signal peptide,
was constructed and used to immunize BALB/c mice through electroporation.
It was demonstrated that the eukaryotic expression vector pVAX-S1 was successfully
constructed by restriction enzyme and sequence analysis. The expressed
protein could be detected specifically by Western blot analysis. The serum
IgG level of the vaccine group mice was significantly higher than that
of the corresponding control group at day 14 after vaccination (P<
0.05). The vaccine group demonstrated significantly higher S1 protein lymphocyte
proliferation index (LPI) than the control groups (P < 0.05).
Furthermore, in the experimental group, a decrease in the ratio of CD4+
to CD8+ T-lymphocytes and an increase level of IFN-g
in serum were observed. However, IL-4 was not detectable in 2 groups. These
results strongly demonstrated that the pVAX-S1 plasmid could induce humoral
and cellular immune responses in mice, and may be a potential candidate
for a DNA vaccine against the SARS coronavirusref.
A single amino acid substitution (R441A) in the full-length S DNA vaccine
failed to induce NAbs and abolished viral entry when pseudoviruses were
generated. However, another substitution (R453A) abolished viral entry
while retaining the capacity for inducing NAbs. The difference between
R441A and R453A suggests that the determinants for immunogenicity and viral
entry may not be identicalref.
SARS coronavirus nucleocapsid proteinref.
Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and
high-level anti-nucleocapsid antibodies are detected in the patients infected
by SARS-CoV. Several studies have shown that there exists an interaction
between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate
whether the expression of membrane protein can affect the immune responses
induced by nucleocapsid DNA immunization. Two recombinant plasmids containing
M and N coding sequence were constructed. Moreover, in order to get the
antigen for ELISA and in vitro stimulation assay, N protein were expressed
and purified from E. coli bacteria. Injection of 20 mg
of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit
the humoral and cellular responses. The ELISA analysis using the N antigen
or inactivated SARS-CoV particles as capture antigen showed that co-injection
of SARS-M could enhance N-induced antibody production, especially IgG2a
subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen,
The CD4+ and CD8+ T cells of N and M plus N group
were increased compared with those of control groups, and the M protein
could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine
ELISA analysis revealed that co-injection of M could enhance the levels
of IFN-g, IL-2 release induced by N antigen.
Further experiments in field mouse also support the claim that membrane
protein can augment the N-specific immune responses. Virus challenge test
was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV
model, and the results indicated that co-immunization of M and N antigens
could reduce the mortality and pathological changes in lung from the virus
infectionref.
It has been reported that after natural infection in humans and immunization
in animals with SARS-CoV vaccine, antibody is produced and persistent for
a long period of time. In the present study, mice were immunized i.m. with
SARS-CoV S DNA vaccine, and 3 different methods (ELISA, ELISPOT and FACS)
were used to evaluate the immune responses when the cells were stimulated
in vitro with a pool of peptides overlapping entire SARS spike protein.
The results show that prime-immunization with SARS-CoV S DNA vaccine can
induce both CD4+ and CD8+ T cell responses. Boosting
with the same vaccine enhances CD4+ and CD8+ T cell
responses in both lymphoid and nonlymphoid organs and were persistent over
2 months. The SARS-CoV S-specific CD4+ and CD8+ T
cells were CD62L-, a marker for memory cells, and -30 to 50%
of the cells expressed IL-7Ra (CD127), a marker
for the capacity of effector cells to develop into memory cells. In addition,
immunization with the DNA vaccine elicited high levels of antibody production.
Taken together, these data demonstrate that immunization with SARS-CoV
S DNA vaccine can generate antigen-specific humoral and cellular immune
responses that may contribute to long-term protectionref.
DNA vaccine : intranasal immunizationref.
Genetic vaccination against TBE has been successful in an experimental
animal model using a plasmid containing a non-structural protein (NS1).
Such vaccines would provide inexpensive protection against disease, without
raising concerns over inducing ADE on subsequent exposure to infectious
virus. Attempts to generate chaemeric plasmids to protect against both
TBE and dengue fever were less successful. Although these chaemeric plasmids
directed the synthesis and secretion of virus protein normally, no protection
was observed against either TBE or dengue fever.
DNA vaccine :
DNA vaccine : antiviral immunity induced by SFV replicons encoding
the RSV fusion (F) and attachement (G) protein is not associated with enhanced
immunopathologyref
DNA vaccine : measles remains a major worldwide problem partly because
of difficulties with vaccination of young infants. New vaccine strategies
need to be safe and to provide sustained protective immunity. Sindbis virus
replicon particles that express the measles virus (MV) hemagglutinin (SIN-H)
or fusion (SIN-F) proteins have been developed. In mice, SIN-H induced
high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination.
SIN-F, or SIN-H and SIN-F combined, induced somewhat lower responses. To
assess protective efficacy, juvenile macaques were vaccinated with a single
dose of 106 or 108 SIN-H particles and infant macaques
with 2 doses of 108 particles. A dose of 108 particles
induced sustained levels of high-titered, MV-neutralizing antibody and
IFN-g-producing memory T cells, and most monkeys
were protected from rash when challenged with wild-type MV 18 months later.
After challenge, there was a biphasic appearance of H- and F-specific IFN-g-secreting
CD4+ and CD8+ T cells in vaccinated monkeys, with
peaks 1 and 3-4 months after challenge. Viremia was cleared within 14 days,
but MV RNA was detectable for 4-5 months. These studies suggest that complete
clearance of MV after infection is a prolonged, phased, and complex process
influenced by prior vaccinationref.
Infants younger than age 9 mo do not respond reliably to the live attenuated
measles vaccine due the immaturity of their immune system and the presence
of maternal Abs that interfere with successful immunization. The immune
responses elicited by Sindbis virus replicon-based DNA vaccines encoding
measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin
and fusion (F, pMSINH-FdU) glycoproteins were evaluated in neonatal
mice born to naive and measles-immune mothers. Despite the presence
of high levels of maternal Abs, neonatal immunization with pMSIN-H induced
long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs,
mainly IgG2a, that also inhibited syncytium formation in CD150+
B95-8 cells. IgG secreting plasma cells were detected in spleen and bone
marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed
the protective level (200 mIU/ml) but were short-lived, had low syncytium
inhibition capacity, and lacked avidity maturation. This vaccine failed
to induce significant PRN titers in the presence of placentally transferred
Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated
immunity, measured by T cell proliferation and IFN-g
production, that was unaffected by maternal Abs. Newborns responded to
measles DNA vaccines with similar or even higher PRN titers and cell-mediated
immunity than adult mice. This study is the first demonstration that a
Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in
neonates bypassing maternal Abs. Such a vaccine could be followed by the
current live attenuated MV vaccine in a heterologous prime-boost to protect
against measles early in liferef.
DNA vaccine :
DNA vaccine : priming with DNA vaccines and boosting with live vaccinia
virus (VacV). DNA plasmids encoding the A4L, A27L and H5R VacV genes were
generated. A considerable increase in antigen-specific IFN-g
responses,
high proliferative and humoral antigen-specific responses were detected
in experimental primed Balb/C mice compared to controls after VacV boost.
The VacV-DNA plasmids elicited IFN-g production
in HLA-A2.1 transgenic mice in response to predicted HLA-A2.1 restricted
peptide epitopes, providing valuable data for further vaccine developmentref,
Crimean
Congo hemorrhagic fever virus (CCHFV),
tick-borne
encephalitis virus (TBEV),
and Hantaan virus (HTNV),
were tested in mice alone or in various combinations. The bunyavirus vaccines
(RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine
(TBEV) expressed the preM and E genes. All vaccines were
delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited
similar levels of antibodies and protected mice from challenge when delivered
alone or in combination with other DNAs. Although in general, the HTNV
and CCHFV DNA vaccines were not very immunogenic in mice, there were no
major differences in performance when given alone or in combination with
the other vaccinesref.
DNA vaccine :
DNA vaccine : pcDNA3-gD (pgD) and pcDNA3-MIP-1a
/ CCL3
(pMIP-1a) were co-injected to examine the modulatory
effects of MIP-1a on immune phenotype and protection
against lethal challenge with HSV-2. Th-cell proliferative responses
were dramatically enhanced by co-injection of pgD and pMIP-1a
compared with injection of pgD alone. The secretion of IL-2 and IFN-g
was also significantly increased by pgD and pMIP-1a
co-injection; however, the production of cytokines IL-4 and IL-10 was not
affected by co-injection. pgD and pMIP-1a co-injection
resulted in a moderate enhancement of systemic gD-specific antibody level,
but mucosal secretory IgA was markedly enhanced. When BALB/c mice were
challenged intravaginally with 100 LD50 of HSV-2 strain Sav,
pMIP-1a co-injection with pgD improved their
survival rate and significantly reduced both the number of mice with lesions
and the lesion severity. Therefore, MIP-1a cDNA
as a HSV-2 DNA vaccine adjuvant drives antigen-specific Th1-type
responses, reducing HSV-2-derived morbidity and mortalityref.
The prime-boost immunization strategies were studied using a DNA vaccine
carrying the full-length glycoprotein D-1 gene and a baculovirus-derived
recombinant glycoprotein D, both expressing HSV glycoprotein D-1 protein.
Immunization with recombinant DNAs encoding antigenic proteins could induce
cellular and humoral responses by providing antigen expression in vivo.
Higher immune response, however, occurred when the recombinant proteins
followed DNA inoculation. While all groups of the immunized mice and positive
control group could resist virus challenge, a higher virus neutralizing
antibody level was detected in the animals receiving recombinant protein
following DNA vaccinationref.
A mouse model was employed to evaluate protective cellular immune responses
induced by an immediate early antigen of HSV-2. Particle-mediated DNA vaccination
of mice with a DNA plasmid-encoding ICP27 resulted in the induction
of ICP27-specific IFN-g and TNF-a
production in Balb/c mice, but little protection to intranasal challenge
with wild type HSV-2. However, when the DNA vaccine was supplemented with
as little as 50 ng of a vector encoding the A and B subunits of the Escherichia
coli heat labile enterotoxin (LT), animals were profoundly protected
from morbidity and mortality. The ICP27+LT-mediated protection was correlated
with a large increase in ICP27-specific IFN-g
and TNF-a production but cytokine-specific monoclonal
antibody treatment at the time of challenge showed that protection was
mediated predominantly by IFN-g. Furthermore,
depletion of T cell subsets prior to infectious challenge demonstrated
that removal of either CD8+ or CD4+ T cells impaired
protection with CD8+ T cells appearing to play a direct effector
role. These data demonstrate that augmented cellular immune responses resulting
from LT vector plus antigen vector administration to the skin are biologically
significant, leading to enhanced protection against mucosal pathogenic
challengeref.
To construct the recombinant eukaryotic expression plasmid of murine MIP-1a
and investigate the effect of MIP-1a as an adjuvant
on immune response induced by herpes simplex virus type II glycoprotein
D (HSV-II gD) DNA vaccine, using total RNA from RAW264.7 cells stimulated
with LPS, the whole code sequence of murine MIP-1a
was amplified by RT-PCR and inserted into pcDNA3 at HindIII/XbaI
restriction sites. The recombinant eukaryotic expression plasmid Pm was
transiently expressed in COS-7 cells and its specificity was demonstrated
by RT-PCR and Boyden chemotaxis chamber assay. BALB/c mice were immunized
with gD DNA vaccine and/or MIP-1a, and the effect
of MIP-1a on gD DNA vaccine was evaluated by
detecting anti-HSV-II antibody, antigen-specific lymphoproliferative responses,
and examining survival rates after mice were challenged intravaginally
with HSV-II. The recombinant eukaryotic expression plasmid of murine MIP-1alpha
was constructed, and it was revealed that immune responses of HSV-II gD
DNA vaccine were enhanced by coimmunization with MIP-1alpha. CONCLUSION:
The murine MIP-1alpha can be used as an adjuvant of HSV-II gD DNA vaccineref
+ anti-HCV
DNA vaccine : a bicistronic vector pcDNA3.0BA was constructed from
pcDNA3.0. HCV PC154 gene and HBV preS2S gene were inserted into this vector
to form bicistronic expression construct pcDNA3.0BAPC154S2S and monocistronic
expression construct pcDNA3.0BAPC154 or pcDNA3.0BAS2S. These plasmids were
transiently expressed in COS-7 cells and injected into muscles of BALB/c
mice. RESULTS: pcDNA3.0BA contains two cistronic units, which can co-express
2 kinds of genes, with the first immunogen gene and the second gene serving
as additional immunogen or as modulator for the immune responses. HBV surface
Ag and HCV core Ag were coexpressed in vitro. The antibody responses and
lymphoproliferation to antigens were similar between bicistronic and monocistronic
expression construct in mice. pcDNA3.0BA is a novel vector, which can coexpress
two proteins and elicit polyvalent immune responsesref.
DNA vaccine : a eucaryotic expression plasmid
encoding the exosporium antigen BclA, which is a collagen-like surface
protein, pSecTag BclA, carries a secretion signal for the recombinant antigen.
Using NMRI mice the effects of immunisation with a combination of PA- and
BclA-encoding plasmid DNA were compared to immunisations with either PA-
or BclA-encoding pDNA. After 3 immunisations the mice were infected with
25xLD50 of B. anthracis Ames spores. The plasmid pSecTag
BclA, induced high BclA-specific antibody responses. Vaccination with a
combination of PA- and BclA-encoding pDNA led to significantly better survival
than immunisation with only PA- or only BclA-encoding plasmidsref.
The current anthrax vaccine in the U.S., the anthrax vaccine adsorbed,
has several serious drawbacks, most notably the very lengthy and complicated
dosing schedule. Thus, there is a critical need to develop an alternative
anthrax vaccine with a simplified immunization schedule. To address this
need, we evaluated the feasibility of topically priming or boosting onto
the skin using an anthrax protective antigen (PA) protein-encoding DNA
vaccine. To this end, topical immunization of mice onto their skin with
a perflubron-based microemulsion incorporated with a PA63-encoding plasmid,
pGPA, led to significant PA-specific antibody responses, which have anthrax
lethal toxin-neutralization activity. Moreover, topical boosting of mice
primed with PA protein with the pGPA-incorporated, perflubron-based microemulsion
significantly enhanced the anti-PA antibody responses induced. This topical
anthrax DNA vaccine has the potential to be combined with a vaccine, such
as the current AVA, to produce a simplified and more convenient dosing
scheduleref.
DNA vaccine : 3 different plasmids each
targeting a fusion of outer membrane proteins (OprF/OprI), a protein regulating
type III secretion system (PcrV), or an appendage (PilA) were prepared
and mice were immunized with single (monovalent) or a combination of these
plasmids (multivalent) via intramuscular electroporation (imEPT) or gene
gun. Immunization with multivalent DNA vaccine via imEPT induced the most
potent protection against lethal pneumonia. Although the serum levels of
IgG binding to whole bacteria cells were comparable between groups, the
strongest immune protection was associated with the serum levels of Th1-dominated
multivalent IgG, the bronchoalveolar levels of MIP-2 and IFN-g,
and the number of neutrophils and macrophages in the bronchoalveolar lavage
following intranasal challenge. These results implied the possible clinical
application of multivalent DNA vaccine against P. aeruginosaref
DNA vaccineref
:
Ankara (MVA) expressing antigen
85A (MVA85A) : in the first phase 1 study of any candidate
subunit vaccine against tuberculosis, it was found to induce high levels
of antigen-specific IFN-g-secreting T cells
when used alone in bacille Calmette-Guérin (BCG)-naive healthy
volunteers. In volunteers who had been vaccinated 0.5–38 years previously
with BCG, substantially higher levels of antigen-specific IFN-g-secreting
T cells were induced, and at 24 weeks after vaccination these levels were
5–30 times greater than in vaccinees administered a single BCG vaccination.
Boosting vaccinations with MVA85A could offer a practical and efficient
strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic
areasref.
65-kDa heat shock protein (hsp65) : 100 mg plasmid in 50 ml sterile PBS
to quadriceps muscle of miceref
hsp65 gene. In mice given drugs alone, bacteria numbers rose after treatment
was stopped. Mice given the combined treatment showed no such flare-up.
In addition, the combination therapy triggered an immune response that
significantly reduced re-infection with tuberculosis bacteriaref.
This is the first study showing that giving vaccination and drugs together
can accelerate the disappearance of bacteria as well as protecting against
re-infection. Although this study involved mice, the combination treatment
may get into the clinic faster than one might think. With the increasing
threat of MDR strains, there is a push to get promising therapies into
clinical trialsref,
IL-23,
or IL-27
were compared as adjuvants for DNA immunization against M. tuberculosis
infection. The genes encoding p19 and p40 chains of IL-23 or EBI3 and p28
chains of IL-27 were cloned on either side of a self-cleaving peptide from
the FMDV2A protein. The secretion of functional cytokines from transfected
cells was detected with bioassays. Supernatant from p2AIL-23-transfected
cells induced the release of IL-17 from activated lymphocytes, confirming
the presence of bioactive IL-23. Further, supernatant from p2AIL-27-transfected
cells stimulated a significant increase in the proliferation of peptide-stimulated
transgenic CD4+ T cells. In initial experiments, M. tuberculosis
infection
of DCs was more potent at inducing IL-12 and IL-23 secretion than infection
with the vaccine strain Mycobacterium bovis bacille Calmette-Guerin
(BCG), and no significant upregulation of IL-27 was observed. Coimmunization
of C57BL/6 mice with DNA expressing M. tuberculosis antigen 85B (Ag85B;
DNA85B) and plasmids expressing IL-23 or IL-12 stimulated stronger Ag85B-specific
T-cell proliferative and IFN-g responses than
DNA85B alone, whereas the addition of p2AIL-27 had no effect. Interestingly,
DNA85B codelivered with p2AIL-12, but not p2AIL-23, reduced the IgG antibody
response. Both p2AIL-23 and p2AIL-12, but not p2AIL-27, enhanced the protective
efficacy of DNA85B against aerosol M. tuberculosis
challenge. Therefore,
both p2AIL-23 and p2AIL-12 are valuable as cytokine adjuvants for increasing
the protective antituberculosis immunity induced by DNA vaccinesref
DNA vaccine : infection of mice with Mycobacterium
avium or immunization with a novel PE gene expressed by M. avium
(MaPE)
showed that a dominant T-cell immune response was elicited. Immunization
with an MaPE DNA vaccine protected mice against an aerosol challenge with
Mycobacterium
tuberculosis, suggesting that mycobacteria express PE antigens with
cross-protective T-cell epitopesref
DNA vaccine :
DNA vaccine : in BALB/c mice infected
with M. ulcerans subcutaneously, Hsp65 appeared to be an immunodominant
antigen eliciting both humoral and cellular responses. However, vaccination
of mice with a DNA vector encoding Mycobacterium leprae Hsp65 only
poorly limited the progression of M. ulcerans infection. In contrast,
a substantial degree of protection was conferred by subcutaneous vaccination
with BCG, suggesting that BCG antigens that are conserved in M. ulcerans,
such as TB10.4, the 19kDa antigen, PstS3 and Hsp70, may be interesting
to consider as subunit vaccines in future prospectsref.
DNA vaccine
DNA vaccine : 2 plasmid DNA constructs were obtained by cloning separately
into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated
protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated
derivative antigen A (agA) gene encoding a known candidate antigen for
dental caries vaccine. The immunogenicity of the two constructs, designated
pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two
groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy
ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA
or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies
to the target antigens after two doses given at 3-week intervals. Higher
salivary IgA level was observed in the mice immunized with the pcDNA-wapA
vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore,
anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting
a potential protection against S. mutans colonization of the tooth, while
anti-AgA had no significant effect. Indeed, prediction and analysis of
protein epitopes showed that WapA contains highly promiscuous MHC-II binding
motifs in addition to those found in AgA. Immunodot assay confirmed that
WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated
that full-length WapA is a better candidate vaccine antigen than the soluble
AgA, which is truncated in the hydrophobic membrane and wall-spanning regionref.
A dual-promoter expression plasmid that harbors the target gene encoding
SBR of Streptococcus mutans can be applied as DNA vaccine especially
suitable for using attenuated Salmonella as delivery vector to elicit
effective mucosal immune responses because of its advantage of possessing
dual-promoter. Genes encoding SBR and eGFP were amplified by PCR and inserted
to the proper sites of vector pCMVnir. Then IRES sequence was inserted
between the genes coding for SBR and eGFP. Furthermore, a DNA fragment
encoding tPA signal peptide was fused to the 5' end of target gene. Thereby,
construction of the dual-promoter expression plasmid pCN-SSIE was completed
and then the plasmid was analyzed with DNA sequencing and endonuclearase
digestion mapping. The expressions of SBR protein by attenuated Salmonella
SL3261 and CHO cell transformed or transfected by the plasmid were tested
respectively. Finally, BALB/c mice were immunized through injecting intramuscularly
with plasmid pCN-SSIE and anti-SBR specific IgG in serum was tested. Both
DNA sequencing and endonuclearase digestion mapping showed that the construction
of pCN-SSIE was successful with its open reading frame being correct. The
expressions of SBR protein in transformed attenuated Salmonella
SL3261 and transfected CHO were detected, and anti-SBR specific IgG levels
in serum of immunized mice were markedly higher than the controlref.
Anti-caries DNA vaccines, pGJA-P and pGJA-P/VAX, encoding Streptococcus
mutans antigens fused to CTLA4, which binds to B7 molecule expressed
on the surfaces of APCs. Rabbits and monkeys were immunized via intranasal
or intramuscular routes. The fusion vaccine induced accelerated and increased
specific antibody responses in serum and saliva compared with non-fusion
DNA vaccine in rabbits. Significant specific serum IgG and salivary IgA
levels could be detected in fusion vaccine-immunized monkeysref.
To investigate and compare the expression pattern and level of targeted
anti-caries plasmids encoding different-size antigens in eukaryotic cells.
The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P
encoding the signal peptide, extracellular domains of human CTLA-4, human
Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding
domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid
pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to
construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was
removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the
recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those
plasmids by using liposome and the expression of fusion protein was observed
with fluorescence microscope. ELISA was used to detect the expression level
of fusion proteins in cultured supernatants. Specific vesicles with green
fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP.
The recombinant fusion protein could be detected in the cultured supernatants
of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of
which the concentration was different. The highest concentration of recombinant
fusion protein was observed in the supernatants of CHO cells transfected
with pGJGLU/VAX. CTLA-4 targeted fusion protein could be expressed and
secreted by eukaryotic cells. The size of antigen may affect the expression
level of CTLA-4 targeted anti-caries DNA vaccineref.
DNA vaccine : blocking the primary stages of Staphylococcus aureus
infection, specifically the bacterial adhesion to cell and the colonization
of the mucosal surface, may be the most effective strategy for preventing
infections. Clumping factor A (ClfA) is considered to be one of
the most important adhesions factors of S. aureus to host cells.
The present study describes the immune response of dairy cattle to a DNA
vaccine against ClfA and evaluates the ability of specific genetic adjuvants,
targeting sequences (GM-CSFand CTLA-4) and transporter molecules (chitosan
and copolymer) to modify the immune response of cows. The results show
that vaccination of cows with fibrinogen-binding region A induced a strong
and specific antibody response to ClfA in comparison with a control group
injected with the pCI vector alone. Although the co-expression of both
genetic adjuvants and the addition copolymer transporter did not augment
the overall antibody response, these approaches decreased the number of
non-responsive cows. Chitosan was the only factor that did not enhance
the immune response. Three months after the last DNA immunization, 3 cows
from each of the pGM-CSF, IRES, pCTLA and pCI groups were injected with
200 mg of recombinant ClfA protein in IFA. A
strong humoral response was observed in all groups following this protein
boost, with the response occurring slightly earlier in DNA-primed protein
boost cows. Sera and milk samples taken from cows after the second DNA
injection or after the protein boost (sera only) were analyzed for their
ability to block adherence and increase phagocytosis. Pre-incubation of
S. aureus with sera or milk from vaccinated cows significantly reduced
the pathogen's ability to adhere to MAC-T cells relative to the sera and
milk samples from the pCI-injected control cows. Similarly, pools of sera
and milk from vaccinated cows increased phagocytosis of S. aureus
by neutrophils. After the protein boost, sera were more efficient promoters
of phagocytosis, reflecting the higher anti-ClfA antibody level of these
sera. DNA-prime/protein boost regimes combined with molecular adjuvants
appeared to be effective in generating a strong immune response to S.
aureus antigens in cattleref.
Staphylococcus
aureus is the most common etiologic agent of mastitis in bovines and
a major cause of economic losses in the dairy industry. Since adhesins,
that anchor bacteria to the extracellular matrix (ECM), are among the most
important S. aureus virulence factors, we used
4 adhesion factors
(fibrinogen binding protein, fibronectin binding protein A, clumping factor
A and collagen adhesin) as target in a DNA vaccine. Intranasal immunization
with a pDNA mixture coding the four adhesins, triggered significant levels
of specific serum and mucosal Ig that inhibited S. aureus adhesion
to cow mammary gland epithelial cells in vitro. Splenocytes of immunized
mice challenged in vitro with S. aureus extracts showed a strong
proliferative response. Finally, immunized mice were significantly protected
against intramammary challenge with S. aureusref
DNA vaccine :
DNA vaccine : Salmonella vaccine strains have been previously
reported to evoke immune response against heterologous antigen cloned in
the flagellin
gene. A non-toxic cholera toxin subunit B epitope was selected by using
computer-based program and genetically fused in single and double copy
in Salmonella typhimurium flagellin gene. The chimeric flagellin
functioned normally as demonstrated by motility assay. Cholera toxin B
epitope cloned in flagellin was expressed at the flagellar surface. The
expression was verified by Western blotting. Mice administered orally and
subcutaneously with aroA flagellin-negative strain of S. dublin
expressing the chimeric flagellin gene resulted in generation of antibody
against cholera toxin. Mice administered intramuscularly and subcutaneously
with naked mammalian expression vector containing the same cholera toxin
epitope could also evoke the antibody response though it was less than
the chimeric flagellinref
DNA vaccine : ureB generef.
The H. pylori urease subunit B construct or vector alone
was administered to mice via the intranasal route. Spleens and stomachs
were examined on day 0 and weeks 3, 6, and 12 after immunization. Proliferation
of spleen cells was assessed using the carboxyfluorescein diacetate succinimidyl
ester-based flow cytometry assay and cytokine secretion from cultured spleen
cells was detected by ELISA, after stimulation with the urease subunit
B recombinant antigen. Total RNA was isolated from stomach and spleen tissue
and the expression of b-defensin and cytokine
genes was monitored by RT-PCR. Immunized mice were challenged with H.
pylori and bacterial DNA quantified by TaqMan PCR. Results: The urease
B subunit DNA vaccine increased INF-g secretion
and splenocyte proliferation without inducing adverse effects in the spleen.
Increase in gastric b-defensin 1 and marked
induction in local IL-10 : IFN-g ratio up to
12 weeks post-immunization suggest a potential role for local innate immune
responses in protection at the site of infection. Although significant
bacterial reduction in the stomachs of urease B subunit DNA-immunized mice
was observed, intermediate reduction was also noted in the vector group.
Increased defensin expression and adjuvant effects of the cytosine preceding
guanosine motifs may contribute to this phenomenon. CpG motifs, even without
coadministration with antigen, can reduce extracellular bacterial load.
In this study, a DNA construct encoding the urease B subunit was assessed
for its immune profile and its ability to reduce bacterial colonization
in the murine stomach. Local innate immune responses may play a greater
role than previously supposed in limiting H. pylori colonization
in the gastric mucosaref.
DNA vaccine : the 56-kD outer membrane protein has previously
been shown to be the immunodominant antigen in scrub typhus infections.
Its gene was cloned into the DNA vaccine vector pVR1012 as a vaccine candidate
(pKarp56). The in vitro expression of this 56-kD antigen by pKarp56
was confirmed in tissue culture by an indirect fluorescence assay and Western
blot analysis. The initial antibody responses of mice immunized with varied
doses of the pKarp56 were barely detected, but increases were observed
after each of three subsequent booster immunizations. Although no protection
was observed with a single immunization of pKarp56, after 4 immunizations,
60% of the mice survived a 1,000 x 50% lethal dose (LD50) challenge.
These results specifically confirm the importance of the 56-kD protein
antigen in protective immunity against O. tsutsugamushi and demonstrate
the feasibility of DNA vaccines for the prevention of scrub typhusref.
The DNA vaccine vector pVR1012 carrying the O. tsutsugamushi Karp
strain 47-kDa protein gene (p47Kp) consistently provides outbred
mice protection against homologous challengeref
DNA vaccine : to construct the recombinant plasmid of eukaryotic expression
containing Gpd gene from Treponema pallidum and study its immunogenicity
in New Zealand White rabbits. Gpd gene was amplified from the genomic DNA
of T. pallidum and cloned into appropriate site of pcDNA3. 1 ( + ) vector.
After verified that the Gpd antigen gene could be expressed in HeLa cells
by Western blot and immunocytochemistry, recombinant plasmids pcDNA3.1
( + )-Gpd, control plasmid pcDNA3. 1 ( + ) or PBS buffer were administered
in 3 groups of New Zeal and White rabbits. Booster immunizations were employed
at 2-week interval for three times. ELISA was used for the quantitative
detection of the specific antibody in the sera of rabbits. The proliferation
response of spleen cells was detected by MTT assay. The results of the
Western blot and immunocytochemistry showed that Gpd gene constructed in
pcDNA3.1 ( + ) vector could express a fusion protein with a calculated
molecular mass of 41kD in HeLa cells and react with positive blood serum
from syphilis patients. The significant specific antibody IgG titers were
observed and the highest titer was 1:1024 in rabbits after three times
with pcDNA3.1 ( + )-Gpd. The proliferation response of spleen cells were
significantly higher than that of rabbits injected with pcDNA3.1 ( + )
(p < 0.05). All above results establish a solid basis for future studying
the biological activities of Gpd and benefit the development of the syphilis
DNA vaccineref
DNA vaccine : DNA vaccination was used in a susceptible BALB/c high-dose
model of infection to screen 100 novel Leishmania major genes as
vaccine candidates. In addition to finding novel protective antigens, several
antigens that reproducibly exacerbated disease were identified. The immune
response to 2 of these antigens, lmd29 and 584C, that were originally identified
in an expressed sequence tag cDNA sequencing project, were examined. In
addition to exacerbating disease in susceptible BALB/c mice, these antigens
retain a propensity to exacerbate disease in resistant C57BL/6 mice. This
ability to exacerbate disease was lost when susceptible BALB/c mice were
rendered resistant by disruption of the genes encoding IL-4 alone, IL-4/IL-13,
or IL-4, IL-5, IL-9, and IL-13. Failure to exacerbate disease was associated
with reduced IL-5 and IL-10 production in IL-4 knockout mice. Treatment
of lmd29-vaccinated mice with anti-IL-10R antibody prior to challenge infection
converted exacerbation in wild-type BALB/c mice into highly significant
antigen-specific protection. These studies demonstrate that some highly
immunogenic antigens of L. major, while having an intrinsic capacity
to exacerbate disease in the context of otherwise Th1-promoting
DNA vaccine delivery, can be rendered protective by the removal of functional
IL-10ref.
Heat shock proteins (HSPs) are highly conserved among different organisms.
A mycobacterial HSP65 DNA vaccine was previously shown to have prophylactic
and immunotherapeutic effects against Mycobacterium tuberculosis infection
in mice. BALB/c mice were immunized with mycobacterial DNA-hsp65 or with
DNA-hsp65 and trehalose dymicolate (TDM), both carried by biodegradable
microspheres (MHSP/TDM), and challenged with Leishmania major. MHSP/TDM
conferred protection against L. major infection, as indicated by
a significant reduction of edema and parasite loads in infected tissues.
Although high levels of interferon-gamma and low levels of IL-4 and IL-10
were detected in mice immunized with DNA-hsp65 or MHSP/TDM, only animals
immunized with MHSP/TDM displayed a consistent Th1 immune response,
i.e., significantly higher levels of anti-soluble Leishmania antigen
(SLA) IgG2a and low anti-SLA IgG1 antibodies. These
findings indicate that encapsulated MHSP/TDM is more immunogenic than naked
hsp65 DNA, and has great potential to improve vaccine effectiveness against
leishmaniasis and tuberculosisref
DNA vaccine : acute visceral leishmaniasis is a progressive disease
caused by Leishmania chagasi in South America. The acquisition of
immunity following infection suggests that vaccination is a feasible approach
to protect against this disease. Since Leishmania homologue of
receptors for activated C kinase (LACK) antigen is of particular interest
as a vaccine candidate because of the prominent role it plays in the pathogenesis
of experimental Leishmania major infection, we evaluated the potential
of a p36LACK DNA vaccine in protecting BALB/c mice challenged
with L. chagasi. In this study, mice received intramuscular (i.m.)
or subcutaneous (s.c.) doses of LACK DNA vaccine. A significant production
of IFN-g was detected by splenocytes from i.m.
vaccinated mice in response to L. chagasi antigen and to rLACK protein.
However, no reduction in parasite load neither in liver nor in the spleen
of vaccinated animals was observed. The lack of protection observed may
be
explained by a significant production of IL-10 induced by the vaccineref.
Vaccination of dogs, the domestic reservoir of Leishmania chagasi,
could not only decrease the burden of canine visceral leishmaniasis (VL),
but could also indirectly reduce the incidence of human VL. Intramuscular
vaccination of foxhounds with a Leishmania multicomponent (10
antigen) DNA vaccine resulted in antigen-induced lymphoproliferative
and IFN-g (but not IL-4) responses. This response
was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants.
The multicomponent DNA vaccine also induced a delayed type hypersensitivity
(DTH) response to viable L. donovani promastigotes and led to a
reduction of parasite burden in an in vitro intracellular infection model,
and in the draining lymph node of dogs early after cutaneous challenge.
Thus, the multicomponent DNA vaccine was effective in priming dogs for
a parasite-specific Th1 cellular immune response, which was
able to restrict parasite growthref.
The nucleoside hydrolase (NH36) of Leishmania (L.) donovani
is a vital enzyme which releases purines or pyrimidines of foreign DNA
to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine,
the VR1012-NH36 was immunoprotective against visceral and cutaneous murine
leishmaniasis. In this work we tested the immunotherapy against Leishmania
(L.) chagasi infection, using 2 doses of 100 or 20 mg
VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous
garlic extract (8 mg/kg/day by the i.p. route), which was effective in
immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was
significantly reduced following treatment with 100 mg
(91%) and 20 mg (77%) of the DNA vaccine, and
by 20
mg DNA vaccine and garlic extract (76%)
(p=0.023). Survival was 33% for saline controls, 100% for the 100 mg
vaccine, and 83 and 67% for the 20 mg vaccine
with and without garlic extract addition, respectively. Garlic treatment
alone did not reduce parasite load (p>0.05), but increased survival (100%).
The NH36-DNA vaccine was highly effective as a new tool for the therapy
and control of visceral leishmaniasis, while the mild protective effect
of garlic might be related to an unspecific enhancement of IFN-g
secretionref
DNA vaccine :
DNA vaccine : immunity depends on CTLsref1,
ref2
DNA vaccine : the ability to generate potent antigen-specific T cell
responses by vaccination has been a major hurdle in vaccinology. Vaccinia
virus and avipox viruses have been shown to be capable of expressing antigens
in mammalian cells and can induce a protective immune response against
several mammalian pathogens. 2 such vaccine constructs, modified vaccinia
virus Ankara (MVA) and FP9 (an attenuated fowlpox virus) both expressing
the pre-erythrocytic malaria antigen thrombospondin-related adhesion protein
and a string of CD8+ epitopes (ME-TRAP). In prime-boost combinations
in a mouse model MVA and FP9 are highly immunogenic and induce substantial
protective efficacy. A series of human clinical trials using the recombinant
MVA and FP9 malaria vaccines encoding ME-TRAP, both independently and in
prime-boost combinations with or without the DNA vaccine DNA ME-TRAP, has
shown them to be both immunogenic for CD8+ T cells and capable
of inducing protective efficacy. The safety profiles of these viral vectors
and show that anti-vector antibody responses induced by the vectors are
generally low to moderate : these vectors are safe and show acceptable
side effect profiles for prophylactic vaccinationref.
An important consideration in the development of a malaria vaccine for
individuals living in areas of endemicity is whether vaccine-elicited immune
responses can be boosted by natural infection. To investigate this question,
Plasmodium
berghei ANKA blood-stage parasites were used for the infection of mice
that were previously immunized with a DNA vaccine encoding the P. berghei
sexual-stage antigen Pbs48/45. Intramuscular immunization in mice with
one or two doses of DNA-Pbs48/45 or of empty DNA vaccine as control did
not elicit detectable anti-Pbs48/45 antibodies as determined by enzyme-linked
immunosorbent assay. An infection with P. berghei ANKA 6 weeks after
DNA vaccination elicited comparable anti-Pbs48/45 antibody levels in mice
which had been primed with DNA-Pbs48/45 or with empty DNA vaccine. However,
a repeat infection with P. berghei ANKA resulted in significantly
higher anti-Pbs48/45 antibody levels in mice which had been primed with
the DNA-Pbs48/45 vaccine than the levels in the mock DNA-vaccinated mice.
In parallel and as an additional control to distinguish the boosting of
Pbs48/45 antibodies exclusively by gametocytes during infection, a separate
group of mice primed with DNA-Pbs48/45 received an infection with P.
berghei ANKA clone 2.33, which was previously described as a "nongametocyte
producer". To our surprise, this parasite clone too elicited antibody levels
comparable to those induced by the P. berghei gametocyte producer
clone. The nongametocyte producer P. berghei clone is in fact a
defective gametocyte producer that expresses Pbs48/45, much like the gametocyte
producer clone, and is therefore capable of boosting antibody levels to
Pbs48/45. Taken together, these results indicate that vaccine-primed antibodies
can be boosted during repeat infections and warrant further investigation
with additional malaria antigensref.
DNA vaccine : cysteine proteinases of C. sinensis are
important virulence factors that induce pathological changes associated
with larval migration and localized biliary epithelial destruction. This
study investigated the immunogenicity and protective efficacy of a DNA
vaccine encoding Clonorchis sinensis cysteine proteinase (CsCP).
The CsCP cDNA sequence displays significant homology to the mammalian or
trematode cathepsin L. Plasmid DNA carrying the CsCP gene (pcDNA3.1-CsCP)
was injected into Sprague-Dawley (SD) rats intradermally. Animals injected
with pcDNA3.1-CsCP developed CsCP-specific antibodies, which exhibited
an IgG2a dominance in sera. In addition, the DNA vaccine elicited
the production of IFN-g, but not IL-4 in splenocytes,
suggesting the induction of a typical Th1 dominated immune response
in rats. The pcDNA3.1-CsCP induced a significant level of protection (31.5%,
p<0.05) in SD rats challenged with C. sinensis metacercariae.
These results indicate that pcDNA3.1-CsCP induces both humoral and cellular
immune responses. The CsCP gene may be a good candidate for use in future
studies of vaccination against clonorchiasisref.
Clonorchis
sinensis, the Chinese liver fluke, resides chronically in the biliary
tract, and fatty acid-binding protein (FABP) is known to play an important
role in the intracellular transport of long-chain fatty acids obtained
from the host. Although FABP has stimulated considerable interest as a
vaccine candidate, the nature of C. sinensis FABP (CsFABP) remains
unclear. The immunogenicity and protective efficacy of a DNA vaccine encoding
CsFABP was investigated. The intradermal injection of plasmid DNA carrying
the CsFABP gene (pcDNA3.1-FABP) into Sprague-Dawley (SD) rats induced both
humoural and cellular immune responses. Animals injected with pcDNA3.1-FABP
developed FABP-specific antibody, which is dominance of IgG2a
in sera. In addition, the DNA vaccine elicited the production of IFN-g,
but not the production of IL-4 in spleen cells stimulated with recombinant
FABP. Moreover, pcDNA3.1-FABP induced a significant level of protection,
decreased worm burden (40.9%, P < 0.05) in SD rats against C. sinensis
metacerariae challenge. These results suggest that pcDNA3.1-FABP induces
a typical Th1-dominated immune response and it is a good candidate
for use in future clonorchiasis vaccination studiesref.DNA
vaccine :
DNA vaccine :
is a neurodegenerative disorder characterized by neuropathological hallmarks
including deposits of the beta-amyloid peptide (AssP). Studies have shown
that immunization with Ab42 peptide
reduces both the spatial memory impairments and AD-like neuropathologic
changes in Alzheimer disease transgenic mice, but can cause side effect
of a cell-mediated autoimmune meningoencephalitis. Recently, some studies
showed that DNA vaccination could be used to generate an antibody response
to Abeta without the adverse cell-mediated immune effect. 4 DNA vaccine
plasmids (pV-GE1, pV-GE2, pV-GE3, and pV-GE4) have been generated against
AD by separately fusing Ab epitope sequences
(coding for EFGH, DAEFGH, EFGH+EFGH, and EFGH+DAEFGH) with IgG heavy chain
coding region of mouse. Meanwhile, the full-length gene Abeta encoding
plasmid (pV-Ab), empty vector (pVAX) and synthetic
AssP were also included as control. The sera of BALB/c mice immunized via
intramuscular with plasmids and peptide were tested by indirect ELISA for
auto-AssP immunoreactivity. The results showed that all the DNA vaccine
plasmids induced AssP-specific antibodies; moreover pV-GE2 and pV-Ab
constructs elicited higher antibody titers than other constructs (P <
0.05). To further enhance the immune response, GM-CSF encoding plasmid
(pGM-CSF) and purified BCG-DNA were used as molecular adjuvants. BCG-DNA
could enhance humoral and cellular immune responses simultaneously and
did not alter the phenotype of the immune responses, whereas pGM-CSF showed
no obvious effect on immune response. These results suggest that this immunization
strategy of using Ab epitope encoding plasmid
+ BCG-DNA adjuvant may serve as the basis for developing anti-AD vaccinesref.
Safe and effective nonviral Ab DNA vaccines
against AD were developed and administered to model (APP23) mice and evaluated
Ab burden reduction. Prophylactic treatments
started before Ab deposition reduced Ab
burden to 15.5% and 38.5% of that found in untreated mice at 7 and 18 months
of age, respectively. Therapeutic treatment started after Ab
deposition reduced Ab burden to approximately
50% at the age of 18 months. Importantly, this therapy induced neither
neuroinflammation nor T cell responses to Ab
peptide in both APP23 and wild-type B6 mice, even after long-term vaccination.
Although it is reported that other anti-Ab therapies
have pharmacological and/or technical difficulties, nonviral DNA vaccines
are highly secure and easily controllable and are promising for the treatment
of ADref.
: rabbits were intramuscularly immunized with the plasmid pCR-X8-HBc-CETP
encoding a B-cell epitope of cholesteryl ester transfer protein (CETP)
C-terminal fragment (CETPC) displayed by Hepatitis B virus core (HBc) particle.
This plasmid also contained immunostimulatory sequences (ISS) which included
eight CpG motifs 5'-GACGTT-3', functioning as immunomodulators. After anti-CETP
antibodies were successfully produced, rabbits were fed with a high-cholesterol
diet for 15 weeks, and then the antiatherogenic effects of this DNA immunization
were evaluated. The fraction of plasma cholesterol in HDL significantly
increased and the fraction of plasma cholesterol in LDL decreased in the
pCR-X8-HBc-CETP immunized rabbits compared with those in the saline control
group and one group treated with the plasmid pCR-X8-HBc containing ISS
but lacking CETP epitope. More importantly, DNA immunization with pCR-X8-HBc-CETP
markedly reduced the average percentage of aortic lesions in the entire
aorta area by 80.60% compared with the saline control (3.78% versus 19.48%)
and the average thickness of hyperplastic coronary artery in this group
was also significantly less than in the saline control group (146+/-11mum
versus 248+/-18mum). CpG DNA alone could be antiatherogenic in this model
because the average % of aortic lesions in pCR-X8-HBc immunized rabbits
was
16.53% lower than that of the saline control group and the average thickness
of hyperplastic coronary artery was also substantially lower than saline
control group (155+/-13 mm versus 248+/-18 mm).
Thus, plasmid pCR-X8-HBc-CETP could significantly inhibit the progression
of atherosclerosis and be potentially developed as a suitable DNA vaccine
against atherosclerosisref
: the use of synthetic Cn2 fragments is not the best strategy to rely on,
since these peptides instead of eliciting a protective response, reveled
an unexpected sensitization phenomena of the immunized animals towards
Cn2ref.
A chimera consistng on the Cn2 gene fused in frame to the DC-SIGN gene
can elicit specific antibody response against Cn2.:
TsNTxP gene expressed as a fusion protein with maltose-binding protein
induced neutralizing antibodies against Ts toxins
: a synthetic nontoxic analog of toxin II
: gene gun immunization with DNA encoding the carboxy-terminal JD9 domain
of jararhagin, a haemorrhage-inducing metalloprotease in the venom of South
America pit viper stimulated high titre IgG1 antibodies that
substantially reduced (-70%) the hemorrhage induced by whole venomref.
Simultaneous delivery with a plasmid encoding murine GM-CSF resulted in
a log fold increase in titre, without immunoglobulin class switching. Increasing
the number of immunisations resulted in a further log fold augmentation
of antibody titreref.
: substitutions in the a-cobratoxin (CTX) cDNA
were introduced at two residues critical for binding to the nicotinic acetylcholine
receptor (Asp27 to Arg, Arg33 to Gly). The mutated CTX expression cassette
was delivered in the context of a replication deficient adenovirus vector
(AdmCTX). To assess whether expression of the mutated CTX in vivo leads
to the development of protective immunity, BALB/c mice were challenged
by IV administration of 2 microg of alpha-cobratoxin protein 21 or 63 days
after administration of AdmCTX or Ad- Null (as a control; both, 10(9) particle
units). Animals receiving AdmCTX but no a-cobratoxin
challenge suffered no ill effects, but > or =80% of naive animals or those
receiving the AdNull control vector died within 10 min from the a-cobratoxin
challenge. In contrast, 100% of animals receiving a single dose of AdmCTX
21 or 63 days prior to a-cobratoxin challenge
survived. The data demonstrates that an adenovirus-based vaccine can be
developed to protect against lethal challenge with a potent snake venom.
The effectiveness of this approach might serve as a basis to consider the
development of a global public health program to protect those at risk
for death by snakebiteref
: introduction of a foreign Th epitope into CCL2
DNA vaccine could boost its immunogenicity by inducing strong neutralizing
autoantibody against the pathogenic chemokine CCL2 sufficiently to be protective
in a classically nonimmune model of disease, Adriamycin nephropathy (AN).
Modification of the CCL2 DNA vaccine by replacing a surface loop region
of CCL2 sequence with tetanus toxoid Th epitope P30 elicited
a strong self-specific CCL2 autoantibody production, as well as an IFN-g-producing
T cell cellular response. The increased immunogenicity of modified CCL2
DNA vaccination but not unmodified CCL2 DNA vaccination was protective
against functional and structural renal injury in rat AN. The protective
effect of the modified CCL2 DNA vaccine was associated with blockade of
glomerular and interstitial macrophage recruitment by neutralizing autoantibody
against CCL2, which plays a critical role in eliciting renal injury in
AN. Therefore, modification with a foreign Th epitope breaks
self-tolerance by inducing a cellular and humoral response against self-protein
and provides a strategy to increase the potency of DNA vaccination sufficiently
to afford protection in toxin-induced chronic renal diseaseref,
HHV-5
/ HCMV,
HIV-1,
HBV
or group B Streptococcus
spp. (GBS),
usually occurs at the end of pregnancy, during birth or by breast feeding.
The vertical transmission of many of these pathogens significantly increases
the risk of neonatal infection. Therefore, active immunization of the fetus
might represent an effective approach to reduce the high risk of neonatal
diseases.
induced detectable immune responses (immune memory and mucosal immunity)
in 80% (12 of 15) of newborn lambs. There was no evidence for the induction
of immune tolerance in nonresponding lambs. Responding lambs displayed
both systemic and mucosal immune responses and reduced virus shedding following
intranasal challenge. Furthermore, strong anamnestic responses were evident
for at least 3 mo after birth. The efficacy of in utero oral DNA
immunization was further demonstrated with the HBsAg, and protective serum
Ab titers occurred in 75% of immunized lambsref.
Fetal immunization was safe and did not affect fetal gestation, neonatal
viability, or significantly alter blood leukocyte populations. In utero
immunization resulted in the induction of protective mucosal immunity and
immune memory in the newborn lamb. Furthermore, there was no evidence that
in
utero DNA immunization induced immune tolerance. The uptake and expression
of the plasmid DNA already occurred within the epithelium of the oral cavity,
correlating with previous findings that local immune responses were found
exclusively in the retropharyngeal lymph nodes draining the oral cavityref.
DNA immunization at two-thirds of pig gestation produces, at birth,
antibody titers considered protective in humans. The boost of antibody
titers in every animal following recall at 4 and 10 months demonstrates
the establishment of immune memory. The safety of in-utero fetus manipulation
is guaranteed by short-term (no fetus loss, lack of local alterations,
at-term spontaneous delivery, breastfeeding) and long-term (2 years) monitoring.
Treatment of fetuses closer to delivery results in immune ignorance without
induction of toleranceref
DNA vaccine : since conventional vaccines have failed in inducing an
effective immune response in sheepref1,
ref2,
ref3,
some proposed to develop a DNA vaccine agaist MVVref.
Plasmid administration by injection into tibialis muscle seems to be more
efficient than injection via the quadriceps route, probably because it
is smaller and so local plasmid concentration is higherref.
DNA vaccine : DNA vectors can be used to deliver vaccine antigens that
stimulate effective protective immunity in mice, but in larger, outbred
animal species, the protective efficacy is lower or large doses of DNA
are required. These data demonstrate that porcine IL-3 when delivered to
pigs by DNA vector or in low doses as recombinant protein, can enhance
antibody responses to classical swine fever virus antigen expressed from
co-delivered DNA, and improve the protective efficacy of the DNA vaccine.
The effect was further enhanced when IL-3 was expressed as a fusion protein
with the potyvirus coat protein. The adjuvant effect of IL-3 was compared
to that of GM-CSF and IL-3 was shown to be at least as efficacious as GM-CSF.
The response to IL-3 is novel and suggests, that at least in pigs, IL-3
could be used as an adjuvant for DNA vaccinesref.
CSFV
E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The
plasmid pVAXE2 was transformed into Salmonella choleraesuis C500
(S. C500) attenuated vaccine strain by electroporation to generate
Salmonella
choleraesuis engineering strain S. C500/pVAXE2. The characterization
of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated
that it retained the same properties as its original strain S. C500 with
exception of kanamycin resistance originated from the plasmid pVAXE2. The
plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits
were vaccinated 3 times at 2 weeks interval with S. C500/pVAXE2 in oral
and intramuscular routes at the dosage of 1 x 108 CFU for mice
and 2 x 109 CFU for rabbits each time. The specific antibody
response against CSFV and Salmonella choleraesuis was detected by
ELISA. 2 weeks after the third boost the immunized rabbits were challenged
with 20 ID50 of hog cholera lapinized virus (HCLV), followed
by a virulent strain of
Salmonella choleraesuis 2 week later than
HCLV challenge. All immunized mice and rabbits produced significant antibodies
against CSFV and Salmonella choleraesuis, and the immunized rabbits
demonstrated the effective protection against the challenge of HCLV and
virulent Salmonella choleraesuis. These results indicated the potential
of developing multiplex swine DNA vaccine by using this bacteria as the
vectorref
DNA vaccine : DNA-based immunisation is cheaper and more versatile
than vaccine prepared with tissue cultureref.
It has been shown recently that dogs can be protected against a challenge
with the rabies virus by injecting a DNA vaccine encoding the RV glycoprotein
(G)ref.
Production, purification and analysis of an experimental DNA vaccine against
rabies has been reportedref.
Rabid dog exposures result in >99% of human rabies deaths worldwide. 98%
of these cases occur in developing countries. Thus, the best protection
against human rabies would be prevention through adequate vaccination of
the reservoir population. The difficulty in re-locating ownerless, freely
roaming dogs for booster vaccinations, in addition to poor coverage with
inadequate vaccines, suggests that a potentially inexpensive vaccine that
elicits long-term protection after a single-dose could improve control
of canine rabies in developing countries. One solution could be a DNA vaccine.
Dogs vaccinated intradermally (i.d.) in ear pinnae with a rabies DNA vaccine
expressing a RV G produce high levels of neutralizing antibody that persist
for at least 6 months. All dogs (100%) vaccinated i.d. in each ear pinna
with a one-time 50 mg of rabies DNA vaccine,
or intramuscular (i.m.) with a commercial canine rabies vaccine survived
a lethal dose of rabies virus given 1 year after. In contrast, 100% of
dogs vaccinated i.m. with 100 mg of rabies DNA
developed rabies, as did 100% of negative control dogs that were vaccinated
i.d. in each ear pinna or i.m. with DNA that did not express the rabies
virus G. The data suggest that a one-time i.d. rabies DNA vaccination into
ear pinnae offers a new approach to facilitate control of endemic canine
rabies in developing countriesref.
A recombinant plasmid pTargeT.rabgp containing the G gene of RV was constructed
and produced for immunogenicity studies on mice and dogs. The mice immunized
twice with 50 mg or 100 mg
of pTargeT.rabgp intramuscularly (i.m.), showed a serum antibody titer
of 256 or 64, respectively 14 days post immunization (p.i.). When the vaccinated
mice were challenged at 14 days p.i. with RV i.c., they were protected
in 81.25% or 87.5%, respectively. A positive cell mediated immune response
was observed by lymphocyte proliferation assay and macrophage migration
inhibition test. These results indicate that the pTargeT.rabgp plasmid
can be used as a rabies DNA vaccineref.
Kinetics of antibody responses and protection against rabies were investigated
after injection of a single dose of rabies DNA vaccine and compared to
those induced by 1 or 2 injections of cell culture-derived vaccine in dogs
issued from the common local breed and reared in experimental conditions.
Rabies DNA vaccine administered intradermally by a jet injector in the
inner face of the ear was by far more efficient in inducing long lasting
high titers of virus neutralizing antibodies compared to cell culture vaccine
Rabisin® administered either subcutaneously or intramuscularly.
Four years after vaccine administration of either DNA or cell culture-derived
rabies vaccines, full protection against a rabies peripheral challenge
was achieved. Vaccine trials targeting dogs living in field conditions
in Tunisia further established that rabies DNA-based vaccination induced
a stronger induction of virus neutralizing antibodies compared to Rabisin®.
This report shows for the first time that DNA vaccination could be more
efficient under experimental or field conditions in large size mammals
than the best commercially available cell culture-derived vaccine. This
improvement will hopefully allow a better rabies control in developing
countries by using a more efficient vaccination with fewer doses and targeting
all categories of dogsref.
Rabies virus (RV) glycoprotein (gp) consists of three domains: cytoplasmic,
transmembrane and ectodomain. It occurs in a complete, membrane-bound form
within the infected cell, but it is released from them in a deleted, secreted
form lacking the transmembrane domain. A study tested the importance of
the transmembrane domain for the capability of the RV gp gene, introduced
into mice via a recombinant plasmid (DNA vaccine), to induce immune response
and protection against challenge. Although the antibody response to the
secreted form of gp was higher than that to complete gp, the protective
efficacy of the respective DNA vaccine against challenge was not better
than that of the DNA vaccine inducing complete gp. This indicates that
the transmembrane domain of RV gp is important for generating protection
against rabies and should be present in RV DNA vaccinesref.
DNA vaccine : genes encoding the nucleocapsid protein (N) and the C-terminal
half of the glycoprotein (G) were amplified by RT-PCR and separately cloned
into the eukaryotic expression vector pcDNA 3.1. The G protein expressed
by transfected cells was detected by western blot analysis. PCR analyses
demonstrated the presence of injected plasmids in fish muscle tissue at
14 days post injection. IHC of muscle tissue injected with the plasmid
DNA showed expression of the target protein in myofibrils and sarcoplasm.
Flounder fry with an average weight of 3 g were injected with 5 mg
of plasmid DNA and challenged at 21 days after immunization. Fish injected
with vector DNA or PBS showed >95% cumulative mortality by 16 days after
inoculation with the virus. In contrast, fish injected with plasmids containing
the N gene, G gene, or N+G gene mixture showed 70, 5, and 2.5% cumulative
mortality, respectively. These results show that the G gene is effective
for the induction of protective immunity against HIRRV infection in injected
fishref.
DNA vaccineref1,
ref2,
ref3.
Pre-clinical research work reported in the literature has clearly demonstrated
that the application of DNA vaccine technology is highly effective for
the control of several viral pathogens in fish. However, the transition
from proof of concept to commercialization has proved elusive for a variety
of reasons, despite the demonstrable purity, potency, safety and efficacy
of early DNA vaccine models. Registration is constrained by a worse-case
risk assessment approach by regulators and vague, often non-existent regulations
for the registration of DNA vaccines and, most importantly, lack of a licensing
precedent. Second, costs to manufacture and secure intellectual property
rights remain prohibitively high. This is compounded by the low unit
value of the target animals, especially where aquaculture is concerned,
and uncertainty with respect to required studies to satisfy regulators.
Third, where food animals are concerned, there is a risk that inappropriate
association with certain recombinant technologies used in agriculture will
confuse consumers and create uncertainty in the marketplace. All combined,
these factors have challenged the commercialization of DNA vaccine technology
for food animal applications, especially in aquaculture. Vaccine plasmid
pIHNw-G, containing the gene for the viral glycoprotein, was detected immediately
after intramuscular injection in all tissues analyzed, including blood,
but at later time points was found primarily in muscle tissue, where it
persisted to 90 days. Glycoprotein expression was detected in muscle, kidney,
and thymus tissues, with levels peaking at 14 days and becoming undetectable
by 28 days. Histologic examination revealed no vaccine-specific pathologic
changes at the standard effective dose of 0.1 mgg
DNA per fish, but at a high dose of 50 mgg an
increased inflammatory response was evident. Transient damage associated
with needle injection was localized in muscle tissue, but by 90 days after
vaccination no damage was detected in any tissue, indicating the vaccine
to be safe and well toleratedref.
The DNA vaccine pIHNw-G encodes the glycoprotein of IHNV. Vaccine performance
in rainbow trout was measured 3, 6, 13, 24, and 25 months after vaccination.
At three months all fish vaccinated with 0.1 mg
pIHNw-G had detectable neutralizing antibody (NAb) and they were completely
protected from lethal IHNV challenge with a relative percent survival (RPS)
of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25
months post-vaccination showed protection with RPS values of 47-69%, while
NAb seroprevalence declined to undetectable levels. Passive transfer experiments
with sera from fish after 2 years post-vaccination were inconsistent but
significant protection was observed in some cases. The long-term duration
of protection observed here defined a third temporal phase in the immune
response to IHNV DNA vaccination, characterized by reduced but significant
levels of protection, and decline or absence of detectable NAb titers.
Examination of multiple tissues showed an absence of detectable long-term
histopathological damage due to DNA vaccinationref.
The DNA vaccine based on the glycoprotein gene of IHNV induces a non-specific
anti-viral immune response and long-term specific immunity against IHNV.
This study characterized gene expression responses associated with the
early anti-viral response. Homozygous rainbow trout were injected intra-muscularly
(I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle
tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray.
Eighty different genes were significantly modulated in the vector DNA group
while 910 genes were modulated in the IHNV DNA vaccinate group relative
to control group. Quantitative reverse-transcriptase PCR was used to examine
expression of selected immune genes at the I.M. site and in other secondary
tissues. In the localized response (I.M. site), the magnitudes of gene
expression changes were much greater in the vaccinate group relative to
the vector DNA group for the majority of genes analyzed. At secondary systemic
sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated
in only the IHNV DNA vaccinated group. The results presented here suggest
that the IHNV DNA vaccine induces up-regulation of the type I IFN system
across multiple tissues, which is the functional basis of early anti-viral
immunityref.
A highly efficacious DNA vaccine against IHNV was mutated to introduce
two stop codons to prevent glycoprotein translation while maintaining the
plasmid DNA integrity and RNA transcription ability. The mutated plasmid
vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish
at high doses, lacked detectable glycoprotein expression in the injection
site muscle, and did not provide protection against lethal virus challenge
7 days post-vaccination. These results suggest that the G-protein itself
is required to stimulate the early protective antiviral response observed
after vaccination with the nonmutated parental DNA vaccineref
DNA vaccineref1,
ref2
: a recombinant glycoprotein vaccine (VHSVg) and a DNA vaccine (pCMV-VHSg)
were injected in Japanese flounder (Paralichthys olivaceus). Each
fish was injected with 10 mg vaccine dissolved
in 50 ml phosphate buffer saline (PBS). One
month after the vaccination, the fish were challenged intraperitoneally
with either 1x102 or 1x103 TCID50 of virus.
Fish that received the VHSg DNA vaccine were highly protected against virus
infection over a 21-day observation period, with cumulative mortalities
ranging from 4 to 10%. However, the recombinant protein vaccine group appeared
to have low survival rates. Using microarray analysis, humoral defense-related
genes such as complement component C3, complement regulatory plasma proteins,
IgM, IgD, MHC class II-associated invariant chain and CD20 receptor were
observed to be up-regulated by the VHSg recombinant protein vaccine at
1 or 21 days post vaccination. On the other hand, cellular defense-related
genes such as CD8a chain, T-cell immune regulator,
MIP1-a and apoptosis-associated protein were
not detected. Specific antibodies against VHSVg protein were detected in
both vaccinated groups at a titer of 1:40 at 28 days post vaccinationref.
Since previous results showed that IL-8 was induced in rainbow trout (Oncorhynchus
mykiss) in response to viral hemorrhagic septicemia virus (VHSV) infection,
we have cloned IL-8 in an expression vector (pIL8+) and studied its possible
adjuvant effect on the early response to a VHSV immunization model, focusing
on the early response of several cytokines induced by a vector coding for
the glycoprotein of VHSV (pMCV1.4-G) in the spleen and head kidney. First,
we demonstrated that the pIL8+ successfully transcribed IL-8,
by induction of IL-8 transcription in the muscle and blood, and by a massive
infiltration of neutrophils at the muscle inoculation site. The effect
of pIL8+ co-administration was studied on the expression of
2 pro-inflammatory cytokines, such as IL-1b
and TNF-a; cytokines that have mainly an inhibitory
role, IL-11 and TGF-b; and a Th1
type cytokine, IL-18. The co-administration of pIL8+ with pMCV1.4-G modulates
the cytokine response that is induced, mainly by having its effect increasing
pro-inflammatory cytokines (IL-1beta and TNF-alpha1), with a greater impact
on the spleen, and to a lesser extent in the head kidney. All these data
suggest that IL-8 is able to modulate the early cytokine immune response
that is produced in response to a DNA vaccine, and therefore, might be
a potential immune adjuvant in fish viral vaccination. More work should
be done to determine if this modulation has a beneficial effect on protection
as seen in other mammal viral modelsref.
DNA vaccine : recently, the Ministry of Agriculture (MOA) of China
after very careful evaluation of their safety and efficacy has certified
...
DNA vaccine : It is known that only the minority of plasmid DNAs effect
a cure or prevention after intramuscular injection into host. But what
is the fate of the majority? And indeed how many of the injected DNAs work?
Till now, little is known about it. To answer these questions, two methods
including PCR and autoradiography were used in distribution study in mice
that had received a single muscular inoculation of plasmid DNA containing
antigenic epitopes of FMDV. The results showed that the plasmid DNAs were
distributed by blood circulation and degraded soon. The degradation ratio
of super coiled plasmid DNA was 20.9% in 10min, 34.1% in 1h, 86.8% in 1
day and 97.8% in 1 week in sera in vivo. And over a half of the
whole were output in urine and faeces. The rest resided most in muscles
as 'antigen pool', next in immune organs, kidney, liver, heart, lung and
little in brain or gonad. About 40% or 0.5% of total plasmid DNAs, inferring
to be effective, resided in muscles or immune organs, respectively. Collective
results suggested that 'nude' DNA, as water injection, was characterized
as quick absorbent, extensive distribution, but low utilization rate. Finally,
the immune mechanism for the DNA vaccine was discussedref.
Various parameters have been examined for the improvement of immune responses
induced by immunisation with FMDV DNA vaccine (pcDNA 3.1/P1-2A3C3D, P1)
in swine : increasing the amount of P1 DNA plasmids and pGM-CSF adjuvant
plasmids induces stronger FMDV specific and neutralising antibody responses,
as well as promoting cytokines IL-8 and IFN-g
secretion, in immunised pigs via multiple inoculation sitesref.
The fungus Agaricus blazei Murill (ABM) is particularly rich in
polysaccharides, which have shown particularly strong results in treating
and preventing cancers. The goal of this study was to investigate whether
co-administration of the ABM extract with FMDV DNA vaccine could increase
the immune responses. Compared with the control mice, which received FMDV
DNA vaccine alone, significant increase in not only the FMDV-specific antibody
response but also T cell proliferation was observed in mice which received
FMDV DNA vaccine plus the ABM extract. Taken together, these results demonstrated
that application of the ABM extract might provide a strategy to improve
the efficacy of DNA vaccinesref.
Despite FMDV being responsible for one of the most devastating animal diseases,
little is known about the cellular immune mechanisms involved in protection
against this virus. In this work we have studied the potential of DNA vaccines
based on viral minigenes corresponding to three major B and T-cell FMDV
epitopes (isolate C-S8c1) originally identified in natural hosts. The BTT
epitopes [VP1133-156-3A11-40-VP420-34]
were cloned into the plasmid pCMV, either alone or fused to ubiquitin,
the lysosomal targeting signal from LIMPII, a soluble version of CTLA4
or a signal peptide from the human prion protein, to analyze the effect
of processing through different antigenic presentation pathways on the
immunogenicity of the FMDV epitopes. As a first step in the analysis of
modulation exerted by these target signals, a FMDV infection inhibition
assay in Swiss outbred mice was developed and used to analyze the protection
conferred by the different BTT-expressing plasmids. Only one of the 37
mice immunized with minigene-bearing plasmids developed specific neutralizing
antibodies prior to FMDV challenge. As expected, this single mouse that
had been immunized with the BTT tandem epitopes fused to a signal peptide
(pCMV-spBTT) was protected against FMDV infection. Interestingly, nine
more of the animals immunized with BTT-expressing plasmids did not show
viremia at 48h post-infection (pi), even in the absence of anti-FMDV antibodies
prior to challenge. The highest protection (50%, 6 out of 12 mice) was
observed with the plasmid expressing BTT alone, indicating that the targeting
strategies used did not result in an improvement of the protection conferred
by BTT epitopes. Interestingly, peptide specific CD4+ T-cells
were detected for some of the BTT-protected mice. Thus, a DNA vaccine based
on single FMDV B and T cell epitopes can protect mice, in the absence of
specific antibodies at the time of challenge. Further work must be done
to elucidate the mechanisms involved in protection and to determine the
protective potential of these vaccines in natural FMDV hostsref
DNA vaccine : in the natural host, the pig, the efficacy of a DNA vaccine
was compared with the efficacy of a conventional modified live vaccine
(MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets.
The priming of the immune response and the degree of protection against
challenge infection was measured for all vaccine types. Piglets with or
without maternal immunity were vaccinated twice, at the age of 5 and 9
weeks, and we assessed protection by challenge infection with virulent
PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines
induced both humoral and cell-mediated immune responses in maternally immune
animals. DNA vaccination seemed not to suffer from suppression by maternal
immunity and resulted in similar or stronger immune responses in maternally
immune piglets as compared in naive piglets. In contrast, vaccination with
conventional vaccines resulted in weaker immune responses in maternally
immune piglets than in naive piglets. Moreover, DNA vaccination provided
better protection against challenge infection in maternally immune piglets
than in naive piglets, whereas vaccination with conventional vaccines did
notref.
A study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant
for glycoprotein B (gB) of PrV in a murine model. The co-injection of GM-CSF
DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold
increase. Moreover, GM-CSF co-injection inhibited the production of IgG2a
isotype. However, it enhanced production of an IgG1 isotype
resulting in humoral responses biased to the Th2-type against
PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced
the T cell-mediated immunity biased to the Th1-type, as judged
by the significantly higher level of cytokine IL-2 and IFN-g
production but not IL-4. When challenged with a lethal dose of PrV, the
GM-CSF co-injection enhanced the resistance against a PrV infection. This
suggests that co-inoculation with a vector expressing GM-CSF enhanced the
protective immunity against a PrV infection. This immunity was caused by
the induction of increased humoral and cellular immunity in response to
PrV antigenref.
Animals were primed with Sindbis virus-derived plasmids that express viral
glycoproteins gC and gD (gC- and gD-pSIN) and subsequently booster immunized
with Orf virus (ORFV; Parapoxvirus) recombinants expressing gC and gD (D1701-VrVgC
and -VrVgD). The prime-boost vaccination induced strong humoral and cellular-like
PRV-specific immune responses. All prime-boost vaccinated pigs survived
the lethal challenge infection without PRV-specific clinical symptoms and
presented excellent body weight loss attenuation. Most notably, nasal shedding
of challenge virus was reduced by more than about 3log10, clearly
reducing the risk of infection of non-immunized pigsref
serovar A strain DNA vaccine : major outer membrane protein (MOMP)
of an (pcDNA1:MOMPA)ref1,
ref2,
ref3.
The DNA vaccine confers partial protection against C. psittaci infection
in turkeys. Turkeys were injected intramuscularly with the DNA vaccine
: a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer
membrane protein (MOMP) of an avian C. psittaci serovar D strain.
Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined
by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR)
and mRNA translation by immunofluorescence staining of muscle biopsies.
The results indicate that the DNA vaccine persists in turkey muscle for
at least 10 weeks. Moreover, during this period of time MOMP was continuously
expressed, as evidenced by the immunofluorescence staining and RT-PCR.
Since C. psittaci infections occur at the age of 3 to 6 and 8 to
12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore,
further research should concentrate on improving the elicited immune response,
more specifically the cell-mediated immune response, rather than prolonging
the lifespan of the plasmidref
DNA vaccineref
: DNA injection + electroporationrefDNA
vaccine : currently, paratuberculosis vaccines are comprised of crude
whole-cell preparations of Mycobacterium avium subsp. paratuberculosis.
Although effective in reducing clinical disease and fecal shedding, these
vaccines have severe disadvantages as well, including seroconversion of
vaccinated animals and granulomatous lesions at the site of vaccination.
DNA vaccines can offer an alternative approach that may be safer and elicit
more protective responses. In an effort to identify protective M. avium
subsp. paratuberculosis sequences, a genomic DNA expression library
was generated and subdivided into pools of clones (approximately 1,500
clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy
by immunizing mice via gene gun delivery and challenging them with live,
virulent M. avium subsp. paratuberculosis. 4 clone pools
resulted in a significant reduction in the amount of M. avium subsp.
paratuberculosis
recovered from mouse tissues compared to mice immunized with other clone
pools and nonvaccinated, infected control mice. One of the protective clone
pools was further partitioned into 10 clone arrays of 108 clones each,
and 4 clone arrays provided significant protection from both spleen and
mesenteric lymph node colonization by M. avium subsp. paratuberculosis.
The nucleotide sequence of each clone present in the protective pools was
determined, and coding region functions were predicted by computer analysis.
Comparison of the protective clone array sequences implicated 26 antigens
that may be responsible for protection in mice. This study is the first
study to demonstrate protection against
M. avium subsp. paratuberculosis
infection with expression library immunizationref
DNA vaccine : currently available live attenuated vaccines, although
relatively efficacious when administered appropriately, remain abortogenic
in livestock, fully pathogenic to humans, and indistinguishable from natural
infections by the currently adopted diagnostic tests. Candidate genes,
identified as homologous to protective antigens and virulence factors in
related pathogens include omp25, flagellin, acvB, Cu,Zn superoxide dismutase
(SOD)ref1,
ref2,
iron-regulated outer membrane protein B (FrpB), lumazine synthaseref,
bacterioferritin or P39 antigenref1,
ref2,
ribosomal L7/L12 generef,
recombinant ribosome recycling factor-homologous proteinref,
glyceraldehyde-3-phosphate-dehydrogenaseref,
and invasion proteins A and Bref1,
ref2.
The outer membrane proteins (Omps) of Brucella spp. have been extensively
characterized as potential immunogenic and protective antigens. Immunization
of BALB/c mice with B. melitensis Omp31 gene cloned in the pCI plasmid
(pCIOmp31) conferred protection against B. ovis and B. melitensis
infection. Mice vaccinated with pCIOmp31 developed a very weak humoral
response, and in vitro stimulation of their splenocytes with recombinant
Omp31 did not induced the secretion of IFN-g.
Splenocytes from Omp31-vaccinated animals induced a specific CTL activity,
which leads to the in vitro lysis of Brucella-infected macrophages.
pCIOmp31 immunization elicited mainly CD8+ T cells, which mediate
cytotoxicity via perforins, but also CD4+ T cells, which mediate
lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets
showed that the pCIOmp31-induced protection against Brucella infection
is mediated predominantly by CD8+ T cells, although CD4+
T
cells also contribute. These results demonstrate that the Omp31 DNA vaccine
induces cytotoxic responses that have the potential to contribute to protection
against Brucella infection. The protective response could be related
to the induction of CD8+ T cells that eliminate
Brucella-infected
cells via the perforin pathwayref
DNA vaccine :
DNA vaccine : many studies have provided evidence that the HBV
core antigen particle is useful as a vaccine carrier for foreign epitopes.
Epitopes KETc1, KETc12, and GK-1 are 3 promising candidates for
designing a vaccine against Taenia solium cysticercosis. In the
present study, epitopes KETc1 and KETc12 were inserted into the immunodominant
loop of the truncated HBc149, and epitope GK-1 was fused to its C-terminus.
The fused protein
DC-3n was expressed and purified
successfully. The polymeric character was tested by SDS-PAGE. After inoculation
of BALB/c mice with DC-3n, antibody titers were
assayed by ELISA, and the antibody specification was analyzed by Western
blot. Dot ELISA was performed to verify the protection of the 3 epitopes.
Results showed that the purified polymeric protein was formed, high antibody
titers were induced in immunized mice and three antibodies different in
molecular weight were induced, serum specific antibody recognized the native
peptide localized mainly in cyst wall cells, and there was no specific
antibody toward the three epitopes in sera of infected pig and humans.
All these revealed that the protein DC-3n was
a potential candidate for vaccine against cysticercosis. So the DC-3n
sequence and the signal peptide sequence of IL-2 were cloned to a vector
pVAX3.0 to construct pVAX-S-DC-3n. Pigs were
immunized with pVAX-S-DC-3n. 2 weeks after the
immunization booster, pigs were introduced to infectious T. solium
eggs. The relative protective rate induced in pigs immunized with the DNA
vaccine pVAX-S-DC-3n was 83%ref.
A DNA vaccine, pcDNA3-B, was developed by using the nucleotide sequence
of Taenia solium B antigen and cloning into pcDNA3.1 plasmid.
The growing pigs were vaccinated by 1 i.m. injection of 200 or 1000 mg
pcDNA3-B. The immunization with 1000 mg of pcDNA3-B
showed 92.6% protection when the pigs were challenged by T. solium
eggs and 4 of the 5 pigs vaccinated had no viable cysts. The results provide
encouraging information on the use of pcDNA3-B vaccination for the prevention
of cysticercosisref
Copyright © 2001-2005 Daniele Focosi.
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