U.S. patent application number 10/484112 was filed with the patent office on 2004-11-25 for vaccine formulation potentiated by the combination of a dna and an antigen.
Invention is credited to Carrera, Santiago Duenas, de Leon, Liz Alvarez-Lajonchere Ponce, Donato, Gillian Martinez, Feyt, Rolando Pajon, Grillo, Juan Morales, Lasa, Alexis Musacchio, Obregon, Julio C. Alvarez, Rivero, Nelson Acosta, Rodriguez, Ariel Vina.
Application Number | 20040234543 10/484112 |
Document ID | / |
Family ID | 40091632 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040234543 |
Kind Code |
A1 |
Carrera, Santiago Duenas ;
et al. |
November 25, 2004 |
Vaccine formulation potentiated by the combination of a dna and an
antigen
Abstract
Formulation of vaccine antigens, containing as main components:
a-) one or several DNA expressing one or several proteins in the
immunized individuals and b-) a viral antigen, in appropriate
proportions. The novelty of the invention is given by the enhancing
effect of at least one component on the immune response generated
against the other one. Development of new formulations, minimizing
the number of components that enhance and diversify the spectrum of
immune response against different pathogenic entities and
generating combined vaccines against pathogens. These formulations
can be applied in the pharmaceutical industry for preventive and-or
therapeutic use in human.
Inventors: |
Carrera, Santiago Duenas;
(Ciudad de la Habana, CU) ; Grillo, Juan Morales;
(Playa Ciudad de la Habana, CU) ; de Leon, Liz
Alvarez-Lajonchere Ponce; (Ciudad de la Habana, CU) ;
Lasa, Alexis Musacchio; (Mariel La Habana, CU) ;
Feyt, Rolando Pajon; (Bauta La Habana, CU) ;
Rodriguez, Ariel Vina; (Arroyo Naranjo Ciudad de La Habana,
CU) ; Obregon, Julio C. Alvarez; (Playa Ciudad de La
Habana, CU) ; Rivero, Nelson Acosta; (10 de Octubre
Ciudad de La Habana, CU) ; Donato, Gillian Martinez;
(Playa Ciudad de La Habana, CU) |
Correspondence
Address: |
Ronald J Baron
Hoffmann & Baron
6900 Jericho Turnpike
Syosset
NY
11791
US
|
Family ID: |
40091632 |
Appl. No.: |
10/484112 |
Filed: |
June 7, 2004 |
PCT Filed: |
July 12, 2002 |
PCT NO: |
PCT/CU02/00005 |
Current U.S.
Class: |
424/189.1 ;
514/44R |
Current CPC
Class: |
A61K 2039/70 20130101;
A61P 31/14 20180101; A61K 39/29 20130101; C12N 2730/10134 20130101;
A61K 2039/53 20130101; A61K 39/292 20130101; C12N 2770/24234
20130101; A61P 31/12 20180101; A61K 39/12 20130101; A61K 2039/55505
20130101; A61P 31/20 20180101 |
Class at
Publication: |
424/189.1 ;
514/044 |
International
Class: |
A61K 039/29; A61K
048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2001 |
CU |
2001-0171 |
Claims
We claim:
1. A vaccine formulation having as components, a DNA that expresses
a protein and a protein antigen, wherein at least one of the
components of this formulation acts as adjuvant of the other
one.
2. A vaccine formulation according to claim 1, having as
components, a DNA that expresses a protein variant that includes
regions of the hepatitis C virus E1 antigen, and a viral
protein.
3. A vaccine formulation according to claim 1, having as
components, a DNA identified by the SEQ ID No. 3, and the hepatitis
C virus capsid protein.
4. A vaccine formulation according to claim 1, having as components
a DNA identified by the SEQ ID No. 3, and the hepatitis B virus
capsid protein.
5. A vaccine formulation according to having as components a DNA
identified by the SEQ ID No. 2 to 4, and the hepatitis B virus
surface antigen.
6. A vaccine formulation according to claim 1, employed as a
therapeutic and/or preventive agent against the hepatitis C
virus.
7. A vaccine formulation according to claim 4, to be employed as a
therapeutic and/or preventive agent against the hepatitis B
virus.
8. A vaccine formulation according to claim 4, to be employed as a
therapeutic and/or preventive agent against both the hepatitis C
virus and the hepatitis B virus.
9. A vaccine formulation according to claim 2, having as
components, a DNA identified by the SEQ ID No. 3, and the hepatitis
C virus capsid protein.
10. A vaccine formulation according to claim 2 having as components
a DNA identified by the SEQ ID No. 3, and the hepatitis B virus
capsid protein.
11. A vaccine formulation according to claim 2, having as
components a DNA identified by the SEQ ID No. 2 to 4, and the
hepatitis B virus surface antigen.
12. A vaccine formulation according to claim 2, to be employed as a
therapeutic and/or preventive agent against the hepatitis C
virus.
13. A vaccine formulation according to claim 11, to be employed as
a therapeutic and/or preventive agent against the hepatitis B
virus.
14. A vaccine formulation according to claim 11, to be employed as
a therapeutic and/or preventive agent against both the hepatitis C
virus and the hepatitis B virus.
Description
FIELD OF THE INVENTION
[0001] The present invention is related with the branch of the
medicine, particularly with a new formulation of vaccine antigens.
The technical objective of the present invention is the development
of new vaccine formulations, minimizing the number of components
that are able to induce an enhanced and diverse immune response
through the interaction among them. Additionally, the development
of combined vaccine formulations is approached in order to increase
the immune response induced against the co-administered
antigens.
PREVIOUS TECHNIQUE
[0002] Several obstacles exist for the obtaining of an effective
vaccine against the HCV. Because its RNA nature, HCV can quickly
mutate in adaptation to the environment. This contributes to the
high diversity of sequences of the multiple viral isolates
identified in the world. The biggest heterogeneity concentrates on
the hypervariable region I of the HCV E2 protein, where a possible
neutralizing epitope has been described. The HCV causes persistent
infection in spite of the existence of an active immune response
(Lechmann et al., Semin Liver Dis 2000, 20, 211-226). Neither an
animal model, nor an in vitro culture system for the efficient
replication of the virus and the study about the occurrence of
neutralizing antibodies exist. The immunologic patterns associated
with the progression of the illness or with the protection have not
been defined. It is probable that potent, multispecific and
long-lasting both, humoral and cellular immune responses are
required for the resolution of the infection (Lechmann et al.,
Semin Liver Dis 2000, 20, 211-226). Several approaches have been
used to develop a vaccine against the HCV. Recombinant proteins,
synthetic peptides, virus like particles, DNA vaccines and
live-viral vectors are the most widely evaluated.
[0003] The development of a vaccine based on protein subunits was
one of the first strategies evaluated for the HCV because for
several flaviviruses, antibodies directed against surface proteins
can confer protection. Some variants based on the HCV structural
antigens have achieved limited protection against the virus in
animal models. Such it is the case of the chimpanzees immunized
with EI-E2 heterodimers. Seven chimpanzees were vaccinated, five
were protected and two developed a self-limiting disease (Choo et
al., PROC NATL ACAD SCI USES 1994, 91, 1294-1298). This protection
has been correlated with the presence of antibodies (Abs) able to
inhibit the E2 binding to human cells (Rosa et al., PROC NATL ACAD
SCI USES 1996, 93, 1759-1763).
[0004] The recombinant E1 protein from an isolate of the genotype 1
b was purified as homodimers self-associating in particles of 9 nm
diameter, approximately (Maertens et al., Records Gastroenterol
Belg 2000, 63, 203). Two chimpanzees chronically infected with HCV
received 9 doses of 50 .mu.g of the recombinant E1 protein. The
vaccination improved the hepatic histology and determined the
disappearance of the viral antigens of the liver. Vaccination with
recombinant E1 protein also reduced the levels of alanine
aminotransferase (ALT). Although the levels of viral RNA in serum
didn't change during the treatment, the liver inflammation and the
levels of viral antigens increased after treatment. An association
was observed between the high levels of antibodies against E1 and
the improvement of the illness (Maertens et al., Records
Gastroenterol Belg 2000, 63, 203).
[0005] Particularly, the formation of virus-like particles from
recombinant proteins and their employment as vaccines is very
attractive because these structures frequently simulate viral
properties. This kind of particles, obtained from insect cells
infected with a recombinant baculovirus containing the sequence of
the HCV structural antigens, have been able to generate both
humoral and cellular immune response against these antigens
(Baumert et al., Gastroenterology 1999, 117, 1397-407; Lechmann et
al., Hepatology 1999, 30, 423-429). Although the results obtained
with vaccines based on protein subunits are encouraging, the immune
response induced by these variants is mainly humoral, short-term
and isolate-specific.
[0006] On the other hand, different recombinant viral vectors have
been evaluated in the development of a recombinant vaccine against
the HCV. Particularly, recombinant adenoviral vectors are
interesting candidates due to their liver tropism, their power to
induce both humoral and cellular immunity, and the feasibility for
oral or systemic delivery. Adenoviruses containing the DNA encoding
sequence for the HCV structural proteins induce an antibody
response against each one of these proteins (Makimura et al.,
Vaccine 1996, 14, 28-36). Moreover, after immunization in mice with
recombinant adenoviruses for C and E1, a specific CTL response is
detected against these antigens (Bruna-Romero et al., Hepatology
1997, 25, 470-477). Although these results have been encouraging,
the recent problems with the use of recombinant adenoviruses in
gene therapy have raised doubts about their employment in humans.
Other recombinant viruses, like vaccinia, canary-pox and fowl-pox,
containing different HCV genes have induced strong CTL and T-helper
immune responses in mice (Shirai et al., J Virol 1994, 68, 3334
-3342; Large et al., J Immunol 1999, 162, 931-938). However, these
recombinant viruses, as well as other variants of alpha virus like
the Semliki Forest Virus are also affected by regulatory issues and
security concerns related with their application.
[0007] The identification of several epitopes for CD4+ and CD8+ T
cells in the HCV polyprotein, which could be important in the viral
elimination, support the evaluation of synthetic peptides as
vaccine candidates against this pathogen. Different peptides,
lipidated or not, containing epitopes of C, NS4 and NS5, have
induced a strong CTL response in mice (Shirai et al., J Infect Dis
1996, 173, 24-31; Hiranuma et al., J Gene Virol 1999, 80, 187-193;
Oseroff et al., Vaccine 1998,16, 823-833).
[0008] Another strategy used to develop a vaccine against the HCV
is based in the possibility of generating Abs against linear
epitopes. This alternative has been evaluated basically to generate
Abs against the HVR-l of the HCV, with some encouraging results in
rabbits and chimpanzees (Esumi et al., Arch Virol 1999, 144,
973-980; Shang et al., Virology 1999, 258, 396-405). Quasi-species
is the main problem of selecting the HVR-l as the target for a
vaccine against the HCV.
[0009] The main obstacle for the peptide vaccines is that those
peptides without epitopes for helper T cells can be poorly
immunogenics. Moreover, the effectiveness of a vaccine is
frequently based on the induction of specific immune response
against a wide range of different antigens. These limitations are
important weaknesses of this strategy.
[0010] The DNA immunization is one of the most recent strategies in
vaccine development. A DNA vaccine consists on a purified plasmid
containing the sequence coding for an antigen of interest, under
the control of a functional transcriptional unit in eucariotic
cells. After injection of the plasmid in muscle or the skin, the
plasmid is taken up by host cells and the antigen is expressed
intracellularly. The expression of the encoded antigens in the host
cells is one of the major advantages of this methodology because is
similar to viral natural infections. The simplicity to manipulate
the DNA, together with the DNA stability that makes possible a
relatively cheap large-scale production of DNA, is other advantage
of DNA vaccination.
[0011] The immune response induced with this kind of vaccines can
be modulated by co-immunization with molecules or genes coding for
co-stimulatory molecules like cytokines. The genetic constructs can
be modified, by insertions or deletions of transmembrane domains,
signal sequences for secretion, or other types of residues
affecting the intracellular trafficking and processing of the
antigen.
[0012] Particularly, the DNA immunization has been largely studied
in the development of vaccines against the HCV. Different
expression vectors encoding full-length or truncated variants of
the HCV capsid protein have been generated (Lagging et al., J Virol
1995, 69, 5859-5863; Chen et al., Vaccine Res 1995, 4, 135-144).
Other constructs also include the HCV 5' non-translated region
(Tokushige et al., Hepatology 1996, 24, 14-20). Plasmids expressing
fusion variants to the hepatitis B virus (HBV) surface antigen or
other envelope antigens of the HBV have been evaluated (Major et
al., J VIROL 1995, 69, 5798-5805). Immunization with these plasmids
has generally induced positive CTL and lymphocyte proliferative
response.
[0013] The HCV envelope proteins have also constituted targets of
interest for this type of technology. In the case of the HCV E2,
the humoral response seems to be mainly directed to the HVR-1 (Lee
et al., Mol Cells 1998, 8, 444-451). Immunization with plasmids
expressing intracellular or secreted variants of the E1 and E2
proteins has rendered similar immune response (Lee et al., J VIROL
1998, 72, 8430-8436). The inoculation with bicistronic plasmids
expressing the GM-CSF and the HCV E1 or E2 proteins increased both
humoral and cellular immune response. Recently, the use of
bicistronic plasmids expressing the E1 and E2 proteins were
generated to investigate the influence of heterodimer formation
between these proteins in vivo on the immune response induced after
DNA immunization. When heterodimers were formed, the antibody
response against HCV E1 and E2 proteins was not obtained. In sharp
contrast, high-level antibody titers, directed to both linear and
conformational epitopes, were obtained after immunization with
plasmids expressing truncated variants of E1 and E2. Therefore, it
seems necessary to avoid the heterodimers formation to obtain a
strong antibody response when constructs including these antigens
are evaluated (Fournillier et al., J VIROL 1999, 73, 497-504).
[0014] The non structural proteins have also been evaluated by this
technology. Good results were obtained when the region coding for
the C-terminal domain of the NS3 protein was included in a vector
that allows the simultaneous and independent expression of this
domain and the IL-2 (Papa et al., Res Virol 1998, 149, 315-319).
The NS4 and NS5 proteins also generate Abs and CTL responses by
this immunization strategy (Encke et al., J IMMUNOL 1998, 161,
4917-4923). Recently, the use of a construction coding for GM-CSF
and the non structural proteins of the virin (NS3, NS4 and NS5)
induced a potent Ab response and a potentiated lymphoproliferative
response against each non structural protein (Cho et al., Vaccine
1999,17, 1136-1144).
[0015] In general, the effective expression of different HCV
antigens, as well as the generation of anti-HCV Abs in levels
ranging from 1:100 to 1:100 000 according to the combination in
study, has been reported for different DNA constructs (Inchauspe et
al., Vaccine 1997, 15, 853-856). Additionally, the development of
specific CTL and lymphocyte proliferative response has been
demonstrated (Inchauspe et al., DNA AND CELL BIOLOGY 1997, 16,
185-195). However, efforts are required to improve this methodology
in order to generate stronger both humoral and cellular response
against different proteins of the HCV. Thus, some variants like
liposomes (Gramzinski et al., Mol Medicine 1998, 4, 109-118) and
saponin QS-21 (Sasaki et to the., J Virol 1998, 72, 4931-4939) have
been evaluated to increase the immune response induced after DNA
vaccination. The dendritic cells as biological adjuvants have been
also studied in DNA immunization. Dendritic cells (CD) derived of
former genetically modified mouse bone marrow to express tumor
antigens, by using viral vectors (Specht et al., J Exp Med 1997,
186, 1213-1221; Brossart et al., J Immunol 1997, 158, 3270-3276;
Song et al., J Exp Med 1997, 186, 1247-1256), or RNA (Boczkowski et
to the., J Exp Med 1996, 184, 465-472), have demonstrated their
capacity to promote T cell response specific for tumor antigens,
and prophylactic immunity mediated by cells against tumors in
mouse.
[0016] At the present, the improvement of vectors for DNA
immunization, including the insertion of CpG motifs to increase the
immune response against the expressed antigens (Hasan et al., J
Immunol Meth 1999, 229,1-22), and the DNA delivery systems is
crucial to overcome the limitations of this technology. Due to the
challenges that outlines the HCV infection, and to the absence of a
clear definition about the immunologic parameters correlating with
the protection against this pathogen, it is possible that an
effective vaccine against the HCV shall require a multispecific
approach stimulating several aspects of the immune response. The
solution of this problem is probably in the combination of several
vaccination strategies explored until the moment. Particularly,
immunization schedules that combine a prime dose with a DNA vaccine
and a booster dose with recombinant proteins or viral vectors (Hu
et al., Vaccine 1999, 17, 3160-3170; Pancholi et al., J Infect Dis
2000, 182, 18-27) have been evaluated with results that, although
positives, require additional investigations to demonstrate if the
prime-boost strategies can really induce a protective immunity
against the HCV.
[0017] Additionally, for the hepatitis B model, a vaccine
composition comprising the complex formed by the hepatitis B
surface antigen, an antibody specific for this antigen, and a DNA
vaccine expressing for this antigen has been evaluated (Wen et al.,
U.S. Pat. No. 6,221,664, 1998). This formulation allowed the
antigen presentation by different means and a quick induction of
immune response that resulted superior regarding to the one
generated by the individual variants.
[0018] In the present invention, a vaccine formulation comprising
as components only a protein antigen and a plasmid expressing one
or several proteins, acting at least one of them as adjuvant of the
other one, is described. Particularly, the capsid antigen of the
hepatitis C or B virus, and a plasmid expressing individual or
polyprotein variants of the HCV E1 protein, are evaluated. Contrary
to the composition previously described for the hepatitis B model,
the presence of antibodies in the formulation is not required to
generate the enhancement of the immune response, thus reducing the
number of components required. Additionally, the biggest
flexibility in the vaccine composition also allows generating
simultaneously potent immune responses against different
antigens.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides the composition and methods
to immunize an individual in a prophylactic or therapeutic way
against the HCV and the HBV, as well as their combination. It is
reported for the first time a vaccine formulation having as
components: (a) a DNA that expresses a protein variant that
includes regions of the E1 antigen of the HCV envelope and (b) a
protein antigen of the HCV or HBV, in appropriate proportions. The
novelty of the invention is given by the adjuvant effect of at
least one component on the immune response generated against the
other one. Antigens coded by the genetic constructs and expressed
by the host cells, as well as the protein antigen comprising the
vaccine the formulation, are interesting targets to generate an
immune response against the HCV and the HBV. Thus, the immune
response can be directed against a wide spectrum of important
antigens.
[0020] The vaccine formulation includes a DNA enhancing the immune
response generated against a protein antigen mixed with him; being
this effect dependent on the expression of one or several proteins
coded by the DNA, in the immunized host. The DNA is obtained from a
bacterial strain and purified according to traditional procedures
(Horn et al., H Gene Ther 1995, 6, 565-573).
[0021] The vaccine formulation comprises in preferred embodiments
at least one of the following plasmids: plDKE1S, plDKE2 and
PAEC-ME, whose DNA sequences coding for the protein variants
expressed are identified with the number of sequence of 2-4,
respectively.
[0022] The plDKE1S plasmid, expressed a protein that comprise the
aa from the 176 to the 363 of the HCV E1 (Sec. Id. No. 2). On the
other hand, the plDKE2 plasmid expressed a protein encompassing the
first 650 aa of the viral polyprotein (C, E1 and a part of the E2)
(Sec. Id. No.3). The pAEC-ME plasmid expresses a chimeric protein
comprising B and T cells epitopes of different HCV antigens (Sec.
Id. No. 4). In these plasmids, the coding sequence for the viral
antigens was obtained from the cDNA of a HCV cuban isolate (Morales
et al., 1998, WO 9825960). The pAEC-ME, plDKE1S and plDKE2 plasmids
contain the coding sequence for the HCV antigens inserted into the
multiple cloning site of the pAEC-K6 plasmid (Herrera et al.,
Biochem Biophys Res Commun. 2000, 279, 548-551). The plasmids
included in the present invention have the regulatory elements able
to direct the antigen expression in human cells. These regulatory
elements include a transcriptional unit functional in mammals,
integrated for example by the human cytomegalovirus promoter and
the polyadenilation signal of the simian virus 40. These plasmids
also contain a replication origin in bacteria and a selection
marker for the resistance to kanamyicin.
[0023] The protein component of the formulation can be a soluble
viral antigen able to form particles, with a purity superior to
90%. In preferred embodiments, are component of the vaccine
formulation the capsid antigens of the HCV and HBV, that enhanced
the immune response generated against the proteins expressed by the
DNA mixed with them.
[0024] The present invention also contemplates the procedure for
the mixture of the DNA with the antigen. The mixture is prepared by
addition of components, DNA and antigen, dissolved in an
appropriate buffer. In preferred embodiments, the formulation can
be prepared by the combination of both components, dissolved in
saline phosphate, in 101 (ww) proportion. The mixture is incubated
at least 2 h between 26.degree. C. and 30.degree. C., with shaking,
before administration to the individuals. This formulation can be
administered by intramuscular, subcutaneous, intraperitoneal,
intramucosal, intravenous sublingual way, or others. The
immunization can be performed by means of syringes, gene gun,
sprays or other delivery devices. Each individual receives a dose
ranging from 0.001 to 10 mg of each component in a volume
determined by the animal species and the immunization method
employed. In the case of vaccine formulations having as components
a DNA mixed with a protein antigen, a superior product can be
obtained compared with each one of the individual components due
to:
[0025] It is possible to generate a stronger and diverse both
humoral and cellular immune response directed to a broader range of
epitopes.
[0026] The toxic effect generated by the injection of the adjuvant
can be eliminated because the antigen 2 is simultaneously the
adjuvant.
[0027] It is possible the employment of these formulations as core
for combined vaccines.
[0028] The process of vaccine formulation doesn't require of
adsorption of the antigen.
[0029] In the case of the formulations containing a DNA that
expressed a protein variant that include regions of the HCV E1
protein, and the HCV capsid protein, a superior product can be
obtained compared with each one of the individual components due
to:
[0030] It is possible to generate a stronger and diverse both
humoral and cellular immune response directed to a broader range of
epitopes.
[0031] The toxic effect generated by the injection of the adjuvant
can be eliminated because the antigen 2 is simultaneously the
adjuvant.
[0032] It is possible the employment of the DNA plus the capsid as
core for combined or multivalent vaccines.
[0033] On the other hand, the immunization with a DNA that
expresses a protein variant that includes regions of the HCV E1
protein increased the immunogenicity of HBV protein antigens,
present in the formulation. Particularly, the mixture with the
HBsAg or the HBcAg, allows superior results to those obtained with
this antigens due to:
[0034] a) The levels of IgG induced against the HBsAg are superior
to those obtained with the inoculation of the HBsAg with aluminum
hydroxide.
[0035] b) Constitutes a potential combined vaccine HBV-HCV.
[0036] c) The formulation process doesn't require of adsorption of
the antigen.
DESCRIPTION OF THE FIGURES
[0037] FIG. 1: Schematic representation of the plasmids pAEC-ME,
plDKE1S and plDKE2.
[0038] FIG. 2: E1ectron microscopy of the particles of the
hepatitis C virus capsid (A), of the hepatitis B virus surface
antigen (B) and of the hepatitis B virus capsid (C).
[0039] FIG. 3: Immunization schedule with the plDKE2 plasmid and
the Core protein. The animals were immunized intramuscularly with
50 .mu.g of DNA and 5 .mu.g of protein.
[0040] FIG. 4: Immunization schedule with different plasmids and
the protein HBcAg. The animals were immunized intramuscularly with
50 .mu.g of DNA and 5 .mu.g of protein.
[0041] FIG. 5: Immunization schedule with different plasmids and
the protein HBsAg. The animals were immunized intramuscularly with
50 .mu.g of DNA and 5 .mu.g of protein.
EXAMPLES
Example 1
Immunogenicity of Formulations Having as Components a DNA that
Expresses a Polyprotein Capsid-E1-E2 of the HCV, and the Protein
Antigen of the HCV Capsid.
[0042] With the objective of demonstrating the enhancement of the
immune response generated against the HCV structural antigens after
the administration of the mixture of the plasmid plDKE2 (FIG. 1),
with recombinant HCV capsid particles (FIG. 2A), 10 BALBc females
mice, 8 weeks old, per group were inoculated intramuscularly. The
schedule included 2 inoculations in the days 0 and 21, except one
of the groups in which the influence of a single dose in day 0 was
studied. Blood samples were taken 14 weeks after the first
immunization. Immunogens were administered in phosphate buffer
saline (PBS). The group 1 was inoculated with 50 .mu.g of the
plDKE2 plasmid (FIG. 1, the plasmid contains the sequence coding
for the first 650 aa of the viral polyprotein, Sec. Id. No.3). The
group 2 was inoculated with 5 .mu.g of the Core protein (comprising
the first 173 aa of the HCV capsid protein). The group 3 received a
first dose with 5 .mu.g of the Core protein and a second one with
50 .mu.g of the plDKE2 (CoreplDKE2). The group 4 was inoculated
under similar conditions to the group 3 but in inverse order
(plDKE2Core). The group 5 was inoculated with the mixture of 50
.mu.g of the plDKE2 and 5 .mu.g of the Core protein in the days 0
and 21 (CoreplDKE2). The group 6 was inoculated in the same way
that the group 5 but only in the day 0 (CoreplDKE2 (1)).
Additionally, a seventh group, negative control, was immunized with
50 .mu.g of the plasmid pAEC-K6 (it doesn't contain sequences
coding for the HCV antigens).
[0043] The antibody response was determined by ELISA to detect the
Ab response against the HCV structural proteins. The Student T test
was employed to analyze the results, statistical differences were
considered for p <0.05.
[0044] The FIG. 3 shows that it is possible to increase the immune
response against the HCV structural antigens by the administration
of two doses of the mixture of the plDKE2 with the Core protein
with respect to the individual components. This formulation (in two
doses) induced Ab titers against the HCV E1 and E2 envelope
proteins statistically higher to those obtained in the remaining
groups (FIG. 3A). These Ab titers were also statistically higher to
the levels of Abs against the HCV capsid protein, generated by the
plDKE2-Core mixture administered in a single dose (FIG. 3A). The
inoculation of the mixture in a single dose always induced the
lower levels of Abs among the immunized groups.
[0045] The evaluation of the lymphoproliferative response against
the HCV structural antigens (FIG. 3B) indicated a significantly
superior response against the capsid in the group of animals
immunized with the plDKE2-core in 2 doses, with respect to the
remaining groups. The results are shown as the stimulation index of
spleen cells obtained from immunized animals. The stimulation index
was determined by the (H.sup.3) Thymidine uptake. It is possible to
conclude that the immunization with the mixture of plDKE2 and the
Core protein generates a synergic stimulation of the immune
response induced against the HCV structural antigens.
Example 2
Immunogenicity of Formulations Having as Components a DNA that
Expresses a Polyprotein Capsid-E1-E2 of the HCV, and the Protein
Antigen of the HBV Capsid.
[0046] With the objective of investigating the behavior of the
immune response generated by the mixture of the plDKE2 plasmid with
protein antigens of other pathogens, 10 females BALBc/mice, 8 weeks
old, per group were inoculated intramuscularly with the mixture of
the above referred plasmid with recombinant particles of the HBV
capsid (HBcAg, FIG. 2C). The schedule included 2 inoculations in
the days 0 and 21. Blood samples were taken at 9 and 19 weeks after
the first immunization. Immunogens were prepared in phosphate
buffer saline (PBS). The plasmids were administered in dose of 50
.mu.g , and the HBV capsid protein in dose of 5 .mu.g . The group 1
was inoculated with the plasmid pAEC-K6 (negative control). The
group 2 was administered with the HBcAg protein. The group 3 was
vaccinated with plDKE2. The groups 4 and 5 were vaccinated with the
mixture of the HBcAg with the plasmids plDKE2 and pAEC-K6,
respectively. The Student T test was employed to analyze the
results statistically, a significant difference was considered for
p<0.05.
[0047] The FIG. 4 shows the antibody response induced in mice 19
weeks after primary immunization. FIG. 4A shows that the mixture of
the plDKE2 plasmid with the HBcAg induced Ab titers against the
HBcAg, statistically higher to the observed in the rest of the
vaccinated animals. No statistical differences were detected
between the groups immunized with HBcAg alone or mixed with the
pAEC-K6. Therefore it is possible to conclude that the plasmid
plDKE2 enhance the immune response against the HBcAg.
[0048] On the other hand, the FIG. 4B shows that the mixture of the
plDKE2 plasmid with the HBcAg induces antibody titers against the
HCV structural antigens higher to those generated in the animals
immunized with the plDKE2 alone. Therefore, the HBcAg is also
capable of enhance the immune response induced against the HCV
structural antigens induced after the administration of the
plDKE2.
Example 3
Immunogenicity of Formulations Having as Components Plasmids
Expressing Variants of the HCV and HBV, and the Protein Variant of
the HBV Surface Antigen.
[0049] With the objective of demonstrating the enhancement of the
immune response generated against other protein antigens observed
after the co-administration with the plDKE2 plasmid, and to study
other plasmids with similar adjuvant properties, 10 female BALBc
mice, 8 weeks old, per group were inoculated intramuscularly with
the mixture of the plasmid with recombinant particles of the HBsAg
(FIG. 2B). The schedule included 3 inoculations in days 0, 21 and
50. Blood samples were taken at week 16, after the primary
immunization. All the immunogens were prepared in phosphate buffer
saline (PBS), except a group formulated with Aluminum hydroxide.
The group 1 was inoculated with the mixture of 50 .mu.g of the
plasmid plDKCo, containing the sequence coding for the first 176 aa
of the HCV capsid protein (Dueas-Carrera et al., Vaccine
2000;19(7):992-997), and 5 .mu.g of the HBsAg (plDKCo-HBsAg). The
groups 2 to 7 were inoculated with mixtures of DNA and HBsAg in
same quantities but using the following plasmids: group 2
(plDKE1S-HBsAg), the plasmid plDKE1S (FIG. 1, containing the
sequence coding for the aa 176-363 of the HCV polyprotein, Sec. Id.
No.2); group 3 (pAEC-ME-HBsAg), the plasmid pAEC-ME (FIG. 1,
containing the sequence coding for a protein that includes
different epitopes of the HCV antigens, Sec. Id. No.4); group 4
(plDKE2-HBsAg), the plasmid plDKE2 (FIG. 1) containing the sequence
coding for the aa 1-650 of the HCV polyprotein, Sec. Id. No.3;
group 5 (plDKE1Sm-HBsAg), the plasmid plDKE1Sm is identical to the
plDKE1S except that it includes 2 nucleotide insertions in the
region coding for the HCV E1 that changes the open reading frame
and impedes the expression of this protein (Sec. Id. No.5); group 6
(pAEC-d2-HBsAg-HBsAg), the plasmid pAEC-d2-HBsAg contains the
sequence coding for the HBV HBsAg (Musacchio et al., Biochem Bioph
Res Commun 2001, 282, 442446); group 7 (pAEC-K6-HBsAg), the plasmid
pAEC-K6 (negative control, doesn't contain coding sequence under
the control of the transcriptional unit). Finally, the groups 8 and
9 were inoculated with 5 .mu.g of HBsAg formulated in Aluminum
hydroxide or alone, respectively. The Student T test was employed
to analyze the results statistically, a significant difference was
considered for p <0.05.
[0050] The FIG. 5 shows the Abs titers generated against the HBsAg,
16 weeks after primary immunization. The levels of Abs induced by
the HBsAg alone in PBS were statistically inferior to the rest of
the variants evaluated except for the mixture formed by the HBsAg
and the pAEC-K6. On the other hand, the mixtures of HBsAg with the
plasmids plDKCo, plDKE1S, pAEC-ME and plDKE2 induced Ab titres
against the HBsAg statistically higher to those induced by the
immunization with the HBsAg formulated in Aluminum hydroxide or
mixed with the pAEC-K6. The immunization with the HBsAg formulated
with aluminum hydroxide or mixed with pAEC-K6, plDKE1Sm and
pAEC-d2-HBsAg induced similar levels of Ab titers against the
HBsAg. It is possible to conclude that the expression in the host
cells of protein variants that include the amino acid regions of
the HCV E1 antigen, from the plasmids administered, enhance the
immune response generated against the protein antigen mixed with
the DNA construct.
Sequence CWU 1
1
5 1 531 DNA Artificial sequence gene (1)..(528) Includes the
sequence coding for aa 1 to 176 of the HCV core protein 1
atgagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccg ccgcccacag
60 gacgtcaagt tcccgggcgg tggtcagatc gttggtggag tttacctgtt
gccgcgcagg 120 ggccccaggt tgggtgtgcg cgcaactagg aagacttccg
agcggtcgca acctcgtgga 180 aggcgacaac ctatccccaa ggctcgccgg
cccgagggca ggtcctgggc ccagcccggg 240 tacccttggc ccctctatgg
taacgagggc atgggatggg caggatggct cctgtcaccc 300 cgtggctctc
ggcctagttg gggccccact gacccccggc gtaggtcgcg taatttgggt 360
aaggtcatcg ataccctcac atgcggcttc gccgacctca tggggtacat tccgctcgtc
420 ggcgcccccc tagggggcgc tgccagggcc ctggcgcatg gcgtccgggt
tctggaggac 480 ggcgtgaatt atgcaacagg gaatctgccc ggttgctctt
tctctctcta a 531 2 567 DNA Artificial sequence gene (1)..(564)
Includes the nucleotide sequence coding for the aa 176-363 on HCV
polyprotein, mainly corresponding to the E1 protein. 2 atgttccttt
tggctttgct gtcctgtttg accatcccag tttccgccta tgaagtgcgc 60
aacgcgtccg gggtgtacca tgtcacgaac gactgctcca actcaagcat tgtgtatgag
120 gcagacgaca tgatcatgca cacccccgga tgcgtgccct gcgttcggga
ggacaacacc 180 tcccgctgct gggtagcgct cacccccaca ctcgcggcca
ggaatgccag cgtccccacc 240 acgacaatac gacgccacgt cgatttgctc
gttggggcgg ctgctctctg ctccgctatg 300 tacgtggggg atctctgcgg
atctgttttc ctcgtttccc agctgttcac cttctcgcct 360 cgccggcatg
agacagcaca ggactgcaac tgctcaatct atcccggcca cgtatcaggt 420
caccgcatgg cctgggatat gatgatgaac tggtcacctt caacagccct agtggtatcg
480 cagttactcc ggatcccaca agccgtcgtg gacatggtag cgggggccca
ctggggagtc 540 ctagcgggcc ttgcctacta ctcctaa 567 3 1953 DNA
Artificial sequence gene (1)..(1950) Includes the nucleotide
sequence coding for aa 1-650 on HCV polyprotein, encompassing the
capsid, E1 and a portion of the E2 protein. 3 atgagcacga atcctaaacc
tcaaagaaaa accaaacgta acaccaaccg ccgcccacag 60 gacgtcaagt
tcccgggcgg tggtcagatc gttggtggag tttacctgtt gccgcgcagg 120
ggccccaggt tgggtgtgcg cgcaactagg aagacttccg agcggtcgca acctcgtgga
180 aggcgacaac ctatccccaa ggctcgccgg cccgagggca ggtcctgggc
ccagcccggg 240 tacccttggc ccctctatgg taacgagggc atgggatggg
caggatggct cctgtcaccc 300 cgtggctctc ggcctagttg gggccccact
gacccccggc gtaggtcgcg taatttgggt 360 aaggtcatcg ataccctcac
atgcggcttc gccgacctca tggggtacat tccgctcgtc 420 ggcgcccccc
tagggggcgc tgccagggcc ctggcgcatg gcgtccgggt tctggaggac 480
ggcgtgaatt atgcaacagg gaatctgccc ggttgctctt tctctctctt ccttttggct
540 ttgctgtcct gtttgaccat cccagtttcc gcctatgaag tgcgcaacgc
gtccggggtg 600 taccatgtca cgaacgactg ctccaactca agcattgtgt
atgaggcaga cgacatgatc 660 atgcacaccc ccggatgcgt gccctgcgtt
cgggaggaca acacctcccg ctgctgggta 720 gcgctcaccc ccacactcgc
ggccaggaat gccagcgtcc ccaccacgac aatacgacgc 780 cacgtcgatt
tgctcgttgg ggcggctgct ctctgctccg ctatgtacgt gggggatctc 840
tgcggatctg ttttcctcgt ttcccagctg ttcaccttct cgcctcgccg gcatgagaca
900 gcacaggact gcaactgctc aatctatccc ggccacgtat caggtcaccg
catggcctgg 960 gatatgatga tgaactggtc accttcaaca gccctagtgg
tatcgcagtt actccggatc 1020 ccacaagccg tcgtggacat ggtagcgggg
gcccactggg gagtcctagc gggccttgcc 1080 tactactcca tggtggggaa
ctgggccaag gttttgattg tgatgctact ctttgccggc 1140 gttgacggga
cgggaaccta cgtgacaggg gggacggcag cccgcggcgt cagccagttt 1200
acgggcctct ttacatctgg gccgagtcag aaaatccagc ttgtaaatac caacggcagc
1260 tggcatatta accggactgc cctgaactgc aacgactccc tccagaccgg
gttccttgct 1320 gcgttgtttt acgtgcacag gttcaactcg tccggatgct
cagatcgcat ggccagctgc 1380 cgccccattg atacgttcga ccaggggtgg
ggccccatta cttacgctga gccgcgcagc 1440 ttggaccaga ggccctattg
ctggcactac gcacctcaac cgtgtggtat cgtacccgcg 1500 gcggaggtgt
gtggtccagt gtattgtttc actccaagcc ccgttgtcgt ggggaccacc 1560
gatcgttccg gcgtccctac gtataactgg ggggagaatg agacggacgt gctgctcctt
1620 aacaacacgc ggccgccgct gggcaactgg tttggctgta catggatgaa
tagcactggg 1680 ttcaccaaga cgtgcggggg ccctccgtat aacatcggag
gggtcggtaa caacaccttg 1740 acctgcccta cggattgctt ccgcaagcac
cccgaggcca cttacaccaa atgtggtttg 1800 gggccttggt tgacacctag
gtgcttggtc gactacccat acaggctttg gcattacccc 1860 tgcactgtca
actttaccat cttcaaggtt cggatgtatg tggggggcgt ggagcacagg 1920
cttaccgctg catgcaactg gactcgagga taa 1953 4 1194 DNA Artificial
sequence gene (1)..(1191) Includes the nucleotide sequence coding
for different epitopes of HCV proteins. 4 atgacgggaa cctacgtgac
aggggggacg gcagcccgcg gcgtcagcca gtttacgggc 60 ctctttacat
ctgggccgag tcagaaaatc cagcttgtaa ataccaacgg cagctggcat 120
attaaccgga ctgccctgaa ctgcaacgac tccctccaga ccgggttcct tgctgcgttg
180 ttttacgtgc acaggttcaa ctcgtccgga tgctcagatc gcatggccag
ctgccgcccc 240 attgatacgt tcgaccaggg gtggggcccc attacttacg
ctgagccgcg cagcttggac 300 cagaggccct attgctggca ctacgcacct
caaccgtgtg gtatcgtacc cgcggcggag 360 gtgtgtggtc cagtgtattg
tttcactcca agccccgttg tcgtggggac caccgatcgt 420 tccggcgtcc
ctacgtataa ctggggggag aatgagacgg acgtgctgct ccttaacaac 480
acgcggccgc cgctgggcaa ctggtttggc tgtacatgga tgaatagcac tgggttcacc
540 aagacgtgcg ggggccctcc gtataacatc ggaggggtcg gtaacaacac
cttgacctgc 600 cctacggatt gcttccgcaa gcacggatcc acccacgtga
ccggcggcag ccaggcccgc 660 accacccaca gcttcacctc cctgctgcgc
cagggcgcca agcagaacgt gcagctgatc 720 gccgacctga tgggctacat
cccactggtg ggcgccccac tgggcaagaa gggccacgtg 780 agcggccacc
gcatggcctg ggacatgatg atgaactggg ccagcaagaa ggccgccagc 840
cgcgccgccg gcttgcagga cagcaccatg ctggtgagcc acacccgcgt gaccggcggc
900 gtggccggcc acgtgaccag cggcctggtg tccctgttca gccctggcgc
cagccagaag 960 atccagctgg tgggctccag cttcagcctg ttcctgttgg
ccctcctgag cagcttgacc 1020 atcaagaaga tgagctactc ctggaccggc
gccctggtga ccccaagcgc cgccgagaag 1080 aagctgttgt tcaacatcct
gggcggctgg gtgaagaaga gcatggtggg caactgggcc 1140 aaggtgaaga
agtacaccgg cgacttcgac agcgtgatcg actccaggcc ttaa 1194 5 569 DNA
Artificial sequence Description of the artificial sequence pIDKE1Sm
5 attgttcctt ttggctttgc tgtcctgttt gaccatccca gtttccgcct atgaagtgcg
60 caacgcgtcc ggggtgtacc atgtcacgaa cgactgactc caactcaagc
attgtgtatg 120 aggcagacga catgatcatg cacacccccg gatgcgtgcc
ctgcgttcgg gaggacaaca 180 cctcccgctg ctgggtagcg ctcaccccca
cactcgcggc caggaatgcc agcgtcccca 240 ccacgacaat acgacgccac
gtcgatttgc tcgttggggc ggctgctctc tgctccgcta 300 tgtacgtggg
ggatctctgc ggatctgttt tcctcgtttc ccagctgttc accttctcgc 360
ctcgccggca tgagacagca caggactgca actgctcaat ctatcccggc cacgtatcag
420 gtcaccgcat ggcctgggat atgatgatga actggtcacc ttcaacagcc
ctagtggtat 480 cgcagttact ccggatccca caagccgtcg tggacatggt
agcgggggcc cactggggag 540 tcctagcggg ccttgcctac tactcctaa 569
* * * * *