U.S. patent application number 10/735601 was filed with the patent office on 2004-10-21 for multi-antigenic alphavirus replicon particles and methods.
Invention is credited to Caley, Ian, Dryga, Sergey, Kamrud, Kurt, Smith, Jonathan F..
Application Number | 20040208848 10/735601 |
Document ID | / |
Family ID | 32600124 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040208848 |
Kind Code |
A1 |
Smith, Jonathan F. ; et
al. |
October 21, 2004 |
Multi-antigenic alphavirus replicon particles and methods
Abstract
Viral replicon selected nucleic acid expression libraries are
useful for analyzing multiple antigens associated with a parasite,
pathogen or neoplasia or for preparing immunogenic compositions for
generating immune responses specific for the parasite, pathogen or
neoplasia. Alphavirus replicon particles representative of the
nucleic acid expression library are preferred. The nucleic acid
library can be a random library, or it can be prepared after a
selection step, for example, by differential hybridization prior to
cloning into the replicon vector.
Inventors: |
Smith, Jonathan F.; (Cary,
NC) ; Kamrud, Kurt; (Apex, NC) ; Dryga,
Sergey; (Chapel Hill, NC) ; Caley, Ian;
(Durham, NC) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
5370 MANHATTAN CIRCLE
SUITE 201
BOULDER
CO
80303
US
|
Family ID: |
32600124 |
Appl. No.: |
10/735601 |
Filed: |
December 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60433299 |
Dec 13, 2002 |
|
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60433058 |
Dec 13, 2002 |
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Current U.S.
Class: |
424/93.2 ;
435/457 |
Current CPC
Class: |
A61K 48/00 20130101;
A61P 31/00 20180101; A61K 2039/5256 20130101; A61P 33/00 20180101;
A61P 35/00 20180101; C12N 2770/36134 20130101; C12N 2770/36143
20130101; C12N 15/86 20130101; C12N 2740/16222 20130101; C12N
2770/36152 20130101; C12N 7/00 20130101; A61P 37/04 20180101; A61P
37/00 20180101; A61K 2039/70 20130101; A61P 31/12 20180101; A61P
35/02 20180101 |
Class at
Publication: |
424/093.2 ;
435/457 |
International
Class: |
A61K 048/00; C12N
015/861 |
Claims
What is claimed is:
1. A method for preparing alphaviral replicon particles (ARPS)
encoding and expressing a plurality of antigens, said method
comprising the steps of: a) introducing a plurality of alphaviral
replicon nucleic acids into a plurality of cells, wherein said
cells are permissive for alphavirus replication and packaging,
wherein said replicon nucleic acid comprises at least a virus
packaging signal and at least one heterologous coding sequence
expressible in said alphaviral replicon nucleic acid, wherein said
cell comprises at least one helper function, to produce a modified
cell, and wherein the plurality of alphaviral replicon nucleic
acids encode a plurality of antigens, to produce a plurality of
modified cells; b) culturing said plurality of modified cells of
step (a) under conditions allowing expression of the at least one
helper function, allowing replication of said alphaviral replicon
nucleic acid and packaging of said alphaviral replicon nucleic acid
to form ARPs; c) contacting the modified cells after step (b) with
an aqueous solution having an ionic strength from 0.2M to 5M to
release the ARPs into the aqueous solution to produce a
ARP-containing solution; and d) collecting ARPs from the
ARP-containing solution of step (c).
2. The method of claim 1 wherein the at least one helper function
in the host cell of step (a) is encoded by a nucleic acid sequence
stably integrated within the genome of said host cell.
3. The method of claim 1 wherein the at least one helper function
in the cell is introduced on at least one helper nucleic acid which
encodes a capsid protein capable of binding said alphaviral
replicon nucleic acid, and at least one alphaviral glycoprotein,
wherein said alphaviral glycoprotein associates with said
alphaviral replicon nucleic acid and said capsid protein, wherein
the at least one helper nucleic acid molecule is introduced into
the cell together with said alphaviral replicon nucleic acid.
4. The method of claim 1, wherein the at least one helper function
is encoded by at least two helper nucleic acid molecules wherein
each of said two helper nucleic acid molecules encodes at least one
alphaviral helper function.
5. The method of claim 1, wherein the at least one helper nucleic
acid molecule and the alphaviral replicon RNA are RNA
molecules.
6. The method of claim 5, wherein the at least one helper nucleic
acid molecule is not capped.
7. The method of claim 1, wherein at least one helper nucleic acid
molecule is a DNA molecule.
8. The method of claim 1, wherein the replicon nucleic acid is
introduced into said host cell by electroporation.
9. The method of claim 8, wherein the cell density in the
electroporation milieu is from 10.sup.7 to 5.times.10.sup.8 per
mL.
10. The method of claim 8, wherein the electroporation is carried
out in an electroporation cuvette.
11. The method of claim 1, wherein step (d) is followed by an ion
exchange chromatography step or a heparin affinity chromatography
step.
12. The method of claim 1, wherein the alphavirus is an attenuated
alphavirus.
13. The method of claim 12, wherein the attenuated alphavirus is
Venezuelan equine encephalitis virus (VEE).
14. The method of claim 13, wherein the attenuated VEE is strain
3014.
15. The method of claim 1, wherein the wash step employs NaCl, KCl,
MgCl.sub.2, CaCl.sub.2, NH.sub.4Cl, (NH.sub.4).sub.2SO4, NH.sub.4
Acetate or NH.sub.4 Bicarbonate.
16. An alphavirus replicon particle preparation prepared by the
method of claim 1.
17. The alphavirus replicon particle preparation of claim 16,
wherein the plurality of encoded antigens are derived from tumor
cells.
18. The alphavirus replicon particle preparation of claim 16,
wherein the plurality of encoded antigens are derived from a
parasite or a pathogen.
19. The alphavirus replicon particle preparation of claim 18,
wherein the plurality of encoded antigens are derived from a
pathogen selected from the group consisting of viruses, fungi,
yeasts, bacteria and protozoans.
20. A method for immunizing a human or animal against a parasite,
pathogen or cancer, said method comprising the step of
administering an amount of a virus replicon particle preparation of
claim 15 effective for generating an immune response to at least
one antigen of said parasite, pathogen or cancer.
21. The method of claim 20, wherein the pathogen is a virus, a
bacterium, a yeast, a fungus or a protozoan.
22. The method of claim 21, wherein the virus is an influenza
virus, a herpes virus, a parainfluenza virus, respiratory syncytial
virus, cytomegalovirus, human papilloma, or human immunodeficiency
virus.
23. The method of claim 21, wherein the protozoan is Plasmodium
falciparum.
24. The method of claim 21, wherein the bacterium is Mycobacterium
tuberculosis.
25. The method of claim 20, wherein the cancer is selected from the
group consisting of pancreatic cancer, kidney cancer, sarcoma,
neuroblastoma, glioma, colon cancer, melanoma, breast cancer,
ovarian cancer and prostate cancer.
26. A method for preparing alphaviral replicon particles (ARPs)
encoding and expressing a plurality of antigens, said method
comprising the steps of: a) introducing a plurality of alphaviral
replicon nucleic acids into a plurality of cells, wherein said
cells are permissive for alphavirus replication and packaging,
wherein said replicon nucleic acid comprises at least a virus
packaging signal and at least one heterologous coding sequence
expressible in said alphaviral replicon nucleic acid, wherein said
cell comprises at least one helper function, to produce a modified
cell, and wherein the plurality of alphaviral replicon nucleic
acids encode a plurality of antigens, to produce a plurality of
modified cells, wherein the step of introducing the nucleic acids
is by electroporating said cells at a density from 5.times.10.sup.7
to 5.times.10.sup.8 per mL of electroporation mixture; b) culturing
said plurality of modified cells of step (a) under conditions
allowing expression of the at least one helper function, allowing
replication of said alphaviral replicon nucleic acid and packaging
of said alphaviral replicon nucleic acid to form ARPs; c)
contacting the modified cells after step (b) with an aqueous
solution having an ionic strength from 0.2M to 5M to release the
ARPs into the aqueous solution to produce a ARP-containing
solution; and d) collecting ARPs from the ARP-containing solution
of step (c).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Nos. 60/433,299 and 60/433,058, both filed Dec. 13,
2002.
ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to recombinant DNA technology,
and in particular to introducing foreign nucleic acid(s) into a
eukaryotic host cell, and more particularly to producing infective,
propagation-defective virus-like particles which collectively
direct the expression of a representative set of immunogenic
proteins (an expression library) of a pathogen (virus, fungus,
bacterium or protozoan), parasite or tumor cell. These libraries
have applications in human and veterinary medicine.
[0004] A vaccine is one of the most efficacious, safe and
economical strategies for preventing disease and controlling the
spread of disease. Conventional vaccines are a form of
immunoprophylaxis given before disease occurrence to afford
immunoprotection by generating a strong host immunological memory
against a specific antigen. The primary aim of vaccination is to
activate the adaptive specific immune response, primarily to
generate B and T lymphocytes against specific antigen(s) associated
with the disease or the disease agent.
[0005] Similarly, cancer vaccines aim to generate immune responses
against cancer tumor-associated antigens. Cancers can be
immunogenic and can activate host immune responses capable of
controlling the disease and causing tumor regression. However,
cancer at the same time can be specifically and nonspecifically
immunosuppressive and can evade the host's immune system. Many
protein/glycoprotein tumor-associated antigens have been identified
and linked to certain types of cancer. Her-2-neu, PSA, PSMA,
MAGE-3, MAGE-1, gp100, TRP-2, tyrosinase, MART-1, .beta.-HCG, CEA,
Ras; B-catenin, gp43, GAGE-1, BAGE-1, MUC-1,2,3, and HSP-70 are
just a few examples.
[0006] Multiple approaches are being assessed in immunizing cancer
patients with tumor-associated antigens (TAAs). Vaccines in
clinical use fall into several categories determined by their
components, which range from whole cells to immunogenic peptides.
Whole cell and cell lysate vaccines can be autologous or allogeneic
vaccines, depending on the host origin of the cancer cells. An
autologous whole cell cancer vaccine is a patient-specific
formulation made from the patient's own tumor. To date, many
autologous cancer vaccines have not been clinically successful
unless they are modified to increase their intrinsic
immunogenicity, for example by the co-expression of lymphokines
such as GM-CSF (Ward et. al., 2002. Cancer Immunol. Immunother.
51:351-7). Because they are patient-specific, they can also be
costly and limited to those patients from whom cancer cells can be
obtained in sufficient quantity to produce a single-cell
suspension. In addition, the inherently limited number of cells is
problematic with respect to the need for modification or for
multiple vaccinations, making an autologous formulation impractical
for prophylaxis or treatment of early disease. Some of these
problems are solved with allogeneic whole cell vaccines or
genetically engineered whole cell vaccines where instead of
supplying immunostimulatory agents such as lymphokines exogenously
with the tumor vaccine, the tumor cells are genetically modified to
express the lymphokine endogenously. However, these methods may be
time consuming and prohibitively expensive to produce.
[0007] Natural and recombinant cancer protein antigen vaccines are
subunit vaccines. Unlike whole cell vaccines, these subunit
vaccines contain defined immunogenic antigens at standardized
levels. The key problem with such vaccines is finding the right
adjuvant and delivery system. In addition, purification of natural
or recombinant tumor antigens is tedious and not always
logistically practical. Protein cancer vaccines require culturing
tumor cells, purifying tumor antigens, or producing specific
peptides or recombinant proteins. In addition, vaccines that are
made solely from tumor protein/peptides pose intrinsic problems in
that they can be limited in the ability to be directed into the
correct antigen presentation pathways or may not be recognized by
the host due to host major histocompatibility complex (MHC)
polymorphisms. For these reasons, whole cell, or vector delivered
tumor vaccines expressing a large array of tumor antigens are
anticipated to be preferred vaccination methods. Vaccines which
include nucleic acid encoding the tumor antigens rather than
vaccines comprising the antigen itself, address some of these
problems. To date these approaches have shown the most promise in
pre-clinical and clinical testing. Amongst the current technologies
being applied to cancer vaccination, two particular systems have
shown significant potential for application in this field. The
first is delivery of TAAs using viral vectors, including but not
limited to adenoviral, adeno associated virus, retroviral,
poxviruses, flaviviruses, picornaviruses, herpesviruses and
alphaviruses (see WO 99/51263). The second is vaccination with
tumor cell protein or RNA using ex vivo derived dendritic cells as
the delivery vehicle for transfer and expression of the TAAs into
the host (Heiser et al., 2002. J. Clin. Inv. 109:409-417 and
Kumamoto et al., 2002. Nature Biotech. 20:64-69).
[0008] A limiting factor in many tumor vaccine approaches appears
to be the limited availability of known tumor-specific antigens.
These tumor-specific antigens can vary not only between tissue type
from which the tumor originated, but may even vary from
cell-to-cell within the same tumor. A confounding problem
associated with using only a limited number of tumor antigen
targets in a vaccine is the potential for "tumor escape" where the
tumor essentially evades detection by the vaccine induced immune
effector cells by deleting certain tumor associated antigens.
[0009] This observation prompted investigators to design cancer
vaccines expressing multiple antigens to reduce the propensity of
tumor escape. Unfortunately due to the limited number of antigens
that have been identified to date, this is not a feasible approach
for the majority of tumors. Therefore, a more recent evolution of
cancer therapy has been the use of entire tumor antigen libraries.
This combines multiple beneficial characteristics one would want in
a cancer vaccine. A vaccine encoding an entire tumor antigen
repertoire negates the need for antigen identification and
isolation; essentially the vaccine recipient's immune system is
allowed to make this choice in determining which TAAs the
individual will respond to. The second distinct advantage of this
approach is that, since the repertoire of antigens being expressed
is so broad, the chance of tumor escape is minimized or eliminated
entirely. Currently this approach is most actively being pursued
using dendritic cells to deliver tumor antigen libraries. These
cells, which function as antigen presenting cells by presenting the
tumor antigens to the immune system, are isolated from each cancer
patient, cultured and expanded in vitro, loaded with tumor antigen
either in the form of protein or nucleic acid; see U.S. Pat. Nos.
5,853,719 and 6,306,388. This approach has generated promising
clinical data in human testing and has shown the ability to retard
tumor growth in some individuals, and even to drive tumor
regression in a number of patients (Sadanaga et al., 2001, Clin.
Cancer Res. 7:2277-84). The major drawback for this technology is
the need for in vitro culture, expansion and antigen loading of the
patient derived dendritic cells prior to vaccination of each
individual. This is a time consuming and expensive process, and can
be highly variable since the dendritic cell population from
individual to individual can vary widely in its phenotype, growth
characteristics and activity.
[0010] To date, naked DNA, RNA, viral and bacterial vectors have
been tested for their ability to induce cancer specific responses
against a tumor antigen library. An alternative approach is the use
of viral vectors to deliver a tumor antigen library to a cancer
patient. To date, some success has been achieved with naked nucleic
acid expression libraries; e.g., see U.S. Pat. Nos. 5,989,553 and
5,703,057. Attempts to augment the immune responses elicited to
naked nucleic acid vectors include the use of self-replicating
viral vectors delivered in the form of naked RNA or DNA (Ying et
al., 1999, Nature Medicine, 5:823-827).
[0011] Viral vectors have shown great promise in pre-clinical and
clinical testing for prevention of a number of infectious disease
targets. One of the most pressing issues for development of viral
vectors for prophylactic and therapeutic vaccine uses in humans is
the ability to produce enough particles in a regulatory acceptable
form. For many viral systems, this goal is within reach and a
number of vector systems have produced positive immune response and
safety profiles in clinical trials. However, most production
schemes for vaccine vector platforms are focused on production of
large quantities of vaccine particles expressing single or at the
most two or three known antigens for specific disease targets e.g.
the gag, pol and env genes of HIV in poxvirus vectors. However, in
most cases, these large-scale manufacturing approaches are not
practical for the manufacture of individual patient-specific
vaccines.
[0012] Alphaviral vector delivery systems have been identified as
attractive vaccine vectors for a number of reasons including: high
expression of heterologous gene sequences, the derivation of
non-replicating (alpha)virus replicon particles (ARP) with good
safety profiles, an RNA genome which replicates in the cytoplasm of
the target cell and negates the chance of genomic integration of
the vector, and finally the demonstration that certain alphaviral
vectors are intrinsically targeted for replication in dendritic
cells and thus can generate strong and comprehensive immune
responses to a multitude of vaccine antigens (reviewed in Rayner,
Dryga and Kamrud, 2002, Rev. Med. Virol. 12:279-296). The
Alphavirus genus includes a variety of viruses, all of which are
members of the Togaviridae family. The alphaviruses include Eastern
Equine Encephalitis Virus (EEE), Venezuelan Equine Encephalitis
Virus (VEE), Everglades Virus, Mucambo Virus, Pixuna Virus, Western
Equine Encephalitis Virus (WEE), Sindbis Virus, Semliki Forest
Virus, Middleburg Virus, Chikungunya Virus, O'nyong-nyong Virus,
Ross River Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus,
Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus,
Babanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan
Virus, Ndumu Virus, and Buggy Creek Virus. The viral genome is a
single-stranded, messenger-sense RNA, modified at the 5'-end with a
methylated cap and at the 3'-end with a variable-length poly (A)
tract. Structural subunits containing a single viral protein, C,
associated with the RNA genome in an icosahedral nucleocapsid. In
the virion, the capsid is surrounded by a lipid envelope covered
with a regular array of transmembrane protein spikes, each of which
consists of a heterodimeric complex of two glycoproteins, usually
E1 and E2. See Pedersen et al., J. Virol 14:40 (1974). The Sindbis
and Semliki Forest viruses are considered the prototypical
alphaviruses and have been studied extensively. See Schlesinger,
The Togaviridae and Flaviviridae, Plenum Publishing Corp., N.Y.
(1986). The VEE virus has also been extensively studied. See, e.g.,
U.S. Pat. No. 5,185,440, and other references cited herein.
[0013] The studies of these viruses have led to the development of
techniques for vaccination against the alphavirus diseases and
against other diseases through the use of alphavirus vectors for
the introduction of foreign DNA encoding antigens of interest. See
U.S. Pat. No. 5,185,440 to Davis et al., and PCT Publication WO
92/10578. The introduction of foreign expressible DNA into
eukaryotic cells has become a topic of increasing interest. It is
well known that live, attenuated viral vaccines are among the most
successful means of controlling viral disease. However, for some
viral (or other) pathogens, immunization with a live virus strain
may be either impractical or unsafe. One alternative strategy is
the insertion of sequences encoding immunizing antigens of such
agents into a live, replicating strain of another virus. One such
system utilizing a live VEE vector is described in U.S. Pat. No.
5,505,947 to Johnston et al. Another such system is described by
Hahn et al., 1992, Proc. Natl. Acad. Sci. USA 89:2679-2683, wherein
Sindbis virus constructs express a truncated form of the influenza
hemagglutinin protein. Another approach is the use of infective,
propagation-defective alphavirus particles, as described in U.S.
Pat. No. 6,190,666 to Garoff et al., U.S. Pat. Nos. 5,792,462 and
6,156,558 to Johnston et al., U.S. Published Application No.
2002/0015945 A1 (Polo et al.), U.S. Published Application No.
2001/0016199 (Johnston et al.), Frolov et al., 1996, Proc. Natl.
Acad. Sci. USA 93:11371-11377 and Pushko et al. (1997) Virology
239:389-401. Alphaviruses have also been shown to be relatively
easy to genetically manipulate, as reflected by a number of
applications using alphaviruses as genomic expression libraries,
e.g., see U.S. Pat. No. 6,197,502. The use of Semliki Forest Virus
(SFV) vectors expressing a library of antigens has also been
explored in animal models where SFV particles expressing a library
of tumor antigens were used to infect dendritic cells in vitro and
the dendritic cells were used to immunize mice showing some
protection in a glioma model (Yamanaka et al., 2001, J. Neurosurg.
94:474-81).
[0014] There is a longfelt need in the art for nucleic acid
sequences encoding foreign antigens which can be used to immunize a
person or an animal against neoplastic conditions or against
parasite or pathogen infection, especially where there is no
attenuated strain or where the neoplasia, parasite or pathogen is
not well characterized at the molecular level, or where it is
recognized that protective immunization requires the expression of
multiple antigens.
SUMMARY OF THE INVENTION
[0015] It is an object of the present invention to provide a virus
replicon particle preparation derived from a neoplastic cell,
pathogen or a parasite and immunogenic compositions comprising
same. The preparation contains a multiplicity of expressible coding
sequences derived from the neoplastic cell, pathogen or parasite,
and expression of the coding sequences in a human or animal patient
to whom the preparation is administered results in the generation
of an immune response to the multiplicity of antigenic determinants
encoded by and expressed from the alphavirus replicon nucleic acid.
The immunogenic composition comprises the alphavirus replicon
particle preparation of interest and a pharmaceutically acceptable
carrier, and advantageously further comprises an immunological
adjuvant and/or a cytokine to improve or stimulate the immune
response. The alphavirus replicon can be any alphavirus replicon
RNA vector derived from VEE, Sindbis virus, South African Arbovirus
No. 86, Semliki Forest virus, among others. In preferred
embodiments, the alphavirus vector contains one or more attenuating
mutations. Suitable mutations, as well as methods to identify them,
have been described (see, for example, U.S. Pat. Nos. 5,505,947;
5,639,650; 5,811,407).
[0016] Routes of administration can include subcutaneous (s.c.),
intraperitoneal (i.p.), intramuscular (i.m.), intradermal (i.d.),
intravenous (i.v.), intratumoral, intracerebral (i.c.), direct
lymph node inoculation (i.n.), and mucosal routes such as nasal,
bronchial, intrarectal, intravaginal and oral routes. Intramuscular
administration is advantageous.
[0017] Dosages in humans and animals can range from about
1.times.10.sup.4 to about 1.times.10.sup.10, advantageously at a
dose of about 1.times.10.sup.6 to about 1.times.10.sup.8 per dose.
For the vaccine-type immunogenic approaches, the present inventors
contemplate weekly, biweekly or monthly doses for a period of about
1 to about 12 months, or longer. This can be followed by booster
vaccinations, on an as needed basis, e.g. annually.
[0018] Especially in the case where the alphavirus replicon
preparation is derived from tumor cells from a specific patient, a
patient specific vaccine preparation is made and administered back
to the same individual; i.e. the autologous vaccine approach. Also
within the scope of the present invention is an allogeneic
approach, in which the viral replicon population derived from one
patient's tumor cells is administered to another patient suffering
from, believed to be suffering from or at high risk for the same
neoplastic condition. An example of a high risk patient is an
individual with a genetic predisposition or proven hereditary
increased risk for cancer. For example, breast cancer is associated
with high familial risk in female family members of patients
suffering from breast cancer. Similarly, one might vaccinate an HIV
positive individual and at the same time, prophylactically
vaccinate their non-infected partner with the same vaccine
preparation to try to prevent the uninfected individual from
becoming infected.
[0019] The present invention further encompasses following the
immune responses elicited by administration of a virus replicon
preparation or an immunogenic composition comprising the same in a
patient to identify those tumor antigens to which the patient has
responded. These responses can be humoral and/or cellular. This
approach allows the identification of novel antigens and enables
the use of a more defined population of antigens with which to
immunize the patient. This can be accomplished by administering
boosts with more limited ARP preparations or by carrying out
subsequent immunizations of other patients or individuals (in a
prophylactic regimen) with the more defined set of antigen-encoding
ARP-containing immunogenic preparations.
[0020] The present invention also relates to the treatment and/or
prevention of infectious diseases and parasite infestations. Using
HIV as an example, a successful multi-antigenic HIV ARP vaccine
derived from a patient-specific HIV gene or genes directly from an
individual's own viral population can be applied to persons
infected with a similar genetic strain of virus or persons exposed,
likely to be exposed or potentially exposed to a similar strain.
Particularly, immunogenic or novel immunogens from the pathogen or
parasite of interest can be identified using the ARPs as a tool to
identify new immunogenic proteins. Similarly, multiple strains of a
disease causing virus (such as the recognized clades of HIV) or
parasite can be combined into the ARP preparation of this invention
to provide robust, immunogenic compositions which are not
strain-specific. For example, several different clades of HIV have
been recognized, and they can be combined to provide a multi-clade
HIV vaccine.
[0021] In the case of cancer patients, the administration of ARPs
carrying expressible cancer cell antigenic determinants' coding
sequences is advantageously accompanied by chemotherapeutic
treatments, especially where chemotherapeutic treatments do not
ablate the ability of the immune system to respond to antigens
expressed after the administration of immunogenic compositions
comprising the ARPs of the present invention.
[0022] The ARP preparations of the present invention, expressing
antigens characteristic of a particular type of tumor or cancer, a
virus, a bacterial, fungal or protozoan pathogen or a parasite can
be administered in prophylactic or therapeutic treatment regimens,
and administration of the ARPs can be carried out in combination
with other immunogenic preparations for priming and/or boosting,
for example, using an ARP vaccine prime and dendritic cell vaccine
boost, or an ARP prime and an adenoviral vector boost. All possible
combinations of DNA, RNA, adenoviruses, picornaviruses,
adeno-associated viruses, poxviruses, retroviruses, aphthoviruses,
nodaviruses, flaviviruses, dendritic cell, peptides, heat shock
proteins, minigenes, whole tumor cells and tumor cell lysate
vaccines can be used in conjunction with the ARPs expressing a
multiplicity of antigens of interest of the present invention.
Adjuvants such as cytokines or chemokines, or ARPs which direct the
expression of chemokines or cytokines, can be utilized in the
preparations of the present invention. The addition of heterologous
prime/boosts in combination with the ARP expressing a multiplicity
of genes would likely be with vector replicons or sets of vector
replicons expressing single or a relatively small number of tumor
antigens. This functions so as to focus the immune system on
specific antigens following or prior to a broader immune response
elicited by the ARP(s). Similar such heterologous delivery systems
may be used in combination with the present alphavirus replicon
expression libraries to enhance and/or maintain addition memory and
longterm immune functions.
[0023] A further object of the present invention is the
administration of the ARP-containing immunogenic compositions of
the present invention to a human not only to treat cancer or other
pathological states in a therapeutic setting when the patient is
positive for tumor, pathogen or parasite, but also once treatment
is successful and the patient is in remission. Such ongoing
periodic (booster) immunization can facilitate maintenance of a
tumor-free, disease-free or parasite-free state and prevent
regression or recurrence of the tumor or disease, respectively.
[0024] A further object of the present invention is the
administration of the ARP-containing immunogenic compositions of
the present invention to an animal (e.g. horse, pig, cow, goat,
primate, rabbit, mouse, hamster, avian) to generate immune
responses, such as antibodies. Sera or cells collected from such
animals are useful in providing polyclonal sera or cells for the
production of hybridomas that generate monoclonal sera, such
antibody preparations being useful in research, diagnostic and
therapeutic applications.
[0025] A further object of the invention is a method for preparing
alphaviral replicon particles (ARPs) which collectively encode a
multiplicity of antigens from a tumor, a tumor cell, pathogen or
parasite. The method includes the steps of preparing DNA or cDNA
from the tumor, a tumor cell, pathogen or parasite of interest and
cloning into the virus/alphavirus replicon nucleic acid to produce
a modified virus/alphavirus replicon nucleic acid, introducing the
modified viral/alphaviral replicon nucleic acid into a permissive
cell, said modified viral/alphaviral replicon nucleic acid
containing at least a virus packaging signal to produce a modified
permissive cell, culturing the modified permissive cell under
conditions allowing expression of at least one helper function and
allowing replication of said modified viral/alphaviral nucleic acid
and packaging to form ARPs, and desirably contacting the cultured
permissive cells with a Release Medium to release cell- and
debris-bound ARPs. The modified viral/alphaviral replicon nucleic
acid can be introduced into permissive cells which already contain
and express coding sequences required for packaging, or one or more
"helper" DNA or RNA molecules carrying packaging genes can be
introduced together with the modified viral/alphaviral replicon
nucleic acid. Optionally, the Release Medium step can be preceded
by a wash step which does not result in the release of the ARPs
from the cells. Advantageously the wash step includes DNase
treatment, or DNA can be digested in an ARP preparation with DNase.
DNase, for example, from Serratia marcescens, can be used at a
concentration from 10-1000 units per mL, with incubation from 10 to
60 minutes at 37.degree. . The Release Medium is an aqueous medium
which desirably is from about pH 6 to 9, desirably from about 6.5
to about 8.5, and contains from about 0.2 to about 5 M of a salt
including but not limited to ammonium acetate, ammonium chloride,
sodium chloride, magnesium chloride, calcium chloride, potassium
chloride, ammonium sulfate and sodium bicarbonate. It is
advantageous that when modified alphaviral replicon nucleic acids
are introduced into the permissive cells by electroporation, the
cells are present in a density of from about 10.sup.7 to about
5.times.10.sup.8 per mL of electroporation mixture.
[0026] Advantageously, the cells in which the ARPs are to be
produced are synchronized in the G2/M phase of the cell cycle prior
to electroporation with the alphavirus replicon vector and helper
nucleic acid(s). Without wishing to be bound by any particular
theory, it is believed that greater electroporation efficiency and
transfer of nucleic acid to the nucleus (in those embodiments of
the invention that involve nuclear activity) of the electroporated
cell is achieved in such G2/M phase cells.
BRIEF DESCRIPTION OF THE DRAWING
[0027] FIG. 1 is a bar graph depicting antigen-specific immune
responses in animals vaccinated with multi-antigenic ARP.
Antigen-specific immune responses (in the form of humoral immunity)
as measured by either ELISA and presented as reciprocal geometric
mean titer, or Western blot or IFA and presented as the lowest
dilution at which antigen specific signal was detectable. Antigen
specific immune responses in the form of cellular immunity as
measured by ELISPOT detection of IFN-.gamma. secreting cells and
presented as antigen specific IFN-.gamma. secreting lymphocytes per
10.sup.6 lymphocytes. Animals which received the multi-antigenic
ARP preparation either by a subcutaneous (s.c.) or an
intraperitoneal (i.p.) route of inoculation mounted immune
responses to all antigens in the preparation. As a positive
control, one group received HIV-Gag ARP and mounted immune
responses only specific for Gag. Negative control animals had no
detectable response to any antigen.
DETAILED DESCRIPTION OF THE INVENTION
[0028] In the context of the present application, nm means
nanometer, mL means milliliter, .mu.L means microliter, pfu/mL
means plaque forming units/milliliter, iu means infectious units,
VEE means Venezuelan Equine Encephalitis virus, EMC means
Encephalocomyocarditis virus, BHK means baby hamster kidney cells,
HA means hemagglutinin gene, CAT means chloramphenicol acetyl
transferase, .beta.-gal means .beta.-galactosidase, GFP means green
fluorescent protein gene, N means nucleocapsid, FACS means
fluorescence activated cell sorter, ELISA means enzyme-linked
immunosorbent assay, and IRES means internal ribosome entry site.
The expression "E2 amino acid (e.g., Lys, Thr, etc.) number"
indicates designated amino acid at the designated residue of the E2
gene, and is also used to refer to amino acids at specific residues
in the E1 gene.
[0029] The term "alphavirus" has its conventional meaning in the
art, and includes the various species of alphaviruses such as
Eastern Equine Encephalitis Virus (EEE), Venezuelan Equine
Encephalitis Virus (VEE), Everglades Virus, Mucambo Virus, Pixuna
Virus, Western Equine Encephalitis Virus (WEE), Sindbis Virus,
South African Arbovirus No. 86, Semliki Forest Virus, Middleburg
Virus, Chikungunya Virus, O'nyong-nyong Virus, Ross River Virus,
Barmah Forest Virus, Getah Virus, Sagiyama Virus, Bebaru Virus,
Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Babanki Virus,
Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu
Virus, and Buggy Creek Virus. The preferred alphavirus RNA
transcripts for use in the present invention include VEE Virus,
Sindbis Virus, South African Arbovirus No. 86, and Semliki Forest
Virus RNA transcripts.
[0030] Alphavirus-permissive cells employed in the methods of the
present invention are cells which, upon transfection with an
alphaviral RNA transcript, are capable of producing viral
particles. Alphaviruses have a broad host range. Examples of
suitable host cells include, but are not limited to Vero, baby
hamster kidney (BHK), DF1, CHO, 293, 293T, chicken embryo
fibroblast and insect cells such as SF21, Spodoptera frugiperda;
C6/36, Aedes albopictus; TRA-171, Toxorhynchites amboinensis;
RML-12, Aedes aegypti; AP-61, Aedes pseudoscutellaris; and MOS-55,
Anopheles gambiae cells.
[0031] The phrases "structural protein" or "alphavirus structural
protein" as used herein refer to the virally encoded proteins which
are required for encapsidation of the RNA replicon into a replicon
particle, and include the capsid protein, E1 glycoprotein, and E2
glycoprotein. As described herein, the structural proteins of the
alphavirus are distributed among one or more helper nucleic acids.
For example, a first helper RNA and a second helper RNA can be
used, or a single DNA helper encoding all alphavirus structural
proteins, can be used. In addition one or more structural proteins
may be located on the same RNA molecule as the replicon RNA,
provided that at least one structural protein is deleted from the
replicon RNA such that the replicon and resulting alphavirus
particle are propagation-defective. As used herein, the terms
"deleted" or "deletion" mean either total deletion of the specified
segment or the deletion of a sufficient portion of the specified
segment to render the segment inoperative or nonfunctional, in
accordance with standard usage. See, e.g., U.S. Pat. No. 4,650,764
to Temin et al. The term "replication defective" as used herein is
synonymous with "propagation-defective", and means that the
particles produced in a given host cell cannot produce progeny
particles in the other host cell, due to the absence of the helper
function, i.e. the alphavirus structural proteins required for
packaging the replicon nucleic acid. However, the replicon nucleic
acid is capable of replicating itself and being expressed within
the host cell into which it has been introduced.
[0032] The helper cell, also referred to as a packaging cell, used
to produce the infectious, propagation defective alphavirus
particles, must express or be capable of expressing alphavirus
structural proteins sufficient to package the replicon nucleic
acid. The structural proteins can be produced from a set of RNAs,
typically two, that are introduced into the helper cell
concomitantly with or prior to introduction of the replicon vector.
The first helper RNA includes RNA encoding at least one alphavirus
structural protein but does not encode all alphavirus structural
proteins. The first helper RNA may comprise RNA encoding the
alphavirus E1 glycoprotein, but not encoding the alphavirus capsid
protein and the alphavirus E2 glycoprotein. Alternatively, the
first helper RNA may comprise RNA encoding the alphavirus E2
glycoprotein, but not encoding the alphavirus capsid protein and
the alphavirus E1 glycoprotein. In a further embodiment, the first
helper RNA may comprise RNA encoding the alphavirus E1 glycoprotein
and the alphavirus E2 glycoprotein, but not the alphavirus capsid
protein. In a fourth embodiment, the first helper RNA may comprise
RNA encoding the alphavirus capsid, but none of the alphavirus
glycoproteins. In a fifth embodiment, the first helper RNA may
comprise RNA encoding the capsid and one of the glycoproteins, i.e.
either E1 or E2, but not both.
[0033] In combination with any one of these first helper RNAs, the
second helper RNA encodes at least one alphavirus structural
protein not encoded by the first helper RNA. For example, where the
first helper RNA encodes only the alphavirus E1 glycoprotein, the
second helper RNA may encode one or both of the alphavirus capsid
protein and the alphavirus E2 glycoprotein. Where the first helper
RNA encodes only the alphavirus capsid protein, the second helper
RNA may include RNA encoding one or both of the alphavirus
glycoproteins. Where the first helper RNA encodes only the
alphavirus E2 glycoprotein, the second helper RNA may encode one or
both of the alphavirus capsid protein and the alphavirus E1
glycoprotein. Where the first helper RNA encodes both the capsid
and alphavirus E1 glycoprotein, the second helper RNA may include
RNA encoding one or both of the alphavirus capsid protein and the
alphavirus E2 glycoprotein.
[0034] In all of the helper nucleic acids, it is understood that
these molecules further comprise sequences necessary for expression
(encompassing translation and where appropriate, transcription or
replication signals) of the encoded structural protein sequences in
the helper cells. Such sequences can include, for example,
promoters (either viral, prokaryotic or eukaryotic, inducible or
constitutive) and 5' and 3' viral replicase recognition sequences.
In the case of the helper nucleic acids expressing one or more
glycoproteins, it is understood from the art that these sequences
are advantageously expressed with a leader or signal sequence at
the N-terminus of the structural protein coding region in the
nucleic acid constructs. The leader or signal sequence can be
derived from the alphavirus, for example E3 or 6k, or it can be a
heterologous sequence such as a tissue plasminogen activator signal
peptide or a synthetic sequence. Thus, as an example, a first
helper nucleic acid may be an RNA molecule encoding capsid-E3-E1,
and the second helper nucleic acid may be an RNA molecule encoding
capsid-E3-E2. Alternatively, the first helper RNA can encode capsid
alone, and the second helper RNA can encode E3-E2-6k-E1.
Additionally, the packaging signal or "encapsidation sequence" that
is present in the viral genome is not present in all of the helper
nucleic acids. Preferably, the packaging signal is deleted from all
of the helper nucleic acids.
[0035] These RNA helpers can be introduced into the cells in a
number of ways. They can be expressed from one or more expression
cassettes that have been stably transformed into the cells, thereby
establishing packaging cell lines (see, for example, U.S. Pat. No.
6,242,259). Alternatively, the RNAs can be introduced as RNA or DNA
molecules that can be expressed in the helper cell without
integrating into the cell's genome. Methods of introduction include
electroporation, viral vectors (e.g. SV40, adenovirus, nodavirus,
astrovirus), and lipid-mediated transfection.
[0036] An alternative to multiple helper RNAs is the use of a
single nucleic acid molecule which encodes all the functions
necessary for replicating the viral replicon RNA and synthesizing
the polypeptides necessary for packaging the alphaviral replicon
RNA into infective alphavirus replicon particles. This can be
accomplished with an RNA molecule determining the necessary
functions or a DNA molecule determining the necessary functions.
The single DNA helper nucleic acid can be introduced into the
packaging cell by any means known to the art, including but not
limited to electroporation, lipid-mediated transfection, viral
vectored (e.g. adenovirus or SV-40), and calcium phosphate-mediated
transfection. Preferably, the DNA is introduced via the
electroporation-based methods of this invention, with voltage and
capacitance optimized for the cells and nucleic acid(s) being
introduced. The DNA is typically electroporated into cells with a
decrease in voltage and an increase in capacitance, as compared to
that required for the uptake of RNA. In all electroporations, the
value for the voltage and capacitance must be set so as to avoid
destroying the ability of the packaging cells to produce infective
alphavirus particles. The DNA was highly purified to remove toxic
contaminants and concentrated to about 5 mg/mL prior to
electroporation. Generally, it is preferable to concentrate the DNA
to between 1-8 mg/mL, preferably between 5 and 8 mg/mL. The DNA
helper is present in the electroporation mixture at from about
20-500, desirably from about 50 to about 300, for example about 150
.mu.g per 0.8 mL electroporation mixture, desirably containing from
about 5.times.10.sup.7 to about 2.times.10.sup.8 cells, for
example, about 1.2.times.10.sup.8 cells.
[0037] Alternatively, the helper function, in this format and under
an inducible promoter, can be incorporated into the packaging cell
genome prior to the introduction/expression of the viral RNA vector
replicon nucleic acid, and then induced with the appropriate
stimulus just prior to, concomitant with, or after the introduction
of the RNA vector replicon.
[0038] Advantageously, the nucleic acid encoding the alphavirus
structural proteins, i.e., the capsid, E1 glycoprotein and E2
glycoprotein, contains at least one attenuating mutation. The
phrases "attenuating mutation" and "attenuating amino acid," as
used herein, mean a nucleotide mutation or an amino acid coded for
in view of such a mutation which result in a decreased probability
of causing disease in its host (i.e., a loss of virulence), in
accordance with standard terminology in the art, See, e.g., B.
Davis, et al. Microbiology 132 (3d ed. 1980), whether the mutation
be a substitution mutation, or an in-frame deletion or addition
mutation. The phrase "attenuating mutation" excludes mutations
which would be lethal to the virus unless such a mutation is used
in combination with a "restoring" mutation which renders the virus
viable, albeit attenuated. In specific embodiments, the helper
nucleic acid(s) include at least one attenuating mutation.
[0039] Methods for identifying suitable attenuating mutations in
the alphavirus genome are known in the art. Olmsted et al. (1984;
Science 225:424) describes a method of identifying attenuating
mutations in Sindbis virus by selecting for rapid growth in cell
culture. Johnston and Smith (1988; Virology 162:437) describe the
identification of attenuating mutations in VEE by applying direct
selective pressure for accelerated penetration of BHK cells.
Attenuating mutations in alphaviruses have been described in the
art, e.g. White et al. 2001 J. Virology 75:3706; Kinney et al. 1989
Virology 70:19; Heise et al. 2000 J. Virology 74:4207; Bernard et
al 2000 Virology 276:93; Smith et al 2001 J. Virology 75:11196;
Heidner & Johnston 1994 J. Virology 68:8064; Klimstra et al.
1999 J. Virology 73:10387; Glasgow et al. 1991 Virology 185:741;
Polo and Johnston 1990 J. Virology 64:4438; and Smerdou and
Liljestrom 1999 J. Virology 73:1092.
[0040] In certain embodiments, the replicon RNA comprises at least
one attenuating mutation. In other specific embodiments, the helper
nucleic acid molecule(s) include at least one attenuating mutation.
In the embodiment comprising two helper nucleic acid molecules, at
least one molecule includes at least one attenuating mutation, or
both can encode at least one attenuating mutation. Alternatively,
the helper nucleic acid, or at least one of the first or second
helper nucleic acids includes at least two, or multiple,
attenuating mutations. Appropriate attenuating mutations depend
upon the alphavirus used. For example, when the alphavirus is VEE,
suitable attenuating mutations may be selected from the group
consisting of codons at E2 amino acid position 76 which specify an
attenuating amino acid, preferably lysine, arginine, or histidine
as E2 amino acid 76; codons at E2 amino acid position 120 which
specify an attenuating amino acid, preferably lysine as E2 amino
acid 120; codons at E2 amino acid position 209 which specify an
attenuating amino acid, preferably lysine, arginine, or histidine
as E2 amino acid 209; codons at E1 amino acid 272 which specify an
attenuating mutation, preferably threonine or serine as E1 amino
acid 272; codons at E1 amino acid 81 which specify an attenuating
mutation, preferably isoleucine or leucine as E1 amino acid 81; and
codons at E1 amino acid 253 which specify an attenuating mutation,
preferably serine or threonine as E1 amino acid 253. Additional
attenuating mutations include deletions or substitution mutations
in the cleavage domain between E3 and E2 such that the E3/E2
polyprotein is not cleaved; this mutation in combination with the
mutation at E1-253 is a preferred attenuated strain for use in this
invention. Similarly, mutations present in existing live vaccine
strains, e.g. strain TC83 (see Kinney et al., 1989, Virology 170:
19-30, particularly the mutation at nucleotide 3), are also
advantageously employed in the particles purified by the methods of
this invention. An example of an attenuating mutation in the
non-coding region of the replicon nucleic acid is the substitution
of A or C at nucleotide 3 in VEE.
[0041] Suitable helper and viral replicon RNAs are disclosed in
U.S. Pat. No. 6,156,558, which is incorporated herein by
reference.
[0042] Where the alphavirus is the South African Arbovirus No. 86
(S.A. AR86), suitable attenuating mutations may be selected from
the group consisting of codons at nsP1 amino acid position 538
which specify an attenuating amino acid, preferably isoleucine as
nsP1 amino acid 538; codons at E2 amino acid position 304 which
specify an attenuating amino acid, preferably threonine as E2 amino
acid position 304; codons at E2 amino acid position 314 which
specify an attenuating amino acid, preferably lysine as E2 amino
acid 314; codons at E2 amino acid position 376 which specify an
attenuating amino acid, preferably alanine as E2 amino acid 376;
codons at E2 amino acid position 372 which specify an attenuating
amino acid, preferably leucine as E2 amino acid 372; codons at nsP2
amino acid position 96 which specify an attenuating amino acid,
preferably glycine as nsP2 amino acid 96; and codons at nsP2 amino
acid position 372 which specify an attenuating amino acid,
preferably valine as nsP2 amino acid 372. Suitable attenuating
mutations useful in embodiments wherein other alphaviruses are
employed are known to those skilled in the art.
[0043] Attenuating mutations may be introduced into the nucleic
acid by performing site-directed mutagenesis, in accordance with
known procedures. See, Kunkel, Proc. Natl. Acad. Sci. USA 82:488
(1985), the disclosure of which is incorporated herein by reference
in its entirety. Alternatively, mutations may be introduced into
the nucleic acid by replacement of homologous restriction
fragments, in accordance with known procedures, or by mutagenic
polymerase chain reaction methods.
[0044] Once the helper nucleic acid(s) and replicon RNAs for use in
producing ARPs are generated, they are introduced into suitable
host cells, desirably by electroporation. The present inventors
discovered that the electroporation carried out at relatively high
cell density allows efficient uptake of helper nucleic acid and
virus replicon RNAs. The helper and replicon nucleic acids should
be purified for use in electroporation or other protocols for
introducing the nucleic acids into cells for ARP production, but
the helper RNAs need not be capped.
[0045] The step of producing the infectious viral particles in the
cells may also be carried out using conventional techniques. See
e.g., U.S. Pat. No. 5,185,440 to Davis et al., PCT Publication No.
WO 92/10578 to Bioption AB, and the U.S. Pat. No. 4,650,764 to
Temin et al. (although Temin et al., relates to retroviruses rather
than alphaviruses). The infectious viral particles may be produced
by standard cell culture growth techniques improved by procedures
described herein and/or by conventional particle harvesting
techniques or the salt wash procedure described hereinbelow. The
salt wash appears to improve ARP recovery, especially when there
are particular surface charges on the ARP surface. In the case of
VEE, amino acid residues at E2=309 and E2-120 provide good sites
for introducing a positive charge.
[0046] The viral replicon RNAs encode multiple heterologous coding
sequences which are operably linked to promoters and other
sequences required for transcriptional and translational expression
of the coding sequence in the host cell where the ARPS are to be
introduced and expressed.
[0047] Any amino acids which occur in the amino acid sequences
referred to in the specification have their usual three- and
one-letter abbreviations routinely used in the art: A, Ala,
Alanine; C, Cys, Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic
Acid; F, Phe, Phenylalanine; G, Gly, Glycine; H, His, Histidine; I,
lie, Isoleucine; K, Lys, Lysine; L, Leu, Leucine; M, Met,
Methionine; N, Asn, Asparagine; P, Pro, Proline; Q, Gln, Glutamine;
R, Arg, Arginine; S, Ser, Serine; T, Thr, Threonine; V, Val,
Valine; W, Try, Tryptophan; Y, Tyr, Tyrosine.
[0048] As used herein "expression" directed by a particular
sequence is the transcription of an associated downstream sequence.
If appropriate and desired for the associated sequence, there the
term expression also encompasses translation (protein synthesis) of
the transcribed RNA. Alternatively, different sequences can be used
to direct transcription and translation.
[0049] Genomic DNA (where genes are not interrupted by introns
and/or where this is not a significant proportion of the genome
devoted to highly repeated or non-expressed sequences) or cDNA is
cloned into a suitably prepared virus vector nucleic acid
preparation to produce a recombinant vector nucleic acid
preparation. The recombinant vector nucleic acid preparation is
then introduced into cells which allow packaging of the recombinant
vector nucleic acids into infective particles. The recombinant
vector nucleic acid preparation can be electroporated into cells
for packaging together with helper nucleic acids, RNA or DNA, in a
relatively high cell density electroporation, e.g. about 10.sup.7
to about 10.sup.9 cells/per mL electroporation mixture. The cells
are then cultured in growth medium to allow packaging of the
recombinant vector nucleic acids into viral replicon particles.
[0050] After the ARPs have been collected from the cells by salt
wash, and desirably collected from the cell free supernatant, the
ARPs are partially purified by ion exchange chromatography.
[0051] The methods of the present invention are advantageously
applied to viral replicon nucleic acids derived from an alphavirus,
preferably from an attenuated alphavirus. A particularly preferred
alphavirus is Venezuelan equine encephalitis virus (VEE). A
specifically exemplified attenuated VEE is strain 3014, which virus
or ARPs derived therefrom can be purified using heparin affinity
chromatography. VEE strain 3042 is another attenuated virus
suitable for use in ARP methods, but the coat of this virus or ARPs
derived therefrom cannot be purified using heparin affinity
chromatography. The viruses, or ARPs derived therefrom, that carry
mutations conferring glycosaminoglycan-binding ability are
particularly well suited for purification using the salt wash step,
and they can also be further purified using heparin affinity
chromatography.
[0052] Cancers (neoplastic conditions) from which cells can be
obtained for use in the methods of the present invention include
carcinomas, sarcomas, leukemias, and cancers derived from cells of
the nervous system. These include, but are not limited to: brain
tumors, such as astrocytoma, oligodendroglioma, ependymoma,
medulloblastomas, and Primitive Neural Ectodermal Tumor (PNET);
pancreatic tumors, such as pancreatic ductal adenocarcinomas; lung
tumors, such as small and large cell adenocarcinomas, squamous cell
carcinoma and bronchoalveolarcarcinom- a; colon tumors, such as
epithelial adenocarcinoma and liver metastases of these tumors;
liver tumors, such as hepatoma and cholangiocarcinoma; breast
tumors, such as ductal and lobular adenocarcinoma; gynecologic
tumors, such as squamous and adenocarcinoma of the uterine cervix,
and uterine and ovarian epithelial adenocarcinoma; prostate tumors,
such as prostatic adenocarcinoma; bladder tumors, such as
transitional, squamous cell carcinoma; tumors of the
reticuloendothelial system (RES), such as B and T cell lymphoma
(nodular and diffuse), plasmacytoma and acute and chronic leukemia;
skin tumors, such as melanoma; and soft tissue tumors, such as soft
tissue sarcoma and leiomyosarcoma.
[0053] The terms "neoplastic cell", "tumor cell", or "cancer cell",
used either in the singular or plural form, refer to cells that
have undergone a malignant transformation that makes them harmful
to the host organism. Primary cancer cells (that is, cells obtained
from near the site of malignant transformation) can be readily
distinguished from non-cancerous cells by well-established
techniques, particularly histological examination. The definition
of a cancer cell, as used herein, includes not only a primary
cancer cell, but also any cell derived from a cancer cell ancestor.
This includes metastasized cancer cells, and in vitro cultures and
cell lines derived from cancer cells. When referring to a type of
cancer that normally manifests as a solid tumor, a "clinically
detectable" tumor is one that is detectable on the basis of tumor
mass; e.g., by such procedures as CAT scan, magnetic resonance
imaging (MRI), X-ray, ultrasound or palpation. Biochemical or
immunologic findings alone may be insufficient to meet this
definition.
[0054] Pathogens to which multiple antigen immunological responses
are advantageous include viral, bacterial, fungal and protozoan
pathogens. Viruses to which immunity is desirable include, but are
not limited to, hemorrhagic fever viruses (such as Ebola virus),
immune deficiency viruses (such as feline or human immunodeficiency
viruses), herpesviruses, coronaviruses, adenoviruses, poxviruses,
retroviruses, aphthoviruses, nodaviruses, picornaviruses,
orthomyxoviruses, paramyxoviruses, rubella, togaviruses,
flaviviruses, bunyaviruses, reoviruses, oncogenic viruses such as
retroviruses, pathogenic alphaviruses (such as Semliki forest virus
or Sindbis virus), rhinoviruses, hepatitis viruses (Group B, C,
etc), influenza viruses, among others. Bacterial pathogens to which
immune responses are helpful include, without limitation,
staphylococci, streptococci, pneumococci, salmonellae,
escherichiae, yersiniae, enterococci, clostridia, corynebacteria,
hemophilus, neisseriae, bacteroides, francisella, pasteurellae,
brucellae, mycobacteriae, bordetella, spirochetes, actinomycetes,
chlamydiae, mycoplasmas, rickettsiae, and others. Pathogenic fungi
of interest include but are not limited to Candida, cryptococci,
blastomyces, histoplasma, coccidiodes, phycomycetes, trichodermas,
aspergilli, pneumocystis, and others. Protozoans to which immunity
is useful include, without limitation, toxoplasma, plasmodia,
schistosomes, amoebae, giardia, babesia, leishmania, and others.
Other parasites include the roundworms, hookworms and tapeworms,
filiaria and others.
[0055] One of the strengths of the present alphavirus replicon
vector technology is the ability to express more than one foreign
gene. Until now, alphaviral replicon vaccines have been limited to
the expression of single or a handful of heterologous genes. This
ability to express more than one heterologous gene has been
achieved through the addition of multiple promoter units to drive
each individual gene's expression. The number of heterologous genes
a replicon vector can carry is ultimately constrained by the capsid
structure which is limited in the amount of nucleic acid it can
accommodate. An alternate strategy to single replicons expressing
two or three antigens is to administer a cocktail of individual
alphavirus replicon particles, each encoding and expressing
different antigens to elicit an immune response against multiple
antigens and/or infectious agents as described herein. To date,
these approaches have been limited to the expression of only a few
antigens at the same time (.about.3), either in the multi-promoter
or the cocktail replicon setting.
[0056] However, the recent improvements in process technology as
described herein for the generation of alphavirus replicon
particles have opened the door to new opportunities in vaccination
against multiple antigens in the same vaccine preparation. The
process improvements are based on a high cell density
electroporation method (cell concentration of 5.times.10.sup.7 to
1.5.times.10.sup.8 cells/mL of electroporation mixture) and salt
wash techniques. Other improvements include the use of uncapped (or
capped) RNA molecules or DNA molecules in the electroporation
mixture. Yields from these improvements have been increased by 2 to
3 orders of magnitude (up to 10.sup.11 i.u. can be produced from a
single cuvette electroporation). These significant increases in
efficiency of replicon production over the existing art mean a
number of vaccine approaches that were previously not feasible from
a scale standpoint are now enabled. The yield which can be achieved
using the present methods, disclosed herein and in the referenced
provisional applications, in theory, allows the production of ARPs
which express the full range of antigens expressed by the tumor,
tumor cell, pathogen or parasite from which the nucleic acid
inserted into the viral replicon nucleic acid was prepared.
[0057] One such approach is a "patient-specific vaccine" where a
single vaccine preparation is prepared on a patient-by-patient
basis for prophylactic or therapeutic treatment of infectious
diseases or neoplastic condition, e.g., cancer. Because a single
tumor cell is estimated to express up to 5,000 genes, any attempt
to generate an alphaviral replicon tumor library vaccine expressing
this large a number of genes using traditional approaches would
have been significantly limited in the number of replicons
expressing each gene. In addition, the particles would require
purification to be suitable for formulation and administration in a
clinical setting, and purification often results in a significant
additional loss of titer. Using the improved ARP production
techniques, we can now generate a population of replicons where
most, if not all, genes from the tumor cell are likely represented,
on average, at least once in a population of 1.times.10.sup.5
particles. In addition to the high yields from this approach, the
process may provide a purer formulation on a per infectious unit
basis. This means sequential purification steps may not be
required, thus preventing subsequent process losses. In addition,
the increased purity may lower the risk of eliciting anti-vector
and anti-contaminant immune responses in the host. Normally, such a
response could potentially prevent or compromise the efficacy of
booster vaccinations. For approaches such as therapeutic tumor
treatment, the ability to deliver high titers of vaccine in a pure
formulation at frequent intervals is a key desirable characteristic
of a vaccine. The present invention enables a new multi-antigenic
library approach to be taken using alphaviral replicon vectors.
These libraries can encode either multiple antigens, or entire gene
repertoires from pathogenic organisms, parasites or tumor
cells.
[0058] While prior art methods used to produce nucleic acids for
introduction into cells for ARP production are expensive and labor
intensive, the present disclosure describes modifying various
parameters to achieve improved ARP yield while simplifying the
process and decreasing the cost per ARP by orders of magnitude. The
improved alphavirus particle yield has enabled cloning nucleic
acids derived from a tumor cell, pathogen or parasite into an
alphavirus replicon nucleic acid and packaging with sufficient
efficiency such that a representative set of tumor cell, pathogen
or parasite antigens are produced by the ARP "expression library".
The yield of ARPs is also sufficiently high such that a human or
animal patient can be inoculated with an aliquot of such an ARP
preparation, with the preparation optionally further containing an
immunological adjuvant, so that immune responses are generated to a
multiplicity of antigenic determinants encoded within the ARP
library and preparation administered to the patient.
[0059] Table 1 shows titration of multi-antigenic ARP produced from
a pool of cDNAs. Alphavirus replicon constructs expressing 10
different heterologous genes (chloramphenicol acetyltransferase
(CAT), beta-galactosidase (.beta.-gal), Rat/Neu oncogene,
luciferase, HIV Gag, cancer antigen A, and four malarial antigens:
PkMSP1-42, PyHep17, PfAMA1 and PkCSP) were linearized with Not1
restriction endonuclease, pooled and RNA transcripts generated
using T7 RNA polymerase. The pool of RNA molecules were
co-electroporated into VERO cells with alphaviral capsid and
glycoprotein helper RNAs to produce a population of ARP consisting
of individual ARP expressing all 10 different antigens as
determined by ARP titration using immunofluorescence assays
specific for each gene product.
[0060] Table 2 shows titration of multi-antigenic ARP produced from
a pool of RNAs. Alphavirus replicon constructs expressing 7
different heterologous genes (CMV IE1, CMV gB, Influenza HA, HIV
Pol, HIV Gag, Rat/neu, CAT) were individually linearized with Not1
restriction endonuclease. RNA transcripts for each replicon were
generated using T7 RNA polymerase. The seven different RNA
transcription products were mixed at equivalent concentrations and
were co-electroporated into VERO cells with alphaviral capsid and
glycoprotein helper RNAs. A population of ARP was produced which
expressed all 7 different antigens as determined by ARP titration
using immunofluorescence assays specific for each gene product.
[0061] Table 3 provides a summary of antigen-specific immune
responses in animals vaccinated with multi-antigenic ARP (as shown
in FIG. 1). Antigen-specific immune responses in the form of
humoral immunity are measured by either ELISA and presented as
reciprocal geometric mean titer, or Western blot or IFA and
presented as the lowest dilution at which antigen-specific signal
was detectable. Antigen specific immune responses in the form of
cellular immunity are measured by ELISPOT detection of IFN-.gamma.
secreting cells and presented as antigen specific IFN-.gamma.
secreting lymphocytes per 10.sup.6 lymphocytes. Animals which
received the multi-antigenic ARP preparation either by a s.c. or an
i.p. route of inoculation mounted immune responses to all antigens
in the preparation. As a positive control, one group received
HIV-Gag ARP and mounted immune responses only specific for Gag.
Negative control animals had no detectable response to any antigen.
Many samples were not titrated to endpoint, and are presented as
titers equal to or greater than the given value. Notably, the
immune response elicited to the HIV Gag gene protein as part of the
multiantigenic preparations was equivalent on a humoral and
cellular basis as compared to the HIV Gag protein delivered as a
single (homogeneous) standard preparation. This demonstrates coding
sequences expressed as a component of a larger expression library
can still be effectively immunogenic employing the compositions and
methods of this invention.
[0062] The immunological ARP preparations which comprise
expressible nucleotide sequences encoding a multiplicity of tumor
cell, pathogen or parasite antigenic determinants can be
administered as a part of a prophylactic regimen, i.e., to lower
the probability that the human or animal to which the preparation
is administered suffers from the neoplastic condition, pathogen
infection or parasite infection, or as a therapeutic regimen, to
lessen the severity of any conditions associated with an existing
neoplastic condition, pathogen infection or parasite infection or
such that the neoplastic condition, pathogen infection or parasite
infection is prevented due to an immune response generated in the
human or animal to which the preparation has been administered.
[0063] While the generation of an immune response includes at least
some level of protective immunity directed to the tumor cell (or
neoplastic condition), pathogen or parasite, the clinical outcome
in the patient suffering from such a neoplastic condition or
infection with a parasite or a pathogen can be improved by also
treating the patient with a suitable chemotherapeutic agent, as
known to the art. Where the pathogen is viral, an anti-viral
compound such as acyclovir can be administered concomitantly with
ARP vaccination, for example, in patients with herpesvirus
infection, or HAART (highly active anti-retroviral therapy) in
individuals infected with HIV. Where the pathogen is a bacterial
pathogen, an antibiotic to which that bacterium is susceptible is
desirably administered and where the pathogen is a fungus a
suitable antifungal antibiotic is desirably administered.
Similarly, chemical agents for the control and/or eradication of
parasitic infections are known and are advantageously administered
to the human or animal patients using dosages and schedules well
known to the art. Where the patient is suffering from a neoplastic
condition, for example, a cancer, the administration of the
immunogenic composition comprising ARPs capable of expressing a
multiplicity of cancer-associated antigens in the patient to which
it has been administered is desirably accompanied by administration
of antineoplastic agent(s), including, but not limited to, such
chemotherapeutic agents as daunorubicin, taxol, thioureas,
cancer-specific antibodies linked with therapeutic radionuclides,
with the proviso that the agent(s) do not ablate the ability of the
patient to generate an immune response to the administered ARPs and
the antigens whose expression they direct in the patient.
[0064] Pharmaceutical formulations, such as vaccines or other
immunogenic compositions, of the present invention comprise an
immunogenic amount of the infectious, propagation-defective
alphavirus replicon particles in combination with a
pharmaceutically acceptable carrier. An "immunogenic amount" is an
amount of the infectious alphavirus particles which is sufficient
to evoke an immune response in the subject to which the
pharmaceutical formulation is administered. An amount of from about
10.sup.1 to about 10.sup.10 infectious units per dose, preferably
10.sup.5 to 10.sup.8, is believed suitable, depending upon the age
and species of the subject being treated. Exemplary
pharmaceutically acceptable carries include, but are not limited
to, sterile pyrogen-free water and sterile pyrogen-free
physiological saline solution. Subjects which may be administered
immunogenic amounts of the infectious, propagation defective
alphavirus particles of the present invention include but are not
limited to human and animal (e.g., dog, cat, horse, pig, cow, goat,
rabbit, donkey, mouse, hamster, monkey) subjects. Immunologically
active compounds such as cytokines and/or BCG can also be added to
increase the immune response to the administered viral replicon
particle preparation. Administration may be by any suitable means,
such as intratumoral, intraperitoneal, intramuscular, intradermal,
intranasal, intravaginal, intrarectal, subcutaneous or intravenous
administration.
[0065] Immunogenic compositions comprising the ARPs (which direct
the expression of the antigens of interest when the compositions
are administered to a human or animal) produced using the methods
of the present invention may be formulated by any of the means
known in the art. Such compositions, especially vaccines, are
typically prepared as injectables, either as liquid solutions or
suspensions. Solid forms, for example, lyophilized preparations,
suitable for solution in, or suspension in, liquid prior to
injection may also be prepared.
[0066] The active immunogenic ingredients (the ARPs) are often
mixed with excipients or carriers that are pharmaceutically
acceptable and compatible with the active ingredient. Suitable
excipients include but are not limited to sterile water, saline,
dextrose, glycerol, ethanol, or the like and combinations
thereof.
[0067] In addition, if desired, the vaccines may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine. Examples of adjuvants which may be
effective include but are not limited to: aluminum hydroxide;
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alani-
ne-2-(1'-2'-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine
(CGP 19835A, referred to as MTP-PE); and RIBI, which contains three
components extracted from bacteria, monophosphoryl lipid A,
trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be
determined by measuring the amount of antibodies directed against
the immunogenic product of the ARP resulting from administration of
the immunogen in vaccines which are also comprised of the various
adjuvants. Such additional formulations and modes of administration
as are known in the art may also be used.
[0068] One or more immuno-potentiator molecules, such as chemokines
and/or cytokines, can be incorporated into the immunogenic
composition administered to the patient or animal. Alternatively,
alphavirus replicon vectors which contain coding sequence(s) for
the immuno-potentiator molecule can be incorporated in the
immunogenic composition. It is understood that the choice of
chemokine and/or cytokine may vary according to the neoplastic
tissue or cell, parasite or pathogen against which an immune
response is desired. Examples can include, but are not limited to,
interleukin-4, interleukin-12, gamma-interferon, granulocyte
macrophage colony stimulating factor and FLT-3 ligand.
[0069] The immunogenic (or otherwise biologically active)
ARP-containing compositions are administered in a manner compatible
with the dosage formulation, and in such amount as will be
prophylactically and/or therapeutically effective. The quantity to
be administered, which is generally in the range of about 10.sup.1
to about 10.sup.10 infectious units, preferably 10.sup.5 to
10.sup.8, in a dose, depends on the subject to be treated, the
capacity of the individual's immune system to synthesize
antibodies, and the degree of protection desired. Precise amounts
of the active ingredient required to be administered may depend on
the judgment of the physician, veterinarian or other health
practitioner and may be peculiar to each individual, but such a
determination is within the skill of such a practitioner.
[0070] The vaccine or other immunogenic composition may be given in
a single dose or multiple dose schedule. A multiple dose schedule
is one in which a primary course of vaccination may include 1 to 10
or more separate doses, followed by other doses administered at
subsequent time intervals as required to maintain and or reinforce
the immune response, e.g., at weekly, monthly or 1 to 4 months for
a second dose, and if needed, a subsequent dose(s) after several
months or years.
[0071] Standard techniques for cloning, DNA isolation,
amplification and purification, for enzymatic reactions involving
DNA ligase, DNA polymerase, restriction endonucleases and the like,
and various separation techniques are those known and commonly
employed by those skilled in the art. A number of standard
techniques are described in Sambrook et al. (1989) Molecular
Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview,
N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor
Laboratory, Plainview, New York; Wu (ed.) (1993) Meth. Enzymol.
218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.)
(1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)
Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular
Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York; Old and Primrose (1981) Principles of Gene Manipulation,
University of California Press, Berkeley; Schleif and Wensink
(1982) Practical Methods in Molecular Biology; Glover (ed.) (1985)
DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins
(eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK;
Setlow and Hollaender (1979) Genetic Engineering: Principles and
Methods, Vols. 1-4, Plenum Press, New York; and Ausubel et al.
(1992) Current Protocols in Molecular Biology, Greene/Wiley, New
York, N.Y. Abbreviations and nomenclature, where employed, are
deemed standard in the field and commonly used in professional
journals such as those cited herein.
[0072] All references cited in the present application are
incorporated by reference in their entireties to the extent that
they are not inconsistent with the present disclosure.
[0073] The following examples are provided for illustrative
purposes, and are not intended to limit the scope of the invention
as claimed herein. Any variations in the exemplified articles which
occur to the skilled artisan are intended to fall within the scope
of the present invention.
EXAMPLES
Example 1
Generation of Alphavirus Replicon Vectors Expressing a Library of
Tumor Associated Antigens
[0074] Tumor cells are typically obtained from a cancer patient by
resection, biopsy, or endoscopic sampling; the cells may be used
directly, stored frozen, or maintained or expanded in culture RNA
is extracted from tumor cells using standard methods known in the
art, e.g. using commercially available reagents and kits such as
Trizol (Sigma, St. Louis, Mo.) or S.N.A.P. total RNA isolation kit
(Invitrogen, Inc, Carlsbad, Calif.), followed by mRNA purification
on oligo (dT)-Sepharose. mRNA can be further enriched in
tumor-specific sequences by subtractive hybridization or other
method known in the art. First-strand cDNA is synthesized using
oligo (dT) oligonucleotides with a rare restriction site at
5'-terminus. Following purification of the cDNA, the second strand
is produced using any of the standard methods, e.g. using DNA
polymerase I-RnaseH or non-specific amplification. An adaptor is
then ligated to create a cohesive end, and double-stranded DNA is
digested with a rarely recognized restriction endonuclease (such as
DraI) at a site which has been incorporated in the oligo (dT)
primer. This procedure creates a double-stranded cDNA with
non-compatible cohesive ends suitable for directional cloning.
[0075] Alternatively, a strategy described in Example 2 (below) can
be used for generation of cohesive ends for directional cloning. In
an additional embodiment, cohesive ends can be attached by terminal
deoxyribonucleotide transferase. The double-stranded cDNA is then
cloned into a plasmid replicon vector or used to construct
recombinant replicon molecules in vitro in a manner similar to the
one described below. This approach produces recombinant replicon
molecules that contain a biotin label on the 3'-termini and a T7
promoter on the 5'-termini, thus allowing for selection of the
recombinant molecules and generation of RNA in vitro using T7
DNA-dependent RNA polymerase. Additional selective steps can be
implemented to "down-select" the number of antigens present in the
tumor antigen library. Methods such as subtractive hybridization
and differential analysis are well known in the art (See U.S. Pat.
Nos. 5,958,738, 5,827,658 and 5,726,022 and U.S. patent app.
2002-0018766), and such a selection method can be implemented
immediately prior to cloning into the VEE replicon construct. This
approach serves to limit the tumor antigen pool to genes either
exclusively expressed or preferentially up-regulated in a tumor
cell. This selection serves to reduce or eliminate the frequency
and/or presence of normal cellular genes in the antigen library.
Without wishing to be bound by any particular theory, it is
believed that additional benefits include the elimination of
non-tumor specific antigens focusing of the immune response against
tumor-associated antigens, thus maximizing the potential
specificity of the vaccine preparation and reducing the risk of
inducing autoimmune responses. This "down-selection" of the antigen
repertoire is also relevant to prime-boost strategies. In many
instances, it may be advantageous to vaccinate with a broad array
of tumor antigens, and in the subsequent boost inoculations, to
limit/down-select the number of antigens so as to effectively focus
the immune system on specific antigens. This can feasibly be done
by down-selecting antigens also based on identifying which antigens
the host has responded to following the first immunization, and
thus essentially tailoring each subsequent boost to augment the
immune response to antigens the host has demonstrated it can
recognize and to which an immune response has been raised.
Example 2
Generation of Alphavirus Replicon Vectors Expressing cDNAs Specific
for Infectious Disease Organism from a Sample of Infected Tissue or
Blood When the Target Gene Sequences Are Known
[0076] This example describes cloning of a
viral/bacterial/parasitic gene repertoire specific for an
individual with either an acute or chronic infection in instances
where the gene or genes of interest (i.e., the genes which encode
the immunogenic moieties to be expressed by the replicons) are
acquired from an agent of known sequence. An mRNA is isolated from
a tissue or blood sample following standard methods known in the
art, e.g. S.N.A.P. total RNA isolation kit (Invitrogen, Inc,
Carlsbad, Calif.). First-strand cDNA is synthesized by any standard
methods known in the art, e.g. cDNA cycle kit (Invitrogen, Inc,
Carlsbad, Calif.), or using AMV reverse transcriptase and random
primers. The gene(s) of interest are amplified from cDNA using
target gene-specific primers, following which the amplicon is
purified using a PCR purification kit (Qiagen Inc., Valencia,
Calif.) or any other method known in the art. This amplicon can be
cloned into the VEE replicon using methods known to those skilled
in the art, e.g. using G:C cloning, directional cloning following
restriction endonuclease digestion or in vitro recombination
methods such as Gateway (Invitrogen, Carlsbad, Calif.) or the
Cre-lox recombination system.
[0077] In a preferred embodiment, the coding sequence(s) of
interest are amplified using RNA/DNA hybrid oligonucleotides.
Following amplification, the DNA amplicon is treated with NaOH to
digest the RNA portion of the primers, or alternatively, incubated
at 50.degree. C. in the presence of rare-earth metals to
selectively hydrolyze the phosphodiester bond between the
deoxyribonucleotide and the ribonucleotide (Chen et al., 2000,
Biotechniques; 28(3):498-500, 504-5 and Chen et al., 2002,
Biotechniques, 32:516, 518-20) in order to create a 3'-overhang
required for ligation. A complementary 3'-overhang in the vector
sequences is created in a similar fashion or by using a restriction
endonuclease. In this manner the two fragments of the replicon
molecule are prepared: the left arm and the right arm. The left arm
includes a T7 promoter operatively linked to VEE specific
sequences, up to and including a convenient cloning site. The right
arm contains the 3'-untranslated region of VEE. The right arm also
contains a biotin label at the 3'-terminus. The amplified fragment
with a 3'-overhang is linked to the left and right arms of the
vector using T4 DNA ligase. The assembled molecule is separated
from the ligation reaction mixture using magnetic
streptavidin-coated beads, or any other similar solid-phase
absorption technique. Full-length replicon RNA is produced from
purified recombinant vector DNA by in vitro transcription using T7
DNA-dependent RNA polymerase. This step results in production of
only full-length recombinant molecules, since incomplete molecules
do not bind to streptavidin, or are not transcribed due to the lack
of T7 promoter sequences. The resulting recombinant replicon RNA
molecules encode a comprehensive repertoire of the target gene(s),
which represent the genotype of the target which is infectious in
the patient. An advantage of this method is the ability to have
representation of all variants for a particular gene population
from an individual, e.g. amplification of the HIV-1 envelope gp160
gene sequence isolated from an HIV-infected patient using the
methods outlined above generates an ARP population encoding the
majority or all of the envelope variants from that particular
patient. If the patient is infected with multiple strains of virus
or distinct variants originating from an original parental
circulating strain, the technique above captures all variants and
they are represented in the final ARP vaccine population.
Example 3
Generation of Alphavirus Replicon Vectors Expressing Infectious
Disease Specific cDNA From a Sample of Infected Tissue/Blood When
the Target Gene Sequences Are Not Known
[0078] This example describes cloning of a
viral/bacterial/parasitic gene repertoire in cases where the gene
or genes of interest are not of a known sequence. Viral, bacterial
or parasitic mRNA is isolated from a field sample or a stock
culture or purified preparation using MICROBExpress kit (Ambion,
Austin, Tex.) or any other method known to those skilled in the
art. First strand cDNA is synthesized using random primers, or
random primers with a rare restriction site at the 5'-terminus,
followed by second-strand cDNA synthesis with DNA polymerase I and
RNase H using standard methods known to one skilled in the art.
Double-stranded cDNA is subsequently cloned into a VEE vector after
ligation of an adaptor or a linker sequence as follows. In cases
when the cDNA is synthesized with a random primer containing a rare
restriction site, a linker is used to attach a second different
rare restriction site at the 5'-terminus of double-stranded cDNA.
Digestion of the cDNA pool with these two restriction endonucleases
results in the generation of cDNA fragments with different cohesive
ends, which facilitates directional cloning into the replicon
vector using methods known in the art. In the case that cDNA is
generated with a random primer lacking an additional unique
restriction site, double-stranded cDNA is methylated using EcoRI
methylase to protect internal sequences from subsequent digestion
with EcoRI restriction enzyme. The EcoRI linker is then attached
using T4 DNA ligase, followed by digestion with EcoRI restriction
endonuclease. This produces a cDNA fragment with cohesive ends,
which can be cloned into a replicon. A cloning strategy similar to
the one described in Example 2 can be used for the generation of a
pool of replicon molecules labeled with biotin at the 3'-terminus
and containing a T7 DNA-dependent RNA polymerase promoter at the
5'-terminus. Again, as described in the previous examples,
subtractive hybridization or differential display can be used as
additional subsequent screening steps to positively or negatively
select pathogen specific genes/sequences in a manner similar to
that described for the tumor specific approaches. Again, this can
be done with all vaccinations or on a "real-time" basis where the
host is monitored during vaccinations and the vaccine is tailored
to contain antigens to which the host demonstrates recognition and
response.
Example 4
Multi-Antigenic ARP Packaging
[0079] Generation of a population of ARPs in which each ARP
expresses a different antigen or antigens from a single
electroporation event were performed in two alternate manners. The
first method consisted of combining 0.5 .mu.g of DNA from 10
different replicon vector constructs, each containing a single
heterologous coding sequence (Table 1). The DNAs were linearized
with NotI restriction enzyme, and RNA was transcribed from the
replicon DNA pool with T7 RNA polymerase. The multiple-replicon RNA
transcription reaction was then purified using an RNEasy column
(Qiagen Inc., Valencia, Calif.). ARP were produced by
electroporation using 30 .mu.g of multiple-replicon RNA combined
with 30 .mu.g each of purified capsid (C) helper and glycoprotein
(GP) helper RNAs into 1.0.times.10.sup.8 Vero cells in a 0.8 mL
volume cuvette. After electroporation, the cells were suspended in
200 mL of Opti-pro media (Invitrogen, Carlsbad, Calif.) and seeded
into 4, 175 cm.sup.2 culture flasks. Approximately 26 hr post
electroporation the media from each flask was discarded and
replaced with 5 mL of a salt wash solution (1 M NaCl in 20 mM
phosphate buffer (pH 7.3). The flasks were incubated at room
temperature for 10 minutes, the salt wash was collected and
filtered through a 0.2 micron syringe filter. The titer of
individual ARP was determined in Vero cells using antigen-specific
antibodies by standard immunofluorescence methods. The titer of
each ARP in the pool produced from a single electroporation is
shown in Table 1. The titer of the ARP preparation was
4.1.times.10.sup.9 infectious units per mL, resulting in a total of
8.2.times.10.sup.10 i.u. total ARP generated from a single cuvette
electroporation. Representatives of all 10 antigens were present in
the ARP population. This example demonstrates that not only can
multiple different antigens be expressed from a single ARP
preparation, but that the range of antigen type can be extremely
varied. In this preparation antigens were derived from viral
infectious disease origin (HIV), from parasitic origin (malaria),
or from cancer origins (rat/Neu and cancer antigen A) as well as
enzymes (CAT, luciferase and .beta.-gal).
[0080] The second method consisted of generating RNA transcripts
for each replicon vector independently rather than as a pool. The 7
replicon vectors used in this experiment are listed in Table 2. 10
.mu.g of each purified replicon RNA was combined with 30 .mu.g each
of purified C-helper and GP-helper RNAs for a total of 130 .mu.g of
RNA. The RNA mix was then electroporated into 1.0.times.10.sup.8
Vero cells. Electroporated cells were suspended in 200 mL of
Opti-pro media and seeded into 2 300 cm.sup.2 culture flasks.
Approximately 24 hr post electroporation the media from each flask
was collected and replaced with 10 mL of salt wash (1M NaCl in 20
mM phosphate buffer, pH 7.3). The flasks were incubated at room
temperature for 5 minutes, and the salt wash was collected. Both
the media and salt wash material were filtered through a 0.2 micron
syringe filter. The individual ARP in both the media and salt wash
were titrated in Vero cells using antigen specific antibodies for
IFA. The titer of each ARP found in either the media or salt wash
is shown in Table 2. The titer of the ARP recovered in the media
was 5.3.times.10.sup.7 i.u./mL resulting in 1.1.times.10.sup.10
i.u. total ARP generated (5.3.times.10.sup.7 i.u./mL.times.200
mL=1.1.times.10.sup.10 i.u.). The titer of the ARP recovered in the
salt wash was 4.05.times.10.sup.9 i.u./mL resulting in
8.1.times.10.sup.10 i.u. total ARP generated per single cuvette
electroporation. The material in the salt wash and the media were
combined for a total of 9.2.times.10.sup.10 i.u. ARP.
Representatives of all 7 antigens were present in the ARP
population. The pooled ARP were then purified on a HiTrap Heparin
HP 5 mL column (Amersham Bioscience, Uppsala, Sweden) for use in
animal vaccination studies.
[0081] ARP preparations were all evaluated by standard safety
testing to confirm the absence of replication competent virus
(RCV). Briefly, 1.times.10.sup.8 i.u. of each preparation was
inoculated onto VERO cell monolayers at an m.o.i. of less than 1
for 1 hour. Growth media was applied to the cell monolayers after a
1 hour infection period and cells cultured for 24 hours. After 24
hours, the entire supernatant was harvested, clarified and applied
to fresh VERO cell monolayers for a further 48 hours. Cell
monolayers were monitored for the presence of any cytopathic effect
(CPE) indicative of the presence of contaminating replication
competent virus particles. In all cases, no RCV was detected in any
multi-antigenic ARP vaccine preparations.
Example 5
Animal Studies With Multi-Antigenic Virus Particles
[0082] Five to six week-old female BALB/c mice were obtained from
Charles River Laboratories and were acclimatized for one week prior
to any procedure. Mice were fed ad libitum water (reverse osmosis,
1 ppm Cl) and an irradiated standard rodent diet (NIH31 Modified
and Irradiated) consisting of 18% protein, 5% fat, and 5% fiber.
Mice were housed in static microisolators on a 12-hour light cycle
at 21-22.degree. C. (70-72.degree. F.) and 40%-60% humidity. All
animal studies comply with recommendations of the Guide for Care
and Use of Laboratory Animals with respect to restraint, husbandry,
surgical procedures, feed and fluid regulation, and veterinary
care. The animal care and use program is AAALAC accredited.
[0083] For prime and boost injections, groups of mice were each
inoculated in both rear footpads under isoflorane anesthesia with
multi-antigenic ARP in diluent (PBS with 1% v/v human serum albumin
and 5% w/v sucrose). Footpad subcutaneous (s.c.) injections were
performed with a 30.5 G needle and a 0.10 mL Hamilton syringe by
injecting 20 .mu.L in each footpad. Intraperitoneal (i.p.)
inoculations were administered by the same syringe/needle but in a
volume of 0.1 mL. Animals were inoculated on days 1, 23 and 44.
Serum samples were obtained by retro-orbital bleeding under
isoflorane anesthesia before the first inoculation on days -7 and 0
(pre-bleed), days 30 and 35 (after the primary inoculation) and
days 51 and 56 (7 and 12 days after the boost). Spleens were
harvested at least 7 days post-boost for IFN-.gamma. ELISPOT
assays.
[0084] Immunofluorescence assay (IFA) of ARP-infected Vero cells
was used to measure the potency or infectious titer of each of the
vaccine preparation. All ARP vaccines were titered prior to
inoculations. On the day of each injection residual inocula were
back-titrated. ARP vaccine inocula were kept at 4.degree. C. during
the time following vaccination to maintain titer. Test groups
included the following vaccine preparations: high and low dose
multi-antigenic ARP preparations administered as dosages of
1.times.10.sup.8 or 1.times.10.sup.6 i.u., respectively. As a
control for monitoring the immune response as compared to a single
ARP preparation expressing a single antigen ARP expressing HIV Gag
alone were administered at a dosage equivalent to the number of
HIV-Gag ARP in the multi-antigenic mix. Negative control animals
were sham immunized with diluent alone.
Example 6
Measurement of Humoral and Cellular Immune Responses after
Multi-Antigenic ARP Administration
[0085] Detection of HIV Gag specific antibodies by ELISA. Purified
recombinant histidine-tagged (his)-p55 from HIV-1 subtype C isolate
DU-422 was used as antigen coat. Briefly, BHK cells were
transfected with VEE replicon RNA expressing his-p55 and Triton-X
100 lysates were prepared. Protein was purified by ion metal
affinity chromatography, in accordance with the suppliers'
recommendations.
[0086] Sera from Day 51 (7 days post boost) were evaluated for the
presence of Gag-specific antibodies by a standard indirect ELISA.
For detection of Gag-specific total Ig, a secondary polyclonal
antibody that detects IgM, IgG and IgA was used for end point titer
determination. Briefly, 96-well Maxisorp ELISA plates (polystyrene
multiwell plates with modified surface to increase affinity for
polar molecules, i.e., antibodies; Nunc, Naperville, Ill.) were
coated with 50 .mu.L of 0.05 M sodium carbonate buffer, pH 9.6
(Sigma, St. Louis, Mo.) containing 40-80 ng his-p55 per well.
Plates were covered with adhesive plastic and incubated overnight
at 4.degree. C. The next day, unbound antigen was discarded and
plates were incubated for 1 hour with 200 .mu.L blocking buffer
(PBS containing 3% w/v BSA) at room temperature. Wells were washed
6 times with PBS and 50 .mu.L/well of test serum, diluted serially
two-fold in buffer (PBS with 1% w/v BSA and 0.05% v/v Tween 20),
was added to antigen-coated wells. Mouse anti-p24 monoclonal
antibody (Zeptometrix, Buffalo, N.Y.) was included in every assay
as a positive control. Negative controls in each assay included
blanks (wells with all reagents and treatments except serum) and
pre-bleed sera. Plates were incubated for one hour at room
temperature, and then rinsed 6 times with PBS. 50 .mu.L /well of
alkaline phosphatase (AP)-conjugated goat anti-mouse poly-isotype
secondary antibody (Sigma) diluted to a predetermined concentration
in diluent buffer was added to each well and incubated for 1 hour
at room temperature. Wells were rinsed 6 times with PBS before
addition of 100 .mu.L p-nitrophenyl phosphate (pNPP) substrate
(Sigma). The serum antibody ELISA titer was defined as the inverse
of the greatest serum dilution giving an optical density at 405 nm
greater than or equal to 0.2 above the background (blank wells).
Positive antibody (immune) responses were detected in mice
vaccinated with the multi-antigenic ARP preparation and in mice
that received the ARP HIV-Gag.
[0087] Gag and Pol antigen-specific Interferon-gamma (IFN-.gamma.)
secreting cells are detected by IFN-.gamma.ELISPOT Assay.
Single-cell suspensions of splenic lymphocytes from ARP-immunized
BALB/c mice were prepared by physical disruption of the splenic
capsule in R-10 medium (RPMI medium 1640 supplemented with 100 U/mL
penicillin, 100 .mu.g/mL streptomycin, 0.1 mM MEM non-essential
amino acids solution, 0.01 M HEPES, 2 mM glutamine and 10% heat
inactivated fetal calf serum). Lymphocytes were isolated by
Lympholyte M density gradient centrifugation (Accurate Scientific,
Westbury, N.Y.), washed twice and resuspended in fresh R-10 medium.
Total, unseparated splenic lymphocyte populations were tested.
[0088] A mouse IFN-.gamma. ELISPOT kit (Monoclonal Antibody
Technology, Nacka, Sweden) was used to perform the assay. Viable
cells were seeded into individual ELISPOT wells in a Multiscreen
Immobilon-P ELISPOT plate (ELISPOT certified 96-well filtration
plate with high protein-binding PVDF membranes; Millipore,
Billerica, Mass.) that had been pre-coated with an anti-IFN-.gamma.
monoclonal antibody, and incubated for 16-20 hours. Cells were
removed by multiple washes with buffer and the wells were incubated
with a biotinylated anti-IFN-.gamma. monoclonal antibody, followed
by washing and incubation with Avidin-Peroxidase-Complex
(Vectastain ABC Peroxidase Kit, Vector Laboratories, Burlingame,
Calif.). Following incubation, the wells were washed and incubated
for 4 minutes at room temperature with substrate (Avidin-Peroxidase
Complex tablets, Sigma, St. Louis, Mo.) to facilitate formation of
spots, which represent the positions of the individual
IFN-.gamma.-secreting cells during culture. Plates were enumerated
by automated analysis with a Zeiss KS ELISPOT system.
[0089] To enumerate Gag-specific IFN-.gamma. secreting cells in
lymphocytes from mice immunized with HIV GAG ARP and
multi-antigenic HIV ARP constructs expressing gag, lymphocytes were
stimulated with the immunodominant CD8 H-2K.sup.d-restricted
HIV-Gag peptide, or an irrelevant Nef peptide pool (Nef peptide
containing 10 15-mers overlapping by 11 made from Clade C HIV
strain DU.sub.151), for 16-20 hours (5% CO.sub.2 at 37.degree. C).
The Gag peptide was tested at 10 .mu.g/mL and the Nef control was
tested at 20 .mu.g/mL. Cells minus peptide serve as a background
control. As a positive control, cells were stimulated with 4
.mu.g/mL concanavalin A for a similar time period. Peptides were
synthesized and purified to >90% (New England Peptide, Gardner,
Mass.).
[0090] To enumerate Pol-specific IFN-.gamma. secreting cells in
lymphocytes from mice immunized with multi-antigenic ARP constructs
expressing pol, the protocol above was used with the following
modifications. HIV-1 Pol epitopes for both CD8 and CD4 T cells have
been recently identified in the H-2.sup.d background (Casimiro et
al., J. Virology 76:185, 2002). Cell populations were stimulated
with a pool of 3 Pol epitope-containing peptides and with an
irrelevant antigen peptide pool as a negative control (nef pool 1).
The three peptides below were selected after a literature search to
identify the known murine Pol CTL epitopes.
1 VYYDPSKDLIA (SEQ ID NO:1) (Casimiro et al, J. Virol. 76:185,
2002) ELRQHLLRWGL (SEQ ID NO:2) (Casimiro et al, J. Virol. 76:185,
2002) ELREHLLKWGF (SEQ ID NO:3) (homologue to number 2, identical
to our sequence).
[0091] These three peptides were mixed together at a concentration
of 10 .mu.g/mL each (total peptide concentration was 30 .mu.g/mL)
and added to triplicate wells. The ELISPOT assay results presented
were performed 26 days post the second boost.
[0092] Detection of Rat/neu specific antibodies was by ELISA.
Rat/neu antigen for use as an ELISA reagent was prepared as
follows: a histidine tag was added by PCR to the C-terminus of the
Rat/neu coding sequence in pRAT/neu #14. This PCR amplified product
was digested and ligated into the VEE replicon plasmid, pERK. BHK
cells were electroporated with RNA generated from the pERK
Rat/neu-his construct. At 16 hours post-electroporation cell
lysates were prepared and purified over a nickel affinity column,
achieving 60-70% purity of the his-tagged Rat neu antigen.
[0093] Sera from Day 51 (7 days post boost) were evaluated for the
presence of Rat/neu-specific antibodies by an indirect ELISA. Nunc
high binding plates were coated at 4.degree. C. overnight with 75
ng/well of his-tagged Rat neu in carbonate-bicarbonate coating
buffer. The next day plates were blocked with 200 .mu.l/well of 3%
BSA in PBS for 1 hour at 30.degree. C. After 6 washes in PBS, 50
.mu.l of mouse serum samples were diluted in 1% BSA, 0.05% Tween 20
in PBS and added to each well and the plates were incubated for 1
hr at 30.degree. C. Pre-bleeds at 1:40 and 1:80, as well as
two-fold dilutions from 1:40-1:1280 of day 51 sera were tested for
each experimental animal. Plates were then washed 6 times with PBS,
followed by the addition of 50 .mu.l/well of a 1:500 dilution of
goat anti-mouse HRP and incubated for 1 hr at 30.degree. C. Plates
were washed as before and developed with 100 .mu.l/well of ABTS
(KPL), and the absorbance was read at 405 nm using a standard ELISA
reader. The cut off value to determine a positive sample was
determined by averaging the OD (absorbance) value of all the
pre-bleed serum samples diluted 1:40 and multiplying that value by
two. Any sample with an OD greater than the cut off value was
considered positive.
[0094] Detection of anti-CAT specific antibodies was by ELISA. An
anti-CAT antibody ELISA was developed to detect anti-CAT immune
responses in multi-antigen ARP vaccinated mice. ELISA microplates
coated with sheep anti-CAT polyclonal antibodies (Roche,
Indianapolis, Ind.) were loaded with 0.15 ng of purified CAT
protein suspended in CAT ELISA sample buffer (Roche) in a volume of
50 .mu.l per well. The ELISA plates were incubated at 37.degree. C.
for 45 min and washed three times with 0.2 mL of CAT ELISA wash
buffer (Roche). 50 .mu.l of mouse serum, two-fold serially diluted
in sample buffer, was loaded per well and the plates were incubated
at 37.degree. C. for 45 min. After incubation, the ELISA plates
were washed three times as described above. Goat anti-mouse
HRP-conjugated secondary antibody (Kirkegaard and Perry
Laboratories (KPL), Gaithersburg, Md.) diluted 1:500 in sample
buffer was added to each well (0.1 mL per well) and incubated at
37.degree. C. for 45 min. After incubation, the plates were washed
three times as described above, and 0.1 mL of ABTS peroxidase
substrate (2,2'-azino-bis 3-ethylbenzthiazoline-b-sulfonic acid;
KPL) was added per well. Color development was ended by addition of
0.1 mL stop solution (KPL) and the absorbance in the plates were
read at 405 nm using a Molecular Devices Versamax microplate
reader. The cut off value to determine a positive sample was
determined by averaging the OD value of all the pre-bleed serum
samples diluted 1:40 and multiplying that value by two. Any sample
with an OD greater than the cut off value was considered
positive.
[0095] Detection of CMV gB specific antibodies was by Western blot.
Analysis of anti-gB immune responses in multi-antigen ARP
vaccinated animals was by Western blot. Purified, recombinant,
histidine-tagged gB protein was electrophoresed through 4-10%
Bis-Tris NuPAGE gels (Sodium dodecyl sulfate-polyacrylamide gel;
Invitrogen, Carlsbad, Calif.) and transferred to PVDF membranes
using a Novex mini-cell (Invitrogen) electrophoresis unit.
Pre-bleed and Day 51 post-vaccination sera were diluted 1:40 or
1:80 for each animal in blocking buffer (Invitrogen) and incubated
on strips of PVDF membranes after gB protein transfer. Goat
anti-mouse alkaline phosphates conjugated antibody (Sigma, St.
Louis, Mo.) diluted 1:10,000 in blocking buffer was used as the
secondary antibody. Western blots were developed using BCIP/NBT
(5-bromo,4-chloro,3-indolylphosphate/nitroblue tetrazolium; Bio
Rad, Hercules, Calif.), and color development was arrested by
washing with distilled water. Positive samples were identified by
visual detection of immunoreactive bands with electrophoretic
mobility matching the expected molecular weight of gB on the
immunoblot.
[0096] Detection of Influenza HA specific antibodies was by
immunofluorescence assay (IFA). Analysis of anti-HA immune
responses in multi-antigen ARP vaccinated animals was determined by
IFA. Vero cells were electroporated with a VEE replicon vector that
expressed the H1N1 influenza HA gene and 1.times.10.sup.4
electroporated cells per well were seeded into 96 well tissue
culture plates. Electroporated Vero cells were fixed with methanol
16 hr post-electroporation. Pre-bleed and day 56 post-vaccination
sera were diluted two-fold from 1:40 to 1:160 in blocking buffer
(PBS:FBS (1:1)) for each animal and incubated on HA protein
expressing Vero cells. A goat anti-mouse Alexa Fluor 488 conjugated
antibody (Molecular Probes, Eugene, Oreg.) diluted 1:400 was used
as the secondary antibody. Cells were analyzed on a Nikon Eclipse
TE300 UV microscope for HA specific fluorescence. Titer was
determined by visual detection of immunofluorescent cells at the
lowest detectable serum dilution value.
[0097] Detection of anti-CMV IE1 specific antibodies was by ELISA.
Purified recombinant histidine-tagged (his)-IE1 from CMV was used
as antigen coat. Briefly, BHK cells were transfected with VEE
replicon RNA expressing his-IE1 and Triton-X 100 lysates were
prepared. Protein was purified by ion metal affinity
chromatography.
[0098] Sera from Day 51 (7 days post boost) were evaluated for the
presence of CMV-IE1-specific antibodies by a standard indirect
ELISA. For detection of CMV-IE1-specific total Ig, a secondary
polyclonal antibody that detects IgM, IgG and IgA was used for end
point titer determination. Briefly, 96-well Maxisorp ELISA plates
(Nunc, Naperville, Ill.) were coated with 2 .mu.g IE1 in a volume
of 50 .mu.L in citrate/phosphate, pH 8.3, per well. Plates were
covered with adhesive plastic and incubated overnight at 4.degree.
C. The next day, unbound antigen was discarded and plates were
incubated for 1 hour with 200 .mu.l blocking buffer (PBS containing
3% w/v BSA) at room temperature. Wells were washed 6 times with PBS
and 50 .mu.l of serum, diluted serially two-fold in buffer (PBS
with 1% w/v BSA and 0.05% v/v Tween 20), was added to
antigen-coated wells. An .alpha.-IE1 monoclonal antibody
(Rumbaugh-Goodwin Institute for Cancer Research, Inc, Plantation,
Fla.) was included in every assay as a positive control. Negative
controls in each assay included blanks (wells with all reagents and
treatments except serum) and pre-bleed sera. Plates were incubated
for one hour at room temperature, and then rinsed 6 times with PBS.
Fifty .mu.L/well of alkaline phosphatase (AP)-conjugated goat
anti-mouse poly-isotype secondary antibody (Sigma) diluted to a
predetermined concentration in diluent buffer was added to each
well and incubated for 1 hour at room temperature. Wells were
rinsed 6 times with PBS before addition of 100 .mu.l p-nitrophenyl
phosphate (pNPP) substrate (Sigma). The serum antibody ELISA titer
was defined as the inverse of the greatest serum dilution giving an
optical density at 405 nm greater than or equal to 0.2 above the
background (blank wells).
[0099] Summary of Immune Response to Multi-antigenic ARP
[0100] As shown in FIG. 1 and Table 3, animals vaccinated with
multi-antigenic ARP mounted immune responses to all seven antigens
present in the ARP population. These immune responses included both
humoral and cellular responses, indicating this type of approach
can stimulate both arms of the immune system. The strength of the
immune response to a specific antigen was also measured in the
context of the multi-antigenic ARP and compared to a single-antigen
ARP preparation. Anti-Gag antibody and cellular immune responses
were equivalent whether the HIV-Gag ARP was alone or in a
multi-antigenic formulation, indicating that addition of a
plurality of different antigens does not appear to diminish the
immune response to each individual component of the preparation.
This multi-antigenic preparation was intentionally composed of
genes from infectious disease agents (HIV and CMV), cancer antigen
(Rat/neu) and bacterial enzyme (CAT) to demonstrate that the host
immune system can be stimulated with multi-antigenic ARP to respond
to a broad array of antigen types within a single ARP
preparation.
Example 7
Animal Studies with Multi-Antigenic ARPs Expressing a Tumor cDNA
Library
[0101] A cDNA library is generated from a B16F10(B16) [Gold et al.,
(2003) J. Immunol. 170:5188-5194) pigmented mouse melanoma cell
line originally derived from C576BU6 mice. This library is
directionally cloned into the alphaviral replicon cDNA construct so
that the heterologous cDNA is expressed from the replicon upon
infection of a target cell. ARP are generated and purified as
described above to produce a population of ARP particles expressing
an entire library of cDNAs from the B16 tumor cells. Expression of
representative genes such as .beta.-actin can be analyzed by
quantitative PCR to determine whether the library expresses known
gene standards. Subtractive hybridization or differential display
against a non-tumorigenic genetically matched cell line can be used
to enhance the proportion of tumor-specific sequences in the
library.
[0102] C57BL/6 mice are vaccinated with the B16 library ARP
preparation one, two or three times on days 0, 21 and 42. Doses of
between 10.sup.5-10.sup.9 i.u. in ARP are administered via a
subcutaneous (sc.) route delivered both rear footpads of the mouse.
Control groups of mice receive placebo vaccinations or ARP
expressing irrelevant antigens. An additional set of animals can be
included which receive ARP expressing single known melanoma
specific tumor antigens such as TYR, TRP-2, gp100, MAGE-1 or
MAGE-3, or a combination of said antigens as comparators to the
multi-antigenic approach.
[0103] Mice are injected intradermally (id.) with 10.sup.4,
10.sup.5 or 10.sup.6 B16 melanoma cells on the right flank 5 days
after the final ARP immunization. The mice are then followed for
tumor onset by palpation every other day. Tumors are scored as
present once they reach a diameter of equal to or greater than 2
mm. Mice are sacrificed once it is assured that the tumor is
progressing (usually at a size of 1 cm). Kaplan-Meier tumor-free
survival curves are constructed and log rank analysis performed to
determine statistical significance of protection from tumor
challenge between each group.
[0104] Prior to tumor challenge, sera and lymphocytes are harvested
from mice for immunoassay. The presence of humoral or cellular
responses to known tumor antigens expected to be present in the ARP
B16 library can be assayed using standard methods and techniques
known in the art.
[0105] Canine malignant melanoma (CMM) is a spontaneous, aggressive
and metastatic neoplasm which occurs in dogs. CMM is a relatively
frequently diagnosed tumor and accounts for about 4% of all canine
tumors. CMM is initially treated with local therapies including
surgery and/or fractionated radiation therapy; however, systemic
metastatic disease is a common sequela. CMM is a chemo-resistant
neoplasm. All these properties are common to human melanoma, and on
the basis of these similarities, CMM serves as a clinical model for
evaluating new treatments for human melanoma [Bergman et al. (2003)
Clin. Cancer Res. 9:1284-1290).
[0106] Dogs are screened for the presence of histologically
confirmed spontaneous malignant melanoma. Pre-trial evaluation
includes complete physical evaluation, complete blood count and
platelet count, serum chemistry profile, urinalysis, LDH,
anti-nuclear antibody, and three-dimensional measurements of the
primary tumor if present (or maximal tumor size from medical
records if patient has been treated before pretrial
considerations). For the evaluation of metastatic disease, 3-view
radiographs of the thorax are obtained and regional lymph nodes are
evaluated with fine needle aspiration/cytology and/or
biopsy/histopathology. All dogs are staged according to the WHO
staging system of stage II tumors (tumors 2-4 cm diameter, negative
nodes), stage III (tumor >4 cm and/or positive nodes) or stage
IV (distant metastatic disease). Dogs from all three of these
stages of disease are included in the study, provided they have not
received any other form of therapy in the previous three weeks.
[0107] Fine needle aspiration or biopsy is used to confirm
malignant melanoma in each animal by cytology or histopathology,
respectively. These samples, taken from either the primary tumor
mass or from metastatic masses, are used as the source of the tumor
cDNA library. For each animal, tumor RNA is isolated form the tumor
cell population. A cDNA library is prepared from each sample.
Multi-antigenic ARP preparations are generated for each animal as
described herein.
[0108] Cohorts of dogs receive multiple vaccinations of canine
patient-specific ARP preparations with a range of dosages. Dogs are
vaccinated between 3-12 times over a period of 1-3 months. Dosages
of ARPs administered via either a subcutaneous, intradermal or
intramuscular route range from 10.sup.6 to 10.sup.9 i.u. In
addition to administering patient-specific (autologous) ARP
vaccines, some cohorts can receive ARP preparations from other
patients (allogeneic) in order to determine if a vaccine
preparation from an alternate melanoma provides clinical
benefit.
[0109] The clinical status of each patient is monitored throughout
the vaccination regime and for up to two years following treatment.
Patients are physically, radiologically and biochemically examined
on a frequent basis for clinical evidence of tumor presence and
progression or regression. If euthanasia is requested by owners in
the event of degradation in the quality of life due to advanced
disease, a full necropsy is performed with subsequent necropsy
examination to determine gross and histopathological status of the
tumor at primary and metastatic sites. Statistical analysis is
performed to determine the effect of multi-antigenic ARP
vaccination on survival and disease progression. Statistical
analysis tools include the Kaplan-Meier product limit method, Cox
proportional hazard analysis, Mann-Whitney U test, and a Spearman
rank correlation.
2TABLE 1 Titration of Multi-antigenic ARPs (Pool of 10 constructs)
Replicon vector ARP titer CAT(chloramphenicol acetyltransferase)
3.6 .times. 10.sup.8/mL .beta.-gal 1.3 .times. 10.sup.5/mL Rat/neu
5.2 .times. 10.sup.8/mL Luciferase 6.8 .times. 10.sup.6/mL
PkMSP1-42 4.5 .times. 10.sup.8/mL PyHep17 2.0 .times. 10.sup.8/mL
PfAMA1 4.0 .times. 10.sup.7/mL PkCSP 5.7 .times. 10.sup.8/mL HIV
Gag 1.5 .times. 10.sup.9/mL Cancer Antigen A 4.5 .times.
10.sup.8/mL Total/ml 4.1 .times. 10.sup.9/mL Total from single
cuvette electroporation 8.2 .times. 10.sup.10
[0110]
3TABLE 2 Titration of Multi-antigenic ARPs Produced from a Pool of
Seven RNAs ARP titer ARP titer in Replicon vector in media salt
wash CMV IE1 2.9 .times. 10.sup.6/mL 1.9 .times. 10.sup.8/mL CMV gB
2.9 .times. 10.sup.5/mL 5.8 .times. 10.sup.7/mL Influenza HA 1.3
.times. 10.sup.5/mL 1.9 .times. 10.sup.7/mL HIV pol 3.4 .times.
10.sup.6/mL 3.3 .times. 10.sup.8/mL HIV Gag 4.2 .times. 10.sup.7/mL
2.9 .times. 10.sup.9/mL Rat/neu 1.9 .times. 10.sup.6/mL 2.6 .times.
10.sup.8/mL CAT(chloramphenicol acetyltransferase) 2.3 .times.
10.sup.6/mL 2.9 .times. 10.sup.8/mL Total/mL 5.7 .times.
10.sup.7/mL 4.1 .times. 10.sup.9/mL Total from single cuvette 1.1
.times. 10.sup.10 8.2 .times. 10.sup.10 electroporation Total
Pooled ARP Titer 9.3 .times. 10.sup.10
[0111]
4TABLE 3 Antigen-specific Immune Responses in Animals Immunized
with Multi-antigenic ARPs Vaccination HIV GAG HIV GAG HIV POL FLU
HA CMV gB CAT Rat/neu CMV IE1 Group ELISA ELISPOT ELISPOT IFA
Western ELISA ELISA ELISA Multi-Ag ARP 8192 475 614 160.sup.a
80.sup.a 1280.sup.a 1280.sup.a 1280 s.c. footpad Multi-Ag ARP
40960.sup.a 439 105 160.sup.a 80.sup.a 1280.sup.a 320 2560.sup.a
i.p. HIV GAG ARP 5120 500 0.sup.b 10.sup.b 10.sup.b 10.sup.b
10.sup.b 10.sup.b s.c. footpad Negative 10.sup.b 0.sup.b 0.sup.b
10.sup.b 10.sup.b 10.sup.b 10.sup.b 10.sup.b control s.c. footpad
.sup.aSamples not tested to full endpoint, actual titers are all
equal or greater than this measurement .sup.bAt or below limit of
detection of the assay
[0112] References Cited in the Present Application
[0113] Casimiro D R, Tang A, Perry H C, Long R S, Chen M, Heidecker
G J, Davies M E, Freed D C, Persaud N V, Dubey S, Smith J G, Havlir
D, Richman D, Chastain M A, Simon A J, Fu T M, Emini E A, Shiver J
W. Vaccine-induced immune responses in rodents and nonhuman
primates by use of a humanized human immunodeficiency virus type 1
pol gene. J. Virology. 2002. 76:185-195, 2002
[0114] Chen G J, Qiu N, Karrer C, Caspers P, and Page M G.
Restriction site-free insertion of PCR products directionally into
vectors. Biotechniques. 2000; 28(3):498-500, 504-5.
[0115] Chen G J, Qiu N, Page M P. Universal restriction site-free
cloning method using chimeric primers. Biotechniques. 2002;
32(3):516, 518-20.
[0116] Heiser, A., Coleman, D., Dannull, J., Yancy, D., Maurice,
M., Lallas, C., Dahm, P., Niedzwiecki, D., Gilboa, E. and J.
Vieweg. J. Clinical Investigation. 2002. 109(3):409-417.
[0117] Kumamoto, T., Huang, E., Paek, H-J., Morita, A., Matsue, H.,
Valentini, R., and A. Takashima. Nature Biotechnology. 2002.
20:64-69.
[0118] Rayner, J O, Dryga, S. A. and Kurt I. Kamrud. Alphavirus
vectors and vaccination. Rev. Med. Virol. 2002. 12:279-296.
[0119] Sadanaga N, Nagashima H, Mashino K, Tahara K, Yamaguchi H,
Ohta M, Fujie T, Tanaka F, Inoue H, Takesako K, Akiyoshi T, Mori M.
Dendritic cell vaccination with MAGE peptide is a novel therapeutic
approach for gastrointestinal carcinomas. Clin. Cancer Res. 2001
August; 7(8):2277-84.
[0120] Yamanaka, R., Zullo, S. A., Tanaka, R., Blaese, M., and K.
G. Xanthopoulos. Enhancement of antitumor immune response in glioma
models in mice by genetically modified dendritic cells pulsed with
Semliki forest virus-mediated complementary DNA. J. Neurosurg.
2001. 94(3):474-81.
[0121] Ward S, Casey D, Labarthe M C, Whelan M, Dalgleish A, Pandha
H, Todryk S. Immunotherapeutic potential of whole tumour cells.
Cancer Immunol. Immunother. 2002. 51(7):351-7.
[0122] Ying, H., Zaks, T. Z. Rong-Fu, W., Irvine, K. R., Kammula,
U. S. Marincola, F. M. Leiner, W. W. and N. P. Restifo. Cancer
therapy using a self-replicating RNA vaccine. Nature Medicine.
1999. 7(5):823-827
Sequence CWU 1
1
3 1 11 PRT Artificial Sequence peptide epitope of murine Pol CTL 1
Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala 1 5 10 2 11 PRT
Artificial Sequence Peptide epitope of murine Pol CTL 2 Glu Leu Arg
Gln His Leu Leu Arg Trp Gly Leu 1 5 10 3 11 PRT Artificial Sequence
Peptide epitope of murine Pol CTL 3 Glu Leu Arg Glu His Leu Leu Lys
Trp Gly Phe 1 5 10
* * * * *