JP2002518996A - Recombinant cytomegalovirus expression system targeting immediate gene region - Google Patents

Abstract

(57) Abstract: The present invention provides a cytomegalovirus comprising a gene cassette containing at least one promoter adjacent to at least one DNA sequence and a polyadenylation termination sequence in the immediate-type (IE) gene region of the virus genome. A genome comprising: (1) the promoter is configured to express the DNA sequence; and (2) the promoter, the DNA sequence, and the polyadenylation termination sequence are heterologous to the virus into which the cassette has been inserted. Consisting of a recombinant cytomegalovirus.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to recombinant cytomegalovirus (CMV) vectors and, in particular, to recombinant CMV comprising at least one heterologous DNA sequence within a genomic region encoding an immediate protein.

General [0002] It will be appreciated by those skilled in the art that the invention described herein is open to variations and modifications other than those specifically noted. It is understood that the present invention includes all such variations and modifications. The present invention also relates to all steps, features, compositions, and compounds described or indicated herein individually or collectively, and any and all combinations or any two or more steps or features. Including.

[0003] The present invention is not limited in scope by the specific embodiments described herein, but is intended for purposes of illustration only. Functionally equivalent products, combinations,
It is clear that the methods and methods are within the scope of the invention described herein.

[0004] The details of an inventory of many of the publications mentioned herein are summarized at the end of the specification. All references, including patents or patent applications, are incorporated herein by reference. None of the references is allowed to constitute prior art.

[0005] As used herein, the term "derived from" is understood to indicate that a particular whole can be obtained from a particular source, although not necessarily obtained from that source. Should.

[0006] Throughout this specification, unless the context requires otherwise, the term "compri"
"se" or its variants ("comprises" or "comprising"
)) Are intended to include the recited integer or group of integers, and not to exclude any other integer or group of integers.

BACKGROUND OF THE INVENTION [0007] Cytomegalovirus (CMV) is a Herpesviridae (generally,
(Known as the herpes virus). Because of the narrow range of mutations in the host and the long duration of the replication cycle, CMV promotes slow lysis of cells in cell culture and often produces large cells (giant cells) in vitro and in vivo.

[0008] CMV has a different genomic organization than other herpesviruses. Human CMV (
Approximately 1990 of the 230 kb dsDNA genome of HCMV) has been sequenced and has at least 200 open reading frames (ORFs). The function of some HCMV proteins is known or predicted by homology to other viral and cellular proteins. However, the function of the protein encoded for most of the HCMV ORF is unknown. Mouse CMV (M
CMMV) has also been sequenced and is closely related to HCMV. 170 MC
78 of the MV ORFs show sequences similar to HCMV.

[0009] In beginning vaccinia virus recombination experiments, virus delivery systems have been used for many years. In these studies, vaccinia virus was genetically engineered to express a heterologous antigen. While the concept of using viruses as a delivery system for heterologous genes and / or antigens is evident in these studies, what was not obvious was the answer to the more practical question of optimal candidate viral vectors. . The answer to this question is to select the details of the pathogenicity of the virus, its replication site, the type of immune response elicited, the potential of the virus to express foreign antigens, and its suitability for genetic manipulation. The above factors. For example, a viral vector carrying a therapeutic agent needs to target the correct cell type for delivering the therapeutic agent. One of the viral subfamilies that has received little attention as a possible vector system is the cytomegalovirus.

The present invention provides a CMV that improves some or all of the problems associated with the prior art.
Explore providing a vector.

SUMMARY OF THE INVENTION [0011] The present invention relates to a recombinant cytomegalovirus, comprising a gene cassette comprising at least one promoter flanked by at least one DNA sequence and a polyadenylation termination sequence, in the immediate form of said viral genome. (IE) a cytomegalovirus genome contained within the gene region, (1) the promoter is configured to express the DNA sequence, and (2) the promoter, the DNA sequence, and the polyadenylation termination sequence are The cassette consists of a recombinant cytomegalovirus that is heterologous to the inserted virus.

In a preferred embodiment, the present invention relates to a recombinant cytomegalovirus, comprising a gene cassette comprising at least one HCMV IE-1 promoter flanked by at least one DNA sequence and a polyadenylation termination sequence. A cytomegalovirus genome comprising within the immediate-early (IE) gene region of the genome,
1) the HCMV IE-1 promoter is configured to express the DNA sequence; and (2) the DNA sequence and the polyadenylation termination sequence are heterologous to the virus into which the cassette has been inserted. Consists of

[0013] In another embodiment of the invention, a cassette is inserted into the IE gene region in a manner that inactivates at least one gene encoding an IE protein. Insertion of the cassette into the viral genome may lead to either activation of an identifiable phenotype or deletion of that phenotype from the viral genome in the virus, thereby providing a means of selecting the transformed virus. Is desired.

The heterologous DNA sequence in the cassette can encode part or all of any native or recombinant protein that is heterologous to the CMV species from which the recombinant virus was formed. Preferably, the heterologous DNA sequence encodes one or more of the following:
Zona pellucida (ZP) proteins, malaria surface antigen, β-galactosidase, major antigen virus antigens (eg, hemagglutinin (HA) from influenza virus), eukaryotic cell polypeptides, enzyme inhibitors, hormones, lymphokines , Cytokines, chemokines, plasminogen activators, or natural, modified, or chimeric immunoglobulins or fragments thereof.

[0015] In a highly preferred embodiment of the invention, the heterologous DNA sequence comprises one or more ZPs.
Encodes a protein or virus antigen-like HA.

The present invention also provides a prophylactic or therapeutic substance comprising the recombinant viral vector prepared according to the present invention in a pharmaceutically acceptable vehicle. In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient may also include one or more excipients, adjuvants, stabilizers, or other similar substances. This type of medicinal product should be prepared and used by standard techniques known in the art.

The present invention also provides a) a step of growing the recombinant CMV described herein in a suitable host cell line; and b) an effective immunizing amount of the resulting virus and a pharmaceutically acceptable addition. And a method of producing a prophylactic or therapeutic substance.

DETAILED DESCRIPTION OF THE INVENTION The present invention uses CMV as a vector for the delivery of polypeptides and therapeutic agents. CMV is ubiquitous in nature, a DNA virus, non-mutagenic, non-integrative, stable, has benign potency in healthy individuals, and CMV-specific species are: Very suitable for this role as it is found in most animal populations. The immunoglobulin response to the expressed antigen predominates the IgG2a isotype and is presumed to be a type 1 immune response. In addition, inoculation of animals with CMV results in a prolonged IgA response in the salivary glands.

The present inventors have found that CMV produced according to the present invention has a booster response in the salivary glands to the expression of an antigen that causes a long lasting IgA response. The response can detect the absence of significantly detectable viral replication in the immunologically competent animal.

Accordingly, the present invention relates to a cytomegalovirus genome comprising a gene cassette comprising at least one promoter flanked by at least one DNA sequence and a polyadenylation termination sequence in the immediate early (IE) gene region of said virus genome. (1) the promoter is configured to express the DNA sequence, (2)
Consist of a recombinant cytomegalovirus wherein said promoter, said DNA sequence, and said polyadenylation termination sequence are heterologous to the virus into which said cassette has been inserted.

While not wishing to be limited to any mode of action, we have found that expression of a foreign protein via a recombinant virus (rMCMV) made according to the present invention results in an immune response against the protein, resulting in an immune response to the protein. It is believed that the recognition and clearance of host cells expressing the membrane-bound protein is increased, and therefore the virus is also eliminated. That is, foreign proteins probably act as additional viral antigens, overriding the immune evasion mechanisms expressed by that virus. This "immunological attenuation" is caused by the expression of a heterologous antigen on the membrane of the infected host cell. This increases the recognition of virus-infected cells by the immune system.

We have determined that rMCMV-ovalbumin (OVA) and rMCMV-H
A was found to be replicated in the salivary glands of IFN-α / γ receptor knockout mice to a level comparable to that achieved by parental (RM427 + or K181-WT) MCMV in immunocompetent animals. rMCMV
-OVA is also replicated in the salivary glands of IFN- [alpha] R ko mice. These mice are deficient in their immune response. This suggests that rMCMV can replicate in the salivary glands once the host immune response is deficient. More specifically, this appears to be an early or innate immune response responsible for regulating viral replication.

Both rMCMV-OVA and rMCMV-HA are immunocompetent BA
Generates an immune response against OVA and HA antigens in LB / c mice, respectively. The preferential antibody response is of the IgG2a isotype, indicating a TH1-type response. This requires low levels of rM to generate an antibody response to the expressed foreign antigen.
Indicates that CMV replication is sufficient.

When the IFN-αR ko mouse or BALB / c weaning mouse is subcultured for the purpose of SGV production, the salivary gland rMCMV- derived from the IFN-α / γR ko mouse is passaged.
OVA does not display the virus. These experiments show that, although rMCMV can replicate in the salivary glands of immunodeficient mice, it cannot replicate in the BALB / c salivary glands, indicating that any rMCMV on the MCMV genome responsible for reduced replication is present. Shows that no harmful mutations exist. These mutations can be introduced and selected during the process of homologous recombination by tissue culture-based techniques and subsequent isolation of the recombinant.

[0025] The cassette is preferably inserted into the IE gene region in such a manner as to inactivate at least one gene encoding the IE protein. In a more preferred embodiment of the invention, the recombinant cytomegalovirus into which the cassette has been inserted is lacZ.
Or identifiable phenotypes such as other antibiotic resistance markers. Insertion of the cassette into the viral genome leads to either activation of an identifiable phenotype or deletion of that phenotype from the viral genome in the virus, thereby providing a means for selecting said transformed virus. Is desirable.

The cassette used in the present invention can be inserted into the CMV genome at any position that does not interfere with virus growth and replication. Preferably, the cassette is inserted into the CMV genome within or between the IE genes in the selected CMV. By inserting the cassette into one or more IE regions in the viral genome, the insert does not interfere with viral growth and replication. For example, in a mouse, I
Deletion of part or all of the E-2 gene does not appear to significantly interfere with MCMV growth or replication patterns. However, in humans, deletion of that gene causes HC
The MV replication characteristics change significantly. To the extent that a cassette is inserted between IE genes in the viral genome, the insert does not interfere with viral growth or replication. In mice, this could be between the genes IE-1 and IE-2 or between the genes IE-2 and IE-
3 can be achieved by inserting the cassette anywhere.
In humans, this can be achieved by inserting the cassette either between the genes IE-1 and IE-2 or after the IE-2 gene.

The designation “promoter” herein is understood in the broadest sense and includes:
Transcriptional regulatory sequences and other regulatory elements of the classical genomic gene containing the TATA box that require accurate transcription initiation in eukaryotic cells with or without CCAAT box sequences (ie, upstream activation sequences, enhancers, and silencers) Is included. The promoter may also lack a TATA box motif, but may contain one or more "initiator" or, in the case of a yeast derived promoter sequence, one or more "upstream activator sequences" or "UAS" Contains elements. For prokaryotic (eg, bacterial) expression, the promoter will include at least the -35 box and -10 box sequences.

[0028] Usually, the promoter is located upstream (ie, 5 ') of the heterologous DNA sequence and its expression is regulated. In addition, regulatory elements, including promoters, are usually
It is located within 2 kb from the transcription start site of the gene.

In the context of the present invention, the term “promoter” is used to describe a synthetic or fusion molecule, or derivative, that confers, activates, or enhances the expression of a heterologous DNA sequence in a cell. Preferably, the promoter can include additional copies of one or more specific regulatory elements that further promote expression of the gene and / or alter spatial and / or transient expression.

Placing a heterologous DNA sequence operably under the control of a promoter sequence means that
It means placing the sequence such that its expression is controlled by the promoter sequence. In the construction of heterologous promoter / structural gene combinations,
It is preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between the promoter and the gene controlled in its native location (ie, the gene from which the promoter is derived). As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the position of a regulatory sequence element with respect to a heterologous DNA sequence placed under its control is preferably defined by placing the element at its natural location (ie, the gene from which it is derived). In addition, there are also some changes in this distance, as known in the art.

By way of example, suitable promoters for use in regulating the expression of a heterologous DNA sequence include:
Includes promoters from viruses, bacteria, yeast, insects, animals, and plants. Preferably, the promoter is capable of conferring expression on eukaryotic cells (particularly mammalian cells). Promoters can regulate gene expression that participates in the organization in which expression occurs, or in differentiation, or relates to the stage at which expression occurs, or responds to external stresses such as environmental stress or hormones, etc. .

It will be appreciated by those skilled in the art that the choice of promoter will depend on the cell to be transformed and the nature of the heterologous DNA sequence to be expressed. One of skill in the art can readily identify a minimal combination of promoter and operator in the cell type in which the method of the invention is performed.

In a particularly preferred embodiment, the promoter is a viral, mammalian, bacterial, or bacteriophage promoter sequence. In a further preferred embodiment of the invention, the promoter is a CMV promoter sequence, more preferably a CMV-IE promoter sequence, or an SV40 promoter sequence, especially the SV40 late promoter sequence. These and other promoter sequences suitable for gene expression in mammalian cells are well-known in the art.

Thus, in one embodiment, the present invention relates to a recombinant cytomegalovirus, wherein at least one HCMV IE- adjacent to at least one DNA sequence.
A cytomegalovirus genome comprising a gene cassette comprising one promoter and a polyadenylation termination sequence in the (IE) gene region of said virus genome;
1) comprising a recombinant cytomegalovirus, wherein the HCMV IE-1 promoter is configured to express the DNA sequence, and (2) the DNA sequence and the polyadenylation termination sequence are heterologous to the virus.

Although the cassette used in the present invention possesses its own promoter, it need not include other regulatory elements that promote gene expression (eg, enhancers). If the cassette does not contain such a regulatory element, the cassette
Preferably, the endogenous viral regulatory elements are inserted into the viral genome via the HCMV IE-1 promoter in a manner that ensures that the expression of the heterologous DNA sequence in the cassette is affected. By way of example, high levels of construction expression are MCMV
This can be achieved by inserting the HCMV IE-1 promoter adjacent to the IE-1, IE-2, IE-3 enhancers. In a preferred embodiment of the invention, the expression cassette contains all of the regulatory elements that promote expression via the HCMV IE-1 promoter. Such elements are well-known to those skilled in the art. Techniques used to insert such sequences into viral vectors and other techniques in viral DNA (eg, inserting linker sequences, etc.)
Is well known to those skilled in the art. Accordingly, certain disclosures (heterologous DNs) contained herein may be used.
Construction of a CMV vector suitable for expression of the A sequence) is within the skill of the art.

[0036] To maximize the stability and effective termination of the message encoded by the heterologous DNA sequence, the cassette also includes a heterologous polyadenylation termination sequence. One skilled in the art will recognize that any polyadenylation termination sequence can be used in the present invention. The polyadenylation termination sequence may be derived from an SV40 polyadenylation termination sequence, human or bovine growth factor p [A], a retrovirus 3 'LTR, any other long-lived hormone (eg, cytokine) polyadenylation termination sequence. desirable.

According to one aspect of the invention, the heterologous DNA sequence encodes at least one polypeptide. As used herein, the term "polypeptide" is within the scope of the invention, as well as parts of polypeptides, such as peptides, oligopeptides, as well as complete or substantially complete proteins.

The heterologous DNA sequence used in the cassette can encode some or all of any native or recombinant protein that is heterologous to the CMV species from which the recombinant virus is formed. For example, when expressed in a protein, the heterologous DNA sequence can be used to form a polypeptide consisting of a plurality of antigenic and / or immunogenic peptides linked together in such a way that each peptide retains its immunological identity. Can be code. Alternatively, the heterologous DNA sequence is, for example, a ZP protein,
A malaria surface antigen, β-galactosidase, one or more of the major antigenic virus antigens (eg, HA from influenza virus),
In the case of human immunodeficiency virus (HIV), the heterologous DNA sequence is HIV gp 1
DNA sequences of 20 and / or HIV gag proteins, eukaryotic polypeptides (eg, mammalian polypeptides), enzymes (eg, chymosin or gastric lipase), enzyme inhibitors (eg, tissue inhibitors or metalloproteinases). TIMP)), hormones (eg, growth hormone), lymphokines (eg, interferon), cytokines (eg, interleukin-2, IL-4, IL-6, etc.), chemokines (eg, macrophage inflammatory protein-). 2), a plasminogen activator (e.g., tissue plasminogen activator or pro-urokinase), or a dual-acting key such as a natural, modified, or chimeric immunoglobulin or antibody-enzyme or antibody-toxin chimera. Fragments thereof, including melanoglobulin, can be encoded.

In a more preferred embodiment of the present invention, the heterologous DNA sequence comprises one or more CMV
It encodes either a ZP protein from an animal species specific for the virus or a viral antigen such as HA from an influenza virus.

The zona pellucida is the extracellular matrix surrounding growing oocytes and ovulated eggs. Antibodies to these proteins have been shown to have a contraceptive effect in some animals. For example, if MCMV is used as the viral vector into which the cassette will be inserted, the heterologous DNA sequence may contain one or more mouse ZP1, ZP2
, Or a ZP3 protein. Desirably, the DNA sequence encodes ZP3, which is required for immunocontraception in mice.

A heterologous DNA sequence can encode only a single polypeptide sequence in a cassette, but can be ligated with a plurality of heterologous DNA sequences and inserted into the cassette to produce a plurality of polypeptides. One skilled in the art will recognize what can be done. Such a polypeptide can be produced as a fusion protein, or can be manipulated in such a manner to obtain two different polypeptide sequences. When the polypeptides are fused, it is preferred that at least one polypeptide sequence is capable of membrane binding. In such an environment, the polypeptide is preferably linked to a polypeptide expressed on the cell surface. For example, the polypeptide can be a transferrin receptor (eg, a human transferrin receptor) or any other polypeptide sequence that has a native membrane-bound anchoring sequence. For example, hemagglutinin and ZP-3 have their own anchoring sequences.

For example, when a CMV cassette is engineered to express multiple antigens (protective antigens) along with cytokines or other immuno-immunomodulators to enhance an immune response, multiple expression of the polypeptide also occurs. Can be useful. Alternatively, when the expression vector is used to deliver an immunogenic polypeptide to a host to achieve a therapeutic effect against a particular type of viral infection, the cassette is used to correlate with a range of epitopes that contribute to T cell activity. An immunogenic polypeptide can be encoded. In such an environment, the cassette may contain a T helper cell response or cytotoxic T cells (
Preferably, it encodes an epitope capable of eliciting either a CTL) response or both.

In addition to the above, the heterologous DNA sequence can also encode one or more proteins that act to promote the effect of the expressed protein. For example, ubiquitin binding of a viral protein expressed from a DNA vector promotes induction of cytotoxic T lymphocytes and antiviral protection after immunization. Thus, in a preferred embodiment of the invention, the heterologous gene sequence encodes a ubiquitin associated with the protein to be expressed, and the resulting fusion of the protein with the proteosome for efficient processing and the MHC class I complex Uptake can be targeted.

Infection by CMV causes downregulation of cellular MHC class I heavy chains.
ation). In this context, down-regulation relates to a reduction in MHC class I heavy chain synthesis, stability, or surface expression. Recent studies have shown that IE polypeptides are more efficiently presented by interferon gamma-treated HMCV-infected cells than by uninfected cells. Interferon gamma is
It is a factor that increases the surface expression of MHC class I protein. Therefore, CMV
Increased expression of class I heavy chains in infected cells is due to polypeptide IE-specific CTL
Is preferred for effective production of Thus, in a preferred embodiment of the present invention, those genes capable of down-regulating the major histocompatibility complex in CMV are preferably deleted from the recombinant virus. This can be accomplished using the method described in US Pat. No. 5,720,957 [ii], which is hereby incorporated by reference. The patent teaches a method for identifying gene sequences that can down-regulate the major histocompatibility gene complex and a method for deleting the identified gene sequences from the cytomegalovirus genome.

The present invention also provides a prophylactic or therapeutic substance comprising a recombinant viral vector prepared according to the present invention in a pharmaceutically acceptable excipient. In a preferred aspect of the invention, the pharmaceutically acceptable excipient may also include one or more additives, adjuvants, stabilizers, or other similar substances. This type of medicinal product should be prepared and used by standard techniques known in the art.

Preferably, the prophylactic or therapeutic agent comprises a pharmaceutically acceptable excipient and an effective immunizing amount of a recombinant CMV prepared according to the present invention. Effective immunizing amount of recombinant CMV
Can be identified by techniques well known in the art.

The present invention also provides a) a recombinant CM as described herein in a suitable host cell system.
A method for producing a prophylactic or therapeutic substance, comprising the steps of: growing V; and b) mixing an effective immunizing amount of the obtained virus with a pharmaceutically acceptable additive.

[0048] Host cell lines that are believed to be useful in the methods of the present invention can be immortalized and support CMV replication without significant reduction in growth rate or protein production, ie, Any eukaryotic cell line that can survive multiple passages (eg, 50 generations or more) is included. Useful cell lines are also easily transfected, can stably maintain intact foreign RNA, and contain the cellular components required for efficient transcription, translation, post-translational modification, and secretion of proteins. Should have. Presently preferred cells are those that require simple media components and are adaptable to suspension culture. Most preferred are mammalian cell lines that can accommodate growth in low serum or serum-free media locations. Representative host cell lines include human fibroblasts (for human cells) or fibroblasts from species specific to the CMV vector, immortalized NIH3T3 cells, E6 / E7 immortalized cells, astrocytomas Cell lines (such as U373-MG), stromal cells, and macrophage-like cell lines are included. Other useful cells and cell lines are American Ty.
pe Culture Collection (ATCC), Rockville
e, Md. Or European Collection of Anima
l Cell Culture, Porton Down, Salisbury
SP40JG, U.S.A. K. Can be obtained from

Regarding the transfection treatment used in the practice of the present invention, the method of transferring a viral vector into cells includes CaPO ₄ co-precipitation, electroporation,
It is intended to include, but is not limited to, DEAE-dextran mediated uptake, protoplast fusion, microinjection, and lipofusion. Furthermore, the present invention contemplates either simultaneous or sequential transfection of host cells with a vector containing the RNA sequence to be integrated into the genome.

[0050] Recombinant CMV can be purified by any protein purification method known in the art. Purification can be achieved, for example, by techniques such as salt fractionation, chromatography on ion exchange resins, affinity chromatography, and centrifugation. For example, for methods of purifying various proteins,
method in Enzymology or Current Protocol
ols in Protein Chemistry [iii] [iv]. Preferably, the protein is purified by a combination of gradient sucrose and cesium chloride gradient centrifugation using methods described in the literature.

[0051] Methods of administering CMV particles to an uninfected individual or infected patient are well known to those of skill in the art.
However, the selection method for the most effective response needs to be identified empirically, and is described below by way of example, without limiting the delivery method.

Typically, therapeutics are prepared as either injectable liquid solutions or suspensions, and solid forms suitable for solution in, suspension in, pre-injection also can be prepared. . Alternatively, the preparation can be emulsified or the particles can be encapsulated in liposomes.

It is preferred to formulate the therapeutic agent with the viral particles together with excipients that are pharmaceutically acceptable and compatible with the active ingredient.

By way of example, additives that can be used in such formulations include water,
Physiological saline, ethanol, dextrose, glycerol, and the like, and combinations thereof. In addition, if desired, the virus-like particle formulations may also contain minor amounts of auxiliary substances such as adjuvants, wetting agents, pH buffering agents, or emulsifiers, which enhance vaccine efficacy. Suitable adjuvants that can be included in such formulations include, for example, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3) -Hydroxyphosphoryloxy) methylamine (MTP-PE) and three components extracted from bacteria, monophosphoryl lipid A trehalose dimycolase, and the cell wall skeleton (MPL + TDM + C
WS).

The virus particles can also be formulated into a therapeutic agent in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free radicals of the peptide) and inorganic acids (eg, hydrochloric or phosphoric acid) or organic acids (acetic, oxalic, maleic, etc.) Salt is included. Salts formed with free carboxyl groups can also be used with inorganic bases (eg, sodium, potassium, ammonium, calcium,
Ferric hydroxide etc.) can be derived from organic bases (isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine etc.).

The viral particle formulation can also be administered in a manner compatible with the dosage formulation, and in such amount as to have a prophylactic and / or therapeutic effect. The amount of virus particles administered will generally depend on the regulatory elements involved in the half-life of the cassette and polyadenylation site. In addition, the dosage will also depend on the subject being treated, the ability of the subject's immune system to respond, and the degree of protection desired. Precise amounts of active ingredient required for administration depend on the judgment of the practitioner and may be specific to each subject.

The formulation may be administered intradermally, subcutaneously, or intramuscularly, or orally, by aerosol,
It can be administered by other routes, including parenteral, intravenous, intraperitoneal, rectal, or vaginal administration. For example, the virus-like particles can be administered by parenteral (eg, subcutaneous or intramuscular) injection. All of the above formulations are commonly used in the pharmaceutical industry and are known to appropriately recognized practitioners.

For oral administration, the viral particles can be delivered with a diluent (water, saline, etc.) and / or delivery vehicles (tablets, capsules) that do not interfere with the activity of the particles. Oral formulations can include excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such formulations may also take the form of solutions, suspensions, tablets, pills, capsules, depot preparations or powders.

Rectal or vaginal administration also requires special formulation in an acceptable form including lubricants and / or emulsifiers. For example, such formulations typically include traditional binders and carriers such as polyalkylene glycols or triglycerides.
Is included.

Further, the therapeutic agent may be administered on a single dosing schedule, preferably on a multiple dosing schedule. A multiple dose schedule is one in which the first route of delivery is administered in, for example, 1 to 10 separate doses, followed by the time interval necessary to maintain and / or enhance the immune response (eg, 1 to 10 doses). Four months later, if necessary,
(Months later). The dosage regimen will also determine, at least in part, the required interval, which is left to the discretion of the practitioner.

Further features of the present invention are described in more detail in the following figures and examples.

EXAMPLES Further, features of the present invention will be described in more detail in the following Examples. It should be understood that the following examples are intended to be illustrative only of the present invention, and are not intended to limit the foregoing broad description in any way.

Cells Primary mouse embryonic fibroblast (MEF) cultures were prepared by trypsin dispersion of 15-day-old embryos from BALB / c mice [V], and 10% (v / v) bovine neonatal serum Were grown in minimal essential medium (MEM) containing The MEF culture was grown at 37 ° C.
It was incubated with 5% CO _2.

Virus The K181 MCMV used throughout this study was a wild-type virus. O
The parent MCMV used for the construction of VA recombinant MCMV was purchased from Prof. Ed. Moca
r181, K181 (smith) MCMV from Stanford USA
RM427 + lacZ. This virus was constructed by inserting it into the HpaI site of the IE2 gene with respect to the K181 (Smith) strain of MCMV containing the lacZ gene. The construction and characterization of the RM427 recombination is described in Mning et al.
1992 [vi]. This virus contains the lacZ gene under the transcriptional control of the human CMV immediate-early promoter-enhancer (positions -219 to -19 compared to the transcription start site). RM427 + rescues the sgg1 gene. The presence of sgg + 1 was confirmed by PCR. All viruses used in this study were grown in MEF tissue culture in cell culture medium supplemented with 10% newborn calf serum.

[Mice] Five- to six-week-old female BALB / c mice were subjected to Animal Resources.
Center, Murdoch, Western Australia, Aus
obtained from tralia. Mice were maintained under SPF conditions.

[Plasmid] Plasmid cloning was performed by a standard method [vii]. All restriction enzymes were obtained from commercial suppliers and used according to the manufacturer's instructions using the supply buffer. Recombinant plasmids designed to replace the lacZ and OVA genes in the HindIII L fragment of RM427 + were constructed as described.

The pGEM11Zf (+) plasmid carrying the MCMV K181 HindIII L fragment at the HindIII site was cloned into Scalzo et al., Depar.
tment of Microbiology, University of
Western Australia, Western Australia,
Obtained from Australia (see FIG. 1). With this plasmid,
pK181-H3L was designed (see FIG. 5). The pBlueRIP-TfROVA plasmid containing the OVA gene with the transferrin receptor cytoplasmic tail sequence (TfR) fused to the amino terminus is described in Prof. F. Carbo
ne, Monash University, Melbourn, Victor
ria, contributed favorably by Australia. T to OVA gene
Fusion of the fR sequence confirms expression of the OVA protein on the cell membrane of infected cells. The TfROVA sequence was excised from pBlueRIP-TfROVA with HindIII and made blunt ended by a transient reaction with Klenow DNA polymerase. The resulting fragment was ligated into the blunt-ended site of the Smal site of pMV11 located between the human CMVIEl promoter and the polyadenylation termination sequence (see Figures 2 and 3). The HindIII fragment containing the regulatory elements and TfROVA was further cut to blunt ends,
The TATA box and transcription start site of the IE2 gene are removed by insertion into the two HpaI sites of the HindIII L fragment on pK181-H3L (see FIG. 4). With this plasmid, pK181-H3L / M
V11 / OVA / TfR was designed (see FIG. 6A).

[Construction of Plasmid Containing Surrogate Gene] [Plasmid Containing Mouse ZP-3] (See FIG. 6B) Plasmid pUC19 Containing Mouse ZP-3 cDNA
/ ZP-3 with Dr Ron Jackson VBCRC, Canberra
, ACT, Australia. CDNA encoding ZpP-3 is
Excision was performed using the commercially available restriction endonucleases Bam HI and Eco RI. The cDNA was then transferred to the BamHI / EcoRI of the pMV11 vector.
Site and ligated to the competent E. coli using this DNA. coli bacteria were transformed. After transformation, putative clones were screened and clones were evaluated by standard molecular biology quality control methods. CDN encoding ZP-3
The expression cassette containing A was excised from pMV11 using HindIII, the fragment was blunt-ended using DNA polymerase (Klenow), and the blunt-end ligated to the pK181-H3L plasmid. pK181-H3L
The plasmid was first completely digested with the restriction enzyme HpaI and the larger fragment was purified by standard techniques such as ethanol precipitation, agarose gel electrophoresis, and gel purification. Again, the ligation product is transferred to competent E
. E. coli and putative clones were evaluated by standard molecular biology techniques. The resulting plasmid DNA (pK181-H3L containing ZP-3) was extracted and purified and used for a co-transfection reaction.

The plasmid pZP-3 / 1 containing the ZP-3 cDNA was extracted from the ovaries of BALB / c mice, using random priming and DNA synthesis, primers containing restriction sites and sites specific for ZP-3. CDN amplified by PCR
Obtained by A. The resulting DNA was cloned into pUC19 and the DNA sequenced for confirmation. The cDNA encoding ZP-3 was excised using the commercially available restriction endonuclease BamHI / EcoRI. The cDNA is then:
The DNA was ligated to the BamHI / EcoRI site of the pMV11 vector,
Using competent E. coli bacteria were transformed. After transformation, putative clones were screened and clones were evaluated by standard molecular biology quality control methods. An expression cassette containing the cDNA encoding ZP-3 was inserted into Hind.
The fragment was excised from pMV11 using III and the fragment was blunt-ended using DNA polymerase (Klenow) and the blunt-end ligated to the pK181-H3L plasmid. The pK181-H3L plasmid was first transformed with the restriction enzyme Hp
Digested completely with aI and the larger fragment was purified by standard techniques such as ethanol precipitation, agarose gel electrophoresis, and gel purification. Again, the ligation product was transferred to competent E. coli. E. coli and putative clones were evaluated by standard molecular biology techniques. Obtained plasmid DN
A (pK181-H3L containing ZP-3) was extracted and purified and used for the co-transfection reaction.

A plasmid containing influenza virus (PB8) hemagglutinin (FIG. 7 (
See i)). Influenza virus (PB8) hemagglutinin cD
Plasmid pKG4 containing the NA. Bernadette Scott
, University of Western Australia, Wes
obtained from tern Australia, Australia. The cDNA encoding HA was obtained from the commercially available restriction endonucleases Bam HI and Bgl II.
It cut out using. The cDNA was then transferred to the BamHI vector of the pMV11 vector.
Site and ligated to the competent E. coli using this DNA. coli bacteria were transformed. After transformation, putative clones were screened and clones were evaluated by standard molecular biology quality control methods. The expression cassette containing the cDNA encoding HA was excised from pMV11 using HindIII, the fragment was blunt-ended using DNA polymerase (Klenow), and the blunt-end ligated to the pK181-H3L plasmid. The pK181-H3L plasmid was first completely digested with the restriction enzyme HpaI, and the larger fragment was purified by standard techniques such as ethanol precipitation, agarose gel electrophoresis, and gel purification. Again, the ligation product was transferred to competent E. coli. c
oli, and putative clones were evaluated by standard molecular biology techniques. The resulting plasmid DNA (pK181-H3L containing HA) was extracted and purified and used for a co-transfection reaction.

[Plasmid containing mouse IL-6] (see FIG. 7 (ii)). Plasmid p containing mouse IL-6 cDNA
CD-mlL6 was added to Dr. Alistair Ramsay, Austral
ian National University, Canberra, Aus
obtained from tralia. The cDNA encoding IL-6 was excised using the commercially available restriction endonuclease Nla IV. The cDNA was then transferred to pMV11
The vector was ligated to the SmaI site of the vector, and this DNA was used to competent E. coli. coli bacteria were transformed. After transformation, putative clones were screened and clones were evaluated by standard molecular biology quality control methods. IL-
The expression cassette containing the cDNA encoding No. 6 was converted into pMV using HindIII.
The fragment was excised from 11 and the fragment was blunt-ended using DNA polymerase (Klenow) and the blunt-end ligated to the pK181-H3L plasmid. The pK181-H3L plasmid was first completely digested with the restriction enzyme HpaI, and the larger fragment was subjected to ethanol precipitation, agarose gel electrophoresis,
And purified by standard techniques such as gel purification. Again, the ligation product was transferred to competent E. coli. E. coli and putative clones were evaluated by standard molecular biology techniques. Obtained plasmid DNA (p containing IL-6)
K181-H3L) was extracted and purified and used for the co-transfection reaction.

Isolation of MCMV DNA Infected MCMV DNA was prepared from RM427 + grown in tissue culture and extracted from both supernatant and infected cells by protein kinase digestion, phenol / chloroform extraction and ethanol precipitation.

[Construction of Recombinant Virus] Recombinant MCMV was added to 10 to 50 μg of MEF cells at 60% to 70% confluence.
K181-RM427 + DNA and 2 μg of the HindIII linearized transfection plasmid. Cells were washed with calcium phosphate co-precipitation (C
elfect, Pharmacia) and the reaction was allowed to proceed for 7 hours, osmotic shock. The transfected MEF monolayer is incubated for 4 days, sonicated, and the supernatant is returned to a fresh confluent MEF monolayer. The monolayer was coated with carboxymethylcellulose (CMC) during the development of the viral plaque. Six days later, potential recombinants were selected by the unstained plaque phenotype after X-gal addition. Remove half of CMC coating and replace with X-gal CMC,
The color reaction was allowed to proceed at 37 ° C. for 24 hours. Take out the transparent plaque, Mann
Two rounds of plaque purification as described in ing and Mocarski, 1988 were performed to remove all contained parental RM427 + [viii]. This was confirmed by screening MEF monolayers containing more than 1000 plaques using X-gal staining of the killed bacteria. Briefly, MEFs were fixed in 0.5% glutaraldehyde, permeabilized, and ^placed in a solution containing Fe ²⁺ / Fe ³⁺ with X-
gal was added and blue plaques were screened. Thereafter, a high titer tissue culture growth virus shock of OVA recombinant MCMV was prepared.

The OVA recombinant MCMV (rMCMV-TfROVA) was converted to RM427 + DNA
Generated by co-transfection of pH3L / MV11 / TfROVA with IE3
Recombinant MCMV (rMCMV-IE2) in which a part of the gene was deleted was transformed into RM427.
+ DNA and co-transfection of pK181-H3L IE2,
The cZ-containing revertant (rMCMV-TfROVA-REV) was transformed with rMCMV-T
Created by co-transfection of fROVA DNA with pON427. The outline is shown in FIG. 7B.

[PCR] After immunostaining, putative recombinant virus plaques were treated with 1% sarkosyl and 1%
The cells were digested with TE containing 00 μg / ml proteinase K, incubated at 56 ° C. for 2 hours, extracted with phenol / chloroform, and the DNA was precipitated with ethanol. This DNA was resuspended in ddH ₂ O and used for the PCR reaction to confirm the presence of the insert of the predicted size in the IE2 region. Primers were used for virus IE2
It was designed to be adjacent to the gene cloning site. Forward primer Im2
AF is CATTAAAAAACTATTGGTTCTA and reverse primer Im2AR is CCCATAGCCGAGCCCAATGCA. The expected size of lacZ contained in the virus was 4.1 kb, the recombinant MCMV clone containing the TfROVA construct was 2.6 kb, and the IE2 construct had a 79 bp difference compared to wild-type MCMV.

The primers used to confirm the intact sgg1 gene are shown below. Forward primer H3JSG1F: ACAAGAGTCTGTCCGACCAC and reverse primer H3JSG2R: GCGGTACGTATACTGCCCGT
TA. These are MCMV H3s that span a potential 323 bp deletion of RM427.
Amplify the 843 bp fragment within the J fragment. For all PCR reactions, the Geneamp kit (perkin Elmer) was used according to the manufacturer's instructions. The template was amplified in a 2400 PCR instrument for 30 cycles (94 ° C. for 30 seconds, 52 ° C. for 30 seconds, 72 ° C. for 45 seconds) and the product was amplified to 0.
Analyze at 9% AGE.

RFLP Analysis and Southern Blot Analysis Viral DNA was digested with the restriction endonuclease HindIII for 3 hours,
Fragments were analyzed by pulse field electrophoresis on a 1.0% gel (CHEF, BioRad). After electrophoresis, the DNA fragment was subjected to Manni
Nylon membrane (Hybond) as described in atis et al., 1989 [i].
N, Amersham). Probes were generated by PCR of the IE1 and IE2 genes using the PCR primers described above. OVA-specific probes were generated by excising the OVA fragment from pBlueRIP-TfROVA. Probes were labeled with ³² P via random primer synthesis.

[In Vitro Propagation of Recombinant MCMV] MEF cells were isolated from rMCMV-TfROVA, RM427 +, rMCMV-Tf
ROVA-REV, rMCMV IE2, K181-HA, K181-HA-R
Infections were carried out at a multiplicity of 5 using EV and K181 MCMV. One hour after the first incubation, the cell monolayer was washed with 40 mM citrate, 10 mM KC
1 and citrate buffer (pH 3.0) containing 135 mM NaCl to remove reversibly bound particles. At specific times post-infection, virus was harvested from the monolayer and supernatant, sonicated, and virus titer quantified by plaque assay on MEF cells. The detection limit for each assayed monolayer was 10 p
fu / ml. The results are shown in FIGS. 10 (a), (b) and (c).

Generation of Hyperimmune Serum Against OVA, HA, ZP-3, and MCMV Adult BALB / c mice were immunized i.v. with OVA-adjuvant emulsion. p. Two consecutive boosts were obtained every two weeks after inoculation. The whole blood was collected (heart puncture), allowed to clot at room temperature for 1 hour, and then centrifuged for 1 minute. Serum until use -2
Stored at 0 ° C.

A similar experiment was performed to generate hyperimmune sera against ZP-3 and HA. HA hyperimmune serum was produced from mice infected with influenza PR8, and ZP-3 hyperimmune serum was raised against purified B cell epitopes in ZP-3.

Detection of OVA expression by recombinant MCMV. OVA expression by the recombinant virus was measured by immunostaining viral plaques infected with the recombinant virus using hyperimmune sera raised in mice against purified OVA protein.

The expression of OVA by rMCMV-TfROVA was confirmed by in vitro immunostaining with anti-OVA hyperimmune serum. 50% virus-infected cells
/ 50% methanol / acetone, blocking with normal mouse serum, then phosphate buffered saline (MO
Primary antibody in BS) was added. After 1 hour incubation at 37 ° C., the primary antibody was removed, the monolayer was washed with MOBS, a secondary alkaline phosphatase-conjugated goat anti-mouse antibody was added and incubated as above. Add the substrate,
The presence of stained plaques was observed and recorded.

The enzyme immunoassay (ELISA) used was a modification of the method described in [ix]. For detection of anti-MCMV antibodies, plates were prepared using MCMV prepared by infecting MEF monolayers with MCMV until almost 100% CPE was observed.
Coated with antigen, the cultures were clarified by centrifugation at 4500 g for 20 min at 4 ° C. and ultracentrifuged at 30000 g for 2 h at 4 ° C. [I] The virus pellet was resuspended in MOBS and the antigen was titrated for optimal dilution.
Plates were coated overnight with antigen in carbonate / bicarbonate buffer (pH 9.6) at 4 ° C. Primary antibody was added at 1/10 dilution and serial doubling dilutions of serum were made with 10% bovine serum albumin (BSA, CSL) and 0.05% Tween 20 (S
igma). The secondary antibody used was a biotinylated goat anti-mouse anti-IgM, IgG1, or IgG2a (Southern Biotec).
hnology, Birmingham, USA). Streptavidin alkaline phosphatase (Pierce, USA) was added in a further step. The substrate solution was prepared by adding 5 mg of p in 5 ml of diethanolamine buffer (pH 9.8).
-Nitrophenyl phosphate tablets (Sigma). The reaction was washed after 20 minutes with BioRad Model 3550 Microplate.
reader (BioRad, Hercules, California, US)
405 nm was read using A). All incubation steps were performed at 37
C. for 1 hour. Absorption values (y-axis) were plotted against the log of the dilution factor (x-axis). Serum titers are shown as the values above for the intersection of the linear region of the curve with NMS + 3 standard deviations with the x-axis and as the reciprocal of the dilution. ELISA for detection of anti-OVA or anti-β-galactosidase antibody was 1.0 μg each.
Performed as above using plates coated with / VA OVA (grade VI, Sigma) or 0.5 μg / ml β-galactosidase.

[Immunofluorescence Microscopy] Eight-well glass microscope slides (LAB-TEK, Nalge Nun)
c International), rMEMV-TfROV
Infected with A or K181-WT for 4 hours, washed with MOBS / 1% FCS, and fixed with methanol / acetone (50% / 50%) for 2 minutes. The fixed cells are
Washed with OBS, 1% FCS and at room temperature, 10% normal mouse serum, MO
Blocked in BS, 1% FCS for 30 minutes and washed again with MOBS, 1% FCS. This was followed by a 45-minute reaction time with the primary antibody (MOBS, 1% F
Rabbit anti-OVA polyclonal antibody (ICN Bi) diluted 1/100 in CS
medicals, Inc)). Again, single layer MO
The cells were washed with BS, 1% FCS and incubated with anti-rabbit immunoglobulin biotin conjugate (Silenus) diluted 1/200 with MOBS, 1% FCS for a reaction time of 1 hour. The third incubation step consists of MOBS, 1
Fluorolink with a reaction time of 1 hour diluted 1/1000 with 1% FCS
Incubate with Cy3-labeled streptavidin (LIFESCIENCE), then wash with MOBS, 1% FCS, and embed in MOBS, 50% glycerol. Monolayers were examined by immunofluorescence microscopy using a Leitz Wetzlar microscope.

[Detection of ZP-3 Expression Expression by Recombinant MCMV] ZP-3 expression was infected with a recombinant virus using hyperimmune sera raised in mice against purified B cell epitopes. It was determined by immunostaining of viral plaques.

[Detection of Expression of HA Expression by Recombinant MCMV] Influenza virus HA was expressed in a virus plaque infected with the recombinant virus using hyperimmune serum produced in mice infected with influenza virus PR8. Measured by immunostaining.

[Detection of Expression of IL-6 Expression by Recombinant MCMV] Serial dilution of supernatant of cell culture in which indicator cells dependent on the presence of IL-6 are infected with IL-6 containing recombinant MCMV is performed. The expression of IL-6 was measured in the assay. The result is shown in FIG.

In Vivo Growth of Recombinant MCMV Three-week-old female BALB / c mice were grown in 2 × 10 ⁴ pfu tissue culture (TCV
) MCMV was inoculated intraperitoneally (ip). 3, 7, 14, 28, and 42 of infections
On day three mice were sacrificed per hour and salivary glands, spleen, and liver were collected. Similarly, IFNα / β and γ receptor knockout mice (129Sv
(Ev)) and control mice with 2 × 10 ⁴ pfu TCV MCMV
Was inoculated. Weigh each organ, homogenize in 2 ml MEM + 2% NCS,
Centrifuged at 2000g for 20 minutes at 4 ° C and stored at -70 ° C until use. [X]
Plaque assays were performed on MEFs using 4-fold serial dilutions of these homogenates, as described in. Cultures were grown at 1.75% methylcellulose (Si
gma) and left at 37 ° C. with 5% CO ₂ for 4 days. Plaques were treated with 24% in 0.5% (w / v) methylene blue containing 10% formaldehyde.
Counting was performed after time staining. Organ virus titers were expressed as pfu / g organ. The limit of detection of virus in these assays is 200 pfu / salivary gland or spleen.
g and 20 pfu / g in the liver.

[0089] RM427 + was used as a control. Scalzo et al., 1990 [x
The plaque assay was performed as described under [i].

2 × 10^FourIn BALB / c mice inoculated with pfu TCV, replicated K
181-OVA / TfR is an organ that maintains infectivity against MCMV, saliva
It was not detected in glands (see FIG. 8). The virus was
Only was detected. However, K181-OVA / TfR was not used at higher doses (5 × 10 ^Four pfu TCV) i. p. Non-fluorescent RM427 + during the initial infection time of inoculation
It was replicated in viscera at levels comparable to MCMV (see FIG. 9). K18
1-OVA / TfR is 10⁸BALB / c administered with pfu TCV ip
It was not detected by plaque assay in the salivary glands of mice. Liver and
5x10 for in vivo replication in the spleen^Four3 days after infection using pfu
And 5 days later the organs were harvested.

BALB mice were given 2 × 10 ⁴ pfu of K181-OVA / TfR or K1
81 wild-type (wt) were inoculated. Spleens were harvested after infection, restimulated in vitro and used in CTL assays against K181-wt infected mouse embryonic fibroblast (MEF) targets. These experimental data are shown in FIG. 11 and FIG.

FIG. 13 shows that 2 × 10 ⁴ pfu of tissue culture derived (MEF) recombinant virus was ip
1 shows a summary of the in vivo replication of recombinant MCMV containing the different gene cassettes described above in inoculated BALB / c mice.

[ELISA] For detection of anti-MCMV antibodies, plates were coated with MCMV and MEF monolayer 9
Prepared by infection with 0% CPE, cultures were clarified by centrifugation at 3000 RPM and ultracentrifugation at 40,000 RPM for 3 hours. The virus pellet was resuspended in PBS and used to coat the plate. Primary antibody (1
Time, washing), secondary antibodies conjugated with either IgM / IgG or anti-IgG1 or IgG2a. Serum titers are shown at the intersection of the linear regions of the curve with MNS + 3SD and shown as the reciprocal of the dilution.

[0094] Either anti-OVA or anti-β-galactosidase was measured. Plates were dissolved at 1.0 mg / ml or 0.5 mg / ml in alkaline phosphate buffer
Coated with any of the proteins.

FIG. 14 shows 2 × 10 ⁴ pfu tissue culture-derived recombinant MCMV-bound OVA, HA
ELISA performed on sera from BALB / c mice inoculated with OVA or UV-inactivated (non-replicating) recombinant MCMV with solubility and adjuvant
2 shows a table summarizing the results obtained from the assays. Recombinant MCMV OVA or HA preferentially stimulates an immune response to a heterologous antigen of the IgG2a isotype when compared to administration of the antigen by other means, and exhibits the ability to switch a separately occurring antibody response. A serum boost of IgG2a response was detected using MCMV expressing HA
Occurs after (boost).

FIG. 15 shows the antibody isotypes (IgG1 and Ig) against ZP-3 from animals inoculated 100 days before with 2 × 10 ⁴ pfu of recombinant MCMV-ZP3 virus.
G2a) Summary of ELISA titer results.

FIG. 16 and FIG. 17 show recombinant M as measured by ELISPOT assay.
Stimulation of mucosal immune response in salivary glands induced by CMV-expressing membrane-bound OVA
Intense. Antibody-secreting cells from the cervical lymph node of BALB / c mice were ^Four RMCMV-OVA were infected ip. In this method, IgA
No secretory cells were detected on inoculation with OVA / adjuvant.

FIGS. 18 (A), 18 (B), and 18 (C) show K181-OVA / TfR replication in interferon receptor gene knockout mice. K181-OVA TfR in IFN-γR gene knockout mice
Although replication did not differ significantly from the results obtained in parenteral 129 / Ev / Sv mice, IFN-α appears to play a role in regulating viral replication in the spleen. Loss of IFN-α and IFN-γ functions significantly increased viral replication in the gut, indicating that K181-OVA / TfR is not deficient in salivary gland replication. Mice were challenged with 2 × 10 ⁴ pfu TCV ip
Inoculated.

FIG. 19 (A), FIG. 19 (B), and FIG. 19 (C) show the replication of K181-OVA / TfR in interferon α receptor gene knockout mice. Compared to the results in FIG. 18, 12 inoculated with 2 × 10 ⁵ pfu TCV
Although a dose-dependent effect was observed in 9 / Ev / Sv control mice, K181-OVA / TfR replication is detected at higher levels not only in the gut but also in the salivary glands. IFN-α plays a role in regulating K181-OVATfR replication in these organs. Parenteral RM42 in IFN-αR gko mice
7+ replication increased on the same order.

FIG. 20 shows a table summarizing the replication of MCMV-IL6 (non-membrane antigen expression) and replication of the virus tropism in salivary glands in CMV organs.

In the following experiments, BALB / c, C57 / BL6, and ARC at 6-8 weeks of age were used.
Female mice were inoculated ip with 2 × 10 ⁴ recombinant viruses containing the gene encoding ZP-3. Male mice were added 21 days after infection in the following ratios.

Uninfected, 1 male + 3 females in 3 groups (9 females), infected with RM427 +, 1 male + 3 females in 3 groups (9 females), infected with rMCMV-mZP3, 1 male 3 groups of 3 animals + 3 females (9 females).

Next, pregnancy was measured. Nine female BALB / c mice infected with rMCMV-ZP3 did not give birth 90 days after mating. In contrast, nine control female mice gave birth to 141 offspring. rMCMV-ZP3-infected female C5
7BL / 6 mice had a reduced number of births compared to controls. BALB / c
And a marked reduction in follicles in C57BL / 6 was first seen on day 7 post-infection, with C57BL / 6 recovered by day 35 post-infection. Seroconversion to MCMV by one of the three male CBA mice showed rMCMV-ZP
3 infected female mice.

FIG. 21 shows a summary and graph of data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2 × 10 ⁴ pfu virus in C57BL / 6 mice.

FIG. 22 shows a summary and graph of data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2 × 10 ⁴ pfu virus in BALB / c mice.

FIG. 23 shows a summary of data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2 × 10 ⁴ pfu of virus in outbred ARC / s mice.

FIG. 24 shows a summary of data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2 × 10 ⁴ pfu virus in wild type outbred mice.

Mice inoculated with wild-type parent MCMV and boosted twice with MCMV-ZP3 were
As shown in FIG. This figure shows the immunocontraceptive effect in wild type outbred mice.

Groups of mice were vaccinated with MCMV and challenged with a lethal dose of live influenza virus either 6 days or 21 days after infection. These experimental results highlight the protective effects of recombinant McMV-HA (see FIGS. 27 and 28).

[Alternative Method for Insertion of Heterologous DNA into CMV Vector] In order to develop a unique method for inserting a heterologous antigen into the MCMV genome, a desired gene or antigen sequence of interest can be transferred to a bacterially transportable element sequence Tn7L. And Tn
Clone into the transfer vector adjacent to 7R.

The bacterial plasmid (pCMH151) was transformed with the bacterial origin of replication, kanamycin resistance (
Km ^R ) marker gene and bacterially transportable element Tn7 (att:
Tn7) was constructed by standard cloning techniques involving the lacZ gene containing the attachment sequence. This plasmid is then digested with an appropriate restriction enzyme (NotI) to give a second plasmid (pK181H3) that is ampicillin resistant (Amp ^R ).
L-NotI) and was engineered to include a NotI restriction site (formerly an HpaI site) in the IE2 gene of the HindIII L fragment. The resulting clone (pK181H3L / pCMH151) contains pCMH151 elements flanked by MCMV sequences homologous to the IE1 and IE2 regions of the MCMV genome.

[0112] This transfer vector can be transformed into bacterial cells containing the MCMV genome that has been cloned as a bacterial artificial chromosome (BAC). In the presence of the bacterial translocation protein provided by the helper plasmid, the desired antigen sequence in the transfer vector is replaced by a specific binding site (designated on the MCMV BAC plasmid).
att: Tn7). Positive MC containing inserted antigen sequence
The MV BAC plasmid is identified by disruption of the lacZ gene associated with the att: Tn7 site.

After characterizing the clone by standard techniques, the MCMV BAC plasmid can be purified and transfected into eukaryotic cells to recover the progeny of the infectious virus containing the foreign antigen sequence.

To confirm that it is possible to replace a genetic element at the att: Tn7 site of the engineered plasmid pK181H3L / pCMH151, a model for the procedure for transposition in vitro was developed. Bacterial cells (DH10B) were translocated with plasmid pK181H3L / pCMH151. Positive transformants were identified by their bright blue color on L-agar plates containing X-gal, kanamycin, and ampicillin. Positively transformed colonies were grown in culture medium and the cells were subsequently transformed with plasmid pMON7124, a helper plasmid containing the gene for the bacterial translocation protein and the tetracycline resistance marker (Tc ^R ).

To indicate transposition, the transfer vector pFASTBA containing a gentamicin resistance marker (Gm ^R ) flanked by transposable element sequences Tn7R and Tn7L
C was transformed into host bacteria containing pK181H3L / pCMH151 and pMON7124k. Several white colonies were seen growing on L-agar containing Km, Gm, Amp, and Tc, indicating that pFASTBAC
This shows that the transposable element from the strain was inserted into pK181H3L / pCMH151 and disrupted the lacZ gene. When only host bacteria were plated on L-agar plates containing Km, Gm, Amp, and Tc, no colonies were observed.

These results indicate that transposition of the Gm ^R gene occurred at a specific binding site of pK181H3L / pCMH151, indicating that it is possible to translocate the specific binding site to the desired sequence. Is shown.

It will be appreciated by those skilled in the art that the invention described herein is subject to variations and modifications other than those specifically noted. It is understood that the present invention includes all such variations and modifications. The present invention also relates to all steps, features, compositions, and compounds described or indicated herein individually or collectively, and any and all combinations or any two or more steps or features. Including.

[References]

[Table 1]

[Brief description of the drawings]

FIG. 1 shows a plasmid and a cloning vector used for construction of a recombinant MCMV expressing the following genes. Mouse zona pellucida, chicken ovalbumin (OVA) linked to human transferrin cytoplasmic tail, influenza virus hemagglutinin and mouse interleukin-6 gene from PR8 strain containing transmembrane junction region. Plasmids containing the above genes were constructed in a similar manner as described in the Examples below, but using different restriction enzyme sites.

FIG. 2 shows the plasmid pMV11 containing the human CMV promoter and the SV40 polyadenylation sequence. Shows multiple cloning sites.

FIG. 3 shows the nucleotide sequence of the OVA / TfR construct cloned into pMV11.

FIG. 4 is a diagram showing the sequence of the MCMV IE-2 region showing the position of the HpaI restriction enzyme site excluding the TATA and AUG start regions of IE-2, into which the gene cassette has been inserted. Specific restriction sites that may be appropriate for insertion of the cassette are also highlighted.

FIG. 5 shows the plasmid pK181-H3L containing the 7.5 kb fragment of the MCMV K181 strain after being digested with the restriction enzyme HindIII and cloned into the promega plasmid pGEM11Zf.

FIG. 6A shows a restriction map and salient features of a complete plasmid containing the OVA / TfR gene for co-transfection and production of recombinant MCMV.

FIG. 6B shows a restriction map and salient features of a complete plasmid containing the ZP-3RJ gene for co-transfection and production of recombinant MCMV.

FIG. 7A (i) shows a restriction map and salient features of a complete plasmid containing the HA (PR8) gene for co-transfection and production of recombinant MCMV.

FIG. 7A (ii) shows a restriction map and salient features of the complete plasmid containing the mlL-6 gene for co-transfection and production of recombinant MCMV.

FIG. 7b shows the process and recombinant virus and vector constructs that produced recombinant MCMV.

FIG. 8 is a diagram showing the replication of K181-OVA / TfR in the salivary glands of BALB / c mice.

FIG. 9 shows the replication of K181-OVA / TfR in the internal organs of BALB / c mice.

10A-C show the replication of recombinant MCMV in tissues as measured by plaque assay.

FIG. 11: K181-OVA / TfR or K of 2 × 10 ⁴ plaque forming units (pfu)
FIG. 9 shows the results of a CTL assay of BALB / c mice inoculated with 181 wild-type (wt). Spleens are harvested after infection, restimulated in vitro, and K181-wt
Used in CLT assays against infected mouse embryo fibroblast (MEF) targets.

FIG. 12. 2 × 10 ⁴ pfu of K181-OVA / TfR or K181 wild type (wt)
FIG. 9 shows the results of CTL assay of BALB / c mice inoculated with E. coli. Spleens were harvested after infection, restimulated in vitro and used for CLT assays against K181-wt infected mouse embryonic fibroblast (MEF) targets.

FIG. 13. B inoculated ip with 2 × 10 ⁴ pfu of tissue culture derived (MEF) recombinant virus
FIG. 3 shows a summary of the in vivo replication of recombinant MCMV containing the different gene cassettes described above in ALB / c mice.

FIG. 14. 2 × 10 ⁴ pfu tissue culture-derived recombinant MCMV-bound OVA, OVA with HA solubility and adjuvant or UV-inactivated (non-replicating) recombinant MCMV
FIG. 7 is a table showing a summary of results obtained from an ELISA assay performed on serum obtained from BALB / c mice inoculated with E. coli.

FIG. 15 shows a summary of the ELISA results of antibody isotype (IgG1 and IgG2a) titers against ZP-3 from animals inoculated 100 days before with 2 × 10 ⁴ pfu of recombinant MCMV-ZP3 virus.

16A-C. Recombinant MCMV-expressed membrane-bound OVA as measured by ELISPOT assay.
FIG. 3 shows the stimulation of mucosal immune response in salivary glands induced by liposome.

FIG. 17. Recombinant MCMV-expressing membrane-bound OVA measured by ELISPOT assay
FIG. 3 shows the stimulation of mucosal immune response in salivary glands induced by liposome.

18A-C. K181-O in interferon receptor gene knockout mice.
FIG. 4 is a diagram showing replication of VA / TfR.

Figures 19A-C. K181- in interferon alpha receptor gene knockout mice.
FIG. 3 is a diagram showing OVA / TfR replication.

FIG. 20 shows a table summarizing replication of MCMV-IL6 (non-membrane antigen expression) in salivary glands and replication of affinity for virus in CMV organs.

FIG. 21 shows a summary and graph of data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2 × 10 ⁴ pfu virus in C57BL / 6 mice.

FIG. 22 shows data summaries and graphs for immunocontraception delivered by recombinant MCMV ZP3 in BALB / c mice infected with 2 × 10 ⁴ pfu virus of virus.

FIG. 23 shows a summary of data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2 × 10 ⁴ pfu of virus in outbred ARC / s mice.

FIG. 24 shows a summary of data on immunocontraception delivered by recombinant MCMV-ZP3 in mice infected with 2 × 10 ⁴ pfu virus in wild type outbred mice.

FIG. 25. Non-recombinant MCMV or MCMV-La prior to infection with MCMV-ZP3.
FIG. 3 shows a summary of data on immunocontraception in BALB / c mice infected with cZ.

FIG. 26 is a graph showing a graph of titration of IL-6.

FIG. 27. rM from lethal influenza virus 6 days after inoculation of rMCMV-HA.
It is a figure which shows the summary of the data of the protective effect of CMV-HA.

FIG. 28 shows a summary of data on the protective effect of rMCMV-HA from lethal influenza virus 21 days after inoculation with rMCMV-HA.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Commonwealth Scientific and Industrial Research Organization COMMONWEALTH SCIENT IFIC AND INDUSTRIAL RESEARCH ORGANIZAT ION Australia 2601 Australia Capital Territory Campbell Limestone Avenue No address (71) Applicant Grains Research and Development Corporation Australian Capital Territory 2601, Burton, Bly Street, Bly House, Second Floor (71) Applicant Australian National University Australian Capital Territory 2601, Canberra ( 71) Applicant Agricultural Protection Board of Western Australia Baron-Hay Court, South Perth, Western Australia, 6151, Australia (71) Applicant Executive Director of the Department Store Ment of Conservation and Land Management 6009, Western Australia, Crawley, Hackett Drive (72) Inventor Lawson, Malcolm Andrew Austra Evandale Street, Florito, Western Australia 9614, 96 (72) Inventor Van Domelen, Serani Lee Henry 6057, Western Australia, Maida Vale, Woodland Grove 9 (72) Inventor Lloyd, Megan Lewis 6053, Bayswater, Laurens Street, Western Australia, 6053, Australia (72) Inventor Sheram, Jeffrey Randolph 6009, Western Australia, Australia, Nedlands, Stanley Street 28 F-term ( (Reference) 4B024 AA01 BA26 BA80 CA03 CA04 DA02 FA02 GA11 HA12 HA15 4B065 AA91Y AA95X AB01 AC14 BA02 CA24 CA44 4C084 AA13 NA14 4C085 BA78 DD62 4C087 AA01 AA02 BC83 NA14

Claims (29)

[Claims] 1. A cytomegalovirus genome comprising a gene cassette comprising at least one promoter flanked by at least one DNA sequence and a polyadenylation termination sequence in the immediate early (IE) gene region of the virus genome,
1) a recombination site wherein the promoter is configured to express the DNA sequence; and (2) the promoter, the DNA sequence, and the polyadenylation termination sequence are heterologous to the virus into which the cassette has been inserted. Megalovirus. 2. The method according to claim 2, wherein the gene cassette is inserted into an IE region in a manner to inactivate at least one gene encoding an IE protein.
A recombinant cytomegalovirus according to claim 1. 3. The recombinant cytomegalovirus according to claim 1, wherein the gene cassette contains an identifiable phenotype. 4. Insertion of the cassette into the viral genome leads to either activation of an identifiable phenotype or deletion of that phenotype from the viral genome in the virus, thereby transforming the transformed virus. 4. The recombinant cytomegalovirus according to claim 3, wherein a selection means is obtained. 5. The method according to claim 1, wherein the gene cassette is inserted into a cytomegalovirus genome within or between the IE genes in the selected cytomegalovirus. The recombinant cytomegalovirus according to claim 1 or 2. 6. The recombinant cytomegalovirus according to claim 5, wherein the gene cassette is inserted between genes IE-1 and IE-2. 7. The recombinant cytomegalovirus according to claim 5, wherein the gene cassette is inserted after the IE-2 gene. 8. The recombinant cytomegalovirus according to claim 1, wherein the promoter is a viral promoter sequence. 9. The recombinant cytomegalovirus according to any one of claims 1 to 7, wherein the promoter is a mammalian promoter sequence. 10. The recombinant cytomegalovirus according to any one of claims 1 to 7, wherein the promoter is a bacterial promoter sequence. 11. The recombinant cytomegalovirus according to any one of claims 1 to 7, wherein the promoter is a bacteriophage promoter sequence. 12. A cytomegalovirus comprising a gene cassette comprising at least one HCMV IE-1 promoter flanked by at least one DNA sequence and a polyadenylation termination sequence inserted into the (IE) gene region of the viral genome. (1) the recombination site, wherein the HCMV IE-1 promoter is configured to express the DNA sequence, and (2) the DNA sequence and the polyadenylation termination sequence are heterologous to the virus. Megalovirus. 13. The recombinant cytomegalovirus according to any one of claims 1 to 12, wherein the polyadenylation termination sequence is derived from an SV40 polyadenylation termination sequence. 14. The recombinant cytomegalovirus according to any one of claims 1 to 12, wherein the polyadenylation termination sequence is derived from human or bovine growth factor. 15. The recombinant cytomegalovirus according to any one of claims 1 to 12, wherein the polyadenylation termination sequence is derived from bovine growth factor p [A]. 16. The method according to claim 16, wherein the polyadenylation termination sequence is a retrovirus 3′LT.
The recombinant cytomegalovirus according to any one of claims 1 to 12, wherein the recombinant cytomegalovirus is derived from R. 17. The method of claim 1, wherein said heterologous DNA sequence encodes part or all of any native or recombinant protein that is heterologous to the cytomegalovirus from which said recombinant virus was formed. The recombinant cytomegalovirus according to any one of claims 16 to 16. 18. The set according to claim 1, wherein the heterologous DNA sequence encodes a zona pellucida protein from an animal species specific for the CMV virus. Change cytomegalovirus. 19. The method according to claim 19, wherein the heterologous DNA sequence is H
The recombinant cytomegalovirus according to any one of claims 1 to 16, which encodes a virus antigen such as A. 20. The set according to claim 1, wherein there are a plurality of heterologous DNA sequences inserted in such a way as to produce a plurality of polypeptides. Change cytomegalovirus. 21. The method of claim 1, wherein the heterologous DNA sequence encodes an epitope capable of eliciting either a T helper cell response or a cytotoxic T cell response, or both. The recombinant cytomegalovirus according to any one of claims 16 to 16. 22. The method according to claim 1, wherein the heterologous DNA sequence encodes a ubiquitin associated with the protein to be expressed.
Item 6. The recombinant cytomegalovirus according to item 4. 23. The method according to claim 1, wherein the gene capable of down-regulating the major histocompatibility complex in CMV is preferably deleted from the recombinant virus. The recombinant cytomegalovirus as described. 24. A prophylactic or therapeutic substance comprising the recombinant viral vector according to any one of claims 1 to 23 in a pharmaceutically acceptable excipient. 25. The method according to claim 24, wherein the pharmaceutically acceptable excipient comprises one or more additives, adjuvants, stabilizers, or other similar substances. Prophylactic or therapeutic substance. 26. The method according to claim 26, wherein the prophylactic or therapeutic substance comprises a pharmaceutically acceptable additive and an effective immunizing amount of the recombinant CMV prepared according to the present invention.
A prophylactic or therapeutic substance according to claim 24. 27. A method comprising: a) growing recombinant CMV in a suitable host cell line; b) mixing an effective immunizing amount of the resulting virus with a pharmaceutically acceptable excipient. A method for producing a prophylactic or therapeutic substance. 28. The method of claim 27, wherein said host cell line is a mammalian cell line that is capable of adapting to growth in low serum or serum free media. 29. The recombinant virus according to claim 1, characterized essentially as described in the working examples herein.