HK1151829B - Replication-defective arenavirus vectors - Google Patents

Description

Replication-defective arenavirus vectors

Technical Field

The present invention relates to genetically modified arenaviruses (arenavirus) suitable for use as vectors for vaccines or gene therapy and methods of vaccination and disease treatment using these viruses.

Background

Prophylactic vaccines represent one of the most successful chapters of modern medicine, bringing smallpox away worldwide, and controlling polio, measles and many other devastating infectious diseases. Recently, vaccines for the prevention of cancer have emerged and efforts are underway to develop "vaccines" in therapeutic form, which are expected to be useful for infections and malignancies. Historically, vaccination strategies have included a variety of approaches: from the very beginning of the use of wild-type infectious agents and of the self (re) inoculation of tumor cells, followed by attenuated live infectious agents and dead tumor tissues, clinical medicine has increasingly tended over time to use (inert) proteins and/or other extracts (generally referred to as "antigens") from infectious agents or tumors, respectively. This gradual process represents the process of finding safer vaccine formulations, however, this is often accompanied by a relative loss of vaccine efficacy. In recent years, the development of biotechnology has made possible another approach, which is now widely regarded as one of the most promising approaches: sources of infection used as "boating" (referred to as "vectors") are equipped with antigens from selected pathogens or tumors. Thus, in the context of the strong immune enhancement ("immunogenicity") conferred by the vector, the immune response of the vaccinee recognizes the antigen of interest.

This "vector method" has been used to introduce a foreign gene directly into living cells at the tissue culture level, and also to introduce a foreign gene directly into living cells in multicellular organisms (including humans), and therefore, the vector can also be used to express a gene in cultured cells or in gene therapy.

There are many vectors currently used in experimental research, including vaccination and gene therapy, with the ultimate aim of optimizing efficacy and safety for clinical applications (vaccines and gene therapy) or biotechnology (gene transfer in cell culture).

It is generally accepted that vectors often have the general characteristics of the organism from which they are derived (e.g., a virus). Thus, the development of new virus families for vector design offers the prospect of discovering new combinations of traits that could confer unprecedented capabilities on such new vectors and corresponding applications in biomedical applications. However, vector design requires taking into account the safety of the organism used, so strategies must be proposed how to eliminate the pathogenic potential of the organism in a way that does not interfere with the desired traits, such as immunogenicity for administration as a vaccine.

For more than 70 years, arenaviruses in general, and lymphocytic choriomeningitis virus (LCMV) in particular, have been known to elicit exceptionally strong and long-lasting humoral and cell-mediated immune responses. It is noteworthy that, despite this, protective neutralizing antibody immunity against the viral envelope Glycoprotein (GP) is extremely low, and antibody-mediated protection against reinfection by infection is extremely low, if at all. Moreover, it has been firmly established for decades that, due to its non-cytolytic (cell-sparing properties), arenaviruses can maintain long-term antigen expression in animals without causing disease under certain conditions. Recently, reverse genetics systems for manipulating infectious arenavirus genomes have been described (L.Flatz, A.Bergthaler, J.C.de la Torre, and D.D.Pinschewer, Proc Natl Acad Sci USA 103: 4663-containing 4668, 2006; A.B.Sanchez and J.C.dela Torre, Virology 350: 370, 2006), but, to date, arenaviruses have not been used as vaccine vectors. This is mainly due to two main obstacles: i) arenaviruses can cause very severe infections, which can subsequently lead to severe disease and immunosuppression. ii) selected foreign antigens cannot be introduced.

Summary of The Invention

The present invention relates to an infectious arenavirus particle engineered to contain a genome capable of amplifying and expressing its genetic information in infected cells but incapable of further producing infectious progeny particles in normal, non-genetically engineered cells.

More specifically, the invention relates to arenavirus particles that contain additional ribonucleic acids that can encode proteins of interest or modulate host gene expression.

The arenavirus of the invention comprises a modified genome wherein:

i) one or more of the four open reading frames of arenaviruses, i.e., Glycoprotein (GP), Nucleoprotein (NP), matrix protein Z, and RNA-dependent RNA polymerase L, are removed or mutated to prevent transmission of infectivity in normal cells, but still allow gene expression in these cells;

II) introducing exogenous ribonucleic acids encoding one or more proteins or regulating host gene expression and transcription by one or more of the four arenavirus promoters (5 'UTR and 3' UTR of the S-segment and 5'UTR and 3' UTR of the L-segment) or by additionally introduced promoters which can be read out by viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III, respectively, wherein the ribonucleic acids encoding the proteins or regulating host gene expression can be transcribed by themselves or read-through by fusion with the arenavirus protein open reading frame (read-through); and optionally

iii) introducing one or more internal ribosome entry sites into the viral transcription sequence to enhance expression of proteins in cells infected with arenavirus.

In addition, the invention relates to vaccines and pharmaceutical formulations comprising such genetically engineered arenaviruses and methods of vaccination and gene therapy using these genetically engineered arenaviruses.

Furthermore, the invention relates to expressing a protein of interest in a cell culture or modulating gene expression in a cell culture, wherein the cell culture is infected with a genetically engineered arenavirus.

Drawings

FIG. 1 shows a schematic view of a

The resulting arenavirus vector replicates only in complementing cells (complementing cells) due to the engineering of the wild-type arenavirus genome.

A: arenavirus vectors (1) can infect normal cells (2) or anaplerotic cells (C cells, 3). C cells, when infected, can form further infectious progeny vectors, whereas normal cells, when infected, do not produce vector particles or non-infectious particles.

B: c cells (1) and normal cells (2) were infected with LCMV-based vector expressing Green Fluorescent Protein (GFP) instead of LCMV-GP (rcmv/GFP) and supernatant samples were collected at different time points (3, expressed in hours). Infectivity in the supernatant was measured using a plaque formation assay (4, expressed as PFU/ml).

C: the wild-type arenavirus genome consists of a large segment (L; 1) and a small segment (S; 2). The L segment expresses the L gene (3) and the Z gene (4), while the S segment carries the NP gene (5) and the GP gene (6). One strategy for generating replication-defective arenavirus vectors may be to replace the GP gene with a gene of interest, such as GFP (7) or ovalbumin (OVA; 8).

FIG. 2

Schematic representation of the complementing plasmid (C plasmid), the plasmid for intracellular expression of the trans-acting factor (TF plasmid) and the plasmid for intracellular expression of the arenavirus vector genome segment (GS plasmid).

A: example of plasmid C.

B: examples of TF plasmids expressing the viral NP and L proteins, respectively.

C: examples of GS plasmids expressing S-segment and L-segment of arenavirus vectors, respectively.

1: a polymerase II promoter; 2: viral genes expressed for complementation; 3: an internal ribosome entry site; 4: a mammalian selectable marker, such as a puromycin resistance gene; 5: a polyadenylation signal; 6: an ampicillin resistance cassette; 7: an origin of replication; 8: viral trans-acting factors, such as NP ORF; 9: viral trans-acting factors, such as the L ORF; 10: promoters that drive expression of arenavirus genomic segments in C-cells, such as the polymerase I promoter; 11: the 5' UTR of the S segment; 12: an antigen of interest; 13: an IGR of the S-segment; 14: an NP gene; 15: a 3' UTR of the S segment; 16: a polymerase I terminator; 17: the 5' UTR of the L segment; 18: a Z gene; 19: an IGR of the L-segment; 20: an L gene; 21: 3' UTR of the L segment.

FIG. 3

Arenavirus vectors are cleared within days after vaccination and therefore do not cause immunosuppression in immunized subjects.

A: on day 0, mice were immunized intravenously with rLCMV/GFP (1). Thereafter, the number of copies of the viral genome in the spleen was measured (3; in log10) at different time points (2; in days).

B: as primary immunization/infection (1 ℃), immunization with rLCMV/OVA (5) is minorMice were either uninfected (6) or infected with wild-type LCMV (7). On day 20, all mice were infected intraperitoneally with vesicular stomatitis virus (VSV, 8) (2 °). Blood was then collected for measurement of antiviral and anti-vector T cell responses (3) and antiviral antibody responses (4). On day 28, H-2K in peripheral blood was measured by intracellular staining with gamma interferon after re-stimulation of the peptide^bSIINFEKL (CD 8+ T cell epitope from ovalbumin) specific CD8+ T cells (9) and H-2K^bVSV-NP52-29 (CD 8+ T cell epitope derived from VSV-NP) specific CD8+ T cells (10) (values expressed as percentage of specific cells among CD8+ T cells). Total VSV neutralizing antibody (11) and β -mercaptoethanol resistant IgG (12) (values are expressed as log2 of 40-fold pre-diluted serum) in sera were tested using a 50% plaque reduction assay on days 27 (expressed as "d 7" with reference to the time point after VSV infection), 29 (expressed as "d 9") and 61 (expressed as "d 41").

FIG. 4

Arenavirus vectors induce a high rate of CD8+ T cells with long-term memory and antibodies with long-term memory at high titers.

A: mice were immunized with rLCMV/OVA and blood samples were collected over a period of time (2; expressed in days) for detection of H2K with MHC class I tetramers^b-ratio of OVA/SIINFEKL-specific CD8+ T cells (3, values are expressed as ratio of tetramer-positive CD8+ T cells in CD8+ T cell compartment).

B: mice were immunized with the indicated dose of rLCMV/OVA by subcutaneous injection (s.c.) or intravenous (i.v.) routes (1), and OVA-specific IgG in serum was measured by ELISA on days 14 (5) and 58 (6). Values are expressed as serum dilutions that yield a measurement of twice the background Optical Density (OD).

FIG. 5

Arenavirus vectors do not cause central nervous system disease.

Mice were immunized intracerebrally with rLCMV/OVA (open squares) or wild-type LCMV (black circles) and then monitored for clinical signs of terminal choriomeningitis at the indicated time points (1, expressed in days). The number of healthy animals/number of experimental animals per time point is shown (2).

FIG. 6

Arenavirus vectors confer T cell-mediated and antibody-mediated protection against infectious challenge.

A) On day 0 of the experiment, mice were immunized with rcmv/OVA (AA group) or rcmv control vector expressing an unrelated Cre recombinase antigen (BB group) used as a negative control. After 16 or 58 days (d16, d58) intervals, an intravenous challenge was performed with recombinant listeria monocytogenes expressing OVA. The fourth day after challenge, bacterial titers in the animal's spleen were measured (1) (expressed as log of colony forming units (log10) for each organ). Black circles indicate values for each mouse. The vertical bars represent the average of each group.

B) Mice deficient in class I interferon receptors were immunized (filled squares) or not (open circles) with LCMV vectors expressing antigenic but non-functional variants of vesicular stomatitis virus envelope protein G (modified by insertion of a foreign polypeptide sequence in the extracellular domain). After one month, use 2X 10⁶PFU vesicular stomatitis virus attacks all animals intravenously. Animals were monitored for clinical signs of terminal beam encephalomyelitis at designated time points (2, expressed in days) post challenge. At each time point and group, healthy survival was expressed as number of healthy animals/number of experimental animals (3).

Detailed Description

The present invention relates to infectious arenavirus particles (referred to as arenavirus vectors) engineered to contain a genome capable of amplifying and expressing its genetic information in infected cells but incapable of further producing infectious progeny particles in normal, non-genetically engineered cells. Fig. 1A schematically shows this principle. Example data is shown in FIG. 1B.

Replication of arenavirus vectors requires genetically engineered cells that complement replication-defective vectors. After the cells are infected, the genome of the arenavirus vector expresses not only arenavirus proteins, but also additional proteins of interest, such as antigens of interest. Arenavirus vectors were generated by standard reverse genetics techniques described for LCMV (l.flatz, a.bergthaler, j.c.de la Torre and d.d.pinschewer, Proc Natl Acad Sci USA 103: 4663-4668, 2006; a.b.sanchez and j.c.de la Tore, Virology 350: 370, 2006), but their genomes were modified by one or more of the following methods to obtain the above-mentioned characteristics:

i) four open reading frames of arenaviruses (glycoproteins (GP); nucleoprotein (NP); matrix protein Z; RNA-dependent RNA polymerase L) is removed or mutated to prevent the formation of infectious particles in normal cells, but still allow gene expression in cells infected with arenavirus vectors.

ii) exogenous nucleic acid encoding one or more proteins may be introduced, or alternatively, or in addition, exogenous nucleic acid may be introduced to regulate host gene expression. These exogenous nucleic acids include, but are not limited to, short hairpin rna (shrna), small interfering rna (sirna), micro rna (mirna), and precursors thereof. These foreign nucleic acids can be transcribed by one or more (e.g., two or three) of the four arenavirus promoters (5 'UTR and 3' UTR of the S-segment and 5'UTR and 3' UTR of the L-segment), or by additionally introduced promoter sequences that can be read by viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III (e.g., copies of viral promoter sequences naturally present in the viral UTRs, 28S ribosomal RNA promoters, beta actin promoters or 5S ribosomal RNA promoters), respectively. Ribonucleic acids that encode proteins or regulate host gene expression may be transcribed and translated by themselves or may be read through fusion with an arenavirus open reading frame. One or more (e.g., two, three, or four) internal ribosome entry sites can be introduced at appropriate sites in the viral transcription sequence to enhance expression of the protein in the host cell.

As used herein, "regulating host gene expression" refers to reducing or increasing host gene expression in all cells targeted by the vector (or in a cell-type specific manner). These desired characteristics can be achieved by introducing the nucleic acid sequence into a vector.

Arenavirus vectors are useful for improving general life and health, and can be used to immunize (in prophylactic form) or treat (in immunotherapeutic form) animals (including humans) in a variety of situations, including but not limited to

i) Infection: including but not limited to viruses (such as Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), influenza virus and Respiratory Syncytial Virus (RSV)), bacteria (such as mycobacteria, haemophilus (haemophilus spp) and pneumococci (pneumacococcus spp)) and parasites (such as plasmodium, amoeba (amebia) and philaria) and prions (prion) (such as infectious agents of Creutzfeldt-Jakob disease (Creutzfeldt-Jakob disease) and mad cow disease that can cause both classical and variant);

ii) autoimmune diseases: including but not limited to type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus erythematosus and psoriasis;

iii) neoplastic diseases: including but not limited to melanoma, prostate cancer, breast cancer, lung cancer, and neuroblastoma;

iv) metabolic disorders: including but not limited to type 2 diabetes, obesity, and gout;

v) degenerative diseases: including but not limited to alzheimer's disease and parkinson's disease;

vi) genetic diseases: including but not limited to huntington's disease, severe combined immunodeficiency disease, and lipid storage disorders;

vii) substance dependence: including but not limited to tobacco and alcohol abuse; and

viii) allergic diseases: including but not limited to seasonal or chronic rhinoconjunctivitis, asthma, and eczema.

For the same purpose, arenavirus vectors can be used for introducing genes of interest (e.g., foreign nucleic acids) into cells of living animals (including humans), i.e., gene therapy, or for introducing and expressing gene products of interest in biotechnological applications. Replication of arenavirus vectors can be abolished by deletion of the Z gene required for e.g. viral particle release or GP gene required for infection of target cells from their genome (see fig. 3), the total number of infected cells being limited by the inoculum administered to e.g. the vaccinee or the recipient of gene therapy or accidentally transmitted to humans or animals involved in medical or biotechnological applications. Arenavirus disease and immunosuppression in wild-type arenavirus infections are known to be caused by uninhibited viral replication. Thus, elimination of arenavirus vector replication can prevent pathogenicity resulting from purposeful or accidental transmission of vector particles. In the present invention, an important aspect is to take advantage of the abovedescribed necessity of eliminating replication in a beneficial manner for the purpose of expressing one or more foreign proteins (e.g. antigens of interest): removal (e.g., structural deletion or functional mutagenesis) of one or more genes of the arenavirus releases the corresponding promoter for expression of the selected protein.

The many combined advantages of the arenavirus vector strategy of the present invention are: it is noteworthy that although arenavirus vectors are not transmissible, they retain immunogenicity precisely, which is a great surprise for those immunologists working in arenavirus immunological studies. The presence of considerable viral and antigen loads over a critical period of time is generally considered necessary to account for the superior immunogenic properties of arenaviruses. In terms of safety, the non-lytic behavior of the virus (and the vector) is a major advantage over most available vector systems, as is the general lack of oncogenic potential of arenaviruses. Furthermore, it is also very important in terms of safety that arenavirus vectors are not able to replicate. In particular for use as a vaccine, arenavirus vectors with high levels of resistance to antibody neutralization are highly advantageous. This feature is inherent to many arenavirus envelopes and allows repeated immunizations with the same arenavirus vector to achieve repeated booster immune responses. Likewise, pre-existing immunity to arenaviruses in the human population is very low or negligible.

Arenaviruses are considered to be either old world viruses (e.g., lassa virus, lymphocytic choriomeningitis virus (LCMV), mobara virus, mopenia virus, or epstein-barr virus) or new world viruses (e.g., amapali virus, Flexal virus, melon nreto virus, junin virus, leidero virus, marthala virus, Oliveros virus, paraan a virus, pichinde virus, pirita virus, Sabi a virus, Tacaribe virus, tamiram virus, bercanyon virus, or whitewater virus). Members of the old world virus, such as lassa virus or LCMV, especially LCMV, are preferred.

Foreign nucleic acids encoding one or more proteins of interest are, for example, sequences derived from messenger RNA or RNA corresponding to the transcription product of a primary gene, which, when arenavirus particles of the invention carrying such RNA infect a cell, result in the expression of the protein of interest. In addition, exogenous nucleic acids that modify gene expression in cells infected with arenavirus vector particles by, for example, RNA interference, are also contemplated.

The ribonucleic acid of interest that can be introduced into the genetically engineered arenavirus of the invention is any sequence that encodes a protein or modulates host gene expression, the sequence can be introduced into the arenavirus vector genome by replacing or fusing with the open reading frame of glycoprotein GP, matrix protein Z, nucleoprotein NP or polymerase protein L, i.e., it can be transcribed and/or expressed under the control of the four arenavirus promoters (5 'UTR and 3' UTR of the S segment and 5'UTR and 3' UTR of the L segment) or ribonucleic acids inserted with regulatory elements (e.g., copies of the viral promoter sequence naturally present in the viral UTR, 28S ribosomal RNA promoter, beta actin promoter, or 5S ribosomal RNA promoter) that can be read by viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II, or RNA polymerase III, respectively. The protein or nucleic acid may be transcribed and/or expressed by itself or may be read through fusion with an arenavirus open reading frame and/or in combination with one or more (e.g., two, three, or four) internal ribosome entry sites, respectively. The length of the inserted gene and the nature of the expressed protein are not critical, as demonstrated by the replacement of GP with GFP and ovalbumin genes, which makes it possible to express a variety of proteins of interest.

Preferred proteins of interest are peptide antigens or proteinaceous antigens. The peptide or proteinaceous antigen of the invention may, for example, be selected from (a) proteins or peptides suitable for inducing or modulating an immune response against an infectious disease; (b) proteins or peptides suitable for inducing or modulating an immune response against a neoplastic disease, i.e., cancer cells; and (c) a protein or peptide suitable for inducing or modulating an immune response against an allergen. Combinations of antigens (e.g., a combination of antigens from one or more infectious organisms or tumors or allergens) can be combined to induce or modulate an immune response for protecting or treating more than one infection, tumor type, or allergic disease, respectively.

As used herein, "modulating an immune response" refers to i) increasing in quality or quantity a beneficial immune response in a patient. This is desirable, for example, when enhancing HIV-specific T cell and antibody immune responses in the context of immunotherapy of infected individuals. The term "modulating an immune response" also refers to ii) a process commonly referred to as desensitization, e.g., by suppressing an allergic type immune response (e.g., one of the immunoglobulin E isotypes) to desensitize the allergen with the aim of replacing or boosting a protective immune response or mitigating a pathogenic immune response.

In a particular embodiment of the invention, the antigen is an antigen useful for the prevention of infectious diseases. Specific examples of antigens or antigenic determinants include HIV antigens gp41, gp120, gag, and pol, the Nonstructural (NS) proteins of hepatitis c virus, influenza antigens hemagglutinin and neuraminidase, hepatitis b surface antigen, and the circumsporozoite protein of malaria.

Preferably, the antigen is selected from the group consisting of respiratory syncytial virus antigens, human immunodeficiency virus antigens, hepatitis c virus antigens, varicella zoster virus antigens, herpes simplex virus antigens, cytomegalovirus antigens and antigens derived from mycobacterium tuberculosis.

The choice of antigen for use in compositions and methods for treating cancer is well known to those skilled in the medical arts for treating such diseases. Representative examples of such antigens include: HER2/neu (breast cancer), GD2 (neuroblastomA), EGF-R (glioblastomA), CEA (medullary thyroid carcinomA), CD52 (leukemiA), MUC1 (expressed in hematological malignancies), gp100 protein, MELAN-A/MART1 or the product of the tumor suppressor gene WT 1.

The choice of antigen for use in compositions and methods for treating allergy is well known to those skilled in the medical arts for the treatment of such diseases. Representative examples of such antigens include, but are not limited to, birch pollen Bet v1 and cat allergen Fel d 1.

The choice of antigen for use in compositions and methods for treating obesity is well known to those skilled in the medical arts for treating such diseases. Representative examples of such antigens include, but are not limited to, growth hormone releasing peptide (ghrelin) and Gastric Inhibitory Peptide (GIP).

Design of arenavirus vector genomes

Starting from the wild-type arenavirus genome (fig. 1C), the arenavirus vector genome is designed to retain at least the necessary regulatory elements in the 5 'and 3' untranslated regions (UTR) of the two segments, preferably also the intergenic region (IGR). The minimal trans-acting factors required for gene expression in infected cells remain as expressible open reading frames in the vector genome, but they may be located at different positions in the genome and may be under the control of a promoter different from the native promoter, or may be expressed from an internal ribosome entry site. At least one of the four viral genes (NP, L, GP, Z) is removed or functionally inactivated. One or more additional genes or nucleic acid segments of interest can be inserted into the arenavirus vector genome at positions and orientations that enable their expression in infected cells under the control of four viral promoters (5 'UTR and 3' UTR of the S segment and 5'UTR and 3' UTR of the L segment) or internal ribosome entry sites or promoters that can be read by viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III. FIG. 1C shows an example in which the arenavirus GP Open Reading Frame (ORF) is replaced with an Ovalbumin (OVA) or Green Fluorescent Protein (GFP) ORF.

Generation of anaplerotic cell lines

Since "deletion" (meaning removal or functional inactivation) of one or more viral genes in an arenavirus vector (here, deletion of glycoprotein GP is exemplified), arenavirus vectors must be produced and amplified in cells that provide the deletion of viral genes (e.g., GP in this example) in trans (in trans). Such a complementing cell line (herein referred to as a C-cell) is generated by transfecting a mammalian cell line (such as BHK-21, HEK293, VERO or other cell lines (here exemplified by BHK-21)) with one or more plasmids (complementing plasmids, referred to as C-plasmids) expressing the viral genes of interest. The C plasmid (see, for example, fig. 2A) expresses a viral gene deleted in an arenavirus vector that is produced under the control of one or more expression cassettes suitable for expression in mammalian cells (e.g., a mammalian polymerase II promoter, such as a CMV or EF 1a promoter with a polyadenylation signal). In addition, complementing plasmids are characterized by a mammalian selectable marker, such as, for example, puromycin resistance, under the control of an expression cassette suitable for gene expression in mammalian cells (e.g., the polymerase II expression cassette described above), or by an internal ribosome entry site following viral gene transcripts (e.g., the internal ribosome entry site of encephalomyocarditis virus), followed by a mammalian resistance marker. Other features of the plasmid, for production in e.coli (e.coli), are bacterial selectable markers, such as ampicillin resistance cassettes.

The cells to be used (e.g., BHK-21, HEK293, MC57G or other cells) are maintained in culture and transfected with the complementing plasmid by any commonly used strategy (e.g., calcium phosphate, liposome-based methods, or electroporation). After several days, a suitable selection agent, e.g. puromycin, is added at a suitable concentration. Surviving clones were isolated and subcloned according to standard methods, and highly expressed C cell clones were identified by Western blotting or flow cytometry using antibodies against the viral protein of interest. As an alternative to using stably transfected C cells, transient transfection of normal cells can be used to complement the deleted viral genes in each step following the use of C cells.

Plasmid for recovery of arenavirus vectors

Two types of plasmids are required:

i) two plasmids for intracellular expression of the minimal trans-acting factor of arenaviruses in C-cells, called TF-plasmids (see for example figure 2B), are derived from the NP and L proteins of LCMV, for example, in this example.

ii) a plasmid for intracellular expression of arenavirus vector genomic segments (such as the engineered modified segments described in figure 1C) in C cells, called the GS plasmid (see figure 2C for example). The TF plasmid expresses the NP and L proteins of the corresponding arenavirus vector under the control of an expression cassette suitable for protein expression in mammalian cells (e.g. a mammalian polymerase II promoter such as CMV or EF 1a promoters, either of which is preferably combined with a polyadenylation signal) (fig. 2B). The GS plasmid expresses both small (S) and large (L) genomic segments of the vector. Generally, either a polymerase I driven expression cassette (FIG. 2C) or a T7 phage RNA polymerase (T7) driven expression cassette can be used, the latter being preferentially used with a 3' terminal ribozyme to process the primary transcript, thereby obtaining the correct ends. In the case of using a T7-based system, expression of T7 in C cells must be provided by including an additional expression plasmid in the recovery process that is structurally similar to the TF plasmid and provides T7, or constructing C-cells to otherwise express T7 in a stable manner.

Recovery of arenavirus vectors

The first day: c cells were transfected with a mixture of two TF plasmids and two GS plasmids (typically 80% confluence in M6 well plates). In this regard, any of the usual strategies may be employed, such as calcium phosphate, liposome-based methods, or electroporation.

After 3-5 days: culture supernatants (arenavirus vector preparations) were harvested, aliquoted, and stored at 4 ℃, -20 ℃ or-80 ℃ depending on the length of time the arenavirus vector should be stored before use. Infectious titer of the arenavirus vector preparation on C cells was then evaluated by an immunophagy plaque assay.

Titration of arenavirus vector infectivity

To determine the infectivity of arenavirus vectors, C cells were used in a typical immunophagy assay according to the principles commonly used in virology, as described below:

c cell monolayers (typically 80% confluency in M24 well plates) were infected with 10-fold dilutions of arenavirus vector preparations for 90 min. The cell layer was then overlaid with a suitable cell culture medium supplemented with 1% methylcellulose. After 2-3 days, depending on the permissivity of the C cell line used, the culture supernatant is removed, the cell layer is fixed, usually with ethanol/acetone or 4% formalin, and then a mild detergent is used to increase the permeability of the cell layer. The arenavirus vector infected cell plaques are then identified with a monoclonal or polyclonal antibody preparation directed against one of the proteins in the arenavirus vector to be tested or the introduced antigen. The bound antibody is detected using a suitable reagent, such as an anti-isotype or anti-species antibody conjugated to a chromogenic system, such as horseradish peroxidase, followed by a color reaction with a suitable chromophore, such as o-phenylenediamine. The resulting plaques on the plate were counted to calculate the number of infectious plaque forming units (FFU) per unit volume of arenavirus vector preparation.

Vaccine and pharmaceutical preparation

The invention also relates to a vaccine and a pharmaceutical preparation containing the genetically engineered arenavirus. Vaccines and pharmaceutical formulations for other uses are prepared according to standard methods in the art.

Compositions for enteral administration (e.g. nasal, buccal, rectal or oral administration) and for parenteral administration (e.g. intravenous, intramuscular, intradermal or subcutaneous administration) to warm-blooded animals, especially humans, are preferred. Particularly preferred are compositions for parenteral administration. The compositions comprise the genetically engineered arenavirus alone or, preferably, the genetically engineered arenavirus and a pharmaceutically acceptable carrier. The dosage of the active ingredient depends on the type of immunization, the disease to be treated and the species, age, weight and individual condition, the pharmacokinetic data of the individual and the mode of administration.

The pharmaceutical composition comprises about 10³To about 10¹¹A genetically engineered arenavirus of individual plaque forming units. Unit dose forms for parenteral administration, such as ampoules or vials, e.g. containing about 10³To 10¹⁰Plaque Forming Unit or 10⁵To 10¹⁵A penicillin bottle of a genetically engineered arenavirus physical particle.

Preferably, a suspension or dispersion, especially an isotonic aqueous dispersion or suspension, of the genetically engineered arenavirus is used. The pharmaceutical compositions may be sterile and/or may contain excipients, for example preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and may be prepared in a manner known per se, for example by means of conventional dispersing and suspending methods. The dispersion or suspension may contain a viscosity modifier. The suspension or dispersion is stored at a temperature of about 2-4 ℃, or preferably, can be frozen for long term storage and then thawed shortly before use.

The invention also relates to a method for the preparation of a vaccine in the form of a pharmaceutical preparation and to the use of a genetically engineered arenavirus for the preparation of a vaccine in the form of a pharmaceutical preparation, comprising a genetically engineered arenavirus as active ingredient. The pharmaceutical compositions of the invention may be prepared in a manner known per se, for example by means of conventional mixing and/or dispersing methods.

Administration to vaccinees and gene therapy recipients

The invention also relates to methods of vaccination and gene therapy using genetically engineered arenaviruses, as described above.

The administration of arenavirus vectors is used to improve the quality of life, including but not limited to vaccination, immunotherapy and gene therapy, to prevent, treat or ameliorate:

i) infection: including but not limited to viruses (such as Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), influenza virus and Respiratory Syncytial Virus (RSV)), bacteria (such as mycobacteria, haemophilus and pneumococci), parasites (such as plasmodium, amoeba and philaria), and prions (such as infectious agents of creutzfeldt-jakob disease and mad cow disease that can cause both classical and variant);

ii) autoimmune diseases: including but not limited to type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus erythematosus and psoriasis;

iii) neoplastic diseases: including but not limited to melanoma, prostate cancer, breast cancer, lung cancer, and neuroblastoma;

iv) metabolic disorders: including but not limited to type 2 diabetes, obesity, and gout;

v) degenerative diseases: including but not limited to alzheimer's disease and parkinson's disease;

vi) genetic diseases: including but not limited to huntington's disease, severe combined immunodeficiency disease, and lipid storage disorders;

vii) substance dependence: including but not limited to tobacco and alcohol abuse.

In particular, the present invention relates to a method for preventing infections caused by viruses, bacteria, parasites and prions, comprising administering to a patient in need thereof a vaccine comprising a genetically engineered arenavirus, as well as to a method for preventing neoplastic and degenerative diseases as described above.

Furthermore, the invention relates to a method for treating infections caused by viruses, bacteria, parasites and prions, autoimmune diseases, tumor diseases, metabolic diseases, degenerative diseases, genetic diseases or substance dependence, comprising administering to a patient in need thereof a pharmaceutical preparation containing a genetically engineered arenavirus.

The arenavirus vector can be administered to the vaccinee by one or more available routes including, but not limited to, intramuscular, intradermal, subcutaneous, oral, intranasal, or intravenous routes, for example, as in the experiment set forth in figure 3A. This will result in the cells being infected and the viral genome segment being amplified in these highly identical initially infected cells, for example after intravenous inoculation. This includes dendritic cells in the spleen that can induce a T cell response. Since arenavirus vectors are unable to replicate in cells of the recipient (due to their lack of complementing viral proteins present in C cells), arenavirus vector RNA levels will drop rapidly over time and the viral genome will disappear within days after inoculation with arenavirus vectors (fig. 3A). Since arenavirus vectors are unable to replicate and persist, arenavirus vector immunization does not cause immunosuppression (fig. 3B) or disease (fig. 5) compared to infection with the same dose of wild-type virus. This was tested in mice infected with wild-type LCMV or LCMV-based vectors expressing OVA instead of LCMV-GP (rLCMV/OVA; compare results with FIG. 1C). Subsequently, vesicular stomatitis virus infection elicited a normal CD8T cell and antibody response in animals previously immunized with rcmv/OVA, but was inhibited in animals previously infected with wild-type LCMV. Likewise, when administered intracranially, wild-type LCMV caused lethal choriomeningitis in mice, whereas rcmv/OVA did not cause any clinically detectable signs of disease (fig. 5).

Despite its transient nature, expression of the antigen of interest does induce a strong and long-term T cell response (see fig. 4A) and induces high titers of specific antibodies (fig. 4B). This response was dose-dependent, but was effective even at small doses (fig. 4B). The protective capacity of T cell immune responses induced by LCMV-based vaccine vectors was tested in mice. Immunization with rLCMV/OVA protected against infectious challenge by recombinant Listeria monocytogenes expressing OVA (rLM/OVA). This was shown to be a significant drop or undetectable drop in rLM/OVA titers in the spleen of vaccinated animals (see FIG. 6A). The induction of antibody-mediated protection by LCMV vectors was tested in type I interferon receptor deficient mice. These mice were very sensitive to Vesicular Stomatitis Virus (VSV), with a 50% Lethal Dose (LD) thereof₅₀) Around the 50PFU range. For immunization against VSV, LCMV vectors were utilized that expressed antigenic but non-functional variants of vesicular stomatitis virus envelope protein G (modified by insertion of a foreign peptide sequence in its extracellular domain). The immunized mice can be infected with 2X 10⁵PFU VSV (i.e. > 10000 times LD)₅₀) The mice remained viable, while the non-immunized control mice developed terminal choriomeningitis within 2-3 days after VSV challenge (figure 6). However, it is noteworthy that inactivation of the arenavirus vector genome by UV radiation results in a loss of its immunogenicity, suggesting that viral vector replication and gene expression in infected cells are essential for vaccine efficacy. In addition, T cell and antibody immune responses can be enhanced by recycling the same (homologous) or a different (heterologous) arenavirus vector (i.e., by means of a form of booster vaccination). In a homologous prime-boost regimen, where there is no neutralizing antibody induction, this makes boosting particularly effective.

When used in gene therapy, arenavirus vectors can be administered systemically (e.g., intravenously) or locally (e.g., by localized injection using a suitable device) for targeting and delivery to specific tissues where the antigen of interest should be expressed. Due to its non-lytic nature, arenavirus vectors are not harmful to the cells they infect and can functionally replace genes of interest.

As an alternative to the use of arenavirus vectors to treat multicellular organisms, the implantation of either complementing cells (C cells) or non-complementing (normal) cells coated with a biocompatible material into a recipient prevents immune rejection by the recipient, but still allows the constant release of infectious (implanted infectious C cells) particles or non-infectious (implanted infected normal cells) particles or proteins and/or ribonucleic acids from the coated cells through the coating into the recipient's tissue.

Expression of proteins of interest in cell culture

In addition, the invention relates to the expression of a protein of interest in a cell culture, wherein the cell culture is infected with a cell genetically engineered arenavirus. When used to express a protein or nucleic acid segment of interest (e.g., an antigen of interest) in cultured cells, the following two methods are involved:

i) infection of a cell type of interest with an arenavirus vector preparation at a multiplicity of infection (MOI) of 1 or higher (e.g., 2, 3, or 4) results in production of the protein of interest in all cells shortly after infection.

ii) alternatively, a lower MOI can be used and single cell clones can be selected for their viral driver protein expression levels. Subsequently, a single clone can be amplified indefinitely because the arenavirus vector has non-lytic properties. Regardless of which method is employed, the protein of interest may then be collected from the culture supernatant or the cells themselves, depending on the nature of the protein produced.

However, the present invention is not limited to these two strategies, and other methods of driving expression of a protein or nucleic acid of interest using genetically engineered arenaviruses as vectors are also contemplated.

Claims (10)

1. An infectious arenavirus particle engineered to comprise a genome capable of amplifying and expressing its genetic information in an infected cell but incapable of further producing infectious progeny particles in a normal, non-genetically engineered cell, wherein the arenavirus has its open reading frame Glycoprotein (GP) removed or mutated, and wherein the arenavirus is lymphocytic choriomeningitis virus (LCMV).

2. The arenavirus particle according to claim 1, comprising an additional nucleic acid encoding a protein or peptide of interest.

3. The arenavirus particle according to claim 1, comprising additional nucleic acids that modulate host gene expression.

4. The arenavirus particle according to claim 2 or 3, comprising a modified genome, wherein:

i) exogenous ribonucleic acids encoding one or more proteins of interest or regulating host gene expression are expressed under the control of regulatory elements readable by viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III, either expressed by themselves or read-through by fusion with an arenavirus protein open reading frame.

5. The arenavirus particle according to claim 4, wherein the open reading frame Glycoprotein (GP) of the arenavirus particle is removed and replaced with an exogenous ribonucleic acid encoding one or more proteins of interest or regulating host gene expression.

6. The arenavirus particle according to claim 4, wherein the open reading frame Glycoprotein (GP) of the arenavirus particle is removed and replaced with an exogenous ribonucleic acid encoding a peptide or protein antigen from an infectious organism, tumor or allergen.

7. A vaccine or pharmaceutical formulation comprising an arenavirus particle according to any of claims 2 to 6.

8. A method of expressing a protein of interest or modifying gene expression in a cell culture, wherein the cell culture is infected with an arenavirus particle according to any of claims 2 to 6.

9. The arenavirus particle according to claim 4, wherein the exogenous ribonucleic acid encoding one or more proteins of interest or regulating host gene expression is expressed under the control of one or more of the four promoters of the arenavirus, the 5'UTR and the 3' UTR of the S segment and the 5'UTR and the 3' UTR of the L segment.

10. The arenavirus particle according to claim 4, wherein one or more internal ribosomal entry sites are introduced to enhance the expression of the protein of interest in the cells infected with the arenavirus.