WO2003020322A1 - Vector used to induce an immune response - Google Patents

Abstract

The invention relates to recombinant vectors, comprising a non-mammalian DNA virus, that express one or more antigens. The invention further relates to the use thereof for inducing an immune response in mammals, especially in humans.

Description

Vector to induce an immune response

description

The invention relates to recombinant vectors comprising a non-mammalian DNA virus which express one or more antigens and their use for inducing an immune response in mammals, in particular in humans.

The induction of an immune response is the basis for the production of antibodies and vaccines. Currently, the protein (antigen) against which an immune response is to be achieved is produced recombinantly and then applied in combination with an adjuvant. This method is very complex since it is often difficult to produce the antigen sufficiently in its natural protein structure. In addition, the use of attenuated bacteria or viruses as carriers for the expression of heterologous antigens is known. A disadvantage of this live vaccine, however, is that the pathogen used as the carrier can cause undesirable effects.

The introduction of DNA sequences into mammalian cells and the use of recombinant vectors based on non-mammalian DNA viruses is known (e.g. WO-A-95/23866 or WO96 / 09074). In addition, coat protein-modified non-mammalian DNA virus vectors are known, for example from WO99 / 091 93 and US Patents 6, 1 83.993 and 6, 1 90.887. Reference is expressly made to the disclosure of these documents with regard to suitable viral vectors or the modification of the coat protein.

The present application describes the invention that an unexpectedly strong immune response against antigens is generated when these respond to the Surface of a non-mammalian DNA virus, such as the baculovirus, brought. Furthermore, it was discovered that the expression of a heterologous DNA sequence also results in a strong immune response against the product of the synthesized protein. A combination of both, the presentation of an antigen or any number of antigens on the surface of a non-mammalian DNA virus and the expression of a heterologous DNA sequence which codes for other antigens, allows a very strong humoral (antibody) and cellular immune response. Possible applications of the invention are, for example, the production of antibodies, the vaccination against pathogens or tumors and the isolation of immune response-inducing proteins and others.

A first aspect of the invention relates to a recombinant vector comprising a non-mammalian DNA virus which has at least one heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells, e.g. Promoter, and optionally enhancer, contains.

The heterologous DNA sequence can code for a product against which an immune response is to be induced or / and for a product which enhances an immune response (directed against another product). The DNA virus may optionally be a modified envelope, e.g. as described in one of the aforementioned documents. The envelope is particularly preferably modified by expression of antigens in order to further strengthen the immune response. To express the heterologous DNA sequence, a suitable promoter active in mammalian cells, e.g. Rous-Sarcoma Virus LTR (RSV) promoter or cytomegalovirus (CMV) promoter.

Another aspect relates to a recombinant vector comprising a non-mammalian DNA virus, which is at least one heterologous, for an antigen encoding DNA sequence, fused with a DNA sequence coding for a coat protein of the virus.

Baculovirus gp64 protein or a corresponding coat protein from another virus can be used as suitable coat proteins which can be fused with the antigen. The heterologous DNA sequence can be fused to the viral sequence coding for a coat protein, C-terminally, N-terminally and / or inside the viral sequence.

Yet another aspect of the invention relates to a recombinant vector comprising a non-mammalian DNA virus, the at least one heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells and at least one heterologous DNA coding for an antigen Sequence, fused to a DNA sequence coding for a coat protein of the virus.

Preferred embodiments of the vector according to the invention are the subject of dependent claims 4 to 10.

The invention also relates to a pharmaceutical composition which contains as an active ingredient a viral vector according to the invention together with pharmaceutically customary carriers, auxiliaries and diluents. The composition may further contain a mammalian immune response-enhancing adjuvant, such as aluminum hydroxide or cytosine / guanine-rich sequences.

The composition is suitable for use as a vaccine, for inducing an immune response in mammals, for producing antigens and for isolating proteins which induce an immune response. The composition can be administered by any means, for example by injection, for example subcutaneous, intramuscular or intraperitoneal injection, by oral administration, by inhalation or nasal administration or by any other suitable administration method.

The administration can take place in one or preferably in several doses. The dose amount is preferably 10 ⁷ to 10 ⁹ , in particular 10 ⁸ vector units / kg.

The viral vectors according to the invention are produced by customary methods, with plasmids carrying the corresponding heterologous DNA sequences first being produced as recombination vectors and transfected together with the DNA of the non-mammalian virus in permessive cells and the recombinant vector being obtained by homologous recombination.

In a preferred embodiment, the invention relates to a vector which induces a specific immune response against products of inserted sequences in mammals and / or mammalian cells. Areas of application are e.g. medicine, biotechnology and genetic engineering. The vector preferably consists of an insect virus, preferably a representative of the baculoviruses or a nuclear polyhedrosis virus, which contains at least one component selected from (i) a modified envelope, (ii) a heterologous DNA sequence and (iii) one for the Gene expression suitable promoter.

Furthermore, the present invention is explained in more detail by the following tables and figures and by the following exemplary embodiments: Table 1 shows the detection of the induction of a strong immune response by expression and / or surface presentation. Baculoviruses generated using psCSvac, peCSvac and psCSeCS-vac were injected into BalbC mice and the immune response against the antigen was determined using the antibody in an ELISA test (after 56 days).

Table 2 shows the ratio of immunoglobulins G 1 and G2a (IgG 1 / IgG2a) in mouse serum. This ratio was obtained from the data in Figures 9 and 10. Cytokines regulate the production of classes and subclasses of antibodies. Therefore, a low ratio of IgG 1 / IgG2a indicates a cellular immune response. The low ratio of IgG1 / IgG2a after immunization with the Baculovirus AcNPVsCS / eCS gives a clear indication of a cellular immune response. In contrast, immunization with recombinant CS protein and Alhydrogel ^{® shows} a high IgG1 / IgG2a ratio, which does not indicate a cellular immune response.

Figure 1 shows the recombination vector psCSvac. The antigen (here, for example, the circumsporozoite protein (CS) from Plasmodium falciparum) is presented on the surface of the virus by using this vector and mainly induces a humoral immune response.

Figure 2 shows the recombination vector peCSvac. The antigen (here, for example, the circumsporozoite protein (CS) from Plasmodium falciparum) is expressed in the cell by using this vector and mainly induces a cellular immune response.

Figure 3 shows the recombination vector psCS / eCSvac. By using this vector, the antigen (here, for example, the circumsporozoite protein (CS) from Plasmodium falciparum) is presented on the surface of the virus and expressed and induced in the cell and induces a strong humoral and cellular immune response. Figure 4 shows a schematic of the new vectors. psCS results in a vector that carries the antigen (s) on the surface. peCSvac results in a vector that expresses the antigen (s) in the cell. psCS / eCSvac results in a vector which carries the antigen (s) on the surface and at the same time expresses the antigen (s) in the cell.

Figure 5 shows the evidence of induction of a strong immune response. A baculovirus, which was generated by means of the vector psCSvac, was injected into BalbC mice and mediated a strong and long-lasting immune response against the CS antigen, which could be increased by repeated administration (boost).

Figure 6 shows an overview of the immunization regime and the animal test conditions of Example 2.

Figure 7 shows the malaria CS-specific immunoglobulin M (IgM) immune response.

Figure 8 shows the malaria CS-specific total immunoglobulin G (IgG H + L, heavy and light chain) immune response.

Figure 9 shows the malaria CS-specific immunoglobulin G1 (IgGD immune response.

Figure 10 shows the malaria CS-specific immunoglobulin G2a (lgG2a) immune response.

Figure 1 1 shows the malaria CS-specific cellular immune response from Baculovirus AcNPVsCS / eCS. Example 1: Preparation of a non-mammalian DNA virus that carries antigens on the surface.

A sequence for an N-terminally modified non-mammalian DNA virus coat protein (baculovirus, gp64) was cloned into a recombination vector in a known manner under the control of a baculoviral promoter. The modification was designed by inserting DNA sequences which code for antigens from pathogens, viruses and / or tumors. This sequence is achieved between the signal sequence and the sequence of the baculovirus coat protein gp64 at the DNA level. Using the example of the plasmodium _. falciparum circumsporozoite protein (CS) this is shown below.

The recombination vectors were produced using standard methods. The plasmids are shown in Figure 1-3 and can be modified as desired by replacing the CS with other antigens, such as. B. Tu mo ra ntig en e or er Vi rus / Path og en-Anti g e n e. The

Recombination vectors are shared with the DNA of one

Insect virus transfected in insect cells and by homologous recombination, the viral vector from the supernatant

Insect cell culture obtained. The corresponding virus (Figure 4) is then used in active or inactive form, in mammals

To win antibodies against the inserted antigens

(therapeutic or sterilizing) or isolating immune response-inducing proteins.

Example 2: Immunization regimes and animal testing conditions.

The following agents were injected into balb / c mice, divided into three groups of three animals each under the following conditions:

1 . Each 5x10 ⁸ pfu baculovirus (AcNPVsCS / sCS, see Figure 4), which was generated using psCS / eCSvac, and thus malaria Circumsporozoit (CS) protein carries on the surface as well as is expressed in target cells. 2. In each case 1 0 g recombinant malaria circumsporozoite (CS) protein, which has been previously adsorbed to Alhydrogel ^® (Alhydrogel-CS). 3. PBS (mock) injected animals serve as controls.

The first injection (priming) as well as a further injection (boost) after three weeks was carried out intramuscularly in all experimental animal groups in a total volume of 50 μ \ (Figure 6). Blood was taken retroorbitally at the indicated times to determine the specific CS antibody titer (Ab) in the serum (Figure 6). The antibody titer was determined in the serum of immunized and boosted mice at the times indicated using a standardized ELISA. For this ELISA, recombinant CS protein was adsorbed on 96-well plates, then the plates were incubated with different dilutions of the mouse sera from the treated animals and subsequently by means of an enzyme-coupled anti-IgM- (Figure 7), anti-IgG- ( Figure 8), anti-lgG 1 (Figure 9) and anti lgG2a- (Figure 10) specific antibody quantitatively developed according to standard methods.

The CS-specific anti-IgM antibody titer induced by the baculovirus (AcNPVsCS / eCS) was significantly higher than that in the group of animals treated with Alhydrogel / CS (Figure 7). Furthermore, both (AcNPVsCS / eCS and Alhydrogel / CS) immunizations, in contrast to mock injection, lead to a high anti-IgG antibody titer against the circumsporozoite protein (CS protein) and thus show a strong humoral immune response (Figure 8). In contrast, the CS-specific anti-IgG 1 antibody titer induced by the baculovirus (AcNPVsCS / sCS) was significantly lower than that in the animal group treated with alhydrogel / CS. Furthermore, the CS-specific anti-IgG2a antibody titer induced by the baculovirus (AcNPVsCS / eCS) was significantly higher than that in the group of animals treated with Alhydrogel / CS (Figure 10). This indicates the induction of an additional immune response by baculovirus (AcNPVsCS / eCS) (Table 2).

The spleen was removed after five weeks to determine the specific T cell response in the ELIspot assay (ELI). The ELIspot method detects the specific secretion of interferon-gamma by T cells from the spleen of immunized mice after activation of the T cells with CS protein. T cells from mice immunized with Baculovirus AcNPVsCS / eCS show a clear interferon-gamma secretion in the ELIspot (22 spots at 2 µg CS, 89 spots at 10 µg CS antigen (Figure 1 1)). In contrast, no interferon gamma secretion was found in the mock-infected animals (4-5 spots = background of the method) (Figure 1 1). Concanavalin A activates T cells non-specifically and serves as a positive control in this experiment. This shows that the baculovirus is able to induce a strong cellular immune response that is necessary to protect humans from pathogens such as Plasmodium falciparum.

The results of the preclinical immunization experiment using AcNPVsCS / eCS are shown in Figures 7-1 1 and Table 2. The quantity and quality of the induced humoral and cellular immune response would, when transferred to humans, lead to protective protection against a malaria infection.

Claims

Expectations 1 . Recombinant vector comprising a non-mammalian virus, characterized in that it contains at least one heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells. 2. Recombinant vector comprising a non-mammalian virus, characterized in that it contains at least one heterologous DNA sequence coding for an antigen, fused with a DNA sequence coding for a coat protein of the virus. 3. Recombinant vector comprising a non-mammalian virus, characterized in that it (a) at least one heterologous DNA sequence coding for an antigen in operative linkage with an expression control sequence active in mammalian cells and (b) contains at least one heterologous DNA sequence coding for an antigen, fused with a DNA sequence coding for a coat protein of the virus. Vector according to one of claims 1 to 3, characterized in that the virus is selected from insect viruses, crustacea viruses, Amphibian viruses, fish viruses, plant viruses and / or algae viruses. 5. Vector according to claim 4, characterized in that the virus is selected from (a) Baculoviridae, e.g. Eubaculoviridae or Nudibaculoviridae, (b) Iridiviridae, (c) Parvoviridae, (d) Poxviridae, e.g. Entomoxpoxviridae, (e) Canlimoviridae, (f) Geminiviridae, (g) Phycodnaviridae, (h) .Polydnaviridae. 6. Vector according to one of claims 1 to 5, characterized in that the virus is a baculovirus, in particular a multiples Is a nucleocapsid virus such as Autographa californica, a single nucleocapsid virus such as Bombyx mori S nuclear polyhedrosis virus, a granulovirus such as Plodia interpunctella granulose virus, or a non-enveloped baculovirus such as Heliothis zea virus. Vector according to one of claims 1 to 6, characterized in that the antigen or antigens from pathogens, such as viruses, e.g. Hepatitis viruses, especially HBV, HCV, bacteria, e.g. Pneumococci or meningococci, or parasites such as e.g. Plasmodicum falciparum. 8. Vector according to one of claims 1 to 6, characterized in that the antigen or antigens originate from tumors, for example melanoma. 9. Vector according to one of claims 1 to 6, characterized in that the antigen or antigens originate from autoantigens. 1 0. Vector according to one of claims 1 to 9, characterized in that the antigens are selected from B cell or / and T cell antigens. 1 1. Pharmaceutical composition, characterized in that it contains, as active ingredient, a vector according to one of Claims 1 to 10 together with pharmaceutically customary carriers, auxiliaries and diluents. 1 2. Composition according to claim 1 1, characterized in that it contains an adjuvant enhancing the immune response. 1 3. Composition according to claim 1 1 or 1 2 for use as a vaccine. 14. Use of a vector according to any one of claims 1 to 10 for inducing an immune response in mammals. 1 5. Use of a vector according to any one of claims 1 to 10 for the production of antibodies. 1 6. Use of a vector according to any one of claims 1 to 10 for the isolation of proteins which induce an immune response. 7. Use of a vector according to one of claims 1 to 1 0 for diagnostic purposes.