Antiviral agent comprising recombinant mistletoe lectins

Abstract

An antiviral agent containing recombinant mistletoe lectins for treating virus infections and a medicament and/or pharmaceutical composition for treating virus infections are described. Recombinant mistletoe lectin polypeptides can be a mistletoe lectin A-chain, as well as parts or fragments of the mistletoe lectin A-chain. The antiviral agent can be used for any number of virus infections, such as Herpes simplex, adenovirus, poliovirus, and poxvirus. Also, the antiviral agent can be used for skin virus warts, anogenital warts, mucous membrane warts and malignant tumors such as cervical cancer, penis and vulvar cancer.

Claims

1. A method of treating a viral infection comprising administering to a patient with a viral infection a drug containing a recombinant mistletoe lectin, wherein the recombinant mistletoe lectin is a mistletoe lectin A-chain selected from the group consisting of the amino acid sequences of SEQ ID NO: 1-3, or comprises parts and fragments thereof, or is a combination thereof, and wherein the viral infection comprises Herpes simplex virus infection.

2. The method of claim 1, wherein a cytotoxicity of recombinant mistletoe lectin is less than 0.5 ng/plaque.

3. The method of claim 1, wherein the drug further comprises a pharmaceutically acceptable carrier.

4. The method of claim 1, wherein the drug further comprises interleukins, interferons, a cytostatic agent.

5. The method according to claim 1, wherein the recombinant mistletoe lectin further comprises a mistletoe lectin B-chain selected from the group consisting of amino acid sequences of SEQ ID NOs: 4-12, or comprises parts and fragments thereof, or is a combination thereof.

6. The method according to claim 1, wherein the recombinant mistletoe lectin further comprises a mistletoe lectin A-chain of amino acid sequence of SEQ ID NO: 1 and further comprises the mistletoe lectin B-chain of amino acid sequence SEQ ID NO: 4.

7. A method of reducing a recurrence of a viral infection comprising administering to a patient with a viral infection a drug containing a recombinant mistletoe lectin, wherein the recombinant mistletoe lectin is a mistletoe lectin A-chain selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1-3, or comprises parts and fragments thereof, or is a combination thereof, wherein the viral infection comprises Herpes simplex virus infection.

8. The method according to claim 7, wherein the recombinant mistletoe lectin further comprises a mistletoe lectin B-chain selected from the group consisting of amino acid sequences of SEQ ID NOs: 4-12, or comprises parts and fragments thereof, or is a combination thereof.

Description

EXAMPLES

(1) Vero cells (renal cells from African green monkeys) and BGM cells (renal cells from Borgio green monkeys) were incubated for 2 hours with Herpes simplex viruses type 1 (HSV-1) as part of a log.sub.10 dilution series. The virus was removed after the cells were infected. The recombinant mistletoe lectin was diluted with cell culture medium, which was used both with and without interfering substances. The interfering substances used were 10% fetal calf serum (FCS) and 3% bovine serum albumin with sheep erythrocytes. 200 μl of the solution with recombinant mistletoe lectin was applied per well to the infected cells. Cell culture medium was applied to the infected cells for virus control purposes. Aciclovir (1.5 mmol) was used in place of the recombinant mistletoe lectin as a negative control substance against HSV-1. The cells were incubated at 37° C.+/−1° C. for 2 to 7 days.

(2) After incubation, the cells were analyzed for cytopathic effects (CPE) using an inverted microscope, and the 50% infectious dose, based on a cell culture, TCID.sub.50/ml (tissue culture infectious dose) was determined. If no cytopathic effects were visible, this meant that the viruses had been successfully inactivated by the recombinant mistletoe lectin.

(3) So as to gain a more detailed impression of the virus reduction, a plaque reduction assay was conducted, so as to determine the antiviral effectiveness of the recombinant mistletoe lectin. For this purpose, the Vero cells/BGM cells were placed in 12-well plates.

(4) The semi-adherent cells were incubated for 2 hours with a log.sub.10 dilution series of the HSV-1 (100 μl per well). The virus was removed after the cells were infected. The recombinant mistletoe lectin was diluted with cell culture medium (with and without interfering substances) and mixed 1:1 with warm agarose. The interfering substances used were fetal calf serum and sheep erythrocytes.

(5) 2 ml of the solution comprising the recombinant mistletoe lectin was applied to the infected cells. Cell culture medium was applied to the infected cells for virus control purposes. Aciclovir (1.5 mmol) was used in place of the recombinant mistletoe lectin as a negative control substance against HSV-1. After gelling of the agarose, the cells were incubated at 37° C.+/−1° C. for 7 days. After the incubation period, the cells were fixed for 4 hours with methanol comprising 4% sodium chloride. The cells were then dyed with crystal violet. The virus-induced plaque was counted under the microscope and the TCID.sub.50/ml was determined.

(6) The following validation steps were conducted as part of this analysis: identification of the suitable virus titer with the corresponding cell line; identification of the concentration of recombinant mistletoe lectin that is tolerated by the corresponding cell line by measurement of the cytotoxicity and vitality; identification of the concentration of recombinant mistletoe lectin that leaves the cell line vulnerable to a virus infection.
Results:

(7) The analysis surprisingly showed an antiviral effectiveness of recombinant mistletoe lectin against HSV-1 and against adenovirus type 5. In the suspension assay with HSV-1/BGM cells, a concentration of 50 ng/ml of recombinant mistletoe lectin caused a virus titer reduction of 2.43 log.sub.10 without interfering substances and without the use of MicroSpin columns (see Table 2). A concentration of 500 ng/ml of recombinant mistletoe lectin produced a virus titer reduction of 2.57 log.sub.10 and ≧3.29 log.sub.10 when using MicroSpin columns and FCS as the interfering substance (see Table 2).

(8) In the plaque reduction assay, an average relative inhibition of the HSV-1 infection of 17.04% was shown at a concentration of 0.5 ng/ml of recombinant mistletoe lectin (without MicroSpin filtration), and an average relative inhibition of the HSV-1 infection of 7.41% was shown with MicroSpin filtration (see Table 3).

(9) The average relative inhibition of the adenovirus type 5 infection in the plaque reduction assay was 79.7% at a concentration of 0.5 ng/ml recombinant mistletoe lectin (without MicroSpin filtration), and 22.2% with MicroSpin filtration (see Table 4).

(10) TABLE-US-00001 TABLE 1 Results of the cytotoxicity tests of the test cells BGM BGM with without Vero MicroSpin MicroSpin Vero with without 7 days 7 days Cell Line MicroSpin MicroSpin incubation incubation Recombinant mistletoe cytotoxic cytotoxic n.t. n.t. lectin 5000 ng/ml Recombinant mistletoe cytotoxic cytotoxic cytotoxic cytotoxic lectin 500 ng/ml Recombinant mistletoe cytotoxic cytotoxic negative negative lectin 50 ng/ml Recombinant mistletoe negative cytotoxic negative negative lectin 5 ng/ml Recombinant mistletoe negative negative negative negative lectin 0.5 ng/ml Recombinant mistletoe negative negative negative negative lectin 0.05 ng/ml Recombinant mistletoe n.t. n.t. negative negative lectin 0.005 ng/ml Negative control n.t. negative n.t. negative substance n.t. = not tested

(11) TABLE-US-00002 TABLE 2 Test for antiviral effectiveness of recombinant mistletoe lectin with HSV-1, host BGM cells (cell suspension) Virus titer HSV-1 Virus reduction (log.sub.10) + (TCID.sub.50/ml) 95% conf. limits Virus control 10.sup.−6.64+/−0.46 — 10.sup.−6.93+/−0.36 Recombinant oSS, without 10.sup.−6.79+/−0.36 no virus titer reduction mistletoe lectin MicroSpin  5 ng/ml oSS, with 10.sup.−6.79+/−0.36 no virus titer reduction MicroSpin FCS, without 10.sup.−6.79+/−0.54 no virus titer reduction MicroSpin FCS, with 10.sup.−6.64+/−0.46 no virus titer reduction MicroSpin Recombinant oSS, without 10.sup.−4.21+/−0.54 2.43 +/− 0.71 mistletoe lectin MicroSpin  50 ng/ml oSS, with 10.sup.−6.36+/−0.56 0.28 +/− 0.72 MicroSpin FCS, without 10.sup.−6.36+/−0.54 0.28 +/− 0.65 MicroSpin FCS, with 10.sup.−6.36+/−0.46 0.28 +/− 0.65 MicroSpin Recombinant FCS, with 10.sup.−4.07+/−0.49 2.57 +/− 0.67 mistletoe lectin MicroSpin    500 ng/ml ≦10.sup.−3.64+/−0.28   ≧3.29 +/− 0.54   oSS = without interfering substances

(12) TABLE-US-00003 TABLE 3 Test for antiviral effectiveness of recombinant mistletoe lectin, plaque assay with HSV- 1 on Vero cells Recombinant mistletoe lectin Aciclovir 0.5 ng/ml 0.5 ng/ml 0.5 ng/ml Virus dilution Virus control (1.5 mmol) without Spin with Spin with Spin 10.sup.−5.0 Number of >50, >50, 6 0, 0, 0 >50, >50, 10, >50, cytotoxic plaque >50 >50 Mean value >35 0 >50 >36 StabW n.d. 0 n.d. n.d. n.d. Relative 100 0 100 100 n.d. infectiosity Relative 0 100 0 0 n.d. inhibition 10.sup.−5.48 Number of 5, 5, 4, 4 0, 0, 0, 0 6, 5, 3, 2 3, 4, 4, 3 cytotoxic plaque Mean value 4.5 0 4.0 3.5 StabW 0.57 0 1.82 0.57 n.d. Relative 100 0 88.88 77.77 n.d. infectiosity Relative 0 100 11.12 22.23 n.d. inhibition Number of 2, 3, 3, 2 0, 0, 0, 0 1, 2, 1, 2 2, 3, 1, 4 cytotoxic plaque Mean value 2.5 0 1.5 2.5 10.sup.−5.95 StabW 0.57 0 0.57 1.29 n.d. Relative 100 0 60.0 100 n.d. infectiosity Relative 0 100 40.0 0 n.d. inhibition Average relative n.a. 100 17.04 7.41 n.d. inhibition n.d. = not detectable n.a. = not to be evaluated

(13) TABLE-US-00004 TABLE 4 Test for antiviral effectiveness of recombinant mistletoe lectin, plaque assay with adenovirus type 5 on BGM cells Recombinant mistletoe lectin 0.5 ng/ml Virus without 0.5 ng/ml 0.5 ng/ml Virus dilution control Spin with Spin with Spin 10.sup.−4.0  Number of 5, 5, 8 2, 3, 0 11, 8, 8 cytotoxic plaque Mean value 6 1.66 9 StabW 1.7 1.52 1.73 n.d. Relative 100 27.7 100 n.d. infectiosity Relative 0 72.3 0 n.d. inhibition Number of 3, 4, 2, 3 0, 0, 0, 0 4, 2, 2, 4 cytotoxic plaque 10.sup.−4.46 Mean value 3 0 3 StabW 0.81 0 1.15 n.d. Relative 100 0 100 n.d. infectiosity Relative 0 100 0 n.d. inhibition Number of 1, 1, 2, 2 0, 0, 2, 0 0, 0, 0, 2 cytotoxic plaque Mean value 1.5 0.5 0.5 10.sup.−4.95 StabW 0.57 1.0 1.0 n.d. Relative 100 33.3 33.3 n.d. infectiosity Relative 0 66.7 66.7 n.d. inhibition Average relative n.a. 79.7 22.2 n.d. inhibition n.d. = not detectable n.a. = not to be evaluated