Nano-particles that contain synthetic variants of GM3 ganglioside as adjuvants in vaccines

Abstract

This invention describes ways of obtaining nano-particulated adjuvants formed by different synthetic variants of GM3 ganglioside. Depending on the fine structure of the fatty acid in the ceramide of the synthetic GM3, said adjuvants are able to stimulate specifically and in a specialized way the humoral or cellular immune response against accompanying antigens. Particularly, this invention provides immunogenic vaccine compositions that comprise peptides, polypeptides or proteins and the aforementioned nanoparticles, which are formed through the dispersion of hydrophobic proteins of the outer membrane complex (OMC) of Neisseria meningitidis in solutions containing fully synthetic variants of the GM3 ganglioside.

Claims

1. An adjuvant comprising nanoparticles comprising GM3 ganglioside and hydrophobic proteins of the outer membrane complex of the bacterium Neisseria meningitidis, wherein the fatty acid in the ceramide of the GM3 ganglioside consists of oleic acid (18:1), and wherein the GM3 ganglioside and hydrophobic proteins of the outer membrane complex of the bacterium Neisseria meningitides are in association with each other.

2. A vaccine composition comprising the adjuvant of claim 1 and peptides, polypeptides or proteins as antigen.

3. The vaccine composition of claim 2 which comprises another adjuvant selected from the group consisting of: alum and an oily adjuvant.

4. The vaccine composition of claim 2 wherein the antigen is the extracellular domains of growth factor receptors or portions thereof.

5. The vaccine composition of claim 2 wherein the growth factor receptors are HER1, HER2, HER3 alone or in combination.

6. The vaccine composition of claim 2 wherein the antigen is the PyrGnRHm1-TT peptide.

7. The vaccine composition according to claim 2, wherein the vaccine composition treats cancer.

8. The vaccine composition according to claim 2, wherein the vaccine composition treats chronic viral infections with oncogenic viruses.

9. A method comprising administering the composition of claim 2 to a subject via the subcutaneous, intradermal, intramuscular, intranodal, or intratumoral routes or by direct application to mucous membranes.

10. An adjuvant comprising nanoparticles comprising GM3 ganglioside and hydrophobic proteins of the outer membrane complex of the bacterium Neisseria meningitidis, wherein the fatty acid in the ceramide of the GM3 ganglioside consists of stearic acid (18:0), and wherein the GM3 ganglioside and hydrophobic proteins of the outer membrane complex of the bacterium Neisseria meningitides are in association with each other.

11. A vaccine composition comprising the adjuvant of claim 10 and peptides, polypeptides or proteins as antigen.

12. The vaccine composition of claim 11 which comprises another adjuvant selected from the group consisting of: alum and an oily adjuvant.

13. The vaccine composition of claim 11 wherein the antigen is the extracellular domains of growth factor receptors or portions thereof.

14. The vaccine composition of claim 11 wherein the growth factor receptors are HER1, HER2, HER3 alone or in combination.

15. The vaccine composition of claim 11 wherein the antigen is the PyrGnRHm1-TT peptide.

16. The vaccine composition according to claim 11, wherein the vaccine composition treats cancer.

17. The vaccine composition according to claim 11, wherein the vaccine composition treats chronic viral infections with oncogenic viruses.

18. A method comprising administering the composition of claim 11 to a subject via the subcutaneous, intradermal, intramuscular, intranodal, or intratumoral routes or by direct application to mucous membranes.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Accumulation of CD11b.sup.+GR1.sup.+ cells in the spleen of mice carrying the MCA203 tumor and treated with VSSP nanoparticles containing different GM3 molecular species, measured by flow cytometry.

(2) FIG. 2. Measurement by flow cytometry of the in vivo antigen-specific CTL response induced in mice immunized with OVA/VSSP GM3 (18:1), OVA/VSSP GM3 (18:0) and OVA/VSSP GM3 (natural source).

(3) FIG. 3. Anti-tumor effect in the EG7 model of treatment with OVA vaccines employing VSSP nanoparticles as adjuvants containing different GM3 molecular species. The graph shows the individual values of tumor volume of each group and its average, on day 20 of the experiment. The dotted line represents the value below which the tumors were classified as not in progression. On the right, the frequency of animals in each group that did not have tumor progression with the treatments is shown.

(4) FIG. 4. IgG titers induced by the HER3 vaccine preparation adjuvated in VSSP GM3 (18:0), as measured by ELISA.

(5) FIG. 5a. Comparison of specific Abs titers against HER1 and HER2 induced by the HER1+HER2 bivalent vaccine preparation adjuvated in VSSP GM3 (18:0) against the one adjuvated in VSSP GM3 (natural source), as measured by ELISA.

(6) FIG. 5b. Comparison measured by ELISA of titers of specific Abs against the DEC-HER2 subdomains induced by the bivalent HER1+HER2 vaccine preparation adjuvated in GM3 VSSP (18:0) against the one adjuvated in VSSP GM3 (natural source).

(7) FIG. 6. Comparison measured by flow cytometry of the recognition of tumor lines HER1+/HER2+ by Abs induced with the bivalent vaccine preparation HER1+HER2 adjuvated in GM3 VSSP (18:0) against the one adjuvated in VSSP GM3 (natural source).

(8) FIG. 7. Comparison as measured by MTT colorimetric assay of the effect on the viability of the H125 HER1+/HER2+tumor line of the Abs induced by the bivalent HER1+HER2 vaccine preparation adjuvated in GM3 VSSP (18:0) against the one adjuvated in VSSP GM3 (natural source).

(9) FIG. 8. Comparison as measured by flow cytometry of the recognition of the tumor line MDA-MB468 (HER1+) by the Abs induced with the HER1 vaccine preparation adjuvated in GM3 VSSP (18:0) against the one adjuvated in VSSP GM3 (natural source).

(10) FIG. 9. Comparison as measured by Western Blot of the inhibition of HER1 activation by the Abs induced with the HER1 vaccine preparation adjuvated either in VSSP GM3 (18:0) or in VSSP GM3 (natural source).

(11) FIG. 10. Effect of immunization with PyrGnRHm1-TT peptide with and without VSSP GM3 (18:0) on the weight of Copenhagen rat prostate.

(12) FIG. 11. Evaluation of the tumor growth rate in Copenhagen rats implanted with the Dunning R3327-H tumor line.

EXAMPLES

Example 1. The Administration of VSSP GM3 (18:1) in Mice Bearing the MCA203 Tumor does not Increase the Number of CD11 b+GR1+Cells in the Spleen

(13) Four groups of female C57BL/6 mice (3 animals per group) were inoculated by SC route on day 0 with 1×10.sup.6 cells of MCA203. Subsequently, on days 11, 12 and 18, the VSSP GM3 (natural source), the VSSP GM3 (18:1) and the VSSP GM3 (18:0) (200 μg of OMPC) were injected by SC route, to the mice of three of the groups, the fourth group of mice was left as untreated control and a phosphate buffer was administered to it. On day 22, the animals were sacrificed and the extracted spleens were analyzed individually by flow cytometry to determine the number of CD11b.sup.+Gr1.sup.+cells. Animals treated with VSSP GM3 (18:1) (FIG. 1) showed similar quantity of these regulatory cells if compared with tumor bearing mice just receiving buffer. On the other hand CD11b.sup.+Gr1.sup.+ cells significantly increased in the spleens of the animals injected with VSSP GM3 (natural source) and VSSP GM3 (18:0) (equal letters p>0.05, different letters p<0.05, ANOVA and Tukey tests).

Example 2. Vaccination with OVA/VSSP GM3 (18:1) Induces Three Times More Specific Cytotoxicity of T CD8+ Cells, as Compared to OVA/VSSP GM3 (Natural Source) and OVA/VS3 GM3 (18:0)

(14) The in vivo CTL antigen-specific response was compared in female C57BL/6 mice (3 animals per group) immunized with three different formulations of the OVA antigen: one using VSSP GM3 (natural source) nano-particles, other with VSSP GM3 (18:1) and a third one using VSSP GM3 (18:0) as adjuvants. The vaccines were administered SC on days 0, 1 and 7 (200 μg OMPC). In parallel splenocytes were obtained from naive animals that were differentially labeled with CFSE (5 min at 37° C.). Highly labelled cells (5 μM) were used as target cells after incubation with the SIINFEKL peptide (1 μM, 90 min at 37° C. and 5% CO.sub.2), while lower labelled cells (0.33 μM) without peptide loading were used as control. After the last immunization, a wash was performed to remove the free peptide and both types of splenocytes were mixed in equal proportions and injected into the vaccinated animals. After 16 hours the inguinal lymph nodes of the vaccinated mice were removed and the two fluorescence intensities were measured by flow cytometry. The percentage of specific lysis was calculated according to the formula: 100−[(CFSEhigh/CFSElow)×100]. The OVA/VSSP GM3 (18:1) vaccine (FIG. 2), induces 60% of specific lysis, a value nearly three times higher than the one achieved with the OVA/VSSP GM3 (natural source) and OVA/VSSP GM3 (18:0) vaccines.

Example 3. The Use of Nanoparticles of VSSP Containing GM3 (18:1) as Adjuvant in Therapeutic Cancer Vaccines Improve its Antitumor Effect, Compared to Nanoparticles Including GM3 Obtained from Natural Sources

(15) Three groups of female C57BL/6 mice (10 animals per group) were SC inoculated on day 0 with 3×10.sup.5 cells of EG.7 tumor (a variant of EL4 thymoma genetically modified to express OVA as neo antigen). Subsequently, on days 4, 5 and 11, three successive immunizations by SC route were performed with two different vaccine formulations of OVA: the first (200 μg/dose) was adjuvated in VSSP GM3 (natural source) and the second in VSSP GM3 (18:1) (200 μg/dose). The third group of mice was injected with phosphate buffer with the same scheme and used as control of the experiment. Seven days after the tumor was inoculated, the tumor volume (TV) was measured twice a week. The animals were sacrificed when the tumor showed necrosis or when the tumor size exceeded 17 mm in diameter. On day 20 of the experiment (FIG. 3) superiority of the OVA/VSSP GM3 (18:1) therapeutic vaccine with respect to the OVA/VSSP GM3 (natural source) formulation was observed (TV means: VSSP GM3 (18:1) 560 mm.sup.3; natural 740 mm.sup.3; control 1640 mm.sup.3) (p=0.037 ANOVA and Tukey tests). More importantly, it was found that in the group treated with the OVA/VSSP GM3 (18:1) preparation, 50% of the animals did not show tumor progression in spite of EG7 being an aggressive model (100% of animals with progression was observed in the control group). In animals vaccinated with OVA/VSSP GM3 (natural source), only 20% showed no progression (p=0.012, Chi square test).

Example 4. Induction of High Titers of HER3 Specific Abs by the HER3/VSSP GM3 Vaccine Preparation (18:0)

(16) BALB/c mice (n=5) were SC immunized with a vaccine preparation containing 200 μg of HER3 ECD mixed with 200 μg of GM3 VSSP (18:0). Immunizations were carried out on days 0, 14, 28 and 42. Blood was extracted to process the serum on day 35 (1st extraction) and on day 56 (2nd extraction) and specific Abs titers against the the HER3 ECD were determined by ELISA. The plates were coated with 10 μg/mL of HER3 ECD and incubated at 37° C. After blocking, serum dilutions (1/100, 1/1000, 1/5000, 1/10000) were added. The reaction was visualized using an anti-murine IgG Ab/peroxidase conjugate (Sigma) and the corresponding substrate.

(17) Immunized mice developed specific IgGs, with titers up to 1/5000 (FIG. 4). This result demonstrates the potential of GM3 VSSP (18:0) to activate the humoral immune response against very poorly immunogenic antigens such as autologous tumor antigens.

Example 5. Comparison of the Induction of Specific IgGs by the Bivalent HER1+HER2 Vaccine Either Adjuvated in VSSP GM3 (18:0) or in VSSP GM3 (Natural Source)

(18) BALB/c mice (n=5) were immunized with the vaccine preparations containing the mixture of 100 μg of HER1 ECD and 100 μg of HER2 ECD adjuvated in 200 μg of VSSP GM3 (natural source) (Group 1) or 200 μg of VSSP GM3 (18:0) (Group 2). The immunizations were carried out by SC route on days 0, 14 and 28. Blood extraction and process were on day 35. The specific Abs titers against HER1 and HER2 ECD were determined by ELISA. For this the plates were coated with 5 μg/mL of HER1 ECD or 5 μg/mL of HER2 ECD and incubated at 37° C. After blocking, serum dilutions were added (1/100, 1/1000, 1/10 000, 1/50 000, 1/100 000). The reaction was visualized using an anti-murine IgG Ab/alkaline phosphatase conjugate and the corresponding substrate. Pre-immune serum was used as a negative control.

(19) All immunized mice developed specific IgG Abs. The Abs titers specific for the HER1 ECD were higher in Group 2, the group in which the adjuvant used was GM3 VSSP (18:0), as compared with the Abs titers raised in Group 1, which used VSSP GM3 (natural source) as adjuvant (FIG. 5a). On the other hand, even if in terms of specific Abs raised against the HER2 ECD there were no significant differences between the Abs titers induced in Groups 1 and 2, there was a tendency to an increase in the titers in Group 2, the group in which the vaccine preparation had the GM3 VSSP (18:0) as adjuvant. Given the trend observed in the recognition of HER2 ECD, a titration against the HER2 ECD subdomains (subdomains I, II, III and IV, expressed on filamentous phages) with the purified polyclonal antibodies (PcAb) isolated from the immune sera was performed. To this purpose, the ELISA plates were coated with 10 μg/mL of the PcAb and incubated at 37° C. Then the phage samples expressing the different subdomains of HER2 were added and the colorimetric signal was detected with the anti-phage antibody conjugated to peroxidase (GE-Healthcare). PcAb from animals in which the GM3 VSSP (18:0) was used as an adjuvant, showed more reactivity against the HER2 ECD subdomains I, III and IV than the antibodies purified from animals in which the VSSP GM3 (natural source) was employed (FIG. 5b).

Example 6. Higher Recognition of HER1+/HER2+ Tumor Cell Lines by Abs Induced with the HER1+HER2 Bivalent Vaccine Adjuvated in VSSP GM3 (18:0) as Compared to the Vaccine Using the VSSP GM3 (Natural Source) as Adjuvant

(20) BALB/c mice (n=5) were SC immunized with vaccine preparations containing the mixture of 100 μg of HER1 ECD and 100 μg of HER2 ECD adjuvated in 200 μg of VSSP GM3 (natural source) or adjuvated in 200 μg of VSSP GM3 (18:0). Immunizations were performed on days 0, 14 and 28 and sera corresponding to day 35 was used to evaluate the recognition of tumor cell lines expressing HER1 and HER2 (A431 epithelial carcinoma of the vulva (ATCC-CRL 1555), H125 non-small cell lung carcinoma (ATCC-CRC 5801) and SKBR3 breast carcinoma (ATCC-HTB 30) by flow cytometry. To perform the measurement, 10.sup.5 cells from each cell line were blocked with 2% fetal calf serum in phosphate buffered saline and subsequently incubated with a 1/200 dilution of a mixture of sera from each treated group. The binding of the specific Abs to the HER1 and HER2 receptors in the tumor cells was visualized using an anti-murine IgG Ab/FITC (Sigma) conjugate and by acquiring at least 5000 cells in the flow cytometer. A mixture of pre-immune sera was used as a negative control in each group evaluated. Sera induced by the vaccine preparation adjuvated in VSSP GM3 (18:0) recognized with higher intensity the evaluated tumor cells lines (FIG. 6).

Example 7. The Bivalent HER1+HER2 Vaccine Adjuvated in GM3 VSSP (18:0) Induce Antibodies with Increased Impact on the Viability of the H125 Tumor Cell Line as Compared to the Composition Using VSSP GM3 (Natural Origin)

(21) H125 cells (10.sup.5) were incubated for 72 hours with mixtures of sera obtained from BALB/c mice immunized with three doses every fifteen days of the bivalent vaccine preparations containing the mixture of 100 μg of HER1 ECD and 100 μg of HER2 ECD, adjuvated in GM3 VSSP (18:0) or in VSSP GM3 (natural origin) at 1/20 dilution. As a negative control a mixture of pre-immune sera was used at the same dilution, while as a positive control 10 μM of AG1478 tyrosine kinase inhibitor was employed. The determination of the effect of immune sera on cell viability was performed using MTT colorimetric assay, and the absorbance reading was performed at 540 nm and 630 nm. Cell viability was determined by the formula:

(22) $\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{\left(A540\mathrm{nm}-A630\mathrm{nm}\right)\mathrm{immune}\mathrm{serum}}{\left(A540\mathrm{nm}-A630\mathrm{nm}\right)\mathrm{pre}\text{-}\mathrm{immune}\mathrm{serum}}\times 100$

(23) Sera induced by the vaccine adjuvated in GM3 VSSP (18:0) significantly decreased the cell viability as compared to the composition using VSSP GM3 (natural origin). (FIG. 7)

Example 8. The HER1 Vaccine Adjuvated in GM3 VSSP (18:0) Induce Abs with More Reactivity Against HER1+Tumor Cell Lines, Compared to the Preparation Using VSSP GM3 (Natural Origin)

(24) C57BL/6 mice (n=5) were SC immunized with vaccine preparations containing 200 μg of HER1 ECD adjuvated in 400 μg of VSSP GM3 (natural origin) or in 400 μg of GM3 VSSP (18:0). Immunizations were performed on days 0, 14, 28 and 42, whereas at day 56 animals were bleed in order to evaluate sera reactivity against the intensive HER1 expressing MDA-MB468 breast carcinoma cell line (ATCC-HTB 132) by flow cytometry. Basically, 10.sup.5 cells were blocked with 2% fetal calf serum in phosphate buffer saline and subsequently incubated with a 1/100 dilution of the mixture of the sera from each treated group. The specific Abs binding to the HER1 receptors at the tumor cells was visualized by means of an anti-murine IgG Ab/FITC (Sigma) conjugate and by acquiring at least 5000 cells in the flow cytometer. As a negative control, a mixture of pre-immune sera in each group evaluated was used. Sera induced by the vaccine adjuvated in VSSP GM3 (18:0) reacted more intensively with the tumor cells, as compared to the composition using VSSP GM3 (natural origin) (FIG. 8).

Example 9. The HER1 Vaccine Adjuvated in VSSP GM3 (18:0) Induce Abs with Increased Capability to Inhibit the Activation of HER1 as Compared to the Composition Using VSSP GM3 (Natural Origin)

(25) Four groups of C57BL/6 mice (n=5) were SC immunized with one of the following vaccine preparations: Group 1: 200 μg of HER1-ECD adjuvated in 200 μg of VSSP GM3 (natural origin) Group 2: 200 μg of HER1-ECD adjuvated in 200 μg of GM3 VSSP (18:0) Group 3: 200 μg of HER1-ECD adjuvated in 400 μg of VSSP GM3 (natural origin) Group 4: 200 μg of HER1-ECD adjuvated in 400 μg of GM3 VSSP (18:0)

(26) Immunizations were carried out on days 0, 14, 28, 42 and sera corresponding to day 56 were used to evaluate the capacity of the generated Abs to inhibit HER1 phosphorylation in the presence of EGF. Briefly, H292 lung carcinoma cells (CRL 1848) were incubated for 2 hours with a mixture of sera at a 1/100 dilution. Subsequently, the cells were stimulated with 100 ng/mL of EGF for 10 minutes to induce the activation of HER1. The effect of the sera on the phosphorylation of HER1 was measured by Western Blot, using specific Abs for the detection of phosphorylated HER1 and β-actin. Cells treated with EGF were used as control of HER1 activation. A mixture of pre-immune sera at a 1/100 dilution was used as a negative control of HER1 inhibition, and 10 μM of AG1478 tyrosine kinase inhibitor was used as a positive inhibition control. A photograph of the experiment was submitted to densitometry analysis for data normalization. Sera obtained from animals immunized with the vaccines using GM3 VSSP (18:0) as adjuvant exhibited a stronger inhibition of the HER1 receptor activation in terms of phosphorylation than sera coming from the groups of mice injected with the composition using VSSP GM3 (natural source) at 200 μg and 400 μg dose as well (FIG. 9). This result demonstrates the superiority of the vaccine containing GM3 VSSP (18:0) as adjuvant in terms of the quality of the induced humoral immune response.

Example 10. Effect of the PyrGnRHm1-TT/VSSP GM3 (18:0) Vaccine Formulation on the Prostate of Copenhagen Rats

(27) Male Copenhagen rats of 8-12 weeks of age were used. The animals were divided into 3 groups of 10 animals each. Group 1: Injected with Placebo (Montanide ISA 51 VG/VSSP GM3 (18:0) Group 2: Immunized with PyrGnRHm1-TT/Montanide ISA 51 VG Group 3: Immunized with PyrGnRHm1-TT/Montanide ISA 51 VG/VSSP GM3 (18:0)

(28) For the preparation of the immunogen, the PyrGnRHm1-TT peptide was dispersed in distilled water and VSSP nanoparticles until it reached a final concentration of 750 μg of peptide and 120 μg of VSSP in 250 μL. Then this mixture was proportionally formulated 50:50 (v/v) with Montanide ISA 51 VG. The rats received 4 immunizations with a fortnightly frequency.

(29) The immunization with the PyrGnRHm1-TT peptide emulsified in Montanide ISA 51 VG, produced a significant decrease in the size of the prostate, as compared to immunization with placebo (p<0.05). This difference, however, was much greater when the peptide PyrGnRHm1-TT was emulsified in the presence of VSSP GM3 (18:0) (p<0.01) (FIG. 10).

Example 11. Induction of Antitumor Responses in the R3327-H Dunning Model Using the PyrGnRHm1-TT Peptide

(30) Adult Copenhagen rats were SC transplanted in the distal zone of the right hind limb with (2×2×2 mm) tumor fragments of the Dunning R3327-H murine tumor model. The animals were divided into four groups that received different treatments: Group 1: Placebo: Montanide ISA 51 VG/VSSP GM3 (18:0) Group 2: Castrated Group 3: Immunized with the PyrGnRHm1-TT peptide/Montanide ISA 51 VG Group 4: Immunized with the PyrGnRHm1-TT peptide/Montanide ISA 51 VG/VSSP GM3 (18:0)

(31) Immunizations with the corresponding vaccines started when tumors reached a diameter of approximately 10 mm. A total of 7 immunizations were administered every 15 days. While a pronounced growth of the tumor in the placebo group (Group 1) was observed (FIG. 11), the castrated and immunized groups (Groups 2, 3 and 4) showed a marked inhibition of tumor growth (p<0.01). This effect was more significant in rats immunized with the PyrGnRHm1-TT peptide emulsified in the presence of VSSP GM3 (18:0) (p=0.005).

(32) Nano-Particles that Contain Synthetic Variants of GM3 Ganglioside as Adjuvants in Vaccines

SUMMARY

(33) This invention describes ways of obtaining nano-particulated adjuvants formed by different synthetic variants of GM3 ganglioside. Depending on the fine structure of the fatty acid in the ceramide of the synthetic GM3, said adjuvants are able to stimulate specifically and in a specialized way the humoral or cellular immune response against accompanying antigens. Particularly, this invention provides immunogenic vaccine compositions that comprise peptides, polypeptides or proteins and the aforementioned nanoparticles, which are formed through the dispersion of hydrophobic proteins of the outer membrane complex (OMC) of Neisseria meningitidis in solutions containing fully synthetic variants of the GM3 ganglioside.