MXPA97007295A - Method for improving antibody response to specific antigens with interleucine - Google Patents

Abstract

The present invention relates to a method for improving the immune response of a mammal to a vaccine, the method comprising administering to this mammal an effective amount of IL-10 together with the vaccine. A pharmaceutical composition containing an effective amount of IL-10, a natural, synthetic or recombinant antigen and a pharmaceutically acceptable carrier.

Description

METHOD FOR IMPROVING ANTIBODY RESPONSE TO SPECIFIC ANTIGENS WITH iNTERLZUCINE-10 BACKGROUND OF THE INVENTION Active immunization is the administration of an antigen to an animal to elicit an immune response in the animal. A vaccine against a microorganism is an antigenic preparation that, when inoculated into a non-immune individual, will confer active immunity to the microorganism but will not cause the disease. Specificity and memory, the two key elements of the adaptive immune system are widely used in vaccination. Since the adaptive immune system develops a stronger response in the second encounter with an antigen, this secondary immune response is faster to appear and more effective than the primary response. The principle of the development of the vaccine is to modify a microorganism or its toxins (natural antigens) in such a way that they become innocuous without losing antigenicity. Alternatively, the antigenic polypeptides of the organism in question can be produced by recombinant methods or by synthetic chemistry to produce an effective vaccine.

A problem often encountered during active immunization is that the antigens used in the vaccine are not sufficiently immunogenic to elevate the titre of the antibodies to levels sufficient to provide protection against further challenge or to maintain the potential to develop these levels for extended periods of time. Another problem is that the vaccine may be deficient in inducing cell-mediated immunity which is a primary immune defense against bacterial and viral infection. Still another problem is that an individual patient may be immunosuppressed due to illness or old age. To obtain a stronger humoral and / or cellular response, it is common to administer a vaccine in a formulation containing an adjuvant. An adjuvant is a substance that improves, not specifically, the immune response to an antigen, or that causes an individual to respond to an antigen that otherwise, without the adjuvant does not respond to the antigen. An adjuvant is usually given with an antigen, but it can also be given before or after antigen administration. However, despite the multiple advances of vaccines and vaccine preparation, vaccines very often do not provide the immunogenic response desired especially in immunosuppressed and elderly people. An example is the Pneu-immune neutral pneumococcal vaccine 23. Pneumococcal pneumonia is currently the most frequent cause of bacterial pneumonia in the United States and the rate of this disease is especially high in the elderly, young children, patients with predisposing conditions as asplenia, chronic heart disease, lung and kidney, diabetics and patients suffering from genetic or acquired immunosuppression (Breiman et al., Arch. Intern Med., 150: 1401-1404 (1990)). These groups are at the greatest risk of pneumococcal spread to the blood system and the central nervous system, which is the most common cause of bacterial meningitis. This vaccine has an aggregate efficacy of approximately 75% in immunocompetent adults, but the coverage in high risk groups listed in the above has been discussed, and is certainly much lower (Butler et al., J. Am. Med. Assoc., 270: 1826 (1993) Thus, there is a need for additional adjuvants or adjuvants that can be administered together with a vaccine to produce an immunizing effect in the elderly and those with disorders of the immune system.

SUMMARY OF THE INVENTION Surprisingly it has been discovered that Interleukin-10 satisfies this need as an effective vaccine adjuvant. Accordingly, the present invention provides a method for improving the immune response of a mammal to a vaccine, the method comprising administering to a mammal in need of vaccination an effective amount of IL-10 together with a vaccine. Preferably, the treated mammals will be human and the IL-10 used will be one of the human allotypes. In a preferred embodiment, humans will be immunocompromised.

The present invention furthermore relates to a pharmaceutical composition containing an effective amount of IL-10, a natural synthetic or recombinant antigen and a pharmaceutically acceptable carrier. The dose of IL-10 for mammals will preferably be administered by subcutaneous injection or intravenous injection and will be in an amount of 2 to 150 micrograms (μg) per kilogram (kg) of body weight per day. Most preferably, the dose of IL-10 will be in an amount of 2 to 80 micrograms per kilogram of body weight per day. Alternatively, mammals will be pretreated with IL-10 for 1-4 days before vaccination, and then continued with IL-10 therapy. Preferably IL-10 will be administered simultaneously with the vaccine, from 1 to 14 days before or after administration of the vaccine in an amount of about 2 to 150 micrograms (μg) per kilogram of body weight, preferably , 2μg -80μg per kilogram of body weight.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphic representation of the increase in the specific primary response of spleen platelet-forming cell antibodies to lamb erythrocytes observed in vivo in mice dosed with IL-10. Figure 2 is a graphical representation of the improvement of the secondary specific response of spleen platelet-forming cell antibodies to lamb erythrocytes observed in mice dosed with IL-10. Figure 3 is a graphical representation of the increase in Response of splenic platelet-forming cells to the response to Pnu-immune 23 in old mice (22 months) (higher) compared to young mice (4-5 months) (lower) after IL-10 administration in vivo . Figure 4 is a graphical representation of the concentration-response of platelet-forming cells in vi tro observed when the vessel cells of young (upper) or old (lower) mice were incubated with different concentrations of IL-10 and the vaccine Pnu-immune. Figure 5 illustrates the PFC results in vi tro when the cells of the old mouse vessel were incubated in vi tro with different concentrations of IL-10 and Pnu-immune vaccine.

Figure 6 is a graphical representation of the in vitro effect of IL-10 on the PFC response when unfractionated or depleted splenocytes of T cells from old mice were incubated with IL-10 in the presence of the Pnu-immune vaccine.

DETAILED DESCRIPTION OF THE INVENTION IL-10 was originally described as a product of the T2 helper cell (Th2) that inhibited the production of cytokines such as interferon-? by means of Thl cells (Florentino et al., J. Exp. Med., 1 70, 2081-2095 (1989)) and improved the proliferation of mouse thymocytes in response to IL-2 and IL-4 ( Suda, et al., Cell Immunol., 129: 228-240 (1990)). It was subsequently found that IL-10 inhibits, in the presence of monocytes / macrophages, the proliferation and cytokine synthesis of human T cells and T cell clones (deWaal Malefyt et al., J. Exp. Med., 174, 915-924 (1991)) and mouse T cell clones (Ding and Shevach, J. Immunol., 148: 3133-3139 (1992)).

IL-10 is normally produced by mouse Th2 clones, B cell lymphomas, T cells, activated mast cell lines, activated macrophages, keratinocytes and CD5 + B cells (Florentino et al., J. Exp. Med., 170: 2081-2095 (1989), Moore et al., Science, 248, 1230-1234 (1990), O'Garra e. Al., Int. Immunol., 2, 821-832 (1990), MacNeil et al., J. Immunol., 145, 4167-4173 (1990), Florentino et al., J. Immunol., 147, 3815-3821 (1991), Lin et al., Ann. NY Acad. Sci., 651: 581- 583 (1992) In addition to the effects of IL-10 listed above, IL-10 has been reported to possess an array of stimulatory properties of B lymphocytes in experimental models in vitro B cells play an important role in the immune response of the host producing antibodies in response to the foreign antigen.It was found that IL-10 regulates the surface expression of complex antigens of higher class II histocompatibility in low-density murine B cells. os (Fei Go et al., J. Exp. Med., 172, 1625-1631 (1990)), increases the proliferation of activated human tonsillar B cells and induces their differentiation into antibody-secreting cells (Rousset et al., Proc. Nati. Acad. Sci. , USA 89, 1890-1893 (1992)) capable of secreting immunoglobulin M (IgM) IgG1, IgG3, and, TGFβ, IgA, (Defrance et al., J. Exp. Med., 175: 671-682 (1992); Briere et al. , J. Exp. Med., 1979, 751-162 (1994)). IL-10 has also been found to differentially regulate the production of immunoglobulin in the presence of different cytokines (Pencanha et al., J. Immunol., 148, 3427-3432 (1992)). In vivo administration of anti-IL-10 antibody in mice from newborn to 8 weeks reduced serum IgM and IgA and antibody responses in vivo to two bacterial antigens increased IgG2a and IgG2b levels in serum and decreased the production and function of CD5 + B cells in the peritoneum (Ishida et al., J. Exp. Med., 1975, 1213-1220 (1992)). These in vivo effects of the administration of anti-IL-10 were attributed to an increase in the endogenous levels of interferon-α. Despite this evidence indicating that IL-10 is capable of inducing polyclonal immunoglobulin levels in vi tro, to date there have been no reports on the ability of IL-10 to improve the antigen-specific antibody response in vivo or in vi tro. The production of specific antibodies directed against specific foreign antigens is one of the initial responses of the immune system and is an important factor in the determination of how rapidly infectious agents are eliminated from the host. We have found that the administration of IL-10 in vi and in vi tro improves the response of the antigen-specific antibody to two different antigens in mice, namely lamb erythrocytes and the Pneu-immune pneumococcal vaccine 23. The capacity of IL-10 to improve the humoral response to Pnu-immune 23 is of special interest because pneumococcal pneumonia is currently the most common cause of bacterial pneumonia in the United States, and the rate of this disease is especially high in the United States. elderly, young children, patients with predisposing conditions such as asplenia, chronic heart disease, lung and kidney, diabetics and patients suffering from genetic or acquired immunosuppression (Breiman et al., Arch. Intern. Med. 150: 1401-1404 (1990 )). These groups are at high risk of pneumococcal spread to the blood system and the central nervous system which is the most common cause of bacterial meningitis. This vaccine has an aggregate efficiency of approximately 75% in immunocompetent adults, but the coverage in high risk groups listed in the above has been discussed, is certainly much lower (Butler et al., J. Am. Med. Assoc. 270: 1826 (1993) The results of the examples presented below show that IL-10 restores the antibody response to pneumococcal vaccine in old mice at the levels observed in young mice. can be used to increase the humoral immune response in immunosuppressed patients, the elderly and patients suffering from hypogammaglobulinemia.Therefore, the present invention provides a method for improving the immune response of a mammal, to a vaccine, the method comprises administration to a mammal in need of vaccination, of an effective amount of IL-10 together with a vaccine.The term "together with" as used herein refers to the administration of IL-10 concurrently, before or after administration of the vaccine. As used herein, "interleukin-10 or IL-10" can be human IL-10 (h L-10) or murine IL-10. Human IL-10 is defined as a protein that (a) has a substantially identical amino acid sequence a known sequence of mature hIL-10, ie, lacking a secretory leader sequence) as described in the Patent Application American Series 07 / 917,806 filed July 20, 1992, which corresponds to International Application No. PCT / US90 / 03554, Publication No. O91 / 00349, and (b) has biological activity common to natural hIL-10 . IL-10 can be obtained from culture medium of activated T cells capable of secreting the protein. Preferably, however, recombinant techniques are obtained using isolated nucleic acids encoding the IL-10 polypeptide. General methods of molecular biology are described, for example, in Sambrook et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Publish, Cold Spring, New York, 2a. Edition, 1989 and by Ausubel et al. , (eds.), Current Protocols in Molecular Biology, Green / Wiley, New York (1987 and periodic supplements). Suitable sequences can be obtained from genomic or cDNA libraries. The techniques of polymerase chain reaction (PCR) can be used. See, for example, PCR Protocol: A Guide to Methods and Applications, 1990, Innis et al. , (Ed.), Academic Press, New York, New York. Libraries are constructed from nucleic acids extracted from suitable cells. See, for example, the publication of International Application No. WO 91/00349, which describes the recombinant methods for making IL-10. Useful genetic sequences can be found, for example, in different sequence databases, for example, in the Gene Bank and in the EMBL for the nucleic acid and PIR and S iss-Prot for the proteins, c / o Intelligenetics Mountain View, California or the Genetics Computer Group, Center for Biotechnology, University of Wisconsin, Madison, Wisconsin. Clones comprising the sequences encoding human IL-10 (hIL-10) have been deposited with the American Type Culture Collection (ATCC), Rockv, Maryland under accession numbers 68191 and 68192. Identification of other clones containing the Sequences encoding IL-10 are performed by means of nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. Oligonucleotide probes based on the deposited sequences are described in International Application Publication No. WO 91/00349. Oligonucleotide probes useful for identifying the sequences can also be prepared from conserved regions of related genes in other species. Alternatively, degenerate probes based on the amino acid sequence of IL-10 can be used. The various expression vectors can be used to express the DNA encoding IL-10. Conventional vectors that are used for the expression of recombinant proteins in prokaryotic or eukaryotic cells can be used. Preferred vectors include the pcD vectors described by Okayama et al. , Mol. Ce 11. Biol. , vol. 3, 280-289 (1983); and Takebe et al. , Mol. Cell. Biol. , vol. 8, 466-472 (1988). Other expression vectors of mammals based on SV40 include those described in Kaufman et al. , Mol. Cell. Biol. , vol. 2, 1304-1314 (1982) and in U.S. Patent No. 4,675,285. These SV40-based vectors are particularly useful in C0S7 monkey cells (ATCC No. CRL 1651) as well as in other mammalian cells such as mouse L cells and CHO cells. Standard transfection methods can be used to produce eukaryotic cell lines that express large amounts of polypeptide. The process of the present invention is a process for purifying IL-10 expressed by eukaryotic cells of a cell supernatant in which the protein is expressed. Eukaryotic cell lines include mammalian, yeast and insect cell lines. Exemplary mammalian cell lines include COS-7 cells, mouse L cells and Chinese hamster ovary (CHO) cells. See Sambrook et al. , supra and Ausubel et al. , supra. Methods for purifying biologically active IL-10 are described in International Patent Application Serial No. PCT / US94 701909 filed on March 3, 1994. The adjuvant activity is manifested by a significant increase in immune-mediated protection. the development of an immune response in an individual who would not otherwise respond to a vaccine. The improvement of humoral immunity is usually manifested by a significant increase in the titer of elevated antibodies to the antigen. In accordance with the present invention, an effective amount of IL-10 is administered to mammals simultaneously or before treatment with the indicated antigen of the vaccine to increase the amount of the antibody specific for the particular antigen. The amount of vaccine administered will be in accordance with the manufacturer's instructions. The effective amount of IL-10 is defined as any amount that will increase the amount of an antibody against a specific antigen. The term "effective amount", as used herein, refers to the effective amount of IL-10 that is administered according to the present invention by an amount of IL-10 that produces an increase in the level of antibodies sufficient to give increased protection of an infectious agent if you had administered a vaccine without IL-10. Preferably, the increase will be an increase of at least 25%. Preferably, the mammals will be treated with IL-10 obtained from a human source, i.e., human IL-10 produced by recombinant E. coli or CHO cell techniques. The dosage for mammals will be administered by subcutaneous injection or intravenous infusion and will be in the amount of 2 to 150 μg per kilogram of body weight per day. Preferably, the dosage will be in an amount of 2 to 80 μg per kilogram of body weight per day and more preferably from 2-25 μg. The amount, frequency and period of administration will vary depending on different factors, including serum antibody and patient age, nutrition, etc. Administration will initially be daily and may continue during the patient's lifetime. The amount and frequency of the dose can be determined during initial screenings and the amount of IL-10 in the magnitude of the response. To complement the response of the antigen-specific antibodies, it may be useful to administer IL-10 together with other biologically and / or pharmaceutically active compounds. For example, it can be combined with other agents that show improved B cell responses, such as interleukin-4, interleukin-7, interleukin-13 or interleukin-14. Additionally, the vaccine antigen can be administered in the presence of other adjuvants to promote even greater response. The methods of the present invention for providing administration of IL-10 together with a vaccine have the following advantages. The total antigenic load of the vaccine to be administered can be reduced since less antigen in the presence of IL-10 will develop an immune response at least equivalent to that achieved by administration of the normal amount of the vaccine. Given the, administering IL-10 would require less antigen per vaccine, according to the present invention, the likelihood of undesirable side effects associated with some vaccines currently in use would be reduced. The immune response of certain types of individuals who respond poorly to vaccination would be improved by administering IL-10 along with a vaccine. The types of individuals that would benefit from the methods of the present invention include: (1) those types that have a damaged immune response, due to disease or age, for example, humans 55 years of age or older; (2) individuals who appear normal but who nonetheless do not respond to certain vaccines as well; (3) individuals who undergo immunosuppression therapies such as radiation and chemotherapy. In this way, we have discovered an effective method to (1) improve an effective primary immune response in mammals to the antigens present in a vaccine; (2) to improve an effective level of antibodies in mammals exposed to antigens, in a vaccine in which the immune response of mammals without the administration of IL-10 would not be strong enough or fast enough to avoid the disease. Vaccines contemplated for use in accordance with the present invention include but are not limited to bacterial vaccines, toxoid vaccines (inactivated toxins) and viral vaccines or mixtures thereof that are used for active immunization. See for example chapter 75 entitled "Immunizing agents" in Remington's Pharmaceutical Sciences, 14a. Edition, 1990, Mack Publishing Co. P. 1426-1441 and the antitoxins, toxoids, vaccines and live vaccines approved by the US Food and Drug Administration and listed on page 208-209 (product category index) of Physician's Desk Reference , Edition 46, 1992. Suitable bacterial vaccines include bacterial vaccines against the following entities or disease states: cholera, pertussis, bubonic plague, typhoid fever, meningitis, pneumococcal pneumonia, H. Infl uenzae type B, leprosy, gonorrhea, meningococcal group B and group B streptococci, gram negative sepsis, E. coli sepsis and Pseudomonas aeruginosa. Suitable toxoids include diphtheria toxoid, botulism toxoid and tetanus toxoid. "Suitable multiple antigens include diphtheria and tetanus toxoids, diphtheria toxoids, pertussis and triple antigen tetanus such as those available from Connaught Laboratories Inc. Swiftevater, PA 18370. In addition, IL-10 will ordinarily be used to improve the protection provided by vaccines that are considered "weak" (that is, they provide diminished protection in terms of level, magnitude, and / or duration.) Examples of these vaccines are bacterins such as Bordetella bacterin, E. coli bacterins, bacterins of Haepophilus, Leptospirosis vaccines, Moraxella bovis bacterin, Pasteurella bacterin and Vibrio fetus bacterin, and pneumococcal vaccines IL-10 will normally be administered separately from the vaccine, although it can be administered in combination with the vaccine. -10 is combined with the vaccine, the composition administered contains an univogen that is effective to develop a specific response a to a given pathogen or antigen, a pharmaceutically acceptable vaccine carrier and an immunopotentiating amount of IL-10. The administration of IL-10 can be subcutaneous, intravenous, parenteral, intramuscular or any other acceptable methods. Preferably, IL-10 is administered prior to administration of the vaccine and at the same site where the vaccine is to be administered. The formulations and pharmaceutical compositions contemplated by the above dosage forms can be prepared with conventional pharmaceutically acceptable excipients and additives using conventional techniques. Other adjuvants can be administered with the vaccine or together with IL-10. If multiple doses of the vaccine are to be administered over a period of time, additional IL-10 may be administered along with each subsequent dose of the vaccine. The amount of IL-10 that is administered with each subsequent dose of the vaccine may be more, the same or less than the amount of IL-10 administered together with the initial dose of the vaccine. The amount of IL-10 administered with each subsequent dose of the vaccine will depend on the response of the patient's antibodies after the first dose of the vaccine. The IL-10 solutions to be administered can be reconstituted from lyophilized powders and can additionally contain preservatives, buffer solutions, dispersants, etc. Preferably, IL-10 is reconstituted with any isotonic medium normally used for subcutaneous injection, eg, sterile water without preservative. The effect of IL-10 in improving the immune response of a vaccine is exemplified by the following non-limiting data which should not be considered as limiting the scope of the description.

EXAMPLE 1 To determine the effect of in vitro treatment of IL-10 on the primary response of lamb erythrocyte cell antibodies (SRBC), IL-10 was administered to young DBA / 2 mice by intraperitoneal injection at 0.3, 3 or 10 μg per day for 5 days. The control mice received only the vehicle (10 mM Tris, pH 7.4). Two to four hours after the first injection of IL-10 or vehicle, the mice received an intravenous injection (0.2 ml) of a 20%, 2%, or 0.2% vol / vol dilution of SRBC. After five days, the spleen cells of each mouse were prepared by collecting the intact spleen, macerating the spleen in Dulbecco's phosphate buffered saline with the rounded end of a 5 ml syringe plunger followed by grinding and passage through a nylon mesh of 75 microns. Spleen cells were counted and their viability was determined by exclusion of the trypan blue dye. To measure the response of the spleen platelet-forming cell antibodies, the procedure originally described by Jerne and Nordin (Science, 140, 405-407 (1963)) was used with minor modifications. In short, 200,000 splenic cells in 50 μl were added to tubes previously incubated at 42 ° C with 200 μl of SeaPlaque Agarose 1% v / v solution and 50 μl of a 50% v / v SRBC solution. The tubes were shaken by hand and the contents were poured into the center of a slide and dispersed over two thirds of the slide. After air drying for 5-10 minutes, the slides were inverted, placed in a plate tray filled with RPMI 1640 medium and incubated for one hour at 3"7 ° C with 5% C02 in a humidified chamber. The excess liquid was dried from the slides and placed in a new tray of plates containing guinea pig complement diluted 1:50 in cold RPMI 1640. After incubation at 37 ° C for 4 hours, with 5% C02 in a chamber. humidified, the slides were carefully removed, the excess liquid was dried and incubated overnight at 41C in another plate dish with RPMI 1640. The plates were numbered the next day using an amplifier glass and a Monostat colony counter and the platelet number was normalized by one million splenic cells.The results, as shown in figure 1, demonstrate that the in vivo treatment of DBA / 2 mice with 0. 3-10 μg of IL-10 per day produced a statistically significant increase in the amount of PFC per million spleen cells after immunization with red lamb cells at 20% (upper) or 0.2 and 2% SRBC (lower).

Example 2 To demonstrate the effect of IL-10 on the secondary response of IgG to the SRBC antigen, DBA / 2 mice were injected intravenously with SRBC, twice for four weeks apart, and treatment of IL-10 started at the time of the second immunization as described above. The indirect PFC response was determined five days after the second injection by another modification of the Jerne slide method described above (Nordin et al., J. Immunol., 103, 859-863 (1969). prepared an additional series of slides which were incubated in RPMI 1640 for one hour as in the direct assay.The slides were then placed in a new plate tray and incubated with 0.5 mg / ml of Concanavalin A to block the activity of the IgM After incubation for two hours, these slides were rinsed in D-PBS and placed in a new plate tray containing 100 μg / ml of rabbit anti-mouse IgG for one hour.The slides were then transferred to a new tray of plates with guinea pig complement for three hours.The slides were then dried and stored at 41C until the next day when the counting was done.As shown in figure 2, the treatment was With 3 μg of IL-10 at the time of secondary immunization resulted in a statistically significant increase in the amount of PFC per one million spleen cells compared to mice treated with the vehicle.

Together, the results illustrated in Figures 1 and 2 indicate that the in vivo treatment of IL-10 significantly improves the primary and secondary antibody responses to the SRBC antigen.

Example 3 To determine whether the treatment of IL-10 can also improve the antibody response to the Pnu-immune polysaccharide vaccine 23, young mice (4 to 5 months old) and old mice (22 months old) BALB / c were immunized with 11.5 μg of Pnu-immune 23 vaccine by intraperitoneal injection, were treated daily with IL-10 or vehicle or intraperitoneal injection. 5 days after immunization with Pnu-immune 23, the mice were sacrificed and the splenic cells were isolated as above. The response of the PFCs to the Pnu-immune 23 antigen was tested as previously described (Garg and Subbarao, Infect. Immuni ty, 60, 164-169 (1992)); The SRBCs were washed three times in saline and coupled with the Pnu-immune 23 vaccine in the presence of chromium chloride (CrCl 3). The coupled SRBCs were then washed three times to remove any amount of free vaccine and CrC13. The PFC direct assay was performed as described above. In some experiments, the SRBCs were coupled to the bovine serum alumina using the same CrCl3 concentration to ensure that the PFC response being measured was vaccine-specific, as previously demonstrated (Garg and Subbarao, Infecí. Immuni ty 60, 164-169 (1992)); Garg, Kaplan and Bondada J. Immunol. , 152, 1589-1595 (1993). As shown in Figure 3, IL-10 significantly improved the PFC response to the vaccine in old mice, while there was no effect on the response in young mice. A dose of 0.3 μg of IL-10 was optimal for increasing the response to the vaccine in old mice.

Example 4 The in vivo effect of IL-10 was reproduced in an in vitro culture system to define the cellular requirements of the response to the vaccine Garg, Kaplan and Bondada J. Immunol. , 152, 1589-1595 (1993). For these studies, spleen cells from immunized mice (young and old) were isolated as specified above, and then cultured in a 1: 1 mixture of modified Dulbecco's modified Eagle's medium from Iscove and the F-12 medium from Ham supplemented with 10% calf serum, transferrin, insulin and traces of elements as described in the above (Moiser, J. Immunol., 127, 1490-1494 (1981).

Different doses of Pnu-immune 23 and IL-10 vaccine were added until the start of cultures and the cells were incubated at 37 ° C, with 5% C02 in a humidified atmosphere, for 5 days. The specific PFC numbers were quantified as described above. Previous work has shown that the SRBC-coupled vaccine is effective in detecting the PFC response to 21 of 23 polysaccharides contained in the vaccine (Garg, Kaplan and Bondada J. Immunol., 152, 1589-1595 (1993). of the spleen cells of old mice to elaborate a PFC response to this vaccine is especially compromised under these in vitro culture conditions As shown in Figure 4, 25 and 50 U / ml of IL-10 (equivalent to 6 and 12 μg / ml respectively) were able to restore the PFC response to the splenic cell cultures of old mice treated with 0.1 μg of Pnu-immune 23, and all concentrations of 10.50 U / ml and of IL-10 were able to restore the response of splenic cells of old mice to a? .01 μg of P u-immune 23. In contrast, the response of the PFC in the baseline splenic cells of young mice was much higher than that of old mice and all concentrations of IL -10 tested had no effect on this response. Other studies, as illustrated in Figure 5, show that 1-100 U / ml of IL-10 (equivalent to 0.25 and 25 μg / ml respectively) were able to significantly improve the PFC response of splenic cells of old mice for 1-100 ng of vaccine in culture. These results indicate that IL-10 is able to significantly increase the antibody response to the Pnu-immune 23 vaccine in cultures of splenic cells of old mice. This increase seems to result in the restoration of the response to the levels normally observed in young mice.

Example 5 For a better understanding of the effect of IL-10 to improve or restore the response of antibodies to the vaccine, the ability of IL-10 to increase the PFC response in the absence of T lymphocytes was evaluated. Spleen from old mice were treated with antibodies to the surface markers of specific T cells (Thy 1.2, CD4 and CD8) and rabbit complement to eliminate T cells from the sensitive population. The proliferative response induced by Concanavalin A of the resulting population was reduced 95% after depletion of T cells, indicating that T cells were almost completely eliminated. As shown in Figure 6, these splenic cells depleted in T cells responded to the Pnu-immune 23 vaccine when supplemented with IL-10 in a manner similar to intact splenic preparations. These data suggest that the effect of the adjuvant of IL-10 in old mice does not require the obligatory presence of T lymphocytes. IL-10 can act directly on B cells to favor their response to proliferation and / or differentiation to the vaccine . Alternatively, IL-10 can affect the macrophages or dendritic cells in the sensitive population and thus indirectly improve the response of the B cells. Although the present invention has been described together with the specific modalities established in the above, many alternatives as modifications and variations thereof will be apparent to those skilled in the art. These alternatives, modifications and variations are proposed within the spirit and scope of the present invention, which is limited only by the claims.

Claims (20)

1. A method for improving an immune response of a mammal to a vaccine, the method comprises administering to a mammal in need of vaccination an effective amount of interleukin-10 (IL-10) together with the vaccine.

2. The method of claim 1, wherein the mammal is human.

3. The method of claim 1 wherein the mammal is immunocompromised.

4. The method of claim 3, wherein the mammal is immunocompromised due to age.

The method of claim 1, wherein the IL-10 is administered in an amount of 2 to 150 μg per kilogram of body weight.

The method of claim 5, wherein the IL-10 is administered in an amount of 2-80 μg per kilogram of body weight.

The method of claim 5, wherein the IL-10 is administered in an amount of 25 μg per kilogram of body weight.

The method of claim 1, wherein the IL-10 is administered 2-4 days prior to administration of the vaccine.

9. The method of claim 1, wherein the vaccine is a vaccine-bacterial.

10. The method of claim 1, wherein the vaccine is a pneumococcal vaccine.

11. A method for improving an immune response of a mammal to a vaccine, wherein the mammal is immunocompromised due to age, the method comprising: administering to the mammal an enhancing amount of the immune response of interleukin-10 (IL-10) together with a vaccine.

12. The method of claim wherein the IL-10 is administered in an amount of 2-15 μg per kilogram of body weight.

The method of claim 12, wherein the amount of IL-10 that is administered is 2-80 μg per kilogram of body weight.

The method of claim 11, wherein the mammal is a human.

15. The method of claim 11, wherein IL-10 is administered 2-4 days prior to administration of the vaccine.

16. A pharmaceutical composition containing an enhancing immunological amount of interleukin-10 (IL-10); and a vaccine.

17. The pharmaceutical composition of claim 16, wherein the IL-10 is contained in a sustained release formulation.

18. A kit for improving an immunogenic response of a mammal to antigens present in a vaccine, the kit contains a container of an interleukin-10 (IL-10) pharmaceutical composition, and a pharmaceutically acceptable carrier therefor; and a container of a vaccine.

19. The kit of claim 18, wherein the IL-10 is contained in a sustained release formulation.

20. The kit of claim 18, wherein the vaccine is a pneumococcal vaccine.