MXPA00006071A - Lymphotactin as an adjuvant - Google Patents

Abstract

A method of enhancing immune response by administration of an immune-enhancing effective amount of lymphotactin in a pharmaceutically acceptable carrier.

Description

LYMPOTACTIN AS AN ADJUVANT FIELD OF THE INVENTION This invention relates to the use of lymphotactin as an adjuvant to boost the immune response.

BACKGROUND OF THE INVENTION The mechanisms of immune response involve both systemic and mucosal responses localized to pathogens and to vaccines. The response to the immunogen or pathogen can be cell-mediated or humoral. (See Fundamental Immunology, 3rd edition. (W.E. Paul, Editor), Raven Press, New York, (1993)). For example, many intestinal pathogens require a mucosal immune response to provide effective protection against the disease. The use of an adjuvant as a means to improve immunogenic responses has been known for a long time. Adjuvants can work in several ways. Some act on the immune system to induce a more effective antibody reaction against the antigen by activating host macrophages, dendritic cells, B cells and T cells, or by improving antigen presentation. The adjuvants REF.121282 can improve immune responses by prolonging antigen release, increasing antigen uptake, activating antigen processing, stimulating cytokine release, stimulating B cell activation or change as well as its maturation, and / or eliminating immunosuppressive cells. Currently known adjuvants include aluminum hydroxide and Freund's complete adjuvant. A list of the most effective adjuvants would include bacterial toxins which can be administered with the target immunogen. Sometimes, these molecules that increase the immune response bind to the toxin. However, many of these adjuvants cause serious unwanted effects. The U.S. patent 5,571,515 describes the use of IL-12 as an adjuvant for use for the improvement of cell-mediated immunity. There are four classes of chemokines: CC, CXC, C and Cx3C. The chemokine class C lymphotactin is similar to the CC and CXC chemokine families that are common in mammals and that are chemotactic for lymphocytes. In adult humans, the surface of the mucosa covers more than 300 m2 and requires a significant amount of lymphoid cells such as T cell receptors. (t dTCR), intrahepitelial lymphocytes (IEL), which produce lymphotactin and B cells, which produce secretory immunoglobulins (S-Ig), antibodies A (Abs).

The S-IgA Abs in the mucosa represents the first line of defense against invading pathogens or toxins that, if left unchanged, cause pathologies. Unfortunately, in the context of vaccine development, attempts to induce these protective Abs have so far not been successful.

BRIEF DESCRIPTION OF THE INVENTION This invention provides a means for improving the immune response, particularly the mucosal immune response by administering an effective enhancer amount of lymphotactin immunity, in a pharmaceutically acceptable carrier. Lymphotactin can be delivered to the mucosa together with an antigen. The mucosal medium of application includes oral, intranasal, ocular, intravaginal and / or intraurethral administration, in liquid or particulate form.

DETAILED DESCRIPTION OF THE INVENTION It has been known that lymphotactin has been produced predominantly by NK and CDX cells as well as the T-cell T (TCR) receptor of intraepithelial lymphocytes (IEL).

T t d cells of splenic origin do not produce lymphotactin in the same degree as similar and more abundant lymphocytes of mucosal origin, such as those of the upper and lower respiratory, gastrointestinal and reproductive tracts. The lymphotactin used in the examples is obtained from the DNAX Research Institute. Lymphotactin produced by recombinant technology is available from Research Diagnostics, Inc., Flanders, New Jersey. Lymphotactin is particularly effective in improving immune responses. Although bacterial toxins can reinforce S-IgA, these substrates have harmful side effects in humans and other mammals. Fortunately it is now possible, by the use of lymphotactin according to the teachings herein, to induce antigen-specific, protective S-IgA Abs in mucosal secretions. In addition, the strategy described herein initiates serum IgA, IgM and IgG with mixed T-helper cell responses type 1 and 2 (Th1 / Th2). The cell-mediated and humoral-comparative immune responses have been shown to protect laboratory animals against lethal doses of mucosal and systemic pathogens as well as toxins. C-chemokines such as lymphotactin can be used as an adjuvant in systemic and local vaccine preparations, particularly mucosal. These protein-based vaccines can facilitate mucosal and systemic immunity to immunogens provided they are administered in composition containing chemokine C and the target antigen in combination or if they are administered separately to improve the immune response to the antigen. The best known of these effective mucosal vaccines is the Salk vaccine of polio. Several antigens are available to increase the immune response to intestinal diseases such as diarrhea that occurs from E. coli or Shigella species. In all of these similar instances, the use of lymphotactin to improve the immune response would be appropriate.

MATERIALS AND METHODS Immunization All mice used were 8-10 weeks old and are C57BL / 6 mice (Charles River Laboratories, Wilmington, MA) housed in laminate cabinets. The mice were free of microbial pathogens, determined by systematic histological analysis. Mice are immunized intranasally with 10 μl (5 μl per nostril) of sterile, phosphate buffered saline (PBS), pH 7.5 containing 25 μg of chicken egg albumin (OVA from Sigma Chemical Col, St. Louis, MO) or only (without lymphotactin) or with 0.01, 0.1, 1.0 or 5 μg of murine lymphotactin on days 0, 7 and 14.

Collection of samples Serum samples are collected via retro-orbital puncture using sterile heparinized capillaries. Samples of vaginal discharge are obtained by washing the vaginal cavity with 50 μl of PBS three times for a total volume of approximately 150 μl. The stool is collected, weighed and dissolved in PBS containing 0.1% sodium azide (100 mg of fecal pellet per 1 ml of PBS / sodium azide). These samples are vortexed, centrifuged and the supernatants are collected for analysis. These mucosal and serum samples accumulate at weekly intervals and are analyzed for antigen (eg OVA), IgA, IgM, IgG, IgE, IgG1, IgG2a, IgG2b and IgG3 as specific antibody titers. Mice are sacrificed on day 21 for analysis of OVA-specific antibody forming cells and proliferating T cells as well as cytokine profile responses.

Cell preparation Suspensions of the submandibular and servical lymph nodes (SM / CLN), mesenteric lymph nodes (MLN), Peyer's patches (PP), vaginal ileal lymph nodes (ILN) and spleen suspensions (SP) are made by passing tissue through of a wire mesh. After the extraction of the PP, the small intestine is isolated to determine the secreting cells of Ig in the intestinal tract which are directly related to the protection against intestinal pathogens. The intestinal tissue is then gently cleaned, itched and treated with 1 mM EDTA in PBS at 37 ° C with shaking for 15 to 30 minutes. Then, these tissues are treated with collagenase in RPMI medium for about 1 hour. Finally, the lymphocytes of the lamina propria (LPL) are isolated using a percoll gradient (Pharmacia, Uppsala, Sweden). Tissue from the lower respiratory tract (lung) and salivary gland (SG) are isolated, cleaned, minced and washed in PBS. These tissues are also digested with collagenase, isolated and examined for antigen-specific Tg-secreting cells and T-cell-mediated immunity in the lung and salivary glands, which are important for lower and upper respiratory immunity.

Lymphoid tissue is isolated from the nasal tract and associated with the nasopharynx (NALT) and passed over sterile glass fiber to acquire a suspension of single cells of lymphocytes. The nasal tract and NALT are studied to determine the number of Ig secreting cells in the upper respiratory tract necessary for protection against respiratory pathogens and toxins.

Detection of titres of antibodies specific for antigen by ELISA and ELISPOT assays.

The antibody titres in sera and secretions were analyzed by ELISA to confirm the source of antigen-specific antibodies detected by ELISA, the quantification of antibody-specific point-forming cells for vaccine antigen from SP, PP, MLN, SM / CLN, lung and NALT and were enumerated by ELISPOT analysis.

Enumeration of cell proliferative responses T specific for antigen Spleen cells irradiated, killed in T cells (3,000 rads) of non-exposed mice are used as feeding cells for T cell proliferation assays. SM / CLN T cells, MLN, PP, lung, ILN and SP of immunized mice are purified using a nylon wool column (purified T cells (2.5 x 10 cells / ml) are cultured with or without 0.5 mg / ml OVA plus feeder cells (0.5 x 10b cells / ml) in complete RPMI medium in round-bottom tissue cultures in 96-well plates.The cells are incubated at 37 ° C in 5% C02.After 48 hours of incubation, 10 μl of each well is added to each well. 50 μCi / ml of [methyl-?] -thymidine Proliferation or uptake of thymidine is measured 18 hours later The stimulation index of the various samples is determined and expressed as counts per minute (CPM) of the cultures containing OVA type divided among the CPM of cultures lacking OVA Also studied were CD4 T cells that were isolated using a mouse CD4 isolation column.

RESULTS The administration of lymphotactin together with the vaccine results in an increase in the titers of IgA, IgG, (IgGl, IgG2a, IgG2b, IgG3) and IgM in serum. There is also an increase in fecal IgA and IgG, as well as in vaginal IgA and IgG. Additionally, increased proliferation of antigen-specific T cells of immunized mice is also observed in lymphocytes isolated from SM / CLN, MLN, PP, spleen, lung and ILN. NALT, SP, PP, MLN, lung and SM / CLN antibody-spotting cells are shown to secrete antigen-specific IgA, IgM, and IgG antibodies. Therefore, it can be seen that immune responses increase due to mucosal exposure to lymphotactin. Lymphotactin compositions in cellular immune enhancing amounts can advantageously be administered at very low levels together with vaccines. A suggested dosage such as 1 to 10 μg can be administered in small animals and from 10 μg to 10 mg in larger animals. Lymphotactin can be administered in conventional pharmaceutical carriers such as saline, buffered saline, glucose, etc. Lymphotactin can be administered to the mucosa in any way. Preferred methods of administration involve direct application to mucous membranes. Such compositions can be provided, for example, in the form of drops, such as in the nose, ears or in the eyes, or as sprays. Dry preparations such as lyophilized lyophilized with sprayed carriers can be inhaled, for example, or sprayed onto the mucosa. Such compositions may also be provided in capsules or in the form of tablets for ingestion. The lymphotactin can also be administered on a solid support such as a sponge or fiber material. Such administration is particularly useful for use in environments where access to sterile equipment is limited. The compositions for oral ingestion may be coated with enteric layer. Additionally, adjuvants can be added to liquids or solids for administration by mouth. For example, the adjuvants of the invention can be administered in food or water or on solid supports such as sponges and fabrics. For example, adjuvants can be administered in or on baits. The adjuvants can be administered orally in alkaline solutions containing appropriate antigens to increase the antibodies against organisms which produce intestinal diseases, to increase the antibodies in the mucosa. Alkaline solutions, such as those containing bicarbonates, protect antigens from adjuvants from destruction in the upper gastrointestinal tract. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (6)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for improving the immune response by administering an effective amount enhancing the immune response of lymphotactin in a pharmaceutically acceptable carrier.

2. The method in accordance with the claim 1, characterized in that lymphotactin is administered directly to the mucosa.

3. The method in accordance with the claim 2, characterized in that the lymphotactin is administered in the form of drops or a spray.

4. The method in accordance with the claim 2, characterized in that lymphotactin is administered in powder form.

5. The method according to claim 1, characterized in that the lymphotactin is on a solid support.

6. The method in accordance with the claim 1, characterized in that lymphotactin is administered orally. T.TTJFOTACTTNA AS AN ADJUVANT SUMMARY OF THE INVENTION A method for improving the immune response is described by administering a lymphocytically effective immunocomplex amount in a pharmaceutically acceptable carrier.