There is described a method for selecting microbial isolates for use in oral killed vaccines against abnormal microbial colonization of mucosal surfaces by the microbes. The method comprises evaluating capacity of a plurality of different isolates of a microbe to activate antigen responsive cells to provide activation data for each microbial isolate, and the effectiveness of the isolates in reducing infection of a mucosal surface by the microbe to provide clearance data for each microbial isolate. An isolate, the activation data and clearance data for which correlate and is optimal for generating mucosal immunity against the microbe compared to the, or each, other of the isolates, or an isolate the activation data for which is optimal and a further isolate the clearance data for which is optimal, compared to the, or each, other of the isolates, respectively, is then selected for use in the vaccine. There is also described a method for providing an oral killed vaccine against abnormal microbial colonization of a mucosal surface, comprising evaluating the capacity of a plurality of different isolates of a microbe to induce expression of IL-10 and IL-12 in antigen responsive cells. At least one isolate is selected that induces optimal expression of IL-12 relative to IL-10 compared to the, or each, other of the isolates, respectively, for use in the vaccine.

Description of the Invention

SUMMARY OF THE INVENTION

Broadly stated, the present invention relates to the provision of oral killed vaccines for protecting against abnormal microbial colonization and stems from the recognition that there is a marked variation in the clearance of such infections elicited by oral killed vaccines in an outbred population, reflecting the genetic variation in the population. The variation in mucosal immunity associated with the use of prior art oral killed bacterial vaccines is believed to arise from the use of less optimal or randomly chosen microbial isolates in the vaccines, due to the failure to recognize the significant variability in the capacity of different isolates of a microbe to activate antigen-presenting cells and T-lymphocytes. Given the observed variability, the selection of the isolate(s) is critical for optimizing the degree of activation of the common mucosal system in different individuals in an outbred population. Methodology provided in one or more embodiments of the present invention enables the selection of isolate(s) for optimizing oral killed vaccines.

More particularly, in one aspect of the present invention there is provided a method for selecting a microbial isolate for an oral killed vaccine against abnormal microbial colonization of a mucosal surface, the method comprising: evaluating capacity of a plurality of different isolates of a unicellular microbe to activate antigen responsive cells to provide activation data for each microbial isolate; evaluating effectiveness of the isolates in reducing infection of a mucosal surface by the microbe to provide clearance data for each microbial isolate; and selecting an isolate from the microbial isolates, the activation data and clearance data for which correlate and is optimal for generating mucosal immunity against the microbe compared to the, or each, other of the isolates, or an isolate the activation data for which is optimal and a further isolate the clearance data for which is optimal, compared to the, or each, other of the isolates, respectively, for use in formulating the vaccine.

In another aspect of the present invention there is a method for providing an oral killed vaccine against abnormal microbial colonization of a mucosal surface, the method comprising: evaluating capacity of a plurality of different isolates of a unicellular microbe to activate antigen responsive cells to provide activation data for each microbial isolate; evaluating effectiveness of the isolates in reducing infection of a mucosal surface by the microbe to provide clearance data for each microbial isolate; selecting an isolate from the plurality of isolates, the activation data and clearance data for which correlate and is optimal for generating mucosal immunity against the microbe compared to the, or each, other of the isolates, or an isolate the activation data for which is optimal and a further isolate the clearance data for which is optimal, compared to the, or each, other of the microbial isolates, respectively, for use in formulating the vaccine; and formulating the vaccine using the selected isolate or isolates.

Typically, an isolate for which both the activation data and the clearance data is optimal compared to the other of the isolates will be selected.

Preferably, the mucosal immunity will comprise predominantly a cellular immune response.

Preferably, the isolates utilized for providing the activation and clearance data will be killed isolates of the microbe. However, the invention is not limited thereto and the activation and clearance data may be obtained from live isolates and the selected isolate(s) subsequently killed for use in the vaccine.

The antigen responsive cells activated by the isolate(s) will normally comprise one or both of antigen presenting cells and T-lymphocytes and preferably, will comprise both types of cells. The antigen presenting cells will typically comprise macrophages. Most preferably, the T-lymphocytes will be Th1 cells. The activation of the antigen responsive cells is to be taken in its broadest sense to encompass direct and/or indirect activation by the isolate(s). By "direct" activation is meant the isolate(s) activate at least some of the antigen responsive cells by contact with the cells such as when a microbial isolate is bound or phagocytosed by them. By "indirect" activation is meant at least some of the antigen responsive cells are activated by interaction with the cells such as macrophages that have contacted the isolate or for instance, by cytokine(s) or other chemical messenger(s) the release of which has been elicited or induced by the isolate(s), or by substances such as toxins or antigens secreted by the isolate(s), or a combination of the foregoing possibilities.

The level of activation of the antigen responsive cells can be evaluated by measuring one or more parameters associated with activation of the cells. Preferably, the capacity of the isolate(s) to activate both antigen presenting cells and T-lymphocytes will be evaluated. In particularly preferred embodiments, the activation of the antigen responsive cells achieved by each isolate will be evaluated by measuring at least one parameter indicative of the level of activation of the antigen presenting cells and at least one other parameter indicative of the level of activation of the T-lymphocytes. Most preferably, isolate(s) the activation data for which is optimal for activating both antigen presenting cells and T-lymphocytes compared to the other isolate(s) tested will be selected for use in the preparation of the oral killed vaccine.

Preferably, an isolate the activation data for which is indicative of the capacity of the isolate to elicit a cytokine response characterized by an IL-10:IL-12 ratio of about 30 or less will be selected for use in formulating the oral killed vaccine.

Hence, in another aspect of the present invention there is provided a method for selecting a microbial isolate for an oral killed vaccine against abnormal microbial colonization of a mucosal surface, the method comprising: evaluating capacity of a plurality of different isolates of a unicellular microbe to induce expression of IL-10 and IL-12 in antigen responsive cells; and selecting at least one isolate from the microbial isolates, that induces optimal expression of IL-12 relative to IL-10 compared to the, or each, other of the isolates, respectively, for use in formulating the vaccine.

In still another aspect of the present invention there is provided a method for providing an oral killed vaccine against abnormal microbial colonization of a mucosal surface, the method comprising: evaluating capacity of a plurality of different isolates of a unicellular microbe to induce expression of IL-10 and IL-12 in antigen responsive cells; selecting at least one isolate from the microbial isolates, that induces optimal expression of IL-12 relative to IL-10 compared to the, or each, other of the isolates, respectively, for use in formulating the vaccine; and formulating the vaccine using the isolate.

The unicellular microbe can be any such microbe having the capacity to colonize a mucosal surface of a mammal and may for instance be selected from the group consisting of bacteria, fungi and yeast. Typically, the microbe will be a bacteria and the vaccine will therefore be an oral killed bacterial vaccine. Preferably, the selected isolate(s) will be used in the vaccines of the invention as whole killed organisms. However, the invention is not limited to the use of whole killed organisms and vaccines may be provided comprising particulate matter derived from the outer cellular membrane of the selected isolate(s).

In yet another aspect, the present invention relates to an oral killed vaccine provided by a method of the invention.

A vaccine of the invention may be directed against infection of any mucosal site including chronic and acute such infections. The infection can be the result of transient exposure to a microbial pathogen which does not normally colonise the mucosal site or for instance, an opportunistic infection arising from microbial flora normally found at the site.

Accordingly, in another aspect, there is provided a method for the prophylaxis or treatment of an infection of a mucosal surface in a mammal by a unicellular microbe, the method comprising: administering to the mammal an effective amount of an oral killed vaccine of the invention for generating mucosal immunity against the microbe.

The mammal may be any mammal treatable with an oral killed bacterial vaccine of the invention. For instance, the mammal may be a primate, a member of the rodent family such as a rat or mouse, or a member of the bovine, porcine, ovine or equine families. Preferable, however, the mammal will be a human being.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Vaccines embodied by the invention find particular application in the prophylaxis or treatment of lung and upper respiratory tract infections. However, the invention is not limited thereto and mucosal immunity resulting from activation of the common mucosal system may provide protection or treatment against infections at other mucosal sites of the body including infections of the oral, nasal, oropharyngeal, nasal pharyngeal, pharyngeal, digestive, vaginal, eye associated, and urinary mucosal surfaces. The vaccine may contain bacteria selected for instance from Chlamydia species, Haemophilus species, Non-typeable Haemophilus species, Pseudomonas species, Streptococcus species, Staphylococcus species, E. coli species, Mycoplasma species and Helicobacter species amongst others, or incorporate combinations of different species of bacteria or of other unicellular microbes. Microbes other than bacteria that may be used in oral killed vaccines according to the invention include Candida species such as Candida albicans and yeast species such as Saccharomyces species. Particularly preferred oral killed bacterial vaccines embodied by the invention are vaccines for the prophylaxis or treatment of mucosal infections by bacteria selected from the group consisting of Non-typeable H. influenzae (NTHi), S. aureus, P. aeruginosa, S. pneumoniae and combinations thereof.

While the primary application of a vaccine embodied by the invention is to generate mucosal immunity against the particular bacterial infection(s) for which the vaccine is provided, which may occur at various mucosal sites, the vaccine can also be used for the treatment or prophylaxis of diseases or conditions exacerbated by the infection(s).

P. aeruginosa for instance can colonise not only the respiratory tract but can also infect eye mucosa and the ear cavity. Non-typeable H. influenzae (NTHi) has also been implicated in a range of infectious conditions including otitis media and in the exacerbation of pneumonia and chronic bronchitis. Accordingly, a vaccine containing one or more killed NTHi isolates of this bacteria may be administered for the prophylaxis or treatment of those conditions. Similarly, vaccines of the invention comprising killed H. influenzae, S. pneumoniae or P. aeruginosa may be utilized in the prophylaxis or treatment of bronchitis or pneumonia, and acute infections in cystic fibrosis and chronic obstructive airways disease, sinus disease, compromised lung function and other lung and respiratory tract diseases and disorders. These vaccines also find particular application in the prophylaxis or treatment of superinfection by the corresponding bacteria following infection by influenzae virus or other virus, particularly in the elderly.

While it is preferable to use whole killed isolate(s) in vaccines of the invention, particulate cell surface matter comprising surface antigens of the isolate(s) may be utilized as well, or instead of, whole killed organisms. In a particularly preferred embodiment, the outer cellular membrane fraction of the organisms will be utilized. The particulate matter can be prepared by disrupting killed or viable selected isolate(s) by sonication or other suitable technique and if desired, separating the required fraction from other cellular components such as by centrifugation, filtration and/or other appropriate technique known in the art. Any suitable method which achieves the required level of cellular disruption may be employed including sonication or dissolution utilizng appropriate surfactants and agitation. When sonication is employed, the isolate may be subjected to a number of sonication steps in order to obtain the desired degree of cellular disruption or generation of particulate matter of a desired size. The fraction of particulate matter utilized may be selected by comparing the response of antigen responsive cells to different fractions of the isolate(s) and selecting the fraction which maximizes the immune response by the cells.

To evaluate the capacity of a microbial isolate or particulate matter thereof to activate the antigen responsive cells, any parameter which is indicative of the level of activation of the cells may be evaluated. Particularly, preferred parameters include one or more of cellular proliferation, cell surface antigen expression, measurement of cell effector functions, and cytokine production.

Cellular proliferation and in particular, T-cell proliferation, may be conveniently evaluated by cell counts, .sup.3H-thymidine uptake and/or MTT assays.

Cytokine expression may be measured directly by capture or sandwich enzyme linked immunosorbent assays (ELISA), or indirectly by cell growth assays in which the cytokine of interest acts as a growth factor or inhibitor. Similarly, cytokine expression may be evaluated by determining the level of expression of mRNA coding for the cytokine by employing reverse transcriptase polymerase chain reaction (RT-PCR) or by in-situ hybridization protocols utilizing single cells and specific oligonucleotides probes as is known in the art.

The protective immune response generated by the vaccine will typically be predominantly mediated by Th1 T-lymphocytes which differentiate from proliferating CD4.sup.+ T-lymphocytes in the presence of IL-12 and IFN-.gamma.. IL-12 is produced by antigen presenting cells in the early stages of activation. Th1 T-lymphocytes stimulate infected macrophages through secretion of IFN-.gamma. and interaction of the CD40 ligand expressed by Th1 cells with the CD40 receptor expressed by macrophages. More broadly, Th1 cells stimulate the antibacterial mechanisms of phagocytic cells (eg. neutrophils and macrophages) and release cytokines that attract such phagocytic cells to sites of infection. Besides IFN-.gamma., Th1 cells typically also secrete IL-12 and TNF-.beta..

While both Th1 and Th2 cells secrete IL-3, GM-CSF and for instance TNF-.alpha., the overall cytokine profiles for Th1 and Th2 cells are different. More particularly, activation of Th2 cells results predominantly in a humoral immune response characterized by the activation of B-lymphocytes and the generation of antibodies by the activated B cells, while Th1 cells mediate a predominantly non-antibody cellular immune response. Cytokines characteristic of Th2 cell driven immune response include IL-4, IL-5, IL-10, IL-13 and TGF-.beta.. Hence, detection of the secretion of one or more of IL-12, IFN-.gamma., or other cytokines characteristic of activated antigen-presenting cells and Th1 committed CD4.sup.+ T-lymphocytes, is useful in evaluating the capacity of a given microbial isolate to activate the common mucosal system. Preferably, the level of IL-12 secretion will be measured to provide an indication of the degree of activation of antigen presenting cells by the microbial isolate(s) being evaluated. Similarly, the level of IFN-.gamma. secretion will typically be measured to provide an indication of the level of T-lymphocytes activation achieved by the microbial isolate(s).

In particularly preferred embodiments, the activation data may comprise a ratio indicating expression IL-10 relative to IL-12. IL-10 inhibits the release of cytokines such as IL-12 by macrophages and so inhibits Th1 cell activation. The ratio is therefore indicative of the level of a Th1 lymphocyte response elicited by a microbial isolate. Thus, an isolate selected for activating the antigen responsive cells will desirably elicit a cytokine response characterized by a low IL-10:IL-12 ratio but high expression of IFN-.gamma..

Preferably, the ratio will be less than 30, more preferably less than 20, 15, 10, 5 and even 4.

The vaccine will typically comprise the selected bacterial isolate(s) in an amount of between about 5% to about 80% w/w of the vaccine composition. As will be appreciated, the number of each isolate in the vaccine will be such that an effective dosage will be delivered to the mammal for activation of the common mucosal system taking into account the proposed mode of delivery and nature of the vaccine (eg. powder, liquid, aerosol delivery etc). The dosage of the, or each, bacterial isolate administered will typically be in a range of from about 10.sup.9 to about 10.sup.12 cfu, and more preferably from about 10.sup.10 to about 10.sup.11 cfu, respectively. The optimum dosage of a selected bacterial isolate can be determined by administering different dosages of the isolate to different groups of test mammals, prior to subsequently infecting the animals in each group with the corresponding live bacterial pathogen, and determining the dosage level required to achieve satisfactory clearance of the pathogen as would be well understood by the skilled addressee.

The vaccine itself may be freeze-dried or lyophilized for later reconstitution utilizing a physiologically acceptable buffer or fluid. The vaccine can also contain one or more anti-caking agents, isotonic agents, preservatives such as thimerosal, stablizers such as amino acids and sugar moieties, sweetening agents such sucrose, lactose or saccharin, pH modifiers sodium hydroxide, hydrochloric acid, monosodium phosphate and/or disodium phosphate, a pharmaceutically acceptable carrier such as physiologically saline, suitable buffers, solvents, dispersion media and isotonic preparations. Use of such ingredients and media for pharmaceutically active substances and vaccines is well known in the art. Except insofar as any conventional media or agent is incompatible with the bacterial isolate(s), their use in vaccines of invention is specifically encompassed. Supplementary active agents such as one or more cytokines for boosting the immune response, particularly cytokines characteristic of a Th1 response such as IFN-.gamma., IL-12 and TNF-.beta., can also be incorporated in the vaccine if desired.

In addition, a vaccine embodied by the invention may also comprise one or more adjuvants. Suitable adjuvants, pharmaceutically acceptable carriers and combinations of ingredients useful in vaccine compositions of the present invention may for instance be found in handbooks and texts well known to the skilled addressee such as "Remington" The Science and Practice of Pharmacy (Mack Publishing Co., 1995)", the contents of which is incorporated herein in its entirety by reference.

The oral killed bacterial vaccine may be administered as a dry powder or in liquid form. Administration can for example be achieved by aerosol inhalation, as a dosed liquid, by instillation, or as a spray. Devices for facilitating for delivery of oral vaccines are well known in the art and include metered dose inhalers (MDIs), dry powder inhalers (DPIs) and nebulisers including those which use ultrasonic energy or compressed air or other propellant to achieve atomisation. Propellants which may be used in MDIs include for instance chlorofluorocarbons (CFCs) such as trichlorofluorocarbon (CFC-11) and dichlorodifluorocarbon (CFC-12) and hydrofluoroalkanes.
 

Claim 1 of 42 Claims

1. A method for selecting at least one microbial isolate for use in an oral killed vaccine against abnormal microbial colonization of a mucosal surface, of the selected microbial isolate or isolates, the method comprising: evaluating capacity of a plurality of different isolates of a unicellular microbe to activate antigen responsive cells to provide activation data for each microbial isolate; evaluating effectiveness of the isolates in reducing infection of a mucosal surface by the microbe to provide clearance data for each microbial isolate; and selecting at least one isolate from the microbial isolates, the activation data and clearance data for which correlate and is optimal for generating mucosal immunity against the microbe compared to the, or each, other of the isolates, or an isolate the activation data for which is optimal and a further isolate the clearance data for which is optimal, compared to the, or each, other of the isolates, respectively, for use in formulating the vaccine.
 

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