WO2008109958A1

WO2008109958A1 - Methods for evaluation of oral vaccines

Info

Publication number: WO2008109958A1
Application number: PCT/AU2008/000360
Authority: WO
Inventors: Margaret Dunkley; Robert Clancy
Original assignee: Hunter Immunology Limited
Priority date: 2007-03-15
Filing date: 2008-03-14
Publication date: 2008-09-18

Abstract

There is provided a method for assessing the efficacy of an oral vaccine against infection or colonisation of a mucosal surface of the respiratory tract by a microbial pathogen. The method comprises measuring at least one parameter of at least one individual orally vaccinated with the vaccine. The parameter can be selected from the group consisting of: (i) a level of T lymphocytes in blood from the individual responsive to the pathogen involving antigen induced proliferation of the T lymphocytes without limiting dilution analysis of the T lymphocytes, (ii) IgG level in a body fluid from the individual, and (iii) at least one marker of inflammation at a mucosal surface of the respiratory tract of the individual. The measured level is compared with a respective control level, and the effectiveness of the vaccine is evaluated on the basis that a higher level of (i) and/or a lower level of (ii) and/or (iii) for the vaccinated individual compared to the control level is indicative the vaccine is efficacious against the pathogen. The method is particularly suitable for assessing efficacy of an oral killed vaccine.

Description

METHODS FOR EVALUATION OF ORAL VACCINES

FIELD OF THE INVENTION

The invention relates to methods for assessing effectiveness of oral vaccines. Various embodiments of the invention find application in one or more of the selection of isolate(s) for use such vaccines, comparison of vaccines containing different isolates and for instance, monitoring of vaccines in clinical trials.

BACKGROUND OF THE INVENTION

Oral killed bacterial preparations have been developed to effectively control recurrent acute bronchitis in subjects with damaged airways that have become colonised by non-invasive bacteria such as Non-typable Haemophilus influenzae (NTHi), but less commonly Pseudomonas aeruginosa, Staphylococcus aureus and others. A similar pathogenic process occurs in other areas of the respiratory tract, namely the middle ear, the paranasal sinuses, and the nasal cavity. Development of an optimal oral killed vaccine has been limited by the absence of useful surrogate markers of protection eg., effect on antibody level in serum or secretions. NTHi is the major species involved in endobronchitis in those with damaged airways (eg., smokers) and only highly sensitive assays such as limiting dilution analysis have been able to detect sensitisation of T cells following oral administration of NTHi. The more practical (and less sensitive) assays in particular antigen-induced lymphocyte stimulation have not been considered useful in assessing the efficacy of the less immunogenic bacterial species such as NTHi.

The mechanism of action of orally administered killed bacteria in reducing acute bronchitis involves activating Peyer's patch derived T cells which home to the bronchus mucosa. Upon re- stimulation by colonising bacteria the T cells activate and recruit neutrophils which reduce the numbers of bacteria within the airways. In the case of respiratory infection, secretory IgA and T cell activation of phagocytic cells have been implicated in protection against infection. Animal studies have shown that for Pseudomonas aeruginosa infection, IgG can cross from the blood (serum) into the lower airways and contribute to protection . Finding appropriate surrogate markers of protection for mucosal vaccines is a challenge as sampling at the mucosal surfaces is difficult.

SUMMARY OF THE INVENTION

In an aspect of the invention there is provided a method for evaluating the efficacy of an oral vaccine against infection or colonisation of a mucosal surface of the respiratory tract by a microbial pathogen, comprising:

(a) measuring at least one parameter of at least one individual orally vaccinated with the vaccine, the parameter being selected from the group consisting of: (i) a level of T lymphocytes in blood from the individual responsive to the pathogen, involving antigen induced proliferation of the T lymphocytes in the absence of limiting dilution analysis of the T lymphocytes;

(ii) an IgG level in a body fluid from the individual; and (iii) at least one marker of inflammation at a mucosal surface of the respiratory tract of the individual, the marker being provided in a biological sample from the vaccinated individual;

(b) comparing the measured level to a respective control level; and

(c) evaluating effectiveness of the vaccine on the basis that a higher level of (i) and/or a lower level of (ii) and/or (iii) for the vaccinated individual compared to the control level is indicative the vaccine is efficacious against the pathogen; and when the pathogen is a bacteria and parameter (i) only is measured, the bacteria is Non-typable Haemophilus influenzae (NTHi).

In another aspect of the invention there is provided a method for evaluating the efficacy of an oral vaccine against infection or colonisation of a mucosal surface of the respiratory tract by NTHi, comprising: measuring a level of T lymphocytes responsive to the NTHi in blood from at least one individual orally vaccinated with the vaccine, the measuring involving antigen induced proliferation of the T cells in the absence of limiting dilution analysis of the T cells; comparing the measured level of the T lymphocytes to a control level; and evaluating effectiveness of the vaccine on the basis that a higher level of the T lymphocytes in the blood of the vaccinated individual compared to the control level is indicative the vaccine is efficacious against NTHi.

In another aspect of the invention there is provided a method for evaluating the efficacy of an oral vaccine against infection or colonisation of a mucosal surface of the respiratory tract by a microbial pathogen, comprising: measuring an IgG level in a body fluid from at least one individual orally vaccinated with the vaccine; comparing the measured IgG level to a control level; and evaluating effectiveness of the vaccine on the basis that a lower IgG level in the blood plasma of the vaccinated individual compared to the control level is indicative the vaccine is efficacious against the pathogen.

In another aspect of the invention there is provided a method for evaluating the efficacy of an oral vaccine against infection or colonisation of a mucosal surface of the respiratory tract by a microbial pathogen, comprising: measuring a level of at least one marker of inflammation at a mucosal surface in the respiratory tract of at least one individual orally vaccinated with the vaccine, the marker being provided in a biological sample from the vaccinated individual; comparing the measured level of the marker to a control level; and evaluating effectiveness of the vaccine on the basis that a lower level of the marker in the vaccinated individual compared to the control level is indicative the vaccine is efficacious against the pathogen.

Typically, the measured level of the marker will be compared with that of at least one individual not vaccinated with the vaccine.

Typically also, at least the level of IgG will be measured in embodiments of the invention. The body fluid assayed for IgG level will normally be saliva or blood plasma. The blood plasma will normally be blood serum. In at least some embodiments, the level of IgG and at least one other of the parameters will be measured. The infection can be an acute or chronic infection of the mucosal surface, and can be the result of transient exposure to the pathogen or for example, an opportunistic infection of the microbial surface by the pathogen.

The mucosal surface can be the surface of upper non-gas exchange airways of the individual (e.g., surfaces of the nasal cavity, paranasal sinuses, middle ear and throat), or the lower gas exchange surfaces of the individual (e.g., lung tissue). Typically, the mucosal surface will be a mucosal surface of the non-gas exchange airways.

The microbial pathogen will normally be selected from the group consisting of bacterial and fungal pathogens having the capacity to colonise a mucosal surface of the respiratory tract. Typically, the pathogen will be a non-invasive bacterial pathogen. Usually, the bacterial pathogen will be NTHi.

The pathogen can be utilised in the vaccine as whole killed organism(s). However, the invention is not limited to the use of whole killed organisms and vaccines may be used comprising inactivated (e.g., attenuated) organisms, or soluble or particulate antigen from the pathogen. Moreover, the vaccine can comprise antigen from a single isolate of the pathogen or antigen from a plurality of different isolates of the pathogen. Typically, the vaccine will be an oral killed vaccine comprising onr or more whole killed microbial pathogens. The individual can be any mammal that can be vaccinated with the oral killed vaccine. For instance, the individual can 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. Typically, however, the individual will be a human being.

Advantageously, comparison of the parameter(s) measured for different isolates of the pathogen allows identification and thereby selection of the isolate most effective for vaccine use and likewise, selection of the most effective vaccine for clinical trial use. Moreover, one or more forms of methods embodied by the invention can allow identification of an optimized dose and/or dosing regimen for the vaccine. That is, by administering different dosages of the vaccine and/or vaccinating individuals with the vaccine using different dosing regimens and measuring one or more of parameters (i) - (iii), the most effective of the dose or dosing regimen of the vaccine can be elucidated. Similarly, at least some forms of methods of the invention have application in monitoring the effectiveness of the vaccine in clinical studies on the vaccine, evaluating stability of the vaccine over time and under different storage conditions as well as for instance, evaluation of different formulations of the vaccine including forms of the vaccine employing different whole killed isolates of the pathogen and/or antigen from different isolates of the pathogen. All such methods and applications are expressly encompassed by the invention.

In this specification the terms "T lymphocytes" and "T cells" are used interchangeably.

Moreover, 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 that has been included in this 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 anywhere before the priority date of this application. The features and advantages of methods of the invention will become further apparent from the following detailed description of a number of embodiments thereof.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 is a graph showing NTHi antigen specific proliferation in placebo and treatment groups immunized with NTHi antigen;

Figure 2 is a graph showing mean number of NTHi isolated in gargle of the placebo group;

Figure 3a is a graph showing serum NTHi-specific IgG levels following immunization with NTHi antigen;

Figure 3b is a graph showing saliva NTHi-specific IgG levels following immunization with NTHI antigen; Figure 4 is a graph showing change in serum IgG versus number of visits at which H. influenzae was detected in the throat gargle following immunization with NTΗi antigen;

Figure 5a is a graph showing serum NTΗi-specific IgA levels following immunization with NTΗi antigen;

Figure 5b is a graph showing saliva NTΗi-specific IgA levels following immunization with NTΗi antigen;

Figure 6a is a graph showing saliva lysozyme levels following immunization with NTΗi antigen; and Figure 6b is a graph showing saliva lactoferrin levels following immunization with NTΗi antigen.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Bacteria-specific T cells migrate from Peyer's patches in the gut (the site of vaccine uptake and immune induction in the gut) via thoracic duct lymph and then the blood circulation to mucosal sites including the lung. It is possible to detect these cells by sampling blood, preparing and culturing the lymphocyte in vitro in the presence or absence of vaccine antigen. The antigen- specific T cells proliferate and this can be quantitated thus giving a measure of vaccine efficacy in inducing such cells. The vaccine- specific T cells that migrate to the respiratory tract secrete cytokines that activate phagocytic cells (eg., macrophages and neutrophils) that engulf and kill the infecting bacteria. However, only some of the T cells migrating to the lung can be sampled and the peak flow of the cells in the blood may be missed. Such antigen induced proliferation of T lymphocytes has prior to the present invention not been considered useful for the assessment of the efficacy of oral killed vaccines for less immunogenic bacteria such as NTHi as only low numbers of T cells are responsive to the vaccine antigen.

As a result, more sensitive methods of analysis such as limiting dilution analysis of T cells have been used to identify the frequency of responsive T cells. Limiting dilution analysis employs a statistical function (e.g., the Poisson distribution) and the culturing of serial dilutions of T cells in the presence of vaccine antigen, antigen presenting cells (eg., monocytes) and growth factors. Response to the antigen is assessed by assaying for specific cytokine release or other parameter indicative of lymphocyte activation. The frequency of T cells responsive to the antigen can be determined from the statistical function employed. However, this method of analysis is relatively complicated and not as practical as classical antigen -induced proliferation of T cells not involving limiting dilution analysis.

Surprisingly, the inventors have found that numbers of T cells responsive to NTHi can be readily determined by such antigen-induced proliferation. More particularly, studies by the inventors have shown that the relative contribution of the proliferative response to the vaccine antigen and normal exposure to the pathogen can be determined, allowing the efficacy of the vaccine to be assessed.

The inventors have also found that a reduced level of one or more markers of inflammation at the mucosal surface can be indicative of the effectiveness of an oral vaccine as described herein. A previous study indicated that increased levels of markers of inflammation would be expected to correlate with protection against infection². It would, for example, have been expected that the presence of bacteria at a mucosal surface would result in the recruitment of inflammatory cells to the site of infection resulting in an increase in the production/release of markers of inflammation at the mucosal site. The finding that a lower level of marker(s) of inflammation is indicative of the efficacy of an oral killed vaccine was not expected. Likewise, the finding that a lower IgG level in blood plasma and particularly serum is indicative of the efficacy of the vaccine is also unexpected given that IgG can cross from the blood (serum) into the lower airways and contribute to protection. Hence, it would be assumed that a higher level of IgG in blood plasma/serum would be indicative of vaccine efficacy rather than a lower level of IgG compared to unvaccinated individual(s).

In one or more forms, the bacterial pathogen can for example be selected from the group consisting of Non-typable Haemophilus influenzae (NTHi), Haemophilus species, Pseudomonas species, Streptococcus species, Staphylococcus species, E. CoIi species, Moraxella species, and Mycoplasma species. Specific examples besides NTHi include S. aureus, Ps. aeruginosa, S. pneumoniae, S. Albus, S. pyrogenes, M. pneumoniae, M. tuberculosis, M. catarrhalis and combinations thereof. Fungal pathogens include Candida species, Aspergillus species, and Saccharomyces species, such as Candida albicans, Aspergillus flavus, and Saccharomyces cerevisiae.

While the primary application of a vaccine embodied by the invention is to generate mucosal immunity against the particular pathogen(s) for which the vaccine is provided, the vaccine can also be used for the treatment or prophylaxis of diseases or conditions related to, or exacerbated by, the infection(s). NTHi for instance has been implicated in a range of infection related conditions including otitis media, sinusitis and in the exacerbation of pneumonia, chronic obstructive pulmonary disease (COPD) and bronchitis, particularly COPD and chronic bronchitis . Applicants co-pending

International Patent Application filed concurrently herewith entitled "Treatment or prophylaxis of asthma" and claiming priority from Australian Provisional Patent Application No. 2007901326 filed 15 March 2007, relates to the finding that administration of NTHi vaccine can reduce asthma treatments and the related use of asthma medication, and the contents of that application are incorporated herein in their entirety by cross-reference.

Accordingly, a vaccine containing one or more killed or inactivated NTHi isolates of this bacteria for the prophylaxis or treatment of those conditions can be assessed by methods embodied by the invention. Likewise, vaccines comprising killed or inactivated H. influenzae, S. pneumoniae or P. aeruginosa can be utilised 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 bacteria following infection by influenzae virus or other virus particularly in the elderly, and all such vaccines can be assessed by methods embodied by the invention. Candida albicans for instance is the causative agent of candidiasis.

While it is preferable to use whole killed isolate(s) of the pathogen in the vaccines, soluble or particulate cell surface matter comprising or consisting of surface antigens of the isolate(s) can also be utilised as well as, or instead of, whole killed organisms. In one or more embodiments, the outer cellular membrane fraction of the organisms and/or specific cellular membrane antigens will be utilised. Soluble and/or particulate antigen can be prepared by disrupting killed or viable selected isolate(s) . A fraction of the disrupted organism(s) can then be prepared by centrifugation, filtration and/or other appropriate techniques known in the art. Any suitable method which achieves the required level of cellular disruption may be employed including sonication or dissolution utilising appropriate surfactants and agitation, and combinations of such techniques. When sonication is employed, the isolate can 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. Any parameter indicative of the proliferation of T cells cultured in the presence of vaccine antigen can be measured in a method embodied by the invention. For example one or more of cell counts (ie., increased T cell numbers), H-thymidine uptake and/or for instance MTT assays, up-regulation of cell surface antigen expression, measurement of cell effecter functions, and cytokine production can be measured.

Cytokine expression can 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 can 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 hybridisation protocols utilising single cells and specific oligonucleotides probes as is known in the art.

The T cells stimulated by the vaccine will typically be CD4⁺ and/or CD8⁺ T- cells, and preferably ThI T-lymphocytes which differentiate from proliferating CD4⁺ T- lymphocytes. ThI T-lymphocytes stimulate macrophages through secretion of IFN- γ and are of particular interest in mucosal immunity. More broadly, besides IFN- γ, ThI cells responsive to antigen typically also secrete IL-2, IL- 12, TNF-β and IL-7. Both ThI and Th2 cells secrete IL-3, GM-CSF and for instance TNF-α, while cytokines characteristic of Th2 cells include IL-4, IL-5, IL-IO, IL- 13 and TGF-β, and any of the above cytokines or combinations thereof can be measured.

Peripheral blood mononuclear cells (PBMCs) can be readily isolated from a blood sample for the purpose of assaying for T cells in the blood sample responsive to vaccine antigen, such as by density gradient centrifugation of the sample over Ficoll (a carbohydrate polymer) and metrizamide (eg., Ficoll- Hypaque density gradient centrifugation). The PBMCs contain principally lymphocytes and monocytes, and are depleted of granulocytes and polymorphonuclear white blood cells. The antigen responsive T cell proliferation measured by methods embodied by the invention will normally involve culturing the PBMCs in the presence and absence of the vaccine antigen (eg., whole killed organisms and/or soluble or particulate antigen), and cell counts taken and/or cell culture supernatant analysed (eg., H-thymidine uptake, cytokine production). Markers indicative of an inflammatory response at the mucosal surface include one or more of lysozyme, lactoferrin, cytokines associated with inflammation such as IL-6, IL-8 and TNF-α, leukotriene B4 (LTB4) and interferon-γ (IFN-γ), C-reactive protein (CRP), reactive oxygen species and nitric oxide (NO). Typically, the marker(s) measured will be, or include, one or both of lysozyme and lactoferrin. Any suitable biological sample (such as fluid samples, including samples of secretions) from the individual containing the marker(s) wherein the level of the markers in the sample is reflective of the status of inflammation at the mucosal surface can be assayed. The sample can for instance be sputum or saliva. In one or more forms of methods of the invention saliva will be utilised. The IgG level measured can be total IgG level or the level of one or more IgG subclasses. For example, in humans, the level of IgGl and/or IgG3 can be measured whilst in the mouse, the level of IgGl, IgG2a and/or IgG2b may be measured. The level of the IgG can be determined by any suitable conventionally known assay such as by ELISA or other immunoassay. Moreover, the IgG level can be the level of total IgG, or the level of one or more of the above IgG subclasses, which bind to the pathogen. The level of the measured parameter(s) can for example be a mean or median level in the instance the level is measured in a number of individuals vaccinated with the test vaccine. Similarly, a ratio of respective of one or more of the parameters can used. This also applies to the control level against which the measured level(s) of the parameter(s) is compared. The control level of the parameter measured in accordance with an embodiment of the invention will generally be the level in a corresponding sample obtained from the at least one individual not vaccinated with the vaccine being evaluated.

The vaccine will typically comprise the selected bacterial isolate(s) and/or antigens in an amount of from about 1% to about 100% w/w of the vaccine composition. An effective dosage of the vaccine will take into account the proposed mode of delivery and nature of the vaccine (eg. powder, liquid, aerosol delivery etc). The dosage of respective of the bacterial isolates administered will typically comprise or be derived from about 10⁹ to about 10¹² killed organisms, and more preferably from about 10¹⁰ to about 10¹¹ killed organisms, respectively. The optimum dosage of a selected 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 by a method embodied by the invention.

Similarly, methods embodied by the invention can be employed to determine an optimum dosing regimen for the vaccine and all such methods are also expressly encompassed by the invention. For instance, dosing regimens can comprise a single dosage of the oral killed vaccine, or a course of administration involving a number of dosages of the vaccine over a period of time. Again, the doses of the vaccine can be the same or different. When a course of the vaccine is administered, the interval between doses of the vaccine can also be the same or different and may for instance comprise 1 or more weeks or months, respectively. The optimum dosing regimen can be determined for example, by administering different courses of the vaccine to different groups (eg., the courses differing in one or more parameters such as the time interval between doses of the vaccine and dosage administered each time), and measuring one or more parameters indicative of the response to the vaccine in accordance with embodiments of the invention to determine which dosing regimen provided the most optimal response. The vaccine itself may be a freeze -dried or lyophilised vaccine reconstituted utilising a physiologically acceptable buffer or fluid. The vaccine can also contain one or more anti-caking agents, isotonic agents, preservatives such as thimerosal, stabilisers 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 for boosting the immune response can also be added to the vaccine. In addition, the vaccine can 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. Specific examples of adjuvants include cholera toxin B subunit and conventionally known alum adjuvants. Typically, although not exclusively, the vaccine is non-adjuvanted.

The oral killed bacterial vaccine can be administered as a dry powder or in liquid form. Administration in the form of an enteric coated tablet or capsule to protect the vaccine components during passage through the stomach is particularly preferred.

In at least some forms of methods of the invention, the individual will be a subject with airway - hyperresponsiveness, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis or lung damage (eg., through smoking), or a person deemed at risk or prone to one or more of recurrent bronchitis (eg., recurrent acute bronchitis), otitis media and sinusitis or other airway disease such as asthma. The invention is further described below by way of a non- limiting example.

EXAMPLE

1. Clinical study overview

A placebo-controlled double-blind clinical study was performed in which 64 subjects were recruited on the basis of having smoked at least 10 cigarettes per day for the past two years. Subjects were randomised into placebo and active groups and were given three courses of study medication at monthly intervals. Each course consisted of two tablets per day for three days. The active tablets each contained 45mg of formalin- killed Non-typeable Haemophilus influenzae (NTHi) (equivalent to 10 killed bacteria per active tablet). Blood (for lymphocyte proliferation assay), saliva (for measurement of lactoferrin, lysozyme, other non-specific markers and specific antibody) and gargles, throat swabs, and nasal swabs (for microbiological assessment) were collected at seven fortnightly visits.

1.1 NTHi specific T cells in blood

NTHi specific T cells were detected by in vitro culture of peripheral blood lymphocytes with NTHi antigen. Briefly, heparinised blood was diluted, layered on ficoll-hypaque and centrifuged to separate the leukocytes from the red blood cells. The leukocytes were washed and suspended in AIM-V tissue culture medium. Leukocytes (2x10 ) were cultured in triplicate in 96-well flat-bottom microwell plates with NTHi antigen (NTHi 164 sonicate preparation at 1 or 10 ug/mL), PHA (phytohaemagglutinin) at 5ug/mL, or without antigen or PHA. After incubation for 5 days at 37°C with 5% CO2 cultures were pulsed with H-thymidine (0.5 uCi/well) for 6h. The cultures were then harvested onto glass fibre filter mats. The filter mats were dried, scintillant wax was melted onto the mats, and the mats were placed into plastic sample bags and placed in a Wallac Trilux beta counter for determination of counts per minute (cpm). The mean cpm was determined for each triplicate set of wells. The stimulation index (SI) for cultures with added antigen or PHA was determined by calculating the mean cpm in the presence of antigen or PHA, subtracting the mean cpm in cultures without antigen or PHA, and dividing by the mean cpm in cultures without antigen or PHA. The extent of proliferation of T cells (proliferation index) is considered a measure of the NTHi specific cells in the blood. The 25% of patients in each of the treatment group with the highest and lowest change in stimulation index between visits 7 and 1 (ie., stimulation index at visit 7 minus stimulation index at visit 1) is shown in Table 1. The active treatment group had a higher mean stimulation index than the placebo group. The 25% with the lowest stimulation index in each group is also shown. There is no difference between the low SI active and placebo groups suggesting there are non-responders in the active group.

Table 1

Measurements in the placebo-treated and vaccine-treated patients over the winter period in the present study showed an increase in specific T cells responsive to the antigen in both the placebo group and in the vaccine group over this period (see Fig. 1 which shows the response in vitro to stimulation with 1 ug antigen/niL).

Surprisingly, an increase in specific T cells over the winter period was found in the placebo group indicating exposure to NTHi over the winter months even in the absence of clinical infection. Fig.2 shows the mean level of NTHi in the gargles of the placebo group at each visit. The vaccine treatment group showed a significantly different specific T-cell profile suggesting an effect of the vaccine over-and-above the effect of exposure to environmental NTHi.

1.2 Relationship between NTHi in the gargle and serum and saliva IgG

NTHi-specific IgG was measured in serum and saliva. NTHi-specific IgG in serum and saliva was measured by ELISA assay. Briefly, wells of 96- well Nunc Maxisorp plates were coated with NTHi 164 sonicate antigen preparation. After incubation overnight at 2-8°C the plates were washed and samples of serum or saliva at various dilutions were added. After incubation at room temperature for 60 minutes the plates were washed and horse-radish peroxidise -conjugated anti-human IgG antibody (Chemicon catalogue number API 12P) was added. After incubation for 60 minutes at room temperature the plates were washed and TMB substrate (Biomediq catalogue number 50-76-00) was added. Following a subsequent incubation for 10 minutes at room temperature, the reaction was stopped by addition of IM phosphoric acid and the absorbance was read on a BioRad micriplate reader on dualwavelength mode with a primary filter of 450nm and reference filter of 655nm. A standard curve was used to determine the ELISA units in each sample. Levels of NTHi-specific IgG in serum and saliva are shown in Figures 3a and

3b. Unexpectedly the levels in the placebo group were higher and more variable than the levels in the vaccine- treated group. It was thought by the inventors that the explanation for this is that in the vaccine-treated group the NTHi in the upper airways is prevented from reaching the lower airways and further propose that in the placebo group, the NTHi reaching the lower airways result in systemic production of IgG.

To test this, plots were prepared of relationship between number of visits at which NTHi was detected in the gargle and the Log change in serum IgG between visits 1 and 6 (see Fig 4). Placebo and active subjects were grouped according to whether they had 0-1 visits or 2-4 visits where NTHi was found in the gargle. In the placebo group, positive increases in serum IgG were associated with increased number of NTHi detections. This was not found in the active treatment group. The difference between the placebo and active change in IgG is statistically significant (p=0.0186) indicating the serum IgG is generated by NTHi in the absence of active immunization and may be due to NTHi reaching the lower airways and stimulating a systemic IgG response. In the active treatment group specific immunity in the lung would appear to be preventing this increase in serum IgG possibly by preventing bacteria reaching the lower airways. This also applies to the appearance of salivary NTHi-specific IgG in the placebo group.

1.3 Relationship between oral vaccination and serum or saliva IgA

NTHi-specific IgA was measured in serum and saliva of the placebo and vaccine treatment groups. The NTHi-specific IgA was measured in the same manner as described above for NTHi-specific IgG, except that horse-radish peroxidise-conjugated anti -human IgA (Chemicon catalogue number AP314P) was used instead of horse- radish peroxidise-conjugated anti-human IgG.

IgA is produced by oral vaccination. In the present study, serum and saliva bacteria- specific IgA was measured and an increase in the vaccine -treated group was expected. The specific IgA in serum (Fig 5a) fell in the placebo group but was maintained, or slightly increased, over the study period in the vaccine-treated group thus indicating an effect of vaccination in maintaining a certain level of specific IgA. The specific IgA levels in saliva (Fig 5b) were not significantly different between the active and placebo groups suggesting that serum IgA measurement is more useful than saliva measurement for this parameter.

1.4 Markers of inflammation

Saliva lysozyme and lactoferrin in saliva were measured at each visit. These were measured by ELISA assay in a similar fashion to the assay described above for measurement of specific IgG. In the case of lactoferrin and lysozyme measurement the plates were coated with Goat anti-human lactoferrin (Medos catalogue number NORl 162) or Rabbit anti-human lysozyme (Medos catalogue number NOR 2460). A drop in the drop in mean lysozyme between visits 1 and 7 was observed which was greater for the active treatment group (dropped by 13600 ± 5270 ELISA units) than the placebo group (dropped 2100 ± 6500 ELISA units). The mean lysozyme level dropped by 3700 ± 4530 EU in the active group but increased slightly (increased by 600 ± 3660) in the placebo group. The drop in saliva lysozyme and lactoferrin suggests a decrease in inflammation in the active treatment group as lysozyme and lactoferrin are produced by inflammatory cells such as neutrophils.

2. Evaluation and discussion of results

The finding of responses in the placebo group suggests that bacteria- specific T cells are arising due to exposure to infecting NTHi in the upper airways during the winter period. The differing shape of the response-over-time curve in the active group also indicates an additional effect due to the oral vaccine in this group. Thus, the bacteria- specific T cells in the blood in the human study are reflecting both exposure to infecting bacteria and exposure to the oral vaccine, and the relative contribution of vaccination (and thereby its effectiveness) and infection can be determined by comparison of active and placebo treatment groups.

Serum IgG antibody as a surrogate marker for the efficacy of the vaccine was measured for the oral vaccine. Highly unexpectedly, an apparent lack of an IgG response in the vaccine-treated group was found while the placebo treated group of patients showed an increase in serum IgG. Without being limited by theory, it is thought by the inventors that this increase in IgG in the placebo group is reflecting an immune response to infecting bacteria reaching the lower airways where uptake of the bacteria by antigen-presenting cells and transport to draining lymph nodes induces an anti-bacterial IgG response. In contrast, the lack of such a response in the vaccine- treated group indicates that the bacteria are being essentially prevented (by a mucosal vaccine- specific immune response) from reaching the lower airways.

A comparison of the IgG response in subjects with NTHi detected in the upper airways at 0-1 or 2-4 visits also showed the increase in IgG in the placebo group but not in the active (vaccine) treatment group. This suggests that serum IgG measurement following oral vaccination with NTHi reflects exposure to infection and the degree to which this is prevented by mucosal immunization. Thus, comparison of serum IgG in placebo and active groups provides an indication of prevention of serum IgG induction by oral vaccination. The saliva IgG response reflected that seen in the serum enabling a non-invasive means of obtaining the same data.

Lysozyme and lactoferrin are anti -bacterial proteins found in saliva and are secreted by inflammatory cells. The results of the current study indicate these parameters are reflective of the level of inflammation in the airways. Lysozyme was found to significantly decrease over the winter in the vaccine-treated group compared to the placebo treated group (Fig 6a) while lactoferrin showed a similar non- significant decrease (Fig 6b). Thus, measurement of lysozyme and lactoferrin can be used to provide an indication of the inflammatory response induced by infection and the modulation of this by oral vaccination. Overall, the results show IgG level in serum, lysozyme or lactoferrin levels in saliva, or NTHi specific T-cells in blood, or combinations of the above parameters, may be used to assess efficacy of oral immunization against bacterial infection in humans.

Although the invention has been described with reference to particular embodiments, it will be appreciated by those skilled in the art that numerous variations and/or modifications may be made without departing from the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. REFERENCES

1. Ostergaard, P.A., et al. (1985). Acta Ped. Scand. 74, 713-719. 2. Taylor, D.C., Cripps, A.W., Clancy, R.L. (1995) A possible role for lysozyme in determining acute exacerbations in chronic bronchitis. Clin Exp Immunol 102, 406-416.

3. "Remington" The Science and Practice of Pharmacy (Mack Publishing Co., 1995)".

Claims

1. A method for assessing the efficacy of an oral vaccine against infection or colonisation of a mucosal surface of the respiratory tract by a microbial pathogen, comprising:

(a) measuring at least one parameter of at least one individual orally vaccinated with the vaccine, the parameter being selected from the group consisting of:

(i) a level of T lymphocytes in blood from the individual responsive to the pathogen involving antigen induced proliferation of the T lymphocytes without limiting dilution analysis of the T lymphocytes;

(ii) IgG level in a body fluid from the individual; and (iii) at least one marker of inflammation at a mucosal surface of the respiratory tract of the individual; (b) comparing the measured level with a respective control level, the marker being provided in a biological sample from the vaccinated individual; and

2. A method according to claim 1 wherein the control level is that of at least one individual not vaccinated with the vaccine.

3. A method according to claim 1 or 2 wherein the level of T lymphocytes in blood from the individual is measured.

4. A method according to claim 1 or 2 comprising measuring the IgG level in the body fluid from the individual.

5. A method according to claim 4 wherein the IgG level is total IgG.

6. A method according to claim 4 wherein the IgG level is the level of one or more IgG subclasses.

7. A method according to any one of claims 4 to 6 wherein the body fluid is blood.

8. A method according to claim 1 or 2 comprising measuring the level of the at least one marker of inflammation.

9. A method according to claim 8 wherein the marker of inflammation is selected from the group consisting of lysozyme, lactoferrin, cytokines associated with inflammation, IL-6, IL-8, TNF-α, leukotriene B4, IFN -γ, C-reactive protein (CRP), reactive oxygen species and nitric oxide (NO).

10. A method according to claim 9 wherein the marker of inflammation is selected from the group consisting of lysozyme and lactoferrin.

11. A method according to any one of claims 8 to 10 wherein the level of the marker of inflammation is measured in saliva or sputum from the individual.

12. A method according to any one of claims 1 to 11 wherein the vaccine is an oral killed vaccine.

13. A method according to any one of claims 1 to 11 wherein the vaccine includes soluble or particulate antigen of the pathogen.

14. A method according to any one of claims 1 to 13 wherein the pathogen is selected from the group consisting of bacteria and fungal pathogens.

15. A method according to any one of claims 1 to 14 wherein the pathogen is a bacterial pathogen.

16. A method according to claim 15 wherein the bacterial pathogen is selected from the group consisting of NTHi, Pseudomonas species, Streptococcus species, Staphylococcus species, E. CoIi species, Moraxella species, Candida albicans and Mycoplasma species.

17. A method according to claim 14 wherein the bacterial pathogen is NTHi.