WO2013030670A2 - Tissue targeted fusobacterium antigenic activation of the immune response to treat intestinal cancers - Google Patents

Abstract

Aspects of the invention include methods of treating intestinal, e.g., lower intestinal such as colon and/or rectal, cancers by administering a composition that is antigenically specific for a Fusobacterium, such as Fusobacterium nucleatum.

Description

TISSUE TARGETED FusoBACTER/uMkwnGtmc ACTIVATION OF THE IMMUNE RESPONSE TO TREAT INTESTINAL CANCERS

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 61 /528,707 filed August 29, 201 1 ; the disclosure of which is herein incorporated by reference.

INTRODUCTION

Few infectious agents have been unequivocally linked to cancer. Those that have, such as Human Papilloma Virus, Hepatitis B and C virus, and

Helicobacter pylori alone are responsible for an estimated 15% of the global cancer burden, based on strength of the association and prevalence of infection (Parkin, "The global health burden of infection-associated cancers in the year 2002," Int. J. Cancer (2006) 1 1 8:3030-3044). Metagenomics methods using new sequencing technologies provide a useful approach for detecting tumor-associated microorganisms that may be overlooked by culture or histology based methods.

Colorectal carcinoma (CRC) is the second leading cause of cancer deaths, responsible for approximately 655,000 deaths per year worldwide (World Health Organization fact sheet # 297, 2009). It is also one of the first and best genetically characterized cancers and specific somatic mutations on oncogenes and tumor suppressor genes have been found to be associated with progression from adenomatous lesions (polyps) to invasive carcinoma (Vogelstein, et al, "Genetic alterations during colorectal-tumor development," N. Engl. J. Med. (1 988) 31 9: 525-532). SUMMARY

Aspects of the invention include, among other inventions as summarized below, methods of treating intestinal, e.g., lower intestinal such as colon and/or rectal, cancers by administering a composition that is antigenically specific for a Fusobacterium, such as Fusobacterium nucleatum. Accordingly, a method for treating a human subject for an intestinal cancer, such as a cancer of the lower intestine, e.g., colon and/or rectum, is provided. The method involves administering to the subject a medicament having an effective amount of a Fusobacterium antigenic composition comprising a whole killed bacterial cell composition, wherein the Fusobacterium, e.g., Fusobacterium nucleatum, is pathogenic in the specific organ or tissue of the subject within which the cancer is situated. The medicament may be administered to the subject in an amount and for a time that is effective to modulate an immune response. Optionally, the modulation of the immune response may involve a shift in the activation state of macrophages. Optionally, the modulation of the immune response may involve shifting from a M2-like macrophage response to a M1 -like macrophage response. The modulation of the immune response may involve a shift from M1 - like macrophages to M2-like macrophages, as those terms are defined herein. Optionally and without limitation, the method may further involve measuring a characteristic of the immune response.

Optionally, comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the

macrophages may include any one or more of the following: M1 -like

macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Further and optionally, the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

Further and without limitation, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1 -like

macrophages or M2-like macrophages. Further, cytokines may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. Optionally, the differential gene expression may be produced as a result of a shift in an activation state of the macrophages. Further and optionally, the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. The macrophages may shift from being M1 -like macrophages to being M2-like macrophages. A diagnostic step may be used to identify the target intestinal cancer, prior to producing the Fusobacterium antigenic composition targeted to the site of the cancer.

In any of the above amendments, the antigenic composition may be sufficiently specific that it would be capable of eliciting an immune response in the mammal specific to the microbial pathogen. The antigenic composition may be a bacterial composition, for example derived from a Fusobacterial species. The microbial pathogen may be killed. In alternative embodiments, the microbial pathogen may be live or attenuated.

Immunogenic compositions of the invention may also be formulated or administered with anti-inflammatory modalities, such as an NSAID. The site of administration may be at a site distant from the site of the cancer, for example in an organ or tissue that is not intestinal within which the cancer is situated, for example the skin or subcutaneous tissue.

The antigenic composition may for example be formulated for

subcutaneous injection, intradermal injection or oral administration. In

embodiments for subcutaneous or intradermal injection, the dosing or formulation of the antigenic composition may be adjusted in order to produce a localized immune reaction visible in the skin at the site of administration, for example an area of inflammation from 2mm to 1 00mm in diameter appearing, for example, 2 - 48 hours after administration and lasting, for example, 2 - 72 hours or longer. The antigenic composition may be formulated for repeated

subcutaneous or intradermal administration, for example at alternating successive sites.

In some embodiments, the invention involves methods of treating a mammal for an intestinal cancer, e.g., a cancer of the lower intestine, such as the colon and/or rectum. In alternative embodiments, the treatment may anticipate the development of the cancer in the target tissue, for example if the site of a primary tumor suggests the likelihood of metastasis to a particular tissue or organ, then the patient may be prophylactically treated to prevent or ameliorate metastasis to that tissue or organ. The method may include administering to the subject an effective amount of an antigenic composition comprising antigenic determinants that together are specific for at least one Fusobacterium microbial pathogen. The antigenic composition may be administered, for example by subcutaneous or intradermal injection at an administration site, in successive doses given at a dosage interval, for example of between one hour and one month, over a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer. Each injection dose may for example be metered so that it is effective to cause visible localized inflammation at the administration site, appearing, for example, 1 - 48 hours after injection.

In another aspect, methods are provided for treating cancers of the intestine (or a portion thereof, e.g., the lower intestine or a portion thereof, such as the colon and/or rectum) in a subject by administering one or more antigens of one or more Fusobacterium microbial pathogens, such as Fusobacterium nucleatum, that are pathogenic in the specific organ or tissue.

In alternative embodiments, the Fusobacterium antigen may be administered by administering a whole microbial species. In alternative embodiments, the method may, for example, include administering at least two or more microbial species, or administering at least three or more microbial species, and the microbes may be bacteria or viruses, where at least one of the microbial species in such embodiments is a Fusobacterium. In alternative embodiments, the method may further include administering a supplement or an adjuvant. An aspect of the invention involves administering antigenic

compositions so as to elicit an immune response in said subject.

In alternative embodiments, the Fusobacterium microbial pathogen in the antigenic composition may be killed, and thus rendered non-infectious. In some embodiments, the antigenic composition is administered at a site distant from the cancer site, and in selected embodiments of this kind, methods of the invention may be carried out so that they do not produce infection at the cancer site.

As detailed herein, various aspects of the invention involve treating cancers. In this context, treatment may be carried out so as to provide a variety of outcomes. For example, treatment may: provoke an immune reaction that is effective to inhibit or ameliorate the growth or proliferation of a cancer; inhibit the growth or proliferation of cancer cells or tumors; cause remission of a cancer; improve quality of life; reduce the risk of recurrence of a cancer; inhibit metastasis of a cancer; or, improve patient survival rates in a patient population. In this context, extending the life expectancy of a patient, or patient population, means to increase the number of patients who survive for a given period of time following a particular diagnosis. In some embodiments, treatment may be of patients who have not responded to other treatments, such as patients for whom a chemotherapy or surgery has not been an effective treatment. Treatment in alternative embodiments may for example be before or after onset of cancer. For example prophylactic treatment may be undertaken, for example of patients diagnosed as being at risk of a particular cancer. For example a patient having a genetic or lifestyle predisposition to cancer of a certain tissue or organ may be treated with an immunogenic composition comprising antigenic determinants of a pathogen that is pathogenic in that organ or tissue. Similarly, the prophylactic treatment of metastasis may be undertaken, so that patients having a primary cancer with a propensity to metastasize to a particular tissue or organ may be treated with an immunogenic composition comprising antigenic determinants of a pathogen that is pathogenic in that organ or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Relative abundance of microbial genomes in tumor and control specimens. Numbers of read pairs that matched known microbial sequences were normalized according to sequencing depth for both tumor and matched normal samples. The abundance of bacterial read pairs ranged from zero to a maximum of 46,795 represented by a transition from white to red on a log scale. Fusobacterium nucleatum sequences were present in the tumor samples at levels 2-fold or greater than in normal samples in 9 out of the 1 1 subjects. The mean over abundance across all subjects was 86-fold. Figure 2. Relative Abundance of Fusobacterium in Tumor versus Normal Colorectal Carcinoma Biopsies. Relative amounts of Fusobacterium DNA were determined between tumor and matched normal biopsies in 99 subjects, using quantitative real-time PCR (qPCR). The cycle threshold (Ct) values for the normal samples had a Ct range of 25.5 to 40 and the Ct range for the tumor samples was between 21 .4 to 40. Data shown are mean values from two independent experiments. Fusobacterium load, as determined by qPCR was found to be significantly higher in the tumor samples versus the matched control samples (two-tailed ratio t-test (p = 2.52 x 10^~6)).

Figure 3. Hive plots showing alignment of three Fusobacterium genomes. Approximately 32M high-quality WGS lllumina HiSeq reads (>= 99 consecutive Q30 bases) from Fusobacterium tumor isolate CC53 were assembled with SSAKE (v3.7, default options) into 379 contigs. The contigs were aligned using cross_match (-minmatch 29 -minscore 59 -masklevel 1 01 ) to the complete F.nucleatum susb. nucleatum ATCC 25586 genome and, independantly to the 1 2-contig HMP Fusobacterium sp. 3_1_36A2 assembly, respectively and ordered/oriented based on the highest identity to the latter sequence. Three-way cross_match (www.phrap.org) alignments between each Fusobacterium genomes were performed and represented visually using hive plots (www.hiveplot.com). For each, the top, left and right axes are proportional to genome size and represent the Fusobacterium tumor isolate CC53 (2.07 Mbp), the HMP sp. 3_1_36A2 (2.25 Mbp) and the ATCC 25586 type strain (2.1 7 Mbp), in that order. Synteny between the isolates is depicted by green and red links that show normal and inverted alignments, respectively. Sequence similarity and synteny is highest between CC53 and sp. 3_1_36A2, compared to ATCC 25586. Three regions of sequences present in sp. 3_1 _36A2 but absent from CC53 are apparent as conspicuous gaps on the sp. 3_1_36A2 axis.

Sequence segments unique to CC53 are not visible at this scale.

Figure 4. In the Immunofluorescence micrograph, CC53 shows a very long, flexible cell morphology. Green is actin (Caco-2 cells), orange are invasive and internalized bacteria, purple are bacteria external to the cell. Figure S1. Number of sequencing read pairs that match known microbial genomes are shown for the 25 most abundant genomes.

Figure S2. Distribution of hits from colorectal carcinoma RNA-Seq data to the annotated F.nucleatum subsp. nucleatum ATCC 25586 genome. The total number of read pair hits was 80, 1 18.

Figure S3. Frequency of metastasis increases with higher

Fusobacterium abundance in tumor biopsies. Patients with 5X or greater

Fusobacterium in their tumor biopsies versus matched normal tissue were compared to those patients with less than 5X relative amounts of Fusobacterium. A significantly higher number of patients from the high Fusobacterium group (A) had more tumor spreading in their lymph nodes as measured by their surgical TNM scores than the low Fusobacterium group (B) (one-tailed Fisher's exact test p-value = 0.0035). DETAILED DESCRIPTION

In various aspects, the invention relates to the surprising discovery that administration, for example at a site distant from an intestinal cancer, of a Fusobacterium microbial pathogen, such as a killed Fusobacterium microbial pathogen, that are pathogenic in the intestine or a portion thereof, e.g., the lower intestine or portion thereof, e.g., the colon and/or rectum, is effective in treating cancer situated in that target tissue or organ. Accordingly, the invention provides Fusobacterium antigenic compositions derived from Fusobacterium, including whole killed bacterial species, or components thereof, for the treatment of cancer, and methods for using the same.

Antigenic compositions of the invention may be produced that include antigenic determinants that together are specific for or characteristic of a

Fusobacterium microbial pathogen, such as Fusobacterium nucleatum. In this context, by "specific", it is meant that the antigenic determinants are sufficiently characteristic of the pathogen that they could be used to raise an immune response, such as an adaptive immune response, against the pathogen in the patient, if the antigenic determinants were to be administered in an appropriate manner to have that effect. It will be recognized that the antigenic determinants need not be so specific that they are characteristic of only one particular strain or species of pathogen, since even a specific immune response against a particular pathogen may be cross reactive with other closely related organisms that are also naturally pathogenic in the tissue or organ in which the cancer is situated and that the antigenic composition is formulated or selected to target.

In some embodiments, the compositions of pathogenic microbes may be used for treating primary cancer sites and/or sites of metastasis. Thus, for example, the microbial compositions may be used for the treatment of a cancer at a particular site, regardless of whether the cancer is a primary cancer or a metastasis. The composition may be directed to the treatment of each cancer site, or may be a combined composition for both the primary cancer and the metastatic site(s). For example, to treat colon cancer that has metastasized to the lung and bone, three different compositions, including one or more species that are known to be Fusobacterium pathogens, one or more species that are known to be lung pathogens and one or more species that are known to be bone pathogens, or a combined composition thereof may be used. In some embodiments, the compositions may be administered in different locations at the same time or at different times.

For example, for colon cancer with metastasis to the bone, in alternative embodiments, both a microbial composition including one or more

Fusobacterium bacterial species and a microbial composition including one or more bacterial species (or viruses) which commonly cause bone infection may be used.

In some embodiments, the antigenic compositions may be used for treating or preventing cancers at primary sites or for treating or preventing metastasis. For example, a Fusobacterium antigenic composition may be used to appropriately stimulate the immune system to defend against the development of cancer within the colon. Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention. Cancers

Most cancers fall within three broad histological classifications:

carcinomas, which are the predominant cancers and are cancers of epithelial cells or cells covering the external or internal surfaces of organs, glands, or other body structures (for e.g., skin, uterus, lung, breast, prostate, stomach, bowel), and which tend to metastasize; sarcomas, which are derived from connective or supportive tissue (for e.g., bone, cartilage, tendons, ligaments, fat, muscle); and hematologic tumors, which are derived from bone marrow and lymphatic tissue. Carcinomas may be adenocarcinomas (which generally develop in organs or glands capable of secretion, such as breast, lung, colon, prostate or bladder) or may be squamous cell carcinomas (which originate in the squamous epithelium and generally develop in most areas of the body). Sarcomas may be

osteosarcomas or osteogenic sarcomas (bone), chondrosarcomas (cartilage), leiomyosarcomas (smooth muscle), rhabdomyosarcomas (skeletal muscle), mesothelial sarcomas or mesotheliomas (membranous lining of body cavities), fibrosarcomas (fibrous tissue), angiosarcomas or hemangioendotheliomas (blood vessels), liposarcomas (adipose tissue), gliomas or astrocytomas

(neurogenic connective tissue found in the brain), myxosarcomas (primitive embryonic connective tissue), or mesenchymous or mixed mesodermal tumors (mixed connective tissue types). Hematologic tumors may be myelomas, which originate in the plasma cells of bone marrow; leukemias which may be "liquid cancers" and are cancers of the bone marrow and may be myelogenous or granulocytic leukemia (myeloid and granulocytic white blood cells), lymphatic, lymphocytic, or lymphoblastic leukemias (lymphoid and lymphocytic blood cells) or polycythemia vera or erythremia (various blood cell products, but with red cells predominating); or lymphomas, which may be solid tumors and which develop in the glands or nodes of the lymphatic system, and which may be Hodgkin or Non-Hodgkin lymphomas. In addition, mixed type cancers, such as adenosquamous carcinomas, mixed mesodermal tumors, carcinosarcomas, or teratocarci nomas also exist.

Cancers named based on primary site may be correlated with histological classifications. For example, lung cancers are generally small cell lung cancers or non-small cell lung cancers, which may be squamous cell carcinoma, adenocarcinoma, or large cell carcinoma; skin cancers are generally basal cell cancers, squamous cell cancers, or melanomas. Lymphomas may arise in the lymph nodes associated with the head, neck and chest, as well as in the abdominal lymph nodes or in the axillary or inguinal lymph nodes. Identification and classification of types and stages of cancers may be performed by using for example information provided by the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute, which is an

authoritative source of information on cancer incidence and survival in the United States and is recognized around the world. The SEER Program currently collects and publishes cancer incidence and survival data from 14 population- based cancer registries and three supplemental registries covering

approximately 26 percent of the US population. The program routinely collects data on patient demographics, primary tumor site, morphology, stage at diagnosis, first course of treatment, and follow-up for vital status, and is the only comprehensive source of population-based information in the United States that includes stage of cancer at the time of diagnosis and survival rates within each stage. Information on more than 3 million in situ and invasive cancer cases is included in the SEER database, and approximately 170,000 new cases are added each year within the SEER coverage areas. The incidence and survival data of the SEER Program may be used to access standard survival for a particular cancer site and stage. For example, to ensure an optimal comparison group, specific criteria may be selected from the database, including date of diagnosis and exact stage (for example, in the case of the lung cancer example herein, the years were selected to match the time-frame of the retrospective review, and stage 3B and 4 lung cancer were selected; and in the case of the colon cancer example herein, the years were also selected to match the timeframe of the retrospective review, and the stage 4 colon cancer was selected).

Cancers may also be named based on the organ in which they originate i.e., the "primary site," for example, cancer of the breast, brain, lung, liver, skin, prostate, testicle, bladder, colon and rectum, cervix, uterus, etc. This naming persists even if the cancer metastasizes to another part of the body that is different from the primary site. With the present invention, treatment is directed to the site of the cancer, not type of cancer, so that a cancer of any type that is situated in the lung, for example, would be treated on the basis of this

localization in the lung.

A "cancer" or "neoplasm" is any unwanted growth of cells serving no physiological function. In general, a cancer cell has been released from its normal cell division control, i.e., a cell whose growth is not regulated by the ordinary biochemical and physical influences in the cellular environment. Thus, "cancer" is a general term for diseases characterized by abnormal uncontrolled cell growth. In most cases, a cancer cell proliferates to form clonal cells that are malignant. The lump or cell mass, "neoplasm" or "tumor," is generally capable of invading and destroying surrounding normal tissues. By "malignancy", as used herein, is meant as an abnormal growth of any cell type or tissue that has a deleterious effect in that organism having the abnormal growth. The term "malignancy" or "cancer" includes cell growths that are technically benign but which carry the risk of becoming malignant. Cancer cells may spread from their original site to other parts of the body through the lymphatic system or blood stream in a process known as "metastasis." Many cancers are refractory to treatment and prove fatal. Examples of cancers or neoplasms include, without limitation, transformed and immortalized cells, tumors, carcinomas, in various organs and tissues as described herein or known to those of skill in the art.

A "cell" is the basic structural and functional unit of a living organism. In higher organisms, e.g., animals, cells having similar structure and function generally aggregate into "tissues" that perform particular functions. Thus, a tissue includes a collection of similar cells and surrounding intercellular substances, e.g., epithelial tissue, connective tissue, muscle, nerve. An "organ" is a fully differentiated structural and functional unit in a higher organism that may be composed of different types of tissues and is specialized for some particular function, e.g., kidney, heart, brain, liver, etc. Accordingly, by "specific organ, tissue, or cell" is meant herein to include any particular organ, and to include the cells and tissues found in that organ.

"Pathogenic" agents are agents, such as microbes, such as bacteria or viruses, which are known to cause infection in a host in nature, and in this sense, "pathogenic" is used in the context of the present invention to mean "naturally pathogenic". Although a wide variety of microbes may be capable of causing infection under artificial conditions, such as artificial innoculations of a microbe into a tissue, the range of microbes that naturally cause infection is necessarily limited, and well established by medical practice.

An "infection" is the state or condition in which the body or a part of it is invaded by a pathogenic agent (e.g., a microbe, such as a bacterium) which, under favorable conditions, multiplies and produces effects that are injurious (Taber's Cyclopedic Medical Dictionary, 14th Ed., C.L Thomas, Ed., F.A. Davis Company, PA, USA). An infection may not always be apparent clinically and may result in only localized cellular injury. Infections may remain subclinical, and temporary if the body's defensive mechanisms are effective. Infections may spread locally to become clinically apparent as an acute, a subacute, or a chronic clinical infection or disease state. A local infection may also become systemic when the pathogenic agent gains access to the lymphatic or vascular system (On-Line Medical Dictionary, http://cancerweb.ncl.ac.uk/omd/). Infection is usually accompanied by inflammation, but inflammation may occur without infection.

"Inflammation" is the characteristic tissue reaction to injury (marked by swelling, redness, heat, and pain), and includes the successive changes that occur in living tissue when it is injured. Infection and inflammation are different conditions, although one may arise from the other (Taber's Cyclopedic Medical Dictionary, supra). Accordingly, inflammation may occur without infection and infection may occur without inflammation (although infection by pathogenic bacteria or viruses typically results in inflammation). Inflammation is

characterized by the following symptoms: redness (rubor), heat (calor), swelling (tumor), pain (dolor). Localized visible inflammation on the skin may be apparent from a combination of these symptoms, particularly redness at a site of administration.

Various subjects may be treated in accordance with alternative aspects of the invention. As used herein, a "subject" is an animal, for e.g., a mammal, to whom the specific pathogenic bacteria, bacterial antigens, viruses, viral antigens or compositions thereof of the invention may be administered. Accordingly, a subject may be a patient, e.g., a human, suffering from a cancer, or suspected of having a cancer, or at risk for developing a cancer. A subject may also be an experimental animal, e.g., an animal model of a cancer, as is described in Example 5. In some embodiments, the terms "subject" and "patient" may be used interchangeably, and may include a human, a non-human mammal, a non- human primate, a rat, mouse, dog, etc. A healthy subject may be a human who is not suffering from a cancer or suspected of having a cancer, or who is not suffering from a chronic disorder or condition. A "healthy subject" may also be a subject who is not immunocompromised. By immunocompromised is meant any condition in which the immune system functions in an abnormal or incomplete manner. Immunocompromization may be due to disease, certain medications, or conditions present at birth. Immunocompromised subjects may be found more frequently among infants, the elderly, and individuals undergoing extensive drug or radiation therapy.

An "immune response" includes, but is not limited to, one or more of the following responses in a mammal: induction or activation of antibodies, neutrophils, monocytes, macrophages (including both M1 -like macrophages and M2-like macrophages as described herein), B cells, T cells (including helper T cells, natural killer cells, cytotoxic T cells, T cells), such as induction or activation by the antigen(s) in a composition or vaccine, following administration of the composition or vaccine. An immune response to a composition or vaccine thus generally includes the development in the host animal of a cellular and/or antibody-mediated response to the composition or vaccine of interest. In some embodiments, the immune response is such that it will also result in slowing or stopping the progression of a cancer in the animal. An immune response includes both cellular immune responses and humoral immune responses as understood by those persons skilled in the art.

Bacteria and Bacterial Colonizations and Infections

Most animals are colonized to some degree by other organisms, such as bacteria, which generally exist in symbiotic or commensal relationships with the host animal. Thus, many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these bacteria aid in the normal functioning of the body. For example, in humans, symbiotic Escherichia coli bacteria may be found in the intestine, where they promote immunity and reduce the risk of infection with more virulent pathogens.

Bacteria that are generally harmless, such as Escherichia coli, can cause infection in healthy subjects, with results ranging from mild to severe infection to death. Whether or not a bacterium is pathogenic (i.e., causes infection) depends to some extent on factors such as the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the bacterium; the amount of the bacteria present at the site of potential infection; or the health of the host animal. Thus, bacteria that are normally harmless can become pathogenic given favorable conditions for infection, and even the most virulent bacterium requires specific circumstances to cause infection. Accordingly, microbial species that are members of the normal flora can be pathogens when they move beyond their normal ecological role in the endogenous flora. For example, endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, these normally harmless endogenous bacteria are considered pathogenic. Specific bacterial species and viruses are known to cause infections in specific cells, tissues, or organs in otherwise healthy subjects. Examples of bacteria and viruses that commonly cause infections in specific organs and tissues of the body are listed below; it will be understood that these examples are not intended to be limiting and that a skilled person would be able to readily recognize and identify infectious or pathogenic bacteria that cause infections, or commonly cause infections, in various organs and tissues in healthy adults (and recognize the relative frequency of infection with each bacterial species) based on the knowledge in the field as represented, for example, by the following publications: Manual of Clinical Microbiology 8th Edition, Patrick Murray, Ed., 2003, ASM Press American Society for Microbiology, Washington DC, USA; Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases 5th Edition, G. L. Mandell, J.E. Bennett, R. Dolin, Eds., 2000, Churchill

Livingstone, Philadelphia, PA, USA, all of which are incorporated by reference herein.

Bacterial Strains/Viral Subtypes

It will be understood by a skilled person in the art that bacterial species are classified operationally as collections of similar strains (which generally refers to groups of presumed common ancestry with identifiable physiological but usually not morphological distinctions, and which may be identified using serological techniques against bacterial surface antigens). Thus, each bacterial species (e.g., Streptococcus pneumoniae) has numerous strains (or serotypes), which may differ in their ability to cause infection or differ in their ability to cause infection in a particular organ/site. For example, although there are at least 90 serotypes of Streptococcus pneumoniae, serotypes 1 , 3, 4, 7, 8, and 1 2 are most frequently responsible for pneumococcal disease in humans.

It is understood that a clinical microbiologist skilled in the art would therefore be able to select, based on the present disclosure and the body of art relating to bacterial strains for each species of bacteria (and viral subtypes for each type of virus), the strains of a particular bacterial species (or subtype of a particular virus) to target a specific organ or tissue.

Bacterial Compositions, Dosages, and Administration

The compositions of the invention include antigens of Fusobacterium pathogenic microbial species that are pathogenic in the intestine or a portion thereof, e.g., the large intestine or portion thereof, such as the colon or rectum. The compositions may include whole cells of bacterial species, or may include extracts or preparations of the pathogenic bacterial species of the invention, such as cell wall or cell membrane extracts, or whole cells, or exotoxins, or whole cells and exotoxins. The compositions may also include one or more isolated antigens from one or more of the Fusobacterium pathogenic bacterial species of the invention; in some embodiments, such compositions may be useful in situations where it may be necessary to precisely administer a specific dose of a particular antigen, or may be useful if administering a whole bacterial species or components thereof (e.g., toxins) may be harmful. Pathogenic bacterial species may be available commercially (from, for example, ATCC (Manassas, VA, USA), or may be clinical isolates from subjects having a bacterial infection of a tissue or organ (e.g., pneumonia).

The microbial compositions of the invention can be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans. As used herein

"pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for any appropriate form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal,

intramuscular, sublingual, inhalational, intratumoral or oral administration.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound (i.e., the specific bacteria, bacterial antigens, or compositions thereof of the

invention), use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

If desired, treatment with bacterial antigens according to the invention may be combined with more traditional and existing therapies for cancer, such as chemotherapy, radiation therapy, surgery, etc., or with any other therapy intended to stimulate the immune system, reduce inflammation or otherwise benefit the subject, such as nutrients, vitamins and supplements. For example, vitamin A, vitamin D, vitamin E, vitamin C, vitamin B complex, selenium, zinc, co- enzyme Q1 0, beta carotene, fish oil, curcumin, green tea, bromelain, resveratrol, ground flaxseed, garlic, lycopene, milk thistle, melatonin, other antioxidants, cimetidine, indomethacin, or COX-2 Inhibitors (e.g., Celebrex™ [celecoxib] or Vioxx™ [rofecoxib]) may be also be administered to the subject.

Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to subjects suffering from a cancer. Any appropriate route of administration may be employed, for example, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, intratumoral, sublingual or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.

Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences" (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. For therapeutic or prophylactic compositions, the pathogenic bacterial species are administered to an individual in an amount effective to stop or slow progression or metastasis of the cancer, or to increase survival of the subject (relative to, for example, prognoses derived from the SEER database) depending on the disorder.

An "effective amount" of a pathogenic microbial species or antigen thereof according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of the cancer cells or tumors, prevention of carcinogenic processes, slowing the growth of the tumor, or an increase in survival time beyond that which is expected using for example the SEER database. A therapeutically effective amount of a pathogenic microbial (bacterial or viral) species or antigen(s) thereof may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the pathogenic bacterial species or virus or antigen thereof are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention of cancer, prevention of metastasis, slowing the growth of the tumor, reduction or elimination of the cancer cells, tissues, organs, or tumors, or an increase in survival time beyond that which is expected using for example the SEER database. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of cancer, so that a prophylactically effective amount may be less than a therapeutically effective amount.

For administration by subcutaneous or intradermal injection, an exemplary range for therapeutically or prophylactically effective amounts of one or more pathogenic bacterial species may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1 000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 1 00 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1 000 million to 4000 million organisms per ml, or any integer within these ranges. The range for therapeutically or prophylactically effective amounts of antigens of a pathogenic bacterial species may be any integer from 0.1 nM- 0.1 M, 0.1 nM-0.05M, 0.05 ηΜ-15μΜ or 0.01 ηΜ-10μΜ. Further, the concentration of antigenic compositions utilized herein may be determined by using the OD600. For example, a dosage of an antigenic composition equating with 5.0 OD600 may be utilized herein. The foregoing is provided as an example and is non-limiting in terms of methods and procedures by which a dosage can be determined.

It is to be noted that dosage concentrations and ranges may vary with the severity of the condition to be alleviated, or may vary with the subject's immune response. In general, the goal is to achieve an adequate immune response. For administration by subcutaneous or intradermal infection, the extent of an immune response may be determined, for example, by size of delayed local immune skin reaction at the site of injection (e.g., from 0.25 inch to 4 inch diameter). The dose required to achieve an appropriate immune response may vary depending on the individual (and their immune system) and the response desired.

Standardized dosages may also be used. In the context of subcutaneous or intradermal administration, if the goal is to achieve a 2 inch local skin reaction, the total bacterial composition dose may, for example, range from 2 million bacteria (e.g., 0.001 ml of a vaccine with a concentration of 2,000 million organisms per ml) to more than 20,000 million bacteria (e.g., 1 ml of a vaccine with a concentration of 20,000 million organisms per ml). The concentrations of individual bacterial species or antigens thereof within a composition may also be considered. For example, if the concentration of one particular pathogenic bacterial species, cell size of that species or antigenic load thereof is much higher relative to the other pathogenic bacterial species in the vaccine, then the local immune skin reaction of an individual may be likely due to its response to this specific bacterial species. In some embodiments, the immune system of an individual may respond more strongly to one bacterial species within a composition than another, depending for example on past history of exposure to infection by a particular species, so the dosage or composition may be adjusted accordingly for that individual. However, in some embodiments detailed herein, an immune response will not be monitored by way of a skin reaction. For example, in some mouse models utilized herein, the effective treatment of such animals with antigenic compositions may not result in corresponding skin reactions. A person skilled in the art will understand that there are alternate ways in which an immune response can be monitored besides relying on the presence or absence of a skin reaction.

For any particular subject, the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. For example, in the context of subcutaneous or intradermal administration, the compositions may be administered every second day. An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01 -0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.

Oral dosages may for example range from 10 million to 1 ,000,000 million organisms per dose, comprising antigenic determinants of one or more species. Oral dosages may be given, for example, from 4 times per day, daily or weekly. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.

In some embodiments, the invention may include antigenic compositions administered sublingually or by inhalation, or administered to one or more epithelial tissues (i.e., skin by intradermal or subcutaneous injection; lung epithelium by inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa by sublingual administration) simultaneously or sequentially.

Accordingly, in some embodiments the antigenic compositions of the invention are administered so as to provoke an immune response in an epithelial tissue. In some embodiments, one or more epithelial routes of administration may be combined with one or more additional routes of administration, such as intratumoral, intramuscular or intravenous administration.

In various aspects of the invention, the antigenic compositions that are administered to a patient may be characterized as having an antigenic signature, i.e., a combination of antigens or epitopes that are sufficiently specific that the antigenic composition is capable of eliciting an immune response that is specific to a particular pathogen, such as an adaptive immune response. In some instances, the non-adaptive or non-specific activation of the immune response that is mediated by these specific antigenic compositions is effective to treat cancers situated in the tissues in which the particular pathogen is pathogenic.

Routes of administration and dosage ranges set forth herein are exemplary only and do not limit the route of administration and dosage ranges that may be selected by medical practitioners. The amount of active compound (e.g., pathogenic bacterial species or viruses or antigens thereof) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be

administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral

compositions in dosage unit form for ease of administration and uniformity of dosage.

In the case of antigenic formulations (analogous to a vaccine), an immunogenically effective amount of a compound of the invention can be provided, alone or in combination with other compounds, with an immunological adjuvant. The compound may also be linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance

immunogenicity. An antigenic composition ("vaccine") is a composition that includes materials that elicit a desired immune response. An antigenic composition may select, activate or expand, without limitation: memory B, T cells, neutrophils, monocytes or macrophages of the immune system to, for example, reduce or eliminate the growth or proliferation of cancerous cells or tissue. In some embodiments, the specific pathogenic microbe, virus, viral antigens, bacteria, bacterial antigens, or compositions thereof of the invention are capable of eliciting the desired immune response in the absence of any other agent, and may therefore be considered to be an antigenic composition. In some embodiments, an antigenic composition includes a suitable carrier, such as an adjuvant, which is an agent that acts in a non-specific manner to increase the immune response to a specific antigen, or to a group of antigens, enabling the reduction of the quantity of antigen in any given vaccine dose, or the reduction of the frequency of dosage required to generate the desired immune response. A bacterial antigenic composition may include live or dead bacteria capable of inducing an immune response against antigenic determinants normally associated with the bacteria. In some embodiments, an antigenic composition may include live bacteria that are of less virulent strains

(attenuated), and therefore cause a less severe infection. In some embodiments the antigenic composition may include live, attenuated or dead viruses capable of inducing an immune response against antigenic determinants normally associated with the virus.

An antigenic composition comprising killed bacteria for administration by injection may be made as follows. The bacteria may be grown in suitable media, and washed with physiological salt solution. The bacteria may then be centrifuged, resuspended in saline solution, and killed with either heat or phenol. The suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner. In addition to the pathogenic bacterial species and/or antigens thereof, a killed bacterial vaccine suitable for administration to humans may include 0.4% phenol preservative and/or 0.9% sodium chloride. The bacterial vaccine may also include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and/or other media components.

In some embodiments, the bacterial vaccine may be used in tablet or capsule form or drops for oral ingestion, as an aerosol for inhalation, or as drops, aerosol or tablet form for sublingual administration.

In antigenic compositions comprising bacteria, the concentrations of specific bacterial species in compositions for subcutaneous or intradermal injection may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1 000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 100 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1 000 million to 4000 million organisms per ml, or any integer within these ranges.

In general, the pathogenic bacterial species and antigens thereof of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population).

In some aspects, the invention involves the use of an anti-inflammatory in conjunction with vaccinations. In these embodiments, a wide variety of antiinflammatory treatments may be employed, including effective amounts of non- steroidal anti-inflammatory drugs (NSAIDs), including but not limited to:

diclofenac potassium, diclofenac sodium, etodolac, indomethicin, ketorolac tromethamine, sulindac, tometin sodium, celecoxib, meloxicam, valdecoxib, floctafenine, mefenamic acid, nabumetone, meloxicam, piroxicam, tenoxicam, fenoprofen calcium, flubiprofen, ibuprofen, ketoprofen, naproxen, naproxen sodium, oxaprozin, tiaprofenic acid, acetylsalicylic acid, diflunisal, choline magnesium trisalicylate, choline salicylate, triethanolamine salicylate, COX1 inhibitors, COX2 inhibitors (e.g., Vioxx™, and Celebrex™). A variety of herbs and natural health products may also be used to provide anti-inflammatory treatment, including but not limited to: green tea, fish oil, vitamin D, antioxidant vitamins and minerals {e.g., B carotene, vitamin A, vitamin C, vitamin D, vitamin E, co-enzyme Q10, selenium, etc.), resveratrol, turmeric, bromelain, boswellia, feverfew, quercetin, ginger, rosemary, oregano, cayenne, clove, nutmeg, willowbark. Alternative anti-inflammatory modalities may also include lifestyle modifications, such as: exercise, weight loss, smoking cessation, stress reduction, seeking social support, treatment of depression, stress management, abdominal breath work and dietary change (such as adopting a mediterranean diet, a low glycemic diet, eating non-charred foods, including foods having omega-3 fatty acids).

Additional Embodiments

In one aspect, a method of comparing immune responses is provided. The method involves administering to an animal having an intestine, a

medicament having an antigenic composition having antigenic determinants selected or formulated so that together the antigenic determinants are specific for at least one Fusobacterium that is pathogenic in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum), extracting a quantifiable immune sample from the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum), measuring a characteristic of the immune response in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum), in the quantifiable immune sample following the administration of the medicament, and, comparing the characteristic of the immune response in the quantifiable immune sample to a corresponding characteristic of the immune response in a reference intestinal immune sample, e.g., where the reference immune sample may be obtained from the corresponding intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum) in the animal prior to the step of administering the medicament or may be obtained from the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum) in a second animal. Optionally, the animal may have a cancer situated in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum).

The formulations of the invention thereby facilitate activation of an immune response to a cancer in the intestine (or a portion thereof, e.g., the large intestine or a portion thereof, e.g., the colon and/or rectum). The immune response may be characterized as an immune response that includes a shift in an activation state of macrophages. For example, the shift in macrophages may include a shift from M2-like macrophages to M1 -like macrophages. The compositions disclosed may, for example, include killed or attenuated microbial pathogens, such as whole killed bacterial cells, and may be administered at sites distant from the cancer, for example the skin or subcutaneous tissue. In some embodiments, microbial species of endogenous flora that are known to cause infection in the relevant organ or tissue may be used in the formulation of the antigenic compositions. In alternative embodiments, exogenous microbial pathogens that are known to cause infection in the relevant organ or tissue may be used in the formulation of the antigenic compositions. The administration of the immunogenic compositions may be repeated relatively frequently over a relatively long period of time. In embodiments for intradermal or subcutaneous injection, dosages may be adjusted so that injections reproduce a consistent, visible, delayed inflammatory immune reaction at the successive site or sites of administration.

Comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. Further, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages or a population of macrophages. Optionally, the macrophages or the population of macrophages may shift from being M2-like macrophages or a population of M2- like macrophages to being M1 -like macrophages or a population of M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages or a population of M1 -like macrophages to being M2-like macrophages or a population of M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. As detailed herein, the macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. Optionally, the cytokines are produced as a result of a shift in an activation state of the macrophages. Optionally, the macrophages shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages shift from being M1 -like macrophages to being M2- like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells:

inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like

macrophages or M2-like macrophages. Optionally, the differential gene expression is produced as a result of a shift in an activation state of the macrophages. Optionally, macrophages may shift from being M2-like

macrophages to being M1 -like macrophages. Further and optionally, the macrophages shift from being M1 -like macrophages to being M2-like

macrophages.

Optionally, the medicament may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week. Optionally, the medicament may be administered intradermal^ or subcutaneously. Optionally, the

medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site. Optionally, the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours. Further and optionally, the animal may be a mammal. Optionally, the animal may be a human or a mouse.

In another aspect, a method of selecting a therapeutic preparation suitable for treating an individual for an intestinal cancer, such as a cancer of the lower intestine, e.g., colon and/or rectum, is provided. The method involves providing an animal having a cancer of the intestine (, such as a cancer of the lower intestine, e.g., colon and/or rectum), providing a test preparation having one or more antigenic determinants of a Fusobacterium which is pathogenic in the corresponding specific organ or tissue in a healthy individual, measuring a characteristic of the immune response in a reference immune sample obtained from the organ or tissue of the animal, administering the test preparation to the animal, measuring a characteristic of the immune response in a quantifiable immune sample obtained from a corresponding organ or tissue of the animal, comparing the characteristic of the immune response in the in the reference and quantifiable immune samples, and treating an enhanced characteristic of the immune response in the quantifiable immune sample compared to the reference immune sample as an indication of the suitability of the test preparation as a therapeutic preparation. Optionally, the animal is sacrificed before the quantifiable immune sample has been obtained.

Optionally, comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1 -like

macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages.

Optionally, the cytokines are produced as a result of a shift in an activate state of the macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. Optionally, the differential gene expression may be produced as a result of a shift in an activation state of the macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

In another aspect, a method of selectively targeting an immune response to a cancerous intestine (or portion thereof, such as the lower intestine, e.g., colon and/or rectum), in a human subject is provided. The method involves administering to the subject a medicament having an effective amount of a Fusobacterium (e.g., Fusobacterium nucleatum, antigenic composition, wherein the Fusobacterium may be pathogenic in the specific organ in which the cancer is situated and the antigenic composition comprises antigenic determinants that together are specific for the microbial pathogen. Optionally, the antigenic composition may include a whole killed bacterial cell composition. Optionally, the medicament may be administered to the subject in an amount and for a time that is effective to up-regulate an immune response in the cancerous organ or tissue of the subject. Optionally, the method may further involve measuring a characteristic of the immune response.

In another aspect, a method of monitoring efficacy of a treatment regime in an individual being treated for an intestinal cancer, such as a cancer of the lower intestine, e.g., colon and/or rectum, is provided. The method involves measuring a characteristic of an immune response in a post-treatment immune sample obtained from the specific organ or tissue after the individual has been subject to the treatment regime for a period of time, wherein the presence of a characteristic of the immune response which is greater in magnitude than would be expected had the individual not been subject to the treatment regime, is indicative of the efficacy of the treatment regime; and the treatment regime involves administering a preparation comprising one or more antigenic determinants of a microbial pathogen which is pathogenic in the corresponding specific organ or tissue in a healthy subject.

The method detailed herein may further involve measuring the

characteristic of the immune response in a pre-treatment reference sample, wherein the pre-treatment reference sample was obtained from the intestine or portion thereof, such as the lower intestine, e.g., colon and/or rectum, before, at the same time as or after commencement of the treatment regime, but prior to obtaining the post-treatment immune sample, and comparing the characteristic of the immune response in the pre-treatment and post-treatment samples, wherein an increase in the magnitude of the immune response in the post- treatment immune sample compared to the pre-treatment reference sample is indicative of the efficacy of the treatment regime. Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of inflammatory monocytes in a sample of the organ or tissue.

Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of macrophages in a sample of the organ or tissue. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages.

Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of CD1 1 b+ Gr-1 + cells in a sample of the organ or tissue or determining an indication of the number of dendritic cells in a sample of the organ or tissue. Further and optionally, measuring the characteristic of the immune response may involve determining an indication of the number of CD1 1 c+ MHC class II+ cells in a sample of the organ or tissue or determining an indication of the number of CD4+ T cells in a sample of the organ or tissue or determining an indication of the number of CD8+ T cells in a sample of the organ or tissue.

Optionally, measuring the magnitude of the immune response may involve determining an indication of the number of NK cells in a sample of the organ or tissue. Further and optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1 -like

macrophages or M2-like macrophages. Further and optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. Optionally, the cytokines may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Further and optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD1 1 b+ Gr-1 + cells, dendritic cells, CD1 1 c+ MHC class I I+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1 -like macrophages or M2-like macrophages. The differential gene expression may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1 -like macrophages. Optionally, the macrophages may shift from being M1 -like macrophages to being M2-like macrophages.

As detailed herein in another aspect, the invention provides methods for formulating an immunogenic composition for treating an intestinal cancer, such as a cancer of the lower intestine, e.g., colon and/or rectum, in a mammal, such as human patient. The method may include selecting at least one Fusobacterium that is naturally pathogenic in the intestine or target portion thereof the mammal within which the cancer is situated. An antigenic composition may be produced that includes antigenic determinants that together are specific for or

characteristic of the Fusobacterium microbial pathogen. Additional details regarding the embodiments described above can also be found in United States Application Serial Nos. 13/019,208; 1 2/234,569 and 1 1 /553,972; as well as PCT application Nos. PCT/CA2007/00191 5 and PCT/ CA2005/000812 and also United States Provisional Application No. 60/577,206; the disclosures of which are herein incorporated by reference.

Additional Aspects

In certain of the above embodiments, the medicament may be

administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a treatment duration of at least one week. Optionally, the medicament may be administered intradermal^ or subcutaneously. Optionally, the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site. Optionally, the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours. However, a visible localized inflammatory immune response may not always be present in all circumstances despite an immune response being initiated. Those skilled in the art will appreciate that there are other methods by which the mounting of an immune response can be monitored. For example, the profile (and relative change in characterization) of immune cells from a subject undergoing an immune reaction can be compared with those from a subject that is not undergoing an immune reaction.

Further and optionally with respect to the methods disclosed herein, the animal may be a mammal. Optionally, the animal may be a human or a mouse. The foregoing examples are provided as examples only and are not meant to be limiting.

The following examples illustrate embodiments of the invention. EXPERIMENTAL

I. Introduction An estimated 1 5% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtractions and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. Fusobacterium nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained Fusobacterium isolate from a frozen tumor specimen and this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p=2.5E-6) and we observed a positive association with lymph node metastasis. II. Materials and methods

A. Clinical specimens

For all cases, fresh CRC samples were obtained with informed consent by the BC Cancer AgencyTumor Tissue Repository (BCCA-TTR) (Watson, 2010, The BC Cancer Agent Tumor Tissue Repository, Biopreserve. Biobanking 8:2) which operates as a dedicated biobank withapproval from the University of British Columbia-British Columbia Cancer Agency Research Ethics Board (BCCA REB). Informed consent was obtained from all donors and samples collected

following assessment of fresh bowel specimens by a pathologist and under standardized operating processes, immediately following surgical resection. Clinical-pathological and outcomes data was obtained from the BC Cancer Agency clinical chart including tumor features reported according to the

American College of Pathologists criteria and 'Protocol for examination of specimens from patients with primary carcinoma of the colon and rectum'. This included histological features indicative of inflammatory and immune response (lymphoid and myeloid cell infiltrates) which were assessed as none, mild- moderate, or marked using semi-quantitative scoring as well as the percent area of tumor involved by necrosis, by a pathologist in a representative tumor cross section.

B. Metagenomic library construction and sequencing

Eleven colorectal tumor samples and eleven matched normal samples were processed, as detailed previously (Moore, et al. "The Sensitivity of

Massively Parallel Sequencing for Detecting Candidate Infectious Agents Associated with Human Tissue," PLOS one (201 1 ) in press), using an RNeasy Plus mini kit (Qiagen) to purify total RNA or an AllPrep DNA/RNA mini kit (Qiagen) to purify both DNA and RNA. RNA quality and concentration was assessed using Agilent Bioanalyzer 2000 RNA Nanochips. Ribosomal RNAs were depleted from 1 mg of total RNA using the manufacturer's protocol for the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen). Depletion was assessed using Agilent Bioanalyzer 2000 RNA Nanochips. Samples with < 1 0% residual ribosomal RNA contamination were processed as described previously (Shah, et al. "Mutational evolution in a lobular breast tumor profiled at single nulceotide resolution," Nature (2009) 461 : 809-U67; Morin, et al. "Somatic mutations altering EZH2 (Tyr641 ) in follicular and diffuse large B-cell lymphomas of germinal-center origin," Nat. Genet. (2010) 42: 181 -U124)) for the construction of lllumina libraries, with the following modifications: Each paired-end library was PCR amplified for 15 cycles using the standard lllumina PE1 PCR primer plus one of 12 modified PE2 primers, each including a unique six base insertion as an index sequence. Libraries prepared using indexed primers were then combined in pools of 1 1 each (one tumor pool, one control pool) gel purified, and then sequenced on the lllumina GAIIx platform. One lane of 75 bp paired end sequence was obtained for each of the two pools.

C. Bioinformatics analysis

Paired-end sequence reads from indexed tumor and adjacent normal sample libraries were processed as described. (Moore, et al., 201 1 , supra) Briefly, corresponding human RNA-seq libraries were aligned with bwa (version 0.5.4 [sample -o 1000, default options] (Li, et al. "Fast and accurate long-read alignment with Burrows-Wheeler transform," Bioinformatics (2010) 26:589-595), sequentially against human rRNA, cDNA and genome reference sequences (Flicek, et al., "Ensemble 201 1 ," Nuc. Acids Res. (201 1 ) 39: D800-D806). Pairs aligning logically, or containing reads having either an average base quality below phred 20 (Ewing, et al. "Base-calling of automated sequencer traces use ph red. I. Accuracy assessment," Genome Res. (1998) 8:175-185) and/or more than 20 consecutive homopolymeric bases were subtracted from the original data. Read pairs that remained unaligned to any of the human sequence databases were used to interrogate a custom-built sequence collection of well- characterized bacterial and viral genes and genomes (lAdb) using Novoalign (version 2.05.20 [-o SAM -r A -R 0, default options] (http://novocraft.com)).

Alignments were run on a single 3GHz 8 CPU Intel(R) Xeon(R) 64-bit 61 GB RAM computer running CentOS release 5.4. Multiplexed reads from the tumor and normal libraries were deconvoluted according to sequence tags (i.e.

barcodes) and the number of read pairs that mapped unambiguously to a single location were tallied for each indexed sample and normalized against the sample read count. Ultimately, read pair count was reported for each Genbank accession in our lAdb, sorted in decreasing order by the sum of unambiguous pairs and PERL scripts were developed to mine these data.

D. Quantitative PCR

A custom TaqMan primer/probe set was designed to amplify

Fusobacterium nucleatum DNA that matched the contiguous sequence from the WTSS experiment. The cycle threshold (Ct) values for Fusobacterium were normalized to the amount of human biopsy gDNA in each reaction by using a primer/probe set for the reference gene, prostaglandin transporter (PGT), as previously described (Wilson, et al. "DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling," Hum. Mol. Genet. (2006)15:743-749). The reaction efficiency for the Fusobacterium assay and the PGT assay were found to be 97% and 98% respectively. The fold difference (2 ^ct) in Fusobacterium abundance in tumor versus normal tissue was calculated by subtracting ACtt_umor from ACt_normal where ACt is the difference in threshold cycle number for the test and reference assay. Isolated biopsy DNA was quantified by PicoGreen Assay (Invitrogen) on a

Wallac Victor spectrophotometer (Perkin Elmer). Each reaction contained 5 ng DNA and was assayed in duplicate in 20 ml reactions containing 1 ^χ final concentration TaqMan Universal Master Mix (ABI part number 4304437), 18 mM of each primer and 5 mM probe and took place in a 384-well optical PCR plate. Amplification and detection of DNA was performed with the ABI 7900HT

Sequence Detection System (Applied Biosystems) using the reaction conditions: 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Cycle thresholding was calculated using the automated settings for SDS 2.2 (Applied Biosystems). Primer and probe sequences for each assay are as follows: Fusobacteria forward primer, 5'

CAACCATTACTTTAACTCTACCATGTTCA 3' (SEQ ID NO:01 ); Fusobacteria reverse primer, 5' G TTG ACTTTAC AG A AG G AG ATT ATG T AAA AATC 3' (SEQ ID NO:02); Fusobacteria FAM

probe, 5' G TTG ACTTTAC AG AAG G AG ATT ATG T AAA AATC 3' (SEQ ID NO:03); PGT forward primer, 5' ATCCCCAAAGCACCTGGT TT 3' (SEQ ID NO:04); PGT reverse primer, 5'

AG AG G CCA AG AT AG TCCTG GT A A 3'(SEQ ID NO:05); PGT FAM probe, 5' CCATCCATGTCCTCATCTC 3' (SEQ ID NO:06). The entire qPCR experiment was performed a second time using the same samples and methods as outlined above, for the purpose of replication, and very similar results were obtained. E. Fusobacterium culture

Frozen tumor sections were thawed and immediately placed into 500 ml of pre-reduced phosphate buffered saline, and the tissue agitated and gently broken up using a pipette fitted with a sterile, wide-bore, plugged tip. 100 ml aliquots of this suspension were directly spread onto pre-reduced fastidious anaerobe agar (FAA) plates supplemented with 5% defibrinated sheep blood (DSB), and incubated for 1 0 days in a humidified anaerobe chamber (Ruskinn Bug Box). Plates were inspected every 2 days for growth, and all colonies were picked and streak-purified on further pre-reduced FAA+5% DSB plates. Single colonies were examined by phase microscopy using a Leica ICC50 microscope fitted with a 1 00χ oil immersion objective, looking for slender rods or needle- shaped cells characteristic of Fusobacterium nucleatum. gDNA was isolated from positively identified isolates using a Maxwell 16 instrument with cell DNA cartidges, and aliquots used as template in PCR with primers and conditions as described by Kim et al. (Kim, et al. "Multiplex PCR using conserved and species- specific 16S rDNA primers for simultaneous detection of Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans," J. Microbiol. Biotechnol. (2004) 14:1 10-1 15). A product size of 495bp confirmed that the isolate belonged to the Fusobacterium genus, and a further PCR to partially amplify 16S rRNA gene was carried out using the same DNA template using primers and conditions as defined by Ben-Dov et al. (Ben-Dov, et al. "Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity," Appl. Environ. Microbiol. (2006) 72:6902-6906); this product was sent for Sanger sequence analysis to MWG Operon, and obtained traces confirmed F.nucleatum as the species. In total, 3 clones of the isolated strain were obtained from the tumor specimen from 53, and named CC53 F, G and H respectively. All strains were stored at -80°C in cryoprotectant media (1 2% w/v skim milk powder, 1 % (v/v) dimethyl sulfoxide and 1 % (v/v) glycerol). F. Primer walking PCR primers were designed using primer 3.0 and the F.nucleatum types strain (ATCC 25586) genome as reference. For PCR, 1 ng of extracted gDNA was used as template, Phusion polymerase (NEB) and buffers were used for the PCR. Cycling conditions were as follows: 94°C for 2 minutes, then 94°C 30 seconds, 67°C 30 seconds, 72°C 30 seconds for 30 cycles. PCR products were purified using Ampure magnetic beads. Sequencing reactions were done using BigDye 3.1 and reaction products were run on AB 3730x1. Phred quality 30 trimmed sequences were used in a BLASTN alignment against the HMP reference genome data, keeping the hit with the highest sequence

identity.

G. Whole genome sequencing of a representative strain

Fusobacterium genomic DNA was sonicated and size fractions between 175 to 200 bp and 400 to 450 bp were isolated following PAGE. WGSS Paired- end lllumina libraries were prepared from each size fraction as described previously with the following modifications: the final PCR amplification was increased to 15 cycles and contained the standard lllumina PE1 PCR primer and an indexed PE2 primer as detailed above for RNA-Seq library construction (Shah, et al., 2009, supra; Morin, et al., 201 0 supra). A total of 92.0M paired 1 00 nt reads were obtained from a single lane of the lllumina HiSeq instrument. After quality filtering, keeping only pairs with an average base quality of Q30 or higher, 64.8M paired reads were aligned with Novoalign (www.novocraft.com; -o SAM -r A -R 0) onto the F.nucleatum subsp. nucleatum ATCC 25586 (Genbank accession NC_003454.1 ) and Fusobacterium sp. 3_1_36A2 genome sequences (HMP accessions GG698790-GG698801 ), respectively. Paired read alignments were processed using custom PERL scripts that tracked the proportion of each genome covered and average sequence identity of mapped pairs. Annotation of strain sp. 3_1 _36A2 regions devoid of read alignments was performed by extracting the coordinates of alignment gaps 1 kbp or larger and mining the HMP genbank-format file for existing gene annotations

(http://www.hmpdacc.org/data_genomes.php). Reads that did not align onto the sp. 3_1 _36A2 genome assembly were quality-trimmed to only include those having 70 or more consecutive Q30 bases and assembled with SSAKE (Warren, et al, 2007) (v3.7 -p 1 -m 20 -o 2 -r 0.7) in 67 contigs (mean size=1 ,225bp max size=6,018bp total bases= 82,076bp N50=1 ,359bp). The contigs were annotated using BLASTX (Altschul, et al. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nuc. Acids Res. (1997) 25:3389-3402 (v2.2.25)), reporting the best hit for each highscoring pairs and manually inspecting each alignment.

In a separate analysis, the 64.8M paired QC reads were filtered further, leaving only sequences having 99 or 100 consecutive Q30 bases. This aggressive filter yielded approximately 32M total reads, including 4.5M paired and 22.9M unpaired reads and assembled with SSAKE (Warren, et al.,

"Assembling millions of short DNA sequences using SSAKE," Bioinformatics (2007) 23:500-501 ) (v3.7 -p 1 -m 20 -o 2 -r 0.7) into 379 contigs (mean size=5,460bp max size=31 ,878bp total bases= 2,069,558bp N50=8,680bp). The Fusobacterium sp. 3_1_36A2 genome assembly was aligned onto the type strain using cross_match (www.phrap.org; -minmatch 29 -minscore 59 - masklevel 101 ) and ordered/oriented based on the latter. Fusobacterium tumor isolate contigs were in turn aligned onto the reordered Fusobacterium sp. 3_1_36A2 HMP genome assembly and ordered/oriented according to that genome sequence, using the same cross_match parameters. Threeway cross_match alignments between the ordered Fusobacterium genomes were performed and plotted using hive plots (www.hiveplot.com).

H. Epithelial cell invasion assays

Caco-2 cell invasion assays with CC53 were carried out in triplicate using a differential staining immunofluorescence procedure as previously described (Strauss, et al. "Invasive potential of gut mucosa-derived Fusobacterium nucleatum positively correlates with IBD status of the host," Inflammatory Bowel Disease (201 1 ) in press). Briefly, bacterial cultures were grown to late log phase according to pre-determined growth-curve data, and normalized for cell number using McFarland standards. Caco-2 cells were grown to 80% confluence on glass coverslips in 24-well plates and infected at a multiplicity of infection of 100:1 (bacterial cells:intestinal cells). Infected cells were maintained at 37°C, 5% CO2 for 4 hours following infection, after which time cells were washed with PBS to remove non-adherent bacteria, and then fixed with 2.5% paraformaldehyde, and blocked in 10% (v/v) normal goat serum. Prepared polyclonal antibodies were diluted to 1 /500, applied to coverslips, and incubated for 1 hr at 37°C. Coverslips were then incubated with donkey anti-rabbit (EAV AS1 ) or anti-rat (EAV_AS2) Alexa 350 (1 /1 00) (Molecular Probes), permeabilized by the addition of 0.1 % TritonX100, and then reincubated with prepared polyclonal antibodies, as above. Following this, cells were labeled with donkey anti-rat or anti-rabbit Cy3 (1 /500) for 30 mins at 37^<Ό, as well as Alexa 488 Phalloidin (Molecular Probes) (1 /200). Coverslips were mounted onto glass slides and examined at 40x magnification using a Leica DMIREB2 microscope and an ORCA-ER digital camera. Images were captured using Volocity (Improvision) software. Using this protocol, bacteria external to the host cell were labeled with both Cy3 and Alexa 350, whereas bacteria inside the cells were labeled with Cy3 only. Each invasion assay was carried out on 3 separate occasions using freshly prepared Caco-2 cells and bacterial inocula. Known invasive isolate, EAVG_002 (7 1 ) was used as a positive control for these experiments. III. Results

Total RNA was isolated from frozen sections of eleven matched pairs of colorectal carcinoma and adjacent normal tissue specimens. RNA was purified by host ribosomal sequence depletion, rather than poly-A selection, in order to retain non-polyadenylated sequences of potential microbial origin. In our screen we analyzed RNA rather than DNA in order to detect active, transcribing microorganisms and to allow for the detection of RNA viruses that may be present. Illumina RNAseq libraries were constructed, barcoded, and pooled, and 2 lanes of paired end sequencing data were obtained using the Illumina GAIIx platform. Reads were filtered for base quality and low complexity, then aligned pairwise to human rRNA and cDNA (Flicek, et al. , 201 1 , supra), and genome (hg18) reference sequences using bwa (Li, et al., 2010, supra), as previously described (Moore, et al., 201 1 , supra). Aligned reads were removed from the data set, leaving 34.9M pairs (Table S1 , Appendix A). These residual read pairs were then used to search a custom database containing accessions for all Refseq bacterial and

viral genomes, using Novoalign (http://novocraft.com), which is a slower but more permissive aligner than bwa. Our analysis was alignment based, because the abundance of candidate organisms can be inferred more directly from alignments than from de novo assemblies. For accuracy we tallied only unambiguous alignments where the best match to both the forward and reverse mate pair was to the same genome accession. These alignments identified a total of 670 distinct genome accessions, representing 415 species (Table S1 ). These were predominantly (97%) bacterial, although several herpes virus sequences were detectable at low levels, and one tumor showed over- expression (142 raw read pairs) of human papillomavirus type 107 (Genbank accession EF422221 .1 ). A wide distribution of bacterial species abundance was apparent, with 30 species representing 95% of the sequence data (Fig S1 ). Of the 670 distinct genome accessions hit, 63% were found in both tumor and normal specimens. Alignments specific to tumor or control specimens were due to rare sequences and, therefore, the representation in one group or the other may simply reflect sampling bias. The clear exception was alignment to

Fusobacterium nucleatum subsp. nucleatum (ATCC 25586), a Gram-negative anaerobe. F. nucleatum was the organism with the highest number of hits overall (21 % of all alignments) and nine of the eleven subjects showed at least two-fold higher read counts in tumor relative to corresponding control tissue (Fig 1 ). The mean over-abundance in tumor was 86-fold. The majority of the hits were to highly abundant F.nucleatum ribosomal transcripts but other non- ribosomal F.nucleatum gene products were also detected (Fig S2).

To explore further the observation of disparate F. nucleatum read counts between tumor and matched normal samples in our RNA-seq data set, we developed a targeted qPCR assay to interrogate additional samples. To design the qPCR primers and probe, we gathered the 51 ,677 read pairs from tumor sample 1 that matched F. nucleatum and performed a local de novo assembly using SSAKE (Warren, et al., 2007, supra) to obtain 861 total contigs, ranging in length from 100 to 1 ,433 bp. The majority of these contigs matched genes encoding F.nucleatum ribosomal RNAs and proteins, but we also obtained 82 contigs that gave BLASTN alignments of 80% or greater sequence identity to other F.nucleatum protein coding genes. A 161 bp contig that returned a high quality BLAST match (95% identity) to the nusG gene (Genbank accession AAL94126.1 ) of F. nucleatum, and no match to any gene of any other species, was used as the target for designing a qPCR (Taqman, ABI) primer/probe set. The initial metagenomics screen described above involved interrogation of expressed genes, however, once we established F.nucleatum as a candidate pathogen we switched to analysis of gDNA because a larger amount of high quality DNA than RNA was obtainable from the frozen tissue sections. We conducted qPCR on gDNA isolated from an additional 88 colorectal carcinomas and matched normal specimens and confirmed an over representation of F.nucleatum in tumor versus matched normal specimens (p=2.5E-6, two-tailed ratio t-test) (Fig 2). The overall abundance of Fusobacterium was found to be 21 7 times greater in the tumor samples (n = 99) than in the matched normal samples (n = 99).

We attempted to culture Fusobacteria anaerobically, directly from several of the frozen tumor sections that showed high abundance by qPCR, and we obtained a single isolate (CC53). We purified HMW gDNA from this culture and constructed and sequenced a WGS library using the lllumina HiSeq platform and obtained an excessive number (64,819,1 56) of quality filtered paired 100 nt reads. These reads were aligned to the finished genome sequence of the F.nucleatum type strain (ATCC 25586) and were found to cover 76% of it, with 2,661 -fold mean depth and 95.6 +/- 2.0% (mean +/- SD) identity. Further, we aligned reads from CC53 to 483 additional draft genome sequences available from the Human Microbiome Project (HMP) (Nelson, et al., "A Catolog of Reference Genomes form the Human Microbiome," Science (2010) 328:994- 999), including sixteen as of yet incomplete Fusobacterium genomes. CC53 aligned with highest identity to Fusobacterium sp. 3_1_36A2, covering 91 .6% of the 1 2-supercontig draft assembly with 99.5 +/- 1 .2% (mean +/- SD) sequence identity. Three-way analysis among these strains using cross_match Smith- Waterman alignments (www.phrap.org) confirmed that CC53 is closest in sequence similarity to Fusobacterium sp. 3_1 _36A2 (Fig 3). Some notable differences were apparent, however. We observed 1 9 segments from strain 3_1_36A2 that were missing from CC53. The majority (156/206) of the predicted coding sequences (CDS) on these segments from strain 3_1_36A2 had unknown function, but there were numerous sequences indicative of prophage content, including genes encoding putative helicase, integrase, recombinase, terminase and topoisomerase activity (Table S2, Appendix A). De novo assembly of unmapped CC53 reads yielded 82 kbp of sequence in 67 contigs >500 nt. These contigs aligned with variable sequence identity to one of the sixteen Fusobacterium genome assemblies or the ATCC type strain. BLASTX (Altschul, et al., 1997, supra) searches of Genbank-nr identified 99 coding sequences (Table S3), the most recurrent of which was hemolysin, a bacterial endotoxin. Most, however, had no predicted function (Table S3). Although we were able to culture Fusobacterium from only a single tumor section, we used primer walking to interrogate an additional four samples where qPCR-predicted levels of Fusobacterium were high. Sanger sequences from these amplicons comprised 68,694 total base pairs and each aligned with highest sequence similarity (93-100%) to one of the various Fusobacterium draft genomes, although we could not assign unambiguously a specific best matching strain to any of these samples, due perhaps to within sample strain heterogeneity.

We were interested to determine if CC53 would demonstrate invasiveness in human colonic epithelial cells. We used immunofluorescence and an antibody- based differential staining method, described previously (Strauss, et al., 201 1 , supra), to measure invasion of cultured Caco-2 cells by the Fusobacterium tumor isolate. We identified two previous Fusobacterium polyclonal antibodies, one rabbit (EAV_AS1 ) and one rat (EAV_AS2), which reacted positively to CC53. Caco-2 cells were grown on glass coverslips, infected with CC53 culture (at a multiplicity of infection of 100:1 ), and then differentially stained with anti- Fusobacterium antibodies conjugated to different fluorophores before and after Caco-2 cell permeabilization. We confirmed the invasiveness of CC53 in this model system (Fig 4).

We explored clinical correlates of Fusobacterium overabundance but did not observe any association with tumor stage, tumor site, history of treatment or survival. To explore histopathological correlates, an H&E stained section from a representative cross section clinical block from each tumor was scored for lymphocytic infiltrates, myeloid/neutrophil infiltrates, circumferential involvement, and luminal or geographic necrosis, and these scores were compared to

Fusobacterium relative abundance (tumor versus control). Fusobacterium showed higher relative abundance in tumours with > 50% circumferential involvement (unpaired, two-tailed t-test, p= 0.0023). In addition, we found that subjects with high relative abundance Fusobacterium in tumor relative to matched control tissue were significantly more likely to have regional lymph node metastases, as determined by their TNM scores (one-tailed Fisher's exact test, p = 0.0035) (Figure S3). Specifically, lymph node metastases were present in 29/39 patients in the high abundance Fusobacterium group versus 26/58 in the low abundance group. IV. Discussion

Fusobacterium nucleatum is an invasive (Swidsinski, et al., Acute appendicitis is characterized by local invasion with Fusobacterium

nucleatum/necrophorum," Gut (201 1 ) 60:34-40), adherent (Weiss, et al.

"Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin," Oral Microbiol. Immunol. (2000) 15:371 -377) and pro-inflammatory (Peyret-Lacombe, et al., "TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response," Cytokine (2009) 46:201 - 21 0; Krisanaprakornkit, et al., "Inducible expression of human beta-defensin 2 by Fusobacterium nucleatum in oral epithelial cells:Multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier," Infect. Immun. (2000) 68:2907-2915) anaerobic bacterium. It is common in dental plaque (Ximenez-Fyvie, et al., "Comparison of the microbiota of supra-and subgingival plaque in health and periodontitis," J. Clin. Periodontol. (2000) 27:648:657; Feng, et al., "Human transcriptome subtraction by using short sequence tags to search for tumor viruses in conjunctival carcinoma," J. Virol. (2007) 81 :1 1332-1 13340) and there is a well-established association between F.nucleatum and periodontitis (Signat, et al., "Role of Fusobacterium nucleatum in Periodontal Health and Disease," Curr. Issues Mol. Biol. (201 1 ) 13:25-35). Anecdotally, F. nucleatum has been found to cause cerebral abscesses (Kai, et al., "A rare presentation of ventriculitis and brain abscess caused by

Fusobacterium nucleatum," J. Med. Microbiol. (2008) 57:668-671 ) and pericarditis (Han, et al., "Fusobacterial brain abscess: a review of five cases and an analysis of possible pathogenenis," J. Neurosurg. (2003) 99:693-700) and it is the infectious agent that is primarily responsible for Lemierre's syndrome, a rare form of thrombophlebitis. More recently, various Fusobacteria including F.nucleatum have been implicated in acute appendicitis, where they have been found by IHC as epithelial and submucosal infiltrates that correlate positively with severity of disease (Swidsinski, et al., 201 1 , supra). Further, when isolated from human intestinal biopsy material, F.nucleatum has been found to be more readily culturable from patients with Gl disease than healthy controls, and the strains grown from inflamed biopsy tissue tend to display a more invasive phenotype (Strauss, et al., 201 1 , supra; Strauss, et al., "Phenotypic and genotypic analyses of clinical Fusobacterium nucleatum and Fusobacteriaum periodonticum isolates from the human gut," Anaerobe (2008) 14:301 -309).

F. nucleatum is a well-known pathogen, but it has not been associated previously with cancer of the Gl tract or any other cancer site. Our observation of an over-representation of F.nucleatum in colorectal tumor specimens was largely unexpected, given that F.nucleatum is generally regarded as an oral pathogen, and it is not an abundant constituent of the normal gut microbiota (Weiss, et al., "Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin," Oral Microbiol. Immunol. (2000) 15:371 -377). Based on the above findings, F.nucleatum is indicated to be involved in tumorigenesis, e.g., through pro-inflammatory mechanisms, particularly with colon cancer. In the present study, three of the 99 tumor sections were comprised entirely of the adenomatous component of adenocarcinoma specimens. By qPCR, two out of these three had very high Fusobacterium content and, in fact, one of these gave the highest tumor normal ratio of all samples. These studies indicate that Fusobacterium infection is related to the early stages of tumor progression and is therefore an appropriate target for vaccination and/or antimicrobial therapy. Furthermore, the above results show that an antigenic composition comprised of antigens of Fusobacterium nucleatum could be used to treat cancer of the colon/rectum.

In addition, the above results show that the presence of Fusobacterium infection can be employed for diagnostic purposes, especially given the low prevalence of this species in the fecal microbiota of healthy individuals.

OTHER EMBODIMENTS

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

It is to be understood that this invention is not limited to particular aspects described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each

intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as

antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and aspects of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof.

Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary aspects shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Supplementary Tables

Table S 1. Host sequence subtraction RNA-seq data from eleven colorectal carcinoma and matched normal specimens.

Control Tumour

(mean +/- SD) (mean +/- SD)

Raw read pairs 2,222,539 +/- 355,530 2, 175,063 +/- 439,279

Filtered read pairs¹ 349,354 +/- 212,209 339,935 +/- 182,207

Read pairs matching

bacterial or viral genomes² 17, 154 +/- 22,837 15,681 +/- 29,568

Distinct bacterial or viral

genome matches 546 544

1. Read pairs remaining after removal of low quality reads and reads matching human rRNA, transcriptome or genome reference sequences.

2. Unambiguous alignments where the best match for each mate pair is to the same accession.

Table S2. Gene content of gaps (segments from strain 3_1_36A2 accession that are missing from CC53).

Coordina tes of

unaligned Unaligned region CDS start CDS end

region length Protein id coordinate coordinate Predicted product

160980-1621 19 1139 EEU32070.1 161007 162053 predicted protein

163749-165149 1400 EEU32072.1 162S70 163818 2-nitropropane dioxygenase

163749-165149 1400 EEU32073.1 163997 164458 N-acetylmuramoyl-L-alanine amidase

163749-165149 1400 EEU32074.1 16₄₄90 165128 conserved hypothetical protein

163749-165149 1400 EEU32075.1 166472 165135 conserved hypothetical protein

230196-237851 7655 EEU32133.1 230444 234547 CRISPR-associated protein

230196-237851 7655 EEU32134.1 234572 235450 CRISPR-associated protein casl

230196-237851 7655 EEU32135.1 235440 235760 conserved hypothetical protein

230196-237851 7655 EEU32136.1 235757 236419 conserved hypothetical protein

230196-237851 7655 EEU32137.1 238229 237840 conserved hypothetical protein

413437-423361 9924 EEU32297.1 415519 413525 hemin receptor

413437-423361 9924 EEU32298.1 415901 416818 nickel ABC transporter

413437-423361 9924 EEU32299.1 416839 417642 nickel ABC transporter

413437-423361 9924 EEU32300.1 417655 418416 nickel import ATP-binding protein NikD

413437-423361 9924 EEU32301.1 418421 419113 nickel import ATP-binding protein NikE

413437-423361 9924 EEU32302.1 4191 10 420735 nickel import ATP-binding protein NikE

413437-423361 9924 EEU32303.1 421767 420790 transcriptional regulator AraC family

413437-423361 9924 EEU32304.1 421979 423325 MATE efflux family protein

800252-803854 3602 EEU32662.1 800383 798980 MATE efflux family protein

800252-803854 3602 EEU32663.1 801168 800395 phosphonate C-P lyase system protein PhnK

800252-803854 3602 EEU32664.1 802198 801 161 transport system permease

800252-803854 3602 EEU32665.1 803435 802293 periplasmic binding protein

800252-803854 3602 EEU32666.1 803816 803643 predicted protein

804906-806958 2052 EEU32668.1 806372 805035 conserved hypothetical protein

804906-806958 2052 EEU32669.1 806844 806386 conserved hypothetical protein

854976-876729 21753 EEU32722.1 855099 855521 predicted protein

854976-876729 21753 EEU32723.1 855S50 856117 predicted protein

854976-876729 21753 EEU32724.1 860232 856126 predicted protein

854976-876729 21753 EEU32725.1 862070 860229 predicted protein

854976-876729 21753 EEU32726.1 862942 862052 predicted protein 854976-876729 21753 EEU32727.1 864868 862955 predicted protein

854976-876729 21753 EEU32728.1 865392 864868 predicted protein

854976-876729 21753 EEU32729.1 865593 865405 conserved hypothetical protein

854976-876729 21753 EEU32730.1 866029 865598 conserved hypothetical protein

854976-876729 21753 EEU32731.1 866417 866037 conserved hypothetical protein

854976-876729 21753 EEU32732.1 866976 866473 predicted protein

854976-876729 21753 EEU32733.1 867481 866978 predicted protein

854976-876729 21753 EEU32734.1 868738 867494 predicted protein

854976-876729 21753 EEU32735.1 869S42 868791 predicted protein

854976-876729 21753 EEU32736.1 870369 869653 predicted protein

854976-876729 21753 EEU32737.1 870587 870369 predicted protein

854976-876729 21753 EEU32738.1 872229 870577 predicted protein

854976-876729 21753 EEU32739.1 872706 872239 predicted protein

854976-876729 21753 EEU32740.1 874108 872717 predicted protein

854976-876729 21753 EEU32741.1 874781 874083 predicted protein

854976-876729 21753 EEU32742.1 875192 875425 predicted protein

854976-876729 21753 EEU32743.1 875441 875674 predicted protein

854976-876729 21753 EEU32744.1 875S64 875915 predicted protein

854976-876729 21753 EEU32745.1 876091 875912 predicted protein

854976-876729 21753 EEU32746.1 876444 876091 predicted protein

854976-876729 21753 EEU32747.1 876724 876449 predicted protein

877026-882644 5618 EEU32748.1 877946 877146 replicative DNA elicase

877026-882644 5618 EEU32749.1 878S88 877948 conserved hypothetical protein

877026-882644 5618 EEU32750.1 880025 879648 predicted protein

877026-882644 5618 EEU32751.1 880385 880164 predicted protein

877026-882644 5618 EEU32752.1 880941 880543 predicted protein

877026-882644 5618 EEU32753.1 881131 880952 predicted protein

877026-882644 5618 EEU32754.1 881328 881 131 conserved hypothetical protein

877026-882644 5618 EEU32755.1 882003 881347 LexA repressor

877026-882644 5618 EEU32756.1 882166 882540 predicted protein

883266-886895 3629 EEU32757.1 883275 883967 conserved hypothetical protein

883266-886895 3629 EEU32758.1 883977 884489 gp157

883266-886895 3629 EEU32759.1 884500 885222 predicted protein 883266-886895 3629 EEU32760.1 885219 885518 conserved hypothetical protein

883266-886895 3629 EEU32761.1 885515 885745 phage protein

883266-886895 3629 EEU32762.1 885708 886172 phage protein

883266-886895 3629 EEU32763.1 886162 886734 conserved hypothetical protein

883266-886895 3629 EEU32764.1 886721 886873 predicted protein

887195-890120 2925 EEU32765.1 887406 887561 predicted protein

887195-890120 2925 EEU32766.1 887576 887806 predicted protein

887195-890120 2925 EEU32767.1 887775 888182 conserved hypothetical protein

887195-890120 2925 EEU32768.1 888187 888717 conserved hypothetical protein

887195-890120 2925 EEU32769.1 888731 888922 predicted protein

887195-890120 2925 EEU32770.1 888960 890027 DNA integration/recornbination/invertion protein

965306-1019₄50 54144 EEU32841.1 966788 965571 DNA integration/recornbination/invertion protein

965306-1019₄50 5414₄ EEU32842.1 967072 966854 predicted protein

965306-1019450 54144 EEU32843.1 967701 967090 predicted protein

965306-1019450 54144 EEU32844.1 968005 96781 1 predicted protein

965306-1019450 54144 EEU32845.1 968S58 968203 predicted protein

965306-1019450 54144 EEU32846.1 968932 968690 predicted protein

965306-1019450 54144 EEU32847.1 969592 969347 conserved hypothetical protein

965306-1019450 54144 EEU32848.1 969875 969612 predicted protein

965306-1019450 54144 EEU32849.1 970173 969913 conserved hypothetical protein

965306-1019450 54144 EEU32850.1 970S20 970282 conserved hypothetical protein

965306-1019450 54144 EEU32851.1 970875 970666 predicted protein

965306-1019450 54144 EEU32852.1 971132 970980 predicted protein

965306-1019450 54144 EEU32853.1 971450 971241 predicted protein

965306-1019450 54144 EEU32854.1 971 S49 971443 predicted protein

965306-1019450 54144 EEU32855.1 973026 972361 predicted protein

965306-1019450 54144 EEU32856.1 973736 973035 ATPase

965306-1019450 54144 EEU32857.1 973922 974233 predicted protein

965306-1019450 54144 EEU32858.1 974562 974254 predicted protein

965306-1019450 54144 EEU32859.1 9751 4 974566 lytic transglycosylase

965306-1019450 54144 EEU32860.1 977485 975188 predicted protein

965306-1019450 54144 EEU32861.1 978407 978042 predicted protein

965306-1019450 54144 EEU32862.1 978937 978455 conserved hypothetical protein 965306-1019450 54144 EEU32863.1 979139 978984 predicted protein

965306-1019450 54144 EEU32864.1 979795 979139 resolvass/recombinase

965306-1019450 54144 EEU32865.1 980528 979782 conserved hypothetical protein

965306-1019450 54144 EEU32866.1 980S98 980540 predicted protein

965306-1019450 54144 EEU32867.1 981075 980707 resolvase/recombinase

965306-1019450 54144 EEU32868.1 981730 981062 conserved hypothetical protein

965306-1019450 54144 EEU32869.1 981903 981730 predicted protein

965306-1019450 54144 EEU32870.1 982381 982019 predicted protein

965306-1019450 54144 EEU32871.1 982S53 982450 predicted protein

965306-1019450 54144 EEU32872.1 983480 982896 predicted protein

965306-1019450 54144 EEU32873.1 983895 983494 predicted protein

965306-1019450 54144 EEU32874.1 984215 983892 predicted protein

965306-1019450 54144 EEU32875.1 990789 984421 helicase

965306-1019450 54144 EEU32876.1 992550 990865 conserved hypothetical protein

965306-1019450 54144 EEU32877.1 993259 992810 predicted protein

965306-1019450 54144 EEU32878.1 993786 993256 predicted protein

965306-1019450 54144 EEU32879.1 996192 993802 type I topoisomease

965306-1019450 54144 EEU32880.1 996886 996269 predicted protein

965306-1019450 54144 EEU32881.1 997S13 997140 predicted protein

965306-1019450 54144 EEU32882.1 998S31 997690 predicted protein

965306-1019450 54144 EEU32883.1 999162 998644 conjugative transfer signal peptidase TraF

965306-1019450 54144 EEU32884.1 999454 999164 predicted protein

965306-1019450 54144 EEU32885.1 999851 999642 predicted protein

965306-1019450 54144 EEU32886.1 1000454 1000095 predicted protein

965306-1019450 54144 EEU32887.1 1000741 1000538 predicted protein

965306-1019450 54144 EEU32888.1 1001035 1000859 predicted protein

965306-1019450 54144 EEU32889.1 1001463 1001 170 predicted protein

965306-1019450 54144 EEU32890.1 1002564 1001503 TrbL/VirB6 plasmid conjugal transfer protein

965306-1019450 54144 EEU32891.1 1003026 1002568 predicted protein

965306-1019450 54144 EEU32892.1 1003896 1003048 predicted protein

965306-1019450 54144 EEU32893.1 1006532 1004058 conjugal transfer protein TrbE

965306-1019450 5 144 EEU32894.1 1006825 1006538 predicted protein

965306-1019450 54144 EEU32895.1 1007801 1006845 P-type conjugative transfer ATPase TrbB 965306-1019450 54144 EEU32896.1 1008154 1007801 predicted protein

965306-1019450 54144 EEU32897.1 1010223 1008154 TRAG protein

965306-1019450 54144 EEU32898.1 1010784 1010308 predicted protein

965306-1019450 54144 EEU32899.1 1012013 1010799 conjugation Trbl family protein

965306-1019450 54144 EEU32900.1 1012811 1012023 P-type conjugative transfer protein VirB9

965306-1019450 54144 EEU32901.1 1013518 1012823 conserved hypothetical protein

965306-1019450 54144 EEU32902.1 1013896 1013579 conjugal transfer protein TrbC

965306-1019450 54144 EEU32903.1 1014228 1013893 predicted protein

965306-1019450 54144 EEU32904.1 1015212 1014400 predicted protein

965306-1019450 54144 EEU32905.1 1015636 1015244 conserved hypothetical protein

965306-1019450 54144 EEU32906.1 1016055 1015723 conserved hypothetical protein

965306-1019450 54144 EEU32907.1 1016469 1016074 predicted protein

965306-1019450 54144 EEU32908.1 1016769 1016473 conserved hypothetical protein

965306-1019450 54144 EEU32909.1 1016991 1016851 conserved hypothetical protein

965306-1019450 54144 EEU32910.1 1017888 1017229 conserved hypothetical protein

965306-1019450 54144 EEU32911.1 1019162 1017885 predicted protein

1110825-1134389 23564 EEU32992.1 1111325 1110942 toxin secretion/phage lysis holin

1110825-1134389 23564 EEU32993.1 1111789 1111340 conserved hypothetical protein

1110825-1134389 23564 EEU32994.1 1112159 1111776 conserved hypothetical protein

1110825-1134389 23564 EEU32995.1 1112371 1112174 predicted protein

1110825-1134389 23564 EEU32996.1 1112940 1112431 conserved hypothetical protein

1110825-1134389 23564 EEU32997.1 1113824 1113054 conserved hypothetical protein

1110825-1134389 23564 EEU32998.1 1114533 1113811 conserved hypothetical protein

1110825-1134389 23564 EEU32999.1 1115187 1114537 conserved hypothetical protein

1110825-1134389 23564 EEU33000.1 1116244 1115180 baseplate J-like protein

1110825-1134389 23564 EEU33001.1 1116673 1116245 phage protein

1110825-1134389 23564 EEU33002.1 1117127 1116675 conserved hypothetical protein

1110825-1134389 23564 EEU33003.1 1118167 1117124 predicted protein

1110825-1134389 23564 EEU33004.1 1118618 1118172 conserved hypothetical protein

1110825-1134389 23564 EEU33005.1 1120539 1118632 phage protein

1110825-1134389 23564 EEU33006.1 1121062 1120601 predicted protein

1110825-1134389 23564 EEU33007.1 1121594 1121226 conserved hypothetical protein

1110825-1134389 23564 EEU33008.1 1122044 1121604 conserved hypothetical protein 1110825-1134389 23564 EEU33009.1 1123136 1122057 conserved hypothetical protein

1110825-1134389 23564 EEU33010.1 1123585 1123136 conserved hypothetical protein

1110825-1134389 23564 EEU33Q11.1 1123962 1123582 phage protein

1110825-1134389 2356₄ EEU33012.1 1124317 1123952 conserved hypothetical protein

1110825-1134389 23564 EEU33013.1 1124643 1124314 conserved hypothetical protein

1110825-1134389 23564 EEU33014.1 1124884 1124678 conserved hypothetical protein

1110825-1134389 23564 EEU33015.1 1126087 1124894 phage protein

1110825-1134389 2356₄ EEU33016.1 1126710 1126087 predicted protein

1110825-1134389 2356₄ EEU33017.1 1127074 1126856 conserved hypothetical protein

1110825-1134389 23564 EEU33018.1 1128740 1127058 phage protein

1110825-1134389 23564 EEU33020.1 1130040 1128724 terminase

1110825-1134389 23564 EEU33019.1 1130040 1128724 phage portal protein

1110825-1134389 23564 EEU33021.1 1131871 1131479 phage Terminase Small Subunit

1110825-1134389 23564 EEU33022.1 1132196 1131936 predicted protein

1110825-1134389 23564 EEU33023.1 1133038 1132538 predicted protein

1110825-1134389 23564 EEU33024.1 1133406 1133050 predicted protein

1110825-1134389 23564 EEU33025.1 1134145 1133408 phage antirepressor protein

1110825-1134389 23564 EEU33026.1 1134359 1134150 predicted protein

1134686-1141495 6809 EEU33027.1 1136021 1134807 replicative DNA elicase

1134686-1141495 6809 EEU33028.1 1136792 1136022 conserved hypothetical protein

1134686-1141495 6809 EEU33029.1 1137732 1137460 predicted protein

1134686-1141495 6809 EEU33030.1 1138145 1137930 predicted protein

1134686-1141495 6809 EEU33031.1 1138750 1138265 predicted protein

1134686-1141495 6809 EEU33032.1 1139046 1138882 predicted protein

1134686-1141495 6809 EEU33033.1 1139965 1139114 predicted protein

1134686-1141495 6809 EEU33034.1 1140391 1139975 conserved hypothetical protein

1134686-1141495 6809 EEU33035.1 11 0823 1140404 predicted protein

1134686-1141495 6809 EEU33036.1 1141005 1141391 predicted protein

1142122-1145342 3220 EEU33037.1 1142129 1142821 conserved hypothetical protein

1142122-1145342 3220 EEU33038.1 1142831 1143343 predicted protein

1142122-1145342 3220 EEU33039.1 1143353 1143595 predicted protein

1142122-1145342 3220 EEU33040.1 1143607 1144329 predicted protein

1142122-1145342 3220 EEU33041.1 11 4326 1144604 predicted protein 1142122-1145342 3220 EEU33042.1 1144729 1145166 conserved hypothetical protein

1142122-1145342 3220 EEU33043.1 1145169 1145321 predicted protein

1145607-1149818 4211 EEU33044.1 1145689 1145847 predicted protein

1145607-1149818 4211 EEU33045.1 1145834 1146547 conserved hypothetical protein

1145607-1149818 4211 EEU33046.1 1146612 1147022 predicted protein

1145607-1149818 4211 EEU33047.1 1147025 1147480 conserved hypothetical protein

1145607-1149818 4211 EEU33048.1 1147487 1147777 predicted protein

1145607-1149818 4211 EEU33049.1 11 7777 1148307 conserved hypothetical protein

1145607-1149818 4211 EEU33050.1 1148298 1148429 predicted protein

1145607-1149818 4211 EEU33051.1 1148500 1148697 predicted protein

1145607-1149818 4211 EEU33052.1 1148672 1149727 phage integrase

Table S3. Annotation of CC53 contigs from de novo assembly of unaligned reads.

Fusobacterium nucleatum subsp. vincentii

133 1344 6493 425.34 ZP 00144284.1 99.6 1343 3 Hemolysin ATCC 49256

Histidyl-tRNA Fusobacterium

137 907 3104 306.18 ZP_05551287.1 100.0 485 3 synthetase sp. 3_1_36A2

Recombination factor Fusobacterium

137 907 3104 306.18 ZP_05442395.1 100.0 907 500 protein RarA sp. D11

Fusobacterium nucleatum subsp. vincentii

138 1935 9406 441.42 ZP 00143333.1 99.4 1797 238 Hypothetical protein ATCC 49256

Conserved Fusobacterium

138 1935 9406 441.42 ZP_05550632.1 97.9 1933 1793 hypothetical protein sp. 3 1 36A2

ABC transporter iron

chelate uptake

transporter (FeCT) Fusobacterium

147 6018 36729 545.52 ZP_06750290.1 99.7 1685 693 family sp- 3_1_27

Fusobacterium nucleatum subsp.

Iron(lll) dicitrate- nucleatum

147 6018 36729 545.52 ZP_06869931.1 99.4 2753 1704 binding protein ATCC 23726

Conserved Fusobacterium

147 6018 36729 545.52 ZP 05816109.1 98.9 4431 2779 hypothetical protein sp. 3 1 33

Nitrogenase iron Fusobacterium

147 6018 36729 545.52 ZP_06750286.1 99.6 5210 4473 protein sp. 3_1_27

Fusobacterium nucleatum

Iron(lll) dicitrate subsp. transport ATP-binding nucleatum

147 6018 36729 545.52 NP 603209.1 99.2 6017 5295 protein fecE ATCC 25586

Fusobacterium

151 1516 4712 280.53 ZP_04571805.1 99.4 1515 1 Hemolysin sp. 4 1 13

Fusobacterium

153 1072 32S1 270.7 ZP_06749749.1 97.2 1072 2 Hemolysin sp. 3_1_27

Dipeptide-binding Fusobacterium

154 2052 8508 365.31 ZP_04572815.1 99.8 1997 465 protein sp. 4_1_13

Iron(lll) dicitrate

transport system Fusobacterium

157 2008 15927 706.84 ZP_058161 12.1 100.0 1435 2007 permease fecD sp. 3 1 33

Iron(lll) dicitrate- Fusobacterium

157 2008 15927 706.84 ZP_058161 13.1 99.1 372 1340 binding protein sp. 3 1 33

Conserved Fusobacterium

161 2363 5320 201.38 ZP_04572103.1 99.6 1 1491 hypothetical protein sp. 4_1_13

Fusobacterium nucleatum

Threonyl-tRNA subsp. vincentii

161 2363 5320 201.38 ZP_00144407.1 100.0 1746 2363 synthetase ATCC 49256 Fusobacterium nucleatum subsp. vincentii

164 1072 5821 482.41 ZP 00144406.1 100.0 517 20 Hypothetical protein ATCC 49256

Fusobacterium nucleatum

Threonyl-t NA subsp. vincentii

164 1072 5821 482.41 ZP_00144407.1 100.0 1072 551 synthetase ATCC 49256

Conserved Fusobacterium

167 605 1915 268.06 ZP_06751328.1 100.0 475 248 hypothetical protein sp. 3_1_27

Conserved Fusobacterium

167 605 1915 268.06 ZP 04571297.1 100.0 79 216 hypothetical protein sp. 4 1 13

Fusobacterium nucleatum subsp.

Short-chain alcohol polymorphum

168 1032 2780 240.22 ZP_04971343.1 96.6 446 3 dehydrogenase ATCC 10953

Tetratrico peptide Fusobacterium

168 1032 2780 240.22 ZP_06750238.1 100.0 1030 614 repeat family protein sp. 3_1_27

Fusobacterium

Hypothetical protein; nucleatum Spore photoproduct subsp. vincentii

170 820 992 108.68 ZP_00143341.1 100.0 1 819 lyase ATCC 49256

Outer membrane Fusobacterium

177 550 2139 355.73 ZP 04571419.1 100.0 550 353 protein sp. 4 1 13

Clostridium

Hypothetical protein bartlettii DS

183 598 1195 166.51 ZP_02212678.1 85.2 244 2 CLOBA _02295 16795

Fusobacterium nucleatum subsp.

Hypothetical protein polymorphum

183 598 1195 166.51 ZP_04971346.1 96.3 406 567 FNPJ655 ATCC 10953

2-nitropropane Fusobacterium

184 1002 3195 282.52 ZP_045721 12.1 100.0 1000 287 dioxygenase sp. 4 1 13

Fusobacterium nucleatum subsp. vincentii

185 736 4177 479.89 ZP_00143874.1 93.9 736 638 Hypothetical protein ATCC 49256

Conserved Fusobacterium

186 922 1639 156.79 ZP_06750274.1 99.0 3 599 hypothetical protein sp. 3 1 27

Hypothetical protein Fusobacterium

186 922 1639 156.79 ZP_05442397.1 100.0 620 922 PrD1 1J 1325 sp. D11

Fusobacterium

194 1080 2883 241.44 ZP_06751048.1 100.0 935 3 Formimidoylglutamase sp. 3_1_27

Polysialic acid capsule

expression protein Fusobacterium

196 2546 7657 269.47 ZP_04572326.1 99.6 1730 2545 kpsF sp. 4 1 13

Conserved Fusobacterium

200 1401 781 508.57 ZP_06749764.1 95.0 545 72 hypothetical protein sp. 3 1 27 Fusobacterium

Hypothetical protein gonidiaformans

200 1401 7817 508.57 ZP_05631047.1 80.4 1017 559 FgonA2_04800 ATCC 25563

Fusobacterium

200 1401 781 508.57 ZP_06749761.1 100.0 1399 1106 Hemolysin sp- 3_1_27

Uroporphyrinogen-lll Fusobacterium

20₄ 531 841 141.1 1 ZP_05440307.1 100.0 1 282 synthase sp. D11

Fusobacterium nucleatum subsp. vincentii

212 529 755 120.99 ZP_00143146.1 98.9 528 1 Serine protease ATCC 49256

Fusobacterium

218 906 2602 256.19 ZP_04572999.1 100.0 1 555 ATP-NAD kinase sp. 4_1_13

DNA repair protein Fusobacterium

218 906 2602 256.19 ZP_06750251.1 100.0 537 905 RecN sp- 3_1_27

Fusobacterium nucleatum

Outer membrane subsp. vincentii

224 1039 2609 222.98 ZP_00144496.1 98.3 1038 1 protein family ATCC 49256

Fusobacterium

4- nucleatum hydroxybutyrate:acetyl- subsp. vincentii

225 681 954 123.98 ZP_00144049.1 99.6 2 679 CoA CoA transferase ATCC 49256 t NA (guanine-N1 )- Fusobacterium

232 734 1495 176.87 ZP_06750263.1 100.0 636 1 methyltransferase sp. 3_1_27

Ribosomal-protein- alanine Fusobacterium

235 517 504 87.56 ZP_04572837.1 100.0 180 515 ace tyltransfe rase sp. 4 1 13

Fusobacterium

237 882 1514 154.52 ZP_04574766.1 100.0 273 881 Methyltransferase sp. 7_1

Conserved Fusobacterium

246 1062 1254 100.1 1 ZP_05551301.1 100.0 123 680 hypothetical protein sp. 3_1_36A2

Fusobacterium nucleatum subsp. vincentii

248 689 1443 193.6 ZP_00144645.1 100.0 434 670 Hypothetical protein ATCC 49256

Fusobacterium

249 2756 5701 186.29 ZP_04571805.1 97.3 2 2755 Hemolysin sp. 4_1_13

Dipeptide-binding Fusobacterium

250 581 688 104.33 ZP_06751292.1 99.5 3 569 protein sp. 3 1 27

Fusobacterium

262 631 487 70.78 ZP_04574649.1 100.0 631 29 Membrane protein sp. 7_1

Fusobacterium nucleatum

Aspartate subsp. vincentii

270 1010 2177 194.64 ZP_00143639.1 99.7 2 967 aminotransferase ATCC 49256

Conserved Fusobacterium

271 1 143 1665 129.3 ZP 04574784.1 99.6 463 1143 hypothetical protein sp. 7_1 D-methionine ABC

transporter, ATP- Fusobacterium

273 822 984 106.27 ZP_05550411.1 99.5 598 2 binding protein sp. 3_1_36A2

Fusobacterium

287 1047 2087 174.75 ZP_06524412.1 99.3 1001 180 Export ABC transporter sp. D11

Fusobacterium

29₄ 1460 3081 189.01 ZP_04574771.1 100.0 2 652 Riboflavin kinase sp. 7_1

Conserved Fusobacterium

29₄ 1460 3081 189.01 ZP_05814256.1 100.0 627 1301 hypothetical protein sp. 3_1_33

Conserved Fusobacterium

297 1359 1631 107.16 ZP_04572332.1 100.0 1044 151 hypothetical protein sp. 4_1_13

Conserved Fusobacterium

299 942 1989 192.42 ZP_06750860.1 99.4 3 941 hypothetical protein sp. 3_1_27

Polysaccharide Fusobacterium

303 782 1239 141.49 ZP_04572817.1 98.9 3 782 deacetylase sp. 4 1 13

Fusobacterium nucleatum subsp.

Possible DNA repair polymorphum

305 839 2387 258.12 ZP_04970650.1 98.3 178 2 photolyase ATCC 10953

Fusobacterium nucleatum subsp. vincentii

305 839 2387 258.12 ZP_00143340.1 100.0 475 206 Hypothetical protein ATCC 49256

Fusobacterium nucleatum subsp. vincentii

305 839 2387 258.12 ZP_00143339.1 100.0 749 471 Hypothetical protein ATCC 49256

Iron(lll) dicitrate

transport system Fusobacterium

319 537 136 21.86 ZP_05816107.1 100.0 2 400 permease fecD sp. 3_1_33

Conserved Fusobacterium

334 667 616 81.93 ZP 04572103.1 100.0 1 666 hypothetical protein sp. 4 1 13

Filamentation induced Fusobacterium

346 978 741 68.88 ZP_04572180.1 99.1 978 304 by cAMP protein Fic sp. 4 1 13

Fusobacterium nucleatum subsp. nucleatum

352 1222 1250 88.31 ZP_06871387.1 100.0 3 395 Oxidoreductase ATCC 23726

Conserved Fusobacterium

352 1222 1250 88.31 ZP_04572329.1 100.0 768 466 hypothetical protein sp. 4 1 13

Glycerol-3-phosphate

dehydrogenase Fusobacterium

352 1222 1250 88.31 ZP_05550650.1 100.0 1220 810 (NAD(+)) sp. 3_1_36A2

Uracil-DNA Fusobacterium

367 1482 2576 154.43 ZP_04572735.1 100.0 2 526 glycosylase sp. 4 1 13

Sensory Transduction Fusobacterium

367 1482 2576 154.43 ZP_06750465.1 100.0 1482 667 Protein Kinase sp. 3_1_27 Methylaspartate Fusobacterium

370 708 947 121.38 ZP_06749809.1 99.6 707 6 mutase E subunit sp- 3_1_27

Fusobacterium

ATP synthase F1 beta sp.

390 836 544 57.57 ZP_06748447.1 100.0 1 243 subunit 1_1_41 FAA

Fusobacterium

ATP synthase epsilon nucleatum chain sodium ion subsp. vincentii

390 836 544 57.57 ZP 00144388.1 100.0 256 552 specific ATCC 49256

Dihydrolipoarnide Fusobacterium

395 610 469 68.64 ZP_04572312.1 100.0 610 293 acyltransferase sp. 4_1_13

NAD(FAD)-utilizing Fusobacterium

434 628 548 78.94 ZP_06750801.1 98.6 3 626 dehydrogenase sp- 3_1_27

Fusobacterium

460 590 728 108.91 ZP_05814379.1 100.0 580 2 Membrane protein sp. 3_1_33

RNA polymerase Fusobacterium

480 957 1015 94.61 ZP_04572122.1 98.6 3 434 sigma-54 factor rpoN sp. 4_1_13

Fusobacterium

Bacterial/Archaeal nucleatum Transporter family subsp. vincentii

480 957 1015 94.61 ZP_00144056.1 100.0 842 498 protein ATCC 49256 branched-chain amino

acid transport system II Fusobacterium

499 501 321 55.66 ZP_06750657.1 100.0 3 368 carrier protein sp. 3_1_27

Ribosomal large

subunit pseudouridine Fusobacterium

510 605 636 93.46 ZP_04572207.1 99.0 599 3 synthase B sp- 4_1_13

Fusobacterium nucleatum subsp. vincentii

514 710 267 33.69 ZP_00144400.1 98.0 2 295 Hypothetical protein ATCC 49256

2-nitropropane Fusobacterium

514 710 267 33.69 ZP 045721 12.1 100.0 376 708 dioxygenase sp. 4 1 13

Propionate CoA- Fusobacterium

617 630 9S 14.17 ZP_04574788.1 100.0 2 628 transferase sp. 7_1

WD-repeat family Fusobacterium

658 662 260 34.69 ZP_04572174.1 99.1 7 660 protein sp. 4_1_13

Claims

WHAT IS CLAIMED IS:

1 . A method for treating a human patient for an intestinal cancer, the method comprising:

administering to the patient a medicament comprising an effective amount of an antigenic composition comprising whole killed cells of a microbial pathogen, wherein the microbial pathogen is a Fusobacterium.

2. The method according to Claim 1 , wherein the Fusobacterium microbial pathogen is Fusobacterium nucleatum.

3. The method according to any of the preceding claims, wherein the composition comprises whole killed cells of only a single microbial pathogen, which microbial pathogen is a Fusobacterium.

4. The method according to any of the preceding claims, wherein the intestinal cancer is a cancer of the large intestine.

5. The method according to Claim 5, wherein the cancer of the large intestine is selected from the group consisting of colon cancer, rectal cancer and combinations thereof.

6. The method according to any of the preceding claims, wherein the method comprises administering the medicament in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least two weeks.

7. The method according to Claim 6, wherein the method comprises administering the medicament in a dose so that each dose is effective to cause a localized inflammatory immune response at an administration site.

8. The method according to Claim 6, wherein the method comprises administering the medicament in a manner such that visible localized inflammation at the administration site occurs within 1 to 48 hours.

9. The method according to any of the preceding claims, wherein the method comprises administering the medicament intradermal^ or subcutaneously.

10. The method according to any of the preceding claims, wherein the method comprises orally administering the medicament.

1 1 . The method according to any of the preceding claims, wherein the method further comprises administering to the patient an effective amount of an anti-inflammatory agent.

12. The method according to Claim 1 1 , wherein the anti-inflammatory agent is an NSAID.

13. The method of any of the preceding claims, wherein the method further comprises diagnosing the patient as having suffered from a prior pathogenic exposure to the microbial pathogen.

14. The method of according to any of the preceding claims, wherein the patient is a patient that has been diagnosed as having suffered from a prior pathogenic exposure to the microbial pathogen.

15. A method for selecting an immunogenic composition and treating a human patient, comprising:

diagnosing the patient as having an intestinal cancer;

selecting an antigenic composition comprising whole killed cells of only one microbial pathogen, wherein the microbial pathogen is a Fusobacterium; and administering the antigenic composition to the patient to elicit an immune reaction to treat the intestinal cancer.

16. The method according to Claim 15, wherein the method comprises administering the antigenic composition by subcutaneous injection, intradermal injection or oral administration.

17. The method according to Claim 16, wherein the method comprises administering the antigenic composition by subcutaneous or intradermal injection to produce a localized skin immune response at a site of administration.

18. The method according to Claim 17, wherein the method comprises administering the antigenic composition so that visible localized inflammation at the administration site occurs within 1 to 48 hours.

19. The method according to any of Claims 15 to 18, wherein the method comprises administering the antigenic composition in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least two weeks.