WO2006124630A2 - Compositions and methods for enhancing the efficacy of vaccines - Google Patents

Abstract

This invention concerns compositions for enhancing the efficacy of vaccines such as influenza vaccines. Also included are kits that include these compositions and methods of using the compositions and kits to treat a subject.

Description

COMPOSITIONS AND METHODS FOR ENHANCING THE EFFICACY OF VACCINES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the priority date of U.S. Application Serial No. 60/680,935, which was filed on May 13, 2005. For the purpose of any U.S. application that may issue from the present application, the content of U.S. Application Serial No. 60/680,935 is hereby incorporated by reference in its entirety.

BACKGROUND

Influenza remains a major cause of morbidity and mortality. There are vaccines against the causative viral pathogens, but the people who are at the greatest risk are also those least likely to mount an adequate immune response to the vaccines currently available.

SUMMARY

The present invention is based, in part, on our discovery that the immunomodulatory properties of probiotics can be exploited in the treatment and prevention of infectious diseases. Accordingly, the invention includes compositions that include a probiotic and an immunogen {e.g., an influenza vaccine), kits that include these compositions, and methods of using the compositions and kits to treat a subject (e.g., a human subject). The composition can, for example, elicit an immune response against a virus that causes influenza. For example, the probiotic (e.g., Lactobacillus rhamnosus (LGG)) can serve as an adjuvant for a mucosally administered vaccine (e.g., a live attenuated influenza virus vaccine (LAIV) or an inactivated influenza vaccine (1IV)). The adjuvant can be administered by any route (e.g., orally, mucosally, intramuscularly, subcutaneously, or intraperitoneally). Suitable immunogens will be known to one of ordinary skill in the art, and some of these are specifically mentioned herein (e.g., the A/New Caledonia/20/99 (HlNl) strain that has been included in the influenza vaccine for the last five years). These compositions (e.g., those containing an influenza vaccine and a probiotic) can be used to enhance the efficiency of an influenza vaccine (e.g., when administered to a subject).

Whether or not the efficacy of a vaccine {e.g., an influenza vaccine) has been enhanced can be determined in a number of ways. For example, where the vaccine is an influenza vaccine, efficacy is enhanced when subjects receiving a probiotic have a greater increase in geometric mean nasal anti-influenza hemagglutinin (HA) IgA than subjects receiving a placebo. The IgA can be measured, for example, four weeks after an immunization. The subject may also exhibit a greater increase in the geometric mean serum hemagglutination inhibition (HAI) antibody titer than a subject receiving a placebo. The antibody titer can be assessed, for example, four weeks after IIV immunization. Any cell mediated immune response may be increased as well. To make that determination, one can assess the proliferation of peripheral blood mononuclear cells (PBMCs) and the production of certain cytokines.

Probiotics are living microorganisms (e.g., bacteria or yeast) that exert health benefits beyond inherent nutrition. "Biotherapeutic agent" is an alternative term that is used to describe microorganisms that have antagonistic properties toward pathogenic bacteria. The compositions of the invention include formulations (e.g., pharmaceutically acceptable formulations) of a live Lactobacillus strain such as an L. rhamnosus strain (e.g., Lactobacillus rhamnosus GG (LGG)). The formulations can include not only probiotic microorganisms, but also microorganisms, including any of the Lactobacillus strains, that have been killed (e.g., a "heat killed" Lactobacillus). LGG has proven safe and effective for the treatment of acute diarrhea in children and for the prevention of Clostridium difficile-associdϊQά diarrhea and travelers' diarrhea. The formulations can also include fragments of microorganisms, including any strain of Lactobacillus. For example, the formulations can include viable or killed Lactobacilli that have been disrupted by chemical or mechanical forces and optionally, separated into fractions (i.e., the formulations can include membrane, cell wall, or cytosolic fractions of formerly viable or formerly killed Lactobacilli (e.g., LGG)). It may be preferable to use killed or fractionated bacteria (e.g., heat killed or fractionated LGG) where the target tissue is within a sterile field, such as the lungs, or where there is some other concern about the use of live bacteria. The methods of the invention include delivering formulations, including those describedherein, to a subject identified as a candidate for treatment. The compositions can be formulated for topical administration (e.g., application to the skin or to a mucosal surface of the body) to enhance the efficacy of a vaccine preparation. The compositions can include a probiotic microorganism such as a species of Lactobacillus (e.g., L. rhamnosus (e.g., LGG)). The probiotic microorganism can be a Lactobacillus or a combination of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) species or strains of lactobacilli. For example, the compositions can contain the L. rhamnosus strain LGG deposited in the American Type Culture Collection (ATCC; Manassas, VA) under code ATCC 53103. Alternatively, or in addition, the compositions can contain one or more of the L. rhamnosis strains LC-705, GR-I (ATCC 55826), GR-2 (ATCC 55915), GR-3 (ATCC 55917), GR-4 (ATCC 55916), RC-17 (ATCC 55825), orRC-12 (ATCC 55895), and one or more of the lactobacillus species L. acidophilus, L. casei, L. crispatus, L. bulgaricus, L. fermentum, L.jensenii, L. plantarum, L. reuteri, L. curvatus, L. salivarius, or L. johnsonii. Any of these lactobacilli can be viable or non- viable. For example, they may have been killed by exposure to heat or another damaging stimuli (e.g., radiation or a non- physiological pH). The lactobacilli can also be disrupted (fragmented) by chemical or mechanical forces so that cell fragments are produced, and the fragments can be crudely or finely separated before inclusion in the composition (e.g., the cytosolic and membrane fractions can be separated or more extensive purification can be carried out). Regardless of the precise form used, the lactobacillus-based composition can contain a non-lactobacillus probiotic, such as Bifidobacterium lactis (B. lactis). Other exemplary members of Bifidobacterium that could be used include B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, and B. longum. The lacto-bacillus-based composition can optionally include a prebiotic or synbiotic.

The compositions are preferably physiologically acceptable (e.g., non-toxic and, preferably, associated with minimal undesirable side effects) and, therefore, can also include a physiologically acceptable carrier, buffer (e.g., a citrate buffer, phosphate buffer, acetate buffer, or bicarbonate buffer), excipient, or diluent (many of which are known in the art). More specifically, the carrier, excipient, or diluent may be predominantly oily or predominantly aqueous, and may include petroleum jelly, lanoline, a polyethylene glycol, amino acids, urea, an alcohol, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, glycerol, or a transdermal enhancer. Lactobacillus- containing compositions can also contain adonitol, glycerol or non-fat milk solids (NFMS), which have been shown to stabilize the microorganism during dehydration and rehydration processes (Selmer-Olsen et al, JAppl Microbiol. 87:429-37, 1999). The probiotic may also be included in preexisting vaccine formulations or administered separately. For example, the probiotic may be administered orally in a capsule, pill, tablet, syrup, or suspension.

The probiotic-enhanced vaccine can be administered to any subject (e.g., a human, e.g., a human subject of any age, including children under the age of two and elderly subjects over the age of about 65) who is a candidate for vaccination with vaccines that do not include a probiotic.

The composition {e.g., the probiotic and the influenza vaccine) can be administered at essentially the same time by the same route of administration. Alternatively, the composition {e.g., the probiotic and the influenza vaccine) can be administered at different times and/or by different routes of administration.

Subjects amenable to treatment with the compositions of the present invention may be healthy or apparently healthy except for being colonized by, infected by, or at risk of colonization and infection with a pathogen {e.g., a flu-causing virus). Other subjects amenable to treatment (therapeutic or prophylactic) are those who have a condition (a term we use broadly to refer to a disease, disorder, syndrome, illness, or the like) that impairs the immune system. For example, a subject may have a viral infection {e.g., an infection with a human immunodeficiency virus, a hepatitis virus, or an influenza virus), an autoimmune disease {e.g., diabetes), a respiratory tract infection, heart disease, liver disease {e.g., cirrhosis), kidney disease, or other condition in which the immune system is compromised {e.g., the subject may be elderly or very young (an infant, toddler, or child)). The subject may be critically ill. Other subjects may be receiving an immunosuppressant {e.g., cyclosporine) to, for example, inhibit transplant rejection. Any of these subjects can be identified and treated by administration of an effective amount of a physiologically acceptable composition containing a strain of Lactobacillus {e.g., L. rhamnosus (e.g., LGG)) and an immunogen.

One can treat the subjects by administering or applying the composition to the mucosal surface (e.g., topical administration of a composition (e.g., a probiotic- enhanced vaccine) to the nasal mucosa).

Regardless of the precise formulation, the compositions can be presented in unit dosage form (e.g., in ampoules or in multi-dose containers with, optionally, an added preservative). More specifically, the compositions can be presented in a pack or dispenser that contains one or more (e.g., 2, 4, 6, 8, 10, 12, or 14, or multiples thereof) unit dosage forms containing an active ingredient (at least one species or strain of lactobacillus). The pack can be fashioned, in whole or in part, from metal (e.g., aluminum foil) and/or plastic, such as a blister pack. The pack or dispenser can be packaged and accompanied by instructions for administration or otherwise labeled, as described herein, hi the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

The compositions can also contain one or more of: a preservative, stabilizer, colorant, and/or flavoring. Alternatively, or in addition, the compositions can contain an additional therapeutic agent such as an analgesic, antihistamine, an agent that modifies the consistency of the blood (for subjects with a vascular or clotting disorder), an antibiotic (e.g., penicillin, erythromycin, amoxicillan or clindamycin), or the like. A "first" strain of lactobacillus can be formulated together with at least one other therapeutic agent (e.g., a "second" strain of lactobacillus or a non-lactobacillus probiotic (e.g., Bifidobacterium lactis) or an agent such as an analgesic, antihistamine, clotting or blood-thinning agent, or antibiotic), in which case one could administer the two (or more) agents coincidentally. Some formulations can be in the form of a food product, such as in a milk product (e.g., a powdered or liquid milk), yogurt, or other edible formulation (e.g., a flavored chewy formulation).

The amount of the probiotic (e.g., LGG) that will be effective in treating, inhibiting, or preventing colonization or in treating, inhibiting, or preventing a bacterial infection (or a disease or disorder associated therewith) can be determined by standard clinical techniques. One of ordinary skill in the art can use, for example, in vitro assays to help identify optimal dosage ranges. Animal test systems are similarly useful, as effective doses can also be extrapolated from dose-response curves derived from these systems. As one of ordinary skill in the art would appreciate, the precise dose to be employed may vary depending on the type of infection, the seriousness of any accompanying disease or disorder, the subject's age and general health, and the dosage form and route of administration utilized. If necessary, the frequency and duration of administration, as well as the dosage, can be adjusted according to the judgment of the practitioner and each subject's (e.g. patient's) circumstances.

While the invention is not so limited, exemplary dosage amounts of a probiotic such as LGG in humans are less than about 10 x 10¹⁰ organisms per day (e.g., about 8 x 10¹⁰ organisms per day, about 6 x 10¹⁰ organisms per day, about 4 x 10¹⁰ organisms per day, about 2 x 10 organisms per day, about 1 x 10¹⁰ organisms per day, or about 0.5 x 10¹⁰ organisms per day, delivered in a single bolus, in divided doses, or by infusion lasting from minutes to hours).

The compositions of the invention may be included in kits, which are also within the scope of the present invention. The kits can include the probiotic (e.g., a strain or a combination of strains), of lactobacilli such as Lactobacillus rhamnosus (e.g., LGG), a killed Lactobacillus, or fraction (e.g., a membrane fraction of a formerly viable or non- viable Lactobacillus) formulated for topical use (e.g., the compositions can be formulated as an ointment, cream, lotion, paste, gel, salve, oil, spray (e.g., an aerosol spray)) suspension, partial liquid, nebulae, or mist. The kits can also contain informational material, such as instructions for use (e.g., instructions for administering the composition to a subject). Optionally, the kits can include an applicator device, such as a cotton swab, spray pump, or nebulizer. For example, a kit can include a first container that includes a probiotic and excluding an influenza vaccine; a second container including the influenza vaccine and excluding a probiotic; and instructions for use. DESCRIPTION OF DRAWINGS

Figure 1 is a bar graph showing serum antibody responses to H. influenzae type b conjugate vaccine in infants who received a prebiotic (oligofructose) and in a control group of infants. The antibody response was measured as levels of antibody. Pre: pre- immunization titers of antibody; Post: post-immunization levels of antibody.

Figure 2 is a panel of four line graphs showing serum IgA response over time to C. parvum diarrhea. Antibody levels were measured by enzyme immunosorbent assays (ELISA).

Figure 3 is a bar graph showing the proliferation of human PBMCs in response to C. parvum (Cp) antigens.

Figure 4 is a schematic diagram of a research study designed to assess the immunogenicity of either LAIV or ITV in healthy volunteers.

Figure 5 is a line graph showing a power analysis performed to determine the power to detect a large effect size with a sample size of 26 subjects pretreatment group.

DETAILED DESCRIPTION AND EXAMPLES

Further details regarding compositions within the scope of the present invention are described below. The methods described below are to be considered prophetic examples and are also within the scope of the invention.

The remainder of this page is left blank intentionally.

A. SPECIFIC AIMS ⁸

The overarching goal of this research submitted in response to PAR-04-087 (General Clinical Research Center (GCRC) Complementary and Alternative Medicine Research Program) is to determine whether immunomodulatory properties of probiotics enhance the efficacy of influenza vaccines. Please note that since this application is submitted by the Program Director of the Tufts-New England Medical Center GCRC (Dr Hibberd), the required letters of support are from GCRC Principal Investigator (Dr Selker) and Associate Program Director (Dr Greenblatt).

Influenza remains a major cause of morbidity and mortality in the United States, responsible for almost 40,000 deaths annually during the 1990s (1). Although rates of influenza infection are highest in children, the majority of the mortality occurs in older adults (particularly those age 65 and over) and those with a wide range of chronic medical conditions including immunocompromised states (2-6). Influenza vaccination is the primary way of preventing both illness due to influenza and its complications; However, those at greatest risk of influenza associated complications are also those least likely to mount an adequate antibody titer to the vaccine strains (7-10). In addition, should a pandemic of influenza occur, as a result of bioterrorism or natural emergence of a new influenza A virus, there will be an emergent need for maximally effective mass immunization.

Prompted by 20 years of basic and clinical research by Drs. Gorbach and Goldin (our colleagues at Tufts) on the use of probiotics, particularly Lactobacillus rhamnosus (LGG), our group has turned its attention to developing ways in which the immunomodulatory properties of probiotics can be exploited to prevent infectious diseases. Our focus is on the potential of probiotics as adjuvants to mucosally administered vaccines, particularly the recently approved live attenuated intranasal influenza virus vaccine (LAIV). There are three important reasons why it is plausible and timely to evaluate whether probiotics, such.as LGG,_are effective in enhancing the immune response to the influenza vaccine, particularly LAIV. First, human data support the hypothesis that co-administration with probiotics may enhance the immune response to other mucosally administered vaccines. Vaccine specific serum and mucosal IgA are enhanced by co-administration of probiotics along with oral S typhi, polio and rotavirus vaccines (11-14))'. Second, animal data support that pre-administration of probiotics induces immunity to the influenza virus. Specifically, in the mouse model, administration of probiotics, including LGG, prior to challenge with influenza virus resulted in higher levels of influenza specific IgG and protection against infection after influenza virus challenge (15-18). Third, even though LAIV induces significantly higher levels of vaccine strain specific (mucosal) IgA in nasal wash specimens (19; 20) than the intramuscularly administered inactivated influenza virus vaccine (irV), (IIV elicits better serum antibody responses) immunogenicity to both vaccines wanes with age (21). There is an important need for vaccine adjuvants, such, as probiotics, to boost the immune response to either type of influenza vaccine, although based on previous similar work by our group with prebiotics and parenterally administered vaccines (22), we anticipate a greater effect after administration of LAIV than HV.

Based on these considerations, we propose to conduct two parallel Phase I/II double-blind randomized placebo controlled clinical trials to assess the safety and immunogenicity to either live attenuated or inactivated influenza vaccine receiving oral LGG vs. placebo. We will also obtain preliminary data on the time it takes to achieve a protective immune response, durability of response and occurrence of influenza in the following influenza season. Although in future studies we will focus on the ability of probiotics to boost the immunogenicity of the influenza vaccine in the elderly, LAIV is not currently approved for use in adults aged > 50 (23). Thus for this proposed study, we will study healthy adults aged 18-49, in whom there is still room for improvement in the effectiveness of the influenza vaccine (24). This younger age group has the advantages of including immunologically naive subjects as well as those who have previously been exposed to at least one of current vaccine strains (e.g. A/New Caledonia/20/99 (HlNl) that has been in the influenza vaccine for the last 5 years). Promising results in this age group will provide preliminary data for future studies in the healthy elderly. These studies will be conducted in the Tufts-NEMC GCRC.

Primary Objectives: To measure anti-influenza systemic and mucosal antibody responses 4 weeks after administration of influenza vaccine to healthy subjects aged 18-49 and to compare responses in subjects receiving Lactobacillus GG (LGG) vs. placebo. Subjects receiving LGG capsules will have a greater increase in geometric mean nasal anti-influenza hemagglutinin (HA) IgA four weeks after LAIV immunization than subjects receiving placebo capsules, ii. Subjects receiving LGG capsules will have a greater increase in the geometric mean serum hemagglutination inhibition (HAI) antibody titer four weeks after IIV immunization than subjects receiving placebo capsules.

Secondary Objectives: To assess time course of antibody and cell, mediated immune response (rapidity and durability) after administration of influenza vaccine and to compare responses in subjects receiving LGG vs. placebo.

Hypotheses: i. Subjects receiving LGG capsules and LAIV will have a more rapid response (achieve protective serum HAI and nasal ariti-influenza HA IgA titers by week 2) than subjects receiving placebo capsules and LAIV. ii. Subjects receiving LGG capsules after either LAIV or IIV will have higher geometric mean serum HAI antibody titers and nasal anti-influenza HA IgA at the end of the influenza season (6-9 months after immunization) than those receiving placebo capsules and either vaccine, iii. Subjects receiving LGG capsules after either LAIV or IIV will have a greater cell mediated immune response to the influenza vaccine as measured by greater proliferation of peripheral blood mononuclear cells (PBMC) and cytokine production in response to vaccine-specific influenza antigens than those, receiving placebo capsules and either vaccine. . •

Exploratory Objectives: To report the occurrence of laboratory documented influenza illness and adverse effects after administration of two types of influenza vaccine (LAIV and IIV) in the LGG and placebo capsule groups, and to obtain preliminary information on efficacy in preparation for future studies.

To our knowledge, this is the first study to use the probiotic LGG to enhance the immune response to the influenza vaccine. The goal of this study is to assess the immune response in healthy volunteers, as a prelude to future investigations to enhance immunity in patients with suboptimal responses to currently available vaccines (elderly, immunocompromised and immunosuppressed patient populations). Because of the importance of influenza as a biodefense category C priority pathogen, our study also explores immunogenicity and potential use in emergency situations (time to developing a protective antibody response). This study is innovative because it evaluates a novel approach - exploiting immunomodulatory properties of probiotics - to enhance the immune response of the newly available mucosally administered LAIV and previously available IIV.

Since our study subjects are healthy volunteers, access to the resources of the Tufts-NEMC GCRC is critical to conduct of this study. The study subjects will be evaluated and studied in GCRC outpatient space; the GCRC research nurses will administer the influenza vaccines, monitor the subjects and obtain blood and nasal specimens; the GCRC nutritionist will advise on avoiding foods that may contain probiotics; the GCRC social worker will assist with recruitment of women and under-represented minorities; the GCRC informatics and statistical staff will provide oversight of the data management and statistical approaches; and the GCRC core laboratory staff will process, store and conduct the serum and nasal wash antibody assays and cytokine assays from the PBMC supernatants.

B. BACKGROUND AND SIGNIFICANCE

B.I IMPORTANCE AND BURDEN OF INFLUENZA

Influenza remains a major cause of morbidity and mortality in the United States. Illness occurs in 10-20% of the population each year (25). Young children, people with chronic medical conditions, and persons aged greater than 65 continue to bear the brunt of the disease because they are at higher risk for complications, hospitalizations, and death from influenza (6). The majority of influenza related deaths Q>90%) occur among the elderly (1; 6) and influenza associated mortality appears to be increasing (estimated 19,000 influenza-associated deaths per influenza season from 1976-1990 and 36,000 deaths per season from 1990-1999 (6; 26)). This increase may be due to an increase in the elderly population or the number of influenza seasons in which influenza A (H3N2) predominates (1 ;

Influenza associated hospitalizations tend to concentrate in the young and old. From 1970-1995, of the estimated 3 million excess hospitalizations associated with influenza, rates were highest in the elderly (174/100,000) vs. 49/100,000 in the rest of the population) (27). At the other end of the age spectrum, children < 5.years have rates of influenza associated hospitalization similar to adults aged 50-64 (6), with the highest rates in children <12 months old and those with high risk medical conditions (1; 28-30). Children have the greatest attack rate of influenza infection, but the predominant burden is outpatient visits, and increased antibiotic use (29; 30). Of note, during the 2003-2004 influenza season there was rise in the number of influenza related deaths reported in children Approximately 40% of these cases occurred in children younger than.2 years of age, highlighting the importance of disease prevention in this age group (23).

More recently, influenza has been recognized as a common cause of illness, absenteeism and increased health care use in employed populations (31). Bridges et al reported that Influenza vaccination of healthy working adults can reduce the rates of influenza-like illness, lost work-days and physician visits if the strains in the vaccine are similar to the strains that circulate in the population (32). The authors caution that vaccination of healthy adults may

_.not provide economic benefits every year. Improvements to the influenza vaccine that result in greater efficacy and duration of protection are likely to render influenza immunization, both cost-effective and effective in preventing

- clinical illness,..evenjn healthy, adults (24). ^'

B.2 CURRENT STRATEGIES TO PREVENT INFLUENZA

Influenza vaccination is the primary means of preventing influenza infection. The Advisory Committee on Immunization Practices currently recommends vaccination in persons aged >50 years, children aged 6-23 months, pregnant women, and persons of any age with chronic medical, conditions (23). In addition, the CDC also recommends that health-care workers and household contacts who have frequent contact with persons at high risk should also be vaccinated. Although young children and the elderly suffer the greatest morbidity and mortality, exposure of their caregivers results in a high burden of health care expenditures. Influenza vaccination reduces both direct medical costs, such as physician visits and antibiotics uses, and indirect costs such as work absenteeism (31; 32). Vaccination in the elderly results in reductions in hospitalization, morbidity, and mortality (10). Although vaccination coverage has improved in the elderly, other at risk populations such as young children, health care workers, and adults aged 50-64 are populations which have had sub-optimal rates of use of the influenza vaccine (23; 33; 34).

As of 2003, two types of influenza vaccines are available for use in the United States. Inactivated influenza vaccine (HV) has long been the primary means of influenza prevention. However, the efficacy and immunogencity of the vaccine is sub-optimal in the very young (<6 months), the chronically ill, and adults greater than age 65 (8-10; 35). In addition, since IIV is administered intramuscularly, vaccine coverage levels are at best about 66% in adults >65 years and only 34% in the target population between ages 50-64 (23). More recently, live attenuated influenza vaccine (LAIV) was approved for use in healthy individuals ages 5-49. One of the major advantages of the LAIV is ease of administration to the nares (avoiding the logistics of parenteral administration) as well as stimulation of the local mucosal immune response. The efficacy of LATV has been well documented in children and adults (31; 36; 37). Nichols et al studied 4,561 healthy adults in a placebo controlled trial to evaluate'the effectiveness of LATV (31). LAlV was associated with reduction in febrile illness and upper respiratory tract infections, lower absenteeism, decreased work of health care resources and decreased use of antibiotics. A randomized, double-blind, placebo- controlled trial among 1,602 healthy children demonstrated efficacy of LAIV including a 92% decrease in culture confirmed influenza, as well as significant reductions in otitis media (36; 38). Many studies have shown that LAIV and IIV are generally equivalent in preventing influenza infection. IIV has been shown to produce higher levels of serum anti-HA IgG and IgA antibodies, while LAIV induces higher levels of nasal wash IgA (19; 20; 37). Both IIV

resulted in lower levels of serum HAI antibody responses but higher levels of local IgA antibodies

B.2.a INFLUENZA VACCINE AND IMMUNITY

Immunity to influenza infection is induced by antibody responses to viral surface antigens hemagglutinin (HA) and neuraminidase (NA). The influenza virus is constantly undergoing antigenic drift in these two proteins. The efficacy of influenza vaccination depends on the degree of concordance between the virus strains in the vaccine and those being spread in the population at large. Antibody induce immunity by vaccination in one season is unlikely to be of benefit in the following season.

Both systemic and' local immune responses protect against infection with the- influenza virus. Since the main portal-of-entry for the influenza virus is mucosal tissue, the mucosal immune system is the key first line of defense against infection. The mucosal immune response is primarily reflected by the local production of secretory IgA that can be detected in nasal washings. Levels of IgM and IgG can also be detected in nasal secretions during primary infection. In subjects previously exposed to influenza, the local and systemic IgA response predominates. Brokstad et al found that even in the absence of influenza exposure, much higher levels of influenza specific antibody ^' secreting cells are found in nasal mucosa than in blood (39). One of -the-advantages-of^he -LAIV-is that the vaccine strains replicate in the respiratory epithelium of the nasal mucosa, stimulating nasal IgA and inducing the local humoral immune response. LAIV induces peak of serum IgA and IgM two weeks after immunization and a peak IgG response 4-12 weeks after immunization (40). Following LAIV administration, mucosal antibodies detected in nasal wash specimens appear to have a long half-life and in previously immunized children may persist up to a year (38). Nasal wash IgA levels were a stronger predictor of protection against influenza than serum HAI antibody titers.

Serum production of anti-influenza IgG reflects the systemic humoral response to either LATV or ITV. This is most commonly measured by the serum hemagglutination inhibition (HAI) test, which measures the ability of serum antibodies to inhibit influenza hemagglutinin (HA)-induced agglutination of avian (chicken or turkey) red blood cells (41). The systemic immune response to TJV can be detected within 7 days and most commonly peaks at 10-14 days (42). The systemic acute immune response is characterized by a rise in serum IgA and IgM levels within the first two weeks, followed by IgG levels that persist for up to 6 weeks (42-44). In those previously exposed to influenza vaccine, serum IgG and IgA are the main indicators of immune response .(44).

While humoral immune responses to influenza virus are responsible for the resistance to infection, cell mediated immunity is important for the clearance of the virus, reduction of the severity of the disease, and recovery from infection (45; 46). In in-vitro studies, the cytotoxic T lymphocyte (CTL) response to influenza correlates with decreased viral shedding (47). The CTL response is at least partially dependent on CD8+ T cells that are specific for HA as well as the internal proteins M, NP or B2 (48). ThI cytokines are also important for the cell mediated immune response to influenza. In particular, the cytokine interferon gamma appears to be important for memory T cell responses to influenza in mice and humans (49; 50). The measurement of the CTL response to influenza has been used to study immune responses in different human populations and different vaccine preparations (46; 51 ; 52). Another recently used approach to measure cell mediated immune response to influenza is to measure the proliferation of peripheral blood monocytes and cytokine production in response to influenza antigens (9; 53).

B.2.b INFLUENZA VACCINE COMBINATIONS AND ADJUVANTS

Despite the promise of enhanced mucosal immunity to influenza following LAIV, concerns about vaccine safety and sub-optimal efficacy in the elderly, people with co-morbid conditions and the immunosuppressed have resulted in a timely search for ways to boost the immune response in recipients of both the LAIV and IIV. One promising strategy is administration of both LAIV and IIV - preliminary data suggest that the combination may be safe and

An alternative approach is to boost the immune response to influenza vaccination by administering an adjuvant at the time of vaccination. Cooper et al. used oligodeoxynucleotides containing immuno-stimulatoτy motifs as a vaccine adjuvant to ETV (53). In a placebo, controlled trial, the adjuvant was safe and well tolerated. The adjuvant- vaccine combination did not increase HAI or ELISA titers in this small study, but there was a trend to increased titers in those with pre-existing immunity to one influenza strain. The greatest effect of this vaccine adjuvant was observed in the group receiving a low dose of IIV, raising the possibility that an immune boosting response may allow for a reduction in vaccine dose. MF59 is another vaccine adjuvant (submicron oil in water emulsion of 5% sqiialene, 0.5% Tween 80 and 5% Span 85) that has been safely used in Human Immunodeficiency Virus and Herpes simplex sub-unit vaccine trials. In a recent meta-analysis, MF59 adjuvanted influenza vaccine resulted in greater . immunogenicity than non adjuvanted vaccine especially in those who had not been previously immunized (60). Studies- of MF^P-adjuvanted-influenza-vac-cine-in-health-y- adults-also-resulted in higher KAI -titers -(61).- Boyce et al recently evaluated -MF59 adjuvanted intranasal vaccine in an open label safety study finding similar mucosal IgA responses after administration of both adjuvanted and non-adjuvanted intranasal vaccine (62). Thus, while vaccine adjuvants remain an attractive solution to solving the problem of sub-optimal influenza vaccine efficacy, there is much room for improvement.

B.3 CLINICAL USES AND SAFETY OF PROBIOTICS

Probiotics are living microorganisms that exert health benefits beyond inherent nutrition (63). Biotherapeutic agent is an alternative term that is used to describe microorganisms that have antagonistic properties toward pathogenic bacteria (64). Several probiotics and biotherapeutic agents have been studied for the treatment of antibiotic associated diarrhea, infantile diarrhea, traveler's diarrhea, urinary tract infections, and vaginal infections (63; 65-67). There are numerous commercially available probiotics, both in lyophilized form or fermented food products. These include lactic acid bacteria, predominantly Lactobacillus spp., which have the desired properties of beneficial clinical effects, minimal side effects and are the most studied probiotics.

B.3.a Lactobacillus rhamnosus GG (LGG)

Lactobacillus rhamnosus GG (LGG) was isolated by Drs. Gorbach and Goldin in 1985 from a healthy human (68). LGG has been extensively studied in humans and has been shown to be safe and non-pathogenic. The safety of LGG has been verified in cell culture systems, animal studies and in a number of human studies (69). With studies of invasiveness in animals and in tissue culture, lies evidence of cellular adhesion but not invasion (70). In animal studies of oral toxicity, no treatment-related deaths or toxicities were observed after exposure to high colony counts of the bacteria (71). Salminen et al. recently summarized the safety of LGG after more than 10 years of widespread use in Finland, concluding that use of LGG was not associated with an increase in Lactobacillus bacteremia (72). Similarly, since lactobacilli are normally present in the vagina, there have been no reported adverse events after use of LGG vaginal suppositories (65). In five studies of more than 400 children, LGG was both safe and effective in decreasing the duration and frequency of diarrhea (73), (74). Our group recently demonstrated the safety of 2 years of administration of LGG in immunosuppressed children with Crohn's disease (75).

B.4 EFFECT OF PROBIOTICS ON THE IMMUNE SYSTEM AND BASIS FOR EVALUATING LGG AS AN IMMUNE ADJUVANT TO THE INFLUENZA VACCINE F^^i^^P'-^O^ttfe^r^^^^^^-^V^f^31*"¹'¹¹*¹¹^^{1 : Hibberd, Patricia L}

B.4.a Effects of Probiotics on Systemic and Mucosal Humoral Immune System

Probiotics are widely available in Europe and the United States and although they are advertised as promoting immunity or boosting the immune system, few human studies have evaluated their immunomodulatory properties. In vitro and animal data suggest that probiotics increase levels of intestinal IgA and upregulate cytokine production.

Human studies provide a strong rationale for the study of probiotics as a vaccine immune adjuvant. In a study of healthy Japanese infants, administration of bifidobacteria containing formula resulted in an elevation of total IgA and anti-poliovirus IgA levels in the feces (12). Similarly, 30 healthy volunteers were randomized to LGG, Lactococcus lactis or placebo for 7 days, prior to receiving oral Salmonella typhi vaccine (10 per group). Although there was insufficient power to detect differences between groups, there was a trend towards higher specific IgA anti- Salmonella antibodies in the LGG treated group (11). Other investigators randomized healthy volunteers to receive either fermented milk with L acidophilus and bifidobacteria vs. control for 3 weeks during which oral Salmonella

was admϊnistef^~ed.The experiirrental-group had significantly higher total- serum IgA and specific serum IgA to S typhi (13). Finally, responses to administration of DxRRV rhesus-human reassortant live oral rotavirus vaccine were compared in infants randomized to LGG vs placebo. Infants receiving LGG had higher rates of IgA seroconversion and rises in rotavirus specific IgM secreting cells vs! infants receiving placebo (14).

Further support for the use of probiotics in combination with mucosaily administered vaccine comes from ^"natural studies of rotavirus infection. Several studies have-evaluated the effect of LGG on rotavirus infection. In one - study, 44.previously-healthy children with . acute rotavirus infection were randomized to receive either LGG or pasteurized yogurt without LGG (76). The LGG treated group had^"a decrease m^" duration of symptoms and an increased nonspecific response in IgG, IgM, and IgA compared with placebo treated children. In the convalescent phase, there was a significant increase in the IgA antigen specific antibody secreting cells to rotavirus in the LGG group compared to the placebo treated group. In a s^'eparate study, LGG vs. Lactobacillus, casei subsp. rhamnosus (Lactophilus) or a combination of Streptococcus ihermophilus and Lactobacillus delbruckii subsp. bulgaricus (Yalacta) was administered twice daily for 5 days to children with rotavirus infection. Those receiving LGG had higher serum IgA and specific antibody secreting cells to rotavirus during the convalescent phase of rotavirus infection (77). Kaila et al had a similar result when children with acute rotavirus infection were treated with viable versus inactivated LGG - those who received viable LGG had a significant increase in serum rotavirus specific IgA antibodies and rotavirus specific IgA secreting cells (78).

The majority of research in probiotics has previously focused on pathogens that enter the human host via the enteral route. However, the effects of secretory IgA are not limited to the gastrointestinal tract. Secretory IgA antibodies may be found throughout the mucosal immune system, including the respiratory tract, salivary glands, and lacrimal glands. In addition, the antigen presenting cells of the Peyers patches and the local gut immune system may interact with lymphocytes that induce both an innate and humoral immune responses at distal sites. Animal studies have started to focus on the effect of probiotics on distal mucosal sites. In a mouse model looking at the affects of Lactobacillus on infection with Streptococcal pneumonia, mice were challenged with Lactobacillus fermentum, S pneumoniae, or L fermentum and then S pneumoniae (experimental group) (79). Anti S pneumoniae antibodies were increased in the Lactobacillus treated mice compared with controls, and Lactobacillus treated mice had an increased number of macrophages in the lung and lymphocytes in the trachea. Another study by Alvarez et al. looked at mice who were challenged with aerosolized Pseudomonas aeruginosa (80). Mice fed a diet of lactobacilli had a significant increase in IgA and IgM levels in bronchoalveolar lavage samples after infection with P aeruginosa.

While these animal studies show the potential for immune modulation in the respiratory tract after administration of lactobacilli during bacterial infections, Hori et al. investigated the effect of oral Lactobacillus casei stain Shirota on influenza infection of the upper respiratory tract in mice (16; 17). Those on an L casei diet had a lower titer of influenza in nasal washings and an increase in NK cell function, interferon gamma and TNF-alpha. In a similar study using mouse model of influenza infection of the upper respiratory tract, the titer of virus, in the nasal washings of infant mice receiving L. casei Shirota (L. casei Shirota group) was significantly lower than that in infant mice Jrinpipaj Jjiv_?stigεjta_r/Bropm pirectar Rf$Ml. rniddle):_ JUlDDerα, ratπna JL.

Sole, there is promising indirect evidence in support of the potential to boost immune response to at least LAIV based on administration of other oral live vaccines in humans as well as direct evidence in support of improved immune response to an influenza virus challenge in mice treated with LGG. ^•

B.4.b Effects of Probiotics on the Innate Immune System

Both the innate and adaptive (humoral) immune systems are modulated by probiotics. The initial interaction of orally delivered probiotics with the mucosal immune system occurs by interaction with epithelial cells in the lining of the gastrointestinal tract. These epithelial cells function as immunoregulatory cells. In vitro studies have shown that lactobacillus increases transepithelial resistance and may prevent decreased transepithelial resistance when administered with other pathogenic bacteria (81; 82). The next step is recognition of lactobacillus molecular patterns by antigen presenting cells (macrophages and dendritic cells). Lactobacilli have been shown to upregulate cytokine expression in murine dendritic cells and human peripheral blood mononuclear cells in in vitro studies (83-87). -L-aetobaeilLus-has-been shown in vitro to induce production and expression of Th-I type cytokines TNFα, IL-2, IL- lβ, 11-6 and 11-18 in ^"periprTerarblbod mononuclear cells (83; ^"85; 8^'6; 88). Lactobacillus has also been found to activate transcription factor NF-κB and Toll-like receptors (TLR 2 and 9)(84; 89; 90). The upregulation of Th-I type cytokines by Lactobacillus may play a role in the CTL response to influenza.

B.5 SIGNIFICANCE OF THE PROPOSED PROJECT

• Influenza infections remain a major public health threat every year arid when ^"pandemics occur, the impact is .Teven mόre^~suhstantϊaL^"I7^" . .. _^"Z MIH~Z..IZ^~.I1^" "_.._...

• There is room for improvement in the immunogencity and efficacy of current influenza vaccines across the age spectrum.

• .Concomitant administration of LGG with oral live vaccines (polio, rotavirus and S typhi) in humans -has resulted in enhanced humoral immunity against vaccine strains.

• Administration of LGG prior to influenza virus challenge results in improved immune response and clearing of the infection in the mouse model.

This study will investigate whether LGG is an effective immune adjuvant to the influenza vaccine in healthy volunteers as a first step towards the goal of boosting the immune response in the elderly and other patient populations. LGG has the potential to be an easily accessible, cost effective influenza vaccine adjuvant that can safely be used throughout the age spectrum.

C. PRELIMINARY STUDIES/PROGRESS REPORT Figure 1: Serum Antibody Responses to H influenzae Cl Prebiotics as an Immune Adjuvant type b conjugate vaccine in Prebiotic vs. Control Groups

Dr. Hibberd and her colleagues at Children's Hospital,. Boston (Dr. Duggan) and Massachusetts General Hospital, Boston (Dr. Kleinman) recently completed two large randomized trials of prebiotics at the Instituto de Investigacion Nutricional in Lima, Peru (22). These two trials enrolled 282 and 349 subjects respectively to determine whether prebiotic supplemented formula (oligofructose), with and without zinc fortification reduced the incidence of diarrheal disease. In the first trial, mean (±SD) days of diarrhea were 10.3 ± 9.6 in the nonsupplemented cereal group and 9.8 ± 11.0 in the prebiotic-supplemeπted cereal group (P = 0.66). In the second trial, mean days of diarrhea were 10.3 ±

8.9 in the group consuming cereal fortified only with zinc and 9.5 ± 8.9 in the «• <"°^{« p}" ^p°^sl group consuming cereal containing both zinc and prebiotics (P = 0.35). Figure 1 shows pre and postimmunization titers of antibody to the administered Haemophilus influenzae type B conjugate vaccine in the nόn-zinc fortified study, comparing infants receiving prebiotics vs control. In this studies cereal supplemented with prebiotics was not aggnriatpH wittt anv TPSΠOTIRR tn H mflup-nvπp type B l^'lnmimivatirm ύVTt nst ικ>« #rø ^middle):- Hibberd. Patricia L

Relevance: Although these studies do not support an immune adjuvant effect of oligofructose on systemic immunity to parenterally administered vaccines and toxoids in children aged 6-12 months, the literature supports the probiotic LGG as a potential immune adjuvant for mucosally administered oral vaccines, and its effect on not parenterally administered vaccines has not been studied to the best of our knowledge. These studies are relevant to this proposal as they demonstrate our experience and track record in conducting immune adjuvant studies involving vaccines with prebiotics. We include the published paper in the Appendix (APPENDIX A, Reprint #1).

C.2 Assays to Measure the Immune Response Figure 2: Serum IgA Responses over time to C. parvum Diarrhea

Dr. Ward has extensive experience in the development of enzyme linked immunosorbent assays (ELISA) to measure antibody responses and in collaboration with Dr. Hibberd recently applied these techniques to measure serum IgG, IgM and IgA to Cryptosporidium parvum antigens in children with diarrhea (see Figure 2). We include the published paper in the Appendix (APPENDIX A, Preprint #2) (91).

¹ Dr. Leav has extensive experience in using the cellular proliferation assay in his laboratory to study the human cellular immune response to the

. protozoan parasite Cryptosporidium parvum. In this representative experiment we obtained blood from a patient with serologic evidence of

past Cryptosporidium infection. 5x10⁶ PBMC were cultured in triplicate in Figure 3: Proliferation of Humai the presence of Cryptosporidium antigens as well as the mitogens, PBMC to C. parvum (Cp) antigens lipopolysaccharide (LPS) and phytohemagglutinin (PHA). The samples were pulsed ^"with ³H after 24 hours and analyzed after a total of 48 hours of culture. The mean counts per minute for each sample are shown here (see below). While there was an obvious response to PHA, there was also an increase in proliferation to both f the lectin (HPA)-affinity column-purified and the crude preparations of Cryptosporidium antigens.

Relevance: These studies and data demonstrate that we have the technical expertise necessary to perform the antibody and cellular proliferation assays described in this proposal.

C.3 Randomized Trials with LGG

The LGG Multi Center Study Group (Drs. Bourvaros, Hibberd, Goldin et al) randomized 73 children with small and large bowel Crohn's disease in remission (Pediatric Crohn's Disease Activity Index less than 10 for 2 months) to either LGG (capsules containing 10¹⁰ LGG) or matching placebo capsules, administered twice a day for 2 years, in addition to routine therapy (aminosalicylates, 6-mercaptopurine, azathioprine and/or prednisone) (75). Time to relapse (defined as exacerbation of or surgery for Crohn's disease or increased immunosuppression) was compared using the log rank test. Data analysis is on going, but there were no episodes of bacteremia or adverse infectious complications associated with use of LGG. In addition, our group has been awarded grants for conducting two randomized trials of LGG to evaluate whether LGG is effective in eradicating nasal carriage of S aureus in adults on hemodialysis (1R21AT002133-01, PI - P Hibberd, Project Period 6/04-5/06) and in eradicating oropharyngeal carriage of S aureus in children with cystic fibrosis (1R21AT002388-01, PI - P Hibberd, Project Period 9/04-8/07). Both of these projects also explore the immunomodulatory properties of LGG and its potential to eradicate S aureus and as of 10/04 are in the ramp-up phase of the research. Both funded projects are also being conducted in the Tufts- New England Medical Center GCRC (SPIDs # 0892 and 0925 respectively).

LGG in clinically important settings.

CA Assessment of Respiratory Illness during Influenza Season

Dr. HibbeTd, in collaboration with her colleagues at Children's Hospital Boston has experience in collecting and tracking respiratory and gastrointestinal illness occurrence and transmission in the home using biweekly telephone calls. We tracked 1,545 respiratory and 360 gastrointestinal illnesses in 208 families from November 2000-May 2001 and evaluated the use of alcohol-based hand gels in reducing secondary illness transmission (92; 93).

Relevance: This study is relevant to our proposal as it demonstrates experience and expertise in tracking respiratory jllness by telephone and_retention of subjects throughout an influenza season. We include one published paper from this study (APPENDIX A₅ Reprint #3) and one accepted manuscript (APPENDIX A, Accepted Manuscript, #4).

D. RESEARCH DESIGN AND METHODS D.I STUDYDESIGN

The goal of this study is to assess the immunogenicity to either LAIV or UV in healthy volunteers aged 18-49 receiving oral LGG vs. placebo. We will also obtain preliminary data on time to achieving a protective immune response, durability of response and occurrence of influenza in the following influenza season. These latter data will provide critical preliminary data for future studies to evaluate the effectiveness and safety of co-administration of LGG with either LAIV or EV to prevent laboratory documented influenza illness.

OUTCOMES

- Serum HAI titers at 1, 2, 3, 4 ^■ weeks, 9 months

- Nasal wash anti HA IgA, titers at 1, 2, 3, 4 weeks, 9 months

- Influenza HA antigen-specific lyrnphoproliferative assays at 2 and 4 weeks

- Laboratory documented influenza illness through 9 months

- Adverse events - any and possibly or probably related to study protocol

Legend * LAIV - Live Attenuated Influenza Vaccine; HV — Inactivated Influenza Vaccine; IM - intramuscular

We will recruit 104 healthy adults at the Tufts-NEMC GCRC for the study. All study activities will be designed to coincide with the usual time line for routine administration of influenza vaccine prior to the influenza season. pft©l1ffiv^tiyi(J|^^>βjgjn₍,,Efir^cβ(Sφg-|gj|τiicldie): Hibberd. Patricia L

Based on prior experience, we anticipate that we will need to screen an estimated 150 subjects to achieve a final sample size of 104. After a screening evaluation, those who meet eligibility criteria (i.e. are healthy and have no contra-indication to receiving either vaccine) will first be randomized to receive either LAIV with IM placebo or IIV with intranasal placebo. Patients will then be randomized for a second time to receive either LGG or placebo. Subjects will receive oral LGG or oral placebo twice daily for 4 weeks and will have blood and nasal specimens obtained weekly for 4 weeks and then at 9 months. Subjects will be contacted by telephone every 2 weeks during the influenza season (November through April) immediately following immunization. Those who develop influenza like illness (described in Section D.4.e below), a reported oral temperature of 100° F with at least 2 symptoms of acute respiratory illness will be evaluated in isolation rooms in the GCRC (see Section D.4.e below for procedures for safe evaluation of patients with possible influenza).

D.2 SUBJECT RECRUITMENT AND ELIGIBILITY

^" The GCRC ResearchrSocial- Worker will assist with recruiting both males and females from diverse racial and ethnic backgrounds, using IRB approved procedures. This assistance includes outreach programs to local community groups informing them about research directions and opportunities. As in our previous studies, we will also use ERB approved advertisements in local papers. Interested subjects will be asked to contact us at our study telephone number or to leave a message with call back information. Subjects will contact Dr. Hibberd or her study staff by telephone and will be informed about the study and subjected to an IRB approved "pre-screening" to determine whether they are in the study age range and state that they have no chronic illnesses. Those who are interested will be scheduled for a screening visit in the GCRC outpatient clinic. After going through the informed consent process including an explanation of current CDC recommendations for influenza immunization for healthy adults (23) (at which time the GCRC Research Subject Advocate will be available), Dr. Hibberd/staff and the GCRC research nurses will conduct the screening examination, and obtain the screening laboratory tests (see Section D.5). Subjects will return within 1 week for study results and referral to their primary care physician_ or new provider should abnormalities be detected on screening. Those who remain eligible after screening and are well at the follow-up visit will meet with the GCRC nutritionist for counseling on avoiding foods and other products that contain probiotics during the study period. Dr. Hibberd/staff will re-review the study with eligible subjects and if they are willing to proceed will be randomized first to receive either the LAIV or HV and then to receive either LGG or placebo. Foτ those subjects who are not able to understand English, the consent process and follow-ups will include a translator.

Eligibility Criteria:

• Demographics - male and female subjects aged 18-49, any race and ethnicity

• Medical history - no self report of any medical conditions for which the CDC states should not be vaccinated with LAIV (23) (asthma, reactive airways disease, other chronic disorders of the pulmonary or cardiovascular systems; metabolic diseases such as diabetes, renal dysfunction, and hemoglobinopathies; or persons with known or suspected immunodeficiency diseases)

• Immunization history - no self-reported vaccination with the influenza vaccine for the current influenza season; vaccination with any vaccine within the one month period prior to study enrollment or receive intent to any other vaccine during the study period; hypersensitivity to any influenza vaccine components including thimerosal or egg or Guillain-Barre syndrome

• Medications - no self-reported treatment with immunomodulator/immunosuppressive drugs (interleukins, corticosteroids (oral or inhaled)), G(M)-CSF in 4 weeks before enrollment or self reported history of IL-2 administration within 5 years; use of theophylline preparations or warfarin because of the theoretical possibilities, of enhanced drug effects and toxicities following influenza vaccination; medication use that might affect the immune response to a vaccine or effects of LGG (no antibiotics during the previous 4 weeks)

• Home or Work environment - no health care or day care workers or close contacts with immunosuppressed persons workers because of the theoretical risk that LATV could be transmitted to the immunosuppressed person and cause disease, per CDC recommendations

» Probiotics - no use of LGG or other probiotics in 4 weeks before enrollment. Yogurt consumption is not an exclusion criterion, but subjects will be asked to avoid consumption of any yogurt during the first 4 weeks of the study tn avoid consumption of other lactohaciiii and prnhiotic organisms PHS 398/2590 (Rev. 05/01) Page / ~7

Physical^' Examination^' - nd"aUnomialities; no acute febrile illness on the day of intended immunization - immunization defeπred until illness resolved

• Routine Laboratory tests - hemoglobin ≥l 1.5 g/dL for women; > 13.5 g/dL for men; WBC - 3,300-12,000 cells/mm³; differential within normal range; platelets - 125,000 550,000/mm³; ALT < upper limit of normal; serum creatinine < upper limit of normal; normal urinalysis (negative glucose, negative or trace protein, and negative oτ trace hemoglobin); negative beta-HCG pregnancy test (urine or serum) for women presumed to be of reproductive potential because LAIV is a contraindicated in pregnant women ((23).

• Infectious Disease tests - negative HIV test; hepatitis B surface antigen; anti-HCV

• Able and willing to participate - willing to complete the informed consent process; no self report of current alcohol, substance abuse, or systemic/psychiatric illness that potentially could interfere with compliance and ability to make study visits; able and willing to participate for the planned duration of the study, including availability for follow-up telephone contact

Pregnant-women .are_. excluded because trie jCDC does not recommend use of LAIV (to which they could be randomized) in pregnancy (23). Pregnancy is assessed by a negative serum or urine test in women of reproductive potential on the day of actual immunization with either LAP/ or IIV. Women with no reproductive potential are defined as those who are post-menopausal; or who have had a hysterectomy, bilateral oophorectomy, or tubal ligation, or agrees to be heterosexually inactive or to consistently practice contraception (condoms, diaphragm or cap with spermicide, intrauterine device, contraceptive pills or patch or other FDA approved contraceptive device) for the first 4 weeks of the study. Pregnancy is not a contraindication for use of either LGG or HV. Subjects using LGG or other -probiotics within 4 weeks will be excluded. Yogurt consumption will not be an exclusion criteria, but since ^~rnost yogurts- COntain:acti5ce..cultures-of.probiotie.:organisms_rsubj.e.ctS_rW.ill be asked to avoid eating yogurt for the first 4 weeks of the study.

D.3 STUDY TREATMENTS . . .

D.3.a INFLUENZA VACCINE AND MATCHING PLACEBO

All subjects will receive either LAIV or IIV according to the CDCs recommendations. To avoid bias in reporting of adverse events and influenza like illness, subjects will receive matching placebo for the LAIV and IP/. Subjects randomized to LAIV will receive the nasal vaccine and will have a placebo intramuscular injection (described below) and subjects randomized to IIV will receive, the intramuscular injection and a placebo nasal vaccine (described below). We considered using a sucrose-glutamate stabilizer with egg allantoic fluid without live attenuated virus as the matching placebo for the LAIV, but since our scientific questions relate to the comparison of responses to the influenza vaccine associated with use of LGG, not a direct head to head comparison of LAIV vs. IIV, we elected to avoid exposure of our study subjects to unnecessary placebo contents.

LAIV will be administered as the live, trivalent nasally administered influenza vaccine (FluMist ™, Medimmune Vaccines, Inc.). The precise composition of the vaccine will depend on the US Public Health Service recommendations in the year it is administered. For example, the 2004-5 season vaccine contains the following strains: A/New Caledonia/20/99 (HlNl); A/Wyorning/3/2003 (H3M2) (A/Fujan/411/2002-like); and B/Jilin/20/2003 (B/Shanghai/361/2002-like). LATV will be stored according to the manufacturer's instructions in the Tufts-NEMC research pharmacy. LAIV is supplied in a pre-filled single-use sprayer containing 0.5 mL of vaccine. Approximately 0.25 mL (i.e., half of the total sprayer contents) is sprayed into the first nostril while the recipient is upright. An attached dose-divider clip is removed from the sprayer to administer the second half of the dose into the other nostril. If the vaccine recipient sneezes after administration, the dose should not be repeated. Since the LAIV dispenser is proprietary and not available for us to purchase and fill with the matching placebo (saline), Dr. Decker, (Head of Pharmacy at Tufts-New England Medical Center and co-investigator) will prepare a companion empty nasal applicator to those subjects randomized to receive LAIV. This empty nasal applicator will appear as similar as possible to the LAFV vaccine nasal applicator. In those subjects randomized to LAIV, the GCRC research nurses will be un-blinded and will administer the first dose of LAIV. The nurse will then return to the counter to prepare (Bf iπilipal ilnvέsttøB wrøgrøivόiiip® (MNEf ® middle): Hibberd, Patricia L for administration of the second dose so that neither investigators nor the subject are aware which syringe was used for the second dose. The GCRC nurse will administer the second dose of LAIV.

In those subjects receiving HV, the pharmacy will dispense a placebo nasal vaccine. The placebo nasal vaccine will consist of two nasal applicators each filled with 0.25 ml of saline. As stated above, the design of the nasal applicator will be as similar to the LAIV applicator as possible. The GCRC nurse will administer the first dose of placebo, return to the counter for preparation of the second dose, and will then administer the second dose of nasal placebo. In this way LAIV will be administered according to manufacturer's directions and the placebo vvill appear to be administered in exactly the same way. πV will be administered as inactive divalent influenza vaccine that does not contain Thimerosal, since LAIV does not contain Thimerosal. The inactivated vaccine will be either FluZone^® (Aventis Pasteur) or Fluvirin™ (Chiron). As above, the precise composition of the vaccine will depend on the US Public Health Service recommendations in the year-it js~administered. For the 2004-5_.seaspn, _the vaccine contains the following strains: A/New. Caledόnia/2O/9S>^"TH1N1); A/Wycmϊng/3/2003 (H3M2)-CAVFu]an/4Ϊ^:ϊy2W2^ike);^r-and B/Jilin/20/2003 (B/Shanghai/361/2002-like). IIV will be stored according to manufacturer's instructions at Tufts-NEMC research pharmacy. ITV is supplied in a pre-filled single-use syringe containing 0.5 mL of vaccine that will be administered intramuscularly using a >1 inch needle. The single-use syringes do not contain the preservative Thimerosal.

In those patient randornized-to LATV, a matching intramuscular, placebo yaccine__will be administered. The matching placebo vaccine containing 0.5 mUoTnorrήal saline willlfe prepared in an identical^" appearing syringe by the Tufts-New England-Medical-Center Research -Pharmacy-under Dr Decker's direction. The placebo will be administered in exactly the same way as IIV. The research pharmacist at Tufts-NEMC will dispense either active LAW (with an empty nasal applicator and placebo ITV) or IIV and (two placebo nasal vaccine applicators) according to the randoπφation scheme (see Section D.7.b) and Jabel each subject's syringes, with the study ID number. The label will not identify the content of the syringe to maintain blinding of subjects and study staff.

D.3.b ORAL LACTOBACILLUS GG AND MATCHING PLACEBO

All subjects will receive 4 weeks of LGG or matching placebo capsules, the contents of which will be ingested as capsules oτ prepared as described below. The rationale for using a 4 week period for administration of the capsules is to provide probiotics throughout the entire period to the immunogenicity end-point as is standard in influenza vaccine trials (45; 52; 53; 56; 58; 59; 61; 94-98). Subjects will receive their first dose of capsule immediately after administration of the influenza vaccine, under observation in the GCRC. They will be given their supply of study capsules for the entire 4 weeks and instructed to bring their bottle of study capsules with them to all study visits, as capsule counts will be conducted during each study visit. The importance of adhering to the twice daily dosing will be reinforced at each visit.

ConAgra Foods Inc. manufacture the only LGG capsules available in the. US under the brand name Cultuielle.™ Culturelle™ capsules contain a small amount of Inulin as described below. ConAgra has agreed to supply LGG capsules and identical appearing placebo capsules for the study, without charge. LGG CAPSULES will be administered as a gelatin capsule that contains Inulin (295 mg) and 1 x 10¹⁰ LGG organisms. Two capsules will be administered daily for a total of 2 x IO¹⁰ organisms per day for 4 weeks. This dose has been widely and safely used in numerous other studies including our recent study of immunosuppressed children with Crohn's disease (75) and the recent study evaluating bacterial nasal colonization (99). PLACEBO CAPSULES will be administered as an Inulin-containing capsule (355 mg Inulin) without LGG that will be administered twice daily for 4 weeks. Inulin is a soluble dietary fiber (fructose polymer (beta(2-l)linkage) with a terminal glucose). We recognize that Inulin may have some immunodulatory properties, but the difference between the amount of Inulin in the placebo and active LGG capsules is so small (60 mg) compared with the usual doses of inulin (> 2 grams), this difference is unlikely to be clinically significant. The gelatin in which both capsules are packaged is a hydrolysate of collagen from beef or pork. The weight of the gelatin capsule will be 75 mg, the same capsule will be used for the active drug and placebo. Inulin and gelatin capsules have been standard for studies of LGG to date. The research pharmacist at Tufts-NEMC Principal Investigator/Program Director (Last, first, middle): Hibberd, Patricia L

WfiϊS2ϊs|)efisfellϊϊribllSG'6.»lbi;:^;|)lUfc'l^'M~according to the randomization scheme (see Section D.7.b) and label each subject's capsules with the study ID number. The label will not identify the capsule contents to maintain blinding of subjects and study staff.

Subjects will be counseled to store the capsules (LGG or placebo) in a cool, dry place at or below room temperature and asked about lapses in proper storage procedure during study visits. We will culture the contents of one of each subject's capsules prior to dispensing to obtain a quantitative estimate of LGG in the capsule (LGG or placebo) to evaluate the accuracy of the randomization assignment. On the last day of administration (day 28 or 4 weeks), we will culture an extra capsule dispensed to the subject to obtain a quantitative estimate of LGG or placebo after storage in the study subject's home.

D.4 STUDY OUTCOMES

standard quality control procedures, under Dr Ward's supervision.

D.4.b NASAL WASH ANTI-HA IgA

The primary outcomes of nasal anti HA IgA titers will be measured 4 weeks after receiving the influenza vaccine (either LAIV or IIV) and after co-administration of 4 weeks of LGG or placebo. Secondary outcomes of nasal HA IgA titers will be measured at 1, 2, 3 weeks and 6-9 months (end of the influenza season). Nasal wash specimens will be obtained as described in Section D.5.b below. Specimens will be stored at -8O⁰C and anti -influenza virus HA nasal wash IgA antibody titers will be determined in batches using an end-point ELISA and purified HA homologous to vaccine strains (obtained from Protein Sciences Corporation) as described previously (59; 97) Briefly, Nunc immunoplates will be coated with purified HA antigen overnight at 4°C at a final concentration of 2μg/ml in phosphate buffered saline (PBS). After washing with PBS, non-specific binding will be blocked with blocking buffer, plates washed again and incubated with tripling dilutions of test sera overnight at 37°C. After washing, plates will be incubated with peroxidase-conjugated goat-anti human IgA (Southern Biotechnology) for 2 hours at room temperature. Plates will be washed again and incubated with paranitrophenyl phosphate substrate solution for 1 hour at room temperature. OD 405 nm will be read using a Biorad plate reader. The dilution of the nasal wash specimen with 50% of the maximum absorption (half-maximum) and after subtraction of the control without the nasal wash specimen will be considered to be the ELISA titer. The nasal wash anti-HA IgA antibody titers will be adjusted to total IgA levels (measured by ELISA) of 500mg/L as described previously (50). All procedures will be conducted in the GCRC core laboratory under the supervision of Dr. Ward. ...Pάαe'Rfl, lπy_?stigs,tor(Prpgram Director (Last, first, middle): ²I- Hibbeid. Patricia L w LJik ϊ.-J Il c J-MJlJ!& 1...JmUUL— .ι"-_s!L.i-IiJUiJL!ftj-ai — τ_>-πc-nr_\κicτ_{? C!}

The secondary outcomes of cell mediated immune responses to influenza vaccination will be measured 2 and 4 weeks after receiving either LAIV or ITV^" and after co-administration of 4 weeks of LGG or placebo. At these two visits, 20ml of blood will be obtained in heparinized tubes for this assay. The blood will be centrifuged on a Percoll gradient and the mononuclear cell fraction collected. The cells will then be washed and re-suspended in complete RPMI medium supplemented with human AB serum. After cell enumeration, the samples will be transferred in triplicate into the wells of a microtiter plate. Each of the three strain-specific antigens (obtained from the CDC) will be added to each well with phytohemaglutinin as a positive control and medium alone used as a negative control. The mononuclear cells will be incubated with antigens and mitogens for 3-5 days at 37⁰C in 5%CO2. For the last 24 hours of culture the supematants will be removed and frozen at -7O⁰C and replaced with complete RPMI containing 0.5μCi of ³H-thymidine for 16 hours. The samples will then be transferred to^' a nitrocellulose filter and counted in a scintillation counter. The stimulation index will be calculated by the counts/minute of the influenza stimulated samples divided by those .stimulated_with_medjuπLalone. Supematants from the proliferation studies will be frozen at -8O⁰C. The samples will be thawed and added to -microtiter plates and ELISA for -interferon- gamma will be performed using a commercially available kit (R+D systems). The ELISA plates will be read using a standard plate reader (Biorad). The assays will be performed in the GCRC core laboratory under the supervision of Dr. Leav.

D.4.d LABORATORY DOCUMENTED INFLUENZA ILLNESS

Throughout the entire study period from enrollment (receipt of the influenza vaeeine-and study capsules) to end of the influenza season-following4mmunization,..we.will contact subjects every 2 weeks by phone to remind subjects to^" contact^" us ^"if they develop symptoms consistent with influenza like illness (ILI) as defined by the CDC - temperature >100 F (>37.8 C) plus cough or sore throat. Any subject who reports ILI will be asked to come in for clinical evaluation. ^• Subjects will be met at the hospital's front desk where they will be provided with a mask. then escorted to the single outpatient in the GCRC for clinical evaluation by Dr. Hibberd or a study physician. A nasal and oropharyngeal swab will be obtained for virus culture (specimens will be sent to Tufts-NEMC CLIA approved Virology Laboratory) and nasal anti-HA IgA antibody titers. A serum specimen will be obtained for serum antibody titers as described above. Subjects will be referred to their primary care provider both before and after the GCRC visit for routine care. We will find a primary care provider for subjects that do not have one. All subjects evaluated for ILI will be asked to return to the clinic in 4 weeks for collection of serum for antibody determination. Laboratory documented influenza (LDI) illness (secondary outcome) will be determined based on the evaluation of ILL LDI has been defined previously (56) as either recovery of wild type influenza A or B in nasal and oropharyngeal secretions (per standard clinical practice); and/or a four-fold increase in serum antibody titer to influenza A or B virus reaching a titer > 16 four weeks after onset of ILL

D.4.e ADVERSE EFFECTS AND DISCONTINUATION OF STUDY THERAPY

To assess vaccine safety in conjunction with co-administration of LGG or placebo capsules, subjects will be observed for 30 minutes in the GCRC after administration of the vaccine and the first dose of the; study capsule. Subjects will be provided a diary card to record their temperature (thermometer will be provided) and check off the occurrence of symptoms potentially attributable to the influenza vaccine or placebo every day for the next 28 days. Diary cards will be provided weekly and subjects asked to return the completed diary card for the previous week during the week 1, 2, 3 and 4 study visits. Subjects will be asked about open-ended and specific questions about possible adverse experiences during their study visits and during telephone calls. As described in Section E₅ this study will be monitored by an independent Data and Safety Monitoring Board who will be responsible for monitoring the project, oversight of subject safety, and reviewing quality of study data.

AU adverse events will be recorded on an adverse event case report form and will include a description of all undesirable experiences, required interventions, subject's condition after the event, an estimate of the extent of injury and potential strategies to prevent future occurrences. The principal investigator will classify the relationship of the study protocol to the event (see Section E). The principal investigator is responsible for reporting serious adverse PHβ^piV^ei!lβ®fe^r"^"1Dteβ^r«|aB&^midclle): Hibberd, Patricia L events (death, life threatening, new,^' serious or permanent disability) to the Chair of the DSMB, the Tufts-NEMC

Institutional Review Board within 72 hours of notification. Other adverse events will be summarized quarterly for the DSMB and the IRB. Particular attention will be paid to abdominal symptoms such as bloating, gas, or intestinal rumbling. Based on extensive prolonged use of LGG by children and other populations (70; 73; 74), we do not anticipate that any serious adverse events attributable to the probiotic will occur.

The . study capsules will be discontinued if a subject develops moderate or severe gastrointestinal side effects, Lactobacillus bacteremia, or the subject requests to be withdrawn from the study for any reason. Subjects in whom the study capsules are discontinued will have their "last results carried forward" in the intent-to-treat analysis as described in Section D.7.C. If an enrolled subject begins immunosuppressive medication or events occur which raise questions as to the safety of continued administration of LGG, the subject's primary care physician will have the option to withdraw the subject from the study.

* Complete Blood Count with differential, platelets, alanine aminotransferase, creatinine, urinaπalysis HIV test; hepatitis B surface antigen; anti-HCV; ** β-HCG - pregnancy test; § ILI - influenza like episode defined in Section D.4.e

D.5.a SCREENING VISIT

After obtaining informed consent, demographic data, medical and medication history, vital signs and the physical examination will be obtained and recorded by the Principal Investigator and GCRC Study Nurses using standardized procedures described in the Manual of Operations. Specially designed case report forms will be prepared for this study prior to its implementation. (Samples from prior studies are included in APPENDIX B). Routine screening laboratory tests will be performed in the hospital's CLIA approved laboratory.

The baseline visit will start with a review of the screening tests and check of interim history and vital signs. For those who remain eligible for the study, they will provide serum and nasal specimens. Nasal wash specimens will be obtained according to standard procedures in the GCRC outpatient area. Specifically subjects sit in a comfortable position with the head slightly tilted backward keep their pharynx closed by saying "K" while the washing fluid 1- 1.5 mL of sterile normal saline is applied to each nostril. The subject then tilts his/her head forward and lets the washing fluid flow into a sterile specimen container. Nasal wash specimens will be sonicated then concentrated using standard techniques (46). Subjects will be randomized to receive either LAIV or ITV (administration described in Section D.3.a above) and then randomized to receive their study capsules (LGG or placebo). Study capsules will be administered as a 28-day supply with extra capsules for future analysis of study drug content. Prior to release of study drug, one capsule will be removed from the dispensed therapy and sent to Dr. Goldin's laboratory for quantitative LGG culture, as described below. Subjects will receive their first capsule during the baseline visit and then will continue. iheir_twjce_daily the evening after the baseline visit. During the first 28 days of the study, subjects will complete diary cards recording^" their daily femperature^"aή^"d^~checking symptoms that-occur-.- ^•

D.5.c STUDY FOLLOW-UP

For the first 4 weeks, the subjects will return weekly for a clinical evaluation and to provide serum and nasal specimens, to turn in their study diary and to have their capsules counted. On the 4-week visit, a final extra capsule will be cultured to assess viability of LGG in capsules ^"at completion of the study." At the end-of the-4-week period, they will -be contacted every 2-weeks -to-deteimine-wheiher an.influenza like illness (ILI - see section D.4.e) has occurred. In addition, they will be asked to report occurrence of an TLT. Subjects who report^"!! ILI will be asked to come to the GCRC for an unscheduled visit during which they will provide nasal and oropharyngeal specimens and serum specimens as described in section D.4.e. At the end of the influenza season, subjects will return to provide final serum and nasal specimens to determine whether antibody responses remained durable during the entire season.

D.5.d LGG COLONY COUNTS OF STUDY CAPSULES

Half a gram of powder from the capsule will be weighed in a sterile container. 4.5 ml of sterile PBS will be added resulting in a 1:10 dilution. Serial dilutions will be performed by adding 0.5 ml from the 1:10 concentration solution into 4.5 ml of PBS resulting in a 1:100 concentration solution and continuing to perform such dilutions until the desired dilution of 1: 10,000,000 is reached. Dilutions from 10"¹ to 10^"8 will be plated on Lactobacilli selective agar and incubated in an anaerobic chamber at 37⁰C for 48 hours. LGG colonies, which appear as white and creamy and have a distinct buttery smell, will be counted. Gram stain will distinguish between LGG and other Lactobacilli by the palisading appearance of LGG. The number of colony forming units per gram of powder will be recorded. A representative sample of LGG colonies will be confirmed by biochemical profile using the API Rapid CH system.

D.6 DATA MANAGEMENT

The Division of Clinical Research Resources (DCRR), at Tufts-NEMC, directed by Dr. Hibberd, will manage the study data. DCRR has extensive experience in the design, data management, conduct and analysis of single multi-center trials. It uses a fully featured relational database on a central server networked to data entry and data analysis workstations. DCRR uses conventional data verification systems that are programmed to prevent logic errors and reduce incorrect out of range values. Periodic analysis of each data . field is conducted to examine the expected distributions of the data and to identify outliers for possible data collection or entry errors. The specific procedures used by the DCRR are described in the Resources and Environment Statement. The GCRC informatics staff will review the study data management system prior to its use to ensure that it meets all GCRC informatics standards.

D.7 SAMPLE SIZE AND STATISTICAL ANALYSIS Principal Investigator/Program Director (Last, first, middle): Hibberd, Patricia L

Since we believe that this is the first study to evaluate the use of LGG as an immunoadjuvant to LAIV or HV, we have based our sample size estimates on data and sample sizes used in Fang's recent study of oral attenuated Salmonella typhi Ty 21a vaccine administered with LGG vs. L lactis vs. placebo (11) and other similar studies evaluating the immunogenicity of LAP/ and IIV and other immune adjuvants including MF59 and CPG 7909 (53; 59; 61; 98). In this study, our primary comparisons are change in nasal anti-HA IgA titers after volunteers receive LAIV with LGG vs. placebo and change in serum HAI titers after volunteers receive IIV with LGG vs. placebo. We assumed that the log of the antibody titers will be normally distributed based on bur previous studies (22). Since most other studies have used a sample size of 20-30 per group, we explored the study power that we would have to detect a large effect size (0.8 or greater) with a sample size in this range (see Figure 5). We elected not to adjust our sample size for multiple comparisons of immune response to the three vaccine strains and types of antibody. JPigur€_5^-JPower Analysis (ά=0.05, 2 sided)

As shown in Figure 5, -with 26 subjects per group we will have 80% power to detect an effect size of 0.8 or greater between the two primary comparison groups, using a two-group t-test with a 0.05 two- sided significance level. If the effect size is only 0.6, we will only have 56% power a difference between the LGG and placebo recipients of the LAIV of IIV. With a required sample size of 26 per group in the LAIV group, the total sample size for the study is then 104 subjects - 26 receive LATV+LGG; 26 receive LAIV+placebo; 26 receive irV+LGG; and 26 receive HV+placebo, as shown previously in Figure 4. Sample size calculations were performed using nQuery Advisor 5.0 (Statistical Solutions, Boston, MA). Recruiting 104

healthy subjects over a 2-year period into this study

Sample Size per Group is logistically feasible based on the Dr Hibberd' s experience and the GCRC track record in recruiting healthy volunteers.

D.7.b RANDOMIZATION SCHEME AND PROCESS

Randomization assignments will be niade in permuted blocks of size 4 and 8 for this 1:1:1:1 randomization (I x LAIV+LGG; 1 x LAIV+placebo; Ix ITV+LGG; 1 x IlV+placebo). The block size of 4 and 8 will also be randomly assigned, to minimize correct -prediction of assignments, while preserving approximate balance between groups. Specially designed software will be used to generate the random numbers. Ms. Ruthazer, the statistician, will review lists prior to release and will keep a copy of the randomization list in a locked filing cabinet. The randomization list will be released to the research pharmacist at Tufts-NEMC. The pharmacist will label the capsules and influenza vaccine with the subject's study ID number and dispense according to the randomization schedule, but the study label will not identify whether the treatment contains LGG or placebo, LATV or IIV.

D.7.C STATISTICAL ANALYSIS

Interim Analysis An independent Data Safety Monitoring Board will monitor this pilot study. The trial may be stopped if the DSMB determines that: an excess number of severe adverse events are occurring in either treatment group, adherence to the study protocol and recruitment is well below acceptable goals and the ability of the study to achieve its goals are seriously compromised. There will be no interim analysis for early evidence of efficacy, as this cannot occur with the sample size in the pilot study.

Primary Objectives: To measure anti-influenza systemic and mucosal antibody responses 4 weeks after administration of influenza vaccine to healthy subjects aged 18-49 and to compare responses in subjects receiving Lactobacillus GG (LGG) vs. placebo.

Hypotheses: i. Subjects receiving LGG capsules will ^"have "a^"" greater increase— in- -geometric -mean nasal anti-influenza hemagglutinin (HA) IgA four weeks after LAIV immunization than subjects receiving placebo capsules, ii. Subjects receiving LGG capsules will have a greater increase in the geometric mean serum hemagglutination inhibition (HAI) antibody titer four weeks after IIV immunization than subjects receiving placebo capsules.

Statistical Approach: yfe will use the same analytic approach to test both hypotheses. First we will compare the change in the log geometric mean titers of each vatcine strain serum HAI antibody-and nasal anti-influenza HA IgA .from baseline. to_ 4. weeks after either LAIV or_nV_administration jn the LGG vs. placebo group using a 2- sample t-test. We will also explore the proportion achieve a protective antibody response (45) in the two groups using a Fisher's exact test, although we anticipate have insufficient power to detect differences between groups with a sample size of 26 per group. These analyses will be done using the intent-to-treat principle. If subjects are discontinued from the study or if they drop out, we will carry forward the subject's last antibody titer and assume that it is the 4-week titer to enable results from all subjects to be analyzed. This approach to missing data is conservative, providing that there is no differential drop out from the LGG and placebo treated groups. Finally, we will explore the effect of LGG vs. placebo on influenza antibody levels in subjects without prior exposure to current vaccine strains (no protective titer at baseline - primary response) and those who have a protective titer at baseline (booster response).

Secondary Objectives: To assess time course of antibody and cell mediated immune response (rapidity and durability) after administration of influenza vaccine and to compare responses in subjects receiving LGG vs. placebo. Hypotheses: i. Subjects receiving LGG capsules and LAW will have a more rapid response (achieve protective serum HAI and nasal anti-influenza HA IgA titers by week 2) than subjects receiving placebo capsules and LAIV.

Statistical Approach: We will plot the serum HAI antibody and nasal anti-influenza HA IgA antibody responses over the first 4 weeks in the LGG vs. placebo group, then compare the proportion achieving protective responses by week 2 Using the Fisher Exact test. We will also compare serum HAI antibody and nasal anti-influenza HA IgA responses over the 4 weeks in the LGG vs. placebo group using a general mixed model. We anticipate an autoregressive within-subject correlation structure, but will use the likelihood ratio test and Akadke Information Criterion to select a covariance structure. As above, we will explore the effect of prior immunity to vaccine strains on outcomes in LGG vs. placebo treated groups.

Hypotheses: ii. Subjects receiving LGG capsules after either LAIV or IIV will have higher geometric mean serum HAI antibody titers and nasal anti-influenza HA IgA at the end of the influenza season (6-9 months after immunization) than those receiving placebo capsules and either vaccine.

protection throughout) using the Fisher Exact test.

Hypotheses: iii. Subjects receiving LGG capsules after either LAIV or IIV will have a greater cell mediated immune response to the influenza vaccine as measured by greater proliferation of peripheral blood mononuclear cells and cytokine production in response to vaccine-specific influenza antigens than those receiving placebo capsules and either vaccine.

Statistical Approach: We anticipate that we will need to log transform the counts per minute from the PBMC stimulated with vaccine antigens prior to comparing the responses between the LAIV+LGG vs. placebo and IIV+LGG_ys. placebo using a two sample t-test. Similarly, we will evaluate whether the gamma-interferon levels ■are normally distributed and perform transformations if -necessary prior to conducting- similar analyses-using a 2- sample t-test. ■ .

Exploratory Objectives: To report the occurrence of laboratory documented influenza illness and adverse effects after administration of two types of influenza vaccine (LAIV and IIV) in the LGG and placebo capsule groups, and to obtain preliminary information on efficacy in preparation for future studies.

Statistical Approach: We will have insufficient power to detect differences between LGG vs. placebo in laboratory documented influenza (LDl), but we will estimate-cumulative rates of LDI by group using the Kaplan-Meier method.

D.8 QUALITY CONTROL PROCEDURES ^"

Consistent application of the study protocol and performance of study procedures are critical to acquiring high quality data. Thus, one of the first study tasks is to prepare a detailed manual of operations and set of case report forms (similar to samples provided in Appendix B). The entire study team including GCRC study nurses will undergo a competency-based training program process prior to enrolling subjects, as is standard in the Tufts-NEMC GCRC. The GCRC core laboratory staff will undergo a similar procedure to ensure exquisite attention to detail with tracking of samples and with the analysis of subject specimens. The hospital's Clinical Laboratory Improvement Act and College of American Pathologists approved microbiology laboratory will culture all oropharyngeal specimens for influenza viruses and the chemistry and hematology laboratory will perform the screening tests. Dr. Goldin's laboratory will culture all capsule specimens using standard quality control procedures, including assaying specimens chosen at random in duplicate.

D.9 ANTICIPATED PROBLEMS AND SOLUTIONS

Recruiting 104 subjects over two consecutive influenza seasons is logistically feasible since Dr. Hibberd and the GCRC have extensive experience in recruitment of healthy volunteers. However, should there be any difficulties achieving the required sample size (not anticipated), we will increase our advertising efforts and community outreach programs. In collaboration with the GCRC Research Social Worker, we will proactively track recruitment by gender, race and ethnicity and use directed advertising for mid course corrections to achieve accrual targets, if necessary. Finally, in a "worst case scenario", we could continue to recruit into a third year and still complete the project on time, however this plan would only be operated as a last resort.

We recognize that the composition of the influenza vaccine changes every year (although one vaccine strain A/New Caledonia/20/99 (HlNl)) has been present in every vaccine since 2000. There are two important implications of these changes. First, we will study the effects of LGG over two seasons for greater generalizability, as it seems unlikely that immunodulatory effects of LGG would be associated with strains in the influenza vaccine. (g^g^i^nv^M&^^Kxj^i^ac^πr.SiFectøni^^fc^ratemidciie): Hibberd, Patricia L

Second, healthy subjects over' age ^"lO^'are more likely to have been exposed to previous doses of IFV, according to targeted recommendations from the CDC. In this age group, it may be difficult to find any healthy adults over age 50 who do not have antibodies to A/New Caledonia/20/99 (HlNl)₃ either as a result of natural infection or immunization. Since our goal is to study the effects of LGG in those with and without prior immunity, we are studying healthy adults in the age range 18-49. We also recognize that there is a risk that healthy adults already achieve a maximal response (ceiling effect) to the influenza vaccine, such that an immune adjuvant might appear to have no effect in this age group. However, the S typhi oral vaccine studies in healthy volunteers in this age range did have an enhanced immune response to LGG. •

D.10 TIMELINE

This three-year study requires careful timing of enrollment after the influenza vaccine for the upcoming season is available (typically late September, early October), with a goal of achieving the 4 week antibody response before the influenza viruses start circulating in the community (typically November). Thus the study activities need to be adjusted to accommodate the required enrollment period as follows.

Yl- Yl Yl Yl Y2 Y2 Y2- Y2 Y3 Y3

TASK JuIy- Oct- Nov- May- July- Oct- Nov- May- July- Oct-

Sept Nov April June Sept Nov April June Sept June

Prepare Manual of Operations, Case Report Forms X

GCRC. Labpratorx Preparation X

GCRC Start-up Meeting X

Pilot test Logistics X

Prepare Data Management System, Randomization X • • Assignments

DSMB meeting X X X

Study recruitment X X

Prepare to dispense influenza

X X vaccine and study capsules

Screening, enrollment, influenza immunization, study X X capsules administration

Telephone follow-up every 2 weeks, evaluation for ^• X X influenza like illness

Final study visit Year 1

X subjects x

Batched laboratory testing X X X X X

Data entry and cleaning X X X X X X X X

Statistical Analysis X X X

Manuscript Preparation X

D.ll NEXT STEPS

This research is intended to evaluate whether LGG is an effective immune-adjuvant particularly when administered in combination with the mucosally administered live attenuated influenza vaccine. This study evaluates immunogeniciry in healthy subjects ages 18-49 as a first test of the. principle. Promising preliminary data have the potential to profoundly impact the effectiveness of the influenza vaccine in patient populations who have sub-optimal vaccine efficacy and may have utility for combating influenza in a wide range of situations. Future studies will be directed at both elucidating the immunomodulatory mechanisms of LGG in relation to administration of the influenza vaccine and to conducting a large multicenter study of LGG vs. placebo to prevent influenza.

PHS 398/2590 (Rev.05/01) Pane rf- / Continuation Format Page

1. RISKS TO SUBJECTS

E.l.a Human Subjects Involvement and Characteristics

The human subject population consists of healthy volunteers, ages 18-49. Since 50% of the study subjects will be randomized to receive LAIV, the upper end of the age range is identical to the approved age range for receiving LATV (FDA approval and CDC recommendations). Although a single dose of LAIV the vaccine may also be administered to healthy children aged 9-17, and LGG can be safely administered to all children, we believe that if this Phase LTI study is successful in healthy adults, a separate study with even more focused and limited specimen collection should be conducted in children. (Two doses of LAIV are required for children aged 5-8.) Pregnant women are not eligible to participate as LAIV is contraindicated during pregnancy. However women of reproductive -age-are- eligible to participate providing that they use an acceptable method of contraception as described in ^'Section BI2.^{~" •} . ^' ; ~ — .—

E.l.b Sources of Materials . .

The source of materials obtained from human subjects will include peripheral venous blood samples nasal washes-obtained for-research purposes, nasal and oropharyngeal specimens subjects with influenza like illness and demographic, medical history and physical examination data in the research records. All materials are for research purposes only.

E.l.c. Potential Risks

Both the LAIV and HV are recommended by the CDC (23) for healthy subjects aged 5-49.

LAIV: Vaccine recipients may experience low-grade fever, upper respiratory symptoms and muscle aches. The Advisory Committee on Immunization Practices (23) specifically states that "among adults, runny nose or nasal congestion (28%--78%), headache (16%— 44%), and sore throat (15%— 27%) have been reported more often among vaccine recipients than placebo recipients. In one clinical trial, among a subset of healthy adults aged 18—49 years, signs and symptoms reported more frequently among LAW recipients (n = 2,548) than placebo recipients (n = 1,290) within 7 days after each dose included cough (13.9% versus 10.8%); runny nose (44.5% versus 27.1%); sore throat (27.8% versus 17.1%); chills (8.6% versus 6.0%); and tiredness/weakness (25.7% versus 21.^'6%). " However, these side effects are generally milder than symptomatic influenza infections and often persist between 1-3 days. Individuals with hypersensitivity to any component of virus vaccines including eggs or egg products may experience anaphylactic reactions. Another risk associated with the administration of LAIV is the shedding, and potentially the transmission of, live influenza viruses to others following close contact. However, the estimated probability of acquiring vaccine virus after close contact with a single LAIV recipient was 0.58 - 2.4%. Overall, the rate of adverse events occurring among healthy adults aged 18-49 years is very low (<1%) (23). πV: This vaccine has been used in the United States for many years. The most common side effect is soreness at the vaccination site. This local reaction is typically mild and rarely interferes with the person's ability to conduct usual daily activities. Fever, malaise, myalgia and headache can potentially occur after vaccination. These side effects will often begin 6-12 hours after vaccination and last for 1-2 days. Although occurring rarely, anaphylactic reactions may be induced among vaccine recipients with hypersensitivity to eggs or egg products contained in the vaccine. •

LGG: Potential risks associated with oral administration of LGG include abdominal symptoms such as bloating, gas, or intestinal rumbling. In most trials of oral and vaginal use of LGG to date, there have been no differences in these side effects between LGG and placebo. These adverse effects are generally mild, and subside in one to two weeks (100). Since Lactobacillus bacteremia following administration of Lactobacillus acidophilus by one patient (101) piddle): Hibberd, Patricia L patients with co-morbid conditions has been reported (102; 103), (Lactobacillus bacteremia strains indistinguishable from probiotic consumed) we will ensure that al] blood cultures on study subjects that are obtained during the 48 week study period will be evaluated for presence of Lactobacillus bacteremia. Still, it should be noted that LGG is in use in 33 countries and an estimated 3 million kilograms of LGG-containing products were consumed by a minimum of 40,000 persons in Finland alone in 1992 (100). Throughout the study, all subjects will be asked whether they have experienced any untoward symptoms.

Venipuncture: The risks of venipuncture are considered to be minimal. The total, amount of blood taken for these studies will be no more than 110 mL on an individual subject over the entire study (up to 9 months).

Nasal and Oropharyngeal specimens: The risks of providing nasal and oropharyngeal specimens are also minimal. They require subjects' to sit in a comfortable position with the head slightly tilted backward keep their pharynx closed by saying "K" while the washing fluid 1-1.5 mL of sterile normal saline is applied to each nostril. "The subject then tilts his/her-head forward -and- -lets the washing fluid flow .into a sterile specimen container. Oropharyngeal specirneris^'afe^""only taken^""iή^"~subjects who report influenza like illness. These specimens are obtained according to routine clinical protocols and are considered minimal risk.

Loss of confidentiality: There is a risk of loss of confidentially as a result of participating in the study.

E. 2 ADEQUACY GF PROTECTION AGAENST-RISKS

E.2.1 Recruitment and Informed Consent

The GCRC Research Social Worker will assist with recruiting both males and females from diverse racial and ethnic backgrounds, using IRB approved procedures. This assistance includes, outreach programs to local community groups informing them about research directions and opportunities. As in our previous studies, we will also use IRB approved advertisements in local papers. Interested subjects will be asked to contact us at our study telephone number or to leave a message with call back information.

Subjects will contact Dr. Hibberd or, her study staff by telephone and will be informed about the study and subjected to an IRB approved "pre-screening" to determine whether they are in the study age range and state that they have no chronic illnesses. Those who are interested will be scheduled for a screening visit in the GCRC outpatient clinic. After going through the informed consent process including an. explanation of current CDC recommendations for influenza immunization for healthy adults (23) (at which time the GCRC Research Subject Advocate will be available), Dr. Hibberd/stafF and the GCRC research nurses will conduct the screening examination, and obtain the screening laboratory tests. Subjects will return within 1 week for study, results and referral to their primary care physician or new provider should abnormalities be detected on screening. Those who remain eligible after screening and are well at the follow-up visit will meet with the GCRC nutritionist for counseling on avoiding foods and other products that contain probiotics during the study period. Dr. Hibberd/staff will re-review the study with eligible subjects and if they are willing to proceed will randomize them first to receive either the LAIV or IIV and then to receive either LGG or placebo. For those subjects who are not able to understand English, the consent process and follow-ups will include a translator. We will cover the cost of parking and compensate study volunteers for their time.

E.2.b Protection Against Risk

First, all events potentially associated with study therapies or procedures including administration of either LAIV or IIV or LGG or placebo capsules will be carefully reviewed and monitored by an independent data safety monitoring board as described in Section D.6 and E.3 below. The close monitoring of all study subjects during the first 4 weeks is also intended to minimize any study-associated risks by promptly identifying and reporting adverse events. ωn|iRal.,jnves|galjar/[|m 30 ^lr The" site ofveff FTM!im^midd*:^):- JtiiDDerα, r amcia L. iritirt'ctu?e'^1;^i!lⁱ"Si^riIrefuny- cleansed, and experienced personnel will obtain blood samples in the Tufts-NEMC GCRC. The nasal specimens will be obtained with sterile saline to Teduce the already minimal risk of this procedure. .

Procedures to minimize the chance that there will be loss of confidentiality include the following. AU subject data will be identified with a unique study identification number (randomization number). Study forms and laboratory data will contain this number and will not contain medical record number, name or other identifiable information (per HIPAA guidelines). The same coded study ID will be assigned to all biologic specimens to maintain confidentiality in the laboratory setting. All information in the database will have no identifiable patient data and study databases will be password protected. Any publication arising from this study will maintain the anonymity of study participants. The signed consent form and the log that links the patient's name to the study ID number will be kept in a locked file cabinet in the Division of Clinical Research Resources at Tufts-New England Medical Center. . . .

E.3 POTENTIALBENEFITS OF THEPROPOSED RESEARCH TOTHE SUBJECTAND OTHERS

There is no direct benefit to the_. subject by participating in this research other than close monitoring after receiving LAIV or HV. Both vaccines are easily available outside the study. However, there is potential for direct benefit in the group who receive the LGG capsules, as LGG may improve the immunogenicity of either influenza vaccine.

E.4 IMPORTANCE OF THE KNOWLEDGE TO BE GAINED

Knowledge about the potential immune-adjuvant effects of LGG when co-administered with the influenza vaccine may lead to new ways to improve the immune response in the elderly and in patients with chronic illness and in emergency situations, such, as an influenza pandemic.

Collaborating Sites - None

E.5 TARGETED/PLANNED ENROLLMENT

Since our subjects are normal volunteers likely from Massachusetts, the projected enrollment figures shown here are based on the Gender and Minority/Majority Distribution in Massachusetts, 2000 Census (http://factfuider.census.gov/serylet/QTTable7ds name=DEC 2000 SFl U&geo id=04000US25&qr name=DEC 2000 SFl U DP1\ accessed: 09/24/2004.

Inclusion of Women

Every effort will be made to recruit women into this study, and women of child-bearing age will be asked to use PrirjcjpalJnMestigatgriBraarafH. DJrpctpr χi_ast_f first, middle): Hibberα. Patricia L m approved^' HySnU¹¹OTDiMi cfafebT?^"' Women who aie pregnant will be asked to defer participation until they are no onger pregnant.

Inclusion of Minorities

Subjects who identify themselves in any racial or ethnic group are eligible to participate in this study. Every effort will be made to recruit a similar racial/ethnic breakdown to the Boston population, as there are no eligibility ;riteria related to race. IRB approved recruitment material will extend an invitation for subjects in any racial or ϊthnic group to participate in the study.

Inclusion of Children

Since 50% of the study subjects will be randomized to receive LAIV, the approved age range for receiving LATV (FDA- approval- and CDC recommendations) needs Jo be addressed. LAIV is approved for children aged 5-17, but children aged 5-8 require^" l-doses Of-LAfV-that-cannot-be-accommodated-in -this -study- -Although a single- dose of LAIV the vaccine may also be administered to healthy children aged 9-17, and LGG can be safely administered to all children, we believe that if this Phase I/II study is successful in healthy adults, a separate study with even more focused and limited specimen- collection should be conducted in children under age 18. For example, the studies of cell mediated immunity require 2 blood draws of 20 mL each, which would increase the risk of the study for younger ■Ghildreni- Chi-ldren-ages-L8=21_are included. inihis_study._

-DATAAND SAFETY-MONITORING PLAN

The principal investigator is responsible for assuring safety of participants in this study and the integrity of the study data. Speςific protocols will be develqped for monitoring and reporting of adverse, events, monitoring study progress, assuring protocol compliance and data accuracy, determining appropriateness of continuing or stopping a trial, and preventing biased interpretation of data and conflict of interest.

Data and Safety Monitoring Board: The Principal Investigator, in conjunction with the NIH program officer overseeing this project, will appoint an independent Data Safety Monitoring Board (DSMB). The DSMB is responsible for monitoring the project, oversight of subject safety and reviewing quality of study data. If the DSMB recommends a study change for subject safety or ethical reasons, or if the study is prematurely terminated due to slow accrual, the Principal Investigator will be responsible for implementing the recommendations as expeditiously as possible, according to standard policies of NIH. The board will meet once prior to the start of the study (in person) and at least once during each study year (either in person or by conference call), more frequently as needed. The DSMB will consist of three members: an infectious disease physician (Chair), an immunologist and a statistician. Decisions will be made by a majority vote. Members of the DSMB will not have any affiliation with Tufts-NEMC. The DSMB chair will receive reports of all serious adverse events throughout the conduct of the study.

Adverse Event Monitoring: Study subjects will be monitored for the occurrence of events, defined as any undesirable experience, from the day of enrollment for a maximum period of 9 months, although intense monitoring of adverse events will occur during the first 28 days when the subject is taking either LGG or placebo following influenza immunization. All adverse events will be recorded on an adverse event case report form and will include a description of all undesirable experiences, required interventions, subject's condition after the event, an estimate of the extent of injury and potential strategies to prevent future occurrences. The principal investigator will classify the relationship of the study protocol to the event. The principal investigator is responsible for reporting serious adverse events (death, life threatening, new, serious or permanent disability) to the Chair of the DSMB, trie Tufts-NEMC Institutional Review Boards within 72 hours of notification. Other adverse events will be summarized quarterly for the DSMB and the IRB. The adverse event case report form will include a description of all undesirable experiences or unanticipated benefits, required interventions, patient's condition after the event, an estimate of the extent of Principal Investigator/Program Director (Last, firsϊTmiddle): . Hibbeid. Patricia L pii^tilf^iahlllrf^flillo^sfe^li^ianie principal investigator will classify the relationship of the study protocol to the event as follows:

• Not related: The event is clearly related to factors such as the subject's clinical state, not to therapeutic interventions associated with the study protocol.

• Remote: The event was most likely related to factors such as the subject's clinical state, not to therapeutic interventions associated with the study protocol.

• Possible: The event follows a reasonable temporal sequence from consuming LGG or placebo but is possibly related to factors such as the subject's clinical state.

• Probable: The event follows a reasonable temporal sequence from consuming or LGG or placebo and cannot be reasonably explained by factors such as the subject's clinical state.

• Highly Probable: The event follows a reasonable temporal sequence from consuming or LGG or placebo and cannot be reasonably explained by factors such as the subject's clinical state. In addition, the event occurs immediately following ingestion or application of study therapy or_reappears_ on repeat exposure, if the PI considers it safe to re-expose the patient to study therapy.

The severity of an adverse event in both groups is defined as a qualitative assessment of the degree of intensity of an adverse event as determined by the principal investigator as follows:

• Mild: Does not impact (in any way) the subject's life.

• Moderate: Impacts the subject's life but is not life-threatening or incapacitating.

•

Serious: Fatal, life ffireatening71^eπrianently disabling; severely incapacitating; requires/prolongs inpatient hospitalization.

The principal investigator will follow-up on all serious events on a case-by-case basis. All adverse events will be summarized and reviewed on a quarterly basis with the Chair of the Data Safety and Monitoring Board. Particular attention will be paid to abdominal symptoms such as bloating, gas, or intestinal rumbling. In most trials of oral use of LGG to date, there have been no differences in these side effects between LGG and placebo. These adverse effects are generally mild, and subside in one to two weeks (100). Since Lactobacillus bacteremia following administration of Lactobacillus acidophilus by one patient (101) and Lactobacillus rhamnosus by two patients with co-morbid conditions has been reported (102; 103), (Lactobacillus bacteremia strains indistinguishable from probiotic consumed) we will ensure that all blood cultures on study subjects that are obtained during the 9 month study period will be evaluated for presence of Lactobacillus bacteremia. Still, it should be noted that LGG is in use in 33 countries and an estimated 3 million kilograms of LGG-contaiπing products were safely consumed by a minimum of 40,000 persons in Finland alone in 1992 (100).

We considered whether it was appropriate to have an independent Data and Safety Monitoring Board (DSMB), as part of the Data Safety Monitoring Plan. Although we do not intend to have a formal interim analysis for early evidence of efficacy, the medical risks associated with participation in this trial are minimal, and it is being conducted in only one site where adverse events are relatively easy to monitor, we believe that this study can benefit from external monitoring by an independent DSMB (104).

F. VERTEBRATE ANIMALS

None

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Claims

What is claimed is:

1. A method of enhancing the efficacy of an influenza vaccine administered to a subject, the method comprising administering a probiotic and the influenza vaccine to the subject.

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

3. The method of claim 1 or claim 2, wherein the subject is apparently healthy.

4. The method of claim 1 or claim 2, wherein the subject has a condition that compromises the immune system.

5. The method of claim 4, wherein the condition is an autoimmune disease.

6. The method of claim 5, wherein the autoimmune disease is diabetes.

7. The method of claim 4, wherein the condition is a viral infection.

8. The method of claim 7, wherein the viral infection is an infection with a human immunodeficiency virus, a hepatitis virus, or an influenza virus.

9. The method of claim 1 or claim 2, wherein the subject is being treated with an immunosuppressant.

10. The method of claim 1 or claim 2, wherein the subject is critically ill.

11. The method of claim 1 or claim 2, wherein the subject is an infant or elderly.

12. The method of any of claims 1-11, wherein the composition is formulated as an ointment, cream, lotion, paste, gel, salve, oil, spray, suspension, partial liquid, nebulae, or mist.

13. The method of any of claims 1-12, wherein the probiotic comprises Lactobacillus rhamnosus GG (LGG).

14. The method of any of claims 1-13, wherein the composition comprises a live L. rhamnosus.

15. The method of any of claims 1-13, wherein the composition comprises a killed Z. rhamnosus.

16. The method of any of claims 1-15, wherein the influenza vaccine is a live attenuated influenza virus vaccine (LATV).

17. The method of any of claims 1-15, wherein the influenza vaccine is an inactivated influenza vaccine (IIV).

18. The method of claim 13, wherein the LGG is the LGG deposited with the American Type Culture Collection (ATCC) under code ATCC 53103.

19. The method of any of claims 1-18, wherein the probiotic and the influenza vaccine are administered at essentially the same time by the same route of administration.

20. The method of any of claims 1-18, wherein the probiotic and the influenza vaccine are administered at different times or by different routes of administration.

21. A composition comprising a probiotic and an immunogen that, upon administration to a subject, elicits an immune response against a virus that causes influenza.

22. The composition of claim 21, wherein the probiotic comprises a Lactobacillus strain of bacteria.

23. The composition of claim 22, wherein the Lactobacillus is an L. rhamnosus.

24. The composition of claim 23, wherein the L. rhamnosus is LGG

25. The composition of claim 24, wherein the LGG is the LGG deposited with the American Type Culture Collection (ATCC) under code ATCC 53103.

26. The composition of claim 22, wherein the Lactobacillus is of the species L. acidophilus, L. casei, L. crispatus, L. bulgaricus, L. fermentum, L.jensenii, L. plantarum, L. reuteri, L. curvatus, L. salivarius, or L. johnsonii.

27. The composition of any of claims 21-26, wherein the Lactobacillus is viable.

28. The composition of any of claims 21-26, wherein the Lactobacillus is nonviable.

29. The composition of claim 28, wherein the Lactobacillus has been fragmented.

30. The composition of any of claims 22-29, further comprising a non- lactobacillus probiotic.

31. The composition of claim 30, wherein the non-lactobacillus probiotic is Bafidobacterium lactis (B. lactis).

32. The composition of any of claims 21-31, further comprising a prebiotic or synbiotic.

33. The composition of any of claims 21-32, wherein the composition is formulated as an ointment, cream, lotion, paste, gel, salve, oil, spray, suspension, partial liquid, nebulae, or mist.

34. The composition of any of claims 21-32, wherein the composition is formulated for oral administration.

35. The composition of any of claims 21-32, wherein the composition is formulated for intramuscular, subcutaneous, or intraperitoneal injection.

36. A kit comprising the composition of any of claims 21-35 and instructions for use.

37. A kit comprising (a) a first container comprising a probiotic and excluding an influenza vaccine, (b) a second container comprising the influenza vaccine and excluding a probiotic, and (c) instructions for use.