WO2006124630A2 - Compositions et procedes permettant d'augmenter l'efficacite de vaccins - Google Patents

Compositions et procedes permettant d'augmenter l'efficacite de vaccins Download PDF

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WO2006124630A2
WO2006124630A2 PCT/US2006/018488 US2006018488W WO2006124630A2 WO 2006124630 A2 WO2006124630 A2 WO 2006124630A2 US 2006018488 W US2006018488 W US 2006018488W WO 2006124630 A2 WO2006124630 A2 WO 2006124630A2
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influenza
lgg
vaccine
composition
study
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PCT/US2006/018488
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WO2006124630A3 (fr
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Patricia Hibberd
Sherwood Gorbach
David Snydman
Barry Goldin
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New England Medical Center Hospitals, Inc.
Tufts University
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Publication of WO2006124630A2 publication Critical patent/WO2006124630A2/fr
Publication of WO2006124630A3 publication Critical patent/WO2006124630A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Influenza remains a major cause of morbidity and mortality.
  • 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.
  • 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.
  • the probiotic e.g., Lactobacillus rhamnosus (LGG)
  • LGG lactobacillus rhamnosus
  • 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.
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • 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)).
  • LGG Lactobacillus rhamnosus GG
  • 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).
  • the formulations can also include fragments of microorganisms, including any strain of Lactobacillus.
  • 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)).
  • 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.
  • the compositions can contain the L. rhamnosus strain LGG deposited in the American Type Culture Collection (ATCC; Manassas, VA) under code ATCC 53103.
  • ATCC American Type Culture Collection
  • VA Manassas, VA
  • the compositions can contain one or more of the L.
  • 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.
  • the lactobacillus-based composition can contain a non-lactobacillus probiotic, such as Bifidobacterium lactis (B. lactis).
  • 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.
  • 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).
  • buffer e.g., a citrate buffer, phosphate buffer, acetate buffer, or bicarbonate buffer
  • 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.
  • 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.
  • a 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
  • a probiotic 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
  • composition ⁇ e.g., the probiotic and the influenza vaccine
  • the composition can be administered at essentially the same time by the same route of administration.
  • the composition ⁇ e.g., the probiotic and the influenza vaccine
  • 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).
  • a pathogen e.g., a flu-causing virus.
  • Other subjects amenable to treatment 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.
  • 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.
  • a viral infection ⁇ e.g., an infection with a human immunodeficiency virus, a hepatitis
  • a composition e.g., a probiotic- enhanced vaccine
  • 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.
  • compositions can also contain one or more of: a preservative, stabilizer, colorant, and/or flavoring.
  • 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.
  • 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.
  • exemplary dosage amounts of a probiotic such as LGG in humans are less than about 10 x 10 10 organisms per day (e.g., about 8 x 10 10 organisms per day, about 6 x 10 10 organisms per day, about 4 x 10 10 organisms per day, about 2 x 10 organisms per day, about 1 x 10 10 organisms per day, or about 0.5 x 10 10 organisms per day, delivered in a single bolus, in divided doses, or by infusion lasting from minutes to hours).
  • 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.
  • lactobacilli such as Lactobacillus rhamnosus (e.g., LGG)
  • a killed Lactobacillus e.g., a killed Lactobacillus
  • fraction e.g., a membrane fraction of a formerly viable or non- viable Lactobacillus
  • kits can also contain informational material, such as instructions for use (e.g., instructions for administering the composition to a subject).
  • the kits can include an applicator device, such as a cotton swab, spray pump, or nebulizer.
  • 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.
  • 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.
  • 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))'.
  • animal data support that pre-administration of probiotics induces immunity to the influenza virus.
  • 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).
  • 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).
  • 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.
  • 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.
  • current vaccine strains e.g. A/New Caledonia/20/99 (HlNl) that has been in the influenza vaccine for the last 5 years.
  • 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.
  • 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. . •
  • PBMC peripheral blood mononuclear cells
  • 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).
  • 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
  • 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).
  • the CDC also recommends that health-care workers and household contacts who have frequent contact with persons at high risk should also be vaccinated.
  • 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).
  • 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).
  • Inactivated influenza vaccine has long been the primary means of influenza prevention.
  • 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).
  • 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).
  • live attenuated influenza vaccine LAIV was approved for use in healthy individuals ages 5-49.
  • LAIV 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).
  • 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).
  • 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
  • Immunity to influenza infection is induced by antibody responses to viral surface antigens hemagglutinin (HA) and neuraminidase (NA).
  • HA hemagglutinin
  • NA neuraminidase
  • 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).
  • LAIV induces peak of serum IgA and IgM two weeks after immunization and a peak IgG response 4-12 weeks after immunization (40).
  • 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).
  • HAI serum hemagglutination inhibition
  • 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).
  • 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.
  • the cytokine interferon gamma appears to be important for memory T cell responses to influenza in mice and humans (49; 50).
  • 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).
  • 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-stimulato ⁇ y motifs as a vaccine adjuvant to ETV (53).
  • 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.
  • MF59 adjuvanted influenza vaccine resulted in greater . immunogenicity than non adjuvanted vaccine especially in those who had not been previously immunized (60).
  • 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).
  • 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).
  • 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.
  • Lactobacillus rhamnosus GG 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.
  • 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.
  • 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).
  • secretory IgA is 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Th-I type cytokines TNF ⁇ , IL-2, IL- l ⁇ , 11-6 and 11-18 in " periprTerarblbod mononuclear cells (83; " 85; 8 ' 6; 88).
  • Lactobacillus has also been found to activate transcription factor NF- ⁇ B and Toll-like receptors (TLR 2 and 9)(84; 89; 90).
  • TLR 2 and 9 transcription factor NF- ⁇ B and Toll-like receptors
  • Influenza infections remain a major public health threat every year arid when " pandemics occur, the impact is .Teven m ⁇ re ⁇ suhstant ⁇ aL " I7 " . .. _ " Z MIH ⁇ Z..IZ ⁇ .I1 " "_.._...
  • LGG • .Concomitant administration of LGG with oral live vaccines (polio, rotavirus and S typhi) in humans -has resulted in enhanced humoral immunity against vaccine strains.
  • 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.
  • 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. 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).
  • ELISA enzyme linked immunosorbent assays
  • 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 10 10 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the consent process and follow-ups will include a translator.
  • 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
  • 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 r subj.e.ctS r W.ill be asked to avoid eating yogurt for the first 4 weeks of the study.
  • LAIV will be administered as the live, trivalent nasally administered influenza vaccine (FluMist TM, 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.
  • 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 i ⁇ ilipal ilnv ⁇ stt ⁇ B wr ⁇ gr ⁇ iv ⁇ iiip® (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.
  • 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.
  • LAIV will be administered according to manufacturer's directions and the placebo vvill appear to be administered in exactly the same way.
  • ⁇ V 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 FluvirinTM (Chiron).
  • FluZone ® Aventis Pasteur
  • FluvirinTM Chiron
  • the precise composition of the vaccine will depend on the US Public Health Service recommendations in the year-it js ⁇ administered.
  • the vaccine contains the following strains: A/New. Caled ⁇ nia/2O/9S> " TH1N1); A/Wycm ⁇ ng/3/2003 (H3M2)-CAVFu]an/4 ⁇ : ⁇ y2W2 ⁇ 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.
  • a matching intramuscular, placebo yaccine__ will be administered.
  • the matching placebo vaccine containing 0.5 mUoTnorr ⁇ al 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 rando ⁇ ation 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.
  • All subjects will receive 4 weeks of LGG or matching placebo capsules, the contents of which will be ingested as capsules o ⁇ 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.TM CulturelleTM 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 10 10 LGG organisms. Two capsules will be administered daily for a total of 2 x IO 10 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).
  • 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
  • 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.
  • nasal anti HA IgA titers 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 -8O 0 C 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 2 ⁇ g/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.
  • PBS phosphate buffered saline
  • 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.
  • 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 37 0 C in 5%CO2.
  • the supematants will be removed and frozen at -7O 0 C and replaced with complete RPMI containing 0.5 ⁇ Ci of 3 H-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_medju ⁇ Lalone. Supematants from the proliferation studies will be frozen at -8O 0 C. 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.
  • 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
  • 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 PH ⁇ p iV e i!l ⁇ ®fe r " "1D te ⁇ r «
  • 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.
  • 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 " a ⁇ " d ⁇ checking symptoms that-occur-.- •
  • 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.
  • a final extra capsule will be cultured to assess viability of LGG in capsules " at completion of the study.”
  • they will -be contacted every 2-weeks -to-deteimine-wheiher an.influenza like illness (ILI - see section D.4.e) has occurred.
  • ILI - see section D.4.e an.influenza like illness
  • 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.
  • 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.
  • DCRR Clinical Research Resources
  • Tufts-NEMC Tufts-NEMC
  • Dr. Hibberd 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.
  • Sample Size per Group is logistically feasible based on the Dr Hibberd' s experience and the GCRC track record in recruiting healthy volunteers.
  • 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.
  • 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.
  • HAI geometric mean serum hemagglutination inhibition
  • Consistent application of the study protocol and performance of study procedures are critical to acquiring high quality data.
  • 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 vibrant 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.
  • 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.
  • 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. ⁇ " • . ' ; ⁇ — .—
  • 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.
  • 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.
  • LAIV Another risk associated with the administration of LAIV is the shedding, and potentially the transmission of, live influenza viruses to others following close contact.
  • the estimated probability of acquiring vaccine virus after close contact with a single LAIV recipient was 0.58 - 2.4%.
  • the rate of adverse events occurring among healthy adults aged 18-49 years is very low ( ⁇ 1%) (23).
  • ⁇ V 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.
  • 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 "" i ⁇ " ⁇ 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.
  • 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.
  • 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. . . .
  • 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 ⁇ thnic group to participate in the study.
  • 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.
  • the principal investigator is responsible for assuring safety of participants in this study and the integrity of the study data. Spe ⁇ ific 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.
  • 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.
  • serious adverse events death, life threatening, new, serious or permanent disability
  • 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, firs ⁇ Tmiddle): . 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:
  • 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.
  • 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:
  • 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-contai ⁇ ing products were safely consumed by a minimum of 40,000 persons in Finland alone in 1992 (100).
  • DSMB Data and Safety Monitoring Board
  • McElhaney JE Beattie BL, Devine R 1 Grynoch R, Toth EL, Bleackley RC. Age-related decline in interleukin 2 production in response to influenza vaccine. J Am Geriatr Soc 1990;38:652-658.
  • LGG multicenter study group A multicenter, placebo-controlled double-blind study of Lactobacillus GG in addition to standard maintenance therapy in children with Crohn's disease . NASPGHAN 2002.

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Abstract

La présente invention se rapporte à des compositions qui augmentent l'efficacité de vaccins, tels que les vaccins contre la grippe. L'invention concerne également des kits qui contiennent lesdites compositions, et des procédés d'utilisation des compositions et des kits pour traiter un sujet.
PCT/US2006/018488 2005-05-13 2006-05-12 Compositions et procedes permettant d'augmenter l'efficacite de vaccins WO2006124630A2 (fr)

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US20110070334A1 (en) * 2009-09-20 2011-03-24 Nagendra Rangavajla Probiotic Stabilization
WO2011045471A1 (fr) * 2009-10-13 2011-04-21 Valio Ltd Compositions et procédés et utilisations s'y rapportant
WO2012062780A1 (fr) * 2010-11-11 2012-05-18 Nestec S.A. Micro-organismes probiotiques ne se répliquant pas et protégeant des infections des voies respiratoires supérieures
US20130195917A1 (en) * 2010-10-15 2013-08-01 Dorte Eskesen Immunoadjuvant
CN103421715A (zh) * 2013-08-03 2013-12-04 青岛蔚蓝生物集团有限公司 一种鼠李糖乳杆菌及其应用
US20140093614A1 (en) * 2009-09-20 2014-04-03 Mead Johnson Nutrition Company Probiotic stabilization
WO2020097226A1 (fr) * 2018-11-06 2020-05-14 Kansas State University Research Foundation Compositions pour améliorer l'innocuité et l'efficacité d'un vaccin et leurs méthodes d'utilisation
CN111529703A (zh) * 2020-06-17 2020-08-14 湖南唯乐可健康产业有限公司 一种组合物及其制备方法与在制备免疫佐剂中的应用
US10980269B2 (en) * 2016-12-12 2021-04-20 Mead Johnson Nutrition Company Protein hydrolysates and methods of making same
WO2022103837A1 (fr) * 2020-11-10 2022-05-19 Elanco Us Inc. Compositions probiotiques immunogènes et procédés d'utilisation, y compris à des fins de vaccination

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JP2010518151A (ja) * 2007-02-16 2010-05-27 カンパーニ ジェルヴェ ダノン インフルエンザワクチンによりもたらされる防御を増大させるためのラクトバシラス・カゼイの使用
US8349315B2 (en) 2007-02-16 2013-01-08 Compagnie Gervais Danone Use of Lactobacillus casei for increasing the protection provided by the influenza vaccine
RU2470994C2 (ru) * 2007-02-16 2012-12-27 Компани Жервэ Данон Применение lactobacillus casei для усиления защиты, индуцированной вакцинацией против гриппа
WO2008129148A3 (fr) * 2007-02-16 2009-02-12 Gervais Danone Sa Utilisation de lactobacillus casei pour renforcer la protection induite par la vaccination anti-grippale
FR2912657A1 (fr) * 2007-02-16 2008-08-22 Gervais Danone Sa Utilisation de lactobacillus casei pour renforcer la protection induite par la vaccination anti-grippale.
US9408819B2 (en) * 2007-02-28 2016-08-09 Mead Johnson Nutrition Company Method for reducing or preventing systemic inflammation
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US9408818B2 (en) 2007-02-28 2016-08-09 Mead Johnson Nutrition Company Method for the utilization of and product containing inactivated probiotic
CN105796607A (zh) * 2007-02-28 2016-07-27 Mjn 美国控股有限责任公司 用于儿童或婴幼儿的含有灭活益生菌的产品
WO2008106373A1 (fr) * 2007-02-28 2008-09-04 Mead Johnson Nutrition Company Produit contenant un probiotique inactivé pour des enfants ou des nourrissons
US20080206213A1 (en) * 2007-02-28 2008-08-28 Bristol-Myers Squibb Company Method for reducing or preventing systemic inflammation
TWI494114B (zh) * 2007-02-28 2015-08-01 Mjn Us Holdings Llc 鼠李醣乳酸桿菌gg(lgg)於治療、預防或減輕全身性發炎之用途
US20110070334A1 (en) * 2009-09-20 2011-03-24 Nagendra Rangavajla Probiotic Stabilization
US20140093614A1 (en) * 2009-09-20 2014-04-03 Mead Johnson Nutrition Company Probiotic stabilization
WO2011045471A1 (fr) * 2009-10-13 2011-04-21 Valio Ltd Compositions et procédés et utilisations s'y rapportant
JP2013507431A (ja) * 2009-10-13 2013-03-04 ヴァリオ・リミテッド 組成物および方法、およびそれらに関する使用
CN102665740A (zh) * 2009-10-13 2012-09-12 维利奥有限公司 组合物和方法以及与其相关的用途
US20130195917A1 (en) * 2010-10-15 2013-08-01 Dorte Eskesen Immunoadjuvant
KR101806435B1 (ko) * 2010-10-15 2017-12-07 시에이치알. 한센 에이/에스 면역보강제
WO2012062780A1 (fr) * 2010-11-11 2012-05-18 Nestec S.A. Micro-organismes probiotiques ne se répliquant pas et protégeant des infections des voies respiratoires supérieures
CN103547276A (zh) * 2010-11-11 2014-01-29 雀巢产品技术援助有限公司 保护免受上呼吸道感染的非复制性益生菌微生物
EP2455092A1 (fr) * 2010-11-11 2012-05-23 Nestec S.A. Protection de micro-organismes probiotiques sans réplication contre les infections des voies respiratoires supérieures
CN103421715B (zh) * 2013-08-03 2015-06-10 青岛蔚蓝生物集团有限公司 一种鼠李糖乳杆菌及其应用
CN103421715A (zh) * 2013-08-03 2013-12-04 青岛蔚蓝生物集团有限公司 一种鼠李糖乳杆菌及其应用
US10980269B2 (en) * 2016-12-12 2021-04-20 Mead Johnson Nutrition Company Protein hydrolysates and methods of making same
US11785976B2 (en) 2016-12-12 2023-10-17 Mead Johnson Nutrition Company Protein hydrolysates and methods of making same
WO2020097226A1 (fr) * 2018-11-06 2020-05-14 Kansas State University Research Foundation Compositions pour améliorer l'innocuité et l'efficacité d'un vaccin et leurs méthodes d'utilisation
CN111529703A (zh) * 2020-06-17 2020-08-14 湖南唯乐可健康产业有限公司 一种组合物及其制备方法与在制备免疫佐剂中的应用
CN111529703B (zh) * 2020-06-17 2023-09-15 湖南循天然营养有限公司 一种组合物及其制备方法与在制备免疫佐剂中的应用
WO2022103837A1 (fr) * 2020-11-10 2022-05-19 Elanco Us Inc. Compositions probiotiques immunogènes et procédés d'utilisation, y compris à des fins de vaccination

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