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Title:  Methods of immunizing adults using anti-meningococcal vaccine compositions

United States Patent:  6,413,520

Inventors:  Granoff; Dan (Berkeley, CA)

Assignee:  Chiron Corporation (Emeryville, CA)

Appl. No.:  446347

Filed:  December 16, 1999

PCT Filed:  June 24, 1998

PCT NO:  PCT/US98/13080

371 Date:  December 16, 1999

102(e) Date:  December 16, 1999

PCT PUB.NO.:  WO98/58670

PCT PUB. Date:  December 30, 1998

Abstract

A method for boosting an immune response against meningococcal capsular antigen is disclosed. The method entails administering a first glycoconjugate vaccine composition to a subject to provide an initial state of anti-meningococcal immunity, and then boosting the anti-meningococcal immunity by administration of a second, boosting vaccination. Also disclosed is the use of vaccine compositions in the preparation of anti-meningococcal medicaments. The use entails administering a first glycoconjugate vaccine composition to a subject to provide an initial state of anti-meningococcal immunity, and then boosting the anti-meningococcal immunity by administration of a second, boosting vaccination.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

I. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

An "antigen" is defined herein to include any substance that may be bound by an antibody molecule. An "immunogen" is an antigen that is capable of initiating lymphocyte activation resulting in an antigen-specific immune response. Such activation generally results in the development of a secretory, cellular and/or antibody-mediated immune response against the immunogen. Usually, such a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as IgA, IgD, IgE, IgG or IgM; the proliferation of B and T lymphocytes; the provision of activation, growth and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell and/or .gamma..delta. T cell populations. Immunogens therefore include any molecule which contains one or more antigenic determinants (e.g., epitopes) that will stimulate a host's immune system to initiate such an antigen-specific response.

By "epitope" is meant a site on an antigen to which specific B cells and T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." A peptide epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids. Methods of determining spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Furthermore, the identification of epitopes in a given protein is readily accomplished using techniques well known in the art. See, e.g., Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998 (general method of rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); U.S. Pat. No. 4,708,871 (procedures for identifying and chemically synthesizing epitopes of antigens); and Geysen et al. (1986) Molecular Immunology 23:709-715 (technique for identifying peptides with high affinity for a given antibody). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

As used herein, "treatment" refers to any of (i) prevention of infection or reinfection, as in a traditional vaccine, (ii) reduction or elimination of symptoms, and (iii) reduction or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).

By "mammalian subject" is meant any member of the class Mammalia, including, without limitation, humans and other primates, including such non-human primates as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; and laboratory animals including rodents such as mice, rats and guinea pigs. The term does not denote a particular age or sex. Thus, both adult and newborn individuals, as well as fetuses, either male or female, are intended to be covered.

II. Modes of Carrying Out the Invention

The present invention is premised, in part, on the unexpected discovery that use of an anti-meningococcal conjugate vaccine composition in adults (instead of an unconjugated anti-meningococcal polysaccharide vaccine) induces polysaccharide-responsive memory B cells and long-term immunologic memory in vaccinated subjects, both of which factors contribute to more robust and durable protection against meningococcal disease. In fact, it has surprisingly been found that the anti-meningococcal immune response in adults vaccinated with a conjugate vaccine formulation is readily boostable upon re-vaccination with a second anti-meningococcal vaccine composition.

In contrast, it has also been found herein that vaccination with an unconjugated tetravalent meningococcal A, C, Y, W135 polysaccharide vaccine (MENOMUNE.TM., Connaught Laboratories, Inc., Swiftwater, Pa.) induces immunologic paralysis of toddlers and adults to meningococcal polysaccharides. More particularly, meningococcal C vaccination with a polysaccharide vaccine (unconjugated) in subjects during the first six months of age results in depression of serum antibody responses to a booster vaccination with meningococcal C polysaccharide given 6 months later (when compared to the responses of infants of similar age vaccinated for the first time). In the study described hereinbelow, infants were given two doses of a meningococcal A and C polysaccharide vaccine at 3 and 6 months of age and boosted with a third injection at 18 to 24 months of age. As shown in FIG. 1, the geometric mean antibody response to the booster dose was nearly 10-fold lower than that of control children of the same age vaccinated for the first time. This new information on induction of immunologic tolerance in these vaccinated subjects indicates that such tolerance is not limited to infants less than 6 months of age, but also occurs in toddlers vaccinated at 15 to 23 months, and in adults. In fact, antibody refractoriness in adult subjects was observed 4 years after a polysaccharide vaccination.

The induction of immunologic tolerance to meningococcal species in previously vaccinated subjects is of significant clinical importance. In this regard, data from experimental animals indicate that mice tolerized to pneumococcal polysaccharide show increased lethality from experimental challenge with pneumococci possessing the homologous serotype (reviewed in Halliday, W. (1971) Bacteriol. Rev. 35:267-289). This increased susceptibility may be a result of an impaired ability to mount serum anticapsular antibody responses upon exposure to the encapsulated bacteria. In humans, the contemporary knowledge accepts that unconjugated meningococcal polysaccharide vaccine are protective in adults and, possibly, in older children. However, this knowledge is generally based upon efficacy data in adults that were obtained from studies performed in military recruits in which follow-up was very short (8 weeks). As has been discovered herein, there may be a late-onset increased risk of disease in vaccinated subjects as a result of immune refractoriness to meningococcal polysaccharides, once increased serum antibody concentrations induced by vaccination have declined to sub-protective levels (i.e., after about 3 years). The impaired meningococcal C serum bactericidal antibody responses of toddlers and adults previously vaccinated with the tetravalent polysaccharide vaccine is consistent with this possibility.

Taken together, the data presented herein raise a safety concern for the use of unconjugated anti-meningococcal vaccines, as this product is recommended in the United States for use in children two years of age or older, but the vaccine also can be used in infants and younger children to control outbreaks of disease (Jafari, supra and Perkins, supra).

Accordingly, it is a primary object of the invention to provide a method for boosting in an adult subject an anti-meningococcal immune response against a meningococcal capsular polysaccharide antigen. The method generally entails a primary vaccination using a anti-meningococcal glycoconjugate vaccine composition which comprises meningococcal capsular polysaccharide antigen derived from one or more meningococcal species (i.e., a monovalent or polyvalent vaccine composition) conjugated to an appropriate carrier molecule. The primary vaccination is sufficient to elicit an anti-meningococcal immune response in the vaccinated subject which is specific for one or more meningococcal species. After the immune response elicited by the primary vaccination has declined to subprotective levels, a boosting vaccination is performed in order to provide a boosted anti-meningococcal immune response.

The anti-meningococcal glycoconjugates used for the primary vaccination are prepared using carrier molecules that will not themselves induce the production of harmful antibodies. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Preferably, capsular meningococcal polysaccharide or oligosaccharide molecules containing at least one immunologically relevant epitope are conjugated to a bacterial toxoid carrier molecule, such as, but not limited to, a toxoid from diphtheria, tetanus, cholera, etc. In particular embodiments, capsular polysaccharide molecules are coupled to the CRM197 protein carrier. The CRM197 carrier is a well-characterized non-toxic diphtheria toxin mutant that is useful in glycoconjugate vaccine preparations intended for human use. (Bixler et al. (1989) Adv. Exp. Med. Biol. 251:175, and Constantino et al. (1992) Vaccine). In other embodiments, glycoconjugates are formed with protein carriers known to have potent T-cell epitopes. Exemplary carriers include, but are not limited to, Fragment C of tetanus toxin (TT), and the Class 1 or Class 2/3 OMPs of N. meningitidis. Such carriers are well known to those of ordinary skill in the art.

In particular embodiments of the invention, the primary vaccination entails administration of a meningococcal A and C oligosaccharide-based glycoconjugate vaccine composition as described by Anderson et al. (supra). In other related embodiments, the primary vaccination is given using a meningococcal B oligosaccharide-based glycoconjugate as described in International Publication No. WO 98/086543, which publication is incorporated herein by reference in its entirety. Other vaccine compositions that can be used herein for the primary vaccination include, for example, glycoconjugates based on meningococcal B polysaccharide derivatives (e.g., those described in EP Publication No. 504,202 B and U.S. Pat. No. 4,727,1361 both of which are incorporated herein by reference), and monovalent meningococcal C or trivalent meningococcal A, B and C oligosaccharide-based glycoconjugates.

The secondary (boosting) vaccination can be carried out using any suitable anti-meningococcal vaccine composition; however, the second vaccine composition is preferably also a meningococcal capsular polysaccharide- or oligosaccharide-based conjugate in order to avoid the possibility of immunologic tolerance associated with unconjugated anti-meningococcal vaccine compositions.

As will be known by those skilled in the art upon reading the instant specification, several factors will have an impact on the physical and immunological properties of the above-described glycoconjugates. Specifically, the ratio of oligosaccharide (and/or polysaccharide)-to-protein (hapten loading density), linkage chemistry, and the choice of carrier moiety are all factors that should be considered and optimized in the preparation of the glycoconjugates used in the methods herein. For example, a low saccharide loading density may result in poor anti-saccharide antibody response. On the other hand, a heavy loading of saccharides could potentially mask important T-cell epitopes of the protein molecule, thus abrogating the carrier effect and attenuating the total anti-saccharide immune response.

Accordingly, during glycoconjugate production, aliquots can be withdrawn and analyzed by SEC-HPLC in order to monitor the extent of the conjugation process. The use of a disaggregating buffer, for example EDTA, SDS, deoxycholate, or the like, can be employed to separate components possibly adhering to the preparations by non-covalent interactions. To ensure glycosylation of the carrier, the shift in retention time of the particular protein carrier toward the exclusion volume (V0) of the column can be monitored. In addition, a gradual reduction of the saccharide peak area in a HPLC chromatogram can be used to indicate incorporation of the saccharide onto the carrier.

Post-production characterization of the glycoconjugates can include molecular weight determination using, for example, gel filtration columns. Further characterization may also include electrophoretic mobility on SDS-PAGE separation equipment and analysis of chemical composition of the glycoconjugates with respect to carbohydrate and amino acid components. The identity of product purity, and the absence of residual contaminants (such as nucleic acids, LPS, and free saccharides and/or carrier) can also be verified using known techniques. Confirmation of stable covalent attachment can be accomplished using a combination of analytical techniques, including gel filtration in detergent-containing buffer, SDS-PAGE followed by Western Blot analysis and amino acid analysis. See, e.g., Vella et al. (1992) Vaccines: New Approaches to Immunological Problems, (Ellis, R. W. ed), Butterworth-Heinemann, Boston, pp 1-22, Seid et al. (1989) Glycoconjugate J. 6:489.

The anti-meningococcal vaccine compositions used in the primary and subsequent (boosting) vaccinations can further be administered in conjunction with other antigens and immunoregulatory agents, for example, immunoglobulins, cytokines, lymphokines, and chemokines, including but not limited to IL-2, modified IL-2 (cys125.fwdarw.ser125), GM-CSF, IL-12, .gamma.-interferon, IP-10, MIP1.beta. and RANTES.

The vaccine compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Adjuvants may also be used to enhance the effectiveness of the vaccines. Adjuvants can be added directly to the vaccine compositions or can be administered separately, either concurrently with or shortly after, administration of the vaccines. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX.TM.); (3) saponin adjuvants, such as STIMULON.TM. (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. W093/13202 and W092/19265); and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn -glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

Typically, the vaccine compositions are prepared as injectables, either as liquid solutions or suspensions; or as solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect.

The vaccine compositions will comprise a therapeutically effective amount of one or more meningococcal capsular oligosaccharide or polysaccharide immunogens, and any other of the above-mentioned components, as needed. By "therapeutically effective amount" is meant an amount of a molecule which will induce an immunological response in the individual to which it is administered without stimulating an autoimmune response. Such a response will generally result in the development in the subject of a secretory, cellular and/or antibody-mediated immune response to the vaccine. Usually, such a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; the proliferation of B and T lymphocytes; the provision of activation, growth and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell and/or .gamma..delta. T cell populations.

Preferably, the effective amount is sufficient to bring about treatment, i.e., reduction or complete elimination of symptoms, or prevention of disease symptoms. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the capacity of the subject's immune system to synthesize antibodies; the degree of protection desired; the severity of the condition being treated; the particular molecule selected and its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. A "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials. More particularly, the meningococcal capsular oligosaccharide or polysaccharide immunogens will be administered in a therapeutically effective amount that comprises from about 0.1 .mu.g to about 100 mg, more preferably from about 0.5 .mu.g to about 1 mg, and most preferably about 1 .mu.g to about 500 .mu.g of the oligosaccharide or polysaccharide immunogen delivered per dose.

Once formulated, the vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Alternative formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Claim 1 of 8 Claims

What is claimed is:

1. A method for boosting in an adult subject an immune response against meningococcal C capsular antigen, said method comprising:

(a) administering a first vaccine composition to said adult subject in order to elicit an immune response against a meningococcal species, wherein said first vaccine composition is a meningococcal glycoconjugate vaccine composition that comprises meningococcal oligosaccharides from serogroups A and C, wherein the oligosaccharides are conjugated to a carrier molecule, and further wherein the first composition is administered in an amount sufficient to elicit an anti-meningococcal immune response; and

(b) administering a second vaccine composition to said adult subject in order to boost the anti-meningococcal response, wherein said second vaccine composition comprises capsular polysaccharides from serogroups A, C, Y and W135, and is administered to the subject about three to four years after the first vaccine composition is administered.
 


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