Vaccination against Cryptococcus

Abstract

Vaccines and methods of inoculation for conferring immunity to Cryptococcus infection are disclosed. Strains of Cryptococcus fungi, including Cryptococcus neoformans and Cryptococcus gattii, can be administered to a human or animal subject via inhalation. Cryptococcus fungi that can be used to confer immunity can comprise one or more mutations in genes that contribute to chitosan production, such as genes encoding a chitin deacetylase (cda), a chitin synthase (chs) and/or a regulator of chitin synthase (csr). Inhalation administration of heat-killed Cryptococcus harboring deletions in cda1, cda2 and cda3 genes can confer immunity. In a murine model system, inhalation administration of Cryptococcus neoformans harboring deletions in cda1, cda2 and cda3 genes conferred immunity against subsequent exposure to wild type Cryptococcus neoformans in 100% of test animals. Inhalation administration of heat-killed Cryptococcus grown under conditions leading to reduced chitosan production can also confer immunity.

Claims

1. A method of inducing immunity against a Cryptococcus fungus infection wherein the Cryptococcus is selected from the group consisting of Cryptocoecus neoformans and Cryptococcus gattii, comprising administering to a subject by inhalation an immunity-inducing amount of a composition comprising a Cryptococcus fungus deficient for chitosan, wherein the Cryptococcus fungus deficient for chitosan is selected from the group consisting of a Cryptococcus neoformans fungus deficient for chitosan and a Cryptococcus gattii fungus deficient for chitosan, and wherein the Cryptococcus fungus deficient for chitosan comprises no more than 60% chitosan level compared to wild type grown on yeast extract peptone dextrose (YPD) and wherein the Cryptococcus fungus deficient for chitosan is a heat-inactivated cdaIcda2cda3 Cryptococcus fungus deficient for chitosan.

2. A method of inducting immunity against a Cryptococcus fungus infection in accordance with claim 1, wherein the Cryptococcus fungus deficient for chitosan comprises no more than 50% chitosan level compared to wild type grown on yeast extract peptone dextrose (YPD).

3. A method of inducing immunity against a Cryptococcus fungus infection in accordance with claim 1, wherein the Cryptococcus fungus deficient for chitosan is a Cryptococcus neoformans fungus deficient for chitosan.

4. A method of inducing immunity against a Cryptococcus fungus infection in accordance with claim 1, wherein the Cryptococcus fungus deficient for chitosan is a Cryptococcus gattii fungus deficient for chitosan.

5. A method of inducing immunity against a Cryptococcus fungus infection in accordance with claim 1, wherein the administering by inhalation comprises nasal inhalation.

6. A method of inducing immunity against a Cryptococcus fungus infection in accordance with claim 5, wherein the nasal inhalation is selected from the group consisting of inhaling a nose drop formulation and inhaling a nasal spray formulation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-B illustrate that mice exposed to Cryptococcus neoformans cda1cda2cda3 survive wild type Cryptococcus neoformans infection.

(2) FIG. 2A-B illustrates that mice exposed to heat-killed Cryptococcus neoformans cda1cda2cda3 survive wild type Cryptococcus neoformans infection.

(3) FIG. 3 illustrates an experimental protocol for testing Cryptococcus neoformans cda1 as a vaccine.

(4) FIG. 4 illustrates a survival curve of a cda1 pilot experiment described in FIG. 3.

(5) FIG. 5 illustrates a survival curve of vaccinated mice immunized with C. neoformans cda1 challenged with R265 (C. gattii).

(6) FIG. 6 illustrates a survival curve of vaccinated mice immunized with C. neoformans cda1 challenged with WM226 (C. gattii).

(7) FIG. 7 illustrates that 129 mice immunized with Cryptococcus neoformans cda1cda2cda3 survive when challenged with C. neoformans.

(8) FIG. 8 illustrates that A/J mice immunized with Cryptococcus neoformans cda1cda2cda3 survive when challenged with C. neoformans.

(9) FIG. 9 illustrates that 129 mice immunized with Cryptococcus neoformans cda1cda2cda3 survive when challenged with C. neoformans.

(10) FIG. 10 illustrates fungal burden for surviving mice of different strains after the conclusion of survival experiments.

(11) FIG. 11 illustrates that CBA/J mice immunized with heat-killed wild type cells grown in YNB pH 7.0 medium buffered to pH 7.0 survive when challenged with C. neoformans.

(12) FIG. 12 illustrates chitosan levels present in mouse lungs when inoculated with wild type or cda1 C. neoformans.

(13) FIG. 13 illustrates chitosan levels present in wild type C. neoformans cells when grown on YPD medium or YNB medium buffered to pH 7.0.

DETAILED DESCRIPTION

(14) The present inventors have developed vaccines and administration protocols against infection by Cryptococcus fungi, including Cryptococcus neoformans and Cryptococcus gattii. In various embodiments, a vaccine of the present teachings can provide significant protection against exposure to a virulent Cryptococcus strain, such as a wild type Cryptococcus neoformans or Cryptococcus gattii, up to 100% protection.

(15) As used herein, an inactivated Cryptococcus refers to a Cryptococcus fungus that has been disabled or killed such that it is unable to reproduce upon infection of a host organism, or grow in a standard nutrient medium. Inactivation of a Cryptococcus, including Cryptococcus deficient for chitosan production, can be accomplished by any method known to skilled artisans. In various configurations, an inactivated Cryptococcus fungus of the present teachings can comprise heat-killed or heat-attenuated Cryptococcus, such as but not limited to heat-killed C. neoformans cda1, C. neoformans cda2C. neoformans cda3, C. neoformans chs3, C. neoformans csr2 or any combination thereof, such as, without limitation C. neoformans cda1cda2cda3. In various configurations, an inactivated Cryptococcus fungus of the present teachings can comprise heat-killed or heat-attenuated Cryptococcus, such as but not limited to heat-killed C. gattii cda1, C. gattii cda2 C. gattii cda3, C. gattii chs3, C. gattii csr2 or any combination thereof, such as, without limitation C. gattii cda1cda2cda3. In some configurations, an inactivated Cryptococcus strain deficient for chitosan production can comprise Cryptococcus that is deficient for chitosan production and has been killed by exposure to heat, to electromagnetic radiation such as ultraviolet light, gamma ray radiation, or x-ray radiation, by exposure to nuclear radiation such as exposure to an alpha particle emitting source or a beta particle emitting source, by exposure to toxic levels of one or more chemicals, by photodynamic inactivation (Rodrigues, G. B., et al., Photochemistry and Photobiology 88:440-447, 2012; Fuchs, B. B., et al., Antimicrobial Agents and Chemotherapy 51: 2929-2936, 2007), or any combination thereof. In some configurations, effectiveness of an inactivating treatment can be tested by plating treated samples on nutrient plates under standard conditions; a treatment can be considered inactivating if no colony forming units develop.

(16) Methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), Cold Spring Harbor, N.Y., 2003 and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Methods of administration of pharmaceuticals and dosage regimes, can be determined according to standard principles of pharmacology well known skilled artisans, using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C., et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003. As used in the present description and any appended claims, the singular forms a, an and the are intended to include the plural forms as well, unless the context indicates otherwise.

(17) In various embodiments, a Cryptococcus fungus, including without limitation Cryptococcus neoformans or Cryptococcus gattii comprising mutations in at least one, at least two, or at least three chitin deacetylase (cda)genes and/or chitin synthase genes and/or chitin synthase regulator genes can be obtained and grown by established procedures (See, e.g., Baker, L. G., et al., Eukaryotic Cell 6: 855-867, 2007 and Baker, L. G., et al., Eukaryotic Cell 10: 1264-1268, 2011). A mutation can interfere with, inactivate, or eliminate a gene or its encoded protein product. In various embodiments, a Cryptococcus fungus, including without limitation either Cryptococcus neoformans or Cryptococcus gattii of the present teachings deficient for chitosan can be obtained and grown by established procedures.

(18) In various embodiments, Cryptococcus neoformans strains of the present teachings can comprise the following DNA sequences.

(19) C. neoformans CDA1 (CNAG_05799):

(20) Wild type gene: Bolded regions are predicted introns. The sequences between the double-underlined sequences are deleted in the deletion strain. GGCCCTCTACTCTACCTTCG (SEQ ID No:1) and GCCGCCTCTAACACTTCTTCC (SEQ ID No:2) are the primer sequences used for deletion. The residues in bold and italicized typeface are the sites used for creating active site mutants. In some configurations, selectable markers (not indicated) can be inserted in place of the deleted portions.

(21) TABLE-US-00001 (SEQIDNo.:3) GCCGAACAGCGCAGGTCAGGGAGAGCATTTCTAGCGTGCCTTGGTAGATCGTTATCGCGATT TACTTTCCAGGCCCTGGGCGCTTCCAGCCATCAGCCAAAGGATAAAAGCGCGTGCCCTCTTC TTTTCATCATTAACTTTTATCCTCCTCAGCACCCACGCTCTGTGATTCCATCTCTTCCTCCT CGCATTCAAGCAGCCTCTTCATTTCTTTCCTCCGTCCCGGTGAGTGCGACGCCCGCCGCTGC CATTCCCACACGATGACTTGAGACGCGTCTTCCCGCTATAGCCGACGCCCCTTTTCGTTTTC TTGGCGTTTTGTCACATTGCCACATTGAGCAGCACAGCTTACTTGTCAGCAGCAAAAATCCA ACTTCAAACAGCTCTTCAGCATCAACTCTATCACTCTTTCATCTCTTGTCAACTTCTCTTCC TTCTCGCTCCAAAAGCGGAATTTCGCC TTTACATTCGCTGCCTTCTCTGCTCTTCTAAT TTCCCTCGCTGGTGTGGTGGCGCAGACTACAGGCACATCGGTTGACAGTAGCATCTTAACTA AGACTGCTGACTCTACCGGTCCCTCTGGTTTCTCCATTCCGTGAGTACTCTCGACTTTTCCG TCAACCTCCAGTCTCGCCACAGGCCATAGCGAACAATGAGCCAAGCGCCCACGCGAACCGTG CCCATCATTATCCTCCCACTAATTCCTTTAACCAAACGTAGCTTTTAGAGGCCAAATGCTGA CAAGTGCGTTTTTAGTGCTTTGAGCGAGCTCACGTCTGGTGCCCCCACTGACTCTACTGTGG CCCTCTACTCTACCTTCGCGGCCGGTGCCACACCTACCGTTTCTGGTGCCCCTGTCCTCCCT ACCAGTGCCCTCACCATCGCCGATTATCCAGCTTTAGATGTCACCCCTCCTACCAACTCCTC TTTGGTTAAGGACTGGATGGCCAAGGTGAGTTGTGTTTGAGTCCGAAAAGGCACCAGAAGAG CTAACAGTTGGATTAGATCGACTTGTCCAAGGTGCCCAGTTATAATGTGACAACGGGCGATT GTTCTACTGACGCGGCTGCTATCAGCGACGGTCGATGCTGGTGGACTTGTGGTGGTTGCACT CGGGAAACCGACATTGTCGAGTGTCCTGACAAGAATGTTTGGGGTCTCTCTTAC GG TCTTCGTTGTCGGCTCTCGTGTCCTTTCTCGACCCGAGATGCTCCAAACCGAATACATGTCT GGACACCAGATCTCTATCCACACTTGGTCTCACCCCGCACTTACTACTCTTACCAACGAGGA AATTGTTGCCGAGCTTGGTTGGACAATGAAGGTCATCAAGGACACCCTTGGCGTCACCCCAA ACACTTTC CCCCCTTATGGTGACATTGATGACCGTGTTCGAGCTATTGCTGCTCAGATG GGCTTGACCCCTGTTATCTGGACTTCTTACACTGATGGCTCAACCACTGTTAACTTTGACAC TGTAGGCTTATCTTGACTTTCGCAATAATCTTACTAACGAAATGACAGAAC TGGCACAT CAGTGGTGGTACCGCCACCGGCGCTTCTTCTTATGAGACCTTTGAGAAGATTCTCACCGAAT ACGCCCCAAAGTTGGACACTGGTTTCATCACTCTTGAGCACGACAGTAAGTCTTGTCTATCC GTCTTGCAATAATAATCCTGACGTATACCTTTACAGTCTACCAGCAGAGTGTTGACCTTGCT GTTGGTTACATTTTGCCCCAAGTTCTCGCCAACGGTACCTATCAGCTCAAATCCATCATCAA CTGTTTGGGCAAGGACAGTAAGTTGCCTCCGCTAATCAGAAAAGGTTGTGGGCTAAGATGAT ACACAGCCTCCGAAGCATACATTGAGACTTCATCCAACCAGACTACTACTCAGATCACTGCA GCCACCGGCTCCCAGTCTACCTTCTTCCAGCCCATTGTTGGCACTGCTACCGGTGCTGAAGT CTCTGCACCTTCTGAGGCCACTGGCAGCACTGCCGCTGGCTCTGCTGCCTCCACCACTAGTG GTTCTGGCGCCAGCGCTTCTACAGGCGCCGCCTCTAACACTTCTTCCAGCGGGTCTGGTCGA TCAGCCACCATGGGTGGTGCCCTCATTGCTCTTGCCGCTGTTGCGGTTGGTATGGTATATGT CGCC GTATTTCAAGGCTTTCAATGTAACGATGGATGGGGATGGGTGGTGGGGGGGGAGG GAAGTGTGTCTAATGGGGCTATACTTGGGCTATACTTTGCCTCAAATCCATCAAGTATTAAT AGCTGAACCATCTTTCGTTGAACCGTCTTTCATTGTGAACCATTTGTCTTTTTGATCTTTCA AAGTTTGATCCATTATGAATATCATGGACATTTTGAACGTTTTGAACATCCATGTACTTTTC ATTCGATCGATCTGAACGTGTTGTTGTGCATACCTCGCGAACAAGCTTTCAATGGATGGCTT CAC

(22) Mutant A is a triple point mutant that can be D166, R254 and D 294-catalytically inactive mutant:

(23) The sequence of the mutant gene can comprise the mutated residues:

(24) TABLE-US-00002 (SEQIDNo.:4) GCCGAACAGCGCAGGTCAGGGAGAGCATTTCTAGCGTGCCTTGGTAGATCGTTATCGCGATT TACTTTCCAGGCCCTGGGCGCTTCCAGCCATCAGCCAAAGGATAAAAGCGCGTGCCCTCTTC TTTTCATCATTAACTTTTATCCTCCTCAGCACCCACGCTCTGTGATTCCATCTCTTCCTCCT CGCATTCAAGCAGCCTCTTCATTTCTTTCCTCCGTCCCGGTGAGTGCGACGCCCGCCGCTGC CATTCCCACACGATGACTTGAGACGCGTCTTCCCGCTATAGCCGACGCCCCTTTTCGTTTTC TTGGCGTTTTGTCACATTGCCACATTGAGCAGCACAGCTTACTTGTCAGCAGCAAAAATCCA ACTTCAAACAGCTCTTCAGCATCAACTCTATCACTCTTTCATCTCTTGTCAACTTCTCTTCC TTCTCGCTCCAAAAGCGGAATTTCGCC TTTACATTCGCTGCCTTCTCTGCTCTTCTAAT TTCCCTCGCTGGTGTGGTGGCGCAGACTACAGGCACATCGGTTGACAGTAGCATCTTAACTA AGACTGCTGACTCTACCGGTCCCTCTGGTTTCTCCATTCCGTGAGTACTCTCGACTTTTCCG TCAACCTCCAGTCTCGCCACAGGCCATAGCGAACAATGAGCCAAGCGCCCACGCGAACCGTG CCCATCATTATCCTCCCACTAATTCCTTTAACCAAACGTAGCTTTTAGAGGCCAAATGCTGA CAAGTGCGTTTTTAGTGCTTTGAGCGAGCTCACGTCTGGTGCCCCCACTGACTCTACTGTGG CCCTCTACTCTACCTTCGCGGCCGGTGCCACACCTACCGTTTCTGGTGCCCCTGTCCTCCCT ACCAGTGCCCTCACCATCGCCGATTATCCAGCTTTAGATGTCACCCCTCCTACCAACTCCTC TTTGGTTAAGGACTGGATGGCCAAGGTGAGTTGTGTTTGAGTCCGAAAAGGCACCAGAAGAG CTAACAGTTGGATTAGATCGACTTGTCCAAGGTGCCCAGTTATAATGTGACAACGGGCGATT GTTCTACTGACGCGGCTGCTATCAGCGACGGTCGATGCTGGTGGACTTGTGGTGGTTGCACT CGGGAAACCGACATTGTCGAGTGTCCTGACAAGAATGTTTGGGGTCTCTCTTAC GATGG GCCTTCTCCCTTCACCCCTCTCCTAATTGATTACCTTCAGGAGAAGAACATCAAGACCACCT TCTTCGTTGTCGGCTCTCGTGTCCTTTCTCGACCCGAGATGCTCCAAACCGAATACATGTCT GGACACCAGATCTCTATCCACACTTGGTCTCACCCCGCACTTACTACTCTTACCAACGAGGA AATTGTTGCCGAGCTTGGTTGGACAATGAAGGTCATCAAGGACACCCTTGGCGTCACCCCAA ACACTTTC CCCCCTTATGGTGACATTGATGACCGTGTTCGAGCTATTGCTGCTCAGATG GGCTTGACCCCTGTTATCTGGACTTCTTACACTGATGGCTCAACCACTGTTAACTTTGACAC TGTAGGCTTATCTTGACTTTCGCAATAATCTTACTAACGAAATGACAGAAC TGGCACAT CAGTGGTGGTACCGCCACCGGCGCTTCTTCTTATGAGACCTTTGAGAAGATTCTCACCGAAT ACGCCCCAAAGTTGGACACTGGTTTCATCACTCTTGAGCACGACAGTAAGTCTTGTCTATCC GTCTTGCAATAATAATCCTGACGTATACCTTTACAGTCTACCAGCAGAGTGTTGACCTTGCT GTTGGTTACATTTTGCCCCAAGTTCTCGCCAACGGTACCTATCAGCTCAAATCCATCATCAA CTGTTTGGGCAAGGACAGTAAGTTGCCTCCGCTAATCAGAAAAGGTTGTGGGCTAAGATGAT ACACAGCCTCCGAAGCATACATTGAGACTTCATCCAACCAGACTACTACTCAGATCACTGCA GCCACCGGCTCCCAGTCTACCTTCTTCCAGCCCATTGTTGGCACTGCTACCGGTGCTGAAGT CTCTGCACCTTCTGAGGCCACTGGCAGCACTGCCGCTGGCTCTGCTGCCTCCACCACTAGTG GTTCTGGCGCCAGCGCTTCTACAGGCGCCGCCTCTAACACTTCTTCCAGCGGGTCTGGTCGA TCAGCCACCATGGGTGGTGCCCTCATTGCTCTTGCCGCTGTTGCGGTTGGTATGGTATATGT CGCC GTATTTCAAGGCTTTCAATGTAACGATGGATGGGGATGGGTGGTGGGGGGGGAGG GAAGTGTGTCTAATGGGGCTATACTTGGGCTATACTTTGCCTCAAATCCATCAAGTATTAAT AGCTGAACCATCTTTCGTTGAACCGTCTTTCATTGTGAACCATTTGTCTTTTTGATCTTTCA AAGTTTGATCCATTATGAATATCATGGACATTTTGAACGTTTTGAACATCCATGTACTTTTC ATTCGATCGATCTGAACGTGTTGTTGTGCATACCTCGCGAACAAGCTTTCAATGGATGGCTT CAC

(25) Mutant B is a mutant in which the potential Zinc binding site viz: D167, H216 and H220 are mutated.

(26) TABLE-US-00003 (SEQIDNo.:5) GGAACAATAACAAAGCACAACGCGACAAAAGCCGAACAGCGCAGGTCAGGGAGAGCATTTCT AGCGTGCCTTGGTAGATCGTTATCGCGATTTACTTTCCAGGCCCTGGGCGCTTCCAGCCATC AGCCAAAGGATAAAAGCGCGTGCCCTCTTCTTTTCATCATTAACTTTTATCCTCCTCAGCAC CCACGCTCTGTGATTCCATCTCTTCCTCCTCGCATTCAAGCAGCCTCTTCATTTCTTTCCTC CGTCCCGGTGAGTGCGACGCCCGCCGCTGCCATTCCCACACGATGACTTGAGACGCGTCTTC CCGCTATAGCCGACGCCCCTTTTCGTTTTCTTGGCGTTTTGTCACATTGCCACATTGAGCAG CACAGCTTACTTGTCAGCAGCAAAAATCCAACTTCAAACAGCTCTTCAGCATCAACTCTATC ACTCTTTCATCTCTTGTCAACTTCTCTTCCTTCTCGCTCCAAAAGCGGAATTTCGCC TT TACATTCGCTGCCTTCTCTGCTCTTCTAATTTCCCTCGCTGGTGTGGTGGCGCAGACTACAG GCACATCGGTTGACAGTAGCATCTTAACTAAGACTGCTGACTCTACCGGTCCCTCTGGTTTC TCCATTCCGTGAGTACTCTCGACTTTTCCGTCAACCTCCAGTCTCGCCACAGGCCATAGCGA ACAATGAGCCAAGCGCCCACGCGAACCGTGCCCATCATTATCCTCCCACTAATTCCTTTAAC CAAACGTAGCTTTTAGAGGCCAAATGCTGACAAGTGCGTTTTTAGTGCTTTGAGCGAGCTCA CGTCTGGTGCCCCCACTGACTCTACTGTGGCCCTCTACTCTACCTTCGCGGCCGGTGCCACA CCTACCGTTTCTGGTGCCCCTGTCCTCCCTACCAGTGCCCTCACCATCGCCGATTATCCAGC TTTAGATGTCACCCCTCCTACCAACTCCTCTTTGGTTAAGGACTGGATGGCCAAGGTGAGTT GTGTTTGAGTCCGAAAAGGCACCAGAAGAGCTAACAGTTGGATTAGATCGACTTGTCCAAGG TGCCCAGTTATAATGTGACAACGGGCGATTGTTCTACTGACGCGGCTGCTATCAGCGACGGT CGATGCTGGTGGACTTGTGGTGGTTGCACTCGGGAAACCGACATTGTCGAGTGTCCTGACAA GAATGTTTGGGGTCTCTCTTACGAT GGGCCTTCTCCCTTCACCCCTCTCCTAATTGATT ACCTTCAGGAGAAGAACATCAAGACCACCTTCTTCGTTGTCGGCTCTCGTGTCCTTTCTCGA CCCGAGATGCTCCAAACCGAATACATGTCTGGACACCAGATCTCTATC ACTTGGTCT CCCGCACTTACTACTCTTACCAACGAGGAAATTGTTGCCGAGCTTGGTTGGACAATGAAGG TCATCAAGGACACCCTTGGCGTCACCCCAAACACTTTCGCTCCCCCTTATGGTGACATTGAT GACCGTGTTCGAGCTATTGCTGCTCAGATGGGCTTGACCCCTGTTATCTGGACTTCTTACAC TGATGGCTCAACCACTGTTAACTTTGACACTGTAGGCTTATCTTGACTTTCGCAATAATCTT ACTAACGAAATGACAGAACAACTGGCACATCAGTGGTGGTACCGCCACCGGCGCTTCTTCTT ATGAGACCTTTGAGAAGATTCTCACCGAATACGCCCCAAAGTTGGACACTGGTTTCATCACT CTTGAGCACGACAGTAAGTCTTGTCTATCCGTCTTGCAATAATAATCCTGACGTATACCTTT ACAGTCTACCAGCAGAGTGTTGACCTTGCTGTTGGTTACATTTTGCCCCAAGTTCTCGCCAA CGGTACCTATCAGCTCAAATCCATCATCAACTGTTTGGGCAAGGACAGTAAGTTGCCTCCGC TAATCAGAAAAGGTTGTGGGCTAAGATGATACACAGCCTCCGAAGCATACATTGAGACTTCA TCCAACCAGACTACTACTCAGATCACTGCAGCCACCGGCTCCCAGTCTACCTTCTTCCAGCC CATTGTTGGCACTGCTACCGGTGCTGAAGTCTCTGCACCTTCTGAGGCCACTGGCAGCACTG CCGCTGGCTCTGCTGCCTCCACCACTAGTGGTTCTGGCGCCAGCGCTTCTACAGGCGCCGCC TCTAACACTTCTTCCAGCGGGTCTGGTCGATCAGCCACCATGGGTGGTGCCCTCATTGCTCT TGCCGCTGTTGCGGTTGGTATGGTATATGTCGCC GTATTTCAAGGCTTTCAATGTAACG ATGGATGGGGATGGGTGGTGGGGGGGGAGGGAAGTGTGTCTAATGGGGCTATACTTGGGCTA TACTTTGCCTCAAATCCATCAAGTATTAATAGCTGAACCATCTTTCGTTGAACCGTCTTTCA TTGTGAACCATTTGTCTTTTTGATCTTTCAAAGTTTGATCCATTATGAATATCATGGACATT TTGAACGTTTTGAACATCCATGTACTTTTCATTCGATCGATCTGAACGTGTTGTTGTGCATA CCTCGCGAACAAGCTTTCAATGGATGGCTTCACAGATC.

(27) CDA2: CNAG_01230: chitin deacetylase 2: The sequences between the underlined sequences are deleted in the deletion strain.

(28) TABLE-US-00004 (SEQIDNo.:6) GAAAATCACAGCACAGCAACATAACAAACCGCAAAACAAAAGGTAGAAGTAAAAATAGCAAATAGC GCAGAATAACCGACATCGCCCTCATAAAACGAAGGCTCAAAGCTCGGCTGCTGATTCTCATTTCTCTC ATCTCCATTCTTCTTTCTCATCGTATTACCTTTTCCGCTCTTTATCTCCAAGAACAATAATAATCTTTTCG CCTCTTAATCACACAGGCGAA ATCCCTTCCACCGCCGCCGCCCTCCTCACCCTCACAGCTGGTGC CGCCTTCGCCCATACCGGATGTGGTGGCCACGAGATTGGTCGGCGAAATGTTGGCGGTCCCATGTT GTATCGTCGAGCTGTCACCGATGAAGCTAGTGCTGCTGTCAGTACAGGTAGGTTAATACAATACAAT ACAATCGTATTCTATGACAATGACTGACCATAACGACCACGTAGACATCAACACCGAGTGTACAGCC TACAGTTATGCCCCTGTGACCGAGTTGATATCCTCTTTCCCGACTATTTGGCAGACTGCTTCCATCCCC TCCAATGACACAGAAGCCCAACAACTTTTTGGGAAAATTAACTCCACTCTTAATACCAAGATTCCAAA TGATGTACCCCACGGAACCCCCACGGGTGATTGGACCGGTGTGAACTACTCTAACAGTGACCCGGA CTGTTGGTGGACTCATAACAAGTGCACGACTCCTTCCAACGACACTGGTTTGCAAGCCGATATCTCC ATCGCACCCGAGCCAATGACATGGGGTTTGGGTTTTGACGATGGACCTAACTGTAGTCACAACGCTT TGTATGATCTTCTTTTGGAGAACAACCAGAAGGCTACCATGTACGTGATCATCTCTCTTTATTCATGTC CAAACTTATGTATGTAAAAGGTTTTTCATTGGATCCAATGTCTTGGACTGGCCTCTCCAGGCTATGAG GGCTCACGACGAAGGTCATGAAATATGTGTTCACACCTGGTCTCATCAATACATGACCGCCCTCAGT AACGAGGTCGTCTTTGCCGAATTGTACTACACCCAGAAAGCCATCAAGGCTGTTCTCGGAGTTACTC CCCAGTGCTGGTATGTTGGCACTTTGGTGGAATCGTGTGAGACTATAGCTAATGATGACACAGGCG ACCTCCGTACGGTGATGTCGACAACAGAGTTCGTATGATTGCCGAGGGACTCAACCTGACTACCATC ATCTGGTCAGACGACACCGATGACTGGGCGGCTGGAACCAACGGCGTCACTGAGCAAGACGTCACA AATAACTACCAGTCAGTGATCGACAAGGCTGGTAACGGTACATACACTACTCACGGCCCCGTTGTTC TTAACCACGAGCTCAGTAAGTCTCTCCCAACGACTAAACCGATGTTTGCTCACGATGTCCTCTTCAGC CAACTACACCATGTCTGTCTTCATGACTATGTTCCCCAAGATCAAATCAGCTTTCAACTACATTGTCCC CATTTGCACTGCATACAACATCACCCAACCATATGCCGAAAGTAATATCACTTGTCCCAACTTTGAAA CTTACATCTCTGGTGTCACAAACATCAGCAGCTCTACCACTCAGAAAGATGGAAGCAGCTCAACAAA CACTGCTTCTGGTTCCGGCGCCGCTGGTAGTGCCAGTGCCACTAGCAGCAGCGACGACTCAAGCAG CTCTGGTGGCTCAAGCGGCTCTAGTGGCTCAAACAACGCTAGCAGTGGTGCTTTGGGCATGTTCGAC AGTTTGTCAGGAGTGGGTCTTATTTTGGGGGGTGTAGTTGCTGGTGTGATGCTGCTG TGTGAT GTGCTTTAGCACGAAAAAAATGGACAGTATATCAAACGTCACATGACGTACTATGTATCCAAACGGA GGTTCGGGTGAGACACCCGTCAATCTAATTCIGTTAAGGGCATTTGATGATTGTCAGTTCTACGTAG TGCCGAAACGAAGATTGAGTTTCTGTTATCTAACAGACGAAAAGGAACGCTGATATGCACTGCCATT TATCTGAGATCCAATG

(29) C. neoformans var. grubii H99 (CNA3): CNAG_01239chitin deacetylase: CDA3

(30) The sequences of the primers are underlined. The sequence between the underlined primer sequences are deleted. In CDA3 deleted strains around 3049 bp of chromosomal sequence has been deleted out of which 1528 bp actually belong to CDA3 genomic region.

(31) TABLE-US-00005 (SEQIDNo.:7) GGCATGTCGTGATTAATTATTTGACATTTTGTTCTATTCGTTTCAGATCCAGTAATAGTTTTTTTCTCTT TATCTTTGTTGTGTGAGTTGAAATGAAGTGGGGGGCGTCGACTTGGGTGTAATTCTTCATCGCCTGC TGAACAGGTTGCACTGTTGGCAAGGAGAGCAGTTGGTGTGGATTGGATAGGACTTTTTCCTTGGTCT ATACGACAATCAATAGCCCGAAGATAGTAGATCAGATGATATGATATATGCTGATTATCTTCATTGG GCTAGCACAGCACAGCGTTAGCAGACGGTAGCAGTCATGCTTTTCTCACCTCAGTCCATCGGAGATT GAAGATCGGAGATCCTGTGTGCTACATGCATGAGCTGTTGTAAGGGACAAATTGCAGAACACTGCA ATGAACTAACTATTAACTAGGAGCTTGAAAGTCAGGAGATCAATGATCAGGAAGAAAAATCATCGG AATTCCCATGCTATTTGTGTACATATAATAAGATGCACATTTGTTCTGAGCGATAATTTTGCTTTTTAT ATCTATCCATTTAGGCTAATACCAGTACTACGTACTTCGCATTTCACAGTTCACAGAGTAGTACATAC GTACAGATAACACAGCATCACAAACCCAGATACCACCACAGACTGCATCACAGTAGCAACATGGAGT CTGAGCTTTGGGAGTTGATTGAGAGACTTGTTGAATTGCCTGTGAAGAGCCGTATAATTGACTGTCT GAAATTATACTGTCTTTAATGGGGACTGGAGACTCATCAAATAAAACTTGACTCGCATGTTGAGGAC CATTGATGCCGTGAGCATGTGTTTATTATGGATATGGACGGATACTATGGCTAAGTAAGAATCTGAT GTTCCTGTTATTGCCTCTTTCATCATCATACAGTTCGGCGAACCTTGTGCAAAGATAGCCCTGAGATA AGTGTACGACGGAAACATGTCCCTGAGCGACGGGTATGAAATATTCAGGGTCTTCACAAGTCGCAA TCCGCAATCCGGCATTTTTCACCATTTGCCGTATCCGGCCTCGGCTCCGTACGTATTCCCACCAAGAA TCTAACACAACACCAACAAACACCTATAAACTTCACTGCTTCTTCCCCTCCCCTTCTTCATCAGCGCTG CCCCTCCATCCTCTACCTCCCCTACTGCCTATACCACGATCCTCTTCCCTCACACATAACCTCCTCCTC TCTCTTTCTCCCCTATCTTTTCTTCGGTACGTCTGTTTACTCACCCATCAAGCGGTTTATTGTTGTGTA CCTATGATCCTTCCCAATTCGATCAAATCAGTTGGTGTCTAACTTCGCACGCAGTCTTTCTGTACAAG AAGTTGCTCTCCCTTCTGTCTTTCGGCATAACGTCAGCCTCTTGCCCACCCCCACACAACCACATGTA AGCACATCCTCTAAGCACATCCTCTCCTTTCGTCTACAAACGGAGAAACTATTACGTATACAACAGG ACAAGGGCTGACTTTAACCTATTCAATCAGAACGGCCCGTTAGA TACGGTCATTTATCTCTCTC CGCACTTTCCTTGTTTGCAGTGGTGGCCGCTGCTCCATTCCGGGAGTCATGGCTTCAGCCTAGAGATT CCCCCGTCTCACAGCTGTTCAGACGAAaGCTCCCGATCCCAACTCCAATGGTCAGTACCATCTTCTA CTATCTCTCAATAAGTAGGACGAGATTTCACTCATATTCCTTTATAGATTACATGAGTTACTATCCAG GCCCTGGGTCCACTCCGAACGTCAGCACCATTCCCCAAGCTTGGTTGGATAAACTTGCCACAGTGAA CTTGCCAAATGTTCCAGTAGCTACACCTGATGGTGGTCGTCCTACTTACCCTAATAATGAGGACGAT GGTGACTCGACAATTTGTTCTTTCACCGATCAATGCCGCGTAGAGGACGATCTGTACTCTCCCCCGG GTGAGAAGATCTGGGCCGTGAGTGTTATATACTTCTCCATTCATTGTCATCTAGAGATGTGATTAAC GCGGGTATACAGCTTTCCTTCGACGATGGACCCACAGACGTCAGTCCTGCTCTCTACGACTATCTGG CTCAGAACAACATATCGTCCTCTGCGACTCATTTCATGATCGGCGGTAATGTTATTACTTCGTATGTC TCGACCTGCAGCATGTCCCTGCAGGTGTTGTAATATTGACTTTGTGAAATGTAACAGCCCACAGTC AGTTCTCGTTGCCGTTAAGGCTGGAGGTCACCTTGCCGTCCACACTTGGTCCCATCCTTATAGTGAGT ATTTGTTAAATTAAAGCACGATTAGTTTTTAAGTCATTTCTTGCAGTGACAACTCTTACCAACGAGC AAGTCGTTGGAGAGCTCGGCTGGACCATGCAGGCCCTTAGCGATCTCAATGGTGGCCGAATTCCCA TGTACTGGCGCCCTCCGTATGGAGATGTTGACAACCGTGTCCGAGCTATTGCGAAGGAAGTATTTGG CTTGGTGACTGTCCTTTGGGATTCGGGTGAGCATCTTGCTTCATGCTGTGGATAAATTGCTAATGGA TCATAGACACCAATGATTGGGCTATTACCGACGAGCCAGGCCAGTACTCTGTTGCAAGCGTTGAGG CTTACTTCGACACTTTGGTCACTGGCAATCGAACCCAAGGTCTTTTGCTCTTAGAACATGAGCTGGAT AACAACACTGTTGAAGTCTTCGAAACGGAGTACCCCAAGGCAGTGGGTAATGGATGGACTGTCAAG AATGTGGCCGATGCTTTTAACATGGAATGGTACCTGAACTCTGGCAAGGGCAACAACGACGTTGTCA CAACTATGTCTGTTGCCGGTACCCTGACCACGGCCACGCCAACTAATACTTCTACCTATGTCGCTTCC TCAACTGCAGCCTCCAGTGCTTCAGTCACGGACTCAGCCGGTGTGTCGATTGCCTCTGCTGCGAGCT CCGAAGCGTCTTCTTCGTGGGCCATTGCCAACAGGCCTTCTCACTTCGTCATCGCCATCGCGTGCGGC CTTGCCCTTGCTGCTATAATGGTC TAGATGCCATGTGCACTTTTTTGTCGGTCTTTTTAGATCATG GACTCTCATTCGCATTATATAGGAATCATGGACATATAATTCATTTTTATTGCCATAGACAGTCAAGG ATTGTTAGATTGTAGCAGTACATTGTTTTTTTTCTTTTTTTGTGAATAATGGACAATTTATTTAGTAGTT GTTTAATTAATCGTCATCAACATTCATTAGCTTTTTCATTTAATAGCACAACAAGGCCGGCAACCAAA ATGAGTAGAACATGTATACTGTCTTCACAACA

EXAMPLES

(32) The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or aspect. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

(33) This example illustrates that exposure of mice to a composition of the present teachings confers immunity to Cryptococcus infection in a model system.

(34) In these experiments, illustrated in FIG. 1A, a group of 5 mice were inoculated through nasal inhalation with 10.sup.7 Cryptococcus neoformans cda1cda2cda3. These mice were challenged with 10.sup.5 wild type (strain KN99) at day 38 post inoculation. At 70 days post inoculation, 100% of these mice were alive. In contrast, 100% of a control group of 4 mice that had not been inoculated with Cryptococcus neoformans cda1cda2cda3 were dead within 15 days after being challenged with 10.sup.5 wild type Cryptococcus neoformans (strain KN99). These data demonstrate the effectiveness of inhalation exposure to Cryptococcus neoformans cda1cda2cda3 for conferring immunity to Cryptococcus neoformans infection.

(35) In a second experiment, ten CBA/J mice were vaccinated with 10.sup.7 of a live preparation of cda123 cells. After 40 days post vaccination, the vaccinated mice and a group of ten naTve CBA/J mice were challenged with 100,000 of C. neoformans cells. FIG. 1B shows that 100% of the vaccinated mice survived to 80 days post infection, while 100% of the nave mice were dead by 20 days post infection.

Example 2

(36) This example illustrates preparation and use of heat-killed Cryptococcus deficient for chitosan production for conferring immunity against Cryptococcus infection.

(37) In these experiments, a suspension of Cryptococcus neoformans cda1cda2cda3 in phosphate-buffered saline (PBS) is heated to 80 C. for 30 minutes. Test platings on nutrient medium are used to confirm the loss of viability of these Cryptococcus neoformans cda1cda2cda3. Test mice are exposed to these heat-killed fungi as in Example 1. These animals can survive a challenge infection with wild type Cryptococcus neoformans. Such experiments can show that heat-killed Cryptococcus deficient for chitosan production can be effective for conferring immunity, with efficacy similar to that obtained using live Cryptococcus neoformans cda1cda2cda3.

Example 3

(38) This example illustrates that inactivated Cryptococcus neoformans deficient for chitosan production is effective as a vaccine against C. neoformans infection in a mouse model system.

(39) In these experiments, C. neoformans fungi (strain KN99), and C. neoformans cda1cda2cda3 subjected to heat-killing. For each strain, heating was applied until samples formed no colonies on standard nutrient plates. 5 mice were inoculated by nasal administration with 10.sup.5 heat-killed KN99, and 5 mice were inoculated by nasal administration with 10.sup.7 heat-killed C. neoformans cda1cda2cda3. The inoculated mice and 10 nave control mice were challenged with 10.sup.5 wild type C. neoformans KN99 40 days after inoculation. As shown in FIG. 2A, no nave mice or mice treated with heat-killed KN99 survived more than 18 days after exposure to wild type C. neoformans. In contrast, 100% of mice inoculated with heat-killed C. neoformans cda1cda2cda3 survived more than 70 days after exposure to wild type C. neoformans.

(40) In a second experiment, ten CBA/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of wild type (KN99) cells and ten CBA/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of cda123 cells. Ten phosphate buffered saline (PBS) vaccinated mice served as control. After 40 days post vaccination, all mice were challenged with 100,000 of C. neoformans cells. FIG. 2B shows that 100% of mice vaccinated with heat attenuated C. neoformans cda1cda2cda3 live to 100 days post infection. In contrast, no mice vaccinated with PBS or heat-killed wild type C. neoformans cells live to 20 days post infection.

(41) These data demonstrate that heat-killed C. neoformans cda1cda2cda3 can confer immunity to C. neoformans infection.

Example 4

(42) This example illustrates vaccination of a human subject against infection by Cryptococcus neoformans.

(43) In this example, Cryptococcus neoformans cda1cda2cda3 is grown by standard protocols. The fungi are pelleted and resuspended in phosphate-buffered saline 3 times. Following the final resuspension, the Cryptococcus neoformans cda1cda2cda3 suspension is administered to a human subject via a nasal spray. The subject does not subsequently develop a Cryptococcus neoformans infection for at least one year.

Example 5

(44) This example illustrates vaccination of an animal subject against infection by Cryptococcus gattii.

(45) In this example, Cryptococcus gattii cda1cda2cda3 is grown by standard protocols. The fungi are pelleted and resuspended in phosphate-buffered saline 3 times. Following the final resuspension, the Cryptococcus gattii cda1cda2cda3 suspension is administered to a dog subject via a nasal spray. The dog does not subsequently develop a Cryptococcus gattii infection for at least one year.

Example 6

(46) This example illustrate conferral of immunity to C. neoformans by administration of C. neoformans cda1.

(47) In these experiments, as illustrated in FIG. 3, 4 CBA/J female mice (4-6 weeks of age) were subjected to an immunization schedule using nasal administration of a live preparation of 10.sup.6 Cryptococcus neoformans cda1. At 54 days, the mice were placed in laboratory housing. At 40 days, the mice were each vaccinated with 10.sup.5 C. neoformans cda1. At day 0 (40 days post vaccination), the mice were exposed to 10.sup.5 KN99 (wild-type C. neoformans). As a control, 10 nave mice were subjected to the same schedule but were not vaccinated before the challenge with KN99. Weight of the mice was monitored; animals were euthanized when weight fell below 75% of starting weight.

(48) The results of the challenge are shown in FIG. 4. The data indicate that the 100% of nave mice were dead in less than 20 days after exposure to C. neoformans KN99, but that 100% of vaccinated mice remained alive more than 50 days after exposure to C. neoformans KN99.

(49) These data demonstrate effectiveness of nasal administration of C. neoformans cda1 for vaccination against C. neoformans infection.

Example 7

(50) This example illustrates conferral of immunity to C. gattii by administration of C. neoformans cda1cda2cda3.

(51) In these experiments, 10 CBA/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of C. neoformans cda1cda2cda3 by nasal administration, while an additional 5 nave mice were kept as controls. The vaccinated mice and the nave control mice were exposed to Cryptococcus gattii strain R265. As illustrated in FIG. 5, all of the nave mice were dead by 21 days after exposure to C. gattii R265. In contrast, 100% of mice inoculated with C. neoformans cda1cda2cda3 were alive at 21 days after exposure to C. gattii R265. Survival extended to over 30 days in the vaccinated mice.

(52) These data survival at least partial protection against C. gattii infection by administration of C. neoformans deficient for chitin deacetylase.

Example 8

(53) This example illustrates conferral of immunity to C. gattii by administration of C. neoformans cda1cda2cda3.

(54) In these experiments, 10 CBA/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of C. neoformans cda1cda2cda3 by nasal administration, while an additional 5 nave mice were kept as controls. The vaccinated mice and the nave control mice were exposed to Cryptococcus gattii strain WM266 40 days post vaccination. As illustrated in FIG. 6, all of the nave mice were dead by 21 days after exposure to C. gattii WM266. In contrast, 100% of mice inoculated with C. neoformans cda1cda2cda3 were alive at 21 days after exposure to C. gattii WM266. Survival extended to over 30 days in the vaccinated mice.

(55) These data demonstrate at least partial protection against C. gattii infection by administration of C. neoformans deleted for chitin deacetylases genes.

Example 9

(56) This example illustrates the induction of protective response to C. neoformans infection in 129 mice after vaccination with heat-killed cda123.

(57) In these experiments, five 129 mice were vaccinated with 10.sup.7 heat-killed preparation of wild type (KN99) and five 129 mice were vaccinated with 10.sup.7 heat-killed preparation of cda123 cells by nasal administration. Phosphate buffered saline (PBS) vaccinated mice served as control. After 40 days post vaccination, mice were challenged with 50,000 of C. neoformans cells. FIG. 7 illustrates the survival of 129 mice vaccinated with heat-killed cda123. As with other mouse strains, 100% of the mice vaccinated with cda123 survived for 80 days post challenge, while controls vaccinated with wild type Cryptococcus or PBS died about 20 days after C. neoformans challenge.

Example 10

(58) This example illustrates the induction of protective response to C. neoformans infection in A/J mice after vaccination with heat-killed cda123 cells by nasal administration.

(59) In these experiments, five A/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of wild type (KN99) and five A/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of cda123 cells by nasal administration. Five phosphate buffered saline (PBS) vaccinated mice served as a control. After 40 days post vaccination, all mice were challenged with 50,000 of C. neoformans cells. FIG. 8 illustrates that 100% of mice vaccinated with heat-killed cda123 cells survived for 80 days after challenge with C. neoformans cells. In contrast, controls vaccinated with PBS or heat-killed KN99 died within 20 days of challenge with C. neoformans cells.

Example 11

(60) This example illustrates the induction of protective response to C. neoformans infection in BALB/c mice after vaccination with heat-killed cda123.

(61) In these experiments, five BALB/c mice were vaccinated with 10.sup.7 of a heat-killed preparation of wild type (KN99) and five BALB/c mice were vaccinated with 10.sup.7 of a heat-killed preparation of cda123 cells by nasal administration. Five phosphate buffered saline (PBS) vaccinated mice served as a control. After 40 days post vaccination, mice were challenged with 50,000 of C. neoformans cells. FIG. 9 illustrates that 100% of mice vaccinated with cda123 cells lived at least 40 days post challenge, with over 60% surviving to 80 days. In contrast, 100% of mice vaccinated with PBS or heat-killed wild type cells died shortly after 20 days post challenge.

Example 12

(62) This example illustrates the fungal burden in the lungs of cda123 vaccinated mice that exhibited complete protection when challenged with Cryptococcus neoformans.

(63) In these experiments, several strains of mice were vaccinated by nasal administration with a preparation of 10.sup.7 heat-killed cda123 cells. After 40 days post vaccination, the mice were challenged with 50,000 of wild type (KN99) C. neoformans cells in experiments discussed infra. At the conclusion of each of these experiments, surviving mice were sacrificed. Lungs were homogenized and plated onto fungal media to determine the CFU of C. neoformans still present in the lungs. FIG. 10 illustrates the fungal burden for each strain.

Example 13

(64) This example illustrates the induction of a protective response to C. neoformans infection in CBA/J mice after vaccination with a heat-killed preparation of wild type cells grown in Yeast Nitrogen Base medium (YNB) buffered to pH 7.0.

(65) In these experiments, five CBA/J mice were vaccinated by nasal administration with a preparation of 10.sup.7 heat-killed of wild type cells grown in Yeast Nitrogen Base medium buffered to pH 7.0 with 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS) and five CBA/J mice were vaccinated with 10.sup.7 of a heat-killed preparation of cda123 cells. Five phosphate buffered saline (PBS) vaccinated mice served as control. After 40 days post vaccination, mice were challenged with 50,000 of C. neoformans cells. FIG. 11 illustrates that mice vaccinated with heat-killed wild type cells that were grown in YNB medium pH 7.0 survive to 50 days post challenge at the same rate as cda123 vaccinated mice. Mice vaccinated with PBS in this experiment did not survive to 20 days post challenge.

Example 14

(66) This example illustrates the relative chitosan levels of different C. neoformans strains isolated from mouse lungs.

(67) Wild type (KN99) and cda1 were inoculated (100,000 cells) to mouse lungs by nasal inhalation. The lungs were excised 16 days post infection. C. neoformans cells were isolated from the lung homogenate and used for chitosan determination. FIG. 12 illustrates the chitosan levels present in lungs inoculated with each strain; cda1 chitosan levels were 50% that of KN99 wild type chitosan levels.

(68) Wild type cells (KN99) were grown in Yeast Extract Peptone Dextrose (YPD) or Yeast Nitrogen Base (YNB) buffered to pH: 7.0 with 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS). Equal number of cells were subjected to chitosan measurements. FIG. 13 illustrates that the level of chitosan in KN99 cells grown in YNB pH 7.0 medium is less than 50% that of the level of KN99 cells grown in YPD medium, a similar reduction to that between wild type cells and cda1 cells isolated from mouse lungs.

(69) All references cited herein are incorporated by reference, each in its entirety.