Methods and kits for use in preventing and treating vulvovaginal candidiasis

Abstract

The invention features methods and kits use in for preventing and treating vulvovaginal candidiasis (VVC), in particular, recurrent VVC.

Claims

1. A method comprising the steps of a) administering to a subject having vulvovaginal candidiasis (VVC) an antifungal agent; and b) administering an immunogenic amount of an Als3 polypeptide to said subject, to reduce VVC in said subject, wherein said Als3 polypeptide has at least 80% identity to any one of SEQ ID NOs: 1-5.

2. The method of claim 1, wherein: a) said immunogenic amount of said Als3 polypeptide is administered subsequent to administration of said antifungal agent; b) said antifungal agent is administered from 5 to 8 days after administration of said Als3 polypeptide; or c) said antifungal agent is administered from 13 to 15 days after administration of said Als3 polypeptide.

3. The method of claim 1, wherein said subject has recurrent VVC (RVVC).

4. The method of claim 1, wherein said antifungal agent is fluconazole.

5. The method of claim 4, wherein said fluconazole is administered in a dose of from 100 mg to 200 mg orally or a dose of 150 mg orally.

6. The method of claim 1, wherein a second dose of said antifungal agent is administered from 2 to 4 days after administration of said antifungal agent of step a) and, optionally, a third dose of said antifungal agent is administered from 2 to 4 days after administration of said second dose of said antifungal agent, or wherein said Als3 polypeptide is administered from 7 days to 21 days or 14 days after the administration of said antifungal agent of step a).

7. The method of claim 1, wherein from 5 to 300 micrograms of said Als3 polypeptide is administered.

8. The method of claim 7, wherein from 10 to 200 micrograms, from 20 to 100 micrograms, from 30 to 90 micrograms, from 40 to 80 micrograms, from 100 to 300 micrograms, from 150 to 200 micrograms, from 200 to 250 micrograms, from 250 to 300 micrograms, or 300 micrograms of said Als3 polypeptide is administered.

9. The method of claim 1, wherein one or more booster doses of said Als3 polypeptide is administered.

10. The method of claim 1, wherein said antifungal agent is administered from 5 to 8 days or from 13 to 15 days after administration of said Als3 polypeptide.

11. The method of claim 1, wherein said subject is less than 40 years old.

12. A kit comprising a) an antifungal agent; and b) an Als3 polypeptide, wherein said Als3 polypeptide has at least 80% identity to any one of SEQ ID NOs: 1-5.

13. The kit of claim 12, wherein: a) said antifungal agent is fluconazole; and b) said Als3 polypeptide is in a vaccine composition with or without an adjuvant.

14. A method of treating a subject with, or at risk of developing, recurrent VVC (RVVC), comprising administering to the subject at least 5.0 micrograms of an Als3 polypeptide to reduce recurrences of VVC in said subject, wherein said Als3 polypeptide has at least 80% identity to any one of SEQ ID NOs: 1-5.

15. The method of claim 14, wherein said subject has RVVC.

16. The method of claim 14, wherein the method comprises administering from 5 micrograms to 300 micrograms of the Als3 polypeptide.

17. The method of claim 14, wherein the subject has had at least one, two, or three prior VVC infection during the past 12 months.

18. The method of claim 14, wherein: said Als3 polypeptide is administered in a vaccine composition with or without an adjuvant.

19. The method of claim 14, wherein: a) said method further comprises administering one or more booster doses of said Als3 polypeptide; b) said subject does not have a current diagnosis of VVC, or is not experiencing a current episode of VVC, at the time of said administering; c) said subject does not have a VVC Sign and Symptom Composite Questionnaire Score of 3 at the time of said administering; d) said subject has previously had a diagnosis of VVC or recurrent VVC; or e) said subject is less than 40 years old.

20. The method of claim 1, wherein said Als3 polypeptide has the sequence of any one of SEQ ID Nos: 1-5.

21. The method of claim 1, wherein said Als3 polypeptide is administered as a vaccine composition with or without an adjuvant.

22. The method of claim 1, wherein said Als3 polypeptide is administered in either a single dose primary vaccination regimen or a multi-dose primary vaccination regimen.

23. The kit of claim 13, wherein said Als3 polypeptide has the sequence of any one of SEQ ID NOs: 1-5.

24. The method of claim 18, wherein said Als3 polypeptide has the sequence of any one of SEQ ID NOs: 1-5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B are graphs showing plasma anti-Als3 antibody titers through Day 270 post vaccination. FIG. 1A is a graph showing total IgG titers. FIG. 1B is a graph showing total IgA1 titers

(2) FIGS. 2A and 2B are graphs showing fold-rise increases in plasma anti-Alsip antibody titers through Day 180 post vaccination. FIG. 2A is a graph showing total IgG antibody titers. FIG. 2B is a graph showing total IgA1 antibody titers.

(3) FIGS. 3A and 3B are graphs showing Als3-stimulated cytokine production by PBMCs for all subjects receiving the 30 g dose through Day 270 post vaccination. FIG. 3A is a graph showing Als3-stimulated IFN- production. FIG. 3B is a graph showing Als3-stimulated IL-17A production.

(4) FIGS. 4A and 4B are graphs showing Als3-stimulated cytokine production by PBMCs for all subjects receiving the 300 g dose through Day 270 post vaccination. FIG. 4A is a graph showing Als3-stimulated IFN- production. FIG. 4B is a graph showing Als3-stimulated IL-17A production.

(5) FIGS. 5A and 5B are graphs showing percentage of subjects responding with cytokine-producing PMBCs through Day 270 post vaccination. FIG. 5A is a graph showing percentage of subjects responding with IFN--producing PBMCs. FIG. 5B is a graph showing percentage of subjects responding with IL-17A-producing PBMCs.

(6) FIGS. 6A-6D are graphs showing anti-Als3 antibody levels in serum (FIG. 6A and FIG. 6C) and vaginal wash (FIGS. 6B and 6D). FIG. 6A shows serum IgG titers, FIG. 6B shows vaginal IgG titers, FIG. 6C shows plasma IgA titers, and FIG. 6D shows vaginal wash IgA titers. The 300 g with alum (black triangles) has 2-3 fold higher antibody responses than the same dose without alum (black squares). The 30 g dose administered intradermally (gray diamonds) showed a similar antibody response to the same dose delivered intramuscularly (with alum). Placebo controls are indicated with black circles.

(7) FIGS. 7A and 7B are graphs showing association between plasma anti-Als3 total IgG and IgA1 titers and C. albicans opsonophagocytic killing (OPK) activity. FIG. 7A shows the geomean anti-Als3 antibody titer and C. albicans OPK activity by treatment group. FIG. 7B shows the C. albicans OPK titer versus anti-Als3 IgG plasma antibody titer for day 14 post-vaccination samples.

(8) FIGS. 8A and 8B are graphs showing Th1 (FIG. 8A) and Th17 (FIG. 8B) T-cell stimulation by the Als3 antigen. The 300 g intramuscular dose with or without alum produce similar responses, while the 30 g intradermal injection produced a reduced, but overall similar response.

(9) FIG. 9 is a graph comparing post-vaccination sera (light gray bars) to pre-vaccination sera (dark gray bars, normalized to 100%) of each individual administered the Als3 antigen.

(10) FIG. 10 is a graph showing the geometric mean functional opsonophagocytic killing (OPK) activity (line) in post-vaccination subjects in each treatment group compared to the geomean concentration of anti-Als3 antibodies measured by ELISA among the three treatment groups in the Phase 1 (bars).

(11) FIGS. 11A-11D are graphs showing anti-Als3 antibody levels in serum (FIG. 11A and FIG. 11C) and vaginal wash (FIGS. 11B and 11D). Each graph shows a comparison between antibody responses to NDV-3A (dark gray line) and NDV-3 (light gray line), relative to placebo (black line). FIG. 11A shows serum IgG titers, FIG. 11B shows vaginal IgG titers, FIG. 11C shows serum IgA titers, and FIG. 11D shows vaginal IgA titers.

(12) FIGS. 12A and 12B are graphs showing Th1 (FIG. 12A) and Th17 (FIG. 12B) T-cell stimulation by the Als3 antigen. Each graph shows a comparison between cytokine responses to NDV-3A (dark gray bars) and NDV-3 (light gray bars), relative to placebo (black bars). FIG. 12A shows the percentage of IFN- positive responders, and FIG. 12B shows the percentage of IL-17A positive responders.

(13) FIG. 13 is a graph comparing the anti-Als3 antibody titers between healthy placebo recipients (black dotted line), healthy vaccine recipients (gray dotted line), placebo recipients with RVVC (black solid line), and vaccine recipients with RVVC (gray solid line).

(14) FIGS. 14A-14C are Kaplan-Meier curves showing the time until first recurrence. FIG. 14A shows the proportion of non-recurrence as measured by signs/symptoms score in NDV-3A-treated patients (solid line) in comparison to placebo patients (dashed line) from a cohort of patients of all ages. FIG. 14B shows the proportion of non-recurrence as measured by patient symptom score in NDV-3A-treated patients (solid line) in comparison to placebo patients (dashed line) from a cohort of patients of all ages. FIG. 14C shows the proportion of non-recurrence as measured by patient symptom score in NDV-3A-treated patients (solid line) in comparison to placebo patients (dashed line) from a cohort of patients below the age of 40 years.

(15) FIGS. 15A-15D are graphs showing cumulative recurrence over time. FIG. 15A shows the mean cumulative number of recurrent episodes as measured by signs/symptom score in NDV-3A-treated patients (dashed line) in comparison with placebo patients (solid line) from a cohort of patients of all ages. FIG. 15B shows the mean cumulative number of recurrent episodes as measured by signs/symptom score in NDV-3A-treated patients (dashed line) in comparison with placebo patients (solid line) from a cohort of patients below the age of 40 years. FIG. 15C shows the mean cumulative number of recurrent episodes as measured by patient symptom score in NDV-3A-treated patients (dashed line) in comparison with placebo patients (solid line) from a cohort of patients of all ages. FIG. 15D shows the mean cumulative number of recurrent episodes as measured by patient symptom score in NDV-3A-treated patients (dashed line) in comparison with placebo patients (solid line) from a cohort of patients below the age of 40 years.

(16) FIG. 16 is a graph comparing the anti-Als3 antibody titers between placebo recipients of age 40 years or over (black dotted line), placebo recipients under the age of 40 years (gray dotted line), vaccine recipients of age 40 years or over (black solid line), and vaccine recipients under the age of 40 years (gray solid line).

DETAILED DESCRIPTION OF THE INVENTION

(17) The invention provides methods for use in preventing and treating vulvovaginal candidiasis (VVC), including recurrent VVC (RVVC). The methods include treatment involving the coordinated use of (i) an agglutinin-like sequence 3 (Als3) polypeptide of Candida albicans, and (ii) an antifungal agent. The treatment methods are described further, as follows.

(18) Als3 Polypeptides

(19) The sequence of an agglutinin-like sequence 3 (Als3) pre-protein of C. albicans is set forth as follows and designated as SEQ ID NO: 1.

(20) TABLE-US-00001 (SEQIDNO:1) 1 MLQQYTLLLIYLSVATAKTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTS 61 ASPGDTFTLNMPCVFKFTTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTP 121 SIKALGTVTLPLAFNVGGTGSSVDLEDSKCFTAGTNTVTFNDGGKKISINVDFERSNVDP 181 KGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLND 241 WNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAG 301 GYWQRAPFTLRWTGYRNSDAGSNGIVIVATTRTVTDSTTAVTTLPFDPNRDKTKTIEILK 361 PIPTTTITTSYVGVTTSYSTKTAPIGETATVIVDIPYHTTTTVTSKWTGTITSTTTHTNP 421 TDSIDTVIVQVPSPNPTVTTTEYWSQSFATTTTITGPPGNTDTVLIREPPNHTVTTTEYW 481 SESYTTTSTFTAPPGGTDSVIIKEPPNPTVTTTEYWSESYTTTSTFTAPPGGTDSVIIKE 541 PPNHTVTTTEYWSQSYTTTTTVTAPPGGTDTVLVREPPNHTVTTTEYWSQSYTTTTTVIA 601 PPGGTDSVIIREPPNPTVTTTEYWSQSYATTTTITAPPGETDTVLIREPPNHTVTTTEYW 661 SQSYATTTTITAPPGETDTVLIREPPNHTVTTTEYWSQSFATTTTVTAPPGGTDTVIIRE 721 PPNHTVTTTEYWSQSYATTTTITAPPGETDTVLIREPPNHTVTTTEYWSQSYATTTTIIA 781 PPGETDTVLIREPPNPTVTTTEYWSQSYTTATTVTAPPGGTDTVIIYDTMSSSEISSFSR 841 PHYTNHTTLWSTTWVIETKTITETSCEGDKGCSWVSVSTRIVTIPNNIETPMVTNTVDST 901 TTESTSQSPSGIFSESGVSVETESSTVTTAQTNPSVPTTESEVVFTTKGNNENGPYESPS 961 TNVKSSMDENSEFTTSTAASTSTDIENETIATTGSVEASSPIISSSADETTTVTTTAEST 1021 SVIEQPTNNNGGGKAPSATSSPSTTTTANNDSVITGTTSTNQSQSQSQYNSDTQQTTLSQ 1081 QMTSSLVSLHMLTTFDGSGSVIQHSTWLCGLITLLSLFI.
This pre-protein includes an N-terminal signal sequence of 17 amino acids (underlined above), which is removed in the generation of mature Als3 protein.

(21) Examples of Als3 polypeptides that can be used in the invention include those which include an N-terminal portion of the mature form of C. albicans Als3, and variants thereof. Thus, for example, an Als3 polypeptide can include 416 amino acids of N-terminal sequence of mature Als3.

(22) A specific example of an Als3 polypeptide that can be used in the invention is Als3-2, which is a recombinant polypeptide expressed in Saccharomyces cerevisiae that is based on an N-terminal region (416 residues) of mature Als3 from C. albicans. A sequence of Als3-2 is set forth as follows and designated as SEQ ID NO: 2.

(23) TABLE-US-00002 (SEQIDNO:2) KTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTSASPGDTFTLNMPCVFKF 60 TTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTPSIKALGTVTLPLAFNVG 120 GTGSSVDLEDSKCFTAGTNTVTFNDGGKKISINVDFERSNVDPKGYLTDSRVIPSLNKVS 180 TLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLNDWNYPVSSESFSYTKTCS 240 SNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAGGYWQRAPFTLRWTGYRN 300 SDAGSNGIVIVATTRTVTDSTTAVTTLPFDPNRDKTKTIEILKPIPTTTITTSYVGVTTS 360 YLTKTAPIGETATVIVDIPYHTTTTVTSKWTGTITSTTTHTNPTDSIDTVIVQVPL. 416

(24) Another specific example of an Als3 polypeptide that can be used in the invention is Als3-1 which, similar to Als3-2, is a recombinant polypeptide expressed in Saccharomyces cerevisiae that is based on an N-terminal region (416 residues) of mature the Als3 from C. albicans. In contrast to Als3-2, Als3-1 includes a 15 amino acid sequence containing a six-His tag region on the N-terminal end to facilitate purification. A sequence of Als3-1 is set forth as follows and designated as SEQ ID NO: 3.

(25) TABLE-US-00003 (SEQIDNO:3) HHHHHHGIQKTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTSASPGDTFT 60 LNMPCVFKFTTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTPSIKALGTV 120 TLPLAFNVGGTGSSVDLEDSKCFTAGTNTVTFNDGGKKISINVDFERSNVDPKGYLTDSR 180 VIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLNDWNYPVSSE 240 SFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAGGYWQRAPF 300 TLRWTGYRNSDAGSNGIVIVATTRTVTDSTTAVTTLPFDPNRDKTKTIEILKPIPTTTIT 360 TSYVGVTTSYLTKTAPIGETATVIVDIPYHTTTTVTSKWTGTITSTTTHTNPTDSIDTVI 420 VQVPL. 425

(26) Additional Als3 polypeptides include Als3 (18-324) and Als3 (Ser/Thr-rich sequence).

(27) A sequence of Als3 (18-324) is set forth as follows and designated as SEQ ID NO: 4.

(28) TABLE-US-00004 (SEQIDNO:4) KTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTS ASPGDTFTLNMPCVFKFTTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTP SIKALGTVTLPLAFNVGGTGSSVDLEDSKCFTAGTNTVTFNDGGKKISINVDFERSNVDP KGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLND WNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAG GYWQRAPFTLRWTGYRNSDAGSNG.

(29) A sequence of Als3 (Als3 Ser/Thr-rich sequence) is set forth as follows and designated as SEQ ID NO: 5.

(30) TABLE-US-00005 (SEQIDNO:5) IVIVATTRTVTDSTTAVTTLPFDPNRDKTKTIEILKPIPTTTITTSYVGVTTSYST KTAPIGETATVIVDIPYHTTTTVTSKWTGTITSTTTHTNPTDSIDTVIVQVP.

(31) The invention includes the use of proteins or polypeptides that comprise or consist of Als3 proteins and polypeptides such as those described above (e.g., SEQ ID NO: 1, 2, 3, 4, or 5), as well as fragments thereof. In particular, the invention includes the use of NDV-3, a vaccine formulation that includes Als3-1 (SEQ ID NO: 3). The invention also includes the use of NDV-3A, a vaccine formulation that includes Als3-2 (SEQ ID NO: 2). In addition, the invention also includes the use of variants of the Als3 proteins and polypeptides described above. Variants include proteins and polypeptides (or fragments thereof) that are substantially identical to SEQ ID NO: 1, 2, 3, 4, or 5, as set forth above, and in reference to the definition of substantially identical, as set forth above.

(32) In some instances, a modification to a polypeptide as described herein does not substantially reduce the biological activity, e.g., immunogenic activity, of the polypeptide. The modified polypeptide may have or may optimize a characteristic of a polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, or conjugation properties.

(33) Modifications include those by natural processes, such as posttranslational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains, and the amino- or carboxy-terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification.

(34) A variant or otherwise modified polypeptide can also include one or more amino acid insertions, deletions, or substitutions, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence. For example, the addition of one or more cysteine residues to the amino or carboxy terminus of any of the polypeptides of the invention can facilitate conjugation of these polypeptides. Exemplary polypeptides have an N- or C-terminal cysteine.

(35) Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a non-naturally occurring amino acid can be substituted for a naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).

(36) Polypeptides made synthetically, e.g., using methods known in the art, can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH.sub.2(CH.sub.2).sub.nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

(37) Variants may be generated by substitutional mutagenesis and retain or even increase the biological activity, e.g., immunogenic activity, of the original polypeptide.

(38) The polypeptides described herein can be obtained, e.g., by chemical synthesis using a commercially available automated peptide synthesizer. The synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Alternatively, the proteins and polypeptides can be obtained by recombinant methods that are well-known in the art (e.g., expression in S. cerevisiae).

(39) Conjugates

(40) Polypeptides of the invention may, in certain embodiments, be conjugated to another moiety or particle.

(41) Protein Moieties

(42) In some instances, it may be useful to conjugate a polypeptide to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin (KLH), CRM197 and variants thereof, tetanus toxoid, diptheria toxoid, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor, or a polycation (poly-L-lysine or poly-L-arginine), e.g., using a bifunctional or derivatizing agent as known in the art, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, or succinic anhydride.

(43) In some instances, the conjugate may be a recombinant fusion protein made, e.g., to facilitate expression and purification of the polypeptide.

(44) Particles for Conjugation or Display of Polypeptides

(45) In some instances, polypeptides are conjugated to or displayed on a particle, e.g., a phage, a yeast, a virus, a virosome, or a recombinant virus-like particle.

(46) For example, one or more polypeptides may be conjugated to a phage, a yeast, or a virus particle, e.g., to the surface of the particle. In one embodiment, a nucleic acid molecule encoding the polypeptide is inserted into the phage, yeast, or virus particle, resulting in expression of the polypeptide in the phage, yeast, or virus, e.g., at the surface of the particle. The phage, yeast, or virus population containing the polypeptide may then be isolated and prepared, e.g., as a vaccine, by adding a pharmaceutically acceptable excipient.

(47) In some embodiments, polypeptides as described herein are conjugated to a virosome or virus-like particle (VLP). Virosomes and VLPs generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. Viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p 1). Virosomes are discussed further in, e.g., Gluck et al. (2002), Vaccine 20:B10-B16, which is incorporated by reference in its entirety.

(48) VLPs are discussed further, e.g., in Niikura et al. (2002), Virology 293:273-280; Lenz et al. (2001), J Immunol 166:5346-5355; Pinto et al. (2003), J Infect Dis 188:327-338; Gerber et al. (2001), Viral 75:4752-4760; WO03/024480; and WO03/024481, each of which is incorporated by reference in its entirety.

(49) Antifungal Agents

(50) Antifungal agents that can be used in the invention include those that are standardly used by medical professionals in the treatment of candidiasis including, for example, an azole (e.g., a triazole, such as fluconazole, albaconazole, efinaconazole, epoxiconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole; an imidazole, such as bifonazole, butoconazole, clotrimazole, eberconazole, econazole, fenticonazole, flutrimazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole; and a thiazole, such as abafungin), a polyene (e.g., amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin), an allylamine (e.g., amorolfin, butenafine, naftifine, and terbinafine), an echinocandin (e.g., anidulafungin, biafungin (e.g., CD101), caspofungin, and micafungin), lanosterol demethylase inhibitors (e.g., VT-1161) and other antifungal agents, including, but not limited to, benzoic acid, ciclopirox olamine, enfumafungin (e.g., SCY-078), 5-flucytosin, griseofulvin, haloprogin, tolnaftate, aminocandin, chlordantoin, chlorphenesin, nifuroxime, undecylenic acid, and crystal violet, and pharmaceutically acceptable salts or esters thereof.

(51) In particular, the antifungal agent is fluconazole. Huconazole is commercially available and sold under the name DIFLUCAN in the United States. The chemical name of fluconazole is 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol, and it has the following structure:

(52) ##STR00001##
Methods of synthesizing fluconazole are described in U.S. Pat. Nos. 4,404,216 and 5,710,280.
Patient Identification

(53) Patients can be identified for treatment according to the methods of the invention using standard methods, which are widely used in clinical settings. These methods can optionally include any one or more aspects of the following exemplary methods.

(54) A diagnosis of VVC can include positive mycological results for Candida. Also, standard testing can be done to rule out other infections. For example, a whiff test and a vaginal pH test can be done to rule out bacterial vaginosis (BV). In addition, a wet mount can be done to rule out clue cells associated with BV, as well as to rule out infection due to Trichomonas vaginalis. Samples can be analyzed using DNA amplification assays to rule out Chlamydia tracomatis and Neisseria gonorrhea using, for example, a commercially available system (e.g., BD ProbeTec ET System, BD Diagnostics, Sparks, Md.). In another example, Candida can be positively identified, and Gardnerella vaginalis and Trichomonas vaginalis can be ruled out, using, for example, a commercially available DNA probe test (e.g., BD Affirm VPIII Microbial Identification System; BD Diagnostics, Sparks, Md.).

(55) In addition to these methods, a minimum Composite Questionnaire Score of signs and symptoms can be utilized. Per draft FDA guidance published in 1998, signs and symptoms of VVC are scored both by the subject and by the examining clinician. An example of the VVC Sign and Symptom Questionnaire includes asking a subject to score the following symptoms as absent (0), mild (1), moderate (2), or severe (3): itching, irritation, and burning. The examining clinician will examine the subject and score the following signs as absent (0), mild (1), moderate (2), or severe (3): erythema, edema, and excoriation/fissure formation.

(56) The two scores are combined to create a Composite Questionnaire Score. A Composite Questionnaire Score of 0-2 is not clinically indicative of VVC. The following scoring ranges are clinically indicative of VVC: 3-6=Mild Disease 7-12=Moderate Disease 13=Severe Disease

(57) Alternatively, a diagnosis of VVC or RVVC can be made based solely on a Composite Questionnaire Score of signs and symptoms as completed by the subject.

(58) Recurrent VVC (RVVC) is generally defined as four or more episodes of VVC in the preceding year, with at least one of these episodes being documented with culture (Workowski et al. (2006), MMWR Recomm. Rep. 55(RR-11):1-94).

(59) The invention includes treatment of patients with RVVC, as defined herein, as well as patients with VVC (e.g., patients having a first incident of candidiasis, or patients having one or two prior episodes within the preceding year).

(60) As described further below, the invention also includes treatment of subjects at risk of developing RVVC. Such patients may not have a current diagnosis of VVC, and/or may not be experiencing a current episode of VVC, at the time of treatment. These subjects, for example, may not have a VVC Sign and Symptom Composite Questionnaire Score of 3 at the time of treatment. Furthermore, these patients may have previously had a diagnosis of VVC or recurrent VVC. The diagnosis may be made with or without a positive culture result.

(61) In addition to standard methods of identifying a patient for RVVC treatment, the invention provides a method of identifying a patient based on the age of the patient. As described further below, a patient suitable for treatment by any of the methods or compositions of the present invention can be a female under the age of 40 years.

(62) Vaccine Compositions

(63) Formulations for vaccine compositions as described herein can be prepared using standard pharmaceutical formulation chemistries and methodologies that are readily available to the reasonably skilled artisan. For example, polypeptides or conjugates as described herein can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

(64) Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions may include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (e.g., a powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.

(65) Other parentally-administrable compositions that are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

(66) Alternatively, the polypeptides and conjugates described herein may be encapsulated, adsorbed to, or associated with particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.

(67) The formulated compositions will include an amount of one or more polypeptides or conjugates described herein that is sufficient to mount an immunological response. An immunogenic amount can be readily determined by one of skill in the art. Such an amount will fall in a relatively broad range that can be determined through routine trials. The compositions may contain from about 0.1% to about 99.9% of the polypeptides, conjugates, or antifungal agents, and can be administered directly to the subject or, alternatively, delivered ex vivo, to cells derived from the subject, using methods known to those skilled in the art.

(68) Compositions can include a mixture of distinct polypeptides or conjugates as described herein. For example, vaccines may include, e.g., 2, 3, 4, 5, 6, 7, 8, or more distinct polypeptides or conjugates as described herein, e.g., containing or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, or 5, or a variant sequence thereof having up to three substitutions (e.g., conservative substitutions), deletions, or additions to the amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, or 5.

(69) Substances that stimulate the immune response, e.g., adjuvants, may be included in the compositions, e.g., in vaccines. Examples of chemical compounds used as adjuvants include, but are not limited to, aluminum compounds (e.g., alum, Alhydrogel (aluminum hydroxide), Adjuphos (aluminum phosphate)), oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccarides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

(70) Als3 polypeptides, optionally in combination with an adjuvant (e.g., aluminum hydroxide; also see above), can be formulated in compositions including, for example, a buffer and a salt. Such compositions can thus include, for example, sodium phosphate, sodium citrate, histidine, or sodium succinate (2-20 mM, e.g., 5-15 mM or 10 mM), pH 6.0-8.0 (e.g., pH 6.5-7.5 or pH 7.0), as well as sodium chloride (100-300 mM, e.g., 100-200 mM or 154 mM.

(71) A specific example of a vaccine formulation that can be used in the invention is NDV-3, which includes Als3-1 formulated in 10 mM sodium phosphate, pH 7.0, and 154 mM sodium chloride. Another example is NDV3-A, which includes Als3-2 formulated in 10 mM sodium phosphate, pH 6.5, and 154 mM sodium chloride. The NDV-3 and NDV-3A vaccines can optionally be filled in 2 mL glass vials with a 0.7 mL volume containing 600 g Als3-1 (or Als3-2)/mL, 1.0 mg Al/mL as aluminum hydroxide and phosphate-buffered saline. When withdrawn from the vial with a needle and syringe for injection, approximately 0.5 mL can be injected (e.g., by the intramuscular route), resulting in a delivered dose of 300 g of Als3-1 (or Als3-2).

(72) Methods of Treatment

(73) As noted above, the methods of the invention involve coordinated administration of (i) an agglutinin-like sequence 3 (Als3) polypeptide of Candida albicans, and (ii) an antifungal agent. The Als3 polypeptide and antifungal agent are generally as described elsewhere herein, but can be, as examples, Als3-1, Als3-2, Als3 (18-324), and/or Als3 (Ser/Thr-rich sequence), or variants thereof (with Als3-1 or Als3-2 optionally in the form of NDV-3 or NDV-3A, respectively), and fluconazole.

(74) As will be described further below, there are many different approaches to coordinated administration of an Als3 polypeptide and an antifungal agent that can be used in the invention. For instance, the method may include treatment with an antifungal agent prior to Als3 polypeptide administration. Taking this approach enables treatment of an acute episode quickly with the antifungal agent, while vaccinating with an Als3 polypeptide afterwards, in an effort to prevent recurrence and/or to supplement the action of the antifungal agent in addressing the acute attack.

(75) In one example, a patient is treated with an antifungal agent 1-4, e.g., 2-3, times before vaccination, and the antifungal treatment takes place, for example, within a time frame of 1, 2, or 3 weeks prior to vaccination. Thus, in a specific example, treatment with an antifungal agent can be carried out on days 14, 11, and 8 relative to day 0, which is the day on which vaccination with an Als3 polypeptide takes place. Any of the antifungal treatment and/or vaccination days can vary by, e.g., 1 or 2 days before or after the days noted above.

(76) In other examples, antifungal treatment takes place on the day of vaccination, in addition to (or instead of) prior antifungal treatment according to, for example, a schedule as noted above. Thus, in one specific example, antifungal treatment takes place on days 14, 11, and 8 (1 or 2 days for each day of administration), and also on day 0, the same day as vaccination (1 or 2 days).

(77) In further examples, antifungal treatment takes place after the day of vaccination, in addition to (or instead of) prior antifungal treatment according to, for example, a schedule as noted above, and optionally in addition to antifungal treatment on day 0, as noted above. Treatment after the day of vaccination can take place, for example, at any time within 3 to 4 weeks after vaccination, and can take the form of, for example, 1-4 treatments. In one example, post-vaccination treatment takes place on day 7 and/or day 14 (1 or 2 days). Thus, in one specific example, antifungal treatment takes place on days 14, 11, and 8 (1 or 2 days for each day of administration), further antifungal treatment and vaccination takes place on day 0 (1 or 2 days), and is followed by still further antifungal treatment at day 7 and/or day 14 (1 or 2 days). In the examples described in this paragraph, the vaccination is a single dose primary vaccination regimen or the initial dose of a multi-dose primary vaccination regimen (see below).

(78) Treatment according to the regimens noted above can be varied, as determined to be appropriate by those of skill in the art. For example, in the instance of a particularly acute case, the patient may be treated with a single or double-dose of antifungal agent on the first day of treatment, and daily administration of anti-fungal agent may continue until symptoms have dissipated sufficiently, as determined by those of skill in the art. Thus, for example, daily treatment may continue for 2-6 days, 1 week, or 1-2 weeks, and be followed by vaccination (optionally including further antifungal treatment on the same day and/or further antifungal treatment following vaccination as, e.g., described herein).

(79) In addition to coordinated administration of an Als3 polypeptide and an antifungal agent, as described above, the invention also includes methods involving the administration of an Als3 polypeptide without an antifungal agent, to subjects at risk of RVVC (e.g., subjects who have previously had a diagnosis of VVC or RVVC). Such subjects may have had, for example, one, two, three, or more VVC infections during the past 12 months prior to treatment. Further, at the time of treatment, these subjects may not have a current diagnosis of VVC, and/or may not be experiencing a current episode of VVC. Further, at the time of treatment, these patients may not have a VVC Sign and Symptom Composite Questionnaire Score of 3 at the time (see above).

(80) The methods of the invention also include optional administration of a primary vaccination regimen and subsequent booster doses of Als3 polypeptides as described herein. The primary vaccination regimen is defined by the number of doses administered and time intervals between the doses. The primary vaccination regimen is optimized to achieve an optimal initial protection in newly vaccinated patients. The number of doses in a primary vaccinated regimen is typically 1-4. The time period for the completion of a primary vaccination regimen is 1-12 months. These primary vaccination doses can be administered at, for example, 1-11 months after the initial vaccination, as determined to be appropriate by one skilled in the art. Thus, in various examples, one or more primary vaccination doses can be administered at 2-10, 3-9, 4-8, 5-7, or 6 months after the initial vaccination. Booster doses can be administered following the primary vaccination regimen to increase the longer-term duration of protection. Booster doses can be administered at 1-10 years following the first dose of the primary series and are typically a single dose. The amount of Als3 polypeptide present in all primary vaccination doses and booster doses is typically the same, but can vary and be, for example, an amount as described elsewhere herein, or optionally can be 5-20%, e.g., 10%, of the amount of the initial dose.

(81) The invention also features methods of detecting recurrence of VVC. Recurrence may be detected by the patient's own observation of her symptoms. Additionally, or alternatively, recurrence may be detected by the physician, in which the physician's observations of signs or symptoms of infection determine whether or not a patient experiences recurrence. Additionally, or alternatively to these detection methods, a vaginal mycological culture positive for C. albicans may be used alone or in combination with one or both of the patient's own determination of infection and the physician's assessment of infection to determine whether a recurrence of VVC has occurred.

(82) Compositions as described herein can be delivered to a mammalian subject (e.g., a human or other mammal described herein) using a variety of known routes and techniques. For example, a composition can be provided as an injectable solution, suspension, or emulsion, and administered via intramuscular, subcutaneous, intradermal, intracavity, parenteral, epidermal, intraarterial, intraperitoneal, or intravenous injection using a conventional needle and syringe, or using a liquid jet injection system. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally, or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques.

(83) The compositions described herein can be administered to a subject (e.g., a human patient that has or is at risk of developing VVC or RVVC) in an amount that is compatible with the dosage formulation and that will be prophylactically and/or therapeutically effective. An appropriate effective amount will fall in a relatively broad range but can be readily determined by one of skill in the art by routine trials. The Physician's Desk Reference and Goodman and Gilman's The Pharmacological Basis of Therapeutics are useful for the purpose of determining the amount needed. An adequate dose of the active antifungal drugs described herein may vary depending on such factors as preparation method, administration method, age and body weight of the patient, severity of symptoms, administration time, administration route, rate of excretion, and responsivity. An adequate dose of the vaccines described herein may vary depending on the primary vaccination regimen, administration route, use of adjuvant as well as the age and immunocompetence of the patient. A physician of ordinary skill in the art will easily determine and diagnose the administration dose effective for treatment.

(84) In the case of, for example, fluconazole, a typical dose is 150 mg given orally, but the dosage amount can optionally range from, e.g., 50-200 mg, and thus can be, for example, 50, 100, 150, or 200 mg, as determined to be appropriate by those of skill in the art. If an antifungal agent other than fluconazole is administered, the dosage of the antifungal may be one that is routinely given for that antifungal agent.

(85) In the case of an Als3 polypeptide vaccine, a typical dose for a single-dose primary vaccination regimen is 300 g given by intramuscular injection, but the dosage amount can optionally range from, e.g., 60-300 g. In various examples, for a single-dose primary vaccination regimen the dosage amount is 100-300 g, for example, 150-200 g, 200-250 g, or 250-300 g. In additional examples for a multiple-dose primary vaccination regimen, the dosage amount is 5-60 g, for example, 10-50 g, 20-40 g, or 30 g. The Als3 polypeptide vaccine may be injected into the patient in a volume of 1.0 ml or less, such as 0.75 ml, 0.5 ml, or 0.25 ml (e.g., a dosage of 300 g in 0.5 ml).

(86) Compositions may be prepared into unit-dose or multiple-dose preparations by those skilled in the art using a pharmaceutically acceptable carrier and/or excipient according to a method known in the art.

(87) Kits

(88) The invention also includes kits that can be used to carry out the methods of the invention. Thus, kits of the invention can include one or more Als3 polypeptides (e.g., Als3-2, Als3-1, Als3 (18-324), and/or Als3 (Ser/Thr-rich sequence)), optionally in the form of a vaccine composition including an adjuvant, such as, for example, aluminum hydroxide). In some examples, the Als3 polypeptide (e.g., Als3-2, Als3-1, Als3 (18-324), and/or Als3 (Ser/Thr-rich sequence)) is present in a container (e.g., a glass vial) in liquid form (e.g., in water or a buffered salt solution, such as, 10 mM sodium phosphate, pH 6.5 or 7.0, and 154 mM sodium chloride; see above for other examples of buffer and salt conditions that can be used). In other examples, the Als3 polypeptide (e.g., Als3-2, Als3-1, Als3 (18-324), and/or Als3 (Ser/Thr-rich sequence)) is present in a container (e.g., a glass vial) in lyophilized form. In such examples, the kit may optionally also include a diluent (e.g., water or a buffered salt solution) for reconstitution of the lyophilized polypeptide into liquid form prior to administration. The polypeptide may also be present in another formulation, as described herein, or as is known to be acceptable in the art. The amount of polypeptide and, optionally, adjuvant present in the compositions of the present kits can be, for example, as described above. Thus, for example, the kits can include an Als3 polypeptide (e.g., Als3-2, Als3-1, Als3 (18-324), and/or Als3 (Ser/Thr-rich sequence)) in an amount to facilitate the administration of a dose as described herein.

(89) In addition to the Als3 polypeptide(s), the kits of the invention include one or more antifungal agents (e.g., fluconazole, ketoconazole, butoconazole, miconazole, terconazole, tioconazole, clotrimazole, and nystatin, or any of the other antifungal agents described above). In the case of, for example, fluconazole, the kits can include one or more doses in an amount as described herein, formulated in a tablet for oral administration (e.g., the fluconazole may be present in table form at a dosage of 50, 100, or 200 mg; the tablet may also include the following inactive ingredients: microcrystalline cellulose, dibasic calcium phosphate anhydrous, povidone, croscarmellose sodium, and magnesium stearate). The kit may include a single dose of the antifungal agent (e.g., fluconazole), or 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (e.g., at least 3 doses) of the antifungal agent (e.g., fluconazole). Other agents, such as butoconazole, miconazole, terconazole, tioconazole, and clotrimazole, can be present in the kits of the invention in the form of a cream, while clotrimazole, miconazole, and terconazole can be present in the form of a vaginal suppository, as is known in the art. These agents can be present in single or multiple doses. The antifungal agent may be packaged in a separate container within the kit so that a user (e.g., a physician) can provide the package containing the antifungal agent to a patient.

(90) The kit components can be provided in dosage form to facilitate administration, and can optionally include materials required for administration and/or instructions for patient treatment consistent with, for example, the methods described herein.

(91) For example, the kit can include instructions for use, which guides the user (e.g., the physician) with respect to the administration of the Als3 vaccine (e.g., at the point of care location). The kit can also include instructions guiding the physician to administer a first dose of an antifungal agent (e.g., fluconazole). These instructions, or a separate set of instructions, in the kit may guide a user (e.g., a patient) with respect to the administration of the antifungal agent, which may be separately packaged in the kit so that the antifungal agent can be given to the patient for later home administration. For example, the instructions may guide the user (e.g., the physician or patient) to administer a first dose of the antifungal agent immediately and to administer a second and subsequent doses of the antifungal agent every 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours, or until the antifungal agent consumed.

(92) For example, for uncomplicated VVC, in which the patient has experienced one or fewer episodes in a year, the symptoms are mild or moderate, and there are no significant host factors, such as poor immune function, the instructions may direct the user to take a 150 mg table by mouth as a single dose. For complicated VVC, in which the patient has experienced two or more episodes in a year, when severe symptoms of vulvovaginal inflammation are experienced, or when symptoms are coupled with pregnancy, poorly controlled diabetes, or poor immune function, the instruction may direct the user to take a 150 mg tablet by mouth every 72 hour for 3 doses or more. For recurrent VVC, in which the patient has experienced four or more episodes in a year, the instructions may direct the user to take a 150 mg tablet by mouth every third day until they have taken three doses total.

(93) The kit may be packaged in materials suitable for storage in a refrigerator at a temperature of between 35 F. and 46 F. (2 C. and 8 C.). The desired average refrigerator vaccine storage temperature is 40 F. (5 C.). Exposure to temperatures outside these ranges may result in reduced vaccine potency and increased risk of vaccine-preventable diseases.

EXAMPLES

(94) The following examples are intended to illustrate the invention. These are not meant to limit the invention in any way.

Example 1: Clinical Studies

(95) Methods

(96) Purpose

(97) The purpose of the Phase 1b portion of this study was to compare the NDV-3A vaccine, the NDV-3 vaccine and the placebo head-to-head in the patient population of interest (women with RVVC) to evaluate safety and immunogenicity. The study size for comparing safety and immunogenicity (N=15 per group) is based on the dose comparison design used in NDV3-001 (Schmidt et al. (2012) Vaccine 30(52):7594-7600).

(98) The purpose of the Phase 2a portion of this study is to determine whether the NDV-3A vaccine decreases the recurrence rate of RVVC in female subjects 18-50 years of age compared to placebo. The study size for evaluating efficacy (N=86 per group) is based on a 50% attack rate over 6 months in the placebo group and a 50% vaccine efficacy.

(99) Phase 1a Study Design

(100) The Phase 1a study was a double-blind, placebo-controlled, ascending dose escalation study (30 and 300 g) that enrolled healthy adults at a single study site. Vaccinations occurred on study day 0, with follow up evaluations on study days 3, 7, 14, 28, 90, and 180. A subset of vaccinees was re-consented to receive a second dose of vaccine on study day 180, with follow up visits 7, 14, and 90 days after the second dose. The lower participation rate in the group receiving the second dose (9 of 15 for the 30 g dose, and 10 of 15 for the 300 g dose) was documented as primarily due to the timing of the second dose and follow-up conflicting with mid-summer personal schedules.

(101) Phase 1b/2a Study Design

(102) The Phase 1b and 2a studies were multi-center, double-blind, randomized, placebo-controlled studies to evaluate the safety, tolerability, immunogenicity and efficacy in preventing vulvovaginal candidiasis in subjects with recurrent vulvovaginal candidiasis following administration of single dose of NDV-3A vaccine, NDV-3 vaccine, or placebo. In the Phase 2a portion of the study, to estimate the effect of a single dose of NDV-3A or placebo administered intramuscularly to patients (N=86 per group) diagnosed with recurrent vulvovaginal candidiasis (RVVC) on the safety and tolerability during the 12-months post vaccination period in the NDV-3A vaccine group and the placebo group; the humoral and cellular immune responses at pre-defined time points over a 12-month period in the NDV-3A vaccine group and the placebo group; and the clinical efficacy of NDV-3A in lowering the recurrence rate of VVC in RVVC subjects relative to placebo.

(103) Vaginal cultures for C. albicans were obtained at enrollment and at pre-defined time points during the study, concomitant with collection of clinical signs and symptoms of VVC.

(104) An additional subset of subjects was included to compare the humoral and cellular immune response of the NDV-3A vaccine group, NDV-3 vaccine group, and placebo group. The study size for comparing safety and immunogenicity (N=15 per group) was based on the dose comparison design used in NDV3-001.

(105) Inclusion and Exclusion Criteria

(106) Subjects were informed of the nature of the study and have agreed to and are able to read, review, and sign the informed consent document prior to screening. Female subjects had to be 18-50 years of age, inclusive, at the time of vaccination on an acceptable form of birth control. Subjects had to have a current episode of VVC (at Screening/Day 14) that can be confirmed with acute signs and symptoms of VVC (Composite Questionnaire score 3), a positive KOH smear, a positive vaginal mycological culture for C. albicans. Subjects had to have a history of 2 or more documented episodes of VVC in the 12 months prior to screening, including at least one of the previous episodes confirmed by positive results from a diagnostic lab test specific for the presence of Candida (e.g., DNA/Affirm, PCR, mycological culture.) Additional episodes may be self-reported. Subjects were administered 3 doses of oral fluconazole (150 mg dose given orally 3 days apart during the screening period on Days 14, 11, and 8) and were screened for response to the antifungal, as evidenced by a Composite Questionnaire score of <3 on Day 0. Subjects were required to show no clinically significant abnormalities on Papanicolaou (Pap) smear at study entry or a normal Pap test result from the previous 12 months as judged and documented by the investigator(s). During the study period, subjects were readministered 3 doses of oral fluconazole (150 mg dose given orally 3 days apart) if the subjects experienced a recurrence of VVC.

(107) In general, subjects must be in good health as judged and documented by the investigator(s) in the medical history, physical examination (including but may not be limited to an evaluation of the cardiovascular, gastrointestinal, psychiatric, respiratory and central nervous systems), vital sign assessments, clinical laboratory assessments, and by general observations.

(108) Subjects were ineligible if they reported receiving any systemic or topical vaginal antifungal therapy for four weeks prior to vaccination, other than the oral fluconazole provided by the site during the subject's participation in the study. Subjects were ineligible if mycological results from within 4 weeks prior to vaccination showed other yeast species (e.g. C. glabrata, C. tropicalis, etc.) as the cause of vaginitis. Subjects were ineligible if they had any other active infectious causes of vulvovaginitis (e.g., bacterial vaginosis, Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhea, symptomatic HSV-1, symptomatic HSV-2, or symptomatic human papilloma virus) at screening or other vaginal or vulvar conditions that would confound the interpretation of clinical response as judged by the investigator. Subjects were ineligible if they would be under treatment or surgery at the start of the study for cervical intraepithelial neoplasia (CIN) or cervical carcinoma. Subjects were ineligible if they had any presence or history of a clinically significant disorder involving the cardiovascular, respiratory, renal, gastrointestinal, immunologic, hematologic, endocrine, or neurologic system(s), diagnosed diabetes mellitus (controlled or not) or psychiatric disease that would confound the interpretation of clinical response as judged by the investigator. Subjects were ineligible if they had a history of allergic response(s) or other serious reactions to nickel, aluminum, or yeast products or any contraindications to fluconazole. Subjects were ineligible if they had a history of clinically significant allergies including food or drug allergies, anaphylaxis (or other serious reaction) to vaccines. Subjects were ineligible if they had a known history of or active infection with hepatitis B, hepatitis C, or human immunodeficiency virus (HIV). Subjects were ineligible if they had received or were planning to receive any investigational drug, investigational vaccine, or investigational device within 4 weeks prior to vaccination, and at any other time during their participation in the study. Subjects were ineligible if they had received or were planning to receive any other live vaccine within 3 weeks prior to vaccination. Subjects were ineligible if they had or showed evidence of a recent history of drug or alcohol abuse. Subjects were ineligible if they reported the use or planned use of any immunosuppressive drugs, including systemic or topical vaginal corticosteroids, within 4 weeks prior to vaccination, with the exception of topical steroids (e.g., OTC hydrocortisone) used elsewhere on the body. Subjects were ineligible if they reported the use planned use of any medications or treatments that may alter immune responses to the study vaccine within 3 weeks prior to vaccination (e.g., cyclosporine, tacrolimus, cytotoxic drugs, immune globulin, Bacillus Calmette-Guerin (BCG), monoclonal antibodies, radiation therapy). Subjects were ineligible if they received any blood products within 3 months prior to vaccination and throughout the study. Subjects were ineligible if they reported donating blood/plasma within 4 weeks prior to vaccination. Subjects were ineligible if they were pregnant or intending to become pregnant over the course of the study, if they were breastfeeding, or had any other medical and/or social (e.g., non-compliant) reason which, in the opinion of the investigator, would prevent participation in the study. Subjects were ineligible if they were unable to commit to the follow-up visits and or have unreliable access to a telephone for follow-up contacts, either by self-admission (self-reporting) or in the opinion of the investigator.

(109) Documentation of episodes of VVC prior to vaccination must meet the conditions specified in the inclusion criteria. In addition to a positive vaginal mycological culture for C. albicans, examples of diagnostic lab tests specific for the presence of Candida include, but are limited to, Affirm (BD), NuSwab (LabCorp), SureSwab (Qwest).

(110) Vaccine and Adjuvants

(111) The active component of the NDV-3 vaccine is a recombinant version of the N-terminal region (416 amino acids) of the C. albicans agglutinin-like sequence 3 protein (Als3p) with the addition of a six-His tag and linker sequences (Spellberg et al. (2006) J Infect. Dis. 194(2):256-60). Als3p was produced by batch fermentation of a Saccharomyces cerevisiae expression cell line at 100 L scale, harvested by centrifugation and purified using two chromatography columns (nickel-affinity and hydrophobic interaction resins) followed by concentration, diafiltration into phosphate-buffered saline (PBS), pH 7, and filtration. The purified Als3p bulk drug substance was intact, monomeric, and 99% pure by SDS-PAGE with Coomassie staining and was formulated with aluminum hydroxide at 1.0 mg Al/mL in PBS, pH 7. Two final container vaccine clinical lots were used for this study; lot 0939 (60 g Als3p/mL) and lot 0940 (600 g Als3p/mL). Clinical lots were stored at 2-8 C. post manufacture and monitored for stability. Manufacture of the bulk drug substance and final container lots using cGMPs was conducted by Althea Technologies (San Diego, Calif.). NDV-3 vaccine (0.5 mL dose containing 300 g Als3-1 (SEQ ID NO: 3) formulated with aluminum hydroxide (0.5 mg Al) in isotonic PBS. NDV-3A vaccine (0.5 mL dose containing 300 g Als3-2 (SEQ ID NO: 2) formulated with aluminum hydroxide (0.5 mg Al) in isotonic phosphate-buffered saline (PBS). Subjects were given a 0.5 mL intramuscular (IM) injection of either the study vaccine NDV-3A, study vaccine NDV-3 or a placebo containing aluminum hydroxide (0.5 mg Al) in isotonic PBS.

(112) Immunogenicity Analysis

(113) Blood samples were obtained from subjects on the specified days post vaccination. Plasma and PBMCs were isolated using standardized procedures. Plasma samples were evaluated for anti-Als3 total IgG and for anti-Als3 IgA1 by standardized ELISA methodology. Results are expressed in units of dilution.sup.1. PBMCs were evaluated by ELISpot analysis to determine the portion of cells that could be stimulated to produce IFN- or IL-17A (two separate assays). Results are expressed in units of spot forming units (SFU) per 10.sup.6 cells.

(114) Statistical Analyses

(115) Statistical analysis of assay results used non-parametric analysis using the Wilcoxon rank-sum test (Mann et al. (1947) Ann. Math. Stat. 18:50-60). Evaluation of trends across groups used the Kruskal-Wallis test (Cuzick (1985) Stat. Med. 4:87-90).

(116) Results

(117) Safety

(118) In this study population, NDV-3 was safe and generally well-tolerated after one or two doses. Local injection site reactions to placebo (post dose 1) and vaccine and (post dose 1 and 2) are summarized in Table 1.

(119) TABLE-US-00006 TABLE 1 Systemic and injection site AEs reported Days 0-7 post-vaccination regardless of causality. MedDRA Placebo 30 g Dose 300 g Dose Preferred Dose 1 Dose 1 Dose2 Dose 1 Dose 2 Term (N = 10) (N = 15) (N = 9.sup.a) (N = 15) (N = 10.sup.a) Injection site Erythema 10% 20%.sup.b 11%.sup.d 0 10%.sup.b Induration 10% 0 0 0 20%.sup.b Pain 20% 73%.sup.b 100% 73%.sup.c 100%.sup.b Swelling 0 7% 22%.sup.d 7% 30%.sup.b Systemic AEs Diarrhea 0 7% 11% 7% 0 Nausea 0 13% 0 0 30% Fatigue 10% 7% 11% 7% 40% Myalgia 0 0 11% 20% 20% Extremity 0 0 0 13% 10%.sup.b pain Headache 10%.sup.b 7% 22% 7%.sup.b 30% .sup.aSubjects volunteered to continue in study to receive a 2.sup.nd dose. .sup.bOne graded as moderate. .sup.cThree graded as moderate. .sup.dOne graded as severe. All AEs resolved without sequelae.

(120) The systemic and injection site AEs occurring in at least two study subjects after either the first or the second dose are presented in Table 1. The most common complaint was injection site pain, typically mild, lasting 1-2 days after vaccination and resolving within 2-3 days without sequelae. After dose 1 each of the systemic AEs shown in Table 1 were reflected in 2 of the 40 subjects. After dose 2, the most common systemic AEs were fatigue and headache (5 out of 19 (26%) subjects for each). Systemic AEs were usually mild and occasionally moderate, but all resolved without sequelae within a few days. There were no notable differences in systemic AEs between the two dose levels.

(121) Immune Response

(122) Plasma Anti-Als3p Total IgG and IgA1.

(123) Prior to vaccination (day 0), 36 of the 40 subjects exhibited a detectable pre-existing anti-Als3p total IgG titer ranging from 114 to 2608 dilution.sup.1, with 4 subjects showing IgG titers below the limit of detection (LOD) of the assay (<50 dilution.sup.1). For IgA1 titers, 36 of the 40 subjects exhibited pre-existing detectable anti-Als3p IgA1 titer ranging from 102 to 6473 dilution.sup.1, with 4 subjects showing IgA1 titers below the LOD (<50 dilution.sup.1). Two subjects had no detectable anti-Als3p IgG or IgA1 prior to vaccination.

(124) The geometric mean of anti-Als3p total IgG titers (FIG. 1A) and IgA1 titers (FIG. 1B) rose quickly after the first dose of vaccine, showing a substantial rise by day 7 post vaccination and reaching a maximum by day 14 post vaccination. The IgG and IgA1 titers from day 7 on were significantly higher for the 300 g dose relative to the 30 g dose (Mann-Whitney test, p<0.05) and both were beyond the range of placebo recipient titers (Mann-Whitney test, p<0.001). Antibody titers out to 180 days post vaccination showed roughly a two-fold decline from the maximum titers.

(125) Following the second dose of vaccine, marked increases in anti-Als3p total IgG and IgA1 were noted, with the increase in the geomean IgG titer of the 30 g recipients being greater than that of the 300 g recipients.

(126) The fold-rise of anti-Als3p total IgG titers (FIG. 2A) and IgA1 titers (FIG. 2B) above the pre-vaccination titers all peaked at 14 days post vaccination. The fold-rise for IgG from day 7 to 180 was significantly higher for the 300 g dose relative to the 30 g dose (Mann-Whitney test, p<0.05) and for both dose levels the mean fold-rise remained >7-fold above pre-vaccination titers through day 180 post vaccination. Based on a definition of seroconversion being a 4-fold rise in antibody titer relative to the pre-vaccination antibody titer, a single dose of NDV-3 resulted in 13% and 53% of subjects seroconverting in IgG by day 7 (30 and 300 g dose, respectively) and 100% seroconverting by day 14 in both dose groups (see Table 2). Similar results were observed for IgA1 seroconversion. For the 300 g dose group, seroconversion remained at 100% for IgG through day 180, although for IgA1 there was a decline from 100% after day 28. For the 30 g dose group, the seroconversion rate for IgG declined from 100% after day 28, while IgA1 seroconversion rates declined from 100% after day 14 (Table 2).

(127) TABLE-US-00007 TABLE 2 Fold rise of anti-Als3 antibody titer relative to pre-vaccination (Day 0) titers. % of subjects with >4-fold Dose Ig rise in anti-Als3 antibody titer (g) type Day 7 Day 14 Day 28 Day 90 Day 120 30 IgG 13 100 100 93 73 300 IgG 53 100 100 100 100 30 IgA1 7 100 93 87 87 300 IgA1 60 100 100 87 80

(128) When study subjects were given a second dose of vaccine identical to their first, the increase in antibody titer after 14 days was relatively modest for those receiving the 300 g dose, with increases in the GMT for IgG and IgA1 of 1.8- and 2.0-fold. For those receiving the 30 g dose the GMT of IgG titers after 14 days increased 4.1-fold and the GMT for IgA1 increased 2.4-fold. At either dose level, the kinetics of the decrease in IgG and IgA1 titers appears to resume about the same rate as seen after the first dose (FIG. 1).

(129) Anti-Als3 IFN- and IL-17A Production by Stimulated PBMCs.

(130) FIGS. 3A and 3B provide an overview of the IFN- (FIG. 3A) and IL-17A (FIG. 3B) ELISpot results for subjects receiving the 30 g dose of vaccine. Subjects at each time point were segregated into non-responders and responders, depending on whether or not their result exceeded the empirical cutoff of 25 SFU per 10.sup.6 cells. For both IFN- and IL-17A, the maximum response was seen on day 28 post vaccination. The mean responses (SFU per 10.sup.6 cells) for responders in each assay were similar on days 7 to 180, with a significantly greater number of cells producing IFN- than IL-17A.

(131) FIGS. 4A and 4B provide an overview of the IFN- and IL-17A ELISpot results, respectively, for subjects receiving the 300 g dose of vaccine. In this case subjects receiving 300 g of Als3p responded more rapidly than those receiving 30 g Als3p, i.e., by day 7, and with a greater number of cells producing both cytokines. However, the drop off of response appeared to be more rapid, with a diminished response at day 90 post vaccination for both cytokines. Again, the IFN- producing cells far outnumbered the IL-17A cells in the responders at all time points.

(132) FIGS. 5A and 5B present IFN- and IL-17A ELISpot data, respectively, as the percent of positive responders for each group, including placebo recipients, at each time point. While this presentation reinforces the observations described above, it also makes clear that the IFN- response was more robust than the IL-17A response, with a greater percent of subjects responded at each time point and a greater difference from the placebo response. There was a more rapid rise in the percentage of both IFN- and IL-17A responders in the 300 g dose group versus the 30 g dose group on day 7 post-vaccination. However, by day 28 the 30 g dose group showed slightly higher response rates than seen in the 300 g dose group. Furthermore, administration of the second dose at day 180 markedly increased the percentage of responders, with the IFN- response elevated most by the 30 g dose and the IL-17A response elevated most by the 300 g dose.

(133) FIGS. 6A and 6B show the anti-Als3 IgG antibody levels in serum (FIG. 6A) and vaginal wash (FIG. 6B). FIGS. 6C and 6D show the anti-Als3 IgA1 antibody levels in serum (FIG. 6A) and vaginal wash (FIG. 6B). Responses were similar between IgG and IgA1. The 300 g with alum has 2-3 fold higher antibody responses than the same dose without alum, whereas the 30 g dose administered intradermally showed a similar antibody response to the same dose delivered intramuscularly (with alum). The 300 g dose with AlOH induced significantly higher titers that the 300 g dose without AlOH (Wilcoxon Rank Sum, p<0.001). The vaginal wash IgG and IgA1 titers correlated with the serum titers. Additionally, the mucosal antibody levels detected in vaginal washes reflect a very similar profile to that seen in plasma.

(134) FIGS. 7A and 7B show the functional activity of the anti-Als3 antibodies, as assessed by a C. albicans opsonophagocytic killing (OPK) activity assay. OPK activity correlated with anti-Als3 IgG and IgA1 antibody titers, indicating that the vaccine-induces anti-Als3 antibodies directed an immune response against C. albicans.

(135) FIGS. 8A and 8B show Th1 (FIG. 7A) and Th17 (FIG. 7B) T-cell stimulation by the Als3 antigen. The percent of subjects at Day 7 responding to the 300 g dose give IM, with or without AlOH in the formulation, were significantly higher than that seen for the 30 g Als3 dose given ID. By Day 14 or Day 90, the response to all doses and regimens reached similar levels for IFN- response. For the IL-17A response, the 300 g dose groups are at a maximum at Day 7 and appear to decline at Day 14 and 90. It appears that the 30 g dose given ID may have a low but more persistent activity for both cytokines.

(136) FIG. 9 is a graph comparing post-vaccination sera (light gray bars) to pre-vaccination sera (dark gray bars, normalized to 100%) of each individual administered the Als3 antigen. Three separate assays were performed:

(137) Adherence and Invasion Assays:

(138) Live Candida albicans is mixed with vaccinee sera and then incubated with human endothelial cells. In the presence of post-vaccination sera, the cell-associated (adhered) and endocytosed (invaded) Candida was reduced to 59% and 54%, respectively, of the pre-vaccination levels of adherence and invasion, (p<0.05 for post-versus pre-).

(139) Cell Damage Assay:

(140) This assay measured the cell damage to human epithelial cells by Candida, again comparing levels of protection by post- and pre-vaccination sera. The cell damage in the presence of post-vaccination sera was about 52% of that with pre-vaccination sera (p<0.05 for post-versus pre-).

(141) The blockage of adhesion and invasion of the vaginal mucosa is believed to be particularly important for preventing symptoms of vaginitis in patients with recurrent VVC. Anti-Als3 antibodies also exhibit the ability to target neutrophils to specifically recognize and engulf (phagocytize) Candida, as assessed using an opsonophagocytic killing (OPK) assay, an in vitro assay measuring the ability of sera from Phase 1 subjects post-vaccination to direct neutrophils to kill Candida. These data demonstrate that the anti-Als3 antibodies made in response to the NDV-3 vaccine were not only present in high amounts, but were also functionally active against Candida in vitro.

(142) FIG. 10 shows the geometric mean functional OPK activity (line) in post-vaccination subjects in each treatment group compared to the geomean concentration of anti-Als3 antibodies measured by ELISA among the three treatment groups in the Phase 1 (bars). The correlation between the antibody concentration and OPK activity demonstrates that anti-Als3 antibodies made in response to the NDV-3 vaccine were functionally active in directing neutrophils to kill Candida in vitro. These data demonstrate that the anti-Als3 antibodies made in response to the NDV-3 vaccine were not only present in high amounts, but were also functionally active against Candida in vitro.

(143) A Phase 1b study was carried out to estimate the effect of a single dose of NDV-3A, NDV-3 or placebo administered intramuscularly to patients (N=15 per group) diagnosed with recurrent vulvovaginal candidiasis (RVVC) on the following parameters. First, the safety and tolerability during the 12-months post vaccination period in the NDV-3A vaccine group, the NDV-3 vaccine group, and the placebo group were assessed. FIGS. 12A-12D show the antibody response results for comparison of the NDV-3 vaccine with the NDV-3A vaccine. FIGS. 11A and 11B show the difference between the NDV-3 vaccine and the NDV-3A vaccine in induction of anti-Als3 IgG antibody levels in serum (FIG. 11A) and vaginal wash (FIG. 11B). FIGS. 11C and 11D show the difference between the NDV-3 vaccine and the NDV-3A vaccine in induction of anti-Als3 IgA antibody levels in serum (FIG. 11C) and vaginal wash (FIG. 11D). Responses were similar between IgG and IgA1.

(144) The humoral and cellular immune responses at pre-defined time points over a 12-month period in the NDV-3A vaccine group, the NDV-3 vaccine group, and the placebo group were also assessed. FIGS. 13A-13B show the Th1 (FIG. 12A) and Th17 (FIG. 12B) T-cell stimulation by the Als3 antigen. The results show a comparison between the NDV-3 vaccine and the NDV-3A vaccine. The percent of subjects at Day 14 showing an IFN- response to the NDV-3 vaccine was about 20% higher than the percent of subjects treated with the NDV-3A vaccine. Both groups retained a high (about 60%) IFN- response rate at Day 90. The percent of subjects showing an IL-17A response was lower for both treatment cohorts, with the NDV-3A patients showing a slightly (about 10%) enhanced response at Day 90.

(145) FIG. 13 is a graph comparing the anti-Als3 IgG antibody titers in sera from the Phase 2a study with RVVC with healthy volunteers of the Phase 1a study. There are no remarkable differences between healthy volunteers and Phase 2a patients. Because RVVC patients tested positive for Candida albicans two weeks prior to vaccination (Day 0), the apparent lack of immune memory response to wild-type Candida infection is surprising. However, when recombinant Als3 is administered IM on alum, a memory response is triggered.

(146) Recurrence Studies

(147) Patients treated with NDV-3A were monitored for recurrence of RVVC and compared to that of placebo control patients. Time-to-first recurrence was monitored, as well as the cumulative number of recurrences at each time point. Recurrence was defined by several metrics and results were analyzed with respect to the corresponding recurrence definition. In some studies, recurrence was defined as the patient's observation of symptoms (patient symptom score). In other studies, recurrence was defined as the physician's observation of signs or symptoms of infection (sign/symptom score). In other studies, recurrence was defined as the physician's observation of signs or symptoms of infection in addition to a positive culture test (signs+symptoms & positive culture).

(148) FIGS. 14A-14C are Kaplan-Meier curves showing the proportion of non-recurrence of RVVC over time in NDV-3A-treated patients versus placebo controls. In studies defining recurrence by sign/symptom score among patients of all ages (FIG. 14A), 30 out of 88 (34%) NDV-3A-treated patients experienced disease recurrence over the course of 97 days, while 38 out of 82 (46%) placebo patients experienced disease recurrence. The significance level of the difference between the Kaplan-Meier recurrence curves is 0.10 and vaccine efficacy was 33%.

(149) In studies defining recurrence by patient symptom score among patients of all ages (FIG. 14B), 44 out of 88 (50%) NDV-3A-treated patients experienced disease recurrence over the course of 374 days, compared to 53 out of 82 (65%) placebo patients experiencing recurrence. The significance level of the difference between the Kaplan-Meier recurrence curves is 0.06 and vaccine efficacy was 31%. The lower significance value resulting from defining recurrence by patient symptom score indicates a greater statistical confidence in the results and, more broadly, indicates that the patient symptom score is a more accurate readout in comparison to the signs/symptoms score.

(150) In studies defining recurrence by patient symptom score among patients under the age of 40 (FIG. 14C), 32 out of 68 (47%) NDV-3A-treated patients experienced disease recurrence over the course of 374 days, compared to 40 out of 63 (63%) placebo patients experiencing recurrence. The significance level of the difference between the Kaplan-Meier recurrence curves is 0.02 and vaccine efficacy was 42%. These data show a markedly improved vaccine response in patients under the age of 40. Specifically, NDV-3A increases the time-to-first recurrence (p<0.05) and that the proportion of patients that remain recurrence free out to 12 months (cure rate) increases from about 30% in the placebo group to about 50% in the NDV-3A group. Placebo recipients are 2.5-times more likely to experience a recurrence within 3 months than the placebo group (p<0.05). The odds ratios for first recurrence measured by each scoring metric at the 3-month visit are shown in Table 3, and the odds ratios for first recurrence measured by patient symptom score for each patient cohort at each time point are shown in Table 4, below.

(151) TABLE-US-00008 TABLE 3 Odds ratios for first recurrence at 3-month visit. Number of Signs + subjects symptoms >2 & Signs + included positive culture symptoms >2 Symptoms >2 All subjects 170 0.90 (0.47, 1.75) 0.60 (0.32, 1.11) 0.54 (0.29, 1.01) Subjects <40 139 0.60 (0.42, 1.91) 0.53 (0.26, 1.07) 0.43 (0.21, 0.88) years old Subjects 40 31 0.93 (0.24, 3.62) 0.92 (0.25, 3.42) 1.14 (0.31, 4.16) years old

(152) TABLE-US-00009 TABLE 4 Odds ratios for first recurrence measured by patient symptom score. Odds ratio for first recurrence 95% confidence Age group by visit (vaccine/placebo) interval At 3-month visit All subjects under 40 years of age 0.43 (0.21, 0.88) All subjects 40 years or older 1.14 (0.31, 4.16) All subjects 0.54 (0.29, 1.01) At 6-month visit All subjects under 40 years of age 0.63 (0.32, 1.26) All subjects 40 years or older 1.67 (0.46, 6.05) All subjects 0.79 (0.43, 1.44) At 12-month visit All subjects under 40 years of age 0.54 (0.27, 1.09) All subjects 40 years or older 0.86 (0.23, 3.26) All subjects 0.60 (0.32, 1.11)

(153) FIGS. 15A-15D are cumulative recurrence graphs showing the mean cumulative number of recurrent episodes in NDV-3A-treated patients versus placebo controls. In studies defining recurrence by sign/symptom score among patients of all ages (FIG. 15A), NDV-3A-treated patients experienced fewer cumulative recurrences compared to placebo controls at a significance level of 0.15. In studies defining recurrence by sign/symptom score among patients under 40 years of age (FIG. 15B), NDV-3A-treated patients experienced fewer cumulative recurrences compared to placebo controls at a significance level of 0.02. These results reflect the non-recurrence trends between NDV-3A-treated patients and placebo controls. Specifically, lower significance levels of the under 40-years old cohort relative to all ages indicates that patients under 40 are more likely to experience fewer cumulative recurrences as a result of the vaccine when recurrence is measured by signs/symptom score.

(154) In studies defining recurrence by patient symptom score among patients of all ages (FIG. 15C), NDV-3A-treated patients experienced fewer cumulative recurrences compared to placebo controls at a significance level of 0.20. In studies defining recurrence by sign/symptom score among patients under 40 years of age (FIG. 15D), NDV-3A-treated patients experienced fewer cumulative recurrences compared to placebo controls at a significance level of 0.03. The mean number of recurrences per patient over a 12-month period was reduced by about 30% in response to NDV-3A treatment.

(155) The onset of RVVC occurs mainly in subjects less than 40 years of age. In a study conducted on our behalf, we determined that, at the first age of onset of RVVC, about 90% of the subjects (n=127) were less than 40 years old. The studies presented herein clearly show that patients under the age of 40 years are more responsive to NDV-3A treatment in terms of time-to-first recurrence and number of cumulative recurrences. To determine whether this difference corresponded with a difference in antibody response to the vaccine between the two age cohorts, antibody titers were quantified over time and compared. FIG. 16 is a graph showing the results of the titer comparison. Vaccine recipients under the age of 40 years did not show a significantly higher titer response than vaccine recipients over the age of 40 years.

Example 2: Use of a Kit to Treat Vulvovaginal Candidiasis

(156) A female patient presenting with symptoms including one or more of heavy white curd-like vaginal discharge, a burning sensation in the vagina and vulva and/or an itchy rash on the vulva and surrounding skin can be diagnosed at a point of care location (e.g., a physician's office or a hospital) as having vulvovaginal candidiasis (VVC). The physician can retrieve a kit that includes instructions for use and two containers: a) a container containing an Als3 polypeptide (e.g., Als3-2, Als3-1, Als3 (18-324), and/or Als3 (Ser/Thr-rich sequence)), e.g., in the form of a vaccine composition including an adjuvant, such as, for example, aluminum hydroxide (e.g., the Als3p vaccine may be formulated at a dose of 600 g/ml), and b) a container containing an antifungal agent (e.g., fluconazole, e.g., in the form of a tablet for oral administration and at a dose of, e.g., 50, 100, 150, or 200 mg per tablet). The physician can retrieve a first dose of the anti-fungal agent (e.g., fluconazole) from the kit and administer it to the patient or instruct the patient to self-administer the first dose as soon as possible. Subsequently, or prior to administration of the anti-fungal agent, the physician can administer a volume (e.g., 0.5 ml) of the Als3 vaccine to the patient to provide a total dose of 60-500 g (e.g., 300 g) of the vaccine. The kit allows the patient to begin the antifungal agent course of therapy immediately without having to visit a pharmacy to fill a prescription. This is more convenient for the patient and can lead to higher compliance since they receive the initial doses of antifungal agent and vaccine during the visit, as well as extra instructions from their healthcare provider for subsequent doses.

(157) If the patient is diagnosed with uncomplicated VVC, the physician may send the patient home with instructions to monitor the condition for improvement and to seek additional medical care if symptoms do not improve within 1-3 days. If the patient is diagnosed with complicated VVC, the physician may provide the container containing the anti-fungal agent to the patient with instructions and an amount of the anti-fungal agent sufficient for the patient to take a 150 mg tablet by mouth every 72 hour for 3 doses or more. If the patient is diagnosed with recurrent VVC, the physician may provide the container containing the anti-fungal agent to the patient with instructions and an amount of the anti-fungal agent sufficient for the patient to take a 150 mg tablet by mouth every third day until they have taken three doses total. Additional doses may be included in the kit, if necessary, or the patient may be given a prescription that includes a refill.

OTHER EMBODIMENTS

(158) All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

(159) Other embodiments are in the claims.

(160) TABLE-US-00010 APPENDIX 1 VVC Signs and Symptoms Questionnaire (example) VVC Signs and Symptoms Questionnaire Score*(circle one) Subject Symptom Evaluation (previous 24 hours) Vaginal itching 0 1 2 3 Vaginal irritation 0 1 2 3 Vaginal burning 0 1 2 3 Investigator VVC Evaluation (on gynecological exam) Vulvar or vaginal erythema 0 1 2 3 Vulvar or vaginal edema 0 1 2 3 Vulvar or vaginal excoriation/fissure formation 0 1 2 3 Total Composite Questionnaire Score (Subject and Investigator): (Sum of scores) *Score Key: 0 = Absent, 1 = Mild, 2 = Moderate, 3 = Severe Composite Questionnaire Score Key: 0-2 = not clinically indicative of VVC (asymptomatic) 3-6 = Mild Disease 7-12 = Moderate Disease 13 = Severe Disease