Description:FIELD OF THE INVENTION:
[001] The present invention relates to a generation of a genetically engineered live attenuated vaccine strain against Candida infections.

BACKGROUND OF THE INVENTION:
[002] Candidemia is a bloodstream fungal infection caused by Candida species, majorly by Candida albicans, and is frequently observed in ICU patients [1-3]. It represents the 4th and 6th most common healthcare-associated bloodstream infections in the USA and Europe, respectively [4, 5]. It is estimated to afflict nearly 400,000 patients per year worldwide [6]. The late and poor diagnosis of invasive candidiasis has only contributed to the rise in multi-organ failures by septicaemia-associated fatality. Despite usage of existing antifungal drugs, mortality rate is very high (40–50%). In addition, superficial mucosal Candida infections (candidiasis) is equally dangerous. Majority of women experience vulvovaginal candidiasis (VVC) at least once in their lifetime and unfortunately, many of them suffer from recurrent VVC (RVVC). While there is no definitive cure, antifungal drug therapy is the only available treatment for RVVC. However, due to the limited number of existing antifungal drugs, their associated side effects and the increasing occurrence of drug resistance, other approaches are greatly needed [7]. An obvious prevention measure for candidemia or RVVC relapse would be to immunize at-risk patients with a vaccine effective against Candida infections. In spite of the advanced and proven techniques successfully applied to the development of antibacterial or antiviral vaccines, however, no antifungal vaccine is still available for human use [8].
[003] Live attenuated vaccine is a whole of pathogen that has just been attenuated by genetic manipulation. It causes a very mild infection that ensures a long-lasting protective immune memory. Typically, live vaccines lead to a stronger, more prolonged and robust immune response than the inactivated (like heat-killed) vaccines. Live attenuated vaccine strategy has been successfully used against bacteria and viruses [9, 10]. Such a vaccine against fungal infections is yet to be demonstrated [6, 11]. However, several reports exist to demonstrate attenuated strains of C. albicans that provide no or mild protection against candidiasis. For example, PCA-2 is a mutant derivative of 3153A strain. An adoptive transfer of macrophage cells from PCA-2 immunised mice conferred substantial protection against subsequent infection [12]. Another study reported that CNC13 (deleted for HOG1) strain was able to protect ~60–70% of mice to a subsequent lethal dose wild-type challenge [13]. Similarly, a double-deletion mutant cph1?/efg1? C. albicans is avirulent and its immunization only partially protects mice from systemic infections. Even its booster dose did not improve the degree of protection [14]. Therefore, a potent live attenuated vaccine strain is yet to be identified that will be safe and highly protective against Candida infections. Based on the studies of host-fungal interaction, two potential peptide vaccines have been reported to be effective and safe in initial clinical trials [15, 16]. NDV-3A (alum as an adjuvant and the N-terminus of a recombinant Als3 protein of C. albicans as antigen) is the first vaccine to demonstrate preclinical efficacy in protecting from diseases caused by both fungal and bacterial pathogens (novadigm.net). The phase 2 randomized, double-blind, placebo-controlled clinical trial has also been recently completed, and the study found that a single dose of NDV-3A was also safe and effective in patients with recurrent vulvovaginal candidiasis (RVVC), and the vaccine reduced the frequency of symptomatic episodes of vulvovaginal candidiasis for up to 12 months in women aged <40 years. Another vaccine being conducted in the clinical trial (Phase I) is on the Sap2 protein of C. albicans in virosomal formulation against RVVC (PEV7, Pevion Biotech AG, Switzerland). It appears that PEV7 was safe, effective, and specific and functional B cell memory in 100 % of vaccinated individuals were detected (www.pevion.com). The clinical studies taking larger cohorts including immunosuppressed individuals or at least to individuals receiving corticosteroids and antibiotics are yet to be carried out. Secondly, these potential vaccine candidates seem to protect superficial infection and not blood stream infections, those are more fatal. To sum of it, till today there is no approved vaccine against any fungal infections for clinical use.

OBJECTS OF THE INVENTION:
[004] It is therefore an object of the present disclosure to propose a genetically engineered Candida albicans as a potential whole cell vaccine candidate for treating fungal blood stream infection.

[005] Another object of the present disclosure is to generate a knockout strain with homozygous deletions of POL32 gene in C. albicans.

[006] Yet another object of the present disclosure is to characterize its morphological transition, genome stability, genome sequencing, and macrophage interaction.

[007] Still another object of the present disclosure is to decipher the pathogenic and vaccine potential of the knockout strain in the development of and protection from systemic candidiasis in in vivo model.

[008] These and other objects and advantages of the invention will be apparent from the ensuing description and the accompanying drawings.

SUMMARY OF THE INVENTION:
[009] Accordingly, the present invention provides a genetically engineered Candida albicans, comprising a target gene involved in DNA replication.
[0010] In one of the feature of the present invention, the target gene is POL32.

[0011] In another feature of the present invention, vectors for producing genetically engineered Candida albicans, having SAT1 flipper cassette comprising SAT1 encoding nourseothricin resistance gene under the control of a Maltose promoter and a recombinase FLP gene is provided. The vector comprises the upstream and downstream sequences of POL32 gene flanking the SAT1 flipper cassette.

[0012] In yet another feature of the present invention, set of forward primers and a set of reverse primers of the mentioned genes for producing genetically engineered Candida albicans are provided.

[0013] In still another feature of the present invention, a method of producing a genetically engineered Candida albicans by targeting the mentioned gene is provided.

[0014] In yet another feature to demonstrate avirulency of the knockout strain and compare its pathogenicity with the wild type C. albicans in an in vivo model.

[0015] In yet another feature to demonstrate ability to protect pathogenic challenge by immunization of mice with the knockout strain via different routes.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
[0016] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:

[0017] Figure 1 (a) illustrates two deletion vectors pNA1554 and pNA1559 generated for POL32 gene deletion in C. albicans. (b) illustrates SAT1 flipper cassettes in deletion vector constructs pNA1554 and pNA1559.

[0018] Figure 2 (a) is a representative flow chart depicting generation of CNA25 C. albicans strain. (b) illustrates deletion of both the alleles of POL32 gene in CNA25 strain of C. albicans by PCR.

[0019] Figure 3 illustrates phenotypic comparison between wild type (WT) and genetically modified knockout (CNA25) strains of C. albicans. (a) Susceptibility of strains of C. albicans to temperature (Temp.), hydroxyurea (HU), ultra violet radiation (UV), methyl methanesulfonate (MMS), Tert-Butyl hydroperoxide (TBHP), and cisplatin; (b) Chromosomal instability assay by pulse field gel electrophoresis; and (c) loss of heterozygosity (LOH) assay by 5FOA counter selection method.

[0020] Figure 4 chromosomal map of CNA25 strain of C. albicans determined by YMAP. YMAP (Yeast Mapping Analysis Pipeline) analyses genome sequences to map copy number variations (CNV), single nucleotide polymorphisms (SNPs) and LOH. The X axis represents the chromosomal distance, with 0.2 representing 200 KB. A graph on the left of the chromosome represents allelic ratio. The right hand side number represents the ploidy of that specific chromosome, i.e. 2 for two alleles, 1 for a single allele, and any numbers between >1 and <2 represent cases of aneuploidy. The regions with copy number variations are depicted as prominent black histograms along the length of the chromosome. The dots on the X-axis denotes the positions of the major repeat sequences (MRS). Heterozygous SNPs) are shown as vertical grey bars in the background of each chromosome, and increasing shades of dark grey indicate regions with higher numbers of SNPs. Homozygous SNPs are displayed in cyan or magenta for different alleles.

[0021] Figure 5 depicts morphological transition of WT and CNA25 strains of C. albicans. (a) Filamentation induced on serum containing YPD + agar and spider media; (b) Germ tube induced by 10% serum in liquid YPD media and estimation of germ tubes length.

[0022] Figure 6 shows immunological discrimination of C. albicans strains. (a) WT strain induces macrophages killing more efficiently than CNA25 as evident from increased propidium iodine staining of immune cells; (b) Macrophages clear CNA25 more effectively than WT as less number of CNA25 as opposed to WT C. albicans cells were recovered from macrophages by colony forming units assay.

[0023] Figure 7 shows systemic candidiasis in mice model. (a) Avirulency of CNA25 strain of C. albicans in BALB/c mice; (b) Fungal death caused by WT C. albicans in vital organs of mice kidneys, liver and spleen; and (c) further confirmed by PAS staining of kidney sections.

[0024] Figure 8 shows CNA25 as a vaccine candidate. (a) Immunisation of CNA25 strain via different routes protects WT C. albicans challenge, (a) immunisation of CNA25 through intravenous; (b) immunisation of heat killed CNA25 through intravenous; (c) immunisation of CNA25 through subcutaneous; and (d) immunisation of CNA25 through feeding.

DETAILED DESCRIPTION OF THE INVENTION:
[0025] The term “knock–out strain” implies a strain in which selected gene of the organism is made inoperative or deleted. In the context of the present disclosure the gene is knocked out or deleted from the genome of the organism.

[0026] The term “upstream fragment” implies 5’ end of a DNA strand and “downstream fragment implies the 3’end of the strand of a gene.

[0027] The term “live attenuated” implies a disease-causing microorganism that is weakened in a laboratory so it cannot cause disease. Live attenuated microorganisms are often used as vaccines because, although weakened, they can stimulate a strong immune response.”

[0028] The term “homozygous deletion of gene” implies the deletion of gene in both the copies of the chromosome. In the context of the present disclosure C.albicans has a diploid genome.

[0029] According, to the present disclosure there is provided a vaccine against Candida albicans, characterized in the vaccine comprises a genetically engineered live attenuated strain of Candida albicans.

[0030] The vaccine comprises the strain in phosphate buffer saline at a physiological pH.

[0031] The genetically modified Candida albicans strain is a knock out strain comprising homozygous gene deletion of POL32 gene, a subunit gene of DNA polymerase delta that promotes DNA replication in wild type Candida albicans.
[0032] In eukaryotes, DNA replication is coordinated by three essential DNA polymerases Pol?, Pol?, and Pol?. Extensive genetic analyses in Saccharomyces cerevisiae suggest that Pol? is involved in only leading strand DNA synthesis, whereas Pol? synthesizes both leading and lagging strands of DNA [17, 18]. Pol?-primase provides the RNA-DNA primer to initiate DNA replication from the origin. In addition, Pol? is also required for base and nucleotide excision repair, break induced replication and homologous recombination. Pol? holoenzyme from S. cerevisiae consists of three subunits: the catalytic subunit Pol3 and the accessory structural subunits Pol31 and Pol32 proteins [19]. Pol? has not been characterized yet in pathogenic yeast Candida albicans. According to Candida genome database, Pold possesses genes encoding all three subunits: POL3 (C7_02790C_A/B and orf19.5182), POL31 (C5_04740C_A/B and orf19.3960), and POL32 (C1_05850W_A/B and orf19.2465). Similar to S. cerevisiae, CaPold most likely is a holoenzyme of three subunits. Although POL32 is dispensable in S. cerevisiae, POL32 null strain exhibits severe defects in DNA replication, repair, and mutagenesis [20].

[0033] The present disclosure delineates the role of POL32 gene in genome stability and pathogenicity of C. albicans.

[0034] By using homologous recombination strategy and a SAT1 flipper cassette [21, 22], a nourseothricin marker gene (SAT1) was inserted into POL32 gene in the chromosome. This facilitated loss function of POL32 genes. After obtaining the knockout strain, its morphological, growth, ability to maintain genome stability, other phenotypes and pathogenicity were compared those with the parental C. albicans strain (SC5314).

[0035] In another embodiment of the present disclosure, a method of producing a genetically engineered C. albicans strain CNA25 via homologous recombination strategy and a SAT1 flipper cassette is provided.

[0036] The said method comprises the steps of amplification of the upstream and downstream fragments of POL32 gene by Taq DNA polymerase in a 100?L PCR reaction; preparation of deletion construct vectors pNA1554 and pNA1559; followed by amplification of POL32 –SAT1 cassette from the deletion construct vectors pNA1554 and pNA1559 and final transformation of the linearly digested DNA fragments to wild type SC5314 C. albicans strain by modified lithium acetate. The deletion of target gene POL32 by SAT1 flipper cassette by homologous recombination of the gene specific sequence present in the deletion constructs and the chromosomal locus. A detailed explanation of the method is disclosed in the working examples.

[0037] In another embodiment of the present disclosure, vectors comprising SAT1 nourseothricin resistance gene (SAT1) under the control of a Maltose promoter and a recombinase FLP gene whose expression is driven from the Maltose promoter gene are provided. The flanking sites of the vectors comprise the amplified upstream and downstream sequences of POL32. Figure 1(a).

[0038] In yet another embodiment of the present disclosure, primers for amplifying and confirmation of cloned upstream and downstream sequences of specific genes from SC5314 genomic DNA are provided.
EXAMPLES:
[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0040] Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

Materials and methods:
[0041] The source and geographical origin of the biological materials used in the present invention are given below:

[0042] The wild type strain Candida albicans SC5314 used in the present disclosure is: Candida albicans (Robin) Berkhout (ATCC MYA-2876).

[0043] The genetically engineered Candida albicans has been deposited at Microbial Type Culture Collection and Gene Bank (MTCC) , Chandigarh.

[0044] The vector constructs for target genes were synthesized in the laboratory. Primer sequences were designed based on the genome sequence of SC5314 strain in the Candida genome database.
[0045] The plasmid pSFS2 was obtained from the Institute for Molecular Infection Biology, Germany.

[0046] Restriction enzymes, customised primers, 5FOA, nourseothricin antibiotics were purchased from NEB, IDT and Biotech Desk, respectively.

Example I:
[0047] PCR Amplification of the upstream and downstream fragments of Genomic DNA isolated from Candida albicans SC5314 and construction of plasmids pNA1554 and pNA1559

[0048] C. albicans possesses a diploid genome. To facilitate efficient homologous deletion in C. albicans, a modified protocol as described before by the inventors were employed [21].

[0049] Two deletion constructs for POL32 gene was generated with different length of upstream sequences but having a common downstream sequence (Figure 1).
[0050] Primer sequences were designed based on the genome sequence of SC5314 strain in the Candida genome database.

[0051] First, the downstream fragment of Sequence ID No. 1 of the POL32 gene was amplified from SC5314 genomic DNA by using primers NAP311 of Sequence ID No.4 and NAP407 of Sequence ID No.5 and cloned into the SacI site of vector pSFS2. Two upstream sequences of Sequence ID No. 2 and Sequence ID No.3 were amplified by using forward primer NAP404 of Sequence ID no. 6 with reverse primers NAP405 of Sequence ID No.7 and NAP406 of Sequence ID No.8, respectively, and then cloned into the KpnI-XhoI sites of the same vector which possessed the downstream sequence of Sequence ID No.1 of the POL32 gene to generate constructs pNA1554 and pNA1559, respectively (Figure 1).

[0052] The following PCR condition was used for the amplification of the upstream and downstream fragments of POL32 gene by Taq DNA polymerases in a standard 100?L PCR reaction.
[0053] Initial denaturation- 95°C for 1 min.
[0054] Denaturation-95°C for 15 sec.
[0055] Annealing-52°C for 30 Sec.
[0056] Extension-72°C for 30 sec.
[0057] Final Extension-72°C for 3 min.
[0058] No. of Cycles-35
[0059] In these constructs, the SAT1 antibiotic marker is flanked by upstream and downstream gene sequences of POL32 gene (Figure 1).
Example II:
[0060] C. albicans transformation and construction of genetically engineered C. albicans knock out strain

[0061] Using the plasmid constructs in Example I, a homozygous deletion of POL32 gene was obtained and the strain was coined as CNA25 strain. A flow chart describing the gene deletion strategy is given (Figure 2).

[0062] The methodology used for DNA transformation to Candida albicans is described. A single colony of C. albicans was inoculated in 10ml YPD and grown overnight at 300C 180 rpm followed by inoculation of 50ml YPD medium with the overnight grown culture (1%) and grow at 30 0C until the O.D.600 reaches at 0.8-1.0. This is followed by spinning at 4000 rpm for 5 min at room temperature to pellet down the cells, Thereafter the pellet is washed in 20 ml sterile water followed by LiOAc/TE solution. The pellet is resuspended in 3 volumes of the pellet in LiOAc/TE solution (approx 300?l). The cell suspension is then aliquot in 100?l aliquots for each transformation and the transforming DNA is added. 700?l of PEG solution (filter sterilized) is added and the mixture is incubated at 300C overnight (12-14hrs). Further, heat shock is provided at 420C for 45 min. The pellets were obtained by centrifuging the cells at 4000 rpm for 5min at room temperature. PEG solution is discarded and the pellet is re-suspended in 1ml YPDU (3hour) at 300 C. This is then centrifuged at 3000rpm for 5min at room temperature. The pellet is resuspended in 100ul of sterile water and plate on YPDU+agar plates with nourseothricin antibiotic (100?g/ml) selection marker. The plates are incubated at 300C for 2-3 days to obtain the transformants.

[0063] The methodology used for homozygous gene deletion in C. albicans is described briefly. Briefly, the POL32-SAT1 cassettes were amplified from pNA1554 or pNA1559 using primers NAP404 and NAP407 and transformed into wild type SC5314 C. albicans strain by a modified lithium acetate method at different stages to obtain homozygous deletion of both the copies of POL32 gene [21]. The following PCR condition was used for the amplification of POL32-SAT1 deletion cassettes by Q5 DNA polymerases in a standard 100?L PCR reaction.
[0064] Initial denaturation- 98°C for 1 min.
[0065] Denaturation-98°C for 15 sec.
[0066] Annealing-52°C for 30 Sec.
[0067] Extension-72°C for 3 min.
[0068] Final Extension-72°C for 9 min.
[0069] No. of Cycles-35

[0070] The PCR fragments were cleaned up using GeneJet purification kit from ThermoFisher and transformed to C. albicans as described above. A flowchart of transformation of two different DNA fragments at different time points have been given (Fig. 2). First, the fragment from shorter upstream deletion construct (pNA1554) was transformed into C. albicans cells and selected the transformants on YPD+ nourseothricin (SAT1 encodes for nourseothricin (NAT)) plate. The transformed colonies were screened for integration of deletion cassette to the targeted locus by colony PCR using respective gene specific upstream forward and NAP336 reverse primers. NAP336 of Sequence ID No. 9 (5’-GAA ATC CAG ACA GTC GAG-3’) hybridizes near the MAL2 promoter of the pSFS2 vector. The positive colonies showing the integration of deletion cassette were inoculated in 2ml of 5% YPM for 3-5 days for curing of the cassette. Maltose induces the expression of FLIP recombinase to remove the SAT1 sequence from the genome leaving behind the FRT sequence in the integrated locus. This will facilitate curing of the NAT marker, so that fresh round of transformation can be done with other SAT1 cassettes. The curing of deletion cassette in strains were further confirmed by streaking on YPD media without and with NAT presence. Then colony PCR with ORF specific primers were performed to check deletion. Presence of two PCR products of (one corresponding to a full-length and second smaller one for a deletant) ensured heterozygous deletion in the strain. To this heterozygous deletion strain of C. albicans, fragment of longer upstream deletion cassette (pNA1559) was transformed and steps from screen of the transformed colonies for integration of deletion cassette to the targeted locus by colony PCR for confirmation of deletion were followed to obtain POL32 homozygous deletion (CNA25). The following PCR condition was used for the confirmation of POL32 gene knockout by Taq DNA polymerase in a standard 50?L PCR reaction. Initial denaturation- 95°C for 1 min, Denaturation-95°C for 15 sec, Annealing-52°C for 30 Sec, Extension-72°C for 30 sec, Final Extension-72°C for 3 min, No. of Cycles-35. The primer sets used for the confirmation of POL32?? are – NAP39 of Sequence ID No. 10 and NAP40 of Sequence ID No. 11.

[0071] In conclusion, Maltose inducible Flip-recombinase facilitates recycling of SAT1 cassettes; and two-steps recycling by two different knockout constructs of same gene prompts efficient deletion. In each steps various gene deletions were confirmed by orf specific primers and DNA sequencing.

Example III:
[0072] Growth and genotoxic stress sensitivity analyses of CNA25 strain
[0073] Spot analyses suggested that CNA25 is a high temperature sensitive strain which also exhibits hypersensitive to genotoxic agents such as hydroxyurea (HU), cisplatin, UV radiation, MMS and TBHP in comparison to WT strain of C. albicans (Figure 3 a). Pulse field gel electrophoresis analysis of their total chromosomal DNA did not reveal any noticeable chromosomal alterations (Figure 3 b). However, spontaneous LOH increased by ~5 folds in CNA25 than the WT strain (Figure 3 c) suggesting genome instability in POL32 null strain due to replication stress.

Example IV:
[0074] Whole genome sequencing of CNA25
[0075] Genomic DNA was isolated using phenol-chloroform method, and after quality and quantity checked, libraries were constructed in alignment with microbial whole genome sequencing recommendations of NexteraTM DNA flex library preparation Kit from Illumina Inc. After QC clearance of the libraries, they were diluted to 4 nM, pooled, spiked with 5% PhiX pre-made library from Illumina and loaded on a MiSeq v3 kit. Sequencing was performed for 2X150 cycles. The original raw data obtained from Illumina MiSeq as FASTQ files (contain paired endreads sequences) was processed to obtain adapter free reads from MiSeq. The raw reads were mapped to the publicly available reference genome of C. albicans SC5314 (assembly ASM18296v3) using bowtie2 to calculate overall genome coverage. The overall alignment rate was found to be satisfactory to proceed with variant calling (95.68%). Copy number and allele status was visualized using YMAP [23]. Out of 8 chromosomes, Chr 3 looks very stable. Several cyan and magenta lines in CNA25 strain are indicative of loss of heterozygosity and gain of homozygosity accumulated due to faulty DNA replication. Such variations are more evident in Chr 7, and interestingly, localized segmental aneuploidy was also observed in the larger arm of this chromosome (chromosome number is estimated to lie between 1.2 and 2) (Figure 4).

Example V:
[0076] Morphological analysis
[0077] C. albicans is a multimorphic fungus and morphological transition is considered as one of the critical determinants of its virulence. Normally round shaped yeast cells are exclusively found in healthy individuals, however, a mixture of blastoconidia, pseudohyphae, and hyphae forms are frequently found in infected patients. Thus, strains of C. albicans that undergo morphological transitions have been shown to be pathogenic than those that locked in any of the morphological structures. Serum and spider media induce filamentation in wild type cell. While the wild type C. albicans cells switched their morphology in serum and spider media, CNA25 exhibited reduced filamentation both in solid and liquid cultures (Figure 5 a and b).

Example VI:
[0078] Macrophage-fungal interaction
[0079] To examine the virulence of CNA25, murine macrophages and fungal cells were co-cultured to measure clearance by macrophages and cell death of macrophages induced by fungal invasiveness. Upon encountering C. albicans, macrophages first engulf the pathogen and deliver it to the phagolysosome for pathogen clearance. However, as a counteractive mechanism, C. albicans kills macrophage for their survival and evades innate immunity. We determined the killing of macrophages by PI staining and fungal survival by estimating colony formation units (Figure 6 a and b). As PI stain only enters to the cells with a leaky membrane, an attribute of dead cells, PI positives cells were observed under the fluorescence microscope. At similar culture conditions, within 2 hrs of incubation a high percentage of PI stained macrophages (~14%) were observed when wild type C. albicans were co-cultured. The population of PI positive RAW cells in CNA25 co-culture was same as the control experiment where no fungal cells were added to macrophages (~6%). Consequently, CFU analysis suggested that higher percentage of wild type cells evaded immune system and got escaped from macrophages, whereas CNA25 C. albicans cells could not escape the macrophage killing. Thus, CNA25 is less virulent than WT C. albicans cells.

Example VII:
[0080] Pathogenicity of CNA25 strain
[0081] The intravenous challenge of C. albicans in murine models causes systemic candidiasis and mice succumb to death due to fungal sepsis [24]. To determine the virulence and pathogenicity of CNA25 strain of C. albicans, BALB/C male mice (n-6 x 2) were injected with a fungal dose of 5 x 105 CFU per mouse via the lateral tail vein, and monitored their survival for a period of 30 days. While all animals succumbed to death within 12 days of inoculation in wild type C. albicans injected group, 100 % of mice survived upon CNA25 inoculated group (Figure 7 a, b, and c). The CNA25 strain injected group of mice survived as good as the saline control injected group. Microscopic examination of PAS stained autopsy of kidney sections obtained from the wild type infected BALB/c mice revealed the presence of C. albicans cells in the kidney, and similarly, CFU of C. albicans cells was estimated to be very high in kidneys than liver and spleen. Thus, we claimed that CNA25 is an avirulent strain of C. albicans.

Example VIII:
[0082] Immunization by CNA25 strain

[0083] To determine the vaccine potential of CNA25 strain, BALB/C male mice (n-6) were inoculated with fungal dose of 6 x 106 CFU per mouse via various modes like intravenous with live cells (a), intravenous with heat killed cells (b), subcutaneous with live cells (c), and oral administration with live cells (d), and monitored their survival for a period of 30 days. Control immunised groups of mice were generated as required. After 30 days of immunisation (1?), these mice were subjected to lethal dose of WT challenge by intravenous inoculation (2?) and monitored survival rates (Figure 8). Except the group of mice immunised with live CNA25 strain of C. albicans by I.V. mode, rest of the mice succumbed to death. Oral route administration of CNA25 significantly delayed the death. This result suggested that I.V. administration of live CNA25 provides enough immune response to protect the pathogenic challenge of C. albicans. Thus, we claimed that CNA25 is a potential whole cell vaccine candidate.

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[0083] The crux of the invention is claimed implicitly and explicitly through the following claims.
, Claims:WE CLAIM:

1. A vaccine against Candida albicans, characterized in the vaccine comprises a genetically engineered live attenuated strain of Candida albicans wherein the strain is a knockout strain with a homozygous gene deletion of POL32 gene, a subunit gene of DNA polymerase delta.

2. The vaccine as claimed in claim 1, wherein the POL32 gene promotes DNA replication in wild type Candida albicans.

3. A vector, pNA1554, for producing genetically engineered Candida albicans as claimed in claim 1, comprising:
(i) POL32 upstream shorter sequence of 451 base pair, represented by Sequence ID No.2 flanked between KpnI-XhoI site;
(ii) POL32 downstream sequence of 451bp, represented by the Sequence ID No.1 flanked between SacI site;
(iii) SAT1 flipper cassette, said cassette comprising SAT1 nourseothricin resistance gene under the control of a Maltose promoter and a recombinase flippase gene flanked by short flippase recognition target sites.

4. A vector, pNA1559, for producing genetically engineered Candida albicans as claimed in claim 1, comprising:
(i) POL32 upstream longer sequence of 591base pair, represented by the Sequence ID No.3 flanked between KpnI-XhoI site;
(ii) POL32 downstream sequence of 451bp, represented by the Sequence ID No.1 flanked between SacI site;
(iii) SAT1 flipper cassette, said cassette comprising SAT1 nourseothricin resistance gene under the control of a Maltose promoter and a recombinase flippase gene flanked by short flippase recognition target sites.
5. A set of forward primers represented by Sequence ID No. 4, Sequence ID No.6, Sequence ID No.9, Sequence ID No.10 and a set of reverse primers of Sequence ID No.5, Sequence ID No.7, Sequence ID No.8, Sequence ID No.11 for producing genetically engineered C. albicans as claimed in claim 1.

6. A method for producing the live attenuated vaccine as claimed in claim 1, wherein the method comprising the steps of:
(i) amplification of the upstream and downstream fragments of POL32 gene by Taq DNA polymerase in a 100?L PCR reaction comprising 35 cycles, with initial denaturation at 95°C for 1 min, followed by denaturation at 95°C for 15 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds and final Extension at 72°C for 3 min.
(ii) preparation of deletion construct vectors pNA1554 and pNA1559 as claimed in claims 3 and 4.
(iii) amplification of POL32 –SAT1 cassette from the deletion construct vectors pNA1554 and pNA1559 using primers NAP404 of Sequence ID No. 6 and NAP407 of Sequence ID No. 5 by Q5 DNA polymerases in standard 100?L PCR reaction comprising 35 cycles with initial denaturation at 98°C for 1 minute, denaturation at 98°C for 15 seconds , annealing at 52°C for 30 Sec, extension at 72°C for 3 min followed by final extension at 72°C for 9 min.
(iv) transformation of the linearly digested DNA fragments to wild type SC5314 C. albicans strain by modified lithium acetate.
(v) deletion of target gene POL32 by SAT1 flipper cassette by homologous recombination of the gene specific sequence present in the deletion constructs and the chromosomal locus.

7. The method as claimed in claim 6, wherein the deletion vector constructs are prepared by:
(i) amplification of the downstream fragment of Sequence ID No. 1 of the POL32 gene from SC5314 genomic DNA in step (i) of the method as claimed in claim 6 by using primers NAP311 of Sequence ID No.4 and NAP407 of Sequence ID No.5 followed by cloning into the SacI site of vector pSFS2.
(ii) amplification of two upstream sequences of Sequence ID No. 2 and Sequence ID No.3 of the POL32 gene from SC5314 genomic DNA in step (i) of the method as claimed in claim 6 , by forward primer NAP404 of Sequence ID no. 6 with reverse primers NAP405 of Sequence ID No.7 and NAP406 of Sequence ID No.8, respectively, followed by cloning into the KpnI-XhoI sites of the same vector which possessed the downstream sequence of Sequence ID No.1 of the POL32 gene generating constructs pNA1554 and pNA1559, respectively.

8. The method as claimed in claim 6, wherein the transformation of wild type C. albicans and homozygous deletion of POL32 comprises the steps of:
(i) transforming the amplified fragment from the shorter upstream deletion cassette pNA1554 by modified lithium acetate and selecting the transformants on YPD+ nourseothricin plate;
(ii) screening of the transformed colonies for integration of the deletion cassette to the targeted locus by colony PCR using gene specific upstream forward and NAP336 reverse primer of Sequence ID No. 9;
(iii) inoculation of the positive colonies showing integration of the deletion cassette in 2ml of 5% YPM for 3 to 5 days for curing of the cassette;
(iv) transforming and screening of longer upstream deletion cassette from pNA1559 using steps (i) to (iii);
(v) PCR confirmation of POL32 gene knockout by Taq DNA polymerase in a standard 50?L PCR reaction comprising 35 cycles with initial denaturation at 95°C for 1 minute, denaturation at 95°C for 15 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds, final extension ay 72°C for 3 minutes using primer sets NAP39 of Sequence ID No. 10 and NAP40 of Sequence ID No. 11.