VACCINE EFFICACY

FIELD OF INVENTION

The present invention relates generally to the field of vaccines. In particular, the present invention provides M.tb mimic useful in enhancing BCG vaccine efficacy or as stand-alone vaccine.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis (M.tb), the causative agent of Tuberculosis (TB) affects about one- third of the global population (WHO 2017). Approximately 2 million deaths globally are directly attributed to TB. M.tb infections may produce varied responses in individuals, ranging from asymptomatic infections to progressive pulmonary to extra-pulmonary TB and even death (Sharma et al., Challenges in the diagnosis & treatment of military tuberculosis, The Indian Journal of Medical Research, 2012, 135, 703-730). The rate of progressive in the severity of TB depends upon the status of the host immune system.

Although world's only accepted vaccine against TB, the live attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is very effective against disseminated and meningeal TB in young children, its efficacy in protecting against adult pulmonary TB varies from 0-80% in different populations depending upon ethnicity and geographical regions (Andersen and Doherty, The success and failure of BCG- implications for a novel tuberculosis vaccine, Nature Reviews, Microbiology, 2005, 3, 656-662; Bhattacharya et al., Simultaneous inhibition of T helper and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis. The Journal of Biological Chemistry, 2014a, 289, 33404- 33411; Bhattacharya et al., Small molecule-directed immunotherapy against recurrent infection by Mycobacterium tuberculosis, The Journal of Biological Chemistry, 2014b, 289, 16508-16515; Chaterjee et al., Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner, PLoS Pathogens, 2011, 7, el002378; Singh et al., Blockade of the Kvl.3 K+ channel enhances BCG vaccine efficacy by expanding central memory T lymphocytes, The journal of infectious diseases, 2016, 214, 1456-1464). BCG's limited vaccine efficacy is majorly attributed to its failure to induce a significant population of central memory T-cells (Bhattacharya et al., 2014a; Maggioli et al., Increased TNF-alpha/IFN- gamma/IL-2 and decreased TNF-alpha/IFN-gamma production by central memory T cells are associated with protective responses against Bovine Tuberculosis following BCG vaccination, Frontiers in Immunology, 2016, 7, 421; Singh et al., 2016; Vogelzang et al., Central memory CD+ T cells are responsible for the recombinant Bacillus Calmette-Guerin DelatureC::hly vaccine's superior protection against tuberculosis, the Journal of Infectious Diseases, 2014, 210, 1928- 1937) as animal modes vaccinated with BCG primarily develop antigen-specific CD4 ⁺ effector memory T-cells. Considering the lags in BCG immunization and increased global TB burden, it is crucial to develop improved methods of immune-prophylaxis against TB.

SUMMARY OF THE PRESENT INVENTION

In an aspect of the present invention, there is provided a composition comprising: (a) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (b) at least one TLR1/2 ligand; and (c) at least one TLR9 ligand.

In an aspect of the present invention, there is provided a vaccine comprising a liposomal composition comprising: (a) self-assembling lipids which form a liposome; (b) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (c) at least one TLR1/2 ligand; and (d) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound.

In an aspect of the present invention, there is provided a concomitant vaccine comprising: (a) BCG; and (b) a liposomal composition comprising: (i) self-assembling lipids which form a liposome; (ii) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (iii) at least one TLR1/2 ligand; and (iv) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound. In an aspect of the present invention, there is provided a method of enhancing BCG vaccine efficacy, said method comprising: concomitant subcutaneous administration of BCG vaccine and intranasal administration of a vaccine comprising a liposomal composition comprising: (a) self assembling lipids which form a liposome; (b) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (c) at least one TLR1/2 ligand; and (d) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound. In an aspect of the present invention, there is provided a method of immunization comprising: (a) a first immunization comprising concomitant subcutaneous administration of BCG vaccine and intranasal administration of a vaccine comprising a liposomal composition comprising: (i) self assembling lipids which form a liposome; (ii) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (iii) at least one TLR1/2 ligand; and (iv) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound; and (b) at least one subsequent booster immunization comprising concomitant subcutaneous administration of BCG vaccine and intranasal administration of a vaccine comprising a liposomal composition comprising: (i) self-assembling lipids which form a liposome; (ii) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (iii) at least one TLR1/2 ligand; and (iv) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound.

This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings along with the detailed description of the embodiments presented herein. Fig. 1 depicts the schematic diagram to show the preparation of M.tb mimic in accordance with an embodiment of the present invention.

Fig. 2 depicts the layout showing the experimental plan wherein naive Balb/c mice or mice vaccinated with BCG /M.tb mimic or with combination of BCG and M.tb mimic were challenged with H37Rv via the aerosol route with low-dose inoculum o about 220CFU/mice. Mice were sacrificed at various time points and lungs, spleen, and liver were harvested to look at the bacterial burden as well as profiling of immune responses, in accordance with an embodiment of the present invention.

Fig. 3 depicts (a) bar diagram to show the number of granulomas in all experimental groups; (b) CFU from lung; (c) CFU from spleen, (d) CFY from liver homogenates at 50 days post infection, in accordance with an embodiment of the present invention.

Fig. 4 depicts mice co-immunized with M.tb mimic and BCG infected with H37Rv M.tb followed by treatment with DOTS for 16 weeks. After 30 days of rest, these mice were treated with dexamethasone for 30 days followed by one more period of rest for 30 days. Mice were then sacrificed for CFU estimation to determine the rate of relapse post-treatment, in accordance with an embodiment of the present invention.

Fig. 5 depicts the determination of reactivation rate of latent M.tb. The reactivation experiment was done once with 10 mice in each group. The results shown are representative of three independent experiments. Bars represent mean ± S (n=3). Unpaired Student's t-test was used to calculate the p-values. *, p<0.05.

Fig. 6a-d depicts the intracellular levels of innate cytokines in spleen; (a) IL-Ib; (b) IL-6; (c) TNF- a; (d) IL-10, in accordance with an embodiment of the present invention.

Fig. 7a-d depicts the intracellular levels of innate cytokines in lung; (a) IL-Ib; (b) IL-6; (c) TNF-a; (d) IL-10, in accordance with an embodiment of the present invention.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1-7 depicts the sequence of M.tb antigenic peptides SEQ ID NO: 1 (AWGRRLMIGTAAAVVLPG)

SEQ ID NO: 2 (TAAVVLPGLVGLAGGAA)

SEQ ID NO: 3 (WDINTPAFEWYYQSGLSI)

SEQ ID NO: 4 (KQSLTKLAAAWGGSG)

SEQ ID NO: 5 (TKLAAAWGGSGSEAY) SEQ ID NO: 6 (LDEGKQSLTKLAAAW)

SEQ ID NO: 7 (LARTISEAGQAMASTEGNVTGMEA)

SEQ ID NO: 8 depicts the nucleotide sequence of a class A CpG ODN TLR9 ligand (ggGGTCAACGTTGAgggggg)

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions, and methods referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of features or steps.

Definitions

For convenience, before further description of the present invention, certain terms employed in this specification, examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

As used in the specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The term concomitant used herein refers to administration of a first and second vaccine component (BCG vaccine, M.tb mimic vaccine) at the same time (one after the other, on the same day, etc.).

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only.

The present invention provides a composition comprising: (a) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:

6, and SEQ ID NO: 7; (b) at least one TLR1/2 ligand; and (c) at least one TLR9 ligand.

In an embodiment, there is provided a composition as described herein, wherein said TLR9 ligand is a class A ligand. In an embodiment, said TLR9 ligand is class B ligand. In an embodiment, said TLR9 ligand is a class C ligand. In a preferred embodiment, said TLR9 ligand is class A ligand. In a more preferred embodiment, said TLR9 ligand is CpG ODN ligand having at least 80% sequence homology to a sequence as set forth in SEQ ID NO: 8. In a still more preferred embodiment, said TLR9 ligand is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8.

In an embodiment, there is provided a composition as described herein, wherein said TLR1/2 ligand is Pam ₃ CSI< ₄ .3HCI.

In an embodiment, there is provided a composition as described herein, wherein said composition comprises a first peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 1, a second peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 2, a third peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 3, a fourth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 4, a fifth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 5, a sixth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 6, and a seventh peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 7. In a preferred embodiment, said composition comprises a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, and a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7.

In a most preferred embodiment, there is provided a composition comprising: a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7, TLR9 ligand that is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8, and TLR1/2 ligand that is Pam ₃ CSK ₄ .3HCI.

The present invention provides a vaccine comprising a liposomal composition, said liposomal composition comprising: (a) self-assembling lipids which form a liposome; and (b) a composition comprising: (i) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (ii) at least one TLR1/2 ligand; and (iii) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound.

In an embodiment, there is provided a vaccine composition as described herein, wherein said liposome comprises cholesterol, L-a-phosphatidylcholine, and stearylamine. In an embodiment, said liposome is cationic. In an embodiment, the ratio of molal concentration of cholesterol to stearylamine is in the range of 1:1-1:5, preferably 1:2. In an embodiment, the ratio of molal concentration of cholesterol to L-a-phosphatidylcholine is in the range of 1:5-1:10, preferably 1:7. In an embodiment, the ratio of molal concentration of stearylamine to L-a- phosphatidylcholine is in the range of 1:1-1:5, preferably 1:3.5.

In an embodiment, there is provided a vaccine composition as described herein, wherein said composition comprises a first peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 1, a second peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 2, a third peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 3, a fourth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 4, a fifth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 5, a sixth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 6, and a seventh peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, or 100% sequence homology to SEQ ID NO: 7. In a preferred embodiment, said composition comprises a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, and a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7.

In an embodiment, there is provided a vaccine composition as described herein, wherein in said composition TLR9 ligand is a class A ligand. In an embodiment, said TLR9 ligand is class B ligand. In an embodiment, said TLR9 ligand is a class C ligand. In a preferred embodiment, said TLR9 ligand is class A ligand. In a more preferred embodiment, said TLR9 ligand is CpG ODN ligand having at least 80% sequence homology to a sequence as set forth in SEQ ID NO: 8. In a still more preferred embodiment, said TLR9 ligand is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8.

In an embodiment, there is provided a vaccine composition as described herein, wherein in said composition said TLR1/2 ligand is Pam ₃ CSI< ₄ .3HCI.

In an embodiment, there is provided a vaccine composition as described herein, wherein said vaccine is in lyophilized form. In another embodiment, the vaccine composition is in liquid form. In an embodiment, there is provided a vaccine composition as described herein, wherein said vaccine further comprises suitable adjuvants. In an embodiment, the vaccine is substantially adjuvant free.

In a most preferred embodiment, there is provided a vaccine composition comprising: liposomes comprising cholesterol, L-a-phosphatidylcholine, and stearylamine having molal concentration ratio of 1:7:2, a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7, TLR9 ligand that is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8, and TLR1/2 ligand that is Pam ₃ CSI< ₄ .3HCI, and wherein the said a peptide fragments, TLR1/2 ligand, and TLR9 ligand are liposomally bound.

The present invention provides a concomitant vaccine comprising: (a) BCG; and (b) a liposomal composition comprising: (i) self-assembling lipids which form a liposome; (ii) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (iii) at least one TLR1/2 ligand; and (iv) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein BCG is in attenuated form. In an embodiment, BCG is obtained from attenuated live bovine tuberculosis bacillus, Mycobacterium bovis. In an embodiment, any conventional strain of bovine tuberculosis bacillus may be used.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein said liposome comprises cholesterol, L-a-phosphatidylcholine, and stearylamine. In an embodiment, said liposome is cationic. In an embodiment, the ratio of molal concentration of cholesterol to stearylamine is in the range of 1:1-1:5, preferably 1:2. In an embodiment, the ratio of molal concentration of cholesterol to L-a-phosphatidylcholine is in the range of 1:5-1:10, preferably 1:7. In an embodiment, the ratio of molal concentration of stearylamine to L-a- phosphatidylcholine is in the range of 1:1-1:5, preferably 1:3.5.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein said vaccine comprises a first peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 1, a second peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 2, a third peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 3, a fourth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 4, a fifth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 5, a sixth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 6, and a seventh peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 7. In a preferred embodiment, said vaccine comprises a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, and a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein in said vaccine, TLR9 ligand is a class A ligand. In an embodiment, said TLR9 ligand is class B ligand. In an embodiment, said TLR9 ligand is a class C ligand. In a preferred embodiment, said TLR9 ligand is class A ligand. In a more preferred embodiment, said TLR9 ligand is CpG ODN ligand having at least 80% sequence homology to a sequence as set forth in SEQ ID NO: 8. In a still more preferred embodiment, said TLR9 ligand is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein in said vaccine, TLR1/2 ligand is Pam ₃ CSK .3HCI.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein said vaccine is in lyophilized form. In another embodiment, the vaccine is in liquid form. In yet another embodiment, the vaccine is a combination of lyophilized form and liquid form.

In an embodiment of the present invention, there is provided a concomitant vaccine as described herein, wherein said vaccine further comprises suitable adjuvants. In an embodiment, the vaccine is substantially adjuvant free.

In a most preferred embodiment, there is provided a concomitant vaccine comprising: live attenuated BCG obtained from bovine tuberculosis bacillus, Mycobacterium bovis, liposomes comprising cholesterol, L-a-phosphatidylcholine, and stearylamine having molal concentration ratio of 1:7:2, a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7, TLR9 ligand that is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8, and TLR1/2 ligand that is Pam ₃ CSI< ₄ .3HCI, and wherein the said a peptide fragments, TLR1/2 ligand, and TLR9 ligand are liposomally bound.

The present invention provides a method of enhancing BCG vaccine efficacy, said method comprising: concomitant subcutaneous administration of BCG vaccine and intranasal administration of a vaccine comprising a liposomal composition comprising: (a) self-assembling lipids which form a liposome; (b) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (c) at least one TLR1/2 ligand; and (d) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound.

In an embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein BCG is in attenuated form. In an embodiment, BCG is obtained from attenuated live bovine tuberculosis bacillus, Mycobacterium bovis. In an embodiment, any conventional strain of bovine tuberculosis bacillus may be used.

In an embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein said liposome comprises cholesterol, L-a-phosphatidylcholine, and stearylamine. In an embodiment, said liposome is cationic. In an embodiment, the ratio of molal concentration of cholesterol to stearylamine is in the range of 1:1-1:5, preferably 1:2. In an embodiment, the ratio of molal concentration of cholesterol to L-a-phosphatidylcholine is in the range of 1:5-1:10, preferably 1:7. In an embodiment, the ratio of molal concentration of stearylamine to L-a- phosphatidylcholine is in the range of 1:1-1:5, preferably 1:3.5.

In an embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein said liposomal composition comprises a first peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 1, a second peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 2, a third peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 3, a fourth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 4, a fifth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 5, a sixth peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 6, and a seventh peptide fragment having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to SEQ ID NO: 7. In a preferred embodiment, said liposomal composition comprises a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, and a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7.

In an embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein TLR9 ligand is a class A ligand. In an embodiment, said TLR9 ligand is class B ligand. In an embodiment, said TLR9 ligand is a class C ligand. In a preferred embodiment, said TLR9 ligand is class A ligand. In a more preferred embodiment, said TLR9 ligand is CpG ODN ligand having at least 80% sequence homology to a sequence as set forth in SEQ ID NO: 8. In a still more preferred embodiment, said TLR9 ligand is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8.

In an embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein TLR1/2 ligand is Pam ₃ CSI< ₄ .3HCI.

In an embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein said liposomal composition is a vaccine further comprises suitable adjuvants. In an embodiment, the vaccine is substantially adjuvant free.

In a most preferred embodiment, there is provided a method of enhancing BCG vaccine efficacy as described herein, wherein BCG vaccine comprising live attenuated BCG obtained from bovine tuberculosis bacillus, Mycobacterium bovis, is administered subcutaneously and concomitantly intranasal administration of a vaccine comprising liposomes comprising cholesterol, L-a- phosphatidylcholine, and stearylamine having molal concentration ratio of 1:7:2, a first peptide fragment having sequence as set forth in SEQ ID NO: 1, a second peptide fragment having sequence as set forth in SEQ ID NO: 2, a third peptide fragment having sequence as set forth in SEQ ID NO: 3, a fourth peptide fragment having sequence as set forth in SEQ ID NO: 4, a fifth peptide fragment having sequence as set forth in SEQ ID NO: 5, a sixth peptide fragment having sequence as set forth in SEQ ID NO: 6, a seventh peptide fragment having sequence as set forth in SEQ ID NO: 7, TLR9 ligand that is CpG ODN ligand having sequence as set forth in SEQ ID NO: 8, and TLR1/2 ligand that is Pam ₃ CSI< ₄ .3HCI, and wherein the said a peptide fragments, TLR1/2 ligand, and TLR9 ligand are liposomally bound.

The present invention provides a method of immunization comprising: (a) a first immunization comprising concomitant subcutaneous administration of BCG vaccine and intranasal administration of a vaccine comprising a liposomal composition comprising: (i) self-assembling lipids which form a liposome; (ii) at least one peptide fragment having at least 80% sequence homology to at least one peptide fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; (iii) at least one TLR1/2 ligand; and (iv) at least one TLR9 ligand, wherein the said at least one peptide fragment, at least one TLR1/2 ligand, and at least one TLR9 ligand is liposomally bound; and (b) at least one subsequent booster immunization comprising concomitant subcutaneous administration of BCG vaccine and intranasal administration of a vaccine comprising a liposomal composition as described in step (a).

In an embodiment, there is provided a method of immunization as described herein, comprising two booster immunization doses.

In an embodiment, there is provided a method of immunization as described herein, wherein total peptide dosage for human administration in first administration is in the range of 0.01- lmg/kg, preferably in the range of 0.04-0.08mg/kg, more preferably about 0.06mg/kg.

In an embodiment, there is provided a method of immunization as described herein, wherein total peptide dosage for human administration in booster administration is in the range of 0.01- lmg/kg, preferably in the range of 0.04-0.08mg/kg, more preferably about 0.06mg/kg.

In an embodiment, there is provided a method of immunization as described herein, wherein total TLR1/2 ligand dosage for human administration in first administration is in the range of 0.001-0. lmg/kg, preferably in the range of 0.005-0. lmg/kg, more preferably about 0.009mg/kg. In an embodiment, there is provided a method of immunization as described herein, wherein total TLR9 ligand dosage for human administration in first administration is in the range of 0.0001-0. Olmg/kg, preferably in the range of 0.0009-0. Olmg/kg, more preferably about O.OOlmg/kg.

In an embodiment, there is provided a method of immunization as described herein, wherein BCG vaccine is as substantially described in the detailed description herein, wherein vaccine comprising a liposomal composition is as substantially described in the detailed description herein.

In the present invention, there is provided a vaccine comprising a liposomal composition as described herein, for use in immunization against TB.

In the present invention, there is provided a concomitant vaccine as described herein, for use in immunization against TB. EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of the disclosure, and not intended to take restrictively to imply any limitations on the scope of the present disclosure. 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.

Materials and methods

M.tb infection of mice and estimation of CFU:

Mycobacterium tuberculosis H37Rv and BCG cultures were grown in 7H9 (Middlebrooks, Difco, USA) medium supplemented with 10% OADC (oleic acid, albumin, dextrose and catalase; Difco, USA) and with0.05% Tween 80 and 0.5% glycerol, and cultures were grown to mid-log phase. Aliquots of the cultures in 20% glycerol were preserved at -80°C and cryopreserved stocks were used for infections.

Mice were infected with H37Rv via the aerosol route using a Madison aerosol chamber (University of Wisconsin, Madison, Wl, USA) with its nebulizer pre-calibrated to deposit around of 220 bacilli to the lungs of each mouse (Chaterjee et al., 2011). Briefly, bacterial stocks were recovered from freezer and quickly thawed and subjected to light ultra-sonication to obtain a single cell suspension. 15ml of the bacterial cell suspension (lOxlO ¹⁶ cells per ml) was placed in the nebulizer to deliver the desired number of CFUs to the lungs of animals placed inside the chamber. At day one post infection, three randomly selected mice sacrificed and organs were harvested, homogenized in 0.2pm filtered PBS and plated onto 7H11 Middlebrooks (Difco, USA) plates containing 10% oleic acid, albumin, dextrose and catalase. Undiluted, 10-fold diluted, and 100-fold diluted lung, liver, and spleen cells homogenates were plated in triplicate on the 7H11 plates and incubated at 37°C for 21-28 days. Colonies were counted and CFU was calculated accordingly.

Mouse model of TB-reactivation:

Mice infected with M.tb with low-dose aerosol infection model, were treated with lOmg/kg of 1NH and RIF administered ad libitum (in the drinking water) or treated to mice co-immunized with BCG and M.tb mimic or immunized with BCG alone for 12 weeks starting at 4 ^th week after infection. These mice were then rested for 30 days followed by treatment with dexamethasone (5mg/kg administered intraperitoneally) three times per week for 30 days. 10 mice from each group were then sacrificed and CFUs were estimated from lung homogenates to determine the reactivation rate of M.tb.

Example 1

Identification of M.tb antigenic peptides

A large number of M.tb peptides were screened for their efficacy to induce M.tb specific T-cells activation and INF-gamma responses (Fan et al., Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of Mycobacterium tuberculosis in a mouse model, Microbiological Research, 2009, 164, 374-382; Kruh-Garcia et al., Antigen 85 variation across lineages of Mycobacterium tuberculosis-implications for vaccine and biomarker success, Journal of Proteomics, 2014, 97, 141-150; Lee and Horwitz, T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs, Infection and Immunity, 1999, 67, 2665-2670; Mustafa et al., Identification and HLA restriction of naturally derived Thl-cell epitopes from secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4(+) T-cell lines, Infection and Immunity, 2000, 68, 3933-3940; Panigada et al., Identification of a promiscuousT-cell epitope in Mycobacterium tuberculosis Mce proteins, Infection and Immunity, 2002, 70, 79-85). From a large number of peptides, seven peptides were selected (SEQ ID NO: 1-7) for further characterization.

Peptide screening was done on the basis of their capacity to induce M.tb specific T- cell activation and high level of IFN-gamma production.

Screening Details:

A group of mice were infected with H37Rv strain of M.tb and were subjected to 45 days of DOTS therapy 15 days post infection. The mice were rested for 30 days post-therapy. T-cells from the infected mice as well as DOTS treated mice were isolated and co-cultured with dendritic cells, which were pulsed with T-cell epitopes/peptides. Following which peptide-specific T cell (subsets of CD4 ⁺ and CD8 ⁺ T cells) activation and proliferation was observed. To investigate the role of these peptides in T-cell activation, experiments were performed using individual as well as pooled peptides, and complete soluble antigen (CSA) of M.tb as a control. All the peptides significantly increased the induced expression of early activation marker CD69 on CD4 ⁺ T cells and CD8 ⁺ T Cells in comparison to complete soluble antigen (CSA) of M.tb. Interestingly the expression of CD25, the late activation marker remained comparable across all the experimental groups. Moreover, it was observed that total pool of peptides (combo) significantly induces the activation of both CD4 ⁺ T and CD8 ⁺ T cells as evidenced by CD69 expression, whereas the expression of CD25 was comparable in all experimental groups. Furthermore, an increase in the number of IFN-y and IL-17 producing both CD4+T and CD8+T cells against peptides in comparison to CSA of M.tb was also observed.

Example 2

Preparation of M.tb mimic

Mixture was obtained from Sigma-Aldrich (Cat no. L4395) which was used for preparation of liposomes which can encapsulate the peptides of SEQ ID NO: 1 through 7. M.tb peptides along with TLR1/2 ligand (Pam3Cys-SK-4 obtained from Enzo Life Sciences, Cat .no. ALX-165-066-M002) and TLR9 ligand (class A CpG ODN ligand having sequence as set forth in SEQ ID NO: 8 obtained from Enzo Life Sciences; Cat no. ALX-746-003-C100) were mixed with lipid mixture as per manufacture's protocol to obtain a homogenous mixture of peptides and TLR ligands contained in liposomes, for intranasal delivery (see Fig. 1). For animal (mice) administration, the first immunization dosage comprises 800pg of each peptide per kg of animal weight, 120pg of TLR1/2 ligand per kg of animal weight, and 20pg of TLR9 ligand per kg of animal weight contained in the liposomes. For subsequent booster immunization, the dosage comprises 40opg of each peptide per kg of animal weight, 120pg ofTLRl/2 ligand per kg of animal weight, and 20pg ofTLR9 ligand per kg of animal weight contained in the liposomes.

Example 3

Enhancement of BCG efficacy by M.tb mimic and protection in mice against tuberculosis

Mice: All animals [Balb/c and C57BL.6 mice (6-8 weeks of age)} were maintained in ICGEB animal facility.

Immunization: Mice were immunized with (i) BCG (subcutaneous) (10 ⁶ bacteria), (ii) M.tb mimics (intranasal); (iii) BCG + M.tb mimics, and (iv) vector alone. BCG vaccinated and mimic immunized mice were boosted with once-a-week, three week-long M.tb mimic boosting regimen. Mice were subsequently rested for 20 days and then challenged with M.tb strain H37Rv by aerosol route (~220CFU/mouse). Organs like lungs, livers, and spleens were harvested for determination of bacterial burden. Fig. 2 depicts the experimental layout. Fig. 3a depicts the granuloma score. It can be seen that compared to BCG or M.tb mimics alone, the combination results in significant reduction of granulomas. It is also note that treatment with M.tb mimics alone also provides reduction of granulomas comparable to that of BCG alone. Fig. 3b-d depicts the CFU count from lung tissue, spleen tissue, and liver tissue sample respectively. It can be seen that for the combination of BCG and M.tb mimics, there is a drastic drop in CFU count across all 3 different samples compared to treatment with BCG or M.tb mimics alone.

Example 4

M.tb mimic immunization protects animals against TB recurrence due to relapse

Mice were co-immunized with M.tb mimic and BCG (BCG vaccinated and mimic immunized mice were boosted with once-a-week, three week-long M.tb mimic boosting regimen) and infected with H37Rv M.tb followed by treatment with DOTS for 16 weeks. After 30 days of rest, the mice were treated with dexamethasone for 30 days followed by one more period of rest for 30 days. Mice were then sacrificed for CFU estimation to determine the rate of relapse post-treatment. Fig. 4 depicts the treatment paradigm.

Fig. 5 depicts the reactivation rate of latent M.tb in three different treatment paradigms. As seen in Fig. 5, while in control (no vaccine), relapse rate was about 70%, in BCG vaccine alone, relapse rate was about 45%, while in mice treated with BCG + M.tb mimics, the relapse rate was about 20%. These data clearly show that the concomitant administration of M.tb mimics of the present invention along with BCG significantly decreases TB reactivation rate compared to BCG administration alone.

Example 5

M.tb mimic immunization induces protective cytokine response

M.tb mimic co-immunization (BCG vaccinated and mimic immunized mice were boosted with once-a-week, three week-long M.tb mimic boosting regimen) enhances IL-Ib, IL-6, and TNF-a producing cells (Fig. 6a-c), with no significant decrease in percentage of IL-10 producing cells (Fig. 5d) in spleen. A similar profile was observed in the lungs of co-immunized mice (Fig. 7a-d). These data suggest that M.tb mimic co-immunization enhances antigen specific Thl and Thl7 responses, which impart protection against TB. M.tb co-immunized mice also show a significant increase in the number of CD4 ⁺ CD8 ⁺ double positive T-cells as compared to mice immunized with BCG alone (data not shown). These double positive T-cells have been reported to mount a strong protective response in various disease settings (Overgaard et al., 2015). Overall, these data collective show that the M.tb mimic of the present invention when administered (alone) to animals, provides protection against TB with efficiency similar to that of BCG vaccine alone. Interestingly, when co-administered with BCG, the M.tb mimic enhances the efficacy of BCG by not only reducing bacterial burden but also enabling the host system in mounting a potent immune response by activating protective T-cells along with an enriched central memory T-cell repertoire, thus increasing the efficacy of existing TB vaccine.