DESC:FIELD OF THE INVENTION
[001] The present disclosure relates to the field of biotechnology. More specifically, the present disclosure relates to the use of chemically attenuated malaria merozoites to suppress immunotolerance.

BACKGROUND OF THE INVENTION
[002] RTS, S/AS02 and R-21 are the two WHO approved vaccines against malaria. It has been found that the efficacy of both the vaccines is almost the same. Both the vaccines require three booster doses to confer immunity. A number of studies have been done to explain the suboptimal immunogenicity conferred by RTS, S/AS02 in malaria endemic regions compared to 60% protection in malaria-naïve individuals. In 2012, World Health Organization (WHO) recommended seasonal malaria chemoprevention (SMC), for malaria control in malaria endemic areas with highly seasonal transmission. Despite its high effectiveness, malaria eradication is still a matter of concern. Immunotolerance represents a major challenge to overcome for any anti-malarial strategy to be effective in malaria-endemic regions. Plasmodium sporozoites induce regulatory macrophages. Thus, newer strategies are needed to eradicate malaria in these regions. Blood stage malaria is responsible for clinical infection and associated symptoms like chills, rigor and ultimately death due to cerebral malaria if left untreated. Due to severity associated with blood stage, leaky sporozoite vaccines and ability of blood stage vaccines to confer cross stage immunity, blood stage malaria vaccine has been felt the need of the hour.
[003] In order to overcome the aforesaid problems associated with the existing malaria vaccines, the present disclosure provides an enoxaparin-based formulation useful in administration to suppress blood-stage malaria related immunotolerance. The present disclosure provides a composition comprising Plasmodium antigen expressing cells that have been exposed to Enoxaparin and an immunologically acceptable carrier, diluent or excipient. The composition of the present disclosure is advantageous as it suppresses immunotolerance and generates sterile protective immunity against blood-stage malaria.
[004] The primary object of the present disclosure is to provide an immunotolerance suppressing composition comprising chemically attenuated preferably Plasmodium antigen expressing cells that have been exposed to Enoxaparin and an immunologically acceptable carrier, diluent or excipient.
[005] Another object of the present disclosure is to provide a method for producing an immunotolerance suppressing composition as described herein.

SUMMARY OF INVENTION
[006] The present disclosure relates to an immunotolerance suppressing composition comprising chemically attenuated malaria merozoites as a blood stage malaria immunotolerance suppressant. The present disclosure also relates to a method of preparing an immunotolerance suppressing composition, as described herein.
[007] In an aspect of the present disclosure, there is provided an immunotolerance suppressing composition comprising Plasmodium antigen expressing cells that have been exposed to Enoxaparin, and an immunologically acceptable carrier, diluent or excipient.
[008] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the Plasmodium antigen expressing cells are Plasmodium infected red blood cells (pRBC).
[009] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the Plasmodium antigen expressing cells comprise Plasmodium merozoites.
[0010] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the composition comprises 106 to 103 pRBC.
[0011] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the concentration of Enoxaparin is 6 mg.
[0012] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the composition provides sterile protective immunity against infection by one or more isolates, strains or species of Plasmodium.
[0013] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the composition is useful for use as a blood stage malaria immunotolerance suppressant.
[0014] In another aspect, the present disclosure relates to a process for preparing the immunotolerance suppressing composition as described herein.
[0015] In a preferred embodiment of the present disclosure, there is provided an in vitro method for producing an immunotolerance suppressing composition, wherein the method comprises the step of:
exposing Plasmodium antigen expressing cells to Enoxaparin,
to thereby produce said immunotolerance suppressing composition.
[0016] In an embodiment of the present disclosure, there is provided an in vitro method for producing an immunotolerance suppressing composition as described herein, wherein the Plasmodium antigen expressing cells are Plasmodium infected red blood cells (pRBC).
[0017] In an embodiment of the present disclosure, there is provided an in vitro method for producing an immunotolerance suppressing composition as described herein, wherein isolated or purified blood-stage parasites are treated with Enoxaparin.
[0018] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form.

DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
[0019] The following figures form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the figures in combination with the detailed description of the specific embodiments presented herein.
[0020] Figure 1 illustrates the course of parasitaemia in various groups on challenge 30 days post immunization Course of parasitaemia (mean ± S.D.) in various groups of mice vs. number of days post-infection is plotted. Parasitaemia was determined by examining Giemsa- stained thin blood smears of individual mice weekly after immunization.
[0021] Figure 2 illustrates Kaplan Maeyer curve depicting survival rate in various groups of mice post 30 days of infection.
[0022] Figure 3 illustrates course of parasitaemia in various groups on rechallenge 90 days post immunization Course of parasitaemia (mean ± S.D.) in various groups of mice vs. days post-challenge is plotted. Parasitaemia was determined by examining Giemsa- stained thin blood smears of individual mice weekly after immunization.
[0023] Figure 4 illustrates Kaplan Meyer Survival Curve providing information graphically about number of animals surviving on rechallenge 90 days post immunization in different experimental groups.
[0024] Figure 5 illustrates post-vaccination increase in the frequencies of CD3+, CD4+ and CD19+ T cell populations following biomimetic chemo attenuated immunisation in group I and II mice. Splenic lymphocytes were harvested on Day 30 following vaccination and Day 10 following infection. These were stained ex vivo for phenotypic and activation markers and analysed by flow cytometry. Following number of samples were plotted, 5/5. Comparisons were performed using Student T test. Bars and lines denote medians and standard deviation, respectively.
[0025] Figure 6 illustrates post-vaccination increase in the frequencies of CD11c+ CD8+ T cell populations following biomimetic chemo-attenuated immunisation. Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex vivo for phenotypic and activation markers and analysed by flow cytometry. Gating strategy for definition of splenic dendritic cells in artemether-lumefantrine treated and biomimetic chemo-attenuated mice: splenic dendritic cells as CD11c+ CD8+ within CD3+ T cells and expression of activation markers CD40, CD80 and CD86 on CD3+ CD8+ cells. Within CD3+CD11c+ cells, increase at Day 30 in along with activated markers CD40, CD80 and CD86 in immunised mice was seen compared to treated mice. Following number of samples were plotted Immunised group II mice: Day 30, n=3; Positive control mice: Day 30, n=3. Comparisons were performed using Student T test. Bars and lines denote medians and standard deviation, respectively.
[0026] Figure 7 illustrates quantification of various subsets of T cells from various groups of mice at various time points immunisation and treatment. Biomimetic chemoattenuation induces a more robust CD4+ response than CD8+. Also rapidly induces memory CD4 and CD8 cells Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex vivo for phenotypic and activation markers and analyzed by flow cytometry. Definition of various subsets: live CD3+ T cells, CD4+ and CD8+ T cells within total live T cells, TCentral memory:- CD44+CD62+, TEffector memory :- CD44+CD62- , TEffector :- CD44-CD62- and Tnaive :- CD44-CD62+ cells within total CD4+ or CD8+ T cell populations. Following number of samples were plotted Immunised group II mice: Day 30, n=3; Positive control mice: Day 30, n=3. Comparisons were performed using Student T test. Bars and lines denote medians and standard deviation, respectively.
[0027] Figure 8 illustrates quantification of various subsets of T cells based on CXCR3 and CCR6 receptor expression from various groups of mice at various time points immunisation and treatment. Biomimetic chemo-attenuation induces a robust and balanced Th1-Th2-Th17 immune response. Also upregulates CXCR3 expression on CD8 cells indicative of long-term survival. Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex vivo for phenotypic and activation markers and analyzed by flow cytometry. (A) Gating strategy for definition of CD4+ and CD8+ cells within live, CD3+ (T cell) lymphocyte population, stratification of CD4+ and CD8+ T cells into Th1 (CXCR3+CCR6-), Th2 (CXCR3-CCR6-) and Th17 (CXCR3-CCR6+) on Day 30. Frequencies of total CD4+ T cells (B) and CD8+ T cells (C) were compared within immunised and treated groups. Following number of samples were plotted Immunised group II mice: Day 30, n=3; Positive control mice: Day 30, n=3. Comparisons were performed using Student T test. Bars and lines denote medians and standard deviation, respectively.
[0028] Figure 9 illustrates analysis of splenic CD4+ T regulatory (Treg) phenotype cells following biomimetic chemo-attenuated immunisation and treatment. Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex-vivo for phenotypic and activation markers and analyzed by flow cytometry. Definition of various subsets used for Treg cells: - CD3+CD4+CD25 and Tregs +Foxp3+ within total CD4+ T cells Following number of samples were plotted Immunised group II mice: Day 30, n=3; Positive control mice: Day 30, n=3.Comparisons were performed using Student T test. Bars and lines denote medians and standard deviation, respectively.
[0029] Figure 10 illustrates intracellular cytokine expression analysis on splenic lymphocytes following biomimetic chemo-attenuated immunisation highlights towards increase in alone TNF secreting CD4 and CD8+ T cells and towards both TNF and IFN secreting CD4 and CD8+ T cells in treated mice. Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex vivo for phenotypic and intracellular markers and analyzed by flow cytometry. (A) Gating strategy for definition of TNF and IFN secreting cells within total CD4+ T cells. Following number of samples were plotted Immunised mice: Day 30, n= n=3/3; Treated mice: Day 30, n= 3/3. Comparisons were performed Student T test. Bars and lines denote medians and standard deviation, respectively.
[0030] Figure 11 illustrates biomimetic chemo-attenuation platform robustly activates and increases resident CD4+ and CD8+ frequencies. Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex vivo for phenotypic and activation markers and analyzed by flow cytometry. (A) Gating strategy used to define resident cells as CD69/CD44L+ cells within CD4+ and CD8+ T cells and based on CD154 expression as activated cells within the CD4+ and CD8+ T cell population. Following number of samples were plotted Immunised group II mice: Day 30, n=3; Positive control mice: Day 30, n=3. Comparisons were performed Student T test. Bars and lines denote medians and standard deviation, respectively.
[0031] Figure 12 illustrates quantification of various subsets of B cells from various groups of mice at various time points immunisation and treatment Direct detection of memory B cells through co-staining with CD3, CD19, CD27, IgD and IgM. Splenic lymphocytes were harvested on Day 30 following vaccination and treatment. These were stained ex vivo for B cells identification and then stained with phenotypic markers and analyzed by flow cytometry. Definition of various subsets: - memory B cells as CD3-CD19+CD27+ cells within the live splenic lymphocyte population. Following number of samples were plotted Immunised group II mice: Day 30, n=3; Positive control mice: Day 30, n=3. Comparisons were performed using Student T test. Bars and lines denote medians and standard deviation, respectively.

DETAILED DESCRIPTION OF THE INVENTION
[0032] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the product, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0033] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[0034] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[0035] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.
[0036] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
[0037] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0039] 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. Functionally equivalent products and methods are clearly within the scope of the disclosure, as described herein.
[0040] In an aspect of the present disclosure, there is provided an immunotolerance suppressing composition comprising chemically attenuated malaria merozoites as a blood stage malaria immunotolerance suppressant.
[0041] In a preferred embodiment of the present disclosure, there is provided an immunotolerance suppressing composition comprising Plasmodium antigen expressing cells that have been exposed to Enoxaparin and an immunologically acceptable carrier, diluent or excipient.
[0042] The immunologically acceptable carrier, diluent or excipient well known to a person skilled in the art, were used in the present disclosure.
[0043] P.berghei NK-65 strain was used to study the effect of administration of Enoxaparin and Artemether-lumefantrine on course of parasitemia and various immunological parameters.
[0044] Enoxaparin (Clexane) was used for the study. Enoxaparin was reconstituted in 0.9% normal saline to be used for administering to mice.
[0045] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the Plasmodium antigen expressing cells are Plasmodium infected red blood cells (pRBC).
[0046] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the composition comprises 106 to 103 pRBC.
[0047] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the Plasmodium antigen expressing cells comprise Plasmodium merozoites.
[0048] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the concentration of Enoxaparin is 6 mg.
[0049] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the animal (mice) dosage of Enoxaparin is 6mg/kg b.w.
[0050] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the human dosage of Enoxaparin is 0.5mg/kg b.w.
[0051] In an embodiment of the present disclosure, Enoxaparin (Clexane vials containing 40mg in 0.4ml) is used in the present composition. Enoxaparin was reconstituted in 0.9% normal saline to be used for administering to mice.
[0052] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the composition provides sterile protective immunity against infection by one or more isolates, strains or species of Plasmodium.
[0053] In an embodiment of the present disclosure, there is provided an immunotolerance suppressing composition as described herein, wherein the composition is useful for use as a blood stage malaria immunotolerance suppressant.
[0054] In another aspect, the present disclosure relates to a process for preparing the immunotolerance suppressing composition as described herein.
[0055] In a preferred embodiment of the present disclosure, there is provided an in vitro method of producing an immunotolerance suppressing composition, wherein the method comprises the step of:
exposing Plasmodium antigen expressing cells to Enoxaparin,
to thereby produce said immunotolerance suppressing composition.
[0056] In an embodiment of the present disclosure, there is provided an in vitro method of producing an immunotolerance suppressing composition as described herein, wherein the Plasmodium antigen expressing cells are Plasmodium infected red blood cells (pRBC).
[0057] In an embodiment of the present disclosure, there is provided an in vitro method of producing an immunotolerance suppressing composition as described herein, wherein 106 to 103 pRBC are subjected to the exposure of Enoxaparin.
[0058] In an embodiment of the present disclosure, there is provided an in vitro method of producing an immunotolerance suppressing composition as described herein, wherein isolated or purified blood-stage parasites are treated with Enoxaparin.
[0059] In the present disclosure, phenotypes of various T cell subtypes, dendritic cells and B cells and co-function in Swiss mice post immunisation and artemether-lumefantrine treatment were assessed. Protection is afforded by a fine balance between humoral and cell mediated immunity mediating bridge between innate immunity and adaptive immunity. However, to date no study involving blood stage Plasmodium has been published in those studies different arms of immune response simultaneously. In the present disclosure, in-vivo protective efficacy of biomimetic chemo-attenuation using an invasion inhibitor were initially investigated and then distribution and frequency of dendritic cells, parasite-specific CD4+ and CD8+ T cells and B cells following a single regime were studied. It was observed that biomimetic chemo-attenuation exerts long term homologous protection. Memory resident CD4+ and CD8+ T cells are widely spread in infection-treatment-vaccination regime in the spleen. It was observed that immunized mice exhibit a distinct heightened total T and helper T cell count, balanced and regulated Th1-Th2-Th17 immune response skewed towards TNF-? secreting cells, increased dendritic cells, distinct subset antigen-specific immune response and increased humoral response compared to that in artemether-lumefantrine treated mice. Importantly, it was demonstrated that both CD4+ and CD8+ central and effector memory T cells equally populate the splenic cellular constituent. It was also revealed that memory CD8+ T cell count is significantly enhanced and is stably imprinted following single immunization regime. Combined results demonstrate that in biomimetically chemo-attenuated immunized mice, different subsets of T and B cells are maintained with apparent antigen-specific imprinting post biomimetic chemo-attenuated immunization, and that both humoral and cell mediated immune responses provide protection against future blood stage infection.
[0060] Correlating the cell populations of CD3+CD8+CD44+ and CD8+ CD11b+, the present disclosure confirms that CD8+CD11b+ also represents CD8+ effector population serving as a marker of recent activation. It has also been described in the present disclosure that CD8+CD11b+ subpopulation suppresses helper CD4 population whereas CD11c cells activate CD4 T and CD19 cells. In the immunized mice cohort, the frequency of CD8+ CD11c+ dendritic cells constituted 38?5.45 percent of total CD8+ splenic cellular constituent compared to 23.8? 1.82 percent in treated mice group. The increase in both memory CD8+T cells and CD8+ dendritic cells in immunized mice and decrease in both the counts in treated mice suggest a positive correlation between dendritic cell and T cell responses. Increased dendritic cell count has also been reported in mice and humans immunized with radiation-attenuated sporozoites. Further, the present disclosure provides that on entry into the spleen, or after encounter with parasites, effector CD11bhigh cells undergo further differentiation into memory CD11chighCD11b+ . Recently, the dual expression of CD11b and CD11c has been used as marker to determine the tolerogenic nature of immune response. In conclusion, the present disclosure shows that in immunized mice there was significant decrease in CD11b+ CD8+, CD11c+CD11b+ CD8+ cells and increase in CD8+CD11c+ compared to artemether-lumefantrine treated mice are governing factors responsible for generating sustainable and protective immunity. These results also highlight that apart from immunomodulatory effect of enoxaparin in blood stage malaria, biomimetic chemo-attenuation has an added therapeutic advantage of decreasing the chances of development of cerebral malaria.
[0061] Differentiation of naïve T cells into effector and memory compartments in spleen in biomimetic chemo-attenuated immunized mice compared to artemether-lumefantrine treated mice, can be reasoned to generate protective immunity. Another striking feature observed in the present disclosure has been the steady increase in the splenic CD62+ population post immunization. Studies have shown that this feature represents the capacity of memory cell populations to proliferate and accumulate at effector sites in response to secondary challenge. All this shows that biomimetic chemo-attenuated immunization augments T cell memory responses over time.
[0062] Differential activation of DCs lead to differential priming of Th immune response or induce tolerance. To evaluate Th response in various groups of mice, we evaluated the surface expression of CXCR3 and CCR6 on T cell subpopulations. A regulated immune response was observed in immunized mice compared to artemether-lumefantrine treated mice. CXCR3 also serves as marker of T cell migration and activation as well. Very few studies have observed chemoattractant receptors after immunotherapy or immunomodulation. CCR6 and CXCR3 have been found to regulate T cell homing in different ways. In the present disclosure, increased CXCR3 chemokine regulatory responses in the spleen of immunized mice were found, which indicate the migratory potential of splenic CD8+ T cell. CXCR3 expression has also been linked with differentiation of antigen activated CD8+ T cells into memory phenotype. In the present disclosure it was found effector memory CD8+ population both in immunized as well as treated cohort but central memory CD8+ subset only in immunized cohort. Interestingly, expression of CCR6; albeit low was observed only in immunized mice. Thus, the present disclosure alludes that CXCR3 along with CCR6 regulates differentiation of antigen activated CD8+ T cells into central and effector memory cells.
[0063] Higher TNF expression by CD4 T cells were also found. Expression of TNF on CD4 T cell is a potential immune correlation of protection studied extensively in RTS, S immunized as well as in malaria infected persons. In a similar finding where CD4 T cell expression of TNF was highly induced in immunized but not in treated animals. Increase in proinflammatory cytokine levels and complement activation have also been demonstrated in pregnant women administered enoxaparin. However, so far it has been reported only in females being administered repeatedly. The present disclosure assumes importance firstly because in the present study enoxaparin was not administered repeatedly and a single administration once daily for 4 days as per Peter’s protocol was able to mount heightened CD4+ T cell immune response countering the immune escape mechanism of blood stage Plasmodium infection by hampering CD4+ T cell proliferation.
[0064] Blood stage Plasmodium infection induces regulatory responses. However, the role of regulatory responses in development of immunity remains unclear. The present disclosure observed correlation between enhanced clearance of infected RBCs and decrease in the count of regulatory T cells.
[0065] B cells form another important group of antigens presenting cells mediating humoral response. Humoral response plays an important role in a number of infectious diseases. However, they have been largely understudied in studies of malaria. B cells have been reported in protecting against cerebral malaria. In the present study, increased memory B cell counts were observed in immunised mice in comparison to treated mice. The contribution of various factors determining humoral mediated immune response has been controversial. In the present disclosure, increased Th1, TNF-a and decreased levels of IFN-? were observed in immunized mice in comparison to treated mice. Thus, the results of present study are essentially in accordance with the conclusion drawn in a recent article that excessive Th1 response inhibits humoral mediated immune responses and a single Th1 cytokine alone is not sufficient to exert suppressive effect.
[0066] It needs to be highlighted that the principle of biomimetic chemo-attenuation by attenuating blood stage Plasmodium resulting in enhanced antigenic exposure is akin to natural re-infection. Further, it has been argued that increased antigenic exposure by re-infections in malaria endemic areas results in suppression of CD27+IgD+ non-class-switched memory B cells. However, in the present disclosure it was observed that sustained expression of non-class-switched memory B cells in immunized mice compared to artemether-lumefantrine treated mice. These are surprising and encouraging results which strongly promote clinical evaluation and deployment of this strategy in malaria endemic areas.
[0067] Marginal zone B cells phenotypically characterized by CD19+IgD+CD27+IgM+ expression have been implicated as a pre-circulating or innate memory B cell subset that functions to control blood-borne pathogens and does not undergo antigen exposed clonal expansion. It has also been reported that malaria infected individuals had increased proportions of CD19+IgM+CD27+IgD+ B cells resulting in tolerogenic profile and impaired protective immunity. Interestingly, in present disclosure increase in count of marginal B cells and well-developed germinal centers indicating lymph node hyperplasia in immunized mice compared to treated mice asserts the protective role of marginal zone B cells.
[0068] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES
[0069] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of 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. Although methods and materials similar to or equivalent to those described herein can be used in the practice of the disclosed methods, 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 vary.

Example 1: Biomimetic chemo-attenuation facilitates persistent antigenic exposure
[0070] Female Swiss mice weighing 22-26g were obtained from the Central animal facility, Panjab University, Punjab, India and are used as experimental models. They were maintained on a standard pellet diet and water ad libitum. The ethical clearance was obtained from Institutional Animal Ethics Committee of Panjab University (PU/45/99/CPCSEA/IAEC/2022/708) and experiments were conducted according to Committee for the Purpose of Control and Supervision of Experiments on Animals (45/GO/ReBi/S/99/CPCSEA) guidelines.
[0071] Swiss mice were randomly divided into five experimental groups (n = 5 per group) shown in Table 1 below.
Table 1: Treatment groups
Group Treatment
Normal control Administered feed and water ad-libitum
Infected control Infected with 106 P.berghei infected erythrocytes
Positive control Infected with 106 P.berghei infected erythrocytes and treated with Lumerax (Artemether and Lumefantrine)
Group I Infected with 106 P.berghei infected erythrocytes and treated with Enoxaparin (6mg/kg b.w.)
Group II Infected with 103 P.berghei infected erythrocytes and treated with Enoxaparin (6mg/kg b.w.)

[0072] Group I and II mice were inoculated intraperitoneally with 106 and 103 P.berghei NK-65 infected erythrocytes under the protective cover of Enoxaparin. Routinely used immunologically acceptable carrier, diluent or excipient were also added to prepare the present composition. Mice dose equivalent to recommended dose of enoxaparin was decided according to the formula; mice dose (mg/kg b.w) = human dose (1mg/kg b.w) *12.3. Finally, half of this dose i.e., 6 mg/kg b.w (The human equivalent dose is 0.5mg/kg bw) was administered to each mice for 4 days. Enoxaparin (Clexane) was used for the study. Mice in positive control were administered combination of artemether (6.84 mg/kg b.w.) and lumefantrine (40.8mg/kg b.w).
[0073] Mice were observed daily to monitor changes in body weight. Mice from each group were sacrificed on 30th day except infected group which was sacrificed on 10th day. Parasitemia was noted by observing Giemsa-stained slides according to the formula:

Mean percent parasitemia = Number of infected RBCs x 100
Total number of RBCs

Survival rate was determined by the following formula:

Survival rate = Total number of survived animals x 100
Total number of animals
[0074] Parasitemia was recorded for mice within all groups and data sets are presented as mean ± standard deviation. Statistical comparisons of data were carried out by Student’s t-test. A p-value?