Patent Application: US-82154410-A

Abstract:
no effective vaccine exists for the devastating parasitic disease of schistosomiasis . the present invention focuses on sm - p80 , a functionally important antigen of schistosoma mansoni that plays a pivotal role in the schistosome immune evasion process . when used in a novel vaccine formulation , sm - p80 demonstrates consistent immunogenicity , protective potential , and antifecundity effects . two novel dna constructs were made for immunization purposes . sm - p80 coding sequence was cloned into vr 1020 . additionally , sm - p80 coding sequence was cloned into pcdna3 . 1 with flanking cpg motifs on each end of the sm - p80 sequence . when used in different vaccine formulations , both of the constructs demonstrate the superior antifecundity and anti - worm effects of sm - p80 , which has great potential as an important vaccine candidate for the reduction of the morbidity associated with schistosome infection .

Description:
disclosed herein are compositions and methods for use of a sm - p80 - based vaccine formulation against schistosoma mansoni . the numerous innovative teachings of the present invention will be described with particular reference to several embodiments ( by way of example , and not of limitation ). reference is first made to fig1 , a schematic of the vr1020 / sm - p80 construct . fig1 , the first of two constructs , depicts a dna construct created by cloning schistosome sm - p80 coding sequence into vr1020 . reference is now made to fig2 , a schematic of the pcdna3 . 1 / sm - p80 construct . fig2 , the second of two constructs , depicts a dna construct created by cloning schistosome sm - p80 coding sequence into pcdna 3 . 1 with flanking cpg motifs on each end of the sm - p80 sequence . one having ordinary skill in the art will be able to construct the dna vaccine relying on fig2 . reference is now made to fig3 , a schematic diagram illustrating the first method of construction of vr1020 / sm - p80 and pcdna3 / sm - p80 . reference is now made to fig4 , a schematic diagram illustrating the second method of construction of vr1020 / sm - p80 and pcdna3 / sm - p80 . one having ordinary skill in the art will be able to construct the dna vaccine relying on fig1 in combination with fig3 or fig4 . both of the constructs depicted in fig1 and fig2 were used in different vaccine formulations ( dna alone and prime boost ) which were delivered to mice and baboons . this is first ever use of an sm - p80 - based vaccine formulation in the baboon model of schistosomiasis . table 1 summarizes the 1 . sm - p80 - based vaccine formulation [( a ) dna vaccine alone , ( b ) dna vaccine in which two unmethylated cpg motifs are inserted in the construct because , they act as immunostimulants ( c ) recombinant sm - p80 protein was introduced in presence of oligodeoxynucleotides ( odn ) containing cpg motifs activate host defense mechanisms leading to innate and acquired immune responses ) 2 . vaccine delivery routes and 3 . results obtained . experimental data on antifecundity and anti - worm effects of sm - p80 in both murine and nonhuman primate models clearly indicate that this antigen has great potential as an important vaccine candidate for the reduction of the morbidity associated with schistosome infection . in summary , sm - p80 - based vaccine formulations have three protective effects ( worm reduction , antifecundity effect and protection against acute schistosomiasis ). this is a first report of an anti - schistosome defined vaccine formulation which has shown these three protective effects . the protocols used for immunization of baboons included both naked dna vaccination and prime boost and protein vaccination strategies . protocols using naked dna vaccination in baboons were as follows : group 1 : for this control group , the initial immunization was with 500 or 1000 μg plasmid dna ( without the inserts ). the dna was injected intramuscularly ( im ) in the quadriceps . baboons were boosted with 500 or 1000 μg control plasmid dna at weeks 4 , 8 , and 12 . 500 μg is used in vaccinations across the board because in many nonhuman primate systems , this amount has provided consistent results . group 2 : to determine the protective effect of sm - p80 alone , the initial immunization was done with 500 μg plasmid sm - p80 - pcdna3 . 1 or sm - p80 - vr1020 . the dna was injected im in the quadriceps . baboons were boosted with 500 μg sm - p80 - pcdna3 . 1 or or sm - p80 - vr1020 at weeks 4 , 8 , and 12 . group 3 : to determine if by using il - 2 as genetic adjuvants , the protective effect of sm - p80 can be enhanced , the initial immunization was carried out with 500 μg plasmid sm - p80 - pcdna3 or sm - p80 - vr1020 and 500 μg plasmid porf - hil - 2 . the dna was injected im in the quadriceps . baboons were boosted with 500 μg sm - p80 - pcdna3 . 1 or sm - p80 - vr1020 and 500 μg porf - hil - 2 at weeks 4 , 8 , and 12 . after 4 weeks of the final boost , baboons from all of the groups were challenged with a total of 1000 cercariae of s . mansoni by the abdominal pouch method . eight weeks after the final challenge , the baboons were be immobilized and lightly anesthetized with a mixture of ketamine ( ketaminol — 10 mg / kg body wt ) and xylazine ( 0 . 5 mg / kg ) and then deeply anesthetized by intravenous injection of heparinized sodium pentabarbitol solution . the animals were then euthanized by exsanguination from the heart ventricle . this method of euthanization favors quantitative adult worm recovery by perfusion and post - perfusion inspection of mesenteric veins . the adult parasites were recovered by perfusion from the mesenteric vasculature and hepatic portal system by modifications of published methods . protection ( p ) will be calculated by comparing worm burdens from vaccinated ( v ) and control ( c ) baboons by a standard formula : % p =( c − v )/( c × 100 ). protocols using prime boost and protein vaccination strategies in baboons were as follows : the prime / boost approach which provides the optimal protection results in mice has been used in baboons . animals first immunized with 500 μg plasmid dna ( sm - p80 - vr1020 or immunostimulatory sequences ( iss )- sm - p80 - iss - vr1020 ) and were boosted with 200 μg baculovirus generated recombinant sm - p80 protein in the presence of either odn # 2138 ( 250 μg ) or resiquimod ( 50 μg ). animals in the age - matched control group received similar amounts of plasmid dna without inserts as animals in the experimental group and boosted with irrelevant bevs - generated recombinant protein in the same adjuvant as in the experimental group . antigen dna alone ( without the boost ) and recombinant protein alone ( without the prime ) were also included as controls . note that the iss sequences used for generating the sm - p80 - iss construct as well as cpg odns utilized in this study have consistently been shown to efficiently work in both mice and nonhuman - primates . reference is now made to fig5 - fig . 24 , which , in conjunction with table 6 - table 24 , detail experimental results derived from in vitro testing and in vivo testing in both mice and baboons . these experimental results demonstrate the efficacy of the present invention . reference is now made to fig5 in conjunction with table 6 , demonstrating the reduction in worm burden distribution for groups of mice immunized with control plasmids , vr1020 ( n = 10 ) and with sm - p80 - vr1020 ( n = 10 ). mice immunized with sm - p80 - vr1020 showed 46 . 87 % reduction in worm burden when compared to mice which received only control plasmids , vr1020 . reduction in worm burden was statistically significant in vaccinated animals ( p & lt ; 0 . 001 ). reference is now made to fig6 , a graph of the antibody titers of anti - sm - p80 total igg in immunized mice . table 7 lists serum antibody total igg production induced by inoculation of recombinant sm - p80 vaccine . reference is now made to fig7 , a graph of the antibody titers of anti - sm - p80 total igm in immunized mice . table 8 lists serum antibody igm production induced by inoculation of recombinant sm - p80 vaccine . reference is now made to fig8 , a graph of the antibody titers of anti - sm - p80 igg2a in immunized mice . table 9 lists serum antibody igg2a production induced by inoculation of recombinant sm - p80 vaccine . reference is now made to fig9 , a graph of the antibody titers of anti - sm - p80 igg2b in immunized mice . table 10 lists serum antibody igg2b production induced by inoculation of recombinant sm - p80 vaccine . reference is now made to fig1 and fig1 , in conjunction with table 11 , demonstrating splenocyte proliferation induced by recombinant sm - p80 compared to stimulation induced by concanavalin a after 48 hours of culturing in vitro . reference is now made to fig1 , depicting levels of cytokine production by splenocytes after 48 hours of stimulation with recombinant sm - p80 in vitro ( see also table 12 ). groups of mice were inoculated with vr1020 and vr1020 - sm - p80 . data are shown as mean ± standard deviation . statistical significance ( p ≦ 0 . 05 ) are indicated by (*) compared with vr1020 group using independent sample test . reference is now made to fig1 - fig . 24 , depicting agarose gel electrophoresis of various cytokines ( gapdh , il - 1α , il - 1β , il - 2 , il - 4 , il - 5 , il - 6 , il - 17 , and tnf - α ) estimated in the vr1020 group and vr1020 - sm - p80 group of immunized mice . table 13 quantitatively analyzes the various cytokines estimated in vr1020 and vr1020 - sm - p80 immunized mice . table 14 summarizes production of serum antibody titers in baboons in the control group vaccinated with vr1020 and production of serum antibody titers in baboons in the experimental group vaccinated with vr1020 - sm - p80 . table 15 and table 16 summarize levels of cytokines produced by splenocytes after 48 hours of stimulation with recombinant sm - p80 in vitro . groups of baboons were inoculated with vr1020 ( control group ) and vr1020 - sm - p80 ( experimental group ). data are shown as mean ± standard deviation . table 17 and table 18 summarize levels of cytokines produced by peripheral blood mononuclear cells after 48 hours of stimulation with recombinant sm - p80 in vitro . groups of baboons were inoculated with vr1020 ( control group ) and vr1020 - sm - p80 ( experimental group ). data are shown as mean ± standard deviation . table 19 and table 20 summarize interleukin 4 and interferon gamma spot - forming units ( sfus ) induced by recombinant sm - p80 after 48 hours of culturing in vitro . each baboon was inoculated with vr1020 ( control group ) or vr1020 - sm - p80 ( experimental group ). data are shown as mean ± standard deviation . the disclosed vaccine compositions and methods of use are generally described , with examples incorporated as particular embodiments of the invention and to demonstrate the practice and advantages thereof . it is understood that the examples are given by way of illustration and are not intended to limit the specification or the claims in any manner . to facilitate the understanding of this invention , a number of terms may be defined below . terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention . terms such as “ a ,” “ an ,” and “ the ” are not intended to refer to only a singular entity , but include the general class of which a specific example may be used for illustration . the terminology herein is used to describe specific embodiments of the invention , but their usage does not delimit the disclosed method , except as may be outlined in the claims . it will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention . the principal features of this invention can be employed in various embodiments without departing from the scope of the invention . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , numerous equivalents to the specific procedures and vaccine compositions described herein . such equivalents are considered to be within the scope of this invention and are covered by the claims . all publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains . all publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference . in the claims , all transitional phrases such as “ comprising ,” “ including ,” “ carrying ,” “ having ,” “ containing ,” “ involving ,” and the like are to be understood to be open - ended , i . e ., to mean including but not limited to . only the transitional phrases “ consisting of ” and “ consisting essentially of ” respectively , shall be closed or semi - closed transitional phrases . all of the vaccine compositions and / or methods of use disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the vaccine compositions and methods of use of this invention have been described in terms of preferred embodiments , it will be apparent to those skilled in the art that variations may be applied to the vaccine compositions and / or methods of use and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit , and scope of the invention . more specifically , it will be apparent that certain components which are both related by material and function may be substituted for the components described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope , and concept of the invention as defined by the appended claims . 1 . gryseels b , polman k , clerinx j , kestens l . human schistosomiasis . lancet 2006 ; 368 ( september ( 9541 )): 1106 - 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