THE MAIN TEA ETA USAITOA 20170368167A9 MA MA MA MA MA MAITI ( 19 ) United States ( 10 ) Pub . No .: US 2017/ 0368167 A9 ( 12) Patent Application Publication (48 ) Pub . Date : Dec . 28 , 2017 Agadjanyan et al. CORRECTED PUBLICATION
( 54 ) COMPOSITIONS AND METHODS RELATED (60 ) Provisional application No . 61 /691 ,607 , filed on Aug. TO NEUROLOGICAL DISORDERS 21 , 2012 , provisional application No . 61/ 792 , 770 , filed on Mar. 15 , 2013 . @(71 ) Applicants : The Institute for Molecular Medicine , Huntington Beach , CA (US ) ; VAXINE Publication Classification PTY LTD , Adelaide (AU ) (51 ) Int. Cl. A61K 39 /39 ( 2006 .01 ) @( 72 ) Inventors: Michael Agadjanyan , Huntington A61K 39 /00 ( 2006 .01 ) Beach , CA ( US) ; Anahit Ghochikyan , (52 ) U . S . CI. Huntington Beach , CA ( US ) ; Nikolai CPC ...... A61K 39 /39 ( 2013 . 01) ; A61K 39 / 0007 Petrovsky, Adelaide ( AU ) ( 2013 . 01 ) ; A61K 2039 / 55583 ( 2013 .01 ); A61K 2039/ 55505 (2013 . 01 ) ; A61K 2039/ 575 @(21 ) Appl. No. : 15 / 174 ,709 ( 2013 .01 ) ; A61K 2039 /55561 (2013 .01 ) (57 ) ABSTRACT @(22 ) Filed : Jun . 6 , 2016 The present technology relates to compositions comprising inulin particles for use in the enhancement of immune Prior Publication Data responses to neuronal self -antigens for treating or preventing (15 ) Correction of US 2017 /0239349 A1 Aug. 24 , 2017 neurodegenerative diseases , in a subject. Also provided are See (63 ) and (60 ) Related U . S . Application Data . pharmaceutically acceptable compositions comprising : par ticles of inulin ; a substance comprising one or more patho (65 ) US 2017 /0239349 A1 Aug. 24 , 2017 gen -associated molecular patterns ( PAMPs) ; and a neuronal self- antigen fused to carrier , and methods and uses of the composition for inducing or modulating an immune Related U .S . Application Data response in a subject, such as modulating an immune (63 ) Continuation - in -part of application No . 14 /628 , 023 , response to a neuronal self -antigen as a vaccine . Also filed on Feb . 20 , 2015 , which is a continuation of provided are vaccine compositions comprising inulin par application No. PCT /US2013 /055877 , filed on Aug . ticles , and an antigen - binding carrier material, and methods 20 , 2013 and uses of the vaccine . Patent Application Publication Dec . 28 , 2017 Sheet 1 of 65 US 2017 /0368167 A9
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Mapping of immunodominant 8 cell epitopes of a synuclein in 865 ] mica .
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COMPOSITIONS AND METHODS RELATED reducing amyloid - B ( AB ) levels in the brain , e . g ., by block TO NEUROLOGICAL DISORDERS ing the formation of Aß, promoting its clearance , preventing aggregation and destabilizing its oligomers. Anti - Aß immu RELATED APPLICATIONS notherapy is considered as one of the most promising approaches in AD treatment and is currently being tested in [0001 ] This application is a Continuation - In -Part of pend clinical trials . Unfortunately , none of these attempts reported ing U . S . application Ser. No. 14 /628 , 023 , filed Feb . 20 , to date have shown positive clinical outcome. The first 2015 , as the national stage of International Application clinical trial of an AD vaccine , AN - 1792 , which used PCT /US2013 /055887 , filed in the United States on Aug . 20 , fibrillar AB , formulated in Th1 saponin - based adjuvant 2013 , which in turn claims benefit under 35 USC section ( QS21) was halted when 6 % of the trial subjects receiving 119 ( e ) of provisional application 61 /792 ,770 , filed Mar . 15 , the active vaccine developed some degree of aseptic menin 2013 and of provisional application 61/ 691 , 607 . This appli goencephalitis . It is hypothesized that the vaccine adjuvant cation is also a Continuation - In -Part of pending U . S . appli used in this clinical vaccine trial as well as the associated cation Ser . No. 14 / 127 ,489 , filed Dec . 19, 2013 , as the activation of AB T cell epitopes ( i . e . resulting in potential national stage of International Application PCT/ EP2012 / Th1 autoimmune responses ) , may have been major media 061748 , filed in the EP on Jun . 19 , 2012 . tors of this severe side effect. At the same time, lessons learned from clinical trials indicate that to be effective , BACKGROUND anti- Aß immunotherapy should be initiated before cognitive [0002 ] Traditional vaccination against infectious diseases decline and severe pathological changes have occurred and relies on generation of cellular and humoral immune that clearing Aß at the late stages may be insufficient to halt responses that act to protect the host from overt disease even the progression of AD . In as much as pathological tau though they do not induce sterilizing immunity . More correlates much better with the degree of dementia than AB recently , attempts have been made with mixed success to deposition , targeting tau is now considered a promising generate therapeutic vaccines against a wide range of non approach for the treatment of advanced AD stages . In infectious diseases including neurodegenerative disorders addition , tau is a common pathological marker for several such as Alzheimer ' s disease ( AD ) , Parkinson disease ( PD ) , neurodegenerative disorders other than AD , categorized as Dementia with Lewy Bodies Dementia (DLB ) , Frontotem tauopathies and therapeutics aimed at eliminating pathologi poral Dementia (FTD ), Traumatic Brain Injury ( TBI) , etc . cal tau may also be beneficial these diseases that include Strategies on development of vaccines against neurodegen Amyotrophic Lateral Sclerosis , Frontotemporal Dementia erative diseases are based on the generation of humoral with Parkinsonism linked to chromosome 17 , Pick ' s Dis immune responses against mutated or altered self- proteins ease, Progressive Supranuclear Palsy, Creutzfeldt- Jakob that are hallmarks of the diseases . However, immunological Disease, Dementia Pugilistica , Down ' s Syndrome and oth tolerance to self -antigens , though altered , make difficult the ers . Currently two vaccines targeting Tau entered phase 1 generation of potent immune responses allowing the pro clinical trials , ACI- 35 and AADVac1 . ACI - 35 is a liposome duction of therapeutically relevant concentrations of anti - based vaccine containing MPLA as an adjuvant and activa bodies specific to pathological self- molecules , such as amy tor of innate immune system , whereas AADVacl contain loid - ß ( AB ) , tau , a - synuclein , etc . To achieve this goal and aluminum hydroxide ( Alhydrogel ) as an adjuvant. generate high concentrations of therapeutically potent anti [0007 ]. Another antigen associated with neurodegenerative bodies one should find a safe composition of an immuno diseases is alpha - synuclein ( a -Syn ) that was first implied to genic non -self vaccine platform for delivery of self -antigen neurodegeneration after identification of its presence in with a strong adjuvant. amyloid plaques of AD (Ueda K , 1993 , PNAS) . a -Syn was [0003 ] The most prevalent form of dementia worldwide is found in Lewy bodies (LBS ), distrophic neurites and sur Alzheimer ' s disease ( AD ) . Compared to other deadly dis rounding the core of the amyloid plaques. Synucleinopathies eases, Alzheimer ' s is the only disease that cannot yet be comprise a class of neurodegenerative diseases that share a prevented , cured or slowed . While death rates for other morphologic hallmark , which is pivotally characterized by major diseases, such as heart diseases , cancer , AIDS etc , the involvement of Lewy pathology in a subset of neurons have declined , death rates from Alzheimer ' s disease have and glia . The synucleinopathies include PD , DLB , Multiple risen 66 percent since 2000 (Www .alz . org ) . System Atrophy (MSA ) and Pure Autonomic Failure ( PAF ) . [0004 ] AD is clinically characterized by progressive loss PD and DLB are the most prevalent neurodegenerative of memory , behavior impairment and decline of cognitive disorders, after AD , and it is with these conditions that function . According to the World Health Organization intracytoplasmic LBs and dystrophic LNs are most com (WHO ), approximately 18 million people worldwide have monly associated . Notably, up to 50 % of AD cases exhibit Alzheimer ' s disease . By 2025 , this estimate is projected to Lewy bodies, and the presence of Lewy body pathology in grow to 34 million people , with the highest increase AD is associated with a more aggressive disease course and expected among developing countries . accelerated cognitive dysfunction . Mixed brain pathologies [ 0005 ] Neuropathological features of AD , and other neu account for most dementia cases in community - dwelling rodegenerative diseases , include neurofibrillary tangles, older persons and there are multiple reports on the interac deposition of misfolded proteins in plaques and neuronal tions of amyloidogenic proteins. Overlap of clinical and loss in affected brain regions. These pathological changes neuropathological features of AD and PD are observed in result in a profound loss of neurons and synapses over the dementia with DLB , and molecular interactions between course of the disease , thereby contributing to a progressive a - syn and AB were directly demonstrated by NMR . In reduction in the functional capacity of the patient. addition , it was shown that AB interacted directly with a -syn 10006 ] Since the " amyloid cascade hypothesis ” was pro - and stabilized the formation of hybrid nanopores that alter posed , most therapeutic approaches for AD have focused on neuronal activity and might contribute to AD . Thus, careful US 2017 /0368167 A9 Dec . 28 , 2017 neuropathological studies have shown that aggregations of formulated with vaccines targeting self -molecules involved a -syn , AB and also tau appear in the same neuronal struc - in Alzheimer ' s Disease and Parkinson Disease pathologies tures , providing a pathological basis for the clinical obser induced unexpectedly and incredibly strong T and B cell vations of the overlap between PD /DLB and AD . mediated immune responses compared with other adjuvants [0008 ] Several pre- clinical studies have demonstrated approved by FDA or used in clinical trials so far. To test a - Syn oligomer/ aggregate clearance using immunotherapy, whether inulin particles could enhance immune responses to including active immunization . The first phase I clinical trial vaccines against different neurodegenerative diseases, they for immunotherapy against a - Syn began in 2012 . Developed were mixed with a universal vaccine platform for delivering by AFFiRiS , the AFFITOPE , PD01, improved a - Syn - in self -antigens , such as AB , tau and synuclein . The combina duced pathology, including neuronal loss in mice , although tion of an inulin particle together with a PAMP innate data on human trials are not published yet . immune activator and the vaccine against neurodegenerative [0009 ] The common link between all these neurodegen diseases has been found to result in a surprisingly favorable erative diseases is chronic activation of innate immune and synergistic immune response without generation of responses including those mediated by microglia , the resi detrimental pro - inflammatory reactions. dent CNS macrophages . Along with controlling inflamma [0014 ] Microbial- derived compounds that trigger innate tory processes , and repair and regeneration , activation of immune activation also enhance the adaptive immune microglia can trigger neurotoxic pathways leading to pro response to a co - administered vaccine antigen . Such com gressive degeneration . The adaptive immune response in pounds are now known to comprise or mimic pathogen neurodegenerative diseases may serve as double edge sword associated molecular patterns ( PAMPs ) , where a PAMP is a contributing to tissue damage or resolving inflammation and structurally conserved motif derived from a pathogen that is mediating neuroprotection and repair . In case of vaccina immunologically distinguishable from host molecules , and tion - induced pro - inflammatory immune responses addi is recognized by an innate immune receptor. PAMPs are tional inflammation may be crucial for neurons that have present in certain types of protein , lipid , lipoprotein , carbo only a limited capacity for repair and cannot tolerate long hydrate , glycolipid , glycoprotein , and nucleic acids term inflammation . expressed by particular pathogens and include triacyl lipo [0010 ] This means that , successful adjuvant -antigen vac peptides, porins, glycans, single and double stranded RNA , cine combinations should be found that will be effective in flagellin , lipotechoic acid , N - formymethionine , and bacte generation of humoral responses inducing anti - inflammatory rial or viral DNA , amongst others. PAMPs act as innate responses and avoiding induction of additional pro - inflam immune activators by binding to PAMP - specific innate matory responses. Even after extensive validation in animal immune receptors such as toll - like receptors ( TLR ) , NOD models , adjuvant- antigen combinations that were effective like receptors , RIG ligase receptors and C -type lectins. This in animal challenge models may be ineffective in generation leads to activation of inflammatory gene pathways in of antibody responses to self - antigens or even detrimental immune cells . when administered to humans. An example of the former is [ 0015 ] What these PAMP compounds have in common is the ineffectiveness of alum adjuvants in human AD vaccines that they all activate the innate immune system and induce CAD106 , ADO3, LU AF20513 that are in various stages of inflammatory gene pathways , in particular through activat clinical trials, despite showing enhanced protection in ani ing Nuclear Factor- Kappa B (NFOB ), the master transcrip mal models . Another example is AN - 1792 trial using QS21 tional regulator of inflammatory gene activation . This adjuvant, showing no adverse effects in animal models but inflammation in turn may lead to enhancement of an adap supposedly increased the adverse events in patients after tive immune response to a co -administered antigen , as a adding emulsifier. by - product or downstream effect of the innate immune [ 0011 ] B - D - ( 2 - 1 ) polyfructofuranosyl a - D - glucose ( com activation . The enhancement by a separate substance of an monly known as inulin ) is a polysaccharide that (as dis adaptive immune response to a co - administered antigen is closed by WO 87/ 02679 , WO 2006 /024100 , and WO 2011 / known as an “ adjuvant ” effect. 032229, the contents of each of which are incorporated [0016 ] Without wishing to be restricted by theory , it is herein by reference ) develops useful properties when crys accepted by those skilled in the art that the common factor tallized into stable particulate structures . Inulin has a rela that links all compounds that possess adjuvant activity is that tively hydrophobic , polyoxyethylene - like backbone, and they induce immune “ danger signals ” leading to activation this unusual structure plus its non - ionized nature allows of innate immune and thereby activation of NFOB and other re - crystallization and easy preparation in a very pure state . inflammatory pathways. Danger signals that provide Inulin in its raw state is generally soluble in warm water but, immune adjuvant effects can be generated by local tissue as disclosed by WO 87 / 02679 , WO 2006 /024100 , WO damage , e . g . , induced by injection of inflammatory sub 2011 /032229 and WO 2012175518 can with specific treat stances such as oil emulsions , or more specifically through ments be crystallized into more stable polymorphic forms, binding of PAMPs to innate immune receptors whose role is including the previously described gamma (gIN ) , delta to detect pathogen invasion and tissue damage . PAMP ( DIN ) and epsilon ( eIN ) forms. associated danger signals thereby alert the innate immune [ 0012 ] Such inulin particles (hereinafter collectively system of the need to mount a defensive inflammatory referred to simply as ‘ inulin particles ' ) are largely insoluble response against the perceived threat . Following the activa at normal mammalian body temperature and have been tion of these danger - sensing PAMP receptors , inflammatory found to possess excellent adjuvant properties when formu gene signaling pathways including the key NFkB pathway lated with antigens . are activated leading to secretion by immune cells such as [0013 ] As described further in the present application , monocytes of key inflammatory effectors including tumor when studying the biological effects of inulin particles , the necrosis factor ( TNF ) - a , interleukin ( IL ) - 1 , IL - 6 , IL - 8 and surprising discovery has now been made that inulin particles IL - 12 , amongst others . These inflammatory mediators US 2017 /0368167 A9 Dec . 28 , 2017 released in response to PAMP activation are believed in the alum adjuvants remains the only adjuvants licensed for art to be critical to the ability of PAMPs to enhance antigen human use in many countries . Although alum adjuvants are specific adaptive immune responses , with high - throughput often useful to induce a good antibody ( Th2 ) response to cell -based screening assays designed to identify new adju co - administered antigen (s ), they are largely ineffective at vant compounds reliant upon their ability to induce inflam stimulating a cellular ( Th1 ) immune response , which are matory cytokines such as TNF -a , IFN -Y , IL - 1, IL - 8 or IL12 important for protection against many pathogens. Further as the readout of potential adjuvant activity . more , alum has the potential to cause rare severe local and 100171. Conceptually , two or more such innate immune systemic side effects including sterile abscesses , eosino activators when combined together induce even stronger philia and macrophagic myofasciitis . There is also commu danger signals , generate higher levels of inflammatory gene nity concern regarding the possible role of aluminum salts in activation , and thereby are predicted to show increased neurodegenerative diseases such as Alzheimer ' s disease . adjuvant potency . However , as known by those skilled in the Other licensed adjuvants including MF59 , a squalene oil art , the problem of using PAMPs either singly or, more emulsion adjuvant that is licensed in Europe as part of an particularly , combined together as immune modulators or influenza vaccine and AS04 , a combination of aluminum vaccine adjuvants is that the inflammatory effects are highly hydroxide and monophosphoryl lipid A (MPL ), which is toxic and hence the ability to achieve enhancement of an licensed in Europe in a hepatitis B vaccine . adaptive immune response in this way is hindered by severe [0021 ] However , the biggest single barrier to the devel dose - limiting local and systemic inflammation - associated opment of improved human adjuvants whether used alone or toxicity which is correspondingly magnified as the dose of together is the problem of local and systemic toxicity and the innate immune activator is increased . For example , even adverse reactions. This is a particular problem for develop the combination of a partially detoxified PAMP analogue , ment of childhood vaccines where safety is paramount . monophosphoryl lipid A (MPL ), with aluminum hydroxide Vaccine -mediated adverse reactions include inflammation (“ alum ” ) adjuvant in a hepatitis B surface antigen (HBsAg ) and granuloma formation at the site of injection , pyrogenic vaccine caused significantly more local injection site reac ity , nausea , adjuvant arthritis , uveitis , eosinophilia , allergy , tions , fever and other systemic side effects than HBsAg with anaphylaxis , organ specific toxicity or immunotoxicity , i. e . alum adjuvant alone . the liberation of toxic quantities of inflammatory cytokines. [ 0018 ] Increased vaccine reactogenicity and toxicity when Such extreme toxicity hampers the use of otherwise highly two or more innate immune activators are combined in a potent adjuvants such as complete Freund ' s adjuvant (CFA ) , vaccine formulation is a major barrier to regulatory approval with this toxicity principally reflecting excessive activation of such adjuvant combinations, even where there might be of inflammatory pathways by innate immune activator adju a favorable impact on vaccine immunogenicity . Further vants . Compounds or combined formulations that can suc more , not all combinations of innate immune activators are cessfully enhance adaptive immune responses , yet at the favorable from an immunogenicity standpoint, such that same time are well tolerated , safe and non - toxic to the host some combinations of innate immune activators produce an remain highly elusive , and of the hundreds of compounds adaptive immune response to a co -administered antigen that known to be innate immune activators and possess vaccine is no better than the individual innate immune activator adjuvant potential, less than a handful are approved for use components alone , and some innate immune activator com in humans, and just two compounds , alum and MPL , being binations even result in lower antigen - specific responses approved by the FDA for human vaccine use in the USA than with each individual innate immune activator used market. alone . For example , humans immunized with C - terminal 10022 ]. Ideally , adjuvant formulations should be suited for recombinant malaria circumsporozoite antigen with alum use with a wide range of potential vaccine antigens and be alone achieved higher antigen - specific antibodies than sub safe for use in low responder populations including children , jects receiving the combination of alum with MPL . the elderly and immuno -compromised individuals . Thus, [0019 ] The vaccine art recognizes the use of certain sub one of the major remaining challenges in vaccine research stances called adjuvants to potentiate an immune response remains how to increase vaccine potency without inducing when used in conjunction with an antigen . As used herein , increased local or systemic toxicity . The difficulty of achiev the term “ adjuvant” will be understood to mean any sub ing this objective is exemplified by the fact alum adjuvants, stance or material that when administered together or in 90 years after their discovery , continue to dominate human conjunction with an antigen increases the immune response vaccine use . to that antigen . The problem with pure recombinant or 0023 ] Because for the most part the mechanisms of synthetic antigens used in modern day vaccines is that they adjuvant action are notknown , the art has generally not been have poor immunogenicity when compared to less pure able to predict on an empirical basis whether a particular older- style live or killed whole cell vaccines . This has compound , or mix of compounds, will have adjuvant activ created a major need for development of effective adjuvants . ity . Similarly there is no way provided in the art to predict Adjuvants are further used to elicit an immune response that on an empirical basis whether a particular adjuvant, or mix is faster or greater than would be elicited without the use of of adjuvants , will be safe and well tolerated . the adjuvant. In addition , adjuvants may be used to create an [0024 ] Moreover , each adjuvant- antigen composition may immunological response using less antigen than would be generate a different type of immune response, which may or needed without the inclusion of adjuvant, to increase pro may not provide enhanced protection against a relevant duction of certain antibody subclasses that afford immuno pathogen . For example , different types of adaptive immune logical protection or to enhance particular cellular immune response have been described , for example T helper ( Th ) 1 , responses ( e. g ., CD4 or CD8 T cell memory responses ). Th2 and Th17 responses . For a particular pathogen , one 10020 ] Known adjuvants include aluminum salts (generi - adaptive immune response may be more favorable for cally referred to as “ alum ” adjuvants ). With few exceptions, providing protection than others . For example , for Leishma US 2017 /0368167 A9 Dec . 28 , 2017 nia a Th1 vaccine response is protective whereas a Th2 [0029 ] As described further in the present application , response may cause an unfavorable outcome. For other when studying the biological effects of inulin particles , we pathogens the converse may be true , such that a Th2 vaccine have now made the surprising finding that anti - inflammatory response is beneficial whereas a Th1 response is detrimental, effects are also provided . More specifically, it has been and in even other situations a Th17 vaccine response may be found that , when cultured with human peripheral blood desired . mononuclear cells (PBMC ) or mouse splenocytes , inulin [ 0025 ] This means that, in order to find successful adju particles will upregulate rather than down - regulate expres vant -antigen vaccine combinations, the art has relied on sion of anti - inflammatory genes . Conversely , they will extensive trial and error testing . Even after extensive vali downregulate the expression of many pro - inflammatory dation in animal models, examples abound of adjuvant genes and , in particular, inulin particles did not activate antigen combinations that were effective in animal challenge NFKB expression . models and were ineffective or even detrimental when [0030 ] This was a highly surprising finding as it appears to administered to humans . An example of the former is the contradict the widely accepted ' danger model' whereby all ineffectiveness of alum adjuvants in human influenza vac adjuvants are thought to work via activation of pro - inflam cines, despite showing enhanced protection in animal mod matory innate immune pathways through activation of els . Another example is respiratory syncytial virus (RSV ) NFKB and /or the inflammasome and thereby induce produc vaccine , which when formulated with alum adjuvant, tion of inflammatory cytokines such as TNF - a and IL - 1. The enhanced immunogenicity and protection in animal models danger model was largely developed based on the known of RSV infection but caused worsened disease and increased adjuvant action of PAMPs, for example TLR agonists that deaths from RSV infection when administered to human activate the innate immune system but also directly or children , an effect thought to be mediated by the vaccine indirectly increase adaptive immune responses to co - admin inducing the wrong type of immune response , namely a Th2 istered antigens. PAMP - derived adjuvants all share the prop rather than Th1 response . erty that they induce pro - inflammatory cytokines including 100261 B - D - ( 2 - 1 ) polyfructofuranosyl a - D - glucose (com tumor necrosis factor ( TNF ) -a , interleukin (IL ) - 1 , and IL - 6 monly known as inulin ) is a polysaccharide that ( as dis production . PAMPs induce these cytokines through activa closed by WO 87 /02679 , WO 2006 /024100 , and WO 2011/ tion of NFKB , a master transcription factor that induces 032229) develops useful properties when crystallized into inflammation in immune cells . Similarly , alum adjuvants stable particulate structures. Inulin has a relatively hydro and oil emulsion adjuvants activate the inflammasome, a phobic , polyoxyethylene -like backbone , and this unusual tissue damage sensing mechanism which when activated structure plus its non - ionized nature allows re - crystallization also leads to the production of inflammatory cytokines and easy preparation in a very pure state . Inulin in its raw including IL - 1 . By contrast , inulin particles when incubated state is generally soluble in warm water but, as disclosed by with human PBMC , surprisingly do not activate NFkB but WO 87/ 02679 , WO 2006 / 024100 and WO 2011/ 032229, can instead downregulate pro - inflammatory gene expression with specific treatments be crystallized into more stable including interleukin ( IL ) - 1 , ILIRAP, IL18RAP, cyclooxy polymorphic forms, including the previously described genase (Cox ) - 2 , NALP3 , NLRP3, NLRP12 , CARD12 . gamma ( gIN ) , delta ( DIN ) and epsilon ( eIN ) forms. IFIT1 , IFIT2 , IFIT3 , IDO , CXCL5 , CXCL6 , CXCR7 , [0027 ] Such inulin particles (hereinafter collectively CD14 , TLR4, NOD2 , formyl receptors 1 , 2 and 3 , and referred to simply as ‘ inulin particles' ) are largely insoluble upregulate genes associated with downregulation of innate at normal mammalian body temperature and have been immune responses and with inhibition of the pro - inflamma found to possess excellent adjuvant properties. Without tory IL1 cytokine pathway , including IL - 1 receptor antago wishing to be bound by theory, the stable conformation of nist ( IL - 1RA ) , ILIRN , and IL1R2 as well as IL 18BP, CD33 , these inulin forms are important for inulin particles to ATF3 , TREM1, PPAR - gamma, FCRL2 and CD36 . This data remain intact long enough to bind and interact with immune indicated that inulin particles have anti- inflammatory activ cells . Hence , when suspensions of inulin particles are heated ity , leading to the first aspect of the current technology , as to high temperature so as to dissociate and solubilize the discussed below . The ability of inulin particles to inhibit inulin particles , the resulting inulin solution loses all immu inflammation was thus tested herein , with a view to potential nological and vaccine adjuvant activity . Inulin particles use of inulin particles to treat or prevent inflammatory share properties relevant to their adjuvant action including disease . the ability to enhance antigen processing and presentation by [0031 ] To test whether inulin particles could reduce the appropriate immune cells, properties not shared by more side effects of pro - inflammatory immune activators and soluble inulin formulations . adjuvant formulations , inulin particles were tested , in vitro [0028 ] Without wishing to be bound by theory , we have and in vivo , with a range of PAMPs and innate immune observed that the immune effects of each inulin polymorphic activators including a broad range of TLR agonists , with the form increases in series as its temperature of solubility expectation that the inulin particles would inhibit both the increases, such that particles of diN are more temperature inflammation and also inhibit the adjuvant activity induced stable and adjuvant potent than gIN , and particles of eIN are by the PAMPs and other innate immune activators . The in turn more temperature stable and adjuvant potent than results were unexpected and surprising and led to the second particles of dIN . Thus , gIN , DIN or elN form are progres aspect of the current technology . As predicted , the co sively more adjuvant active . As disclosed by WO 87 / 02679 , administration of inulin particles together with a classical WO 2006 / 024100 , and WO2011/ 032229 , stable inulin for PAMP innate immune activator such as CpG -motif contain mulations comprising glN , DIN or eIN particles of appro ing oligonucleotides (ODN ) , down -modulated the inflam priate size and composition are able to enhance humoral matory gene activation mediated by the CpG ODN . What and / or cellular adaptive immune responses to co -adminis was unexpected , however, was that, paradoxically , despite tered vaccine antigens. successfully inhibiting the inflammatory signals induced by US 2017 /0368167 A9 Dec . 28 , 2017 the PAMP, the inulin particles actually enhanced the adju - subject against a neurodegenerative disease , and methods of vant activity of the PAMP on an adaptive immune response manufacturing a vaccine according to the compositions as measured by their ability to increase the protective herein . memory immune response against a co -administered anti [0036 ] In certain embodiments , the present technology gen . This finding was surprising given that the inulin par relates to products and methods of inducing a favorable ticles were predicted to downregulate the pro - inflammatory therapeutically potent immune response in patients with danger signals ' and innate immune activation induced by neurodegenerative diseases , such as AD , PD , LBD , etc . This the co - administered PAMPs. Under the prevailing danger is based on the surprising discovery that inulin particles signal model of adjuvant action , inulin particles , by inhib combined with vaccine based on neuronal self -antigens can iting inflammatory responses , would have been expected to be used to induce adaptive and innate immune responses that reduce the PAMP adjuvant activity . are much stronger and transcend all types of immune [0032 ] This experiment was subsequently repeated with a responses generated with all known anti -AD / PD /DLB , etc . wide variety of further PAMP adjuvants , and the same vaccines formulated in any known human adjuvants . beneficial effects of inulin particles were consistently [0037 ] Further embodiments of the technology are based observed that is , reduction in inflammation yet enhanced on the unexpected finding that the co -administration of adjuvant activity . In view of the previous lack of predict inulin particles with an innate immune activator results in a ability in the art when combining adjuvants in a single favorable and synergistic modulation of the balance between composition , the consistent results obtained when combin innate and adaptive immune responses , such that, in various ing inulin particles with all tested PAMPs was a further embodiments , a favorable anti - inflammatory and / or immune unexpected result . Without wishing to be restricted by response , or an enhanced immune memory response , is theory , the downregulation by inulin particles of pro - inflam achieved to a co - administered neuronal self -antigen with , if matory innate immune pathways induced by PAMPs, may anything, a reduction of inflammation -associated side paradoxically enhance the ability of the PAMPs to stimulate effects. an adaptive immune memory response , suggesting that [0038 ] Accordingly, in certain embodiments the present pro - inflammatory innate immune cytokines such as IL1 technology provides a composition comprising inulin par induced by PAMPs may , particularly if their levels are too ticles and vaccine targeting neuronal self -antigen for treating high , suppress rather than stimulate an adaptive immune or preventing neurodegenerative disease , in a subject . memory response . Thus , co -administration of inulin par [ 0039 ] In certain embodiments , the present technology ticles and an innate immune activator such as a PAMP provides methods of treating or preventing neurodegenera together with a vaccine antigen , results in a surprisingly tive diseases in a subject . In certain embodiments , this can synergistic enhancement of the immune memory response be accomplished without inflammation -associated side- ef against a co - administered vaccine antigen . The co - adminis fects , the method comprising the administration of a thera tration of inulin particles with an innate immune activator or peutically -effective amount of a composition comprising PAMP also provided a surprising dose -sparing effect on the inulin particles and vaccine targeting neuronal self - antigen innate immune activator, such that the same adjuvant effect to the subject. could be obtained with a reduced dose of the PAMP innate 10040 ] In certain embodiments , the present technology immune activator. Again this effect of inulin particles would provides for the use of a composition comprising various not be predicted by the danger model of adjuvant action . types of inulin particles and vaccine targeting neuronal This provides the opportunity to use inulin particles to self - antigen in the manufacture of a medicament for treating achieve the same adaptive immune enhancement effect with or preventing neurodegenerative disease , in a subject . A a lower dose of the innate immune activator, thereby offering further embodiment is based on the unexpected finding that the opportunity to reduce dose - limiting side effects such as the co - administration of inulin particles with an innate inflammation associated with innate immune activators immune activator results in a favorable and synergistic including PAMPs. Co -administration of the inulin particles modulation of the balance between innate and adaptive has further potential to reduce adverse inflammation - asso immune responses, such that an enhanced immune memory ciated side effects of innate immune activators and PAMPs response is achieved to a co -administered antigen with , if by blocking or attenuating inflammatory gene expression . anything, a reduction of inflammation - associated side effects . [0033 ] The applicants have found , therefore, that the com [0041 ] Accordingly , in certain embodiments , the technol bination of an inulin particle together with a PAMP innate ogy provides a composition comprising inulin particles for immune activator results in a surprisingly favorable and use in the reduction or inhibition of inflammation , and / or for synergistic immune response . treating or preventing inflammatory disease , in a subject for example, a method of reducing or inhibiting inflamma SUMMARY OF THE DISCLOSED tion , or methods of treating or preventing ( including pro TECHNOLOGY phylaxis against ) inflammatory disease , in a subject, the methods comprising the administration of a therapeutically [ 0034 ] In certain embodiments , the present technology is effective amount of a composition comprising inulin par directed to : a vaccine composition comprising : ( a ) inulin ticles to the subject; or use of a composition comprising particles ; ( b ) a pathogen -associated molecular pattern inulin particles in the manufacture of a medicament of (PAMP ); and ( c) an antigen containing a protein or peptide reducing or inhibiting inflammation , or of treating or pre derived from a neuronal self- antigen . venting inflammatory disease , in a subject. [0035 ] In certain embodiments , the present technology [0042 ] In certain embodiments , the reduction or inhibition provides methods of preventing or treating a degenerative of inflammation , or the treatment or prevention of inflam neurological disease in a subject, methods of vaccinating a matory disease, is characterized by up -regulation of the US 2017 /0368167 A9 Dec . 28 , 2017 expression of one or more anti -inflammatory genes and /or such as one or more adverse reactions including but not proteins and /or for the down- regulation of the expression of limited to : headache , fatigue , myalgia , diarrhea , fever , one or more pro - inflammatory genes and / or proteins in the inflammation and granuloma formation at the site of injec subject , or optionally , specifically in the subject' s myeloid or tion , pyrogenicity , nausea , adjuvant arthritis , uveitis , eosino lymphoid cells including monocytes, dendritic cells , granu philia , allergy, anaphylaxis , organ specific toxicity or immu locytes , NK cells and / or lymphocytes . Exemplary pro - in notoxicity, i. e . , the liberation of toxic quantities of flammatory genes for down - regulation in the subject in this inflammatory cytokines. context include interleukin ( IL ) - 1 , ILIRAP , IL18RAP, IL6 , cyclooxygenase (Cox ) - 2 , FPR2 , MYD88 , NALP3 , NLRP3 , [ 0046 ] In certain embodiments , inulin particles can also be NLRP12 , CARD12 , IFIT1 , IFIT2 , IFIT3 , IDO , CXCL5 , used in accordance with the previously discussed embodi CXCL6 , CXCR7 , CD14 , TLR4 , NOD2 , formyl receptors 1 , ments to treat or prevent inflammatory disease in a subject. 2 or 3 , and members of CXCL chemokine family and/ or Types of inflammatory diseases of particular interest for TLR family members . Exemplary anti - inflammatory genes treatment or prevention in this context include , e . g . , inflam for upregulation in the subject in this context include IL - 1 matory diseases that are characterized by, or associated with NFkB activation , elevated IL - 1 gene or protein levels or receptor antagonist ( IL - 1RA ) , ILIRN , and IL1R2 , IL18BP , signaling , or IL - 1 dysregulation . Exemplary inflammatory CD5L , CD33, ATF3 , TREM1, PPAR - gamma, FCRL2 and diseases include but are not limited to : migraine , chronic CD36 . fatigue syndrome, rheumatoid arthritis , asthma, chronic [0043 ] Accordingly, in certain embodiments , particles can obstructive airways disease, inflammatory bowel disease be used to reduce or inhibit inflammation in a subject including ulcerative colitis and Crohn ' s disease , chronic Inflammation in a subject may be caused , for example , by fatigue syndrome, cryopyrin -associated periodic syndromes the exposure to one or more pro - inflammatory substances, including neonatal onset multisystem inflammatory disease including pathogenic infections including bacterial, viral, and Muckle Wells syndrome, inflammasome- associated dis fungal or protozoal infection ; exemplary infections includ orders , psoriasis , atherosclerosis , type 1 or type 2 diabetes ing pandemic or seasonal influenza , inhalational anthrax , mellitus , hereditary fever syndromes, tumor necrosis factor gram negative septicemia , systemic viraemia , encephalitis , receptor -associated periodic syndrome, Schnitzler syn Q fever, tularemia , small pox , chronic hepatitis B or C drome, systemic lupus erythematosis, autoimmune hepatitis , infection , SARS , pertussis, malaria , HIV , tuberculosis , polio , rabies, respiratory syncytial virus (RSV ) , shigella , Behçet disease and idiopathic recurrent pericarditis . mononucleosis , cytomegalovirus and toxic shock syndrome, 10047 ]. Accordingly , subjects for treatment by the methods allergenic substances ; exemplary allergens being insect herein can include those who have been , will be in the sense venom , cat or dog dander , rye grass, dustmite antigen , and that they are scheduled to be, or are at increased risk of pollens, or other pro - inflammatory substances or composi being, in various embodiments within the following month , tions, including , for example , compositions comprising pro week , 6 , 5 , 4 , 3 , 2 or 1 days, or less than 24 , 12 , 6 , 5 , 4 , 3 , inflammatory substances, such as vaccine compositions or 2 or 1 hours ) , or are simultaneously being, exposed to one allergen - desensitization compositions, or anti -cancer treat or more pro - inflammatory substances , including pathogenic ments . In certain embodiments the inulin particles can be infections ( including bacterial, viral , fungal or protozoal administered to the subject before , simultaneously with , or infection ) , allergenic substances , or other pro - inflammatory after the subject’ s exposure to the one or more pro - inflam compositions, including , for example , compositions com matory substances . prising pro - inflammatory adjuvant, such as vaccine compo [ 0044 ] In certain embodiments , the technology is directed sitions or allergen - desensitization compositions ; those suf to the use of inulin particles to reduce or inhibit inflamma fering from or determined to be at risk of suffering from an tion in a subject that is caused by exposure (such as the inflammatory disease , including an inflammatory disease administration of) a pro - inflammatory substance or compo that is characterized by , or associated with , elevated IL - 1 sition that contains a substance comprising an innate levels or signaling ; or IL - 1 dysregulation , e . g . , migraine , immune activator and in particular a pathogen - associated chronic fatigue syndrome, rheumatoid arthritis , inflamma molecular pattern (PAMP ) including functional variants , tory bowel disease including ulcerative colitis and Crohn ' s derivatives or analogs thereof. The pro - inflammatory com disease , chronic fatigue syndrome, cryopyrin -associated position can , for example , be a pharmaceutically acceptable periodic syndromes including neonatal onset multisystem composition comprising a pro - inflammatory component that inflammatory disease and Muckle Wells syndrome, inflam is intentionally administered to the subject, or a pro -inflam masome- associated disorders , psoriasis , atherosclerosis , matory substance (e .g . , biological or pathogenic substance type 2 diabetes, hereditary fever syndromes, tumor necrosis or organism ) to which the subject is intentionally or acci factor receptor- associated periodic syndrome, Schnitzler dentally exposed . In this context, administration of the inulin syndrome, Behçet disease and idiopathic recurrent pericardi particles to the subject before , or simultaneously with (in tis . cluding as a single mixture with ) , administration of or [0048 ] In other embodiments , the present technology pro exposure to the pro - inflammatory composition can be most vides immunological or pharmaceutically acceptable com beneficial in certain embodiments . Thus, the composition positions comprising : ( a ) an anti - inflammatory component , comprising inulin particles can be used to reduce or inhibit such as inulin particles or one or more other anti - inflamma the inflammatory response of the subject to the pro - inflam tory inhibitors of IL - 1 or one or more other anti - inflamma matory substance or composition . tory inhibitors of NFKB activation ; ( b ) a substance compris [0045 ] In embodiments where the pro - inflammatory com ing one or more species of pathogen -associated molecular position is an adjuvant composition that comprises PAMP, pattern (PAMP ); and optionally , further comprising (c ) one the inulin particles can be used to reduce , inhibit or prevent, or more additional substances, for example , an antibody, one or more of a subject ' s adverse reactions to the PAMP, antisense oligonucleotide , protein , antigen , allergen , a poly US 2017 /0368167 A9 Dec . 28 , 2017 nucleotide molecule , recombinant viral vector , a whole [ 0054 ] In certain embodiments , distinct molecular species microorganism , or a whole virus. of PAMP can be structurally distinct. Such a structural [0049 ] In certain embodiments , pathogen - associated distinction can , for example , be determined by known molecular patterns (PAMPs ), as discussed herein , refers to methods of structural analysis, such as mass spectroscopy , molecules having the ability to activate the innate immune nuclear magnetic resonance , FTIR , circular dichroism , or system . PAMPs can be directly or indirectly recognized by differential scanning calorimetry . one or more innate immune receptors, or activate inflam [0055 ] In certain embodiments , distinct molecular species matory gene pathways in immune cells . PAMPs can induce of PAMP can be functionally distinct . Functionally distinct pro -inflammatory gene expression and protein production molecular species of PAMP can be characterized by dis by immune cells including , for example , one or more of playing a different binding profile to innate immune recep lymphocytes , monocytes , granulocytes , NK cells , dendritic tors. This can be assessed , for example , by measuring the cells , pro - inflammatory gene expression including , for binding of PAMP species to a panel of innate immune example , one or more cytokines including TNF - a , G -CSF , receptors which can , for example , comprise receptors GM - CSF , IL - 1 through to IL - 33 and more particularly IL - 1 , selected from TLRs, such as human or animal TLR - 1 , IL - 4 , IL -5 , IL - 6 , IL - 12 , IL - 13 , IL - 18 , IL - 20 , interferons TLR - 2 , TLR - 3 , TLR - 4 , TLR - 5 , TLR - 6 , TLR - 7 , TLR - 8 , including type 1 interferons and gamma interferon , chemok TLR - 9 , murine TLR - 11 ; NOD -1 , NOD -2 , other NOD - like ines including the CXC family of chemokines including receptors (NLRs ) such as NLRP1, NLRP3 , NLRP12 , CXCL1 to CXCL17 , CC family chemokines including NLRC4 ; DECTIN - 1 ; DC -SIGN ; AIM - 2 ; C - type Lectin , CCL1 to CCL28 , CX3C chemokines including fractalkine , MD2 ; CD14 ; LBP ; CD36 ; RIG - I - like receptors including C Family chemokines including XCL1 to XCL2 , with RIG - I, MDA5 , LGP2 and / or ASC . Binding of PAMP species induction of these pro - inflammatory genes typically involv to these receptors can be assessed by routine methods, such ing activation of the NFKB transcription factor. as surface plasmon resonance . The skilled person will be able to determine appropriate conditions under which to [0050 ] As used herein , the term “ PAMP” includes not only assess binding, which in certain embodiments can be those PAMPs found in nature , but also functionally equiva selected to provide an assessment of binding specificity lent mimetics , variants , derivatives and analogs thereof, under moderate to highly stringent conditions. Additionally , including synthetic PAMPs. Numerous naturally - occurring or alternatively, as known by the skilled person , the property and synthetic PAMPs are known in the art , many of which of a PAMP can be detected or quantified in an immune cell are discussed in more detail below . line such as the THP - 1 or RAW cell line , by a functional [0051 ] In certain embodiments , component ( a ) of the assay, for example using an NFkB activation reporter assay composition above is an anti - inflammatory component, such such as the Thermo Scientific Pierce Luciferase Assay Kit or as an anti - inflammatory inhibitor of IL - 1 or anti - inflamma by measurement of inflammatory gene or protein activation tory inhibitor of NFkB . In certain embodiments , the anti in response to incubation of the cell line with the substance inflammatory component comprises inulin particles . Other being tested for PAMP activity . anti - inflammatory inhibitors of IL - 1 of particular interest are functionally - equivalent to inulin particles, in the sense of [0056 ] In various embodiments , the totality of PAMP possessing an essentially equivalent anti- inflammatory prop present in the component ( b ) of the composition (and , erty , activity or specificity or possessing an essentially optionally , all of the PAMP in the composition , in the event equivalentadjuvant property . These can include one or more that component ( c ) contains further PAMP) will not bind to of IL1 receptor antagonists , ILIRA , Anakinra , Rilonacept, more than 20 , 19 , 18 , 17 , 16 , 15 , 14 , 13 , 12 , 11 , 10 , 9 , 8 , 7 , IL - 1R / IL1RacP /Fc - fusion protein , Canakinumab , mass 6 , 5 , 4 , 3 , 2 or 1 of the receptors in the panel of innate IL - 1B blocking antibody, IL1 receptor blockers, IL - 1RII , immune receptors as described above. indomethacin , non - steroidal anti - inflammatory drugs [0057 ] Although capable of indirectly activating the innate (NSAID ) including indomethacin , glucocorticoids, caspase immune system , through lysis of the lysosome in phagocy tosing cells , inflammasome activation , caspase activation , inhibitors including caspase 1 inhibitors , inflammasome induction of immune cell death , and release of endogenous inhibitors , chloroquine , P2X7 receptor inhibitors , ST2 DNA , antigen - binding carrier materials with adjuvant prop receptor inhibitors , curcumin , resveratrol, and eicosanoid erties such as alum (or other metal salts or precipitates such biosynthesis inhibitors . as magnesium , calcium or aluminum phosphates , sulfates , [0052 ] In certain embodiments , component ( b ) of the hydroxides or hydrates thereof) have not been shown them composition above is a substance comprising one or more selves to bind a specific PAMP receptor, and do not mimic pathogen - associated molecular patterns ( PAMP ). In certain a molecular pattern expressed by a pathogen and as they embodiments , the substance comprises no greater than ten thereby act in a different manner to molecularly defined distinctmolecular species of PAMP, e . g ., nine or less , eight PAMPs that bind specific innate immune receptors , they do or less , seven or less , six or less , five or less, four or less , not form part of the definition of PAMPs as used in this three or less, two or less, or only one distinct molecular application . species of PAMP. In certain embodiments, the limitation on [0058 ] It will be appreciated that in certain embodiments , the number of distinct molecular species of PAMP in com optional component ( c ) of the compositions above can , for ponent ( b ) can be applied only in respect of combination example , include one or more additional substances includ with inulin particles comprising a specific type of inulin . ing but not limited to : an antibody, antisense oligonucle [0053 ] Thus, for example , in various embodiments , com otide , protein , antigen , allergen , a polynucleotide molecule , ponent ( b ) comprises no greater than ten , nine , eight , seven , recombinant viral vector, a whole microorganism , or a six , five , four three , two or one distinct molecular species of whole virus , and so component ( c ) may contribute one or PAMP where the inulin particles in component ( a ) comprise more additional PAMPs to the composition . For example , gamma inulin , or delta inulin , or epsilon inulin . whole microorganisms, whole viruses , endotoxin and the US 2017 /0368167 A9 Dec . 28 , 2017 like will contain high numbers ( certainly greater than ten ) of the amount or concentration of PAMP present in the entire molecularly , structurally , physically and / or functionally dis - composition ) is , in certain embodiments , less than the tinct molecular species of PAMP. Thus , in certain embodi amount of PAMP required in an equivalent composition that ments , the total number of distinct molecular species of differs only in that it does not include the inulin particles (or PAMPs in the composition of the second aspect of the other equivalent anti - inflammatory component ) . In other present technology can be greater than ten . But that does not words, the presence of inulin particles (or other equivalent detract from the requirement, in certain embodiments , that anti -inflammatory component) in component (a ) of the com component ( b ) of the composition comprises no greater than positions herein can , in certain embodiments , provide a ten or fewer distinct molecular species of PAMP. Typically , composition that is able to induce or modulate an immune therefore , the substance ( s ) optionally present in component response in a subject using less PAMP in component (b ) than ( c ) will be molecularly, structurally and/ or functionally would be required to achieve the same level or type of different molecules to the molecules present in component induction or modulation compared to an equivalent compo ( b ) . sition that differs only in that it does not include the inulin [0059 ] The one or more PAMPs ( in certain embodiments particles (or other equivalent anti - inflammatory compo all PAMPs ) present in component ( b ) of the compositions nent) . can possess a weight average molecular weight of up to but [0063 ] Accordingly , in certain embodiments , the amount no more than 200 ,000 KDa, such as up to but no more than : or concentration of the one or more PAMPs in component 150 ,000 KDa, 100 ,000 KDa, 50 , 000 KDa, 40 ,000 KDa, ( b ) of the composition ( and , optionally, the amount and /or 20 . 000 KDa , 10 . 000 KDa , 5 , 000 KDa , 2 ,000 KDa, 1 , 000 concentration of the one or more PAMPs present in the KDa, 500 KDa, 450 KDa, 400 KDa, 350 KDa, 300 KDa, entire composition ) can be less than , e . g . , less than 90 % , 250 KDa , 200 KDa, 150 KDa, 100 KDa , 50 KDa , 40 KDa, 80 % , 70 % , 60 % , 50 % , 40 % , 30 % , 20 % , 10 % , 5 % , 4 % , 3 % , 30 KDa , 20 KDa , 10 KDa, 9 KDa, 8 KDa, 7 KDa , 6 KDa, 2 % , 1 % , 0 . 5 % , 0 . 4 % , 0 . 3 % , 0 . 2 % , 0 . 1 % , 0 .05 % , 0 . 04 % , 5 KDa , 4 KDa , 3 KDa, 2 KDa, or 1 KDa or less. 0 .02 % , 0 .01 % or less (by weight ) than the optimal amount [0060 ] In certain embodiments , a composition herein can of the same one or more PAMPs that is required in an be a pharmaceutically acceptable composition . As used equivalent composition that differs only in that it does not herein , a “ pharmaceutically acceptable composition ” refers include the inulin particles ( or other equivalent anti - inflam to a composition that is safe for administration to a subject, matory component) . In certain embodiments , the optimal such as a human subject, by injection , such as intravenous, amount of PAMPs in the equivalent composition is the subcutaneous or intramuscular injection . In one embodi amount that is required to achieve the desired effect of ment, the composition is defined as being safe if it contains induction or modulation of an immune response including no , or substantially no , endotoxin . Endotoxin is often used for example adjuvant enhancement of an immune response synonymously with the term lipopolysaccharide, which is a to a co -administered antigen without being so high as to major constituent of the outer cell wall of Gram -negative cause unacceptable levels of inflammatory and/ or other bacteria . It includes a polysaccharide ( sugar ) chain and a side- effects . This can be determined empirically for each lipid moiety , known as lipid A , which is responsible for the PAMP using routine methods, for example by performing toxic effects observed with endotoxin . The polysaccharide dose - ranging toxicity studies in animal models , or by use of chain is highly variable among different bacteria and deter surrogate measures such as the extent of NFkB activation in mines the serotype of the endotoxin and the lipid compo cell- based functional assays. nents are also highly variable such that a single endotoxin [0064 ] Indeed , such an equivalent composition can be sample may contain 10 's to 100 ' s of distinct molecular entirely incapable of achieving the same level or type of species. Endotoxin is approximately 10 kDa in size but can induction or modulation , no matter how much PAMP is form large aggregates up to 1000 kDa. Endotoxin is typi included , in the absence of inulin particles. In some embodi cally harmful and pyrogenic in therapeutic compositions and ments , these limitations can be applied to the composition of regulatory authorities have imposed strict limitations on the the second aspect of the technology where the inulin par allowable levels of endotoxin within a pharmaceutical com ticles present in component ( a ) comprise just one or two of position . Accordingly , the level of endotoxin in a composi gamma inulin , delta inulin or epsilon inulin . tion according to certain embodiments herein should be [0065 ] In certain embodiments , a suitable or optimal ratio minimized and may be , in various embodiments , less than of inulin particles (or other equivalent anti - inflammatory 100 endotoxin units ( EU ) per dose , such as less than 90 , 80 , component) in component ( a ) to PAMP in component ( b ) of 70 , 60 , 50 40 , 30 , 20 , 10 , 5 , 4 , 3, 2 , 1 or less EU per dose . the composition , in order to achieve a desired effect, can be In various embodiments , the concentration of endotoxin in determined empirically by the skilled person for each spe a composition herein is less than 200 EU /m3 , such as less cific combination of inulin particles and PAMP using routine than 150 , 100 , 90 , 80 , 70 , 60 , 50 40 , 30 , 20 , 10 , 5, 4 , 3 , 2 , methods. In certain embodiments , however, the weight/ 1 or less EU /m3 . In some embodiments , these limitations weight ratio of inulin particles (or other equivalent anti may be applied to the compositions herein where the inulin inflammatory component ) to PAMP is in the range of from particles present in component ( a ) comprise just one or two 10, 000 : 1 to 1 : 1 , from 1000 : 1 to 1 : 1 , from 100 : 1 to 1 : 1 , or of gamma inulin , delta inulin or epsilon inulin . Methods of from 100 : 1 to 10 :1 . measuring endotoxin levels , such as the limulus amoebocyte [0066 ] Accordingly , an immunological composition assay (LAL ) method , are well known in the art . according to certain embodiments herein can include an [0061 ] In certain embodiments , a composition herein can effective amount for inducing a desired immune response of optionally be packaged and /or presented in a convenient or a combination of components , wherein the combination unit dosage form . includes at least one inulin particle ( or other equivalent 10062 ] The amount or concentration of PAMP present in anti- inflammatory component) and at least one PAMP innate component (b ) of the compositions herein (and , optionally , immune activator. The PAMP innate immune activator in the US 2017 /0368167 A9 Dec . 28 , 2017 immunological composition can be of any type of PAMP 60 , 50 40 , 30 , 20 , 10 , 5 , 4 , 3 , 2 , 1 or less EU per dose . The innate immune activator known in the art. For example , the concentration of endotoxin in either or both of the first PAMP innate immune activator can be one or more of any container or second container in the kit can be less than 200 of the group of substances that are known agonists of innate EU /m3 , such as less than 150 , 100 , 90 , 80 , 70 , 60 , 50 40 , 30 , immune receptors . Accordingly , a PAMP innate immune 20 , 10 , 5 , 4 , 3 , 2 , 1 or less EU /m3 . In some embodiments , activator for use in the present technology can bind and be these limitations may be applied to the kit herein where an agonist of any one or more innate immune receptors of, inulin particles present in the first container comprise just TLRs, RNA helicases, NOD1, NOD2, other NOD - like one or two of gamma inulin , delta inulin or epsilon inulin . receptors (NLRs ) such as NLRP1, NLRP3, NLRP12 , [0074 ] In various embodiments , the amount or concentra NLRC4 ; DECTIN - 1 ; DC - SIGN ; AIM - 2 ; C - type Lectin , tion of PAMP present in the second container of the kit is MD2; CD14 ; LBP ; RIG -I - like receptors including RIG -I , less than the optimal amount, such as less than 90 % , 80 % , MDA5 , LGP2 and /or ASC , C - type lectin receptors , comple 70 % , 60 % , 50 % , 40 % , 30 % , 20 % , 10 % , 5 % , 4 % , 3 % , 2 % , ment receptors , Fc receptors , and scavenger receptors . 1 % , 0 .5 % , 0 .4 % , 0 . 3 % , 0 .2 % , 0 .1 % , 0 .05 % , 0 .04 % , 0 .02 % , [0067 ] In another embodiment, the present technology 0 .01 % or less (by weight ) , of PAMP that is required , when provides a kit of parts comprising : ( a ) a first container that used alone to achieve the desired level or type of induction contains a composition comprising an anti - inflammatory component, such particles of inulin and / or one or more other or modulation of the immune response . anti - inflammatory inhibitors of IL - 1 or NFkB ( as discussed [ 0075 ] In certain embodiments the weight/ weight ratio of above ); and ( b ) a second container that contains a substance inulin particles ( or other equivalent anti - inflammatory com comprising a PAMP. ponent ) in the first container to PAMP in the second con [ 0068 ] Thus , in certain embodiments , the substance pres tainer may be in the range of from 10000 : 1 to 1 : 1 , from ent in the second container comprises no greater than ten 1000 : 1 to 1 : 1 , from 100 : 1 to 1 : 1 , or from 100 : 1 to 10 : 1 . distinct molecular species of PAMP, e . g. , nine or less, eight [0076 ] In a further embodiment, the substance comprising or less , seven or less, six or less , five or less, four or less , a PAMP can be an innate immune activator, and can com three or less , two or less , or only one distinct molecular prise one or more a substances that binds and is an agonist species of PAMP. In certain embodiments , the limitation on of one or more of a TLR , RNA helicase , NODI, NOD2 , the number of distinct molecular species of PAMP in the other NOD - like receptors (NLRs ) such as NLRP1, NLRP3 , substance present in the second container can be applied NLRP12 , NLRC4; DECTIN - 1 ; DC - SIGN ; AIM - 2 ; C - type only in respect of kits in which the first container contains Lectin , MD2; CD14 ; LBP ; RIG - I -like receptors including a composition comprising particles of a specific type of RIG -I , MDA5, LGP2 and /or ASC , C - type lectin receptor, inulin , such as only gamma inulin , only delta inulin or only complement receptor, Fc receptor, and scavenger receptor. epsilon inulin . [0077 ] In a further embodiment, one or more PAMP can be [0069 ] In another embodiment, the totality of PAMP that a substance such as diacyl lipopeptide, triacyl lipopeptide, is present in the second container may not bind to more than Pam3CSK4 , lipoteichoic acid , peptidoglycan , HSP70 , 20 , 19 , 18 , 17 , 16 , 15 , 14 , 13, 12 , 11 , 10 , 9, 8 , 7, 6 , 5, 4 , 3 , zymosan , ssRNA , dsRNA , dsDNA , poly ( I : C ) , poly ( I : C 2 or 1 of the receptors in the panel of innate immune LC ) , Hiltono1TM , Polyl: PolyC12 - U , AmpligenTM MPLA , receptors as described above . heat shock protein , fibrinogen , heparan sulfate fragments , [ 0070 ] In certain embodiments , either or both of the first hyaluronic acid fragments , synthetic TLR4 agonist , imida container and second container in the kit can optionally zoquinoline, gardiquimod , loxoribine , bropirimine, CL264 , further comprise one or more additional substances , for R848 , CL075 PolyU , imiquimod , resiquimod , ssPolyU /Ly example , one or more of an antibody , antisense oligonucle oVec , ssRNA40 /LyoVec , unmethylated CpG oligonucle otide , protein , antigen , allergen , a polynucleotide molecule , otide, Class B ODN , Class C ODN , CpG2006 , CpG1826 , recombinant viral vector, a whole microorganism , or a CpG7909 , C12 -iE - DAP, E - DAP, Tri - DAP, muramyl dipep whole virus . tide (MDP ) , L18 -MDP , M - TriDAP, murabutide , PGN 10071 ] In various embodiments , the one or more PAMPS ECndi, PGN -ECndss , PGN - Sandi, porin , lipoarabinoman ( in certain embodiments all PAMPs ) present in the second nan , phospholipomannan , glucuronoxylomannan , container of the kit can possess a weight average molecular glycosylphosphatidylinositol (GPI ) - anchored protein , weight of up to but no more than 200 , 000 KDa , such up to hemozoin , viral dsDNA , synthetic dsDNA , viral dsRNA , but no more than : 150 ,000 KDa, 100 ,000 KDa, 50 , 000 KDa, synthetic dsRNA peptidoglycan containing the muramyl 40 ,000 KDa, 20 ,000 KDa, 10 , 000 KDa, 5 , 000 KDa, 2 ,000 dipeptide NAG -NAM - gamma- D - glutamyl- meso diamin KDa, 1 , 000 KDa , 500 KDa, 450 KDa, 400 KDa, 350 KDa, opimelic acid , peptidoglycan containing the muramyl dipep 300 KDa , 250 KDa , 200 KDa , 150 KDa , 100 KDa, 50 KDa , tide NAG -NAM - L -alanyl - isoglutamine , N - formyl methio 40 KDa, 30 KDa , 20 KDa, 10 KDa, 9 KDa, 8 KDa, 7 KDa, nine, muramyl tripeptide , beta - 1 , 3 - glucan , zymosan , cord 6 KDa, 5 KDa, 4 KDa, 3 KDa, 2 KDa, 1 KDa or less. factor, trehalose -6 , 6 -dibehenate , Poly (dA :dT ) , Poly (dG : 10072 ] In certain embodiments , either or both of the first dC ) , 5 ' ppp - dsRNA , low density lipoprotein (LDL ) , oxidized container or second container in the kit of the third aspect of LDL , chemically modified LDL , hemozoin , ATP . the present technology contains a unit dose of the material [0078 ] In a further embodiment, the inulin particle can contained therein . comprise inulin including but not limited to : gamma inulin , 10073 ] In various embodiments, either or both of the first delta inulin and epsilon inulin , or combinations of any one container or second container in the kit is a pharmaceutically or more of these inulins; optionally with aluminum phos acceptable composition , as defined above . Accordingly, in phate or aluminum hydroxide , including but not limited to : various embodiments, the level of endotoxin in either or phosgammulin , phosdeltin , phosepsilin , algammulin , and both of the first container or second container in the kit can algammulin , aldeltin or alepsilin . Alpha and / or beta inulin or be less than 100 EU per dose , such as less than 90 , 80 , 70 , other modified inulin particles can also be used in addition US 2017 /0368167 A9 Dec . 28 , 2017
to , or instead of, gamma, delta or epsilon inulin , providing ments , the methods include the steps of administering to the they are in a suitable particulate form . subject the immunological composition or kit, wherein the 10079 ] In a further embodiment, the composition compris composition , or each component, is administered in an ing inulin particles comprises particles of at least two inulin effective amount and at an effective time and route for preparations , and the preparations can differ in the polymor inducing a desired immune response or effect. phic form of the inulin present and / or the presence or species [0092 ] Accordingly , additional embodiments provides of an antigen -binding carrier material. For example , in methods of inducing or modulating an immune response in various embodiments , the inulin particles can comprise a subject, wherein said methods comprise administering to [0080 ] gamma inulin ( or a combination of gamma inulin the subject a therapeutically effective amount of the com with aluminum phosphate or aluminum hydroxide ) mixed position , or simultaneously , sequentially or separately with delta inulin ; or administering therapeutically effective amounts of the con [0081 ] gamma inulin ( or a combination of gamma inulin tents of the first or second containers of a kit herein . Further with aluminum phosphate or aluminum hydroxide ) mixed embodiments provide a composition or kit for use in induc with epsilon inulin ; or ing or modulating an immune response in a subject; or the [0082 ] delta inulin (or a combination of delta inulin with use of a composition or kit herein in the manufacture of a aluminum phosphate or aluminum hydroxide ) mixed with medicament for inducing or modulating an immune gamma inulin ; or response in a subject. [0083 ] delta inulin ( or a combination of delta inulin with [0093 ] In other embodiments , the modulation of the aluminum phosphate or aluminum hydroxide ) mixed with immune response can comprise increasing the speed of epsilon inulin ; or development of the immune response , compared to the [ 0084 ] epsilon inulin ( or a combination of epsilon inulin speed of development of the immune response obtained in with aluminum phosphate or aluminum hydroxide ) mixed the subject with an equivalent composition that differs only with gamma inulin ; or in that it does not include the inulin particles. The immune [0085 ] epsilon inulin (or a combination of delta inulin with response in question can be, for example , an adaptive aluminum phosphate or aluminum hydroxide ) mixed with immune response to one or more antigens . In various delta inulin . embodiments , the adaptive immune response can comprise [0086 ] In the forgoing list, any recitation of gamma, delta a response from one or more of T - cells ( including one or or epsilon inulin can optionally also be replaced with alpha more of CD4 + and / or CD8 + T -cells ) or B - cells , and can for inulin or beta inulin . example be determined with respect to the production of one [ 0087 ] In a further embodiment, the compositions herein or more types or subtypes of antibodies , such as any one or can further comprise one or more additional substances, for more of IgA , IgE , IgG1, IgG2a , IgG2b , IgG3, IgG4 or IgM example , an antibody, antisense oligonucleotide , protein , or with respect to the production of one or more types of antigen , allergen , a polynucleotide molecule , recombinant cytokines , such as any one or more of IFN - y , TGF - B , viral vector , a whole microorganism , or a whole virus . 10088 ). Accordingly , in a further embodiment, the compo GM -CSF , TNFa , IL - 1 , IL - 2 , IL4 , IL - 5 , IL - 6 , IL7 , IL - 8 , sition can further comprise one or more antigens . The one or IL10 , IL12 , IL13 , IL - 17 or IL - 20 . more antigens can be any type of antigen known in the art , [ 0094 ] In other embodiments , the modulation of the including but not limited to : proteins , glycoproteins, pep immune response can comprise increasing the specificity of tides , polypeptides , cells , cell extracts, polysaccharides, the subject' s immune response , compared to the specificity polysaccharide conjugates, lipids, glycolipids, nucleic acids of the immune response obtained in the subject with an and carbohydrates , or conjugates of carbohydrates or lipids equivalent composition that differs only in that it does not with protein , polypeptide /peptide antigens , peptide mimics include the inulin particles. The immune response in ques of polysaccharides ; antigens may also be encoded within tion can be , for example , an adaptive immune response . In nucleic acid sequences . In certain embodiments , antigens various embodiments , the adaptive immune response can can be in a crude, purified or recombinant form . Antigens comprise a response from one or more of T - cells ( including can be derived from an infectious pathogen such as a virus , one or more of CD4 + and /or CD8 + T -cells ) or B -cells , and bacterium , fungus or parasite , or the antigen may be derived may for example be determined with respect to the produc from a tumor antigen , an allergen , or self - protein . tion of one or more types or subtypes of antibodies , such as [0089 ] In the embodiments herein where one or more any one or more of IgA , IgE , IgG1, IgG2, IgG3 , IgG4 or antigens, in particular one or more vaccine antigens is / are IgM . Increased specificity can , for example , include increas included , it can also be suitable to further include one or ing the level of specificity of the B - or T - cell response to any more antigen -binding agents in the samemixture as the one antigen that is presented in the administered composition ( s ) . or more antigens . [0095 ] In other embodiments , the modulation of the 10090 ] In certain embodiments , the present technology immune response can comprise increasing the magnitude or also contemplates methods of preparing the compositions increasing the duration of the subject' s immune response , herein . In various embodiments , the methods can comprise compared to the magnitude or duration respectively of the the step of providing the component parts and then bringing immune response obtained in the subject with an equivalent them together to form a composition . composition that differs only in that it does not include the [0091 ] In certain embodiments , the present technology inulin particles . The immune response in question can be, for also contemplates methods of stimulating or modulating an example , an adaptive immune response . In various embodi immune response , including an antigen -specific immune ments , the adaptive immune response can comprise a response , in a subject by administering to the subject a response from one or more of T - cells ( including one or more therapeutically effective amount of an immunological com - of CD4 + and / or CD8 + T - cells ) or B - cells , and can for positions herein or using a kit herein . In various embodi example be determined with respect to the production of one US 2017 /0368167 A9 Dec . 28 , 2017 or more types or subtypes of antibodies , such as any one or prises antigen - binding carrier material , for the manufacture more of IgA , IgE , IgG1, IgG2, IgG3, IgG4 or IgM . of a medicament for inducing or modulating an immune [0096 ] In other embodiments , the modulation of the response to the antigen . immune response can comprise modifying the type of the 10100 ] In certain embodiments , the technology is directed subject' s immune response , compared to the type of the to a method of vaccinating a subject , wherein said method immune response obtained in the subject with an equivalent comprises: administering to a subject a therapeutically effec composition that differs only in that it does not include the tive amount of a composition according to the second aspect inulin particles. The type of immune response in question of the present technology , wherein said composition also can be , for example , an adaptive immune response . In comprises an antigen and , optionally , further comprises various embodiments, the type of adaptive immune response antigen - binding carrier material; or simultaneously , sequen can be characterized by the speed , magnitude , specificity , or tially or separately administering to a subject therapeutically duration of one or more aspects of an adaptive immune effective amounts of the contents of the first or second response relative to other aspects of the adaptive response , containers of a kit herein , wherein said contents of the first including for example , the response from one or more of or second containers of the kit also comprises an antigen T - cells ( including one or more of CD4 + and / or CD8 + and , optionally , further comprises antigen -binding carrier T - cells ; Thi, Th2 , Th17 and Treg cells ) and /or B - cells , and material . Thus , in certain embodiments the technology pro can for example be determined with respect to the produc vides a composition that comprises an antigen and , option tion of one or more types or subtypes of antibodies com ally , further comprises antigen - binding carrier material, for pared to one or more other subtypes , such as any one or more use in vaccinating a subject; and also provides for a kit , of IgA , IgE , IgG1, IgG2, IgG3, IgG4 or IgM compared to wherein the contents of the first or second containers of the any one or more of the others. kit also comprises an antigen and , optionally , further com [ 0097 ] Other examples of modifying the type of the sub prises antigen -binding carrier material, for use in the vac ject' s immune response , in accordance with these embodi cinating a subject. In certain embodiments , the vaccinating ments , include modifying the balance between the innate of the subject is against a neurodegenerative disease . and adaptive immune response ; enhancing the immune [0101 ] In certain embodiments , the technology herein memory response; altering the type of immune response provides for the use of a composition that comprises an such as by enhancing or inhibiting the Th1, Th2 , Th17 or antigen and , optionally , further comprises antigen -binding Treg response compared to the other responses ; suppressing carrier material, in the manufacture of a medicament for the the IgE response , or enhancing one or more of the IgA , IgM vaccination of a subject; and also provides for the use of a or IgG subtype responses . Thus, in certain embodiments , the kit , wherein the contents of the first or second containers of technology provides methods to obtain an optimal immune the kit also comprises an antigen and , optionally , further subclass or subtype response , including the optimal T - or comprises antigen - binding carrier material, for the manu B - cell response to a vaccine antigen , where it could not be facture of a medicament for the vaccination of a subject. achieved to the same extent using an equivalent composition [0102 ] Suitable vaccine antigens for use in accordance or kit that differs only in that it does not include the inulin with certain embodiments herein can include any of those particles (or other equivalent anti - inflammatory compo described elsewhere in this application . The amount or nent ) . concentration of antigen used in certain embodiments herein [0098 ] In other embodiments , the technology provides a can be less than the amount of antigen that is required in an method of inducing or modulating an immune response to an equivalent composition or kit that differs only in that the antigen , wherein said method comprises : administering to a composition or kit does not include inulin particles ( or other subject a therapeutically effective amount of a composition equivalent anti- inflammatory component) . In other words , herein , wherein said composition also comprises the antigen the presence of inulin particles (or other equivalent anti and , optionally , further comprises antigen -binding carrier inflammatory component ) in the compositions and / or kits material; or simultaneously , sequentially or separately can provide for methods and uses that can induce or modu administering to a subject therapeutically effective amounts late an immune response herein with less antigen . of the contents of the first and second containers of a kit [0103 ] Accordingly , in various embodiments , the amount herein , wherein said contents of the first or second contain or concentration of one or more antigens in the compositions ers of the kit also comprises the antigen and , optionally, or kits herein can be less , such as less than : 90 % , 80 % , 70 % , further comprises antigen - binding carrier material. 60 % , 50 % , 40 % , 30 % , 20 % , 10 % , 5 % , 4 % , 3 % , 2 % , 1 % , [ 0099 ] Thus, in certain embodiments , the technology pro 0 . 5 % , 0 . 4 % , 0 . 3 % , 0 . 2 % , 0 . 1 % , 0 .05 % , 0 . 04 % , 0 .02 % , vides a composition that comprises an antigen and , option 0 .01 % or less (by weight) than the optimal amount of the ally , further comprises antigen - binding carrier material, for same one or more antigens that is / are required to achieve a use in modulating an immune response to the antigen ; and corresponding desired immune response , or effective vacci also provides for a kit , wherein the contents of the first or nation of a subject, in an equivalent composition or kit that second containers of the kit also comprises an antigen and , differs only in that it does not include the inulin particles (or optionally , further comprises antigen - binding carrier mate other equivalent anti - inflammatory component) . rial, for use in inducing or modulating an immune response [0104 The optimal amount in the equivalent composition to the antigen . In certain embodiments , the composition also is the amount that is required to achieve the desired effect of comprises an antigen and , optionally, further comprises induction or modulation of an immune response without antigen -binding carrier material, in the manufacture of a being so high as to cause unacceptable levels of inflamma medicament for inducing or modulating an immune tory or other side - effects . This can be determined empiri response to the antigen ; and also provides for the use of a kit, cally by the skilled person for each antigen and PAMP using wherein the contents of the first or second containers of the routine methods . Indeed , in certain embodiments, such an kit also comprises an antigen and , optionally , further com equivalent composition may be entirely incapable of achiev US 2017 /0368167 A9 Dec . 28 , 2017
ing the same level or type of immune induction or modu composition herein , thereby to produce a vaccine composi lation , or vaccination , no matter how much antigen is tion . In certain embodiments , the technology provides for included , in the absence of inulin particles ( or other equiva the use of a composition herein as an adjuvant in a vaccine . lent anti - inflammatory component) . [0110 ] In the examples and embodiments discussed [0105 ] In certain embodiments , the present technology herein , it is demonstrated that the compositions according to also provides methods of down -modulating an existing certain embodiments of the present technology can provide unwanted immune response in a subject , such as an allergy single vaccine dose protection against an otherwise lethal to an allergen , or a chronic inflammatory condition , for condition . Also , compositions of the certain embodiments , example by downregulation of allergen -specific IgE or when formulated as a vaccine against influenza , can provide induction of blocking allergen -specific IgG . Such methods effective single dose protection in a murine model. Single can include the steps of administering to the subject a dose vaccine protection is extremely desirable and , hitherto , composition herein , or the components of a kit herein , and hard to achieve in the field of vaccinology . Yet the compo optionally a further component such as an antigen or aller sitions of certain embodiments herein have been found to gen wherein each component is administered in an effective provide single dose vaccine protection amount and at an effective time and route for inhibiting or down -modulating the unwanted immune response and / or [0111 ] Accordingly , in certain embodiments , the present inducing a favorable counter -regulatory immune response . technology provides a single -dose vaccine composition [0106 ] In certain embodiments , the present technology comprising inulin particles (optionally in the form of a kit ) , provides methods for the allergen desensitization of a sub an antigen and, optionally , an antigen -binding carrier mate ject, wherein said method comprises : administering to a rial. Such a single dose vaccine composition is effective to subject a therapeutically effective amount of a composition provide vaccine protection in the subject with only a single herein , wherein said composition also comprises an allergen administration of a dose of the vaccine . and , optionally , further comprises allergen - binding carrier [0112 ] In certain embodiments , the present technology material ; or simultaneously , sequentially or separately provides a method of vaccinating a subject the method administering to a subject therapeutically effective amounts comprising administering to the subject a dose of a vaccine of the contents of the first and second containers of a kit herein , in certain embodiments a single does . In various herein , wherein said contents of the first or second contain embodiments , the method can comprise one or more addi ers of the kit also comprises an allergen and , optionally , tional steps, or can comprise no additional steps of admin further comprises an allergen -binding carrier material. That istering the vaccine after the initial administration . is , in certain embodiments, a composition also comprises an [0113 ] In certain embodiments , the present technology allergen and , optionally , further comprises allergen -binding provides a single - dose of the vaccine as defined above for carrier material , for use in the allergen desensitization of a use in vaccinating a subject by a method comprising admin subject; and also provides for a kit herein , wherein the istering to the subject a single - dose of the vaccine ; or the use contents of the first or second containers of the kit also of a single -dose of the vaccine as defined above for the comprises an allergen and , optionally , further comprises manufacture of a medicament for use in vaccinating a allergen - binding carrier material, for use in the allergen subject by a method comprising administering to the subject desensitization of a subject . Such embodiments can provide for the use of a composition that comprises an allergen and , a single - dose of the vaccine . optionally , further comprises allergen -binding carrier mate [0114 ] A further advantageous feature of the present rial, in the manufacture of a medicament for the allergen embodiments is that the compositions, substances , kits and desensitization of a subject ; and also provides for the use of methods described herein are particularly effective in treat a kit herein , wherein the contents of the first and / or second ing those subject groups thatmay typically fail to respond at containers of the kit also comprises an allergen and , option all, or adequately , to conventional adjuvant and vaccine ally , further comprises allergen -binding carrier material, for compositions. Such subject groups may include the young, the manufacture of a medicament for the allergen desensi the older population and pregnant women . In some embodi tization of a subject. ments , influenza vaccines of the present technology may be [0107 ] In certain embodiments , the present technology of particular interest for administration to such subjects . provides methods of treating cancer , wherein said method [0115 ] Accordingly , in various embodiments the subject to comprises administering to a subject a therapeutically effec be treated by the compositions, substances , kits and methods tive amount of a composition herein ; or simultaneously , herein can be child , for example a male or female child . The sequentially or separately administering to a subject thera child can be , for example , less than 18 years old , 17 years peutically effective amounts of the contents of the first and old , 16 years old , 15 years old , 14 years old , 13 years old , second containers of a kit herein . Thus , certain embodiments 12 years old , 11 years old , 10 years old , 9 years old , 8 years provide a composition or kit for use in the treatment of old , 7 years old , 6 years old , 5 years old , 4 years old , 3 years cancer ; or the use of a composition or kit herein in the old , 2 years old , 1 year old , 11 months old , 10 months old , manufacture of a medicament for the treatment of cancer. 9 months old , 8 months old , 7 months old , 6 months old , 5 [ 0108 ] In certain embodiments , a composition or the con months old , 4 months old , 3 months old , 2 months old , or 1 tents of the first or second containers of a kit herein further month old , relative to the date of their birth . comprises a cancer antigen . [0116 ]. In other embodiments , the subject to be treated by [0109 ] In other embodiments , the present technology pro the compositions , substances , kits and methods according to vides a method of manufacturing a vaccine, the method the other aspects of the present technology may be an older comprising the step of combining an antigen , and optionally human , for example a male or female . The older human can also an antigen -binding carrier material, with one or more be , for example , at least 40 years old , at least 45 years old , components ( for example , components ( a ) and ( b ) ) of a at least 50 years old , at least 55 years old , at least 60 years US 2017 /0368167 A9 Dec . 28 , 2017 old , at least 65 years old , at least 70 years old , at least 75 Tau B cell epitopes and at least one a -synuclein B cell years old , at least 80 years old , at least 85 years old , or at epitope , and at least one foreign T helper cell ( Th ) epitope . least 90 years old . (0124 ] In certain embodiments , compositions herein are [0117 ] In certain embodiments , the subject to be treated by used to generate an immune response in a subject in need the compositions, substances, kits and methods according to thereof, comprising administering the immunogen to the the other aspects of the present technology can be a pregnant subject. The subject in need may be at risk of developing or female . The female can be up to , or at least, 5 , 10 , 15 , 20 , has been diagnosed with Alzheimer ' s disease or one or more 25 , 30 , 35 or 40 weeks pregnant. conditions associated with abnormal amyloid deposits , Tau [0118 ] In other embodiments , the technology herein pro deposits , and a -syn deposits . The compositions can be used vides a method of identifying optimal concentrations and to prevent, treat or ameliorate a condition associated with ratio of components ( a ) and ( b ) of a composition herein , the deposits of amyloid , tau , and / or a - syn , comprising admin method comprising the optional step of combining an anti istering to a subject in need thereof an effective amount of gen , and optionally also an antigen - binding carrier material , the immunogen . In certain embodiments , the present tech with components ( a ) and (b ) of the composition , adminis nology is directed to tering the combined composition in a range of different doses to a series of subjects and then measuring the resulting BRIEF DESCRIPTION OF THE DRAWINGS immune response and optionally challenging the subject (0125 ] The patent or application file contains at least one with a live pathogen thereby allowing the optimal compo drawing executed in color. Copies of this patent or patent sition to be identified . application publication with color drawing ( s ) will be pro [0119 ] In certain embodiments , the contents of the first or vided by the Office upon request and payment of the second containers of a kit herein form , optionally with an necessary fee . antigen , an assay kit for identification of the optimal com [0126 ] FIGS . 1A - 1D show four graphs showing the immu position for a desired immune application . nogenicity in mice of trivalent influenza vaccine ( TIV ) [0120 ] In another embodiment a method ofmanufacturing formulated with the TLR9 agonist PAMP CpG2006 , high an assay kit is provided , the method comprising the step of lighting the synergistic effect when inulin particles are added combining an antigen , and optionally also an antigen - bind to the CpG -containing TIV vaccine formulation . Female ing carrier material, with components ( a ) and ( b ) of a Balb / c mice at 6 - 8 weeks of age ( n = 5 - 8 per group ) were composition herein , thereby to produce a vaccine assay kit . immunized intramuscularly twice 14 days apart , with 50 ul [0121 ] In various embodiments, the compositions dis of a commercial human TIV at 100 ng HA per dose , closed herein comprise at least one immunogen , wherein combined with either 2 , 7 , 20 or 60 ug of CpG2006 alone or each at least one immunogen comprises a region A coupled mixed with 1 mg PDmix ( 1 : 5 ) . FIG . 1A shows serum anti to a region B ; wherein region A comprises at least one influenza total IgG levels , FIG . 1B shows serum anti amyloid - ß (AB ) B cell epitope or at least one Tau B cell influenza IgM levels , FIG . 1C shows serum anti - influenza epitope or at least one a - synuclein B cell epitope or a IgG1 levels and FIG . 1D shows serum anti - influenza IgG2a combination of at least one amyloid - B ( AB ) B cell epitope levels 42 days after the second immunization as measured and at least one Tau B cell epitope or a combination of at by ELISA . Shown are group mean OD + SD . least one amyloid - ß (AB ) B cell epitope and at least one [0127 ] FIGS . 2A - 2F show six graphs demonstrating the a -synuclein B cell epitopes, or a combination of at least one synergistic effects of a combination of the TLR9 agonist Tau B cell epitope and at least one a - synuclein B cell PAMP (CpG1668 ) and inulin particles (PDmix1 : 36 ) on the epitope , or a combination of at least one amyloid -ß (AB ) B immune response of neonatalmice to TIV vaccine. Neonatal cell epitope and at least one Tau B cell epitope and at least BALB / c mice ( n = 5 - 7 / group ) were immunized i . m . with TIV one a - synuclein B cell epitope , and region B comprises a ( 100 ng totalHA protein ) at 14 days and 23 days of age . Sera plurality of foreign T helper cell ( Th ) epitopes. In another were collected 14 days after the last injection for measure aspect , the composition comprises at least two immunogens, ment of antibodies by ELISA . Groups received either TIV wherein each immunogen is distinct. alone or formulated with PDmix1 : 36 ( 1 mg ) , CPG1668 ( 20 [0122 ] In some embodiments , the immunogen comprises a ug) , or PDmix (1 mg) + CpG1668 ( 20 ug ). FIG . 2A shows the linker domain between region A and region B . In other group receiving TIV + PDmix + CpG ( final column in each embodiments , the immunogen comprises linker domains figure ) had significantly higher anti - influenza total IgG , FIG . between each epitope . In some embodiments , the order of 2B shows higher IgM , FIG . 2C shows lower IgG1, FIG . 2D the regions is A - B and in other embodiments , the order is shows higher IgG2a , FIG . 2E shows higher anti - influenza B - A . In some embodiments , the compositions further com CD4 + T cell and FIG . 2F shows higher CD8 + T -cell memory prise an adjuvant or a pharmaceutical excipient or both . responses. [0123 ] In other embodiments , the composition comprises [0128 ] FIGS. 3A - 3D show four graphs showing the anti at least one nucleic acid molecule encoding an immunogen , influenza IgM , IgG2a , IgG1, and total IgG responses as wherein the immunogen comprises at least one amyloid - ß measured by ELISA in sera from 200 - 300 day old female ( AB ) B cell epitope or at least one Tau B cell epitope or at Balb / c mice ( n = 10 / group ) immunized intramuscularly twice least one a - synuclein B cell epitope or a combination of at 14 days apart with a trivalent inactivated influenza vaccine . least one amyloid - ß (AB ) B cell epitope and at least one Tau FIG . 3A shows serum anti- influenza total IgG levels , FIG . B cell epitope or a combination of at least one amyloid 3B shows serum anti - influenza IgM levels , FIG . 3C shows (AB ) B cell epitope and at least one a -synuclein B cell serum anti - influenza IgG1 levels and FIG . 3D shows serum epitopes , or a combination of at least one Tau B cell epitope anti - influenza IgG2a levels 42 days after the second immu and at least one a - synuclein B cell epitope, or a combination nization as measured by ELISA . Shown are group mean of at least one amyloid - ß (AB ) B cell epitope and at least one OD + SD . The group co - administered inulin particles ( PD US 2017 /0368167 A9 Dec . 28 , 2017 14 mix ) plus a TLR9 agonist PAMP (CpG2006 ) achieved the [0134 ] FIGS. 9A -9B show 2 graphs showing the favorable highest anti - influenza antibody titers. immune enhancing effect of combinations of inulin particles [0129 ] FIGS. 4A -4D show four graphs showing the immu (DIN ) with a TLR9 agonist PAMP, CpG2006 together with nogenicity in mice of trivalent influenza vaccine ( TIV ) H1N1 PR8 vaccine on survival of mice after challenge with formulated with an inulin particle formulation PDmix alone lethal PR8 virus dose. FIG . 9A shows mice receiving or combined with a range of TLR9 agonist PAMPs. FIG . 4A combinations of inulin particles (DIN ) with a TLR9 agonist shows serum anti - influenza total IgG levels , FIG . 4B shows PAMP, CpG2006 together with H1N1 PR8 vaccine had serum anti- influenza IgM levels , FIG . 4C shows serum complete protection with no weight loss or clinical disease , anti - influenza IgG1 levels and FIG . 4D shows serum anti - whereas PR8 + dIN without CpG was only partially protec influenza IgG2a levels , 28 days after the second immuniza tive . FIG . 9B shows again in a separate study that mice tion as measured by ELISA . Shown are group mean receiving combinations of inulin particles ( dIN ) with a OD + SD . The co - administration of TIV with PDmix and TLR9 agonist PAMP, CpG2006 together with H1N1 PRS either CpG1668, CpG2006 or CpG2395 all showed synergy vaccine were protected against death , whereas PR8 + CpG over the individual components in increasing anti - influenza gave no protection . total IgG , IgG2a and IgM titers. CpG2216 and CpG2237 had 10135 ] FIGS . 10A - 10D show four graphs that show the no effect on the antibody response . hemagglutination inhibition titers (HI ) ( FIGS . 10A and 10B ) [0130 ] FIGS . 5A -5D show four graphs showing the immu and microneutralization (MN ) ( FIGS . 10C and 10D ) titers in nogenicity in mice of rabies vaccine (MIRV ) formulated immunized ferrets measured at the time of the booster dose with either of two inulin particle formulations ( dIN or (21 days prior to challenge ) and 14 days after the booster PDmix ) alone or combined with a TLR9 agonist CpG1668 . dose ( 7 days prior to challenge ) . Ferrets vaccinated with two FIG . 5A shows serum anti - rabies total IgG levels , FIG . 5B doses of H5N1 with Ad2 had the highest neutralizing shows serum anti - rabies IgM levels , FIG . 5C shows serum antibody titers , consistent with enhanced immune response anti - rabies IgG1levels and FIG . 5D shows serum anti - rabies when H5N1 antigen was combined with a formulation of IgG2a levels 14 days after the second immunization as inulin particles plus a TLR9 agonist. measured by ELISA . Shown are group mean OD + SD . The [0136 ] FIG . 11 shows a graph showing enhanced (100 % ) combination of either dIN or PDmix with CpG1668 plus survival post lethal H5N1 challenge in ferrets that received MIRV provided the highest anti- rabies total IgG , IgGi, Ad1 - or Ad2- adjuvanted H5N1 vaccine , including the group IgG2a and IgM . that received just one immunization with 22 .5 ug H5N1 [0131 ] FIGS. 6A -6D show four graphs showing the immu vaccine + Ad2 . Each of the 10 groups is denoted by survival nogenicity in mice of trivalent influenza vaccine ( TIV ) percent: vaccine dose (or saline ) + adjuvant identify (or formulated with an inulin particle formulation PDmix alone saline ) . The survival of the five adjuvanted - vaccine groups or combined with a range of TLR2 agonist PAMPs ( zymo were significantly greater than for the two unadjuvanted san , LTA , Lipomannan and PamCSK4 ) as compared to the vaccine groups ( Log - Rank test , p = 0 .05 ) and from the three TLR9 agonist PAMP CpG2006 . FIG . 6A shows serum unvaccinated control groups (Log - Rank test, p < 0 . 001) . anti - influenza total IgG levels , FIG . 6B shows serum anti [0137 ] FIGS. 12A - 12G show seven graphs that show the influenza total IgM levels , FIG . 6C shows serum anti group mean weight change in immunized ferrets post chal influenza IgG1 levels and FIG . 6D shows serum anti lenge with H5N1 virus . Ferrets vaccinated with two doses of influenza IgG2a levels 14 days after the second H5N1 with Ad2 did not lose any weight, consistent with immunization as measured by ELISA . Shown are group enhanced protection when the H5N1 antigen was combined mean OD + SD . with a formulation of inulin particles plus a TLR9 agonist . [0132 ] FIGS. 7A -7C show three graphs showing the [0138 ] FIGS . 13A - 13G show seven graphs that show the favorable immune enhancing effect of combinations of group mean temperature change in immunized ferrets post inulin particles with various PAMPs on immunogenicity in lethal challenge with H5N1 virus . While four ferrets in the mice of TIV vaccine . FIG . 7A shows serum anti - influenza Adl ( inulin article alone )- adjuvanted vaccine groups dem total IgG levels, FIG . 7B shows serum anti- influenza IgG1 onstrated fever , no ferrets in the Ad2 ( inulin particle + CpG ) levels and FIG . 7C shows serum anti- influenza IgG2a levels adjuvanted group experienced fever, consistent with 42 days after the second immunization as measured by enhanced protection when the H5N1 antigen was combined ELISA . Shown are group mean OD + SD . with a formulation of inulin particles plus a TLR9 agonist. [0133 ] FIGS . 8A - 8F show six graphs showing the favor [0139 ] FIGS . 14A - 14C show three graphs that show gIN , able immune enhancing and antigen - sparing effect of com DIN or eIN all had a synergistic enhancing effect with the binations of inulin particles (DIN ) with a TLR9 agonist CpG in the induction of anti -HBsAg IgG1, IgG2a and IgM PAMP, CpG2006 on immunogenicity in mice of a recom consistent with the synergistic effect on PAMP innate binant pandemic influenza vaccine, rH5 . Balb / c mice at 6 - 8 immune activators being a shared property of different weeks of age ( n = 5 - 8 / group ) were immunized intramuscu polymorphic forms of inulin particles. Adult Balb / c mice larly twice 21 days apart, with 50 ulof a vaccine formulation were immunized intramuscularly twice 21 days apart, with containing between 3 ng and 3 ug of influenza recombinant HBsAg together with either glN , DIN or eIN inulin particles H5 (rH5 ) serotype hemagglutinin protein (rH5 ) ( Protein alone or together with the TLRO PAMP, CpG2006 . FIG . 14A Sciences Corp , Meriden , USA ) plus either dIN 1 mg or dIN shows serum anti- HBsAg IgG1, FIG . 14B shows serum 1 mg mixed with CpG2006 5 ug . FIG . 8A shows serum anti -HBsAg IgG2a levels and FIG . 14C shows serum anti anti - H5 total IgG , FIG . 8B shows anti - H5 IgM , FIG . 8C HBsAg IgGM levels after the second immunization as shows anti - H5 IgG1, FIG . 8D shows anti -H5 IgG2a , FIG . measured by ELISA . Shown are group mean OD + SD . 8E shows anti -H5 IgG2b , and FIG . 8F shows anti - H5 IgG3 [0140 ] FIG . 15 illustrates the mechanism of action for an 14 days after the second immunization as measured by epitope vaccine . Adjuvant and delivery systems support the ELISA . Shown are group mean OD + SD . efficient delivery of the vaccine to the immune system . US 2017 /0368167 A9 Dec . 28 , 2017 15
Antigen -presenting cells uptake delivered vaccine and pres - MultiTEP based AV -1959 vaccine and restimulated in vitro ent the antigen to T helper cells specific to Th epitopes with different epitopes from the vaccine . incorporated into the vaccine . B cells recognize the active 10148 ) FIGS. 23A - 23B show responses of individual, out component of the vaccine ( B cell epitope) by B cell recep bred macaques to different Th cell epitopes after immuni tors ( first signal for activation ) and simultaneously present zation . FIG . 23A shows mapping of Th cell epitopes in the Th epitope of the vaccine to the same T helper cells non - inbred macaques with high MHC class II polymor activated by APC creating B cell / T cell synapse . Thus, B phism . FIG . 23B presents the analyses of prevalence of Th cells specific to AB bind the antigen via a B cell receptor epitopes within the NHP population . ( first signal) and get help from activated Th cells (second [ 0149 ] FIGS. 24A - 24C present a schematic representation signal) . B cells that are activated in this way begin to of experimental design (FIG . 24A ) demonstrating the immu produce specific antibodies . nological potential of pre - existing Th cells and results . FIG . [0141 ] FIGS. 16A - 16B show design of exemplary vac 24B shows cellular response and FIG . 24C shows humoral cines . FIG . 16A shows a schematic representation of con response after immunization with multi - TEP protein in structs encoding various types of epitope vaccines . Parental QuilA or QuilA alone and boosted with AV - 1959 . construct (p3AB11 - PADRE ) was modified to express the [0150 ] FIG . 25A shows overlapping peptides of a - syn same three copies of active component, AB11 B cell epitopes used for mapping immunodominant B cell epitopes . FIG . (one epitope with free N - terminal aspartic acid ) fused with 25B shows a schematic representation of epitope vaccine nine ( AV - 1955 ) or twelve ( AV - 1959 ) different, promiscuous based on a - syn B cell epitope fused to MultiTEP platform . foreign Th cell epitopes each separated by a neutral spacer [0151 ] FIGS . 26A - 26B present data of immune responses with few amino acids ( for example , a glycine - serine spacer ) . in mice vaccinated with an a -Synuclein epitope -based vac Using such constructs one may generate appropriate recom cine . FIG . 26A shows antibody concentration following binant proteins . FIG . 16B shows the origin and sequence of immunization with a - Syn36 -69 -MultiTEP or irrelevant pep various CD4 + T cell epitopes forming the Th epitope strings tide . FIG . 26B shows cellular response to MultiTEP and to for AV - 1955 and AV - 1959 vaccines (designated collectively C - synuclein . as the MultiTEP platform ) ( SEQ ID NO : 45 ) . [0152 ] FIGS. 27A - 27C show antibody responses to dif [ 0142 ] FIGS . 17A - 17B are photographs of a Western blot. ferent portions of a - Synuclein . Mice were immunized with Correct cleavage of signal sequence and generation of free epitope vaccine based on K10AKEG14 calpain cleavage site N - terminus aspartic acid in a first copy of AB , in AV - 1955 of a -Synuclein a - Syn10 - 14 -MultiTEP (FIG . 27A ) . Antibody was analyzed in conditioned media (CM ) of CHO cells binding to a -Syn10 - 18 peptide ( FIG . 27B ) and to full length transfected with p3AB11 -PADRE - Thep (Lane 1 ) and a - Synuclein protein (FIG . 27C ). AV - 1955 ( Lane 2 ) by IP /WB . Both proteins were immuno [0153 ] FIG . 28 shows results ofmapping of immunodomi precipitated with 6E10 monoclonal antibodies (Mab ) and nant B cell epitopes in tau protein . Mice were immunized blots were stained with 6E10 (FIG . 17A ) or rabbit antibody with 4R / 2N Tau protein . Binding of generated antibodies to specific to the N - terminus of AB peptide (FIG . 17B ) . 50 -mer peptides comprising tau protein was analyzed by [0143 ] FIGS. 18A - 18B show results of immunization of ELISA . mice by gene gun with MultiTEP based AD epitope vaccines [0154 ] FIGS . 29A -29C present data of immunization of AV - 1959 , AV - 1955 and p3AB -PADRE . FIG . 18A shows B6SJL mice with Tau , 18 fused with a foreign Th cell cellular response measured as IFNy SFC per 100 spleno epitope . FIG . 29A shows titers of antibody specific to tau , 18 cytes ; FIG . 18B shows humoral immune responses mea peptide were determined in serially diluted individual sera . sured by concentration of anti - Aß antibodies in ug /mL . Lines indicate the average of mice . FIG . 29B showsbinding [0144 ] FIGS. 19A - 19C present graphs showing results of of anti - Tau , 18 antibodies to wild /type ( 4R /ON ) , mutated immunization with MultiTEP based AD epitope vaccine P301L and deleted (419 - 29 ) tau proteins of 4R /ON isoform AV - 1959 . FIG . 19A shows that cellular immune responses (dilution of sera 1 :600 . Lines indicate the average of are specific to Th epitopes incorporated into the vaccine but OD450 ) . FIG . 29C showsdetection of IFN -y producing cells not to AB20 , and FIGS . 19B and 19C show anti - AB anti in the cultures of immune splenocytes activated with P30 bodies in mice , rabbits and monkeys . peptide and tau2- 18 . The number of IFNy producing spleno [0145 ] FIGS. 20A - 20C present results of Rhesus cytes was analyzed by ELISPOT assay after ex vivo re macaques vaccinated with MultiTEP based AD epitope stimulation of cells with 10 ug /mL tau2- 18 and P30 peptides. vaccine showing therapeutic potency . Anti - Aß antibody Error bars indicate average = s . d . ( P 0 . 001) . purified from sera of vaccinated monkeys but not irrelevant f0155 ] FIG . 30 presents photographs of immunostaining monkey IgG binds to cortical plaques in AD brain ( FIG . of brain sections of patients with Alzheimer ' s Disease ( AD ) 20A ) and to immobilized AB42 monomeric , oligomeric , or case and normal non - AD case patients . Antibodies include fibrillar forms as measured using the Biacore (FIG . 20B ) . anti- tau2 -18 sera from mice immunized with tau2- 18 -P30 (left Anti- Aß antibody inhibits AB42 fibrils - and oligomer -medi panels ) , known anti- tau antibodies (middle panels ) and ated neurotoxicity ( FIG . 20C ) . control antisera from mice immunized with an irrelevant [0146 ] FIGS . 21A - 21B show data obtained from APP / Tg antigen ( BORIS ) ( right panels ) . mice vaccinated with MultiTEP based AD epitope vaccine . (0156 ] FIGS . 31A - 31B present results of antibody block FIG . 21A shows induced anti - AB u antibody significantly ing brain lysate induction of aggregation of intracellular tau reduced diffuse and dense - core AB -plaques detected by repeat domain (RD ) . FIG . 31A shows brain lysate was either staining with 6E10 and dense - core plaques detected by untreated or treated with anti - tau , 18 antibody and added to staining with ThS . FIG . 21B shows soluble and insoluble AB HEK293 cells co - transfected with RD ( AK ) -CFP / YFP prior detected by biochemical methods. to FRET analysis . Increased FRET signal was detected in [0147 ] FIG . 22 shows T cell responses after re -stimula wells with untreated brain lysate . Treatment of lysate with tion . Inbred mice of H2b haplotype were vaccinated with anti- tau2- 18 antibody decreased FRET signal to the baseline US 2017 /0368167 A9 Dec . 28 , 2017
level due to blocking the full- length tau in brain lysate and ofmice immunized with AV - 1959R formulated in AdvaxCpG inhibition of induction of RD aggregation . FIG . 31B shows using ANOVA test ( * * P < 0 .01 * * * , P < 0 .001 and * * * * P < 0 . confocal microscope images of exemplary binding of anti 0001) . tau2- 18 antibody /brain lysate complexes to HEK293 cells [0161 ] FIGS. 36A - 36C show cellular immune responses transfected with RD - YFP. Secondary anti- mouse immuno in mice vaccinated with AV - 1959R protein formulated with globulin conjugated with Alexa546 was used . CGMP grade adjuvants ( Advax PG , AdvaxTM , Montanide ISA51 , Montanide -ISA720 , MPLA , Alhydrogel® ) and con [0157 ] FIGS. 32A -32B present data of anti -tau2 - 18 anti trol adjuvant, Quil - A . As shown in FIGS. 36A and 36B , body blocking the trans -cellular propagation of tau RD numbers of IFN - Y ( A ) and IL - 4 ( B ) producing T cells were aggregates . FIG . 32A shows HEK293 cells transfected with calculated by ELISpot in splenocyte cultures obtained from RD ( LM ) - HA were co - cultured for 48 h with an equivalent experimental and control animals . ( C ) IL - 4 / IFN - y ratios number of HEK293 cells co - transfected with RD ( AK ) - CFP / were calculated based on data presented in FIGS . 36A and YFP prior to FRET analysis . Increased FRET signal was 36B . Bars represent average : SD (n = 6 - 8 /per group ) . Statis detected in co - cultured cells . Addition of serial dilutions of tical significances were calculated against group of mice purified mouse anti -tau .2 . 18 or rat anti - tau 382 -418 antibody immunized with AV - 1959R formulated in AdvaxCP® using decreased FRET signal due to inhibition of trans- cellular ANOVA test (* P < 0 .05 , * * P < 0 .01 , * * * P < 0 .001 and propagation of aggregated RD . FIG . 32B shows binding of * * * * P < 0 .0001 ) . anti- tau2- 18 antibodies HEK293 cells transfected with [0162 ] FIGS. 37A - 37C show cellular immune responses RD ( AK ) - YFP or were mock - transfected (NT ) was analyzed in mice immunized with epitope vaccines targeting AB by confocal microscope. Anti - tau2- 18 antibody was added to (AV - 1959R ), tau ( AV - 1980R ), and Aß / tau together in one the culture medium for 48 h . Cells were fixed , permeabi construct (dual epitope vaccine ) : AV1953R or a mixture of lized , and stained with an anti- mouse secondary antibody AV1959R and AV - 1980R ) ( AV1959R + AV1980R ) . Numbers labeled with Alexa 546 and analyzed by confocal micros of IFN - Y (FIG . 37A ) and IL -4 (FIG . 37B ) producing T cells copy. Anti -tau2 - 18 /RDA (K ) - YFP complexes were identified were calculated by ELISpot in splenocyte cultures obtained when RDA ( K ) - YFP is expressed but not in its absence (NT ) . from experimental and control animals . As shown in FIG . [0158 ] FIG . 33 shows schematics of exemplary multiva 37C , proliferation of cells was detected by [ 3H ] - thymidine lent DNA epitope vaccines based on MultiTEP platform . incorporation assay in the same splenocyte cultures and AV - 1953 is bivalent epitope composed of 3 copies of AB . expressed as stimulation index . Cellular immune responses and 3 copies of tau , 18 epitopes fused to MultiTEP platform . in control group were at the background level ( INF - y and AV - 1950 and AV - 1978 are trivalent vaccines containing IL - 4 + SFCs were < 15 , and stimulation index was < 1 .6 ). Bars a - syn epitopes KAKEG and a -syn36 -69 , respectively , in represent average : SD (n = 8 per group ) . [0163 ] FIGS. 38A - 38B show humoral immune responses addition to Aß and tau . in mice vaccinated with AV - 1959R , AV - 1980R , AV - 1953R [ 0159 ] FIGS . 34A -34C show data from immunization of and AV - 1959R + AV - 1980R formulated with AdvaxCp6 adju wildtype mice with bivalent and trivalent DNA epitope vant. Concentrations of anti - AB (FIG . 38A ) and anti - tau vaccines . FIG . 34A shows anti - AB . , and anti - Tau antibody (FIG . 38B ) antibodies were measured by ELISA in sera responses generated by bivalent AV - 1953 vaccine . FIG . 34B collected after the 3rd immunization and calculated using shows anti -AB42 , anti - Tau and anti - a -syn antibody calibration curves generated with 6E10 and 1C9monoclonal responses generated by AV - 1978 trivalent vaccine . Ab antibodies , respectively . Lines represent mean values for responses were measured in sera of individual mice by n = 8 / per group ( P < 0 .05 , * * P < 0 . 01 , ANOVA test ) . ELISA and lines represent the average value of Ab . Con [0164 ] FIGS. 39A - 39B show numbers of B cells produc centration of Ab specific to a - syn and AB42 was calculated ing anti -A? and anti- Tau antibodies in mice vaccinated with using a calibration curve generated with mouse anti - a -syn AV - 1959R , AV - 1980R , AV - 1953R and AV - 1959R + AV and 6E10 anti - AB . , antibodies , respectively . Endpoint titers 1980R formulated with AdvaxCpG adjuvant. Detection of of anti - Tau antibodies were calculated as the reciprocal of anti - AB (FIG . 39A ) and anti- tau (FIG . 39B ) antibody the highest sera dilution that gave a reading twice above the secreting cells ( ASC ) , visualized as spots , was done in cutoff. The cutoff was determined as the titer of pre- immune splenocyte cultures obtained from experimental and control sera at the same dilution . FIG . 34C shows trivalent vaccine mice using ELISpot assay . Bars represent average : SD ( n = 8 / AV - 1978 activated Th cells specific to epitopes of MultiTEP per group , * P < 0 .05 , ANOVA test ). platform but not to B cell epitopes. IFNy producing cells in [0165 ] FIGS. 40A - 40F show 3D structural models of the cultures of immune splenocytes were detected by AV - 1980R , AV - 1959R and AV - 1953R synthetic proteins. ELISPOT after in vitro re - stimulation of cells with indicated The surface filled representations of the AV - 1980R (FIG . peptides/ proteins . Error bars indicate average = s . d . (n = 6 ) . 40A ) , AV - 1959R (FIG . 40B ) and AV1953R (FIG . 40C ) are [0160 ] FIGS. 35A - 35B show humoral immune responses presented in the upper panel. Tau and AB epitopes on the in mice vaccinated with AV - 1959R protein formulated with MultiTEP protein are highlighted in pink and red , respec CGMP grade adjuvants ( Advax PG , AdvaxTM , Montanide tively . The GS linker is highlighted in dark grey . In the lower ISA51 , Montanide - ISA720 , MPLA , Alhydrogel® ) and con panel, critical residues on the AV - 1980R epitope (PRQEF ) trol adjuvant, Quil- A . As shown in FIG . 35A , concentrations are highlighted in blue ( FIG . 40D ) and the critical residues of anti- AB antibodies were measured by ELISA in sera on the AV - 1959R epitope ( EFRH ) are highlighted in cyan collected after the 3rd immunization . Lines represent mean ( FIG . 40E ) . In AV - 1953R critical residues on each epitope values . As shown in FIG . 35B , isotypes of generated anti- AB follows AV -1980R and AV - 1959R color cording (FIG . 40F ) . antibodies had been determined by ELISA and IgG1/ IgG2a [016 ] FIGS . 41A - 41C show that immune sera isolated ratio was calculated . Bars represent average _ SD ( n = 6 - 8 /per from mice vaccinated with AV - 1959R , AV - 1980R , group ) . Statistical significance was calculated against group AV - 1953R and AV - 1959R + AV - 1980R formulated with US 2017 /0368167 A9 Dec . 28 , 2017 17
AdvaxCpG adjuvant bound to different forms of AB and tau adjuvant and LU AF20513 formulated in Alhydragel. Mean in the brains from AD cases . Western blots of soluble ( FIG . concentrations of antibodies are shown . 41A ) and insoluble fractions (FIG . 41B ) of brain homoge [0174 ] FIG . 49 shows antibody titers in PS19 , rTg4510 nates containing 50 ug total protein from four AD cases were and T5x mice vaccinated with AV1980R formulated in stained with immune sera normalized to 1 ug/ ml for anti- AB AdvaxCp6 adjuvant. Table compares antibody titers in PS19 and 0 . 4 ug /ml for anti - tau antibodies based on ELISA data . mice after immunization with AV1980R formulated in ( FIG . 41C ) Immune sera were screened for the ability to AdvaxCpG adjuvant and liposome- based vaccine ACI- 35 bind to human AB plaques or/ and tau tangles using 40 um containing MPLA adjuvant brain sections of formalin - fixed cortical tissue from the same 0175 ] FIG . 50 shows a schematic representation of vac AD cases. The original magnification is 60x and the scale cines targeting different B cell epitopes of ha - Syn : aa85 - 99 bar is 20 um . (PV - 1947) , aa 109 - 126 (PV - 1948 ) , aa126 - 140 (PV - 1949 ) and [0167 ] FIGS. 42A -42B show humoral and cellular all three epitopes together with reverse order ( aa126 - 140 + immune responses in mice vaccinated twice with AV -1959R aa109 - 126 + aa85 - 99 ; PV - 1950 ) . and boosted ( single boost ) with AV - 1980R formulated with AdvaxCp6 adjuvant. (FIG . 42A ) Numbers of IFN - y produc DETAILED DESCRIPTION ing cells were detected by ELISpot in splenocyte cultures. [0176 ] The listing or discussion of any apparently prior Bars represent average - SD for n = 4 / per group . ( FIG . 42B ) published document in this specification should not neces Concentrations of anti - tau antibodies were measured by sarily be taken as an acknowledgement that the document is ELISA . Lines represent mean values for n = 10 /per group part of the state of the art or is common general knowledge . (* P < 0 .05 , * * P < 0 .01 , t - test ) . [0177 ] The practice of the present technology will employ, 10168 ]. FIG . 43 shows humoral immune responses in PS19 unless indicated specifically to the contrary , conventional mice vaccinated with AV - 1980R formulated with AdvaxCpG methods of virology, immunology, microbiology, molecular adjuvant. Concentrations of anti - tau antibodies were mea biology and recombinant DNA techniques within the skill of sured by ELISA in sera collected after the 2nd , 3rd and 4th the art , many of which are described below for the purpose immunizations and calculated using calibration curves gen of illustration . Such techniques are explained fully in the erated with 1C9 anti - tau , 18 monoclonal antibodies . literature . See , e . g . , Sambrook , et al. Molecular Cloning : A [0169 ] FIGS. 44A -44B show humoral immune responses Laboratory Manual (2nd Edition , 1989 ) ; Maniatis et al . in T5XAPP / Tau double transgenic mice vaccinated with Molecular Cloning : A Laboratory Manual ( 1982 ) ; DNA AV - 1959R , AV - 1980R and AV1959R + AV1980R vaccines Cloning : A Practical Approach , vol . I & II ( D . Glover , ed . ) ; formulated with AdvaxCpG adjuvant. Concentrations of anti Oligonucleotide Synthesis ( N . Gait , ed . , 1984 ) ; Nucleic Acid AB ( FIG . 44A ) and anti -tau ( FIG . 44B ) antibodies were Hybridization ( B . Hames & S . Higgins, eds. , 1985 ) ; Tran measured by ELISA in sera collected after the 2nd and 3rd scription and Translation ( B . Hames & S . Higgins , eds . , immunizations and calculated using calibration curves gen 1984 ) ; Animal Cell Culture ( R . Freshney , ed . , 1986 ) ; Perbal, erated with anti - AB6E10 and anti -tau2 - 18 1C9 monoclonal A Practical Guide to Molecular Cloning ( 1984 ) , Current antibodies . Protocols in Immunology ISBN 9780471522768 (Publisher : [0170 ] FIG . 45 shows anti- Tau antibody responses in John Wiley and Sons Inc. ) , Vaccine Adjuvants and Delivery rTg4510 transgenic mice immunized with AV - 1980R for Systems (Manmohan Singh ed . 2007 ) , Methods in Molecu mulated in AdvaxCpG adjuvant after 2nd, 3rd and 4th immu lar Biology, ISBN 9781607615842 (Publisher : Springer ), nizations. Concentrations of anti- Tau antibodies were cal History of Vaccine Development 2011 , ISBN : 1441913386 culated using calibration curves generated with 1C9 anti (Publisher : Springer) tau , 18 monoclonal antibodies . (0178 ] Unless defined otherwise , all technical and scien [0171 ] FIG . 46 shows cellular immune anti- Multi TEP tific terms used herein have the same meaning as commonly responses in rTg4510 transgenic mice immunized with understood by one of ordinary skill in the art to which this AV - 1980R formulated in Advax PG adjuvant. Numbers of technology belongs . Although any methods and materials IFN - y producing T cells were calculated by ELISpot in similar or equivalent to those described herein can be used splenocyte cultures obtained from experimental and control in the practice or testing of the technology , the preferred animals and re - stimulated in vitro with cocktail of Th methods and materials are now described . peptides incorporated into MultiTEP platform or with Tau , 10179 ] As known to those experienced in the art, innate 18 peptide. Bars represent average : SD ( n = 6 ) . immune activation can be used to enhance the type or f0172 ] FIG . 47 shows humoral immune responses in magnitude of an adaptive immune memory response . young and old ha -Syn Tg mice vaccinated with AV - 1950R Enhancement or modulation of the adaptive immune epitope vaccine targeting three different epitopes ofha - Syn . response is advantageous during vaccination or during aller Young mice were immunized at the age of 3 mo and titers gen desensitization , as it can provide a means to magnify or of anti -ha - Syn antibodies were determined in sera of vac extend the duration of the immune memory response against cinated mice after 3rd immunization . Old mice were immu a particular pathogen , or alter the type of immune response nized at the age of 12 - 14 mo and titers of anti - ha - Syn to a more beneficial response . For example , for some antibodies were determined in sera of vaccinated mice after pathogens , it may be advantageous to induce a strong 2nd immunization . Endpoint titers of antibodies specific to antibody ( Th2 ) response to the immunizing antigen , while recombinant ha - Syn are calculated as the reciprocal of the for other pathogens , it may be advantageous to induce a highest sera dilution that gave a reading twice above the strong Th1 response or a strong Th17 response . On the other background levels of pre - immune sera at the same dilution hand , for antigens such as allergens it may be advantageous (cutoff ). to suppress the existing IgE response and instead induce a 10173 ] FIG . 48 shows antibody concentrations in Tg2576 Th1 response to the allergen . It has been discovered accord mice vaccinated with AV1959R formulated in AdvaxCpG ing to the current technology that the combination of inulin US 2017 /0368167 A9 Dec . 28 , 2017 particles with an innate immune activator enables a variety a diameter in the size range of 0 . 1 to 5 UM . Most preferably of unique patterns of immune response to be obtained that the inulin particle will have a diameter in the size range of can , for example , be used to modulate the adaptive immune 0 . 5 to 5 uM . memory response to a co -administered antigen to a favored 10185 ] In certain embodiments , inulin particles as used in type or direction . the present technology are stable inulin particles . The term [ 0180 ] A first aspect of the present technology provides a “ stable ” as used herein refers to an inulin particle that is composition comprising inulin particles for use in the reduc totally insoluble or predominantly insoluble or partially tion or inhibition of inflammation , and /or for treating or insoluble at the body temperature of the subject to whom it preventing inflammatory disease, in a subject. is to be administered . In this context, stability may option [0181 ] A second aspect of the present technology provides ally include the meaning that the inulin particles are an immunological and/ or pharmaceutically acceptable com insoluble when incubated at a temperature of up to 25° C . or position comprising ( a ) an anti - inflammatory component , up to 30° C ., 37° C ., 40° C ., 42° C . , 45° C ., 48° C ., 50° C . , such as inulin particles and / or one or more other anti 52° C ., 55° C ., 58° C ., or 60° C . when present at a inflammatory inhibitors of IL -1 ; together with ( b ) a sub concentration of no greater than 0 . 5 mg/ mL or 1 mg /mL or stance comprising one or more species of an innate immune 2 mg/ mL in distilled water or saline or phosphate buffered activator such as a pathogen - associated molecular pattern saline , for at least 10 , 20 , 30 , 40 , 50 , or 60 minutes . The (PAMP ) . Without wishing to be bound by theory , a favorable amount of insoluble inulin can be measured by changes in immune interaction occurs because each of the two compo the optical density of the inulin suspension at 300 nm , 400 nents of the immunological composition regulate transcrip nm , 500 nm , 600 nm , 700 nm wavelength (OD700 ) using a tion of an independent set of immune genes , such that the spectrophotometer and , in this context, an inulin particle can pattern of immune genes expressed in response to the be said to be stable if it remains insoluble at the defined combined immunological composition is unique to the com condition as indicated by the OD 00 not falling below a value bination and different to the patterns of gene expression that is 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , 98 % , 99 % , 99 . 5 % , induced by the individual components . 99. 9 % or substantially 100 % of the OD700 of the particle [0182 ] A third aspect of the present technology provides a preparation in the same solvent and at the same concentra kit of parts comprising : ( a ) a first container that contains a tion prior to incubation at the defined temperature ( prefer composition comprising an anti - inflammatory component, ably when measured at a temperature that is 10° C . or more such particles of inulin and / or one or more other anti below the incubation temperature ) inflammatory inhibitors of IL - 1 (as discussed above in 10186 ]. Other anti - inflammatory components, which may respect of the second aspect of the present technology ) ; and be used in component ( a ) of the composition of the second (b ) a second container that contains a substance comprising aspect , or the kit of the this aspect of the technology , instead a pathogen - associated molecular pattern (PAMP ) . of or as well as, inulin particles, may include [0183 ] Thus, component ( a ) of the composition of the [0187 ] (i ) inhibitors of the IL - 1 pathway genes or proteins, second aspect of the present technology, or the kit of the particularly those that are functionally - equivalent to inulin third aspect of the technology, comprises anti -inflammatory particles , in the sense of possessing an essentially equivalent component, such as an anti -inflammatory inhibitor of IL - 1 anti- inflammatory property , activity and /or specificity and / or an anti - inflammatory inhibitor of NFKB . or possessing an essentially equivalent immunomodulatory [0184 ] In certain embodiments , the anti - inflammatory or adjuvant property , component comprises inulin particles . The term “ inulin [0188 ] ( ii ) one or more of IL1 receptor antagonists , particle ” as used herein refers not only to particles made ILIRA , Anakinra , Rilonacept, IL - 1R /IL1RacP / Fc - fusion from B - D - ( 2 - 1 )polyfructofuranosyl - a - D - glucose ( also protein , Canakinumab , a human IL - 1ß antibody , IL1 recep known as inulin ) but also to derivatives thereof such as tor blockers , IL - 1RII , indomethacin , non - steroidal anti - in B - D - ( 2 - 1 ) polyfructose which may be obtained by enzymatic flammatory drugs (NSAID ) , glucocorticoids , caspase inhibi removal of the end glucose from inulin , for example using tors including caspase 1 inhibitors , inflammasome inhibitors an invertase or inulase enzyme capable of removing the end including NALP3 antagonists, curcumin , resveratrol, chlo glucose . The term inulin particle also refers to any natural or roquine, P2X7 receptor inhibitors, ST2 receptor inhibitors , synthetic particle that is constituted by, contains or is coated and/ or ATP antagonists ; by inulin , or a derivative or mimetic thereof. Suitable inulin [0189 ] ( iii ) agents that up - regulate or activate the anti derivatives included within the scope of this term are inflammatory protein peroxisome proliferator - activated derivatives of inulin in which the free hydroxyl groups have receptor gamma (PPAR - Y ) or upregulate genes or proteins in been acetylated , methylated , etherified or esterified , for the PPAR - y pathway, particularly in monocytes and den example by chemical substitution with alkyl, aryl or acyl dritic cells (PPAR -y pathway genes are also upregulated by groups, by known methods. The stable inulin particle may be inulin particles ). PPAR - y upregulation has been previously solid or hollow and may be wholly comprised of inulin shown to inhibit inflammatory responses including sup molecules or may alternatively have a non - sugar core , pressing LPS - induced IL - 1 and TNFa and conversely IL1 skeleton or shell comprising , for example , carbohydrate and TNFa PPAR - y . Suitable agents may include one or more compounds, metal compounds, proteins or lipids but which of rosiglitazone , pioglitazone , prostaglandin J2 , curcumin , at its surface expresses inulin molecules either covalently or resveratrol, thiazolidenediones , Berberine , perfluo non - covalently bonded to the components comprising the rononanoic acid , RS5444 , free fatty acids, vitamin D , and/ or core . Preferably , the inulin particle will be selected from the eicosanoids . group of gIN , DIN and elN , or modifications thereof. Most [0190 ] ( iv ) anti- inflammatory agents such as aspirin , ibu preferably , the inulin particle will be dIN . Preferably , the profen , and naproxen , salicylic acid , submandibular gland inulin particle will have a diameter in the size range of 20 peptide - T , phenylalanine - glutamine - glycine ( FEG ) , ginger, nM to 20 uM . More preferably , the inulin particle will have turmeric , sesquiterpene lactone , Omega - 3 fatty acids, pros US 2017 /0368167 A9 Dec . 28 , 2017 taglandin - E , prostaglandin - E3 , Curcumin , Mesalazine, immune system . Without limitation , innate immune activa Selective glucocorticoid receptor agonist , Lisofylline, Mof tion may be manifest at the cellular level by one ormore of ezolac , Oleocanthal, Ibuproxam , Cyclopentenone , prosta changes in gene expression or protein production , induction glandin , Cannabidiol, BMS- 345541 , BMS- 470 ,539 , Amlex of cytokine or chemokine production or secretion , changes anox , Amixetrine , Allicin , Actarit , Butylpyrazolidines, for in cell morphology, differentiation , cell division , changes in example , Phenylbutazone ; Mofebutazone ; Oxyphenbuta cell surface protein expression , chemotaxis , phagocytosis , zone ; Clofezone ; Kebuzone ; Suxibuzone ; Acetic acid exocytosis, autophagy, or apoptosis . derivatives and related substances , such as Indometacin ; [0195 ] The term , " vaccine ” is defined as an immuno Sulindac ; Tolmetin ; Zomepirac ; Diclofenac ; Alclofenac ; stimulatory treatment designed to elicit a beneficial immune Bumadizone ; Etodolac ; Lonazolac ; Fentiazac , Acemetacin ; response against a specific antigen , whether administered Difenpiramide ; Oxametacin ; Proglumetacin ; Ketorolac ; prophylactically or for the treatment of an already existing Aceclofenac ; Bufexamac ; Indometacin , Diclofenac , Oxi condition . cams, such as Piroxicam ; Tenoxicam ; Droxicam ; Lornoxi [0196 ] The term “ immunogenic ” refers to the ability of an cam ; Meloxicam ; Propionic acid derivatives , such as Ibu antigen to elicit an immune response, including either profen ; Naproxen ; Ketoprofen ; Fenoprofen ; Fenbufen ; humoral and /or cell -mediated immunity . Benoxaprofen ; Suprofen ; Pirprofen ; Flurbiprofen ; Indopro [0197 ] The term “ immunologically - effective amount" as fen ; Tiaprofenic acid ; Oxaprozin ; Ibuproxam ; Dexibupro used herein in respect to an antigen or an innate immune fen ; Flunoxaprofen ; Alminoprofen ; Dexketoprofen ; activator refers to the amount of antigen or innate immune Naproxcinod ; Naproxen and esomeprazole ; Naproxen and activator sufficient to elicit an immune response as measured misoprostol ; Vedaprofen ; Carprofen ; Tepoxalin . Fenamates, by standard assays known to one skilled in the art . The such as Mefenamic acid ; Tolfenamic acid ; Flufenamic acid ; effectiveness of an antigen as an immunogen , can be mea Meclofenamic acid ; Flunixin , Coxibs, such as Celecoxib ; sured either by T - cell proliferation or cytokine secretion Rofecoxib ; Valdecoxib ; Parecoxib ; Etoricoxib ; Lumira assays , by cytotoxicity assays , such as chromium release coxib ; Firocoxib ; Robenacoxib ; Mavacoxib ; Cimicoxib , assays to measure the ability of a T - cell to lyse its specific Other anti- inflammatory and antirheumatic agents , such as target cell, or by measuring the levels of B - cell activity by Nabumetone ; Niflumic acid ; Azapropazone ; Glucosamine ; measuring the levels of circulating antibodies specific for the Benzydamine ; Glucosaminoglycan polysulfate ; Proqua antigen in serum , or by measuring the number of antibody zone ; Orgotein ; Nimesulide ; Feprazone, Diacerein ; Morni spot- forming B cells , e . g . , by ELISPOT. Furthermore , the flumate; Tenidap ; Oxaceprol, Chondroitin sulfate ; Avocado level of protection of the immune response may be measured and soybean oil , unsaponifiables, Niflumic acid , Feprazone , by challenging the immunized host with a replicating virus , combinations ; Pentosan polysulfate ; Aminopropionitrile ; pathogen or cell containing the antigen that has been immu Anti - inflammatory /antirheumatic agents in combination nized against . For example , if the antigen to which an with corticosteroids , such as Phenylbutazone and corticos immune response is desired is a virus or a tumor cell , the teroids; Dipyrocetyl and corticosteroids; Acetylsalicylic acid level of protection induced by the “ immunogenically effec and corticosteroids; Specific antirheumatic agents including tive amount” of the antigen is measured by detecting the Quinolines , such as Oxycinchophen , Gold preparations, level of survival after virus or tumor cell challenge of the such as Sodium aurothiomalate ; Sodium aurothiosulfate ; animals . Alternatively , protection can also be measured as Auranofin ; Aurothioglucose ; Aurotioprol, and /or Penicil the reduction in viral replication or tumor growth following lamine and similar agents , such as Bucillamine . challenge of the animals . The amount of antigen necessary [ 0191 ] As a general rule , the inulin particle (or other to provide an immunogenic amount is readily determined by equivalent anti - inflammatory component ) can be used in an one of ordinary skill in the art, e. g ., by preparing a series of amount of 0 . 001 mg and 100 mg per kilogram body weight vaccines of the technology with varying concentrations of of the subject to be immunized . For example , the inulin antigen , administering the vaccine formulations to suitable particle ( or other equivalent anti - inflammatory component ) laboratory animals ( e . g . , mice , rats , guinea pigs, or rabbits ) , of a composition of the present technology may be present and assaying the resulting immune response by measuring at a concentration in the range of 0 . 1 mg to 100. 0 mg per serum or mucosal antibody titers , antigen - induced swelling kilogram body weight. In another example , the inulin par in the skin (delayed type hypersensitivity assay ) , T -cell ticle (or other equivalent anti- inflammatory component) of proliferation , cytokine production or cytotoxic activity , pro the composition may be administered to an adult human tection against pathogen challenge and the like . subject in a range of 1 to 100 mg per dose, such as a 20 mg [0198 ] The term “ parenteral ' refers to injection of a vac per dose . cine into any tissue of the body and includes intramuscular, [0192 ] The term “ adjuvant” refers to a substance or mix subcutaneous, intradermal, intraperitoneal and intraocular ture that enhances the immune response to an antigen . Often , routes of vaccine administration , by methods and delivery a primary immunization with an antigen alone, in the devices well known in the art . absence of an adjuvant, will fail to elicit an immune (0199 ] In certain embodiments , the subject is a human . In response . other embodiments, the subject is animal, including but not [0193 ] The term “ agonist” refers to a protein , nucleic acid , limited to a dog , cat, horse , camel, cow , pig , sheep , goat, lipid , carbohydrate or chemical substance that interacts with chicken , hawk , rabbit and fish . The term “ animal” includes a cellular receptor to produce a cellular response . Agonists all domestic and wild mammals , fish , fowl, and includes , that stimulate innate immune receptors may be of particular without limitation , cattle , horses, swine , sheep , goats , cam interest in the present technology . els , dogs , cats, rabbits , deer, mink , chickens, ducks , geese , [0194 ] The term “ innate immune activator” is to be under turkeys , game hens , and the like . stood as referring to any substance that directly or indirectly 0 200 ] In certain embodiments , as an additional compo activates a cell involved in the functioning of the innate nent, the composition of the technology may also optionally US 2017 /0368167 A9 Dec . 28 , 2017 20 include an immunologically -effective amount of a chemical from any of the above groups of agonists or synthetic substance that activates one or more types of innate immune analogues or derivatives thereof. cell, such as a monocyte , dendritic cells , NK cell , lympho [0203 ] In certain embodiments , the substance comprising cyte or granulocyte . As known in the art , examples of one or more pathogen - associated molecular pattern (PAMP ) chemicals that induce activation of innate immune cells may be present, or administered , at an immunologically include leukotrienes, prostaglandins , cytokines , chemok effective amount and / or concentration in the range of 0 .01 to ines, interferons, kinins , vitamin D , phorbol myristate 500 micrograms per kilogram of body weight. acetate , ionomycin , mitogens, opsonins , histamine , brady [0204 ] In certain embodiments , one or more of the sub kinin , serotonin , leukotrienes , CAMP, antimicrobial pep stances comprising a pathogen - associated molecular pattern tides , and pro - drugs or inducers of the aforementioned (PAMP ) is present, or administered , as a pure , distinct and substances. single molecular and chemical entity . [ 0201] In certain embodiments , the PAMP innate immune [ 0205 ] In certain embodiments , the substance comprising activator of the current technology is an immunologically a pathogen -associated molecular pattern (PAMP ) may be effective amount of a substance that binds to an innate present, or administered , in a highly purified state , whereby immune receptor. Currently known innate immune receptors one or more of each distinct and single molecular and include TLR - 1 , TLR - 2 , TLR - 3 , TLR - 4 , TLR - 5 , TLR - 6 , chemical PAMP entity is at a purity of at least 50 % , 60 % , TLR - 7 , TLR - 8 , TLR - 9 , murine TLR - 11 ; NOD - 1 , NOD - 2 , 70 % , 80 % , 90 % , 95 % , 96 % , 97 % , 98 % , 99 % , 99. 9 % , other NOD - like receptors (NLRs ) such as NLRP1, NLRP3 , 99 . 99 % , 99 . 999 % or essentially 100 % . NLRP12 , NLRC4 ; DECTIN -1 ; DC -SIGN ; AIM - 2 ; mannose [0206 ] In certain embodiments, the PAMP of the current receptors including C - type lectins , MD2 ; CD14 ; LBP ; technology is a TLR agonist . There are presently believed to CARD ( caspase activating and recruitment domain )- con be approximately 10 - 15 different types of TLR in most taining proteins , such as RIG - 1 (retinoic acid - inducible mammalian species . The different TLRs bind and are acti gene - 1 ) and MDA - 5 (melanoma differentiation -associated vated by a range of natural and synthetic ligands . Different gene - 5 ) , LGP2 and ASC , scavenger receptors including TLRs signal through different signaling molecules , although CD -36 , CD -68 , and SRB - 1 , C reactive protein , mannose a feature in common is that they all activate the inflamma binding lectin , complement factors including C3a and C4b tory transcription factor NFKB . and complement receptors , and N - formyl Met receptors [0207 ] In certain embodiments , the PAMP of the current including FPR and FPRL1. Of particular interest for the technology is a TLR1 agonist, such as a TLR1 agonist drawn present technology are PAMPs that bind and activate TLR - 1 , from the group of a triacyl lipopeptide and Pam3CSK4 . - 2 , - 3 , - 5 , - 6 , - 7 , and - 9 and NOD - like receptors . More 10208 ] In certain embodiments , the PAMP of the current preferred are TLR3 , TLR9 and NOD2 receptor agonists . technology is a TLR2 agonist , such as a TLR2 agonist drawn 10202 ] In certain embodiments , an immunologically - ef from the group of a glycolipid , lipoteichoic acid , peptido fective amount of one or more PAMPs (pathogen -associated glycan , HSP70 , zymosan , and Pam3CSK4 . molecular patterns) is / are used . A PAMP is a structurally [0209 ] In certain embodiments , the PAMP of the current conserved molecule derived from a pathogen that is immu technology is a TLR3 agonist , such as a TLR3 agonist drawn nologically distinguishable from host molecules , and is from the group of a double -stranded RNA , poly ( I: C ), poly recognized by and specifically binds to an innate immune ( I: C - LC ) (HiltonolTM ), and poly I: polyC12 U ( AmpligenTM ) receptor. PAMPs are present in certain protein , lipid , lipo [0210 ] In certain embodiments , the PAMP of the current protein , carbohydrate , glycolipid , glycoprotein , and nucleic technology is a TLR4 agonist, such as a TLR4 agonist drawn acids expressed by particular pathogens and include TLR2 from the group of monophosphoryl lipid A (MPLA ) , heat agonists including di- and tri - acyl lip peptides , lipotechoic shock proteins, fibrinogen , heparan sulfate fragments , acid , zymosan , peptidoglycan , poring , Lipoarabinomannan , hyaluronic acid fragments , and synthetic TLR4 agonists Phospholipomannan , Glucuronoxylomannan , glycosylphos including E6020 , GLA and LPS peptide mimotopes . Most phatidylinositol (GPI ) - anchored proteins in parasites , TLR3 preferred is a synthetic TLR4 agonist that preferentially agonists including double stranded RNA , including syn signals through the TIR -domain - containing adapter- induc thetic dsRNA for example polyinosinic :polycytidylic acid ing interferon - B ( TRIF ) and not the NFKB pathway . Due to (poly I: C ), TLR4 agonists including mannan , glucuronoxy toxicity and regulatory requirements , lipopolysaccharide lomannan , heat shock protein , fibrinogen and synthetic (LPS ) TLR4 agonists and substances containing LPS (such MPL , TLR5 agonists including flagellin , TLR6 agonists as endotoxin ) should be avoided in the technology . The including lipotechoic acid , TLR7 and TLR8 agonists includ amount of LPS and /or endotoxin in substances and compo ing viral or synthetic single stranded (ss ) RNA , for example , sitions used in the aspects of the present technology may be imiquimod and resiquimod (R848 ) , and TLR9 agonists less than 100 EU per dose , such as less than 90 , 80 , 70 , 60 , including unmethylated cytosine - guanine dinucleotide oli 50 40 , 30 , 20 , 10 , 5 , 4 , 3 , 2 , 1 or less EU per dose . The gonucleotide sequences (CPG ODN ) and hemozoin , RIG - 1 concentration of LPS and /or endotoxin in substances and agonists such as viral or synthetic double - stranded ( ds ) compositions used in the aspects of the present technology RNA , MDA5 agonists such as viral or synthetic dsDNA , may be less than 200 EU /m® , such as less than 150 , 100 , 90 , NOD1 agonists including peptidoglycan containing the 80 , 70 , 60 , 50 40 , 30 , 20 , 10 , 5 , 4 , 3 , 2 , 1 or less EU / m muramyl dipeptide NAG -NAM - gamma - D - glutamyl- meso [ 0211 ] In certain embodiments , the PAMP of the current diaminopimelic acid , NOD2 agonists including peptidogly technology is a TLR5 agonist , such as a TLR5 agonist drawn can containing the muramyl dipeptide NAG -NAM -L -ala from the group of bacterial or synthetic flagellins. nyl- isoglutamine , RIG1 and MDA5 agonists including [0212 ] In certain embodiments , the PAMP of the current SsRNA and dsRNA , N - formylMet receptor agonists includ technology is a TLR6 agonist , such as a TLR6 agonist drawn ing N - formyl methionine . Hence , a PAMP innate immune from the group of diacyl lipopeptides . Most preferred is activator as used by the current technology may be selected diacyl lipopeptide. US 2017 /0368167 A9 Dec . 28 , 2017 21
[0213 ] In certain embodiments , the PAMP of the current [0217 ] In certain embodiments , the PAMP of the current technology is a TLR7 agonist, such as a TLR7 agonist drawn technology is an agonist of a C - type lectin receptor . In from the group of viral single - stranded RNA , imidazoqui another embodiment, the C - type lectin receptor agonist noline , gardiquimod , loxoribine , bropirimine , CL264 , R848 , binds to one of the group of macrophage mannose receptor, and CL075 . Most preferred is R848 . CLEC - 2 , DEC205 /CD205 , DC - SIGN - like , DC -ASGPR (MGL )/ CD301 , Dectin - 1, Langerin /CD207 , Mincle and [0214 ] In certain embodiments , the PAMP of the current CLR BDCA - 2 /CD303 . In certain embodiments , the C - type technology is a TLR8 agonist , such as a TLR8 agonist drawn lectin receptor agonist is drawn from the group of Beta - 1 , from the group of single -stranded RNA , PolyU , imiquimod , 3 - glucan , zymosan , Heat- killed C . albicans , cord factor, and resiquimod , ssPolyU /LyoVec and ssRNA40 /LyoVec . Trehalose - 6 , 6 -dibehenate . [ 0215 ] In certain embodiments , the PAMP of the current [0218 ] In certain embodiments , the PAMP of the current technology is a TLR9 agonist. More preferably , the TLR9 technology is an agonist of nucleotide -binding oligomeriza agonist is a CpG ODN . The term “ CpG ” or “ CpG ODN tion domain - like receptor family (NLR ) proteins including molecule ” , as used herein , is to be understood as referring to the retinoic acid inducible gene -based - 1 - like helicase recep a ODN molecule comprising a motif wherein a cytosine tor family that include RIG - 1 and MDA - 5 . Preferably , it is nucleoside is followed by a guanine nucleoside , linked by a drawn from the group of poly ( I : C ) , Poly (dA : dT ) , Poly ( dG : phosphate molecule in the normal manner seen in polynucle dC ) and 5' ppp -dsRNA . otide sequences ( i . e . a “ CpG motif ” ) , wherein the cytosine [0219 ] In certain embodiments , the PAMP of the current nucleoside is unmethylated . CpG motifs are prevalent in technology is an agonist of a DNA sensing protein drawn bacterial and viral genomes , but are rare in vertebrate from the group of DNA -dependent activator of interferon genomes. Further, CpG motifs are generally unmethylated in regulatory factors (DAI ) and absent in melanoma 2 ( AIM2) , prokaryotic organisms, whereas in eukaryotic organisms, for example , Poly (dA :dT ). DNA methyltransferases generally methylate 70 -80 % of the [0220 ] In certain embodiments , the PAMP of the current CpG motifs present. It also refers to a synthesized oligo technology is an agonist of a class A , B or C scavenger nucleotide molecule comprising at least one unmethylated receptor expressed on innate immune cells , which may, for CPG motif . Frequently, more than one CPG motif is present. example , be drawn from the group of SCARA1, SCARA2 , A variety of CPG oligonucleotide molecules are commer SCARA3 , SCARA4 , SCARA5 , SCARB1, SCARB2 , cially available . They are typically between 18 -24 nucleo SCARB3 ,MARCO , CD36 , SR - B1 , CD68 , and LOX - 1 , e . g . , tides in length , but a person skilled in the art will appreciate low - density lipoprotein (LDL ) , oxidized LDL , acetylated that CpG oligonucleotide molecules of other lengths are also LDL or chemically modified LDL . suitable . The CpG oligonucleotide molecules can comprise [0221 ] In certain embodiments , the PAMP of the current various nucleotide sequences surrounding at least one CpG technology is an agonist of NLRP1 or NALP3 , e . g ., hemo motif , as different nucleotide sequences have been shown to zoin or ATP . stimulate TLR9 to varying degrees . Class B ODN are strong [0222 ] The selected PAMP innate immune activator of the stimulators of human B cell and monocyte maturation . They current technology can be added to the substances and also stimulate the maturation of plasmacytoid dendritic cells composition used in the aspects of the present technology in (PDC ) but to a lesser extent than Class A ODN and induce an “ immunologically - effective” immunopotentiating only very small amounts of IFNa . Class C ODN have amount which , as known to those of ordinary skill in the art, features of both Class A and Class B ODN . Preferably a may vary depending on the species , strain , age , weight and Class B or Class C CPG ODN is used in the current sex of the animal or human being treated with the immu technology . As known to those skilled in the art, the CpG nological composition . backbone can be varied from a natural phosphodiester (0223 ] The term “ immunopotentiating amount” refers to backbone to a synthetic phosphorothioate backbone or a the amount of an immunological formulation needed to mixture of the two types of backbones to increase the effect an increase in immune response , as measured by stability of the ODN . In a preferred embodiment of the standard assays known to one skilled in the art . As can be technology, the CpG PAMP has a natural phosphorothioate appreciated , each immunological formulation containing backbone and is 18 to 28 nucleotides in length . In another inulin particles ( or other equivalent anti- inflammatory com embodiment of the technology , the TLR9 agonist is a Class ponent ) may have an effective dose range that may differ B or C CPG ODN with a synthetic phosphorothioate back depending on the PAMP innate immune activator and spe bone and is 18 to 28 nucleotides in length . In another cific inulin polymorphic form ( or other equivalent anti embodiment, the PAMP is drawn from the group of inflammatory component) used . Thus , a single dose range CpG2006 , CpG1826 and , in another embodiment, cannot be prescribed which will have a precise fit for each CpG7909 . possible inulin particle ( or other equivalent anti- inflamma [0216 ] In certain embodiments , the PAMP of the current tory component ) and PAMP innate immune activator com technology is a NOD - like receptor agonist . In certain bination within the scope of this technology . However , the embodiments , the agonist is to the NOD1 receptor and is immunopotentiating amount may easily be determined by drawn from the group of, Acylated derivative of iE -DAP ) one of ordinary skill in the art . The effectiveness of immune (C12 - iE -DAP ) , D - gamma -Glu -mDAP ( iE -DAP ), L - Ala activation can be measured either by an immune cell pro gamma- D -Glu -mDAP ( Tri- DAP ) . In certain embodiments , liferation assay , or assays measuring changes in the level of the agonist is to the NOD2 receptor and is drawn from the expression of cell surface activation markers , for example , group of muramyl dipeptide (MDP ) , muramyl tripeptide , by flow cytometry or fluorescent microscopy, or cytolytic L18 - MDP, M - TriDAP, murabutide , PGN - ECndi, PGN assays , or by measuring the secretion of cytokines or ECndss, N - glycolylated muramyldipeptide, and PGN - Sandi. chemokines or other substances secreted by activated In certain embodiments , the NOD2 agonist is murabutide . immune cells, or by measuring activation - induced changes US 2017 /0368167 A9 Dec . 28 , 2017 in immune cell gene expression , for example by real time [0226 ] The technology in other aspects includes a method polymerase chain reaction or gene expression arrays. The of modulating including inducing or suppressing a non amount of each component of the immunological formula antigen - specific immune response . In one aspect , the present tion necessary to provide an immunologically - effective technology provides a method of enhancing protection amount is readily determined by one of ordinary skill in the against a pathogen , wherein said method comprises admin art, e. g. , by preparing a series of immunological formula istering to a subject a therapeutically effective amount of the tions of the technology with varying concentrations of compositions or substances of the technology . This may PAMP innate immune activator and inulin particles ( or other provide temporary protection against various pathogens equivalent anti - inflammatory component) then adding these including viruses, bacteria , parasites, fungi and protozoa , for formulations to cultures of immune cells and assaying treatment of cancer, or prevention or treatment of autoim immune cell activation by means known to one skilled in the mune disease , asthma or allergy. The method involves the art , including the assays detailed herein . Similarly , the steps of administering to a subject the immunological com amount of each component necessary to provide enhance position of the present technology in an immunologically ment of the immune response to a vaccine antigen can be effective amount. For longer- term protection , the immuno readily determined by one of ordinary skill in the art , for logical composition may be administered more than once . example , by preparing a series of immunological formula [0227 ] In various embodiments , the immunological com tions of the technology with varying concentrations of position of the technology is intended for treatment or PAMP innate immune activator and inulin particles (or other prevention of a variety of diseases . Thus, in various embodi equivalent anti - inflammatory component ) plus a vaccine ments , the immunological composition is provided in an antigen and administering the vaccine together with inulin amount effective to treat or prevent an infectious disease , a particles ( or other equivalent anti - inflammatory compo cancer, or an allergy . Accordingly , the methods provided nent ) , to suitable laboratory animals ( e . g . , mice or guinea herein can be used on a subject that has or is at risk of pigs ), and then assaying the resulting antigen - specific developing an infectious disease and therefore the method is immune response by measurement of antigen - specific serum a method of treating or preventing the infectious disease . or mucosal antibody titers , antigen - induced swelling in the The methods can also be used on a subject that has or is at skin (DTH ) , or antigen - stimulated T - cell proliferation or risk of developing asthma and the method is a method of cytokine production . treating or preventing asthma in the subject. The method can [ 0224 ] PAMP innate immune activators used in the tech also be used on a subject that has or is at risk of developing nology can be effective in any animal, preferably a mammal, allergy and the method is a method of treating or preventing and most preferably a human . Different PAMP innate allergy . The method can also be used on a subject that has immune activators can cause optimal immune stimulation or is at risk of developing a cancer and the method is a depending on the species. Thus a PAMP immune activator method of treating or preventing the cancer. such as a specific CpG ODN that provides optimal stimu 0228 ) The compositions and substances used in the lation in humans by binding to human TLR9 may not cause aspects of the present technology may be used in some optimal stimulation in a mouse expressing mouse TLR9 , or embodiments to alter the type or magnitude of the immune vice versa . One of ordinary skill in the art can identify the response including in one option to a co - administered anti optimal PAMP innate immune activators useful for a par gen . Accordingly , it is proposed that the compositions and ticular species of interest using routine immune assays substances can be widely used as a vaccine adjuvant, for described herein or known in the art . example , by combining it / them with one or more relevant [ 0225 ] The aqueous portion of the compositions and sub antigens to form a prophylactic or therapeutic vaccine. Thus , stances of the aspects of the present technology may be in certain embodiments , the compositions and substances of buffered in iso -osmotic saline . Because the compositions the aspects of the present technology further comprise a and substances may be intended for parenteral or mucosal vaccine antigen . Alternatively , the subject to be treated is administration , it may be appropriate to formulate these further administered a vaccine antigen at the same time as or solutions so that the tonicity is essentially the same as following the administration of an immunologically effec normal physiological fluids in order to prevent post - admin tive amount of the immunological composition . In various istration swelling or rapid absorption of the composition due embodiments , the antigen may one or more of a microbial to differential ion concentrations between the composition antigen , a self -antigen , a cancer antigen , and an allergen , but and physiological fluids. It may also be appropriate to buffer it is not so limited . In various embodiments , the microbial the saline in order to maintain a pH compatible with normal antigen is one or more of a bacterial antigen , a viral antigen , physiological conditions. For example , the buffered pH may a fungal antigen and a parasitic antigen . In another embodi suitably be in the range of 4 to 10 , in the range 5 to 9 , in the ment, the antigen is a peptide antigen . In another embodi range 6 to 8 . 5 , or in the range 7 to 8 . 5 . Also , in certain ment, the antigen is encoded by a nucleic acid vector. In instances, it may be necessary to maintain the pH at a another embodiment, the composition further comprises a particular level in order to insure the stability of certain cytokine, or the subject is further administered a cytokine . composition components , such as the inulin particles , PAMP [0229 ] The term “ antigen ” refers to any substance , usually or the protein antigens in a formulation . Any physiologically a protein or glycoprotein , lipoprotein , saccharide , polysac acceptable buffer may be used herein , but it has been found charide or lipopolysaccharide , which upon administration that it is most convenient to use bicarbonate buffered saline stimulates the formation of specific antibodies ormemory T ( 1 % ) at a pH of between 6 and 8 . 5 . Suitable preservatives cells . An antigen can stimulate the proliferation of T -lym include benzalkonium chloride ( 0 . 003 - 0 . 03 % w / v ) ; chlo phocytes with receptors for the antigen , and can react with robutanol ( 0 . 3 - 0 . 9 % w / v ) ; parabens ( 0 .01 - 0 .25 % w / v ) and the lymphocytes to initiate the series of responses designated thimerosal ( 0 .004 - 0 .02 % w / v ) . cell -mediated immunity . US 2017 /0368167 A9 Dec . 28 , 2017 22
[0230 ] Suitable antigens for use in this technology include Ross river virus, Venezuelan equine encephalitis virus , substances from microbes (bacteria , fungi, protozoa , or Western equine encephalitis virus ), the genus Flavirius viruses ) or endogenous substances against which a specific (Mosquito borne yellow fever virus , Dengue virus , Japanese immune response can be generated . Antigens may be pre encephalitis virus, St. Louis encephalitis virus, Murray pared from inactivated organisms or may be generated by Valley encephalitis virus, West Nile virus , Kunjin virus, recombinant protein technology or directly synthesized . For Central European tick borne virus , Far Eastern tick borne the purposes of this description , an antigen is defined as any protein , carbohydrate , lipid , nucleic acid , or mixture of virus, Kyasanur forest virus, Louping III virus, Powassan these , or a plurality of these , to which an immune response virus , Omsk hemorrhagic fever virus ) , the genus Rubivirus is desired . The term antigen as used herein also includes (Rubella virus) , the genus Pestivirus (Mucosal disease virus, combinations of haptens with a carrier . A hapten is a portion Hog cholera virus, Border disease virus ) ; the family Bunya of an antigenic molecule or antigenic complex that deter viridae , including the genus Bunyvirus (Bunyamwera and mines its immunological specificity , but is not sufficient to related viruses , California encephalitis group viruses ), the stimulate an immune response in the absence of a carrier. genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley Commonly, a hapten is a relatively small peptide or poly fever virus ), the genus Nairovirus (Crimean - Congo hemor saccharide and may be a fragment of a naturally occurring rhagic fever virus , Nairobi sheep disease virus ) , and the antigen . A hapten will react specifically in vivo and in vitro genus Uukuvirus (Uukuniemi and related viruses ) ; the fam with homologous antibodies or T - lymphocytes. Haptens are ily Orthomyxoviridae, including the genus Influenza virus typically attached to a large carrier molecule such as tetanus ( Influenza virus type A , many human subtypes ) ; Swine influenza virus, and Avian and Equine Influenza viruses ; toxoid or keyhole limpet hemocyanin (KLH ) by either influenza type B (many human subtypes) , and influenza type covalent or non - covalent binding before formulation as a C (possible separate genus ) ; the family paramyxoviridae , vaccine . including the genus Paramyxovirus ( Parainfluenza virus 0231 ] Antigens can be used in vaccines to either treat or type 1 , Sendai virus , Hemadsorption virus , Parainfluenza prevent a disease . They can also be used to generate specific viruses types 2 to 5 , Newcastle Disease Virus, Mumps immune substances , such as antibodies , which can be used virus) , the genus Morbillivirus (Measles virus, subacute in diagnostic tests or kits . The subjects of an antigen sclerosing panencephalitis virus , distemper virus, Rinder containing vaccine are typically vertebrates , preferably a pest virus) , the genus Pneumovirus ( respiratory syncytial mammal, more preferably a human . It is not always neces virus (RSV ) , Bovine respiratory syncytial virus and Pneu sary that the antigen be identified in molecular terms. For monia virus of mice ); forest virus, Sindbis virus, Chikun example , immune responses to tumors can be generated gunya virus , O 'Nyong - Nyong virus , Ross river virus , Ven without knowing either in advance or post -hoc which mol ezuelan equine encephalitis virus , Western equine ecules the immune response is directed against. In these encephalitis virus ) , the genus Flavirius (Mosquito borne cases , the term antigen refers to the substance or substances , yellow fever virus , Dengue virus, Japanese encephalitis known or not known, toward which a specific immune virus, St. Louis encephalitis virus, Murray Valley encepha response is directed . The specificity of the immune response litis virus , West Nile virus, Kunjin virus, Central European provides an operational definition of an antigen , such that tick borne virus , Far Eastern tick borne virus , Kyasanur immunity generated against one type of tumor may be forest virus , Louping III virus , Powassan virus , Omsk hem specific for that tumor type but not another tumor type . orrhagic fever virus ) , the genus Rubivirus (Rubella virus ) , [ 0232] In one embodiment , the encoded antigen may be the genus Pestivirus Mucosal( disease virus , Hog cholera derived from a virus such as influenza , including inactivated virus, Border disease virus ); the family Bunyaviridae , influenza virus or influenza haemagglutinin , neuraminidase including the genus Bunyvirus (Bunyamwera and related or M2 protein or other components of the influenza virus . viruses , California encephalitis group viruses ) , the genus Examples of other RNA viruses that are antigens in verte Phlebovirus ( Sandfly fever Sicilian virus, Rift Valley fever brate animals include , but are not limited to , the following : virus ) , the genus Nairovirus (Crimean - Congo hemorrhagic members of the family Reoviridae, including the genus fever virus , Nairobi sheep disease virus ), and the genus Orthoreovirus (multiple serotypes of both mammalian and Uukuvirus (Uukuniemi and related viruses ) ; the family avian retroviruses ) , the genus Orbivirus (Bluetongue virus , Orthomyxoviridae , including the genus Influenza virus ( In Eugenangee virus , Kemerovo virus, African horse sickness fluenza virus type A , many human subtypes ) ; Swine influ virus, and Colorado Tick Fever virus ), the genus Rotavirus enza virus, and Avian and Equine Influenza viruses; influ (human rotavirus , Nebraska calf diarrhea virus , murine enza type B (many human subtypes) , and influenza type C rotavirus, simian rotavirus, bovine or ovine rotavirus, avian (possible separate genus ); the family paramyxoviridae , rotavirus) ; the family Picornaviridae , including the genus including the genus Paramyxovirus (Parainfluenza virus Enterovirus (poliovirus , Coxsackie virus A and B , enteric type 1 , Sendai virus, Hemadsorption virus , Parainfluenza cytopathic human orphan ( ECHO ) viruses, hepatitis A virus, viruses types 2 to 5 , Newcastle Disease Virus, Mumps Simian enteroviruses , Murine encephalomyelitis (ME ) virus ) , the genus Morbillivirus (Measles virus, subacute viruses, Poliovirus muris , Bovine enteroviruses , Porcine sclerosing panencephalitis virus , distemper virus, Rinder enteroviruses , the genus Cardiovirus (Encephalomyocardi pest virus ), the genus Pneumovirus (respiratory syncytial tis virus (EMC ) , Mengovirus ) , the genus Rhinovirus , the virus (RSV ) , Bovine respiratory syncytial virus and Pneu genus Apthovirus (Foot and Mouth disease ; the family monia virus of mice ) ; the family Rhabdoviridae, including Calciviridae , including Vesicular exanthema of swine virus , the genus Vesiculovirus (VSV ) , Chandipura virus , Flanders San Miguel sea lion virus, Feline picornavirus and Norwalk Hart Park virus ) , the genus Lyssavirus (Rabies virus ) , fish virus; the family Togaviridae, including the genus Alphavi Rhabdoviruses , and two probable Rhabdoviruses (Marburg rus ( Eastern equine encephalitis virus , Semliki forest virus , virus and Ebola virus ) ; the family Arenaviridae , including Sindbis virus, Chikungunya virus, O ’ Nyong - Nyong virus , Lymphocytic choriomeningitis virus (LCM ) , Tacaribe virus US 2017 /0368167 A9 Dec . 28 , 2017 24 complex , and Lassa virus; the family Coronoaviridae, such as envelope glycoprotein B and other cytomegaloviral including Infectious Bronchitis Virus ( IBV ) , Mouse Hepa antigen components ; respiratory syncytial viral antigens titis virus, Human enteric corona virus , and Feline infectious such as the RSV fusion protein , the M2 protein and other peritonitis ( Feline coronavirus) . respiratory syncytial viral antigen components ; herpes sim [0233 ] Illustrative DNA viruses that are antigens in ver plex viral antigens such as immediate early proteins, glyco tebrate animals include , but are not limited to : the family protein D , and other herpes simplex viral antigen compo Poxviridae, including the genus Orthopoxvirus (Variola nents; varicella zoster viral antigens such as gpl, gpll, and major, Variola minor, Monkey pox Vaccinia , Cowpox , Buf other varicella zoster viral antigen components ; Japanese falopox , Rabbitpox , Ectromelia ), the genus Leporipoxvirus encephalitis viral antigens such as proteins E , M - E , M - E (Myxoma , Fibroma ) , the genus Avipoxvirus (Fowlpox , other NS1, NS 1 , NS 1 -NS2A ; rabies viral antigens such as rabies avian poxvirus ), the genus Capripoxvirus (sheeppox , goat glycoprotein , rabies nucleoprotein and other rabies viral pox ), the genus Suipoxvirus (Swinepox ), the genus Parapox antigen components ; West Nile virus prm and E proteins ; virus ( contagious postular dermatitis virus , pseudocowpox , and Ebola envelope protein . See Fundamental Virology , bovine papular stomatitis virus) ; the family Iridoviridae Second Edition , eds. Knipe , D . M . and , Howley P . M . ( African swine fever virus, Frog viruses 2 and 3 , Lympho (Lippincott Williams & Wilkins, New York , 2001 ) for addi cystis virus of fish ) ; the family Herpesviridae , including the tional examples of viral antigens. In addition , bacterial alpha -Herpesviruses (Herpes Simplex Types 1 and 2 , Vari antigens are also disclosed . Bacterial antigens which can be cella - Zoster, Equine abortion virus, Equine herpes virus 2 used in the compositions and methods of the technology and 3 , pseudorabies virus , infectious bovine keratoconjunc include , but are not limited to , pertussis bacterial antigens tivitis virus, infectious bovine rhinotracheitis virus, feline such as pertussis toxin , filamentous hemagglutinin , pertac rhinotracheitis virus, infectious laryngotracheitis virus ) the tin , FIM2 , FIM3 , adenylate cyclase and other pertussis Beta -herpesvirises (Human cytomegalovirus and cytomega bacterial antigen components ; diphtheria bacterial antigens loviruses of swine , monkeys and rodents ); the gamma such as diphtheria toxin or toxoid and other diphtheria herpesviruses (Epstein -Barr virus (EBV ) , Marek 's disease bacterial antigen components ; tetanus bacterial antigens virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus syl such as tetanus toxin or toxoid and other tetanus bacterial vilagus, guinea pig herpes virus, Lucke tumor virus ) ; the antigen components ; streptococcal bacterial antigens such as family Adenoviridae, including the genus Mastadenovirus M proteins and other streptococcal bacterial antigen com ( Human subgroups A , B ,C ,D ,E and ungrouped ; simian ponents ; Staphylococcal bacterial antigens such as IsdA , adenoviruses ( at least 23 serotypes ), infectious canine hepa IsdB , SdrD , and SdrE ; gram -negative bacilli bacterial anti titis, and adenoviruses of cattle , pigs, sheep , frogs and many gens such as lipopolysaccharides, flagellin , and other gram other species, the genus Aviadenovirus ( Avian adenovi negative bacterial antigen components ; Mycobacterium ruses ); and non - cultivatable adenoviruses ; the family Papo tuberculosis bacterial antigens such as mycolic acid , heat viridae , including the genus Papillomavirus ( Human papil shock protein 65 (HSP65 ), the 30 kDa major secreted loma viruses, bovine papilloma viruses , Shope rabbit protein , antigen 85A , ESAT- 6 , and other mycobacterial papilloma virus, and various pathogenic papilloma viruses antigen components ; Helicobacter pylori bacterial antigen of other species ) , the genus Polyomavirus (polyomavirus , components ; pneumococcal bacterial antigens such as pneu Simian vacuolating agent (SV -40 ), Rabbit vacuolating agent molysin , pneumococcal capsular polysaccharides and other (RKV ) , K virus, BK virus, JC virus, and other primate pneumococcal bacterial antigen components ; haemophilus polyoma viruses such as Lymphotrophic papilloma virus ) ; influenza bacterial antigens such as capsular polysaccha the family Parvoviridae including the genus Adeno -associ rides and other haemophilus influenza bacterial antigen ated viruses , the genus Parvovirus ( Feline panleukopenia components ; anthrax bacterial antigens such as anthrax virus, bovine parvovirus, canine parvovirus, Aleutian mink protective antigen , anthrax lethal factor, and other anthrax disease virus, etc ) . DNA viruses also include Kuru and bacterial antigen components ; the F1 and V proteins from Creutzfeldt - Jacob disease viruses and chronic infectious Yersinia pestis ; rickettsiae bacterial antigens such as romps neuropathic agents (CHINA virus) . Each of the foregoing and other rickettsiae bacterial antigen components . Also lists is illustrative , and is not intended to be limiting . included with the bacterial antigens described herein are any [ 0234 ] Other examples of antigens suitable for the tech other bacterial , mycobacterial, mycoplasmal, rickettsial, or nology include , but are not limited to , infectious disease chlamydial antigens. Examples of protozoa and other para antigens for which a protective immune response may be sitic antigens include , but are not limited to , plasmodium desired including the human immunogenicity virus (HIV ) falciparum antigens such as merozoite surface antigens , antigens gag , env, pol, tat , rev , nef, reverse transcriptase , and sporozoite surface antigens, circumsporozoite antigens, other HIV components or a part thereof, the E6 and E7 gametocyte / gamete surface antigens , blood - stage antigen pf proteins from human papilloma virus , the EBNA1 antigen 1 55 /RESA and other plasmodial antigen components ; toxo from herpes simplex virus, hepatitis viral antigens such as plasma antigens such as SAG - 1 , p30 and other toxoplasma the S , M , and L proteins of hepatitis B virus, the pre - S antigen components ; schistosomae antigens such as gluta antigen of hepatitis B virus, and other hepatitis , e . g . , hepa thione - S - transferase , paramyosin , and other schistosomal titis A , B , and C , viral components such as hepatitis C viral antigen components ; leishmania major and other leishma RNA ; influenza viral antigens such as hemagglutinin , niae antigens such as gp63 , lipophosphoglycan and its neuraminidase , nucleoprotein , M2, and other influenza viral associated protein and other leishmanial antigen compo components ; measles viral antigens such as the measles nents ; and trypanosoma cruzi antigens such as the 75 -77 virus fusion protein and other measles virus components ; kDa antigen , the 56 kDa antigen and other trypanosomal rubella viral antigens such as proteins El and E2 and other antigen components . Examples of fungal antigens include , rubella virus components ; rotaviral antigens such as VP7sc but are not limited to , antigens from Candida species , and other rotaviral components ; cytomegalovirus antigens Aspergillus species, Blastomyces species , Histoplasma spe US 2017 /0368167 A9 Dec . 28 , 2017 25 cies, Coccidiodomycosis species , Malassezia furfur and from a cancer cell, such as membrane proteins . Included are other species, Exophiala werneckii and other species, Pie survivin and telomerase universal antigens and the MAGE draia hortai and other species , Trichosporum beigelii and family of cancer testis antigens . other species, Microsporum species, Trichophyton species , [0237 ] In another embodiment, the compositions and Epidermophyton species , Sporothrix schenckii and other methods of the technology include antigens involved in species, Fonsecaea pedrosoi and other species, Wangiella autoimmunity that can be used to induce immune tolerance . dermatitidis and other species, Pseudallescheria boydii and Such antigens include , but are not limited to , myelin basic other species , Madurella grisea and other species , Rhizopus protein , myelin oligodendrocyte glycoprotein and proteo species , Absidia species, and Mucor species . Examples of lipid protein of multiple sclerosis , CII collagen protein of prion disease antigens include PrP, beta - amyloid , and other rheumatoid arthritis , glutamic acid decarboxylase , insulin prion - associated proteins. and tyrosine phosphatase proteins of type 1 diabetes melli [ 0235 ] In addition to the use of the compositions and tus , gliadin protein of celiac disease . substances of the aspects of the present technology to induce [0238 ] In another embodiment, the compositions, sub an antigen specific immune response in humans , the meth stances and methods of the aspects of the present technology ods of certain embodiments are particularly well suited for can be used with antigens known as “ allergens” involved in treatment of horses and other animals . The methods of the allergy to induce tolerance and suppress allergen -specific technology can be used to protect against infection in IgE . An “ allergen ” is any substance that can induce an livestock , including cows, camels , horses, pigs, sheep , and allergic or asthmatic response in a susceptible subject. goats . Horses are susceptible to flaviviruses including Japa Allergens include pollens, insect venoms, animal dander nese encephalitis and West Nile virus . In certain embodi dust, fungal spores and drugs ( e. g ., penicillin ). Examples of ments , the immunological composition of the technology natural , animal and plant allergens include but are not can be administered to horses together with inactivated limited to proteins specific to the following genuses : Canine Japanese encephalitis virus antigen to protect them against (Canis familiaris ); Dermatophagoides (e . g. , Dermatopha Japanese encephalitis and related flaviviruses. goides farinae ); Felis ( Felis domesticus ); Ambrosia (Ambro [ 0236 ] In addition to the infectious and parasitic agents sia artemiisfolia ; Lolium ( e . g ., Lolium perenne or Lolium mentioned above , another area for desirable enhanced multiflorum ) ; Cryptomeria (Cryptomeria japonica ) ; Alter immunogenicity to a non - infectious agent is in the area of naria ( Alternaria alternata ) ; Alder; Alnus (Alnus gulti cancer , in which cells expressing cancer antigens are desir noasa ) ; Betula (Betula verrucosa ) ; Quercus ( Quercus alba ) ; ably eliminated from the body . A " cancer antigen " as used Olea (Olea europa ); Artemisia ( Artemisia vulgaris ) ; Plan herein is a compound , such as a peptide or protein , present tago ( e . g . , Plantago lanceolata ); Parietaria ( e . g ., Parietaria in a tumor or cancer cell and which is capable of provoking officinalis or Parietaria judaica ) ; Blattella ( e . g . , Blattella an immune response when expressed on the surface of an germanica ) ; Apis ( e . g . , Apis multiflorum ) ; Cupressus ( e . g . , antigen presenting cell in the context of an MHC molecule . Cupressus sempervirens , Cupressus arizonica and Cupres Cancer antigens can be prepared from cancer cells either by sus macrocarpa ) ; Juniperus (e . g. , Juniperus sabinoides , preparing crude extracts of cancer cells , for example , as Juniperus virginiana , Juniperus communis and Juniperus described in Cohen , et al. , 1994 , Cancer Research , 54 : 1055 , ashei ) ; Thuya ( e . g . , Thuya orientalis ) ; Chamaecyparis ( e . g . , by partially purifying the antigens , by recombinant technol Chamaecyparis obtusa ); Periplaneta ( e. g ., Periplaneta ogy, or by de novo synthesis of known antigens. Cancer americana ) ; Agropyron ( e . g . , Agropyron repens ) ; Secale antigens include but are not limited to antigens that are ( e . g ., Secale cereale ) ; Triticum ( e . g . , Triticum aestivum ) ; recombinantly expressed , an immunogenic portion of, or a Dactylis ( e . g . , Dactylis glomerata ) ; Festuca ( e . g . , Festuca whole tumor or cancer. Such antigens can be isolated or elatior ) ; Poa ( e . g . , Poa pratensis or Poa compressa ) ; Avena prepared by recombinant DNA expression technology or by ( e . g . , Avena sativa ); Holcus ( e . g ., Holcus lanatus) ; Anthox any other means known in the art . In one embodiment, the anthum ( e . g . , Anthoxanthum odoratum ) ; Arrhenatherum cancer is chosen from biliary tract cancer ; bone cancer ; brain ( e . g ., Parrhenatherum elatius) ; Agrostis ( e . g . , Agrostis and CNS cancer ; breast cancer; cervical cancer; choriocar alba ) ; Phleum ( e . g ., Phleum pratense ); Phalaris (e . g . , cinoma; colon cancer ; connective tissue cancer ; endometrial Phalaris arundinacea ); Paspalum ( e. g . , Paspalum nota cancer ; esophageal cancer; eye cancer ; gastric cancer ; Hodg tum ); Sorghum (e . g. , Sorghum halepensis ); and Bromus kin 's lymphoma; intraepithelial neoplasms; larynx cancer ; ( e . g . , Bromus inermis ). lymphomas ; liver cancer ; lung cancer (e . g ., small cell and [0239 ] In another embodiment, the compositions, sub non - small cell) ; melanoma; neuroblastomas; oral cavity stances and methods of the aspects of the present technology cancer; ovarian cancer ; pancreas cancer ; prostate cancer ; can be used to immunize against antigens involved in rectal cancer ; sarcomas ; skin cancer ; testicular cancer ; thy asthma. Such antigens include, but are not limited to IgE and roid cancer ; and renal cancer . Cancer antigens which can be histamine . used in the compositions and methods of the technology [0240 ] The term “ treatment” as used herein covers any include , but are not limited to , prostate specific antigen treatment of a disease in a bird , fish or mammal, particularly (PSA ) , breast , ovarian , testicular, melanoma, telomerase ; a human , and includes : multidrug resistance proteins such as P - glycoprotein ; [0241 ] (i ) preventing the disease from occurring in a MAGE - 1, alpha fetoprotein , carcinoembryonic antigen , subject which may be predisposed to the disease but has not mutant p53 , papillomavirus antigens , gangliosides or other yet been diagnosed as having it ; carbohydrate - containing components of melanoma or other [0242 ] ( ii) inhibiting the disease, i .e ., slowing or arresting cancer cells . It is contemplated by the technology that its development; or antigens from any type of cancer cell can be used in the [ 0243 ] (iii ) relieving the disease , i. e ., causing regression of compositions and methods described herein . The antigen the disease . ( It should be noted that vaccination may effect may be a cancer cell, or immunogenic materials isolated regression of a disease where the disease persists due to US 2017 /0368167 A9 Dec . 28 , 2017 26 ineffective antigen recognition by the subject 's immune subject that has already been exposed to the antigen . Where system , where the vaccine effectively presents antigen .) the components are administered sequentially, the separation [ 0244 ] The term “ optionally ” means that the subsequently in time between the administrations of the components may described event or circumstances may or may not occur, and be a matter of minutes or longer. In various embodiments , that the description includes instances where said event or the separation in time is less than 7 days , 3 days , 2 days or circumstances occurs and instances in which it does not less than 1 day . occur. [0248 ]. The compositions or substances of the present [0245 ] The term " modulation of the immune response ” is technology may be used to enhance a vaccine response in to be understood as the induction of any induced change in association with use of a DNA vaccine . In certain embodi an immune cell, which can be measured in a manner known ments , the compositions or substances of the aspects of the to those of ordinary skill in the art . Preferably , the measured present technology with a protein or other physical antigen parameter to indicate a change in the behavior or function of is / are administered as a boost dose following one or more immune cells will be selected from the group of a change in prime doses of an effective immunogenic amount of a DNA gene expression , protein expression , cell morphology , dif vaccine encoding one or more antigens . In a further embodi ferentiation , cell division , cell surface protein expression , ment, the composition or substances of the aspects of the chemotaxis , phagocytosis , exocytosis , autophagy , present technology is / are administered with a protein or chemokine secretion , cytokine secretion and apoptosis . other physical antigen at the same time as a DNA vaccine [ 0246 ] In a further embodiment of the technology , the encoding one or more antigens is administered either at a co -administration of an inulin particle ( or other equivalent different injection site or mixed together and administered at anti- inflammatory component ) with a PAMP innate immune the same injection site . activator allows dose - sparing of the PAMP innate immune 10249 ]. The compositions or substances of the present activator. Hence in the presence of a inulin particle (or other technology with or without the addition of a physical antigen equivalent anti - inflammatory component) , a lower dose of a may also be administered together with a vector encoding an PAMP innate immune activator can be used to obtain the antigen . In its broadest sense , a “ vector” is any vehicle same level of immune activation . Given the different actions capable of facilitating the transfer to and expression by the of a inulin particle ( or other equivalent anti- inflammatory infected cell of an encoded or enclosed antigen . In general, component) and a PAMP innate immune activator, the the vectors useful in the technology include , but are not dose -sparing effect of inulin particles (or other equivalent limited to , plasmids, phages , viruses , and other vehicles anti- inflammatory component) allows a lower dose of derived from viral or bacterial sources that have been PAMP immune activator to be used to achieve a desired manipulated by the insertion or incorporation of the antigen immune response or adjuvant effect and thereby provides a nucleic acid sequences . Viral vectors are a preferred type of means to reduce any dose -related side effects or toxicity of vector and include, but are not limited to , nucleic acid the PAMP innate immune activator , while still achieving the sequences from the following viruses: retrovirus, such as desired immune outcome. As dose - related toxicity from moloney murine leukemia virus, harvey murine sarcoma excess PAMP innate immune activation and inflammation virus , murine mammary tumor virus , and rouse sarcoma are the main dose - limiting side effects of PAMP innate virus ; adenovirus , adeno - associated virus ; SV 40 - type immune activators, the technology provides a novel means viruses; polyoma viruses; Epstein - Barr viruses ; papilloma to reduce the dose - related side effects of PAMP innate viruses ; herpes virus ; vaccinia virus ; polio virus ; retrovirus ; immune activators . lentivirus and sendai virus . It is known in the art how to [ 0247 ] The composition and substances of the present readily employ other vectors in a similar fashion to deliver technology may optionally be administered in its / their sepa antigens to cells . See, e . g ., Sanbrook et al ., " Molecular rate components simultaneously or sequentially but prefer Cloning : A Laboratory Manual, ” Second Edition , Cold ably the inulin particle component ( or other equivalent Spring Harbor Laboratory Press, 1989 . anti- inflammatory component) is administered together with [0250 ] One or more of the preparations of the composi or prior to the antigen rather than following the antigen . tions substances of the present technology may include an When the components of the composition or substances of antigen - binding carrier material or allergen - binding carrier the aspects of the present technology are administered material. The antigen - binding carrier material or allergen simultaneously they can be administered in the same or binding carrier material may comprise , for example , one or separate formulations, and in the latter case at the same or more metal salts such as aluminum hydroxide , aluminum separate injection sites , and at the same time as the vaccine phosphate , aluminum sulphate , calcium phosphate , calcium antigen . The PAMP innate immune activator component can sulphate , ferrous and ferric phosphate , ferrous and ferric be administered before , after, or simultaneously with the sulphate , chromium phosphate and chromium sulphate . inulin particles ( or other equivalent anti - inflammatory com Other suitable antigen -binding carrier materials and aller ponent) and the antigen component. For instance, the PAMP gen -binding carrier materials include proteins , lipids and innate immune activator component may be administered carbohydrates ( e . g . , heparin , dextran and cellulose deriva prior to or after the administration of the inulin particle (or tives ) , and organic bases such as chitin (poly N - acetylglu other equivalent anti- inflammatory component) component cosamine ) and deacetylated derivatives thereof , as known to together with a priming dose of antigen . The boost dose of those of ordinary skill in the art . antigen may subsequently be administered with either or 10251 ) In certain embodiments , the PAMP innate immune both of the PAMP innate immune activator and the inulin activator in the immunological composition is physically particle component (or other equivalent anti - inflammatory bound to the inulin particle ( or other equivalent anti - inflam component ) . A " prime dose ” is the first dose of antigen matory component) or to the antigen -binding carrier mate administered to the subject. A " boost dose ” is a second , rial incorporated with the inulin particle ( or other equivalent third , or subsequent dose of antigen administered to a anti - inflammatory component) . In certain embodiments , the US 2017 /0368167 A9 Dec . 28 , 2017 27
PAMP innate immune activator is bound to the inulin often observed with some innate immune activators alone , particle ( or other equivalent anti- inflammatory component) for example with TLR9 agonists , is reduced or no longer by a bond selected chosen from covalent , hydrostatic , and evident when TLR9 agonists are formulated with inulin electrostatic bonds. Alternatively , the PAMP innate immune particles with or without an antigen -binding alum . In the activator can be sterically trapped inside the inulin particle presence of inulin particles , both Thl and Th2 immune (or other equivalent anti - inflammatory component) . In cer responses develop in parallel, resulting in an improved tain embodiments , a linker sequence can be used to join the immune response against a co - administered antigen not PAMP innate immune activator to the inulin particle ( or achievable with use of the individual components alone . The other equivalent anti -inflammatory component) . inulin particle (or other equivalent anti -inflammatory com [ 0252] Further, where the compositions or substances of ponent) combined with the antigen - binding carrier material the present technology include an antigen -binding material, may comprise a relative amount by weight of the inulin (or in certain embodiments the inulin particles (or other equiva - other equivalent anti - inflammatory component ) to the anti lent anti- inflammatory component) are combined with or gen - binding carrier material in the range of 1 : 20 to 200 : 1 , bound to the antigen - binding carrier material. Co -crystals of such as 1 : 5 to 50 : 1 , or 1 : 2 to 20 : 1 . inulin particles and an antigen - binding carrier material may [0259 ] In another embodiment, the compositions or sub be prepared by, for example , a method comprising : stances according to the present technology may further [ 0253] ( a ) preparing a suspension of the inulin particles ; comprise a therapeutic agent such as an anti- microbial agent, [0254 ] (b ) heating the suspension until the inulin particles an anti -cancer agent, and an allergy or asthma medicament, dissolve ; or the subject is further administered a therapeutic agent [ 0255 ] ( c ) adding to said solution an amount of an antigen selected from the same group . In a related embodiment, the binding carrier material ; anti- microbial agent is one or more of an anti -bacterial [ 0256 ] ( d ) re- precipitating the inulin particles from said agent, an anti- viral agent, an anti - fungal agent, or an anti suspension , and parasite agent . [ 0257 ] (e ) isolating formed particles comprising inulin [0260 ] In a related embodiment, the anti -cancer agent particles and one or more antigen - binding carrier material included with the immunological composition is one or [0258 ] In a development of this work , the inulin particles more of a chemotherapeutic agent, a cancer vaccine , or an can be formulated with an antigen -binding carrier material , immunotherapeutic agent. in particular, aluminum hydroxide or aluminum phosphate [0261 ] In a related embodiment, the allergy or asthma ( collectively referred to as " alum ” ) gel. Alum gel has been medicament included with the immunological composition widely used as an adjuvant in vaccines wherein it is known is one or more of PDE -4 inhibitor, bronchodilator/ beta - 2 to induce a strong antibody ( Th2 ) immune response but only agonist, K + channel opener, VLA - 4 antagonist , neurokin a poor cellular ( Th1) immune response . Thus, it has been antagonist, TXA2 synthesis inhibitor, xanthanine, arachi found possible to form co - crystallized particles of gIN , DIN donic acid antagonist , 5 lipoxygenase inhibitor, thromboxin or eIN together with aluminum salts ( for example aluminum A2 receptor antagonist, thromboxane A2 antagonist, inhibi hydroxide or aluminum phosphate ) , to form , respectively , a tor of 5 - lipox activation protein , or protease inhibitor. gIN / alum preparation (also referred to as “ Algammulin " ) [0262 ] The compositions or substances of the present ( see WO 90 /01949 , WO 2006 / 024100 ), a dIN /alum prepa technology may be formulated for parenteral administration ration ( also referred to as “ Aldeltin " ) or an eIN / alum or may be formulated in a sustained release device . The hydroxide preparation ( also referred to as “ Alepsilin ” ). sustained release device may be a microparticle , a matrix or While in vivo studies have shown that vaccines containing an implantable pump , but it is not so limited . complexes of inulin particles and aluminum salts are well [0263 ] In another embodiment, the compositions and sub tolerated , their ability to increase antibody responses to stances of the aspects of the present technology is / are co -administered antigens over and above the inulin particle formulated for delivery to a mucosal surface . In related or alum adjuvant formulation alone are generally modest embodiments , the compositions and substances of the and additive rather than synergistic , and like alum adjuvants aspects of the present technology is / are provided in an alone , the formulation of inulin with alum biases the resul amount effective to stimulate a mucosal immune response . tant immune response towards a Th2 rather than a Th1 The mucosal surface may be an oral, nasal, rectal, vaginal , response . This may not be desirable for particular vaccines and ocular surface , but is not so limited . In one embodiment, where it is sought to induce Thl immunity to a co -admin the compositions and substances of the present technology istered antigen . In particular , withoutwishing to be restricted is / are formulated for oral administration . by theory , adjuvants that enhance Thl immunity tend to [ 0264 ] The compositions and substances of the present inhibit the magnitude of a Th2 response and vice versa , via technology may also be formulated as a nutritional supple a complex array of feedback pathways involving factors ment . In a related embodiment, the nutritional supplement is such as the Th1 cytokine IFN - y , which inhibits Th2 formulated as a capsule , a pill , or a sublingual tablet . In responses, whereas the Th2 cytokines, IL - 4 and IL - 10 , another embodiment, the immunological composition is inhibit Thl responses . A bias towards a Th2 response may be formulated for local administration . undesirable if it means that less of a Th1 response can be [0265 ] In embodiments relating to the treatment of a achieved and vice versa . In one embodiment of this tech subject , themethod or use may further comprise isolating an nology , it has been found that the Th2 bias seen when inulin immune cell from the subject, contacting the immune cell is co - crystallized with aluminum salts , as in the case of with an immunologically - effective amount of the composi Algammulin , Aldeltin or Alepsilin , or phosgammulin , phos tions and substances of the aspects of the present technology deltin or phosepsilin is reduced or no longer evident when to thereby produce an ex vivo activated immune cell ; and the inulin particle - alum particles are combined with a PAMP optionally then re -administering the activated immune cell innate immune activator. Conversely , the strong Th1 bias to the subject . In one embodiment, the immune cell is a US 2017 /0368167 A9 Dec . 28 , 2017 monocyte and in another embodiment the immune cell is a technology is / are able to modulate the cytokines induced by dendritic cell . In another embodiment, the method or use a PAMP innate immune activator , and thereby lead to a more may further comprise contacting the immune cell with an favorable immune response . antigen in the presence of, before or after the addition of an 0271 ] In other aspects the technology includes a method immunologically -effective amount of the compositions or of preventing in a subject excess polarization of the immune substances of the aspects of the present technology response otherwise caused by administering to the subject a [0266 ] In still another aspect, the technology provides a combination of an antigen and a PAMP innate immune method of identifying an optimal immunological composi activator such as a TLR agonist . It has been previously tion by measuring a control level of activation of an immune shown that the combination of a PAMP innate immune cell population contacted with a composition or substances activator such as CPG ODN , a TLR9 agonist, resulted in a of the aspects of the present technology , then comparing this Th1 bias and suppression of the Th2 arm of the response . It with the level of activation of an immune cell population was thus a surprising finding that when inulin particles are contacted with a test composition , wherein a test level that combined with a Th1 -biasing PAMP innate immune activa is equal to or above the control level is indicative of a tor such as CpG ODN , it is possible to maintain a strong Th2 suitable immunological composition . response while at the same time also inducing a Th1 immune [0267 ] The immune response may comprise immune acti response to a co -administered antigen , thereby resulting in a vation as manifest by changes in gene expression or protein synergistic increase in both the Th2 and Th1 response to the production such as induction of cytokine or chemokine antigen , to an extent that the components in the absence of production or secretion , changes in phenotype , proliferative the inulin particles could not produce . or survival capacity or modulation of immune effector [0272 ] The compositions and substances of the present properties . The immune response may further comprise technology may be formulated in a pharmaceutically accept induction , enhancement or modulation of an adaptive able carrier , diluent or excipient in a form suitable for immune response with induction of antibody production or injection , or a form suitable for oral, rectal, vaginal, topical, induction of a T -cell effector or memory response against an nasal, transdermal or ocular administration . The composi endogenous or exogenous antigen . tions and substances of the aspects of the present technology [ 0268 ] In a further aspect, the present technology provides may also comprise a further active component such as , for a method of modulating an immune response , wherein said example , a vaccinating antigen ( including recombinant anti method comprises administering to a subject a therapeuti gens ) , an antigenic peptide sequence , or an immunoglobulin . cally effective amount of the compositions or substances of Alternatively , the active component may be a macrophage the aspects of the present technology. stimulator, a polynucleotide molecule (e . g ., encoding a [0269 ] As used herein , the term “ effective amount ” refers vaccinating agent) or a recombinant viral vector. to a non - toxic but sufficient amount of the compositions and [0273 ]. The components of the vaccine and adjuvant com substances of the aspects of the present technology to positions of the technology may be obtained through com provide the desired effect. The exact amount required will mercial sources , or may be prepared by one of ordinary skill vary from subject to subject depending on factors such as the in the art . The inulin particle formulations may be prepared species being treated , the age and general condition of the by the processes disclosed in U . S . Provisional Patent Appli subject, the severity of the condition being treated , the cation No . 61/ 243 , 975 and international Patent Applications particular composition or substances of the aspects of the PCT/ AU86 / 00311 (WO 87 / 02679 ) , PCT / AU89 / 00349 (WO present technology being administered and the mode of 90 /01949 ) and PCT/ AU2005 / 001328 (WO 2006 /024100 ) or administration . Thus, it is not possible to specify an exact may be obtained commercially from Vaxine Pty Ltd , " effective amount ” . However, for any given case , an appro Adelaide , Australia . PAMP innate immune activators for use priate “ effective amount” may be routinely determined by in the technology may be obtained commercially or made persons of ordinary skill in the art . using methods well known in the art. For example , synthetic [ 0270 ] In certain embodiments , the technology further triacylated lipoprotein , Pam3CSK4 ( 0 .25 ug /mouse ) , heat provides a method of modulating the patterns of cytokines killed Listeria monocytogenes ( 2 .5x10e7 cells/ mouse ) , produced in response to a vaccine. The term “ modulate ” lipoarabinomannan from M . smegmatis ( 0 . 25 ug /mouse ) , envisions the suppression of expression of a particular LPS - PG ultrapure lipopolysaccharide from P . gingivalis ( 2 . 5 cytokine when lower levels are desired , or augmentation of ug /mouse ) , standard lipoteichoic acids (LTA - SA ) from S . the expression of a particular cytokine when higher levels aureus ( 2 ug/ mouse ), peptidoglycan from Staphylococcus are desired . Modulation of a particular cytokine can occur aureus (PGN -SA ) ( 2 ug /mouse ), synthetic diacylated lipo locally or systemically . PAMP innate immune activators protein ( 0 . 25 ug /mouse ), zymosan ( 1 mg/ mouse ) , and used as vaccine adjuvants can directly activate macrophages CpG2006 (20 ug /mouse ) as used in the current technology and dendritic cells to secrete cytokines such as TNF - a and were all purchased from Invivogen , San Diego , USA . Syn IL - 1 . Cytokine profiles induced by PAMPs innate immune thetic CpG ODN synthesized with a native or modified activators determine T- cell regulatory and effector functions phosphorothioate backbone was purchased from in immune responses and may also contribute to vaccine Geneworks, Australia and can be obtained from other com adverse reactions . In general, PAMP innate immune activa mercial suppliers . MPLA may be purchased from Sigma, tors induce cytokines associated with inflammation and USA or Invivogen , San Diego , USA . Plasmid DNA may also fever including TNF and IL - 1 , but may also induce sup be prepared using methods well known in the art, for pressive cytokines such as IL - 10 , which provide inhibitory example using the Quiagen procedure (Quiagen Inc, USA ) , feedback and may thereby limit or inhibit the adaptive followed by DNA purification using known methods. The immune response to a co - administered antigen . The com - inactivated or recombinant antigens used for immunization positions and substances of the aspects of the present can be obtained through commercial chemical or protein US 2017 /0368167 A9 Dec . 28 , 2017 suppliers such as Sigma, USA or may be prepared using [0278 ] In certain embodiments , the immunological com methods well known in the art. positions are prepared and administered in dose units . Liq [ 0274 ] Biological activity of a vaccine may be assayed uid dose units are vials or ampoules for injection or other using standard laboratory techniques, e . g ., by vaccinating a parenteral administration . Solid dose units are tablets, cap standard laboratory animal ( e . g ., a mouse or guinea pig ) with sules and suppositories . The administration of a given dose a standard antigen ( e . g ., tetanus toxoid ) using a test immu can be carried out both by single administration in the form nological formulation . After allowance of time for boosting of an individual dose unit or else several smaller dose units . the vaccination , and time for immunization to occur, the Multiple administration of doses at specific intervals of animal is bled or the spleen removed and the response to the weeks or months apart can be used for boosting antigen vaccine measured . The response may be quantified by any specific immune responses . measure accepted in the art for measuring immune [0279 ] The compositions and substances of the aspects of responses , e . g . , serum , saliva , vagina , stool antibody titer the present technology , or antigens useful in the technology , against the standard antigen ( for measurement of humoral may be delivered in mixtures of more than two components . immunity ) and T -cell proliferation , cytokine ELISPOT or A mixture may comprise the immunological composition cytokine ELISA assay (for measurement of T -cell immu including one or more types of inulin particles ( or other nity ) . equivalent anti- inflammatory component ) together with one or more PAMP innate immune activators and one or more [0275 ] It will be apparent to one of ordinary skill in the art antigens . that the precise amounts of protein antigen and immuno [ 0280 ] Immunogenic Compositions logical composition needed to produce a given effect will [0281 ] In certain embodiments , disclosed herein is com vary with the particular compounds and antigens, and with positions of immunogens, wherein the immunogens com the size , age , species , and condition of the subject to be prise a region A coupled to a region B . Region A is an active treated . In certain embodiments , these amounts can be component of vaccine that is responsible for induction of determined using methods known to those of ordinary skill therapeutic antibodies . Region B is a helper component that in the art . In general , one or more vaccinations with the is responsible for induction of cellular immune responses desired antigen are initially administered by intramuscular , that help B cells to produce antibodies . subcutaneous or intradermal injection to prime the immune [0282 ] In certain embodiments , region A comprises ( i) at response . The vaccination is then “ boosted ” after a delay least one Amyloid - ß (AB ) B cell epitope or ( ii ) at least one ( usually from 1 - 12 months, for example, 6 months ) using the Tau B cell epitope or (iii ) at least one a - synuclein ( a - syn ) immunological composition of the technology preferably by B cell epitope or ( iv ) at least one Amyloid - B (AB ) B cell administering on one or more occasions the antigen com epitope and at least one Tau B cell epitope or ( v ) at least one bined with the immunological composition by parenteral Amyloid - B (AB ) B cell epitope and at least one a -synuclein injection for systemic immune boosting . Generally the anti ( a -syn ) B cell epitope or ( vi) at least one Tau B cell epitope gen dose used for an adult human will be in the range of and at least one a - synuclein ( a - syn ) B cell epitope or (vii ) 0 .001 - 0 . 1 mg and most commonly 0 . 001- 0 . 1 mg, or 0 . 005 at least one Amyloid - ß ( AB ) B cell epitope and at least one 0 .05 mg per dose . Tau B cell epitope and at least one a -synuclein ( a -syn ) B 10276 ]. In various embodiments , 0 . 1 to 5 . 0 mL or 0 . 1 to 1 cell epitope . In certain embodiments , when multiple mL of a vaccine is administered in the practice of the epitopes are present in Region A , the epitopesmay comprise technology such as to a human subject. the same epitopic sequence ( e . g . , multiple copies of AB ) or [ 0277] The compositions and substances according to the different epitopic sequences ( e . g . , AB and tau , 12 ) . When aspects of the present technology is / are , in various embodi Region A has different epitopes, the order of the epitopes ments , administered by intramuscular or intradermal injec may be arbitrary or optimized based on in vitro or in vivo tion , or other parenteral means , or by ballistic application to tests . the epidermis . They may also be administered by intranasal [0283 ] In certain embodiments , region B comprises at application , inhalation , topically , intravenously , orally , or as least one foreign T helper cell ( Th ) epitope . In certain implants , and even rectal or vaginal use is possible . Suitable embodiments , when multiple T cell epitopes are present in liquid or solid pharmaceutical preparation forms are , for Region B , the epitopes may comprise the same epitopic example , aqueous or saline solutions for injection or inha sequence (e . g ., multiple copies of PADRE ) or different lation ,microencapsulated , encochleated , coated onto micro epitopic sequences ( e . g . , PADRE and tetanus toxin p23 ) . scopic gold particles , contained in liposomes , nebulized , When Region B has different epitopes , the order of the aerosols , pellets for implantation into the skin , or dried onto epitopes may be arbitrary or optimized based on in vitro or a sharp object to be scratched into the skin . The pharma in vivo tests . ceutical compositions also include granules , powders, tab - (0284 ] In certain embodiments , when two or more immu lets, coated tablets , (micro ) capsules, suppositories , syrups , nogens are present in a composition , the immunogens are emulsions , suspensions , creams, drops or preparations with distinct ( i. e . , not identical) in region A or region B or both . protracted release of active compounds , in whose prepara For the purposes of this disclosure , if two regions contain the tion excipients and additives and / or auxiliaries such as same number of epitopes and the same sequence of epitopes , disintegrants , binders , coating agents , swelling agents , lubri if the arrangement varies then the regions , and hence the cants , flavorings , sweeteners or solubilizers are customarily immunogens, are distinct . That is , a region comprising used as described above . The pharmaceutical compositions epitope 1 and epitope 2 in the order 1- 2 is distinct from the are suitable for use in a variety of drug delivery systems. For order 2 - 1 . a brief review of present methods for drug delivery , see [0285 ] In another aspect, the composition comprises Langer , Science 249 : 1527 - 1533 , 1990 , which is incorpo nucleic acid molecules that encode immunogens that com rated herein by reference . prise a region A coupled to a region B . In certain embodi US 2017 /0368167 A9 Dec . 28 , 2017 30 ments , region A comprises ( i ) at least one Amyloid - ß (AB ) embodiments , the B cell epitopes herein may comprise B cell epitope or ( ii ) at least one Tau B cell epitope or ( iii ) additional sequence, such as amino acids that flank the at least one a - synuclein ( a - syn ) B cell epitope or ( iv ) at least epitope in the native protein . For example if the minimal one Amyloid -ß ( AB ) B cell epitope and at least one Tau B sequence of a B cell epitope is amino acids 5 - 11, a B cell cell epitope or ( V ) at least one Amyloid - ß (AB ) B cell epitope epitope herein may comprise additional amino acids such as and at least one a -synuclein ( a - syn ) B cell epitope or ( vi ) at residues 3 - 15 . Typical B cell epitopes are from about 5 to least one Tau B cell epitope and at least one a - synuclein about 30 amino acids long . In some embodiments , the ( a - syn ) B cell epitope or at least one Amyloid - ß (AB ) B cell sequence of the at least one AB B cell epitope is located epitope and at least one Tau B cell epitope and at least one within SEQ ID NO : 1 , wherein the epitope is less than 42 a - synuclein ( Q - syn ) B cell epitope. Region B comprises at amino acids long . In some embodiments , the epitope is 15 least one foreign T helper cell ( Th ) epitope . When multiple amino acids in length and in other embodiments , it is less epitopes are present in Region A , the epitopes may comprise than 15 amino acids in length , i. e ., 14 , 13 , 12 , 11, 10 , 9, 8 , the same epitopic sequence ( e . g ., multiple copies of AB 1 - 11 ) 7 , 6 , 5 , or 4 amino acids . In some embodiments , the epitope or different epitopic sequences ( e . g ., AB 1 - 11 and tau2- 18 ) . When Region A has different epitopes, the order of the comprises the sequence DAEFRH (SEQ ID NO : 7 ) . epitopes may be arbitrary or optimized based on in vitro or 102921 In some embodiments , the sequence of at least one in vivo tests . Tau B cell epitope is located within SEQ ID NO : 2 . [0286 ] In certain embodiments , region B comprises at Typically , the epitope will be from about 5 to about 30 amino least one foreign T helper cell ( Th ) epitope . When multiple acids long . In some embodiments , the epitope is 12 amino T cell epitopes are present in Region B , the epitopes may acids in length and in other embodiments , it is less than 12 comprise the same epitopic sequence ( e . g ., multiple copies amino acids in length , i . e . , 11 , 10 , 9 , 8 , 7 , 6 , or 5 amino acids . of PADRE ) or different epitopic sequences ( e .g . , PADRE In some embodiments , the epitope comprises the sequence and tetanus toxin p23 ) . When Region B has different AKAKTDHGAEIVYKSPWSGDTSPRHLSNVSSTGSID epitopes, the order of the epitopes may be arbitrary or ( SEQ ID NO : 8 ) . In other embodiments, the epitope com optimized based on in vitro or in vivo tests . prises the sequence RSGYSSPGSPGTPGSRSR ( SEQ ID [ 0287 ] In certain embodiments, when two or more immu NO : 9 ), or the sequence NATRIPAKTPPAPKTPPSSGEP nogens are encoded , the immunogens are distinct ( i . e . , not PKSGDRSGYSSPGS (SEQ ID NO : 10 ) , or the sequence identical ) in region A or region B or both . For the purposes GEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPP of this disclosure , if two regions contain the same number of TREPKK (SEQ ID NO : 11 ) , or the sequence KKVAWRT epitopes and the same sequence of epitopes , if the arrange PPKSPSS (SEQ ID NO : 12 ) , or the sequence AEPROEFE ment varies then the regions, and hence the immunogens , are VMEDHAGTY ( SEQ ID NO : 13 ). In certain embodiments , distinct. That is , a region comprising epitope 1 and epitope the epitope comprises at least 5 contiguous amino acids of 2 in the order 1 - 2 is distinct from the order 2 - 1 . Multiple SEQ ID NOs: 8 - 13 . immunogens may be encoded by a single nucleic acid [0293 ] In some embodiments , the sequence of at least one molecule or a single immunogen may be encoded by a single a -syn B cell epitope of region A is located within SEQ ID nucleic acid molecule . In some embodiments , at least two NO : 3 . The epitope will often be about 5 to 50 amino acids immunogens are encoded on a single nucleic acid molecule . long. In some embodiments , the epitope is about 50 amino In other embodiments , each of the immunogens is encoded acids long; in other embodiments , the epitope is less than by separate nucleic acid molecules. In yet other embodi about 50 amino acids , in still other embodiments, the epitope ments , more than one immunogen is encoded by a single is less than about 30 amino acids , or less than about 20 nucleic acid molecule and at least one other immunogen is amino acids , or less than about 15 amino acids, or less than encoded by a separate nucleic acid molecule . about 12 amino acids. In certain embodiments , the fragment [ 0288 ] In various embodiments , the at least one epitope in comprises the sequence : Region A and Region B can be about 1 to about 18 , or about 1 to about 15 , or about 1 to about 12 , or about 1 to about 9 , or about 1 to about 6 , or about 1 to about 3 , or 1 , or 2 , or 3 , SEQ ID NO : or 4 , or 5 , or 6 , or 7 , or 8 , or 9 , or 10 , or 11 , or 12 or 13 or KTKEGVLYVGSKTKEGVVHGVATVAEKTKEOV 14 14 or 15 or 16 or 17 or 18 amino acids . When there is more TNVGGAWTGVTAVAQK than one epitope , the epitopes may all be different sequences , or some of them may be different sequences . AGSIAAATGFVKKDQ 15 [ 0289 ] In some embodiments , the at least one Th epitope 16 of region B is capable of being recognized by one or more QEGILEDMPVDPDNEAYE antigen -experienced T helper cell populations of a subject. EMPSEEGYQDYEPEA 17 The composition is normally capable of activating a humoral immune response in a subject . In some embodiments , the KAKEG 18 humoral immune response comprises one or more antibodies GKTKEGVLYVGSKTKEGVVH + specific to pathological forms of AB , or Tau, or a - syn m proteins. EGWHGVATVAEKTKEQVTNVGGA +4 [ 0290 ] 1 . Structure of B Cell Epitopes EQVTNVGGAVVTGVTAVAQK 44 [0291 ] AB cell epitope is a peptide comprising a sequence that can stimulate production of antibodies by B cells that bind to the epitope or protein containing the epitope . More [0294 ] In certain embodiments , the epitope comprises at over , the B cell epitope within the context of this disclosure least 5 contiguous amino acids of SEQ ID NOs: 14 - 18 and preferably does not stimulate a T cell response . In certain 42 - 44 . US 2017 /0368167 A9 Dec . 28 , 2017 31
[ 0295 ] In some embodiments , region A comprises a plu thus potentially cause a helper T cell immune responses in rality of B cell epitopes . In certain embodiments , region A subject receiving the composition . comprises 1 , 2 , or 3 B cell epitopes . In other embodiments , region A comprises as many as 18 epitopes , e . g . , 1 , 2 , 3 , 4 , [0299 ] 2 . T Cell Epitopes (MultiTEP Platform for Vac 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 15, 16 , 17 or 18 . The plurality cines ) of epitopes can have identical sequences or different [0300 ] In certain embodiments , the T cell epitopes of the sequences. Furthermore, the plurality of epitopes can be all immunogens are “ foreign ” , that is , they are peptide one typei. e . , all having a tau sequence , all having an AB sequences or encode peptide sequences that are not found in sequence, or all having an a - syn sequence . In some embodi the mammals and in the subject to receive the composition . ments, the plurality of epitopes are from a combination of A foreign T cell epitope can be derived from a non - self tau , Aß , and a -syn . In some embodiments , Region A com non -mammalian protein or be an artificial sequence . PADRE prises three AB , three tau , and three a - synuclein epitopes. In is an example of an artificial sequence that serves as a T cell particular embodiments, the Aß epitopes comprise residues epitope . A “ promiscuous T cell epitope” means a peptide 1 - 11, the tau epitopes comprise residues 2 - 13 , and a - sy sequence that can be recognized by many MHC - II ( e. g ., nuclein epitopes comprise residues 36 - 39 . In other embodi human DR ) molecules of the immune system and induce ments , Region comprises three AB and three tau epitopes . In changes in immune cells of these individuals such as, but not particular embodiments , the Aß epitopes comprise residues limited to production of cytokine and chemokines . The T 1 - 11 and the tau epitopes comprise residues 2 - 13 . When cells specific to these epitopes help B cells , such as B cells region A comprises a plurality of B cell epitopes (or encodes specific to amyloid or tau or a - synuclein to produce anti a plurality of B cell epitopes) , the epitopes are typically bodies to these proteins. It is desirable that antibody pro present in a tandem array with linkers between them . The duced be detectable and moreover produced at therapeuti linkers may be of any length and sequence, although short cally relevant titers against pathological forms of these sequences of flexible residues like glycine and serine that proteins in the sera of vaccinated subjects . allow adjacent protein domains to move freely relative to [0301 ] As discussed herein , in certain embodiments the T one another are typically used . Longer linkers may be used cell epitope is foreign to the subject receiving the compo in order to ensure that two adjacent domains do not sterically sition . In some embodiments , the at least one Th epitope of interfere with one another. An exemplary linker sequence is one or more of the immunogens is from 12 to 22 amino acids GS ( glycine -serine ) . in length . Region B may comprise a plurality of Th epitopes , [ 0296 ]. In some embodiments , an AB B cell epitope may be either all having the same sequence or encoding the same encoded by a sub - sequence shown in SEQ ID NO : 4 or a sequence , or a mixture of different Th epitopes . In some nucleic acid sequence that encodes the amino acids . Simi embodiments , region B comprises from 1 to 20 epitopes , in larly , a Tau B cell epitope may be encoded by the sequence other embodiments , region B comprises at least 2 epitopes , or sub - sequence shown in SEQ ID NO : 5 , or by a nucleic in yet other embodiments region B comprises from 2 to acid sequence that encodes the same amino acids, or an about 20 epitopes . Exemplary B regions are illustrated in the a - syn B cell epitope may be encoded by the sequence or a Figures and Examples. When region B comprises a plurality sub - sequence shown in SEQ ID NO : 6 , or by a nucleic acid of T cell epitopes ( or encodes a plurality of T cell epitopes ) , sequence that encodes the same amino acids . the epitopes are typically present in a tandem array with [ 0297] B cell epitopes of Aß, tau and a - syn may be linkers between them . The linkers may be of any length and identified in a variety of ways, including but not limited to sequence , although short sequences of small amino acids computer program analysis , peptide arrays , phage display will usually be used . An exemplary linker sequence is GS libraries, direct binding assays , etc . Computer programs, as (glycine -serine ). Collectively the string of Th epitopes is well as other tests are commercially or freely available , can called MultiTEP platform : be used to predict or directly show B cell epitopes . Candi date sequences can be synthesized and coupled to a carrier ( SEQ ID NO : 45 ) protein that is used to immunize an animal, e . g ., a mouse . AKFVAAWTLKAAAGSVSIDKFRIFCKANPKGSLKFIIKRYTPNNEIDSGS Sera may then be tested by ELISA or other known method for the presence of antibodies to the candidate . In addition , IREDNNITLKLDRCNNGSFNNFTVSFWLRVPKVSASHLEGSQYIKANSKF the epitopes may be tested by any method known in the art IGITEGSPHHTALRQAILCWGELMTLAGSFFLLTRILTIPOSLDGSYSGP or described herein for stimulation of T cells . [0298 ] In certain embodiments , suitable epitopes do not LKAEIAQRLEDVGSNYSLDKIIVDYNLQSKITLPGSLINSTKIYSYFPSV stimulate T cells . Some peptides of AB are known to act as ISKVNQGSLEYIPEITLPVIAALSIAES * . a T cell epitope . These include the sequences , OKLVF FAEDVGSNKGAIIGLMVGGWIA (SEQ ID NO : 19 ) , [0302 ] There are many suitable T cell epitopes. Epitopes VFFAEDVGSNKGAII (SEQ ID NO : 20 ), QKLVFFAED can be identified by a variety of well -known techniques , VGSNKGAIIGL ( SEQ ID NO : 21) , LVFFAEDVGSNKGA including various T cell proliferation assays as well as using (SEQ ID NO : 22 ) , QKLVFFAEDVGSNKG (SEQ ID NO : computer algorithms on protein sequences and MHC -bind 23 ) , and GSNKGAIIGLMVGGVVIA (SEQ ID NO : 24 ) . ing assays , or chosen from myriad databases, such as Other B cell epitope candidates can be assayed for T cell MHCBN (hosted at EMBL -EBI ) , SYFPEITHI (hosted by epitope function using one of the assays described herein or the Institute for Cell Biology, BMI-Heidelberg and found at known in the art, such as [ 3H ] thymidine incorporation upon (www .syfpeithi . de ) , IEDB (Vita R , et al. Nucleic Acids Res . stimulation , MHC - binding assays, intracellular staining , 2010 38 ( Database issue ): D854 -62 . Epub 2009 Nov . 11 , and ELISPOT, flow cytometry of CFSE - stained proliferating found at www . iedb .org ) , and SEDB (hosted at Pondicherry cells , MTA proliferation assay , that can be used to identify University , India , and found at sedb .bicpu .edu . in ) . T cell epitope sequences that elicit helper T cell proliferation and epitopes presented by MHC class I molecules are typically US 2017 /0368167 A9 Dec . 28 , 2017 32 peptides between 8 and 11 amino acids in length , whereas used without further purification or purified by any of a MHC class II molecules present longer peptides , typically variety of protein purification methods, including HPLC and 13 - 17 amino acids in length . affinity chromatography. [ 0303 ] In some embodiments , the at least one Th epitope [0308 ] Coupling of Regions (peptide binding to MHC class II and activating Th cell) is [ 0309 ] In certain embodiments , the A and B regions of the one or more of a Tetanus toxin epitope, a diphtheria toxin at least two immunogens are coupled . When two or more epitope , a Hepatitis B surface antigen epitope , an influenza immunogens are used , the two or more immunogens may virus hemagglutinin epitope , an influenza virus matrix pro also be coupled . Coupling may be through a chemical tein epitope, one or more synthetic promiscuous epitopes , or linkage or peptide linkage ( e . g . , a fusion protein ) or elec mixtures thereof. For example , suitable Th epitopes include trostatic interaction ( e . g ., van der Waals forces ) or other type a P23TT Tetanus Toxin epitope comprising the sequence of coupling . VSIDKFRIFCKANPK (SEQ ID NO : 25 ) , a P32TT Tetanus [0310 ] When the linkage is peptidic , the C -terminus of Toxin epitope comprising the sequence LKFIIKRYTPN region A may be linked to the N - terminus of region B or vice NEIDS (SEQ ID NO : 26 ) , a P21TT Tetanus Toxin epitope versa . Alternatively , C -terminus of one B region may be comprising the sequence IREDNNTLKLDRCNN ( SEO ID coupled to N - terminus of A region and N -terminus of NO : 27 ) , a P30TT Tetanus Toxin epitope comprising the another B region may be coupled to the C - terminus of the sequence FNNFTVSFWLRVPKVSASHLE (SEQ ID NO : same A region . Moreover, region A may be coupled to region 28 ) , a P2TT Tetanus Toxin epitope comprising the sequence B via a linker domain . Linker domains can be any length , as QYIKANSKFIGITE (SEQ ID NO : 29 ) , a Tetanus Toxin long as several hundred amino acids, but more typically will epitope comprising the sequence LEYIPEITLPVIAALSI be 2 - 30 amino acids or equivalent length . Linkers are often AES ( SEQ ID NO : 30 ) , a Tetanus Toxin epitope comprising composed of flexible residues like glycine and serine that the sequence LINSTKIYSYFPSVISKVNQ (SEQ ID NO : allows adjacent protein domains to move freely relative to 31 ), a Tetanus Toxin epitope comprising the sequence one another. Longer linkers are used in order to ensure that NYSLDKIIVDYNLQSKITLP (SEQ ID NO : 32 ), a HBV two adjacent domains do not sterically interfere with one nuclear capsid epitope comprising the sequence PHHTAL another. Some exemplary linkers include the sequences GS , RQAILCWGELMTLA (SEQ ID NO : 33 ) , a HBV surface GSGSG (SEQ ID NO : 37 ) , or YNGK ( SEQ ID NO : 38 ) . In antigen epitope comprising the sequence FFLLTRILT some embodiments , one or more of the linkers comprise a IPQSLD (SEQ ID NO : 34 ) , a MT Influenza matrix epitope helix - forming peptide , such as A ( EAAAK )nA (SEQ ID NO : comprising the sequence YSGPLKAEIAQRLEDV (SEQ ID 39 ) , where n is 2 , 3 , 4 , or 5 . Alternatively , two immunogens NO : 35 ) , a PADRE epitope comprising the sequence may be synthesized as a multiple antigen peptide (MAP ) AKFVAAWTLKAAA (SEQ ID NO : 36 ) and a PADRE coupled through 4 or 8 lysine branch . epitope comprising the sequence aK -Cha - VAAWTLKAAa, [ 0311] Chemical cross - linking is an alternative to coupling ( SEQ ID NO : 40 ) where “ a ” is D alanine and Cha is regions A and B or the at least two immunogens . Linkers and L -cyclohexylalanine . In some embodiments , the MultiTEP cross - linkers are well -known and commercially available platform is encoded by a nucleic acid molecule . from e . g ., Aldrich Co . and ThermoScientific . [0304 ] Construction / Preparation of Immunogens 0312 ] Formulations and Delivery 10305 ] When the immunogens are to be delivered as a [0313 ] In certain embodiments , the immunogen or immu DNA composition , the composition will typically be an nogens is typically formulated with a pharmaceutically expression vector. In some embodiments , the vector is acceptable excipient. Excipients include normal saline , other capable of autonomous replication . In other embodiments, salts , buffers , carriers, buffers , stabilizers , binders , preser the vector is a viral vector or a bacterial vector . The vector vatives such as thimerosal, surfactants , etc . and the like . can alternatively be a plasmid , a phage , a cosmid , a mini Such materials are preferably non - toxic and minimally inter chromosome, or a virus . The sequence encoding an immu fere ( or not interfere at all ) with the efficacy of the immu nogen will be operatively linked to a promoter that is active nogen . The precise nature of the excipient or other material in host cells . There will typically also be a polyA signal can depend on the route of administration , e . g . oral, intra sequence , one or more introns, and optionally other control venous, cutaneous or subcutaneous , nasal, intramuscular , sequences , such as an enhancer . The promoter can be a intraperitoneal routes . In some embodiments , compositions constitutive promoter, an inducible promoter, or cell - type are formulated in nano particles and liposomes . specific promoter. Such promoters are well known in the art . 0314 ] In some embodiments , the composition further comprises an adjuvant . Suitable adjuvants include aluminum [ 0306 ] The nucleic acid constructs may also be used to salts , such as aluminum hydroxide , aluminum phosphate produce a polypeptide immunogen . In this case, the con and aluminum sulfates, saponin adjuvants ( e . g . , QS - 21 ) , 3 struct ( s ) are transfected or introduced into host cells in vitro De - O - acylated monophosphoryl lipid A (MPL ), Montanide , and protein is isolated . Protein may be purified by any of a CpG adjuvant , MF59 , Inulin -based adjuvant , nanoparticle variety of techniques , including precipitation , affinity chro and liposomal adjuvants . They may be formulated as oil in matography, and HPLC . Suitable host cells include bacteria , water emulsions, such as with squalene , or in combination yeast cells, insect cells , and vertebrate cells . The choice of with immune stimulants , such as MPL . Adjuvants can be a host cell depends at least in part on the backbone of the administered as a component of a therapeutic composition construct. Affinity tags , such as FLAG and hexa -His may be with an active agent or can be administered separately , added to the immunogen to facilitate isolation purification . before, concurrently with , or after administration of the [0307 ] Also disclosed herein is a method of making a immunogenic therapeutic agent . Other adjuvants include composition disclosed herein , comprising: obtaining chemokines (e . g. ,MDC ) and cytokines, such as interleukins sequence data representing the sequence of the composition ; (IL - 1 , IL - 2 , IL4, and IL - 12 ) , macrophage colony stimulating and synthesizing the composition . Resulting proteins may be factor (M -CSF ), tumor necrosis factor ( TNF) , etc . US 2017 /0368167 A9 Dec . 28 , 2017
[ 0315 ] The compositions herein can be administered by [0321 ] The actual amount administered , and rate and time any suitable delivery route, such as intradermal, mucosal course of administration , will depend on the nature and ( e . g . , intranasal, oral ) , intramuscular , subcutaneous, sublin severity of protein aggregation disease being treated . Pre gual , rectal , vaginal . The intramuscular ( i. m . ) route is one scription of treatment, e . g . , decisions on dosage is within the such suitable route for the composition . Suitable i. m . deliv responsibility of general practitioners and other medical ery devices include a needle and syringe, a needle - free doctors, and typically takes account of the disorder to be injection device ( for example Biojector, Bioject , Oreg . treated , the condition of the individual patient, the site of USA ), or a pen - injector device , such as those used in delivery , the method of administration and other factors self- injections at home to deliver insulin or epinephrine. known to practitioners . Examples of the techniques and Intradermal (i . d .) and subcutaneous (s . c .) delivery are other protocols mentioned above can be found in Remington ' s suitable routes. Suitable devices include a syringe and Pharmaceutical Sciences , 16th edition , Osol, A . (ed ) , 1980 . needle , syringe with a short needle , and jet injection devices , [0322 ] The compositions disclosed herein can be admin etc . The composition may be administered by a mucosal istered as sole treatment or provided in combination with route , e. g ., intranasally .Many intranasal delivery devices are other treatments (medical and non -medical ) , either simulta available and known in the art . Spray devices are one such neously or sequentially dependent upon the condition to be device . Oral administration can be as simple as providing a treated . solution for the subject to swallow . [0323 ] Also disclosed herein are, in certain embodiments , [ 0316 ). In certain embodiments , the composition may be methods of inducing an immune response in a subject in administered at a single site or at multiple sites . If at multiple need thereof, comprising administering a sufficient amount sites , the route of administration may be the same at each of a composition disclosed herein . The term " sufficient site , e . g . , injection in different muscles , or may be different, amount” is used herein to mean an amount sufficient to e . g ., injection in a muscle and intranasal spray. Furthermore , produce a desired effect, e . g ., an amount sufficient to modu it may be administered i . m . , s . c , i . d ., etc . at a single time late protein aggregation in a cell or raise an immune point or multiple time points . Generally if administered at response . The composition may comprise one or more of the multiple time points, the time between doses has been immunogens. Additives, such as adjuvants , are optional . determined to improve the immune response . Usually , the composition administered is a pharmaceutical 103171 Pharmaceutical compositions for oral administra composition comprising one ormore immunogens . In some tion can be in tablet, capsule , powder or liquid form . A tablet aspects , the subject has been diagnosed with Alzheimer ' s can include a solid carrier such as gelatin or an adjuvant. disease or one or more conditions associated with abnormal Liquid pharmaceutical compositions generally include a amyloid deposits , Tau deposits , or a - syn deposits or will be liquid carrier such as water, petroleum , animal or vegetable at risk of getting Alzheimer 's disease or one or more oils, mineral oil or synthetic oil. Physiological saline solu conditions associated with abnormal amyloid deposits , Tau tion , dextrose or other saccharide solution or glycols such as deposits , or a - syn deposits . An immune response is gener ethylene glycol, propylene glycol or polyethylene glycol can ated by administration of one of the compositions disclosed be included . herein . An immune response can be verified by assay of T [ 0318 ]. For intravenous, cutaneous or subcutaneous injec cell stimulation or production of antibodies to the B cell tion , or injection at the site of affliction , the active ingredient epitope (s ) . Immunoassays for antibody production are well will be in the form of a parenterally acceptable aqueous known and include ELISA , immunoprecipitation , dot blot, solution which is pyrogen - free and has suitable pH , isoto flow cytometry, immunostaining and the like . T cell stimu nicity and stability . Those of relevant skill in the art are well lation assays are also well -known and include proliferation able to prepare suitable solutions using , for example , iso assays, cytokine production assays, detection of activation tonic vehicles such as Sodium Chloride Injection , Ringer' s markers by flow cytometry and the like . Injection , Lactated Ringer ' s Injection . Preservatives, stabi 103241. Also disclosed herein are , in certain embodiments , lizers , buffers , antioxidants and /or other additives can be methods of treating or ameliorating a condition associated included , as required . with deposits of amyloid , tau , or a - syn , comprising admin [0319 ] Compositions comprising nucleic acid may be istering to a subject in need thereof an effective amount of delivered intramuscularly , intradermally by e .g ., electropo a composition disclosed herein . In general, amelioration can ration device , intradermally by e . g . , gene gun or biojector, be determined when the total amount of amyloid , Tau by patches or any other delivery system . protein , or a - syn deposits is decreased post - administration , [ 0320 ] Whether it is a polypeptide or nucleic acid that is relative to a control. Other biochemical tests or neuropa to be given to an individual , the amount administered is thology tests can be used , such as determination of ratio of preferably a “ therapeutically effective amount” or “ prophy phosphorylated and unphosphorylated tau to AB 42 peptide in lactically effective amount” . As used herein , “ therapeuti CSF , PET- scan with dyes ( e . g ., Pittsburgh compound B or cally effective amount ” refers to an amount that is effective 18F -FDDNP ) binding to B - Amyloid plaques in brain , less to ameliorate a symptom of a disease . A therapeutically aggregation of the proteins, prevention or slowing of the effective amount can be a “ prophylactically effective development of dystrophic neurites , and reduced astroglio amount” as prophylaxis is also therapy . The term " amelio sis . Other methods of determining amelioration include rating ” or “ ameliorate ” is used herein to refer to any thera cognitive function assays. Amelioration may be manifest as peutically beneficial result in the treatment of a disease state a delay of onset of cognitive dysfunction or memory impair or symptom of a disease state , such as lessening the severity ment, a significantly slower rate of decline of cognitive of disease or symptoms, slowing or halting disease progres functions and an improvement in the activities of daily sion , causing a remission , effecting a cure , delaying onset , or living. effecting fewer or less severe symptoms of a disease when [0325 ] Methods of treatment of AB , Tau , and a - syn related it occurs . diseases are also encompassed . B -Amyloid (AB ) , tau , and US 2017 /0368167 A9 Dec . 28 , 2017 34
a - synuclein ( a - syn ) are the primary components of amyloid the dIN - enriched suspension dissolved completely at 80 -85° plaques (AB - plaques) , neurofibrillary tangles (NFT ) , and C . The refractive index indicated a concentration of 48 Lewy bodies (LBS ) , respectively . Many neurodegenerative mg/ mL . The Deltin suspension used in these experiments disorders are characterized by the presence of one or more had a concentration of 5 % weight/ volume of water . of these lesions. For example , Alzheimer ' s disease (AD ) is [ 0333 ] Phosdeltin ( DIN / aluminum phosphate preparation characterized by the accumulation of AB plaques and neu ( PD ) ) : To produce Phosdeltin (PD ), a 5 % suspension of rofibrillary tangles . A subtype of AD also displays asyn Deltin as described above was first dissolved in water by bearing LBs. heating at 80 - 85° C . then mixed with a fine suspension of [0326 ] Said methods of the invention include administer aluminum phosphate gel ( Adju - PhosTM Aluminum Phos ing a therapeutically effective amount of a composition phate Gel Adjuvant 0 .44 % , BrenntagBiosector, Freder and / or compositions disclosed herein . ickssund , Denmark ) in a proportion to give an dIN : Adju [0327 ] In order that the nature of the present technology PhosTM weight/ weight ratio of between 2 and 200 . The may be more clearly understood , preferred forms thereof suspension was then crystallized at 5° C . , then converted to will now be described with reference to the following gIN ( 1 hr at 459) then to dIN ( 1 hr at 55° C . ) to yield non - limiting examples. The entire contents of all of the Phosdeltin hybrid particles , and washed and formulated as references ( including literature references, issued patents , appropriate. published patent applications, and co -pending patent appli [0334 ] Aldeltin (DIN /aluminum hydroxide preparation ): cations ) cited throughout this application are hereby To produce Aldeltin (AD ) , the same procedure was followed expressly incorporated by reference . as above for Phosdeltin except that a fine suspension of aluminum hydroxide gel ( AlhydrogelTM Aluminum Hydrox EXAMPLES ide Gel Adjuvant, Al ( calc ) 3 . 0 % , BrenntagBiosector, Fred erickssund , Denmark ) was used instead of aluminum phos [ 0328 ] The use of inulin particles in vaccines for neuro phate gel. In brief , a 5 % suspension of Deltinin water as degenerative diseases, either alone or in combination with described above was first dissolved by heating at 80 - 85° C . other immune activators , was evaluated . Recombinant hepa then mixed with a fine suspension of AlhydrogelTM in a titis B virus surface antigen (HBsAg ) and influenza virus proportion to give an dIN :AlhydrogelTM weight/ weight ratio antigen were used as exemplary model systems in some of between 2 and 200 . The suspension was then crystallized examples set forth below . Alzheimer ' s disease ( AD ) epitope at 5° C ., then converted to gIN ( 1 hr at 459) and then to dIN vaccines based on amyloid - ß , tau or combination of amy ( 1 hr at 55° C . ) to yield Aldeltin hybrid particles, and washed loid - ß and tau , as well as Parkinson disease (PD ) epitope and formulated as appropriate . vaccine based on a - syn , were also used as exemplary model [0335 ] Epsilin ( IN ) : Epsilin was prepared from dIN as systems in the examples set forth below . described in PCT /AU2010 /001221 titled " A novel epsilon Example 1 polymorphic form of inulin and compositions comprising same” . In brief , EI was prepared by heating a concentrated [0329 ] Preparation of Adjuvant Compositions suspension of greater than 50 mg/ mL of dIN at 60° C . for [0330 ] Inulin particle formulations referred to in the fol one hour. lowing examples were prepared as described below . [0336 ] Phosepsilin (PE ) : To produce Phosepsilin (PE ) , a [0331 ] Gammulin (Gamma inulin ; gIN ) , Algammulin 5 % suspension of elNin water as described above was first ( AG ) and Phosgammulin ( PG ): Gamma inulin ( gIN ) and dissolved by heating at 80 - 85° C . then mixed with a fine Algammulin were prepared as previously described in PCT/ suspension of aluminum phosphate gel ( Adju - PhosTM Alu AU86 / 00311 (WO 87 /02679 ) titled " Immunotherapeutic minum Phosphate Gel Adjuvant 0 . 44 % , BrenntagBiosector, treatment” , and PCT/ AU89 /00349 (WO 90 /01949 ) titled Frederickssund , Denmark ) in a proportion to give an eIN : “ Gamma inulin compositions ” , which are hereby expressly Adju - PhosTM weight/ weight ratio of between 2 and 200 . The incorporated by reference . To produce Phosgammulin (PG ) , suspension was then crystallized at 5° C . , then converted to a 5 % suspension of giN in water was first dissolved by gIN ( 1 hr at 459 ) then to the dIN form ( 1 hr at 55° C .) then heating at 80 - 85° C . then mixed with a fine suspension of to the eIN form to yield Phosepsilin hybrid particles, and aluminum phosphate gel (Adju -PhosTM Aluminum Phos washed and formulated as appropriate . Alepsilin (AE ) was phate Gel Adjuvant 0 .44 % , BrenntagBiosector, Freder similarly made by substituting AlhydrogelTM instead of ickssund , Denmark ) in a proportion to give an inulin :Adju Adju -PhosTM in the above process for making Phosepsilin . PhosTM weight/ weight ratio of between 2 and 200 . The [0337 ] PGmix , PDmix and PEmix : Phosdeltin (dIN / alu suspension was then crystallized at 5° C . , then converted to minum phosphate ) and dIN formulations , as described the gamma form ( 1 hour at ) 45° to yield Phosgammulin above, were admixed to form a mixed suspension of par hybrid particles , and washed and formulated as appropriate . ticles some containing pure inulin and others containing [ 0332] Deltin ( Delta inulin ; dIN ) : Deltin ( DIN ) was pre inulin with aluminum phosphate ( PDmix ) . For the experi pared from gIN as previously described in WO 2006 / ments described herein , the PDmix Phosdeltin :Deltin com 024100 , which is hereby expressly incorporated by refer bination adjuvant was prepared in various ratios ranging ence . Briefly , a standard formulation of gIN in water (200 from 1 : 1 to 1 : 36 weight for weight of inulin content of mL at 50 mg /mL ) was incubated for 1 hour in a water bath inulin - alum amalgam particles and inulin particles , respec at 55° C ., which was then raised to 60° C . for 30 min . The tively , hereinafter referred to as PDmix1 : 1 to PDmix1 : 36 ) particles were then centrifuged , resuspended in water at 55° This enabled the amount of aluminum phosphate containing VC . , re - incubated at 55° C . and washed again in the same particles to be varied relative to the number of non - alumi manner , before being finally resuspended in 50 mL cold num salt containing din particles . PGmix and PEmix were water. This treatment is sufficient to remove much of the prepared in the same manner. The ratio of Phosdeltin to inulin present in the alpha and gamma forms. A sample of Deltin particles is expressed as x : y PD : D ). This means that