NZ620865B2 - Parenteral norovirus vaccine formulations - Google Patents

Abstract

Disclosed is the use of a vaccine composition comprising genogroup I or genogroup II Norovirus virus-like particles (VLPs) in the manufacture of a medicament for eliciting protective immunity against Norovirus in a human, wherein said genogroup I or genogroup II Norovirus VLPs comprise a capsid protein derived from a genogroup I or genogroup II viral strain, and wherein the medicament is formulated for: (a) administration of no more than a single dose of the vaccine composition; (b) administration parenterally; and (c) inducing at least a three-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the medicament. ein derived from a genogroup I or genogroup II viral strain, and wherein the medicament is formulated for: (a) administration of no more than a single dose of the vaccine composition; (b) administration parenterally; and (c) inducing at least a three-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the medicament.

Description

PAREN'l‘ERAL NOROVERIJS VACCENE FGRM NS CROSS VCh TO RELATED APPLICATIONS {0001} This application claims priority to US. Provisional Patent Application 6l/506A47, ti ed on July ll, 20l l, the entire contents of which are incorporated herein by reference.

FlELD OF lHE lNVEN'l‘lON {0802} The invention is in the field of vaccines, particularly vaccines for Noroviruses. In addition, the invention relates to methods of preparing vaccine compositions and methods of inducing and evaluating protective immune responses against Norovirus in humans, BACKGROUND OF THE INVENTION {0003} ruses are non—cultivata‘ole human Caliciviruses that have emerged as the single most important cause of epidemic outbreaks ofnon‘oacterial gastroenteritis (Glass 6! all“ 2000; Hardy er a! l999). The. clinical significance ot‘Noroviruses was appreciated prior to the development of sensitive molecular diagnostic assays“ The cloning ofthe prototype genogroup l Norwalk virus (NV) genome and the production of lilte particles (VLPS) from a recombinant Baculovirus expression system led, to the development of assays that revealed widespread Norovirus infections (Jiang at all l990; l992)r {0004} Noroviruses are singlewstranded, positive sense RNA viruses that contain a non- segmented RNA genome. The viral genome encodes three open reading frames, of which the latter two specify the tion of the mai or capsid protein and a minor structural protein, respectively (Glass at all. 2000’). When expressed at high levels in eulraryotic expression s, the capsid protein of NV, and certain other Noroviruses, self—assembles into VLl’s that structurally rnirnic native rus vir‘ions. When viewed by transmission electron microscopy, the VLl’s are logically indistinguishable from infectious virions ed from human stool samples. {0005} lmnrune responses to Noroviiuses a e complex, and the correlates of protection are just now being elucidated. Human volunteer studies performed with native virus demonstrated that mucosallywderived memory immune responses provided shortnterm tion from infection and suggested that vaccine—mediated protection is "easihle (Lindesmith er a]. 2003; Parrino at al. 1977; Wyatt ct aZ., 1974). {0006} Although Norovirus cannot be cultivated in vitro, due to the bility of VLl’s and their ability to be produced in large quantities, considerable progress has een made in defining the antigenic and structural topography of the Norovirus capsid. VLPs preserve the authentic confirmation of the viral capsid protein while lacking the infectious genetic material.

Consequently, VLPs mimic the functional interactions of the virus with cellular receptors, the Tehv eliciting an appropriate host immune response while lacking the ability to reproduce or cause ion. In conjunction with the Nil—1, Baylor College of Medicine studied the humoral, mucosal and cellular immune responses to NV VLPs in human volunteers in an academic, investigator—sponsored Phase l clinical trial. Orally administered VLPs were safe and immunogenic in healthy adults {Ball :22 at. 1999; Tacket e: at. 2003). But, multiple doses ofa relatively high amount of‘v’LPs were ed to observe an immune se, At other academic centers, preclinical experiments in animal models have demonstrated enhancement of immune responses to VlPs when administered intranasally with bacterial exotoxin adjuvants ero at at. 2001; Nicollieralamot er a1. 2004; Periwal c: all. 2003; Souza et a]. (2007) Vaccine, Vol. :8448-59). However, protective immunity against Norovirus in humans remains elusive because the tors of a protective immune response in humans have still not been c1ear1y fied (Herbst-Kralovetz er a3. (2010) Expert Rev. es 9(3), 299-30HmII SUMMARY OF THE 1NVENTiON {0007} The present invention is based, in part, on the discoveryr that a sing1e dose of a Noroviius vaccine elicits a, rapid, robust protective immune response against rus in humans when administered parenterally. Accordingly, the present in rention provides a method of e1iciting protective immunity against Norovirus in a human comprising stering parenterally to the human no more than a, single dose of a vaccine composition, said composition comprising genogroup 1 and/or genogroup 11 Norovims VLl’s, wherein said composition induces at least a three~fo1d increase in Norovirusnspecific serum antibody titer as compared to the titer in the human prior to administration of the composition. in n embodiments, the increase in Norovirus—specitic dy titer is d within seven days of administration of the single dose of the composition. In some embodiments, the vaccine composition is administered to the human via an intravenous, subcutaenous, intradermai, or intramuscuiar route of administration.

In one embodiment, the vaccine composition is administered to the human by an intramuscular route of administration. {33%} The singie dose vaccine compositions can comprise. doses of about 5 pg to about ISO pg of genogroup I rus VLPs, gen group II N oi‘ovirus VLI’s, or both. In embodiments in which the single dose vaccine compositions comprise both genogroup I and genogroup II Norovims VLI’s, the dose of each VLF can be the same or different, In one embodiment, the composition comprises no more than 50 pg of genogroup I Norovirus VLPs. In another embodiment, the composition comprises no more than 25 pg of oup l Noi‘ovirus VLI’s. In yet another embodiment? the composition comprises no more than ISG pg of genogroup II Norovirus VLPs. In stili another ment, the ition comprises no more than 50 pg of roup II Norovirus VIPs, The Norovirus VLPs can he monovalent VLPs or multivalent VLl’s.

{MIME In some aspects of the invention, genogi‘oup I Norovirus VLI’s in the e compositions comprise a capsid protein derived from a genogi‘oup I vii‘aI strain. in one embodiment, the genogi‘oup I‘Norovirus VLI’s comprise a capsid protein from a genogroup I, genotype I N us. In another embodiment, the genigroup l Norovirus VLI’s comprise a capsid protein from Norwalh virus. In other aspects of the invention, genogroup II N orovirus \"Ll’s in the vaccine compositions comprise a capsid protein derived from a genogroup II viral strain. In some e b ‘idiments, the genigroup II N orovirus VLI’s comprise a capsid protein from a genogroup II, genotype 4 Norovirus. In certain embodiments, the genogroup II Norovims VLI’s are VLPs generated from expression of a consensus sequence of genogroup II Norovims.

In one particular embodiment, the genogroup II Norovirus VLPs se a capsid, protein having a sequence of SEQ ID NO: I.

{MIMI} In ceitain embodiments the vaccine composition further comprises at ieast one adjuvant.

'I'he adiuvant is preferably not a bacterial exotoxin adj u vant. In one ment, the adjuvant is a toli~like or agonist, such as monophosphoryi lipid A (MIDI)? flagellin, or CpG. In another embodiment, the adj u vant is aluminum ide (tag alum). In certain embodiments, the vaccine ition comprises two adjuvants, such as MPL and aluminum hydroxide, In some ments, the vaccine composition may further comprise a buffer, such as L~histidine, irnidazoim succinic acid, tris, and citric acid. The e composition can be formulated as a dry powder or;-.liquid. In one embodiment, the VIaccine ition is formulated as a liquid 1:eg. aqueous formulation).

BRIEF DESCRTFTTON OF THF. GS {@8141} Figure ‘1, Results of pan—ELISA assays measuring combined serum lgG, lgA, anLl TgM levels fiom human eersimmunized intrainusLularly with o (saline) L11 a VIacLine formulation Lonta1n111115, 15, 50, 01 1540 ug each of a genogroup 1.41 Norovirus VLF and a4 genogroup 11.4 ovirus VLF. The geometric mean titer for antinGlJ (A) and antinGllflr (B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and;" and 28 Llavs after the second immunization. Volunteers receiVed immunizations on studv days 0 and 28. {191912} Figure 2. s of pan—ELISA assays measuring ed serum lgG, lgA, anLl lgM levels fiom human volunteersimmunized intrainusLularly with placebo (saline) L11 a vacLine formulation Lontain11111Qs, 15, 50, L11 150 11g each of a genogroup 1.41 Norovirus VLF and a4 genogroup 11.4 Norovirus VLF. The geometric mean fold rise for anti—Gl.l (A) and anti—(3111.4 (3} antibodies is shown for each of the dosage levels at 7 and 21 days after the first zation and? and 28 days after the second immunization Volunteers received immunizations on study days 0 and 28. {191913} Figure 3. Results of pan—ELISA assays measuring combinedsserum lgG, lgA, anLl lgM levels from human volunteers immunized, intramuscularly with placebo (saline) or a e formulation Lontain11111Qs, 15, 50, L11 150 11g each of a genogroup 1.41 Norovirus VLF and a4 genogroup 11.4 Norovirus VLP. The percent seroresponse rates (1.9. four—fold increase in antibody titer compared to ore-immunization titers} for anti-Gll (A) and anti—Gil.4 (1%) antibodies are shown for each of the dosage levels at 7 and 2l days after the first immunization and 7 and 28 days after the second immunization. Volunteers ed immunizations on study days 0 and 28. {1111111} Figure 4. s of ELISA assays measuring serum lgA from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 ug each of a genogroup 1.1 Norovirus VLF and a genogroup 11.4 Norovirus VLF. The geometric mean titer for anti-Gl.l (A) and anti-(311.4 (3) antibodies is shown for each of the dosage levels at r-v I I and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28. {111115} Figure 5. Results of ELISA assays measuring serum lgA from human volunteers immunized intramuscularly with placebo (saline) or a vaccine ation containing 5, 15, or 50 ug each of a genogroup 1,1 Norovirus VLF and a genogroup 11.4 Noroyirus VLF. The geometric mean fold rise for 11.1 (A) and 111.4 (8) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. eers ed zations on study days 0 and 28. {111116} Figure 6. Results of ELlSA assays measuring serum IgA from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5., 15, or 50 ug each of a genogroup 1.1 Norovirus VL1’ and a genogroup 11.4 Norovirus VLP. The percent ser aresponse rates (in. fourufold increase in antibody titer compared to ore-immunization titers) for anti-(31.1 (A) and anti-(111.4 (1%) antibodies are shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 2.8 days after the second zation.

Volunteers received zations on study days (I and 28. {1111171 Figure 7° Results of ELlSA assays measuring serum IgG from human volunteers immunized intramuscularly with o (saline) or a vaccine formulation containing 5, 15, or 50 ug each of a genogroup 1.1 Norovirus VL1) and a genogroup 11.4 Norovirus VLP. The geometric mean titer for antisGIJ (A) and anti 4 (E) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and '28 days alter the second immunization. Volunteers received immunizations on study days 1) and '28. 3111113} Figure 8. Results of ELISA assays measuring serum 1gG from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, air 50 ug each of a oup 1.1 Norovirus VLP and a oup 11.4 Norovirus VLP. The geometric mean fold rise for anti —GI.1 (A) and anti-{111.4 (3) antibodies is shown for each of the dosage levels at 7 and '21 days alter the first immunization and 7 and 28' days after the second in'imunization. Volunteers received immunizations on study days 0 and 28' {1111191 Figure 9. Results of ELISA assays measuring serum IgG from human volunteers immunized intarnuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 511 ug each of a genogroup 1.1 Norovirus VLE? and a genogroun 11.4 Norovirus VLF. The percent seroresponse rates (lie, lounfold increase in dy titer compared to pre—immunization WO 09849 titers) for antinl.l (A) and ll.4 (Bi) antibodies are shown for each of the dosage levels at r-v I and El days after the first immunization and 7 and 28 days after the second immunization.

Volunteers received immunizations on study days (i and 28. {8920} Figure ll}. Results of pan—ELISA assays measuring combined serum lgG, lgA, and lglvl levels from human volunteers immunized with either a Norovirus intranasal, monovalent vaccine as described in El Kamary at a]. (Elli (l) l lnl‘ect Dis, Vol. 2020 l): l649~l658 (lerOlrlOS groups) or a Norovirus uscular, bivalent vaccine as descrihed in Example l (l..rV()3~lOI-l groups) at the indicated time points. Human volunteers received either placebo or two doses of either the intramuscular or intranasal vaccine tormulation. The intramuscular, bivalent Norovirus vaccine contained 5 ug each of a genogroup lrl Norovirus VLF and a genogroup ll.4 Norovirus VLF. ’l‘he intranasal, alent e contained lOO ug of a genogroup l.l Norovims. Volunteers receiving the intranasal e or placebo were challenged with live Norovirus following the second immunization. {0(3le Figure ll, FACS analysis of peripheral blood mononuclear cells obtained from human volunteers on Day 0 prior to zation With either a Sug dose of rus intramuscular, bivalent vaccine {A} or placebo (B) and Day 7 postuimmunization. (3319+ PBMC are mucosally targeted as ced of expression of alpha 4/beta7 homing receptor and chenrokine CCRl 0 receptor.

DETAIL. D DESCRlPl‘lON OE lHE lNVEN'l‘lON {@922} "l‘he present invention relates to methods of eliciting a protective immunity to Norovims in "ections in a subject. in ular, the t invention provides methods of eliciting a protective immunity t Norovirus in a human by parenterally administering to the human no more than a single dose of a vaccine comprising Norovirus VLPs and optionally at least one adjuvant, wherein the e s tion from or amelioration of at least one symptom of Noro virus infection. The inventors have surprisingly discovered that intramuscular administration of no more than a single dose of a vaccine composition comprising Norovirus VLPs to humans induces a rapid (17.6. Within 7 days ofimmunization) serum serocon version (it). at least a three—fold increase in antigen—specific serum antihody titers ahove pre—vaccination levels) that is indicative ofa protective immune response against Norovirus infection and illness.

The immune responses induced by this single dose vaccine composition plateau at high antibody titers similar to that observed with natural infection by administration of live virus in human challenge studies. interestingly, a boost dose of the vaccine is not required, as the immune response is not increased upon further administration of an additional vaecine dose. {0023} The invention provides a vaccine composition comprising one or more Norovirus antigens, By “Norovirus,” “Norovirus (NOR),” “norovirus,” and grammatical equivalents herein, are meant members of the genus Norovirus of the family Caliciviridae. In some embodiments, a Norovirus can include a group of related, positive—sense single~stranded RNA, nonenveloped s that can be infectious to human or non~human mammalian species, in some embodiments, a Norovirus can cause acute gastroenteritis in humans Noroviruses also can be referred to as small round, structured viruses (SRSVs) having a defined surface structure or ragged edge when vicwcd, lav on microscopy, ll} included within the Noroviruses are at least five genogroups (Cal, Gil, Gill, GIV, and (3V). Cal, (HI, and GlV Noroviruses are infectious in humans, while Gill ruses primarily infect bovine species. (3V has recently been isolated from mice ('Zheng> er a5. (2006) Virology, Vol 346: 3l 23-323), entative ofGlll are the Jena and Newbury strains, While the Alphatron, Fort Lauderdale, and Saint Cloud strains are representative of GIV, The GI and Gil groups may he further segregated into genetic clusters or pes based on genetic classification (Ando e! a]. (2000) J. Infectious Diseases, Vol, ppZ):S336-S348; l :22 a]. (2005) J. Clin. l‘dlierobiol, Vol. 43(3): 092). As used herein, the term genetic. clusters is used interchangeably with the term genotypes. Within genogroup l, there are 8 (iii clusters known to date (with prototype virus strain name): Gill lk (N \/’-USA93)); G12 (Southhanipton BR93)); (31.3 (Desert Shield (DSV—USAQBD; GL4 (Cruise Ship virus/Chiba (ChibaanNOGB; G15 ’lvlusgrovc (h’lusgroanBROOD; GL6 (Hesse (Hesse— DEUQS»; GL7 (Wnehest—GBRGO); and Gill (BoxernUSAOZ). Within genogroup ll, there are l9 Gll clusters known to date (with ype virus strain name): Gll.l (Hawaii (HawaiinUSA94fi; (3112 (Snow h'lountain/Melksham (Mshani—GBR95D; Gll.3 (Toronto ('l‘orontonCANElBD; (311.4 (Bristol/Lordsdale ol—63393)); Gllfs 290/Hillingdon (Hilingd—GBROODL Gilli (269/Seacroft (SeacrofiGBROQD; Gil? (NB/Leeds (LeedsnGBR00)); Gilli (53 /Amsterdam (Anistdam—NLDQQD; Gll.9 (378 (VABeach~USA0l)), Gll.l0 (Erfurt—DEUOl); Gll.ll (SW9l801PN0l); (211,12 (\IVortl,ey~Gl3R00); Gill 3 (Faytvil—USA02); Gll.l4 (M’LU814,03); GlllS (i’23—USA02); Gll.16 (Tiffin—USAOS); (311.17 (CSELUSAOE; Glll8 2003mm and (311.19 (QW l 3/US). {9925} By "Norovirns" also herein is meant recombinant Norovims Virusnlike particles (rNOR VLPs). in some embodiments, recombinant expression of at least the Norovirus capsid protein encoded by ORFZ in cells? e.g., from a baeulovirus vector in St}? cells, can result in spontaneous seltlassernbly of the capsid protein into VLPs. In some embodiments, recombinant expression of at least the Norovirus proteins encoded by ORFl and ORFE in cells, erg from a baculovirus vector in Sf9 cells, can result in neous selflessembly ot‘tne capsid protein into VLPS.

VLPs are structurally similar to ruses but lack the viral RNA genome and therefore are not infectious Accordingly, "Noi‘oviriis" includes Virions that can be infectious or non— infectious particles“, which include ive les. lilith’i} Nowlimiting examples ot‘Noroviruses include Norovirtis oup l strain lituNoV/West Chester/200} .I’USA, GenBank Accession No. AY502016; Chiba VlI‘US (CHV, GenBank ABtMZSOS); Norovirns genogroup 2 strain V/Braddock Heights/l999/USA, GenBank Accession No. AYSO2ill 5; Norovirus genogroup 2 strain ltln/NoV/Fayette/l 999/11 SA, GenBank Accession No. l 4; Norovirus genogroup 2 strain Hit/NoV/‘Fairtielrl/l999/”USA, GenBanl: Accession No. AY502tl13; Norovirns genogroup 2 strain Hu/NoV/Sandusky/l999/US/‘i GenBanlc Accession No. AYSOZOlZ; Norovirns genogronp '2 strain l-lu/NoV/Canton/l999/USA, GenBank Accession No. AYSGZOI l; Norovinis oup 2 strain l-lu/NoVl’l‘iffin/1999/USA, GenBank Accession No. AYSOZOl O; Norovirus onp 2 strain l-lur’NoV/CS-El x’2002/USA, GenBanlc Accession No. AYSOZOO; Norovims genogronp 1 strain l-lu/NoV/Wisconsin/2()0l/USA, GenBank Accession No. AYSOZOOS; Norovirus genogroup l strain Hii,I’NoV/’CS—841/200l/USA, GenBank Accession No. AY502007; Norovirus genogroup 2 strain Hw’NoV/Hiram/Zmill/USA, GenBank Accession No. AY502006; Norovinis genogroup 2 strain Hit/NoV/Tontoga,ny./1999/USA GenBank Accession No. AYSOZOOS; Norwall; ViI‘US, complete genome GenBank Accession No. o.w001959; Norovirus Hu/Gl/Otofiike/l979/3? genomic RNA, complete , GenBank Accession No. ABIS7514; Norovirns Hu/Holrlraido/l33/2003/31). GenBank ion No. A3212306; Norovirus Sydney 2212, GenBank Accession No. AY588 l 32; Norwalk Virus strain SNZOQQIA, GenBank Accession No, ABlElOI-‘l-57; Lordstlale Virus te. genome, GenBa/nk Accession No. ; Nonvalk—lilre virus genomic RNA cause, GenBank Accession No. ABO45603; Norwalk Virus strain Vietnam 0316, complete genome, k Accession No. 71; Norovii‘us Hu/Gll4/2004,"th GenBank Accession No. AY883096; Norovims Hn/Gll/Hokushin/U3/3?, enBank Accession No. A3195227; Norovirus HWGll/Kamo/OS/JP, Ganank Accession No.

AB 1 9522 S: N orovirus Hu/Gll/Sinsiro/97/JR GenBanl; Accession No. AB 1 95226; N Ol‘OVll‘US Hu/Gll/ina/OE/JR GenBank Accession No. AR] 95225; Norm/nus Hu/Nl..V/GII/Neustrelit2260/2000/DE, GenBa/nk Accession No. AY772730; rus Di‘esdenl 74/pUS~Noi‘H/l 997K313, GenBa/nk ion No. AY74181 i ; Norovirus Hu/Nlfi’i/Oxford/32$16/2002/UK, Ganank Accession No. AY58’7989; Noxiovii'us Hie/N11 UOxfoi‘d/B487/2002/UK, GenBa/nk Accession No. AYS'87987; Norovirus Hu/Nlfi’i/Wiiney/‘BYSZ/ZGOB/UKa, GenBank Accession No. AY588030; Norovinis HuNLV/Banbcry/B9SZS/ZOOS/UK, GenBank Accession No“ AY588G29; Noi‘ovirus lEl'u/NLV/Cl'iippingNofiory’2003/UK, GenBank ion No. AY588028; Norovims Hm YLVC/DidcoU8982/2(NB/UK, GenBanlc Accession No. AY58’8027; Norovirus lElu/NLX/Khford/B885/20021in GenBank Accession No. AY588026; Norovims Hm, YL‘Wflxfoiii/B6i‘2113(./"',r‘;()().“5MK, Gananlx: ion No. AYSSSOZS; Noni-Virus lElu/NLX/Khford/B685/2003/UK, GenBank Accession No. AY588024; Norovims Hm, YLV/Qxfoiii/BSS23/2003/UK, GEHBEEUK Accessimi No. AY588023; Norovirns LX/Khford/3682/2003/UK, GenBank Accession No. AY588022; Norovims i-lLL/NLV/OXfOid/B6Sol/ZOOB/UK, GenBank Accession No. AYSSSOZE; Norwallc—like Virus isolate Bo/"l‘hirskl O/UU/UK, GenBank Accession No. 68; k—lilce Vinis isolate Bo/Penrithfv5,I’()()/UK, k ion No. AY126476; Nom’alk—like Virus isolate Bo/Aberysmych4/00/UK, GenBank Accession No. AY126475; Noxwalk-like Vims e Bo/Dumfries/94/UK, k Accession No. AY126474; Norovirus NLVHF}:G36/2003/1raq, enBank Accession No. AY6’75555; Noro virus NLV/ll: l 998.1"2003/lrag GenBank Accession No. AY675554; Norovirus NLV/BUDS./2002/USA., GenBank Accession No. AY660568; Norovirns NLV/Paris lsland/ZOOB/USA, GenBank Accession No. AY652979; Snow Mountain Virus, complete genome, GenBank Accession No“ AY134748; Norwalk—like Virus NLV/Fon Lauderda le/5 60/19981’US, k Accession No. AF414426; Hu/NoroV'ii‘us/hii‘oshiina/l999,1’J'P(9912n02f')c GenBanl; Accession No. A80443 66; N om’aiknlike vims strain llN/iSU—MWQ GenBank Accession No. AY27l-‘i-8ZO; Norwalk—like Virus strain B~ l SVD, GenBank Accession No“ AY274819; rus genogroup 2 strain HLi/NoV/Farming'ton Hii1s/2002/USA, GenBank Accession No. 23; Norovirus genogroup 2 strain Hu/NoV/CS—G4/2002/USA, Gannnk Accession No. AY'S:02022; Noi‘ovii‘us genogroup 2 strain Hw’NoV/CS—G2/2002/USA, GenBank Accession No. E 1; Norovirus genogi‘onp 2 strain Hu/NoV/CS—G12002/USA, GenBank Accession No. AY502020; Norovii‘us genogroup 2 strain Fin/NoV/Anchorage/2002/118 A, GenBank Accession No. AY502019; ms genogi‘oup 2 strain Hu/NoV/CS—Di /CAN, k Accession No. AYSOEOl 8; Norovirns genogronp 2 strain HuxNoV/Gei‘inanton/ZOOE”USA, k Accession No A ’59201’7; Human calici‘vims GH/Langeni061/2992/013, complete genome, k Accession No, AY485642; Murine noiovirus 1 otein, GenBa/nk Accession No. AY228235; Norwa1k Virus, Ganank Accession No. ,A13067536; Human caiicivinis Nich/‘R’RX7976/‘1 999, GenBank Accession No. 13117542 (199; Human caiicivirus NLV,1/01361‘11311‘5611 455/91/013, GenBank ion No; 440; Human caiicivirus NLV/Hei‘zberg 3.85/01/DE, GenBank Accession No. AF539439; Human calicivirns NLV/Boxen’ZGGi .I’US GenBank Accession No. AF538679; Norwailc—Eike Virus genomic RNA, complete genome, GenBank Accession No. A8081723; Norwalk-iike Virus c RNA, complete genome, isoia‘iezSaitama U201, GenBank Accession No. A3039782; Norwaiklike Virus c RNA, conipiete genome, isoiatefiaitama L118, GenBank Accession No. ABG39781; Nom'a1ksiike Virus genOi'nic RNA, compiete genome, isolatezSaitama U25, GenBank Accession No. ABU39780; k Virus strain:U25611, GenBank Accession No.

A13067543; NciWaEk Virus strain:U2()1 (311, GenBank Accession No. A8067542; Norwalk—like Viruses strain 416/97003156/199i'S/LA, GenBank Accession No. AF()80559; NOiwnik-er Viruses strain 408/97003012/1996/191, GenBank Accession No. AFOSOSSS; NDFWZiik-iikfi Virus NLV/Bum’ash Landing/331/1995/US, Geannk Accession No. AF414425; Nonva1k-1ike Virus NL‘yfx’i‘s/iiaini Beac11/326/1995/US, Geannk Accession No, AF414424; Norwa1k—1ikc Virus NLV/White Rivei‘x’290/1 994/1478, GenBank Accession No. AF414423; knhkc Virus NLV/Ncw Orleans/'306/1994/US, Ganank Accession No, AF414422; Noiwalk—iike Vinis NLV/Port Canaverab’SOl/l 994/US, Ganank Accession No. AF414421; Noiwaikw1ike Vims NLV,I"Honoiu1u/314/1994/US, GenBank ion No. AF414420; Neiwa1kn1i1§e Virus NLV/Ricnmond/283/1994/US, Ganank Accession No, AF414419; Nonva1k—1ikc Vims REV/"Westovein/302M994/138, GenBank Accession No. AF414418; Norwaiknhkc Virus NLV/UKS—l 7/1 2799/1 992K313, Ganank Accession No. A174 14417; Noiwaik—er Virus Elev/’R/Iiami/S 1 f1 986/1}S, Ganank ion No. A174 144 16; Snow in strain, GenBank Accession No. U70059; Desert Shield Virus DSV395, GenBank Accession No. U04469; Norwalh Virus, complete genome, GenBank Accession No. AF093797; Hawaii calicivirus, enBank Accession No. U076l l; Southampton Virus. GenBank Accession No. L07418; Noiwalls Virus (SRSXLKY—89/89/3"). GenBank Accession No. L23828; Noiwallt virus (SRSV— SMA/Tl’ti/US), GenBank Accession No. 1423831; Cainberweli virus. GenEank Accession No.

U46500; Human caliciyirus strain Melksharn. GenBanh ion No. X8l 879; Human calicivirus strain h/lX, GenBank Accession No. [122498; Minireovirus . GenBank Accession No. U02030; and Noiwalk—lihe virus Nl.\I’//'Gwynedd/’Z73/lWily/US GenBank Accession No. AF414409; sequences of all of which (as entered by the date of tiling of this application) are herein incorporated lay reference. Additional Norovirus sequences are sed in the following patent. publications: . WO 9280. JP2002020399.

US2003l2958’8, US. Pat. No. 6,572,862, Wt.) 1994/05700, and WO 05/032457, all ofwhich are herein incorporated by reference in their entireties. See also Green :22 all. (2000) J. Infect. Dis, Vol. 181(Suppl. 2):S322—330; Wang et a1. (1994) .l. Virol, Vol. 68:5982~5990; Chen et ai. (2004) J. Virol., Vol. 78: 6469-6479; Chaltravarty at a]. (2005) J. Virol. Vol. 79: 554668; lElansrnan et a}. (2006) J. Gen. Virol, Vol. 919; 81115 er a1. (2006) .3. Clin. Where, Vol. 32'7~333; ga, of ai. (2007) J. Virol, Vol. E<Il (l 8):9932~9941. and Fankhauscr et a]. (1998) J. . Dis, Vol. l78: l 5'7l ~l 578; for sequence isons and a discussion etic diversity and phylogenetic analysis of Noroyiruses. The nucleic acid and corresponding amino acid sequences of each are all incorporated by re ‘erence in their entirety. in some embodiments, a cryptogram can be used for identification purposes and is organized: host species from which the Virus was isolated/genus abbreviation/species ahhreviation/Strain ear of occurrence/country of origin. (Green er al, Human Caliciviruses, in Fields Virology Vol. l 841" 874 (Knipe and Howley, editors—innchief, 4th ed, Lippincott Williams & Wilkins 2001)).

Genogroup ll, pe 4 ((311.4) Viral strains (tag. Houston, Minewa (also known as Den Haag). and Laurens (also known as Yerseke) strains) are preferred in some embodiments. As new s are identified and their genetic sequences are made availahlet one skilled in the art would he able to employ VLPs using these contemporary strains in the compositions and methods of the present invention using ordinary skill. Thus, the present invention contemplates Vl.Ps made. from such s as le antigens for use in the compositions and methods described herein. {@927} The Norovirus antigen may be in the form of peptides, proteins, or ViI'LlS'lilifi particles . ln a preferred ment, the Norovirus antigen comprises VLPS. As used herein, “virusnlike particlets) or VLPs” refer to a virusnlike particler’s), fragiiient(s), aggregates, or pOl‘tiOl’l(S) thereof ed from the capsid protein coding sequence of Noroyirus and comprising antigenic characteristict’s) similar to those ofint‘ectious Norovirus les. ms antigens may also be in the form of capsirl monomers, capsid ers, protein or e fragments ot‘VLPs, or aggregates or mixtures thereof. The Norovirus antigenic proteins or peptides may also he in a denatured form. produced using methods known in the art. {@328} The VLPs of the present invention can he formed from eithe ‘ the full length Norovirus capsid protein such as VPl and/or VPZ proteins or certain VPl or VPE tives using standard methods in the art. Alternatively, the capsid protein used to form the VLP is a truncated capsid protein. in some embodiments, for example, at least one of the VLPS comprises a truncated VPl protein. in other ments, all the VLPs comprise truncated VP1 proteins.

The truncation may he an N“ or (Lterminal truncation Truncated capsid proteins are suitably onal capsid protein derivatives. Functional capsid protein derivatives are capable of raising an immune response (if necessary, when ly adiuvanted) in the same way as the immune response is raised by a VLF consisting of the full length capsid protein.

} VLPs may contain niajor VPl proteins and/or minor VPZ proteins. in some embodiments, each VLF contains VPl and/or VP?) protein from only one Norovirus genogroup giving rise to a monovalent VLF. As used herein, the term “monovalent” means the antigenic proteins are derived from a single Norovirus oup. For example, the VLPS contain VPl and/or VPZ from a virus strain of genogroup ,5 (tag Vl’l and VPZ from Nonvalk virus).

Preferably the VLF is comprised of inantly Vl’l proteins. in one embodiment of the in rention, the antigen is a mixture of monovalent VLPs wherein the composition includes VLl’s comprised of "V’Pl and VPZ from a single Noroviius genogroup mixed with VLPs comprised of VPl and VPZ from a different Norovirus genogroup (cg. Norwalk virus and n virus) taken from multiple Viral strains. Purely hy way of example the composition can contain monovalent VLPs from one or more strains of Norovirus oup ltogether with monovalent VLPs from one or more strains ofNorovirus genogroup ll. Strains may he selected based on their predominance of circulation at a given time. in certain embodiments“, the Norovirus VLF mixture is composed of Gil and Gil-4 viral strains More preferably, the Norovirus VLF WO 09849 mixture is composed of the strains ofNorwaik and a sus capsid sequence derived from genogroup H iuses. Consensus capsid sequences derived from circulating 1‘».orovirus sequences and VLPs made with such sequences are described in , which is herein orated by reference in its entirety. For instance, in one embodiment, a consensus capsid sequence derived from genogroup ii, genotype 4 (011.4) viral strains ses a sequence ot‘SEQ 1D NO: 1. Thus, in some embodiments, the vaccine composition comprises a mixture ot‘monovaient VLPs, wherein one monovaient VLF comprises a capsid protein from a genogroup irus (sag Norwaik) and the other monovaient VLF comprises a consensus capsid protein comprising a sequence of SEQ TD NO: 1 , M K M ‘3 C D A N P S T: C— S m A F TJ V P 3' t" V N N F V M A L J'— P V V G A A T A A P V A G 2 ,. N V T. F! T W T 9x 17 N F V 2 A T‘ G G E F T V :3 P R N A P C E‘ T L W S A P L C P D L =_ t P ‘1 L S H L A E id Y N C— Y A ‘3 C— F F; V Q V T T V 3 t A G N A F T A G T T F A A V P N F P T T: G T_ S P 3 Q V T M F T‘ H T T V D V R Q T. E P V L T f? L P D ‘ r R N i E f H \j N 'Q S N :1 P T ._ K L l A E’i L I T P L E A Li N A C.- 3 i / V F T V S C R V TJ 'T' i P t: P D F T: F T F T V P P T VT F; C R T’ K P F T V P T L T V I E M T 1‘ S R F P T T‘ L F K L F T’ a P S G A V V Q P Q N G R 'i T' 'T :3 a5 ‘ r L L (— T T Q L S P V N I C T F R C A V T H T A G T Q E V 'T' M N L A S Q N W N N Y D P T J'— F T P A P L G T P D F V G F T Q C V L T Q T T P G L G S T R G H K A 1‘ V S T C :3 V L F T P K L C S V Q E S T D 'T :3 N D F F. T' C Q N T K E T' P V G V V Q D G S T T’ 1 Q N E P Q a,‘ N V T. P D Y 9 G i D S H N V pi L A i: A ‘\ / A P T E i: C 1 Q L L E F P S 'T M D G x: S G 1’ P N i" . N L E’ C L L P Q 3 \v\‘ V Q _ F ‘1 Q E A A P A Q S E’ V A L L R E V N P F! T G R V L F F; C K L 4T K S G Y V T’ V A H T G 2 H F L V T T P N C ‘1 E R F D 3 W \7 F Q F Y 'T L A P M C F C T G R R P A L C 5‘ n; C i D L ; 1 ) {9931)} However, in an alternative embodiment of the invention, the VLPs maybe multivaient VLPs that comprise, for example, VP} and/or VPZ proteins from one Noroviius genogroup WO 09849 intermixed with VPI and/or VPZ proteins from a second Norovirus oup, wherein the different \I’I’l and VPZ proteins are not chimeric \I’I’l and VPZ proteins, but associate together Within the same eapsid ure to form immuno genic VLI’s. As used herein, the term “multivaient” means that the antigenic proteins are derived from two or more Norovirus genogroups or strains. a/Ient VIPs may contain VIP antigens taken from two or more yiraI strains, Purely by way of exampie the composition can contain 1nuItivaIentVI.Ps comprised of capsid monomeror ers trom one or more strains ofNorovirus genogroup I (egg. NorwaIk Virus) together With capsid monomers or inuItimers from one or more strains of rus genogroup II (62g. Houston . Preferahiy, the inuItivalent VIPs contain capsid, proteins from the strains ot‘NorwaIk and Houston Noroyiruses,t1rother predominantly circulating strains at a given time, {IIIISI} The combination ot‘rnonovaIent or inuItivalent VIPs Wi thin the composition preferably would not reduce the innnunogenieity ofeach VLI’ type. In particular it is preferred that there is no interference between Norovirns VIII—’3 in the combination of the invention, such that the combined VLP composition of the invention is able to elicit irnrnunity against infection by each Norovirus genotype ented in the vaccine. Suitabiy the immune response against a given VLP type in the combinationis at least 50/0 or the1nnnune response of that same VI.I3 type when measured individuaily preferably WOW(3 or substantiaily 100‘?“ The irnrnune response may suitably be measured, for example, by dy responses, as illustrated in the exampies herein.

ZE As used herein,“ nogroup I Norovirus VLPs reIer to either monovalent or rnultivalent VLPs that comprise a capsid protein derived from one or more genogronp I rus strains. In some embodiments, genogroup I Norovims VLPs comprise a full length eapsid protein from a genogroup I Norovirus (9.4,> 0 No1vvalh virus) In other ments I Norovims , genogroup VLI’sseomprise a consensus capsid protein derived from various genogroup I strains. The genogroup I strains from which the sus capsid sequence is derived can be within the same pe or genetic eluster or from dif"ere“11tg1iotypes or genetic clusters. Similarly, as used herein gtnom”111p II Noroyirus VLI’s” refer to either monoyalent or multivalent VLPs that comprise a capsid protein derived from one or more genogroup II nrs strains. In some embodiments, genogroup II Norovirus VIPs comprise a full length capsid protein from a genogroup II Noro virus (e15; Laurens or Minerva virus), In other ments, genogronp II Norovirus VLPs comprise a consensus capsid, protein derived from various genogroup ll strains.

The genogroup ll strains from which the consensus eapsid ce is derived can be within the same genotype or c r or from different genotypes or genetic clusters. in one embodiment, the genogroup ll Norovirus VLPs comprise a capsid consensus sequence of genogroup ll, genoty e I-‘l- (Gil-4) Norovirus, Thus, in some embodiments, the. genogroup ll Noroviius VIPs comprise a eapsid sequence of SEQ if) NO: l. {@333} h/lultivalent VLPs may he produced by separate expression of the individual capsid, proteins followed, by combination to form VLPs. atively multiple capsid proteins maybe expressed within the same cell, from one or more DNA constructs, For example, multiple DNA constructs may be transformed or transiected into host cells, each vector encoding a different capsid protein, Alternatively a single vector having multiple eapsid genesg controlled by a shared promoter or le individual prornoters, maybe used. 'ERES elements may also he incorporated into the , where appropriate. Using such expression strategies, the co expressed capsid proteins may he eo-puril‘ied for subsequent VLF formation, or may neously forrn valent VLPs w hi eh can then be purified lllllfid} A preferred process for niultivalent VLF production comprises preparation ol’VLlr’ eapsid proteins or derivatives, such as VPl proteins, frorn different Norovirus genotypes, mixing the proteins, and assernhly of the proteins to produee valent VLPs. The VPl proteins may he in the form of a crude extract, be partially ed or purified prior to . Assembled monovalent VLPs of different genogroups may be disassembled, mixed together and reassembled into multivalent VLPs. Preferably the proteins or VLPs are at least partially purified hefore heing ed. Optionally, further purification of the multivalent VLPs may be carried out after assembly. {@935} ly the VLPs of the invention are made by disassemhly and, reassembly of VLPs, to provide homogenous and pure VLPs. In one embodiment inultivalent VLPs may be made by disassembly of two or more VLPs, followed by combination of the disassembled VLF components at any suitable point prior to reassembly. This approach is suitable when VLPs spontaneously form from expressed VPl protein, as occurs for exan‘rplew in some yeast s.

Where the expression of the VPl protein does not lead to spontaneous VLF formation, ations of VPl proteins or oapsoniers may be combined before assembly into VLPs, {9936} Where multiyalent VLPs are used. ably the components of the VLPs are mixed in the tions in which they are desired in the final mixed VLF. For example, a mixture of the same amount of a partially purified Vl’l protein from Norwalh and Houston Viruses (or other Noroyirus strains) provides a multivalent VLF with approximately equal amounts of each protein. {@837} Compositions comprising multivalent VLPs may be stabilized by solutions known in the art, such as those ofWO 98/449441? W0 00/415841, incorporated herein by reference {8838} Compositions of the invention may comprise other ns or protein ents in addition to rus VP l and VPZ ns or derivatives. Other proteins or es may also be (so—administered with the composition of the invention. Optionally the composition may also be formulated or co—administered, with non~Noroyirus antigens. Suitably these antigens can provide protection against other diseases. {93939} The VPl protein or firnctional protein derivative is suitably able to form a VLF, and VLF formation an be assessed by standard techniques such as for example, size exclusion chrornatography, electron microscopy and dynamic laser light scattering. llllldll} The antigenic molecules of the present invention can be prepared by isolation and purification from the organisms in which they occur natu “ally, or they rn ay be ed by recombinant techniques. Preferably the Norovirus VLF antigens are prepared from insect cells such as Sf9 or HS cells, although any suitable cells such as E. coli or yeast cells. for example, S. cerevisiae, S. pombe, Pickle: pasrori or other Pie/72in expression s, mammalian cell expression such as (El-10 or HEK systems may also he used. When prepared by a recombinant method or by sis, one or more insertions, deletions, inversions or tutions of the amino acids constituting the peptide may he made. Each of the aforementioned ns is preferably used in the substantially pure state. {8941} The procedures of production of noroyirus VLl’s in insect cell culture have been previously disclosed in US. Patent No. 6341865, which is incorporated herein by reference in its entirety. Briefly; a cDNA from the 3' end of the genome containing the viral capsid gene (ORE?) and a, minor structural gene (ORES) is cloned. The recombinant baculoviruses carrying the Viral capsid genes is constructed from the cloned cDNAs. Noroyiius VLl’s are produced in St? or H5 insect cell cultures. {0042} in some embodiments, the e composition comprises one or more adjuvants in combination with the Norovims antigen. Most adjuvants n a substance designed to protect the n from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as Borders/7hr perms is or Mycobactcrium tuberculosis derived proteins. Suitable adj u vants are commercially available as, for example, ’s incomplete.

Adjuvant and Complete. Adjuvant (Pitco Laboratories, Detroit, Mich); Merck Adjuvant 65 (Merck and Company, Jnc., , NJ); um salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine. acylated sugars; cationicalljvl or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A. {0043} Suitable adjuvants also include, but are not limited to, toll—like. receptor (TLR) agonists, particularly toll-like or type 4 (TEE-4) agonists (cg, monophosphoryl lipid A (MP1), synthetic lipid A, lipid A n'iirnetics or analogs), aluminum salts, nes, saponins, rnuramyl dipeptide (MD?) derivatives, CpG oligos, lipopolysaccharide (LPS) ot‘grarnwnegative ia, polvphosphazenes, emulsions, virosornes, cochleates, poly(lactidesco—glyeolides) (PLGJ rnicroparticles, poloxarner particles, microparticles, liposomes, oil—in-water emulsions, MF59, and squalene. in some embodin'ients, the adjuvan ts are not bacterially—derived exotoxins.

Preferred nts include adjuvants which stimulate a Thl type response such as SDMPL or QSZl . {0044} Monophosphoryl Lipid A (MPL), a nonutoxic derivative of lipid A from Salmonella, is a potent TLR—4 agonist that has been developed as a e adjuvant (Evans at a]. 2003). in pre- clinical murine studies intranasal MPL has been shown to enhance secretory, as well as systemic, humoral responses (Baldridge er (7.5/7. 2000; Yang et a]. 2002). it has also been proven to be safe and effective as a vaccine adjuvant in clinical s of greater than 0 patients (Baldrick at 0/3., 2002; Baldridge ct til. 2004). MPL stimulates the ion of innate immunity through the él receptor and is thus capable of eliciting cific immune responses against a wide range of infectious pathogens, including both gram negative and gram positive bacteria, viruses, and parasites (Baldridge at Hz, 2004; Persing at at. 2002). inclusion of MEL in vaccine formulations should provide rapid induction of innate responses, eliciting nonspecific immune responses from viral challenge while enhancing the specific responses generated by the antigenic components of the vaccine.

WO 09849 {@945} in one ment the present invention provides a composition comprising monophosphoryl lipid A (MPL) or 3 cylated monophosphoryl lipid A (3Dnl‘v’ll’L) as an enhancer of adaptive and innate immunity. Chemically 3Dan’L is a mixture of 3 De~0n acylated monophosphoryl lipid A with 4, 5: or 6 acylated . A preferred form of 3 De-O~ acylated, monophosphoryl lipid A is disclosed, in European Patent 0 689 454 Bi (Smithlslline Beecham Biologicals SA), which is incorporated herein by reference. in another embodimenh the present invention provides a composition comprising synthetic lipid, A“, lipid, A mimetics or analogst such as BioMira’s PET Lipid A3 or synthetic derivatives designed to on like TLR— 4 agonists. {85346} in certain embodiments, the vaccine composition comprises two adjuvants. A, combination of adj uvants may be selected from those described above. In one particular embodiment, the two adiuvants are MPL and um ide (tag, alum). in another particular embodiment, the two adiuvants are MPL and oil. lililéfi} The term "ettiective adjuvant amount" or “effective amount of nt” will be well understood by those skilled in the art, and includes an amount of one or more adios/ants which is capable of stimulating the immune response to an stered antigen, to, an amount that increases the immune response of an administered antigen composition, as measured in terms of the lgA levels in the nasal wasl'iings, serum 'lgG or lgh/l levels, or B and T—Cell proliferation.

Suitably effective increases in immunoglobulin levels e by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen composition without any adiuvant. {9943} in one embodiment, the present invention provides a vaccine composition formulated for parenteral administration, wherein the composition includes at least two types of rus VLPs in combination with um hydroxide and a buffer. The bufler can be sel(Dcted, from the group consisting of L-histidine, imidazole, succinic acid: tris, citric acid: bis~trist pipest mes, hepes, glycine aniide, and tricine. In one embodiment, the buffer is Lwhistidine or ole.

Preferably the buffer is present in a tration from about 15 lel to about 50 ml‘v’l, more preferably from about 18 mM to about 40 mlvl, or most preferably about 20 mlvl to about 25 inM. In some embodiments, the pH of the antigenic or vaccine composition is from about (3.0 to about 720, or from about 6.2 to about 63, or about 6.5: The vaccine composition can be an WO 09849 aqueous formulation. In some embodiments, the vaccine composition is a lyopliilized powder and reconstituted to an aqueous formulation. {994%} ln certain embodiments, the vaccine composition r comprises at least one adjuvant in addition to the two or more types ofNoroviius VLPs, um ide, and a buffer. For instance, the adjuvant can be a toll—like receptor agonist, such as MPL, tlagellin, CpG oligos, synthetic lipid A or lipid A cs or analogs. In one particular embodiment, the adjuvant is MPL. {885%} The Noroviius VLPs included in the vaccine itions of the ion can be any of the VLPs described herein, in one embodiment, the. two types ot‘Norovirus VLPs each se a capsid protein from different genogroups (cg, genogroup l and genogroup ll), For instance, one type ot‘Norovirus VLF comprises a capsid protein derived from a genogroup l Noroviius and the other type ofNorovirus VLP comprises a capsid protein derived from a genogroup ii rus. in one embodiment, one type ofNorovirus VLP comprises a capsid protein from Nor’t'ralk virus and the other type ot‘Norovirus VLF comprises a consensus capsid n derived from oup H, genotype 4 Noroviruses (eg, a capsid protein comprising a sequence of SEQ ll) NO: l). The vaccine composition can comprise about 5 pg to about 200 pg of each Norovirus VLF, more preferably aboutl 5 pg to about 50 pg ot‘each Norovirus VLF, In some embodiments, the dose of one type of Norovirus VLP is different than the dose of the other type ofNorovirus V’Ll’. For instance, in certain ments, the vaccine composition comprises about 5 pg to about l5 pg of a genogroup l VLP and about 15 pg to about 50 pg of a genogroup ll VLF. in other embodiments, the vaccine composition ses about 15 pg to about 50 pg ot‘a genogroup l VLF and about 50 pg to about 150 pg of a genogroup H VLF. {@951} ln some embodiments, the vaccine compositions further comprise a pharmaceutically acceptable salt, including, but not limited to, sodium chloride, potassium chloride, sodium sulfate, ainonium sulfate, and sodium citrate. ln one embodiment, the pharmaceutically acceptah e salt is sodium de. The concentration of the pharmaceutically acceptable salt can be from about l 0 niM to about 200 mM, with preferred concentrations in the range of from about l 00 niM to about 150 mM. Preferably, the vaccine compositions of the invention contain less than 2 mM offrec phosphate. in some embodiments, the vaccine compositions comprise ess than l mM of free phosphate The vaccine compositions may also further comprise other pharmaceutically accept‘ ble excipients, such as sugars (6.}??1101‘056, trehalose, ol) and surfactants. {9952} As discussed herein, the compositions of the invention can be formulated for administration as vaccines formulations. As used herein, the term “vaccine” refers to a formulation which contains Norovirus VLPs or other Norovirus antigens of the present invention as described above, which is in a form that is e of being administered to a vertebrate, particularly a human, and which induces a tive immune response. sufficient to induce immunity to prevent and/or ameliorate a Norovirus ion or Norovirusvinduced illness and/or to reduce at least one symptom of a Norovirus infection or illness. {85353} As used herein, the term “immune response” refers to both the humoral immune response and the cell—mediated immune response. The humoral immune response involves the stimulation of the production of antibodies by B lymphocytes that, for example, neutralize infectious , block ious agents from entering cells, block replication of said infectious agents, and/or protect host cells from infection and destruction The cell—mediated immune response refers to an immune response that is mediated by T~lymphoeytes and/or other cells, such as macrophages, against an ious agent, exhibited by a vertebrate tag, a human), that prevents or ameliorates infection or reduces at least one symptom thereof In particular, “protective inununity” or “protective immune response” refers to immunity or eliciting an immune response against an infectious agent, which is exhibited by a vertebrate (eg, a human), that prevents or ameliorates an infection or reduces at least one symptom thereof. Specifically, induction of a protective immune response from administration of the vaccine is evident by ation or reduction of the presence of one or more symptoms of acute gastroenteritis or a reduction in the duration or severity of such symptoms. Clinical symptoms of gastroenteritis from Norovirus include nausea, diarrhea, loose stool, vomiting, fever, and l malaise. A protective immune response that reduces or ates disease symptoms will reduce or stop the spread of a Norovirus ak in a population. Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M. F. a; Newman M. l.) ) Plenum Press New York). The compositions of the present invention can be formulated, for example, for delivery to one or more of the oral, gastro~intestinal, and respiratory (tag nasal) mucosa. The itions of the t invention can be formulated, for e, for delivery by injection, such as parenteral injection (erg, intravenous, aneous, intradermal, or intramuscular injection). {9954} Where the composition is ed for delivery] to the respirator")i (6g. nasal) mucosa, typically it is formulated as an aqueous on for stration as an l or nasal drops, or alternatively, as a dry powder, eg. for rapid tion within the nasal passage.

Compositions for stration as nasal drops may contain one or more excipients of the type usually included in such compositions, for e vatives, Viscosity adjusting , tonicity adjusting agents, hutl'ering agents, and the like, Viscosity agents can be microcrystalline cellulose, chitosan, starches, polysaccharides, and the like. Compositions for administration as dry powder may also contain one or more excipients usually included in such itions, for example, mucoadhesiye agents, bulking agents, and agents to deliver appropriate powder flow and size characteristics. Bulking and powder tlow and size agents may include mannitol, sucrose, trehalose, and xylitol. lllllSS} Whe‘e the composition is intended, for parenteral injection, such as intravenous (iv), subcutaneous (so), intradermal, or intramuscular (int) injection, it is typically formulated as a liquid suspension (:28, aqueous formulation) comprised of at least one type ot‘Norovirus VLF and optionally at least one adjuvant. in one embodiment, the adjuyant may be MP1,. in another embodiment, liquid, yaecine formulated for parenteral administration may have more than one adjuyant. in a preferred, embodiment, a parenterally-tormulated (erg, i.m,, igyx, or s.c.- formulated) liquid vaccine comprises Norovirus genogroup l and/or genogroup ll VlgPs with aluminum hydroxide (cg alum) and monophosphoryl lipid A (MPL) as adjuvants. in one embodiment, a liquid formulation for parenteral administration comprises Norovirus genogroup antigen(s’), such as one or more types ofNorovirus VLPs as described , MPL, aluminum hydroxide, and a buffer. in another embodiment, a liquid formulation for parenteral stration comprises Noroyirus genogroup antigerds), MPL, oil, and a buffer. In certain embodiments, the huffer in the parenteral vaccine formulations is L—histidine or imidazole, Parenteral administration of liquid vaccines can he by needle and syringe, as is well known in the art.

{M956} in certain ments, a vaccine composition of the invention for eliciting a, protective immune response against Norovirus in humans comprises genogroup l and/or genogroup ll rus VLPs at a dose of no more than l50 pg. For instance, in some embodiments, the vaccine composition comprises no more than lSG ug, no more than 100 gig, no more than 50 pg, no more than 25 pg, no more than l5 ug, or no more than 10 ug of genogroup l Norovirus VLPS. ln other embodiments, the e composition comprises no more than 150 rig, no more than 100 pg, no more than 50 ug, no more than 25 pg, no more than 15 pg, or no more than ll) rig of genogroup ll Norovirus VIPs. In certain embodiments, the vaccine composition comprises no more than l50 pg of each genogroup l and gene-group ll rus VLPs. in such embodiments, the dose of genogroup l Norovirus VLl’s and genogroup ll VLPs can be the same or different.

For instance, in one embodiment, the e ition may comprise no more than 50 ug of genogroup l Noroyirus VLPs and no more than lSG ug of genogroup ll Norovirus VLPs. in another embodiment, the yaccine composition may comprise no more than 25 pg of genogroup l Noro virus VLPs and no more than 59 ug of genogro up ll Norovirus VLPs, in other embodiments, the vaccine composition may comprise no more than l5 rig of genogroup l Noroyirus VLPs and no more than 50 pg of genogroup ll Norovirus VLPs. In still other embodiments, the vaccine composition may comprise no more than 25 pg of genogroup l Noroyirus \"LPs and no more than lSO ug of genogroup ll Noroyirus VLPS.

{M357} The genogroup l and genogroup ll rus VLPs can be derived from any of the Norovirus strains described herein. In one embodiment, the genogroup l Norovii'us VlgPs are genogroup l, genotype l (Gil) VLPs (i8. se a capsid protein from a fill Noi'ovirus). in another ment, the genogroup l Norovirus VLPs are Norwallc VLPs. ln another ment, the genogroup ll Norovirus VLPS are oup ll, genotype 4 (Gilli) VLl—‘s. in still another embodiment, the genogroup ll Norovirus VLPS are VLPs generated from expression ofa consensus sequence of genogroup ll Norovirus. In a particular embodiment, the gene-group ll Noroviius VLPs comprise a capsid protein having a ce of SEQ lD NO: 1.

{MPSSE The vaccine compositions hereinbefore described may be lyophilized and stored anhydrous until they are ready to be used, at which point they are reconstituted with diluent.

Alternatively, different components of the composition may be stored separately in a kit (any or all ents being lized). The components may remain in lyophilized form for dry formulation or be reconstituted for liquid formulations, and either mixed prior to use or administered separately to the t. in some embodiments, the vaccine compositions are stored in kits in liquid formulations and may be accompanied by delivery devices, such as syringes equipped with needles. ln other embodiments, the liquid vaccine compositions may be stored Within the delivery devices in a kit. For example, a hit may comprise pre~filled syringes, autoiniectors, or injection pen devices containing a liquid formulation of a vaccine composition described . {995%} The lyophilization of vaccines is well known in the art. Typically the liquid antigen is freeze dried in the presence of agents to protect the antigen during the lyophilization s and to yield a cake with desirable powder characteristics. Sugars such as sucrose, mannitol, ose, or lactose (present at an initial tration of" 0 nag/ml.) are commonly used, for ciyoprotection of" protein antigens and to yield lyophilized cake with desirable powder chaiacteristics. Lyophilizing the compositions theoretically results in a more stable composition {uses} The amount of antigen in each vaccine composition is selected as an amount which induces a rohust immune response without significant, adverse side effects. Such amount will vary depending upon which specifi c n(_s) is employed, route ofadministration, and adiuvants used. in general, the dose administered to a patient, in the context of the present invention should be sufficient to effect a protective immune response in the patient over time, or to induce the production of antigen~speci tic antibodies. Thus, the composition is administered to a t in an amount sufficient to elicit an immune response to the specific antigens and/or to prevent, alleviate, reduce, or cure symptoms and/or complications from the disease or infection, and thus reduce or stop the spread of a Norovirus outbreak in a population. An amount adequate to lish this is defined, as a "therapeutically effective dose." {Willi The e compositions of the present ion may be administered via a non" mueosal or mucosal route. These administrations may include in viva administration via parenteral ion (cg. intravenous, subcutaneous, intradermal, and intramuscular) or other traditional direct routes, such as 'buccal/suhlingual, rectal, oral, nasal, topical (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or directly into a ic tissue. Other suitable routes of administration include transcutaneous, suhdermal, and via suppository. In one embodiment, the vaccine is administered by a, parenteral route of administration, such as intra 'enous, subcutaneous, intradermal, or intramuscular. in certain embodiments, the vaccine is stered by an intramuscular route of administration. Administration may be accomplished simply by direct administration using a needle, catheter or d device (eg. pre~filled syringes or autoiniectors), at a single time point or at multiple time points. Other parenteral formulations may be delivered subcutaneously or intradermally hy microinjection or skin patch delivery methods. {9962} The t invention provides methods for eliciting protective immunity against Noroviius in a subject comprising parenteraliy administering to the subject no more than a single dose of a vaccine composition of the invention, wherein said vaccine ses genogroup 1 and/or genogroup ll Noroviius VLPs as described herein and optionally at least one adj uvant. in such embodiments, the single dose vaccine composition induces at least a three—fold se in ius~specitic serum dy titer as compared to the titer in the human prior to administration of the composition, in some embodiments, the single dose vaccine composition induces at least a ld increase in Norovirus~specific serum antibody titer as compared to the titer in the human prior to administration of the composition. in other embodiments“, the single dose vaccine composition induces a Norovirus—specitic serum antibody titer comparable to the antibody titer induced by exposure to live Norovirus in a natural infec'tion~i.e,, a greater than ten— fold increase in Norovirus-specifie serum antibody as con'ipared to the titer in the human prior to administration of the composition in n ments, the single dose vaccine composition induces the increase in Norovirus~specitic serum antibody titer within seven days of stration of the composition Preferably, the single dose e composition is administered by an intravenous, subcutaneous, or intramuscular route of adn'iinistration. in a certain embodiment, the single dose vaccine composition is administered intramuscularly to the human, {WME As described herein, in some embodiments, the single dose vaccine compositions suitable for use in the method comprise no more than 150 pg each of genogroup 1 and/or genogroup ii Noroviruses, For instance, in some embodiments, the vaccine composition comprises no more than l50 rig, no more than 100 pg, no more than 50 ug, no more than '25 pg, no more than l5 pg, or no more than 10 gig of genogroup l Noro virus VLPs. In other embodiments, the vaccine composition comprises no more than 150 pg, no more than 100 tig, no more than 50 ug, no more than 25 ug, no more than l5 ug, or no more than l0 ug of genogroup H Norovirus VLPS.

In certain embodiments, the vaccine composition comprises no more than 50 pig of each genogroup l and genogroup H Norovirus VLPS. in embodiments in which the single disc vaccine composition comprises both gen group i and genogroup ll Norovirus s, the dose of genogroup iius VLPs and genogroup ll V Li’s can be the same or different. For instance, in one embodiment, the e composition may comprise no more than 50 pg of genogroup l Noroviius VLPs and no more than 150 ug of gen agroup ii Norovirus VLPs. in another embodiment, the vaccine composition may comprise no more than 25 ug of genogronp l Norovirus VLPs and no more than 50 pg of gen ‘igroup ll Noroviius VLPs. in other embodiments, the vaccine composition may comprise no more than l5 pg of genogroup I Norovirus VLPs and no more than 50 pg of genogroup ll N’orovirus VLPs. In still other embodiments, the e composition may comprise no more than 25 ug of genogroup l Norovirus VLPs and no more than lSO pg of genogroup ll Norovirus VLPs. {titled} in one embodiment of the , the subject is a human and the vaccine confers protection from one or more symptoms ofNorovirus infection. Although others have reported methods of inducing an immune response with Norovirus antigens (see US. Patent Application Publication No. US 70070707576), the indicators of a protective immune response t Norovirus in humans have still not been clearly identified (Herhst—Kralovetz at all (2010) Expert Rev. es 9(3), 299-307). Unlike several vaccines currently d1n the U S. where iveness of the vaccine correlates with serum antibodies, studies have shown that markers of an immune response, such as increased titers of serum lgG antibodies against Norwalk virus, are notaassooeiated with protective immunity in humans (Johnson at 5:17. (i 990) J, inieetious Diseases 161: l8—2l ). Moreover, another study examining Norwalk viral challenge in humans indicated that susceptibility to Norwall: ion \ as inultifaetorial and included s such as seeretor status and memory al immune response (Lindesmith e! a]. (2003) Nature Medicine 9: 548— 553). {9965} Because Norovirus is not able to be cultured in vitro, no viral neutralization assays are currently available. A functional assay which serves as a substitute for the neutralization assay is the heniagghitination inhibition (ll/Xi ) assay (see Example l)lEl/Xl measures the ability of Noroyirus vaccineinduced antibodies to inhibit the agglutination ot antigencoated red blood cells by N oroviius VLPs because Norovirus VLPs bind to red blood cell antigens (rag histo~ blood group antigens). This assay is also known as a carbohydrate blocking assay, as it is indicative of the onal ability of antibodies to block binding of the virus or VLPs to blood group antigen carbohydrates on a red blood cell. in this assay, a fixed amount of rus VLPs is mixed with a fixed amount of red blood cells and serum from immunized subjects if the serum sample contains functional antibodies, the antibodies will bind to the VLl’s, thereby inhibiting the agglutination of the red blood cells. As used herein, “functional antibodies” refer to antibodies that are e of inhibiting the interaction between Norovirus particles and red blood cell antigens. in other words, functional dy titer is equivalent to histo~blood group antigen (HBGA) or carbohydrate blocking antibody titer. The serum titer of Noroviius~specific functional antibodies can be measured by the HA1 ass‘ y described above. The serum titer of Noroviius~specific functional antibodies can also be measured using an ELISAnbased assay in which a carbohydrate H n is bound to microtiter wells and Norovirus VLF binding to H antigen is detected in the presence ot‘serum (see Example 1 and Reeck er a]. (2m 0) J infect Dis, Volt : 1 2] 24218) An increase in the level ofNorovims—specific functional antibodies can be an indicator ofa protective immune response. Thus, in one embodimenh the administration of the vaccine elicits a protective immunity comprising an increase in the serum titer of rus~specific functional antibodies as ed to the serum titer in a human not receiving the vaccine. The serum titer of Norovirus—specitic functional dies indicative of a protective immune response is preferably a geometric mean titer greater than 40, 50, 75, 100, 1259 150, 175, 200 as measured by the HA! assay or blocking titer (BT)5.3 (50% inhibition ofll antigen binding by Norovirus VLPs) geometric mean titer of greater than 100, 150, 200, 250, 300, 350, 400, 450, or 500 as measured by the H antigen binding assay. in one embodiment, the serum titer ofNorovirus—specii‘ic functional antibodies is a geometric mean titer greater than 40 as ed by the HA! assay. in another embodiment, the serum titer of Norovirus-specitic timctional antibodies is a geometric rnean titer greater than 100 as measured by the HA3 assay In another embodiment. the serum titer of Norovirus—specific functional dies is a Sign geometric mean titer greater than 100 as measured by the H antigen binding assay. in still another embodiment, the serum titer ofNorovirus-specif'ic functional antibodies is a Big; geometric mean titer r than 200 as measured by the H antigen g assay.

{M966} in a r aspect, the administration of the e elicits a protective immunity comprising an igA niucosal immune response and an lgG ic immune response by administering parenterally (preferably intramuscularly) to the subject no more than a single dose of an antigenic or vaccine composition comprising one or more types of rus antigens and optionally at least one effective adjuyant. The ors have surprisingly found that parenteral administration of the Noroyirus vaccine compositions described herein induces a robust lgA response in addition to a strong lgG response. Typically, strong lgA responses are only observed when vaccines are administered through a mucosal route of administration. {0067} in certain embodiments, the administration of the vaccine elicits a protective immunity comprising an increase in the level of lgA Norovirusnspeeific antibody secreting cells in the blood as compared to the level in a human not receiving the vaccine. in some embodiments, the administration of the vaccine elicits a tive immunity comprising an increase in the level of lgA, Norovirus—specil‘ic antibody secreting cells in the blood as compared to the level in the human before receiving the vaccine, ln one ment, the lgA Noroviius—specific antibody secreting cells are CCR10+, (3919+, (1327+, CD62L+, and (1467+, Antibody secreting cells with this marker profile are capable of homing to both peripheral lymphoid tissue, such as Peyerls patch in the gut, and mucosal lymphoid tissue, such as the gut mucosa. In one embodiment, the number of CCR10+, (3319+, (3327+, Clittiillnh and 3407+ lgA antibody secreting cells is greater than about 500, about 700, about L000, about l,500, or geater than about 2,000 cells per 1 X 106 peripheral blood monocytes. in r embodiment, the lgA Norevirus—speeific antibody ing cells are CCRIt)--t~, CDIQr, CDZ7-r, CDti'ZL—, and (1467+.

Antibody secreting cells with this r profile generally exhibit homing only to niucosal sites and can be indicative ofa memory Bscell response ln some embodiments in which the vaccine is administered intramuscularly, the number of CCRlllt, CDl 9+, CD27+, C1362 L-, and (1,407+ lgA antibody secreting cells is greater than about 5,000, about 6,500, about 7,000, about l0,000, about , about 15,000, or greater than about 20,000 cells per l x l 06 peripheral blood monocytes {0068} Similar gs have been observed with vaccines for other s, such as rotavirus.

For rotaviius vaccines, there is controversy over whether serum antibodies aie di fectly involved in protection or merely rel‘lect recent infection , 2002; Franco, 2006). g such correlates of tion is particularly difticult in the context ot‘diarrbeal diseases such as rotavirus or norovirus, Where nical studies inferring protection maybe multifaceted with contributions front al immunity (such as intestinal lgA), cytokine elaboration, and cell mediated immunity. The difficulty in measuring such immune responses during clinical development, and the lack of correlation to serum antibody measurements, requires that the effectiveness of a vaccine for these types of viruses can only he demonstrated through human clinical challenge experiments. {005% As mentioned above, administration of a vaccine composition of the present in n prevents and/or reduces at least one symptom of Norovirus infection, Symptoms of Norovirus 2HII infection are vvell known in the art and include nausea, vomiting, diarrhea, and stomach cramping. Additionally, a patient with a, Norovirus infection may have a ade "ever, headache, chills, muscle aches., and fatigue. The invention also encompasses a method of inducing a protective immune response in a subject experiencing a, Norovirus infection by administering to the subject a e formulation of the invention such that at least one symptom associated with the Noro virus infection is alleviated and/or d. A reduction in a symptom may be determined subjectively or objectively, e. ;., self assessment by a subject, by a clinician’s assessment or by conducting an appropriate. assay or measurement (6.g. body ature} including cg, a quality of life assessment, a slowed progression of a Norovirus infection or additional symptoms a reduced severity ofNorovirus symptoms or suitable assays (62g, antibody titer, RT—PCR antigen detection, and/or B—cell or T—cell activation assay). An effective response may also be determined by ly ing (rag, RTwPCR) virus load in stool samples, 'Whicl'i reflects the amount of virus shed from the intestines). The objective assessment comprises both animal and human assessrnents. {limit} The invention also provides a method of generating antibodies to one or more Norovirus antigens, said method comprising administ "ation ofa vaccine ition of the invention as described above to a t. These dies can be isolated and puri tied by routine methods in the art. The isolated antibodies specific for Norovirus antigens can be used in the development of diagnostic immunological assays. ”l‘hese assays could be employed to detect a Norovirus in clinical s and identify the particular virus causing the infection (cg.

Norwallt, l-iouston, Snow Mountain, etc}. atively, the isolated antibodies can be administered to subjects susceptible to Norovirus infection to confer passive or short—term immunity.

{M71} The ion will now be illustrated in r detail by reference to the specific embodiments described in the following examples. The examples are ed to be purely illustrative of the invention and are not intended to limit its scope in any way.

EXAMPLES Example 1. flose Escalation, Safety and lmmunogenicity Study of intramuscular Norovirus Bivalent VirnsnLilteml’artiele (VLF) Vaccine in Humans (L‘tltl3nlll4 Study), Cohort A {@872} This example describes Cohort A of a randomized multi—site, dosenescalation study of the safety and immunogenicity of four dosage levels of an intramuscular (lM) Norovirus Bivalent VLF Vaccine nted with monophosphoryi lipid A (MPL) and aluminum hydroxide (AlOH) compared to placebo in adult subjects. Approximately 48 subjects l8 to 49 years ot‘age were enrolled in the . Subjects received two doses ot‘ the e or placebo, by intramuscular (1M) injection, 28 days apart using a l5 inch (38mm) needle.

{M73} The Norovirus Bivalent VLF Vaccine contained genogroup la genotype. 1 (GL1) and genogroup 119 pe lV (Gilli) VLPs as the antigens, and hllonophosphoryl Lipid A (MPL) and aluminum hydroxide (AlOH) as adj u vants, sodium de (NaCl) and L~liistidine (lnHis) as buffer (pl—l 7), ethanol and water for injection. The ition of the intramuscular Norovirus Bivalent VLP Vaccine is summarized in Table l . The 811.4L VLPs comprised a capsid sequence ot‘ SEQ lD NO: l which was derived front three (333.4 strains.

Table l . Final Drug t ition for Four llVl Norovirus Bivalent VLF Vaccine Formulations per 0.5 ml“, Formulation Gil-VLF Gll.4 MPL Al* (its) VLPts-g) (its) ‘ ‘ as Aluminum ide {@874} o was sterile normal saline for injection (0.9% NaCl and preservative—lice). The dose escalation of the vaccine was conducted as follows: after appropriate screening for good health, subjects in Cohort A were enrolled sequentially into each ot‘l’our dosage groups of~l '2 subjects each (Dosage Groups Al, A2, A3, and, A4). Dosage Groups Al and A4 , A2, AB, represent bivalent antigenic dosages old/5 pg, lS/l 5 pg, 50/50 pg, and lSll/lSll pg, respectively, of the G 3.1 and Gilli norovirus. Subjects in each dosage group were randomized 5:1 to receive vaccine or placebo. Subjects in Dosage Group A1 received their respective randomized treatment (10 subjects received 5/5 pg vaccine and 2 subjects received placebo}. Subjects were followed for safety assessment by1eV1ieVV1 of the svmptoms recorded on the memory aid (Days 0— 7) and interim medical histories from the Day 7, 2i and 56 Visits. , 28, 35, Safety data was ed by the Central Safety Monitor (CSM). After the 7nday post Dose 2 safety data (Study Day35) were available for review from subjects in Dosage Group Al and considered able, subjects in Dosage. Group A2 were eligible to receiVe their initial dose The same rule applied for dosing in the subsequent dosage groups; that is, after the 7—day post Dose2 safety data (Study Day 35) were available. for review from a dosage group, the next dosage group was eligible to rec Ve their initial dose, {@375} At the end of enrollmentin Cohort A approximately 19 subjects in each Dosage. Group rece1Ved Vaccine (total of 40 Vaccinees) and2 subjects in each group received saline (total of approximately 8 saline control recipients). lllll7t’i} The subjects kept a daily memory aid cited syn'iptoms including four local injection site reactions, such as pain, tenderness, redness, and swelling, and id ic signs or symptoms including daily oral temperature, headache, fatigue, muscle aches, , joint aches and gastrointestinal symptoms of nausea, vomiting, diarrhea, abdominal cran'ips/pain for Days 0 through '7 after each dose ofli‘vi Noroyirus Biyalent VLP Vaccine or control. The s and swelling at the injection site was measured and "ecorded daily for '7 days after each injection. 7} lnterirn rnedical histories were obtained at each follow~up Vi sit on Days 7+3, 21 +3, 28+3, -+3,' 56-~-7,18tlt 14 and 393H4 and at the follow—up telephone call1‘n Day 265 +14; subjects were queried about interim illness, doctor’s Visits, any serious adverse events (SAEs), and onset of any significant new medical conditions. Subjects had a CBC. with W8C differential and platelet count and serum BUN, creatinine, glucose,A1AS}, and AL as ed at screening and on Days 2i and35 (7 daysa er each dose) toa ses continuing eligibility and safety, respectively. {9978} Blood from subjects V 'as ted before vaccination on Day 0 and on Days 7+3, 21+3, 28+3, 35+3, 56+”, l80+l4, and 393 +l4L to measure serum antibodies (lgG, lgA, and lglVi separately and combined) to IM rus BiV'alent VLF Vaccine by enzyme~linl§ed immunosorhent assays (ELISA). Seium carbohydrate blocking activity and serum HAl antibodies were also measured. For subj ects in Cohoit A, antibodv secreting cells , homing markers, memory B cells and ar immune responses VVere. assayed {M79} The ing methods were use.d to e the blood samplesscollected from immunized individuals or individuals receiving the placebo, Serum Antibody Measurements bv ELlSA {9989} Measurement of antibodies to noroyiius by ELlSA was performed for all ts, using purified recombinant Noroyirus VLl’s (GL1 and Gil/l separately) as target antigens to screen the coded specimens, Briefly, noroyirus VLPs in carbonate coating buffer pH 96 were used to coat rnicrotiter plates Coated plates were washed, blocked, and incubated with serial twovfold ons of test serum followed by washing and incubation with enzyinc—conjugated secondary antibody reagents spot11% tor human total lgG, 1ng(1LlgG2, Ig63,lg0G4 IgA and lgM Appropriate substrate solutions were added, color developed, plates read and the lgG, lgA and lgM endpoint titers determined in comparison to a reference standard curve for each dy class. Geometric rnean titers (GMTs), geometric mean fold rises (GMFRs) and sponse rates tbr each group was determined. Sent-response was defined as a 4*fold increase in antibody titer compared to pre~irnniunization ti ters, Norovirus Carbohydrate Histo~blood~2rou1Antirens HBGA Blockino Activity ll} ng assays to measure the ability ot‘serum antibodies to inhibit NV VLF binding to lltypel or 1 type 3 synthetic carbohydrates were perforrned as previously described (Reeck at all, (20W) 3 t Dis, Vol 202(8): l212 l2l8). Biiet‘ly NV VLPs tor the blocking assays were incubated with an equal volume of serum andserially twcutcld diluted from a starting dilution of 1:25. Neutravidin-coated, 96—well titer plates were washed and coated with 2.5 ug/rnL of either synthetic polyyalent H type l-l’AA—biotin or polyvalent H type biotin. The sera— VLF solutions were added Plates were washed and rabbit pclyclonalsera specitic to NV V’le’ was added washed and followed by incubation with horseradish peroxidasecco1niugated goat a11ti~rabbit lgG. The color was deyeloped with tetrainethylbenzidine peroxidase liquid substrate and stopped with 1M phosphoric acid. Optical density was ed at 450. Positive and negative controls were med Fiftypercent blocking titers (3150) were dterrnined“, defined as the titer at which OD readings (after subtraction of the blank) are 50% of the positive control. A Value of 12.5 was assigned to samples with a BT50 less than '2“. Geometric rhean titers (Glyl'l‘s), geonretric niean fold rises (GMFRs) and seroresponse rates for each group were determined, sponse was defined as a 4v~tbld increase in antibody titer compared to pre— immunization titers, A, blocking control serum sample was used as an al control An assay to confirm the specificity of the blocking was performed using the same protocol for the blocking assay with the following exceptions: after coating with carbohydrate, sera was incubated ly on the plate without first pre~incuhating with V’Ll). After g, VLPs were incubated on the plate and detected as for the blocking assay.

Norovirus Hema' ' lutination antibody inhibition HA1 Assav {@382} e—induced antibodies were examined for the. capacity to inhibit hemagglutination of O—type human RBCs by the. norovirus VLPs as previously bed (El Kamary at all. (2010) l infect Dist VOL 202(1 l): 1649—58): HA1 titers were calculated as the inverse oft‘ne t dilution that inhibited liemagglutination with a compact ve REC pattern and are presented as GMTs, GMFRs and Fr: 4—fold rises; {£1983} Norovirus Gil and 611.4 VLPs were separately serially diluted and incubated with an equal volume ot‘a 0.5% human RBC sion in a 96~well V bottom plate. The amount of noroyirus VLP antigens corresponding to 4 HA units were determined and confirmed by back titration. Test sera were heat inactivated at 56 C for 30 minutes and treated with freshly prepared % Kaolin suspension. To eliminate serum inhibitors, test samples were pre~adsorhed with RBCs. The HA1 assay were performed as follows: ated seia ( diluted 2~fold in PBS pH .5) were added to 96 well V-plates and incubated with an equal volume oi‘Norovirus (ill and Gilli VLF antigen, respectively, containing 4 HA units. A suspension of (1.5% RBCs was added to each well and plates ted for an additional 90 minutes at 4 C. Wells containing only PBS or antigen without serum served as negative and positive controls, respectively. Geometric mean titers (Git/11‘s), geometric mean fold rises ('GMFRs) and seroresponse rates for each group were determined. Seroresponse was defined as a 4~fold se in antibody titer compared to rnunization titers.

Antibody SecretinO Cell Assa “ {M984} PBMCs were isolated from approximately 60 rnL of anti~coagulated blood on Days 0, 7+3, 28+3, and 35+3 after administration of 1M Norovirus Biyalent VLF e or placebo.

Approximately 25 mL of blood for fresh PBMC assays and 35 mL of blood for cryopreservation ofPBMCs was obtained. ASC assays detect cells secreting antibodies to noroyirus VLPs (Tacket 6!: all (2000) 1. lni‘ect. Dis:’ Vol. 182302—305; Tacket at a}. (2003) Clin. 1mmunol., Vol. 1081241n247; El Kamaiy et a2. (2010) l lnfect Dis, Vol. 202(11): ). Fresh PBMCs were evaluated for ASC ncy and determination of homing marliers from a subset of subjects.

Cryopreserved PBMCs from subjects participating in Cohort A were evaluated for ASC frequency. The response rate and mean number ofASC per 106 l)Bl\/le at each time point for each group are descrihed. A positive se is defined as a post—vaccination ASC count per l06 PBMCs that is at least 3 standard deviations (SD) above the mean pie—vaccination count for all subjects (in the log metric) and at least 8 ASC spots, which corresponds to the mean of rnediuin~stimulated negative control wells (2 spots) plus 3 SD as determined in similar assays.

MSidilalisms:2.1l._QILEQIQXE2.11.§__Kl£l1§:.§l?aElli£19..llifl.§£119.ll‘zi..l3:.§§.ll§ {M385} Anti—coagulated blood was collected only in Cohort A subjects (approximately 25 rnL on Days 0, 28, 56 and, 180) to measure memory B cells on days 0, 28, 56 and, 180 after vaccination using> an EU Spot assay preceded by in vitro antigen ation ( Crotty’ at a]. (2004) .l. ln'nnunol.

Methods, Vol. 2861 l l~l22.; Li et a5. (2006) .l. lrnmunol. Methods, Vol. 3.13:1 10-l 18).

Peripheral blood mononuclear cells (5x106 cells/mitt, l mL/well in 24*well ) were incubated for 4 days with rus Gil and 811.4 VLF antigens separately to allow for clonal expansion ot‘antigenaspecitic memory B cells and differentiation into antibody ing cells.

Controls included cells incubated in the same conditions in the absence of antigen and/or cells incubated with an unrelated antigen. Following stimulation, cells were washed, counted, and trans “erred to ELI Spot plates coated with Norwalk VLF. To determine frequency of Virus— specific memory B cells per total lg-secreting B lymphocytes? ed B cells were also added to wells coated with anti-human lgG and anti —hunian ,l gA antibodies. Bound antibodies were revealed with HRl’nlabeled anti—human lgG or antinhurnan lgA followed by appropriate substrate. Conjugates to lgA and lgG subclasses (lgAl, 1gA2 and lgGlnfl) are also used to ine antigen~specific subclass responses that may be related with distinct effector mechanisms and ons of immune priming. Spots were d with an ELlSpot reader.

The expanded cell populations for each subject were examined by flow cytoinetiy to confirm their memory B cell phenotype, tic. CD19+, CD27+, lgG+, 1glvl+, CD38+, igl), among others (Crotty 61‘ Hi. (2004) J. lmmunol. s. Vol. 286:111—122.; Li er (Til. 2006) .l. linrnunol.

Methodsi Vol. 3131ll0~118). \w 1)) Cellular Immune Res. {8986} Antimoagulated blood ximately 25 rnL on Days 0., 28 56, and 180) from subjects in Cohort A were collected as coded specimens and the PBMCS isolated and eserved in liquid nitrogen for possible future evaluation of Civil responses to noroviius Oil and Gil/l VLF antigens. Assays that are med include PBMC proliferative and cytokine responses to norovirus Gil and Gll.4 VLF antigens by measuring interteron (lFN)~y and interleukin (lL)~/—l levels among others according to established techniques (Santandari of: al. (2000) J. ola Vol. 164223—2232; Tacket at al. (2003) Clint ol., Vol. l08:24l—247). T cell responses are also evaluated. {9987} Safety assessment included local and systemic solicited symptoms for '7 days and unsolicited symptoms for 28 days after each dose. Serious e Events are monitored for 12 months. lnini'unogenicity was assessed with serum obtained prior to and alter each vaccination for Pan~ELlSA antibodies (lgG, lgA and lgl‘vl combined) and, peripheral blood monon'uclear cells (PBh/le) for lgG and lgA antibody secreting cells (ASC) via Eli spot. {@9881 All four dosage groups have been enrolled for Cohort A with post dose two safety data available from all four dosage groups (40 vaccinees total). Among the 40 vaccinees, pain or tenderness were the most common local symptoms reported after either dose, whereas swelling or redness was infrequent. No severe local ms were reported. Systemic symptoms of headache, myalgia, or malaise after either dose were reported by less than half of the vaccinees.

No vaccinees reported fever. l\ o related SAEs were reported. {@989} As shown in Figures l~3. robust anamnestic PannELlSA antibody responses (combined lgG, lgA. and lgM) were ed to both VLF antigens 7 days after the first dose of the lowest dosage (5 pg Gil + 5 pg of (311.4 VLPs’). The s cond dose did not boost the post dose one(D ses. Similar results were observed for antigensspecific serum lgG and serum lgA responses measured separately (Figures 4—9). ependent responses were observed for the antibody responses to both antigens (Figures 1—9). However, the maximal se to the Gil VLF appeared to be achieved with a lower dose than the l response to the Gillél VLP ('15 ug vs. 50 pg). interestingly, the single dose of the intramuscularly administered Norovirus bivalent vaccine induced a surprisingly, significantly greater an tigen—specific antibody titer than the. titer induced by two doses of an intranasally administered nionovalent VLF vaccine comprising a 20 fold higher VLF dose (Figure it); compare LViBnitM 5 pg group to L‘s/M403 100 pg . Moreover, the low dose (5 ttg), lM nt Noroviius vaccine produced a Norovirttsepeeitie antibody titer r to that induced in humans exposed to the native Norovirus e 10) } Robust lgG and lgA Elispot responses were also observed at ’7 days after the tirst dose of the lowest dosage (5 rig) for both VLF antigens (Table 2). Notably, the antibody secreting cell (ASC) responses were biased to lgA V‘s, lgG and ASCs exhibited a mneosal homing (alpha 7) and chemokine (CCRl 0) receptor phenotype as assessed by flow eytonietry (Figure ll; Table 3): As shown in Table 3, a greater number ofASCs exhibit rnueosal homing markers (beta 7+, CD62L) as compared to dual inucosal/peripheral homing markers (beta 7+,” CD62L+). Table 4- shows the percentage of memory B cells per l06 peripheral blood monocytes that respond to the VLF antigens A larger percentage ot‘antigenaspeeitie memory B cells also express m’ucosal homing markers as, compared to the dual rnueosal/peripheral or peripheral hornin g rnarlrers.

Similar responses were also observed in recipients who received the 15 pg and 50 pg doses (Tables 24).

Table 2. Day 7,9 Characterization of PBMC se Approximation of dy Secreting Cells (ASCs)/million CDl9+ cells.

Vaceine Response Vaceine ASC/mlllion CD19+ cells n Day 7 Percent Speeific Specific to Specific to Percent of Total Norovirnsnspecifie B 1ng lgG Cirenlating cells G114 GHA rarer: Geometric mean Al pg dose (r125) Deviation Al pg dose 2012/046222 Vaccine Respnnse Vaccine ASC/miltinn CDléH- cells — Day 7 Percent Specific Specific tn Specific to Percent of Total Nerovirns—speeifie B ‘ < < < Cireniatmg cells * * o, o o PnMC Geometric mean A2 ng dose (11:4) Standard Deviation A2 pg dose Geometric mean A3 50 #3"; dose (11:4) Standard Deviatinn A3; 50 gig dose Genmetrie mean A4 34153 % pg dose (11:4) Standard Deviation A4 32938 .7 * ,, . 8976.9 159 “g dose Egg L393: 95mm ”seam «3% 233$ 28.5mm magma? ”38:2 gamma 1.! ummuamm Jammy Sam . 36%», Em “35» 2:3 “imam Aivfiimgau Jimuu “Sea. imam Sam .flwflsmbuemowmo “593m a? m+329 «izmuu Asfirigmu “35 “imam 39m .. >53 Axv 5&5 igmu $3 .3 Axv ,+,..‘Nmu i. mEuESB ES. Jammy oawumfi g 59.8 2 :35 smvom g “imam figmyfimnfi 5... ofiuum», ea “39“. m 5: Mm. .3 $39 $39 :32 «in: E 3 £8 imam mpmxhfim Ami: ”Egg in 383E Um?“ 3% 3% Q fiwwnfi 3% amw flhmflmmwm $335 3% £398ch mm m1 Hm m1 3% m mm ”#3.me am mm Q Qua; gauge w: m mm Umhwvgamw 2 flhflflmmwm 2 @ni Mymmflfimwm 2012/046222 mmmgmm L35 93% .3 3:5va yaw—mum? Egan” MEEES “383% ummuamm Emma in ,+wmfiu Sum @383», gag Each» 2:3 “i‘mflu Jammy)» $§+E$mu “Sea. “$3M.“ Sam “593% a? miwa Jammfi E§+E$mu “3:9 “+wmflu gram wao rigmflv +EKUU 0&6? ES. #fimu g $8.0m E ASE. .xw “imam Samfizmww in miwafighom 3a Em “$9“. m was“. a. momd .3 mo {a +3m9 was inmmu $me .flwammg Q QQQE 3% v4 «NEE 23 Egg 3% um.“ u: 3% Emu um a; wag: a": am cum” 3‘ @HE mmww mafifiim 32 osgfim 383mg“ 333m 92mm uwmumam mésghw wmmE Bang,» iafi “$853M Ema “$30 5% Saga.» umgnm hem “Saw. gamma iamu. “ETmeU rfimuu in fifiificnu ”fink E3892 a+wm‘mmu ,+© «595% 3.»me «+wmmu in \, mmww 39m ,Vfitflfiwmmw \mmmifiufisfi ”33% £39 wae Eumliw ofioflm .3 $339 3 range gamma ESQ in Ax? #1. $3 £60.? “E .3 Ax? “59.“. “imam“ Jammu ,+meU. +fimUU «3m mewflu mum‘commg 2m “33% m +mmflw 22:.

AK. mm?“ kimmw 6&me +wm‘mmu @0802 gown ENE 3% Nd ENE % 3% me‘ mi mi sq gmbafigw m1 3% m aflfifiauw my £an m1 in Md Nfi 3i; 3%:me am aflfifiauw mw may 3i; whammfim maflmfiofl WO 09849 llllll \3G $3.2 foamNm .owtommmifizmitem mflvmflqwfio EWSFE s38 3% E ofi gig uEmEch u: 3% 3H5 Qua Emfimfiw Mn 3. EEEQ am” 333m mmfismm‘q‘ {Will} in the absence of an available direct Viral neutralization assay due to the inability to culture Noroyinls in vitro, functional ass‘ ys which some as substitutes for Viral neutralization ass‘ ys were conducted to measure functional antibodies in vaccinees. {@092} Using the carbohydrate ll antigen blocking activity assay described above, the tion of fill VLF binding to H antigen mediated by yaccine~induced serum antibodies \ 'as measured.

Data are presented as geometric mean fold rise (GMFR) and sponse (Ill-~l‘old rise) in Table , and as geometric mean titer (GMT) in Table 6. Surprisingly, after just one intramuscular injection of the vaccine formulation, significant carbohydrate blocking ty was observed in all dose groups; in fact, the administration of a second dose cine did not significantly increase blocking activity compared to post—dose l . The inhibition of binding actiyity was maintained throughout the testing period, up to 56 days post dose l .

Table 5. Carbohydrate Blocking ty (HBGA BT50), oroyirus Gll Geometric Mean Fold Rise (GMFR) and Seroresponse (ll—Fold Rise) Study Day 28 Days Post Dose 2 28 Days Post Dose 1 ’7 Days Post Dose 2 (56 Days Post Dose 7 Days Post Dose 1 21 Days Past Dose 1 )ose 2) (35 Days Post Dose 1) 1) E4--FoldE ' 4.97911 Ali-Fold 4.190111 4.97911 GMFR Rise. E GMFR Rise GMFR Rise (men Rise GMFR Rise TreatmentE (95% (95% (95% (95% (95% (95% (95% (95% E (95% (95% Group EN CI) = (11,1 EN er) Ci) N or) (.71) EN Cl) en ENE CT) on 575 mcg 9 -51 889 9 19.71000 E9 2.0(7',7 88.9 9 1-51 778 VLF (51s, (:89 (51s, (82 (66.4, 51.7)- (51.s, (5.7, (40.0, Vaccine 99% 70.3) E 99.7) 7.1) 100.03: E 99.7) 48.1) 97.2) /15 meg 8 100.0 Es 25.5 1000 s 18.5 1000 E7 100.0 7 34(24 571 VLF (-53.1, (10.5. (63.1, (8.4, (6.5.1, (59.0, 29.6) (1.4, Vaccine 100.0) "1.8) E 1-000) 40.6) 100.03: E 100.0) 90.1) 50/50mcg i 38.6 100.0 E1 279 100,0 1 20.9 1000 E1 19(99, 100.0 9 10.2 77.8 VLF E0 (18.3, (-592, E0 (134, (69:, 0 (10, (692, E0 3-5.4)- (69.2, = (4.6, (0.0, Vaccine 211.6)- 100ml 59) 100.0) 43.5) 1019.0) 100.0) 22.8) 97.2) 1507150 7 30.5 1000 Es 194 100,0 .3 15.3 1011.11 E Es 13s 100.0 s 23s 1000 mchl.,P (1-5.3 (59.0, (13. , (63.1, (11.7, (65,1, (n.8, (63.1, (17, (63.1, Vaccine 5’7’.6) E1090); 28.5) 100.0) 22.6) 1019.0) 17.5)- 100.0> 33.3) 100.0) Placeho 8, d9ros 0.0 Es cs (11 7 0.0 (0.0, s usage, 0, Es 03(07E 0093.11, 3 E96 (013, no l) E (0.0, E ' 1.1;; 36.9) 1.1) 36.9) 1.1 313.9) 1.2) (0,0, 3.13.9) 36.9) Results based on all subjects receiving both doses of study product.

Two subjects’ data points are excluded due to a possible mix-up of specimens; one ot'these data points is a baseline en ing in the subject not having fcld rise at vailable for anytime point.

Table 6. Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovims Gl.l Geometric Mean Titer (GMT) Study Day 7 Days Post Dose 28 Days Post 28 Days Post Dose 2 Dose 2 7 Days Post 21 Days Post 1 (35 Days Post (‘36 Days Post (Fret-Dose Z) GMT 5 (95%(11) = (95%(11) w: ) '= w' (ass/oer) 768.5 (344.1. lS/lSmchLP ' ' j , : . .. 2 23090052, e ' 1.. ' / i ' i if, 506.7) 50/50mchLP .. . . . ., . . . ., .

\ . ; Vaccine 50 ' i‘ . . .’ . , i Z i ..: 348.3) lSO/lSOnichll’ > 2.1..x; ' 291.50.71.55 > . «90338 Vaccine V ’ .‘ u . i . f, § 495.4) 584.6) Placebo > , 24.6(84 > J8.3(J0.l, 92.S) 726) 33.3,: Results based on all subjects receiving both doses of study product Two subjects’ data points are exeluded due to a possible niix-up of specimens. {0093} rly, carbohydrate blocking activity of serum antibodies against Gllflr VLPs was measured. A significant response 3 'as obsewed in all dosing groups as measured by GMFR and seroresponse (Table 7) as well as GMT (Table 8). Similar to the antibody’~rnediated ng of Gll binding described above, robust blocking of Gilli VLF carbohydrate binding activity was detected after just one close. and a second dose did not appear to enhance the blocking activity.

Table 7. Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovims GHA Geometric Mean Fold Rise (GMFR) and Seroresponse (ALFold Rise) Study Day l 5 8 Days Post Dose 2 28 Days Post Dose 1 i 7 Days Post Dose 2 (56 Days Post Dose ’7’ Days Post Dose ll 21 Days Post Dose ll -lhpse 2) l (35 Day Post Dose 1 1) Rise Git/rm Rise ‘ GMFR (95% i (95% g (95% i (95% e1) * ' i .. . on , , /5 meg VLP " ' . . . ‘. 55.6 Vaccine . . (21.2, 86.3) Study Day 28 Days Pest Dose 2 ’28 Days Past Dose 1 7 Days Post Base 2 (56 Days Post Dose aW Ba ‘8 Post Dose 1 3 (Prelfiose ’2) (35' Days Past Base 1) 1) 4~Fcid ______________________ 44901;: 4~Fcid GMFR Rise GMFR GMFR; Rise GMFR Rise Treatment (95% (95% (959a (95% (95% (95% (95% (,1) J 5/ 1 5 inc g 9.2 (3, VLP Vaccine 27.9) 50/50 mcg 12.

VLP Vaccine (3.8, 39.4) 150/150 incg U. 'JI A ... > x2 VLP Vaccine _. .0 u.\~ V Placebo 8% 1 (0‘9, ..d ' 1.1) /—~\ Resuits based on {1” subjects receiving both doses of study product: Two subjects’ data points are excluded due to a ie mix—up of specimens; one se data points is a baseiine specimen resulting in the subject not having fold rise date a vaiiabie fer any time point.

Table 8. Carbohydrate ng Activity (HBGA BT50)? Arfii-Noroyirus {311.4 Geometric Mean Titer (GMT) Study Day 7 Days Post Dnse 28 Days Past 28 Days Past 2 Dose 2 7 Days Post 21 Days Post Base 1 (35 Days Past (56 Days Past 059 1 Base 1 Dose 1 (I’VE—130819 2) Dose 1) Base 1) GMT GMT GMT GMT GMT GMT Treatment Group " (95% en; (95% CI) (95% (.71) (95% Cl) N (95% <31) (95% <31) /5 meg VLP 40.3 (18, 2021 (106.3, 2016 Vaccine r- w#1),- 3843) '2 /15 meg VLP 2601 (931, Vaccine 50/50 [[11 5272 (271,1, Vac ' 1025) 50/1 50 meg VLP Vaccine Placebo 22.3 (12.6, 41.6) Results based on all subjects receiving both doses oi‘study product. 'i'wa subjecns" data points are excluded due {(7 a possibie mix—up ofspecimenfa.

{M94} Hen-laggiutination lnhihition assays (HA1) were also utilized to test the response of serum antibodies from vaccinated subjects against target NoroVirus VLF antigens. Siiniiar to carbohydrate H antigen binding studies, j 11st one dose of VLF vaccine induced antibodies that inhibited hernaggiutination in all dosing groups, as ed by GMFR (Table 9’), 4—fold rise (Table 9), and GMT (Table 10). Though the level ofinhibition of hemaggiutination was maintained through the last day tested (28 days post dose 2, 56 days post dose l), the second dose of VLF vaccine did not appear to enhance vaccine—induced dy—mediated inhibition of hemaggiutination.

Tabie 9. Heniagglutination tion Assay AntinNoroyirus Gil Geometric Mean Fold Rise (GMFR) and esponse (4—Fold Rise) StudyDay ’28 Days Post Dose 2 i i 28 Days Post Dose 1 7 Days Post Dose 2 (56 Days Post Dose 7 Days Post Dose 1 21 Days Post Dose 1 (Pre~Dose 2) {35 Days Post Dose 1) i 1) 4--Fold 4-Foia eaten; 4-Foid 4--Fold a a GMFR Rise g GMFR Rise g GMFR Rise GMFR Rise = GMFR Rise Treatment t 5% ('95% g (95% (95% g (95% (95% (95% (95% g (95% (95% Group N Cl) C1) g 1" C1) e1) :19 c1) or) N C1) or) N on CD /5 meg 9 5.4-(3, 77y 9 7(46, ; 88.9 9 6.1 (4.1. 88.9 9 e(3.7, 77.8 9 63 (3.9, 880 VLP 9.3) (40.0, 10.7) (51.8, 9.3) (51.8, 9.5) (40.0, 1o 3) (5i 5, Vaccine 97.2) i 99.7) 99,7) 97.2.) 99 '1) /15meg 29 33.94944, 1000 a :9 7.1 (3.1, 75.0 "i 13.5-31.1, 55 i 7E81(-4~.4, 00.0 VLP 1:9) (63.1, 16.1) (34.9, 17.7 (42 1 15.2) (59.0, Vaccine i000) 96 a) 99m i000) 50/5oineg 1 22.4 100.0 1 i is 9 100.0 1 14.5 9 11.8 100.0 VLP o (11.6, (69.2, o 0 (3 1,24) (59.2, o (9.3 (6.3, (66.4, Vaccine 43) i000) 100.0) 22 '7) 21.9) moo) 150/150 7 12.6 85.7 s 9 s 4 (5, 100.0 s s 4 (5, :9 7.3 (4.5, 1000 HE}; VLP (5.7. 23) (42.1, :4) (53.1 14) 11.9) (63.1, Vaccine = 99.6)- 100,o) i000) o.t_)(0.0,i 0.0(00, :_ ‘ 1 09-3)], .9) 36.9) . , 1.1) s based on aii subjects receiving both doses 0 ‘ Two subjects’ data points 5 '3 ed due to a possible mix—up of specimens; one oi'these data points is a baseline specimen resulting in the subject not having fold rise data a vailable for any time point.

Table 10. Hentaggiutination Inhibition Assay Anti—Norovirus GM Geometric Mean Titer (GMT) Study Day 28 Days Post 7 Days Post Dose 28 Days Post 7 Days Post 21 Days Post Dose 1 (Pt‘e— ’Z (35 Days Post Dose 2 (5'6 Days Pre~Dose 1 Dose 1 Dose 1 Dose 2) Dose 1) Post Dose 1) (95% GMT GMr GMT GMT GMT Treatment Group Cl) (95% Cl) N (95% c1) (95% C1) (95% Cl) (95% c1) /5 mcg VLP 26.4 143.5 (5.36 :. 157 (230.3, 1157.4 (88.4, Vaccine (14.1, 383.8) ' 307.1) 316.8) 49.4) ‘15/l5 meg VIP 11.9 99.2 (411.9, Vaccine (6.1, 209.7) 23.4) 50/50 meg VIP 17.2 (5.3, 160 (79.11, 1195.1. (60.4, 99.7 (51.23, 103.8 (513.4, 81.1 (38.8, e 9.1;) 322.6) 235.1) 191.8) 184.5) 169.5) 150/150 9.2 (7455, 114.1 (509 101.6 (112, 77.7. (5i .8, 77,; ’ 111139; VLF . 67.3 (411.7, e 255.7) ) 115) 101.3) Placebo 16.6(11.7, E, 16.1 (111,7, 16.6(11, 113.2 (108 23.7) 24.1) 24.4) Results based on all subjects receiving both doses of study product.

Two ts’ data points are ed due to a possible mix—up of specimens. {llllQ‘SE Inhibition of hernagglutination was also achieved when the target VLE? was a niisrnatched Virus. Vaccine—induced serum antibodies inhibited henragglntination by a Houston Virus strain VLF, as measured by GMFR and seroresponse (Table l l) as well as GMT (Table l2). In this case, the higher VLF vaccine doses ed stronger responses, particularly as measured by 4— fold rise or GM’l'. GMFR and 4—fold rise were also significantly increased when the target VLl’ was the 2003 Cincinnati Virus strain, as measuredjnst 7 {lays post Close 1 (Table 13’).

Table l l. Hernagglutination inhibition Assay (Houston Virus Strain VLF), AntiuNoroyirus G114 Geometric Mean Fold Rise (GMFR) and Seroresponse (49F0ld Rise) ..........................................r...................................... ...........................................r........................................ 28 Days Post Dose ’2 21 Days Post Dose 28 Days Post Dnsc l 7 Days Post Dose 2 l (56 Days Post Dnsc 7 Days Post Dose 1 (Pro—Dose 2) (35 Days Post Dose 1 1) 1 4—Fold 4—F0ld GMFR Rise GMFR 1 GMFR Rise GMFR 12151.1 ' l (959@ (95% (95% (95% (95% (95% (95% 31: Group (:1) Study Day 28 Days Post Dose 2 21 Days Post Dose 28 Days Post Dose 1 7 Days Post Dose 2 (5'6 Days Post Dose '7 Days Post Dose 1 1 (Fire—Dose 2) (35 Days Post Dose 1) g 1) 4—9013 4-Foid 4-Foid 4—Foid 4—9013 GMFR Rise ' GMFR Rise Rise GMFR Rise ' GMFR Rise = 5 Tmntme (95% 195% (959g (95% (95% (959% g 195% tit Group Vaccine 150/] 50 meg VLF Vaccine Resuits based on {1” subjects re' 'Vin'g, both doses of study t: TW-I) subjects‘ data points are ed due to a possible mix»up of specimens; one of these data points is a baseline specimen resulting in the subjec: not having fold rise 1 available for an y time point.

Table 12. Hemagglutination Inhibition Assay (Houston Virus Strain VLF), Antinorovims (311.4 Geometric Mean Titer (GR/1T) Study Day 7 Days Post 23 Days Post 28 Days Post Dose 2 Dose 2 Dose 1 (35 Days Post (56 Days Fost (I’m—Dose 2) Dose 1) 'H‘reatment GMT Group (95% CI} (95% CI) /5 meg VL; /15 meg VLF Vaccinc 50/50 meg VLF Vaccinc 150/150 meg 853.5 (297.4, VLF Vaccinc 150.1 (80.1, 281.1) Results based on 3” subjects ing both doses of study product.

Two subjects’ data points are excluded due to a possible mix—up ofspeeimens.

WO 09849 Table l3. Inhibition of Hemagglutination in placebo versus 50/50 itg VLF vaccine lelemagglutination tion Assay (2003 Cincinnati Virus Strain VLF), Anti—Norovirus (311.4 Geometric Mean Fold Rise ((IS'MFR) and ric Seroresponse (4— Fold Rise) Results by Treatment Group 7 Days Post Dose 1 Treatment Group . GMFR (95% on arms Rise (ass/o en ..........................................................................+................................. ..................................................................... ............................................................ o 1.0 0.6, lull 0,0 030, 84.2) {0096} The results from this study demonstrated that the Bivalent 1M Norovirus VLF vaccine was generally well tolerated. The imrnunogenicity data suggested that a single vaccine dose may be sufficient to protect seropositive human adults. The results from the carbohydrate blocking activity and liemagglutination inhibition assays provided further evidence that a single vaccine dose induced serum antibodies with potent anti-Norovirus activity. The magnitude and rapidity of the observed immune responses following a single parenteral dose in humans were dramatic when compared to earlier immune responses reported by multiple nasal VLF vaccine administrations at much higher VLF dosages (El Kamaiy or at. (20l0) J lnfect Dis, Vol. 202(l l ): l649—1658). These ses were also superior to those induced by orally administered Norovirus VLPs (Tacket at at. (2003) Clin lmmunol 108241—247; Ball at at. (1999,.

Gastroenterology 117240—48) as well as those d by Norovirus VL/Ps produced by transgenic plants (Tacket of a2. (2000) .l infect Dis l82i302u305). in particular, this intramuscular vaccine formulation produced anamnestic responses within seven days of zation and maximal serum antibody responses were observed after a single dose, including a significant lgA response and functional carbohydrate blocking activity and hemagglutination inhibition activity.

Thus, this Norovirus bivalent vaccine induced a strong, protective immune se in humans that was superior to immune responses induced, by any currently ble Noroviius vaccine. e 2. Dose Escalation, Safety and lmmnnogenicity Study of intramuscular Norovirns Bivalent Virus~Like—Particle (VLF) Vaccine in Humans (L‘thSdM Study) {@097} The following e provides the remaining planned portion of the clinical study described in Example 1, n a randomized, multi—siten, dose~escalation study is conducted in adults 33: l8 years of age of the safety and immunogenicity of four dosage levels of an uscular (TM) rus Divalent VLF Vaccine adjuvan ted with monophosphoryl lipid A WO 09849 (MPL) and aluminum hydroxide (AlOH), ed to placebo. Subjects will receive two doses of the yaecine or placebo, by intramuscular (1M) injection, 28 days apart using a l.5 inch (38mm) needle“ This example is intended to further illustrate the principles of the present invention. {3098} Cohort A has completed enrollment in the study and was described above in Example 1.

Cohort B contains ~20 subjects 50~6I¢l years of age. Cohort C contains ~30 subjects 65~85 years ofage Approximately 98 subjects are enrolled in the study as a whole {8899} in Cohort B, ~20 subjects 5064 years of age are enrolled and randomized l :1 to receive vaccine (N=l (l) or placebo ($140). After the 7—day post Dose 2 safety data (Study Day 35) are available for review from subjects in Cohort B, subjects in Cohort C are eligible to receive their initial dose, in Cohort C, ~30 subjects 65 to 85 years of age are enrolled and randomized l:l:l to receive vaccine adj uvan ted with MPL and, AlOll (N: l 0), or vaccine adju’vanted with AEOlEl alone, is. no MPL (Neill), or placebo (Niiilll). The n concentrations of the norovirus VLPs and ot‘ the AlOH in the two vaccine formulations to be evaluated in Cohort C are identical; only the presence or absence ot‘h/l PL is different. llllll till} The Norovirus Bivalent VLF Vaccine contains genogroup l, genotype l (Call) and, genogroup ll, genotype lV ( G114) VLPs as the antigens, and h’lonophosphoryl Lipid A (MPL) and aluminum ide (MOE) as adjuvants, sodium chloride (NaCl) and Lshistidine (bill's) as bufler (pl-l 6.3—6.7), l and water for injection. The Gll4 VLl’s comprised a capsid sequence of SEQ 11) N0: 1, which was derived from three Gilli strains.

{MIME The single dosage of vaccine selected for further evaluation in Cohorts l3 and C is the lowest dosage in Cohort A that results in the rnost robust and reproducible immune response that is also generally well tolerated. The Day 56 safety and immunogenicity data from subjects in Cohort A is reviewed hy the CSM/SMC and the nt dosage is ed for evaluation in Cohorts B and C. {99192} The subjects keep a daily memory aid of ted symptoms including four local injection site reactions: such as pain, tenderness, redness, and swelling, and 10 systemic signs or ms including daily oral temperature, headache fatigue: muscle aches, chills, joint aches and gastrointestinal ms of , vomiting, diarrhea, ahdominal cramps/pain for Days 0 through 7 after each dose oflM Norovirus Bivalent VLF Vaccine or control, The s and swelling at the injection site are ed and recorded daily for 7 days after each injection {@9193} lnterini rnedical histories are obtained at each follow—up visit on Days 7+3, 21+3, 28+3, +3, 56+7, , and 393+14 and at the nup telephone call on Day 265 +14; ts are d about interim illness, doctor’s visits, any serious adverse events (SAEs), and onset of any significant new medical conditions. Subjects have a CBC with WBC differential and platelet count, and serum 31% creatinine, glucose, AST, and ALT assessed at screening and on Days El and 35 (N7 days after each dose) to assess continuing eligibility and safety, respectively, {llllllléll Blood from subjects is collected before vaccination on Day 0 and on Days 7+3, 21+3, 28+3g 35+3, 56+7r l80+l4, and 393 +l4 to measure serum antibodies (lgGi lgA, and lgM separately and combined) to IM Norovirus Bivalent VLF Vaccine by enzyine~linl<ed iniinunosorbent assays (ELISA). Serum carbohydrate blocking activity and serum HAl antibodien are also ed, llllll {PS} The methods described above for Cohort A are used to analyze the blood samples collected from immunized individuals or individuals receiving the placebo. The results of the study will be employed in the development ofa clinical protocol for administration of the vaccine formulations of the invention, llllll {96} The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual s of the invention, and flinctionally equivalent methods and components are within the scope of the invention, lndeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings using no more than routine experimentation. Such modifications and equivalents are intended to fall within the scope of the ed claims. {(98107} All ations, patents and patent applications mentioned in this specification are herein orated by reference into the specification to the same extent as if each individual ation, patent or patent application was specifically and dually indicated to be orated herein by reference. {@8108} Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention. hflNCl:S 1. Glass, R1 .13 Neel, '1" Ando, RL Fanlrliauser, G Beiloit, A Mounts, UD Parashei‘, IS Bi‘esee and SS Monroe. The Epidemiology of Enterie Calicivinises from Human: A Reassessment Using New Diagnostics. JIrzject Dis 2099; 181 (Sup 2): $254-$261 . 2. Hardy, ME. Norwalk and “Ncnvalk—iike Viruses” in Epidemic Gastroenteritis. Clin Lab Med 1999,1990): 675~90. 3. Jiang, X, DY Graham, KN Wang, and MK Estes. Norwaik Virus Geneme Cloning and Characterization. Science 1999; 250: 1580—1583. 4. Jiang, X, M Want, DY Grahan'i, and MK Estes. Expression, Self~.Assernbly, and Antigenieity ofthe Norwaik Virus Capsid Protein. J Viral 1992; 66: 6527—6532.

. Glass, P, U White, JM Ball, 1 iare—Goffart, ME Hardy, and MK Estes. Norwaik Virus Open Reading Frame 3 Encodes a Minor Structural Protein. J Viral 2990; 74: 6581*6591. 6. Lindesrnith, L, C Moe, S Marionneau, N Ruvoen, X Jiang, L Lintlblad, P Stewart, J LePendu, and R Bai‘ie. Human Susceptibility and ance to Norwalk Virus infection. Nat M’ea’ 2003; 9: 548u553. 7. Parrinn, TA, DS Sehrei’ber, IS Trier, AZ Kapikian, and NR Blaeklow. Clinical Immunity in Acute Gastroenteritis Caused 13y Nciwallr Agent. NEngZ Jilted 1977; 297: 8689. 8. Wyatt, RG, R Delin, NR Blaeklow, HL DuPont, RE? , TS 'l‘hornliill, AZ Kapikian, and R1Vi Clianoek. Comparison of Three Agents of Aeute infectious eterial Gastroenteritis by eliallenge in eers. Jim’écr Dis 1974; 129: 709. 9. Ball, 1M, DY Graham, AR Opekum, MA Gilger, RA ro, and MK Estes. Recombinant Norwalli lilie Particles Given Orally to Volunteers: Phase I Study. Gastroememlogy 1999; 117: 4048.

. Tacket, CO, MB Sztein, GA l..osonl<.y, SS Wasserman, and MK Estes llumoral, Mneosal, and Cellular Immune ses to Oral Noiwalk Virus—like Particles in Volunteers. Clin Immunol 2003; 108: Ml 1 1 . ro, RA,” 3M Ball, SS Krater, SE Pacheco, JD ts, and MK Estes. Recombinant Norwalk Virus—like Particles Administered lntranasally to Mice induce Systemic and Mueosal (Fecal and l) Immune Responses. J Virol 2001; 75: 9713. 12. Nicollier—Jamot, B, A , L Piroth, P Pothier, and E Kohli. Recombinant Virusslike Particles of a Norovirus (Caenogroup ll Strain) Administered lntranasally and Orally with Mucosal Adjuvants LT and1_iT(Rl92G) in BALB/c Mice Induce Specific lél'uinoral and Cellular Thl,/’Tl12~lil«:e lmn'rune Responses. e 2004; 2110794 086. 13. Periwal, SB KR Konri e, N Rarnachandaran, SJ ey, S De Bruin, D Zhu, T3 Zainl), L Smith, S Udeni, Ill Eldridge, KE Shroff, and PA Reilly. A Modified Cholera l-lolotoxin Cl”— EZ9l-l Enhances Systemic and Mncosal immune Responses to Recombinant Norwalk Virns- like Particle Vaccine. lhcciite 2003; '21: 376—385. l4. lsaka, M, Y Yasuda, S Kozulta, 'l" 'l‘aniguelii, K Matano, J Macyama, "1‘ Korniya, K Ohliuma, N Goto, and K chhiltubo. Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminummonwadsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an atliuvant. Vaccine 1999; 18: 1.

. Kozlowski, PA, S CunUvin, MR Neutra, and T? Flanigan. Comparison of the oral, rectal, and vaginal immunization routes for induction of dies in rectal and genital tract secretions of women. Infect Immun 1997; 65: 1387—1394. l6. Mestecky, 5, SM Miehalek, Z eanu, and MW Russell. Routes of zation and antigen deliveiy systems for optimal mucosal immune responses in humans. Behring Inst M'irt l997; 33'43. l7. Wu, HY, and MW Russell, Nasal lymphoid , intranasal immunization, and tmentaliza‘tion of the common mneosal immune . i’mmunoi Res l997; 16: l87~ }. 18. Evans, JT, CW Cluff, DA Johnson, Ml Lacy, DH g, and JR Baldridge. Enhancement of antigen—specific immunity Via the TLR4 ligands MPL adjnvant and Ribi 529. Expert Rev Wiccines 2003; 2: 219229. 19. Baldridge, JR, Y Yorgensen, JR Ward, and JT Ulrich. I‘vlonophosphoryl lipid A enhances rnucosal and systemic immunity to vaccine antigens following intranasal administration {In Process Citation}. Vtwcine 2000; 18: 24l 6-2425.

. Yang, QB, M Martin, SM Michalek, and J Katz. Mechanisms ofmonophosphoryl lipid A augmentation of host responses to recombinant HagB from Porpheronionas gingivaiis. infect Immun 2002; 70: 3557—3565. '21. ck, P, D Richardson, G Elliott, and AW Wheeler. Safety evaluation of nionophosphoryl lipid A (MPL): an immunostirnulatory adjuvant. Regal T035560! Pharmacol 2002; 35: 39845. 22. Baldridge; JR, l) n; J'l' Evans, C Cluffi S Mossman, D Johnson, and D l’ersing.

Taking a toll on human disease: Toll—like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol 1717912004; 4: 11294138. 23. Persing, DH; RN Coler, ME Lacy, DA Johnson, JR Baldridge, RM Hershherg, and SG Reed.

Taking toll: lipid A minieties as adjuvants and rnodnlators. Trends zlficrobiol 2002; ll): WO 09849 (I) t»:

Claims (39)

CLAIMS :

1. Use of a vaccine composition comprising genogroup I rus virus-like particles (VLPs) in the cture of a medicament for eliciting protective immunity against Norovirus in a human, wherein said genogroup I Norovirus VLPs comprise a capsid protein derived from a genogroup I viral , and wherein the ment is ated for: (a) stration of no more than a single dose of the vaccine composition; (b) administration parenterally; and (c) inducing at least a three-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the medicament.

2. The use of claim 1, n the composition ses no more than 150 g of said genogroup I Norovirus VLPs.

3. The use of claim 1, wherein the composition comprises no more than 50 g of said genogroup I Norovirus VLPs.

4. The use of claim 1, wherein the composition comprises no more than 25 g of said genogroup I Norovirus VLPs.

5. The use of claim 1, wherein the composition comprises no more than 15 g of said genogroup I Norovirus VLPs.

6. The use of claim 1, wherein the medicament is formulated for inducing at least a sixfold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the medicament.

7. The use of claim 1, wherein said Norovirus VLPs are monovalent VLPs or multivalent VLPs.

8. The use of claim 1, wherein said composition further comprises genogroup II Norovirus VLPs, said genogroup II Norovirus VLPs comprising a capsid protein derived from a genogroup II viral strain.

9. The use of claim 8, wherein said composition comprises no more than 150 g of genogroup II rus VLPs.

10. The use of claim 8, n said composition comprises no more than 50 g of genogroup II Norovirus VLPs.

11. The use of claim 8, wherein said composition comprises no more than 25 g of genogroup II Norovirus VLPs.

12. The use of claim 8, wherein said composition comprises no more than 15 g of genogroup II Norovirus VLPs.

13. The use of claim 8, wherein said genogroup I rus VLPs are Norwalk virus VLPs and said genogroup II rus VLPs are VLPs generated from expression of a consensus ce of genogroup II Norovirus.

14. Use of a vaccine composition comprising genogroup II Norovirus virus-like particles (VLPs) in the manufacture of a medicament for ing protective immunity against Norovirus in a human, wherein said genogroup II Norovirus VLPs comprise a capsid protein derived from a genogroup II viral strain, and wherein the medicament is formulated for: (a) administration of no more than a single dose of the vaccine composition; (b) administration parenterally; and (c) inducing at least a three-fold se in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the medicament.

15. The use of claim 14, wherein the composition comprises no more than 150 g of said genogroup II Norovirus VLPs.

16. The use of claim 14, n the composition comprises no more than 50 g of said genogroup II Norovirus VLPs.

17. The use of claim 14, wherein the composition comprises no more than 25 g of said genogroup II Norovirus VLPs.

18. The use of claim 14, wherein the composition comprises no more than 15 g of said genogroup II Norovirus VLPs.

19. The use of claim 14, wherein said genogroup II Norovirus VLPs se a capsid protein derived from genogroup II, pe 4 viral s.

20. The use of claim 14, n the medicament is formulated for inducing at least a sixfold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the medicament.

21. The use of claim 14, wherein said Norovirus VLPs are monovalent VLPs or multivalent VLPs.

22. The use of claim 14, wherein said composition further ses genogroup I Norovirus VLPs, said genogroup I Norovirus VLPs comprising a capsid protein derived from a genogroup I viral .

23. The use of claim 22, wherein said composition comprises no more than 150 g of genogroup I Norovirus VLPs.

24. The use of claim 22, wherein said composition comprises no more than 50 g of genogroup I Norovirus VLPs.

25. The use of claim 22, wherein said composition comprises no more than 25 g of genogroup I Norovirus VLPs.

26. The use of claim 22, wherein said composition comprises no more than 15 g of genogroup I Norovirus VLPs.

27. The use of claim 14, wherein said genogroup II Norovirus VLPs are VLPs ted from expression of a consensus sequence of genogroup II Norovirus.

28. The use of any one of claims 1 to 27, n the composition further comprises at least one adjuvant.

29. The use of claim 28, wherein said at least one adjuvant is a toll-like receptor agonist.

30. The use of any one of claims 1 to 27, wherein said composition further comprises two adjuvants.

31. The use of claim 30, wherein said two adjuvants are monophosphoryl lipid A and aluminum hydroxide.

32. The use of any one of claims 1 to 31, wherein said composition further comprises a

33. The use of claim 32, wherein said buffer is selected from the group consisting of L- histidine, imidazole, ic acid, tris, and citric acid.

34. The use of any one of claims 1 to 33, wherein the medicament is formulated for administration to the human by an intravenous, subcutaneous, intradermal, or intramuscular route of administration.

35. The method of claim 34, wherein the ment is formulated for stration to the human by an intramuscular route of administration.

36. The use of any one of claims 1 to 35, wherein the medicament is formulated as a liquid.

37. The use of any one of claims 1 to 35, wherein the medicament is formulated such that the increase in Norovirus-specific antibody titer is induced within seven days of administration of the single dose of the composition.

38. The use of any one of claims 1 to 35, wherein the medicament is formulated to confer tion from one or more symptoms of Norovirus infection.

39. The use of any one of claims 1 to 38 substantially as herein described with reference to any one or more of the examples.