EA042694B1 - BUFFER-CONTAINING COMPOSITIONS, VACCINES CONTAINING BUFFER-CONTAINING COMPOSITIONS AND WAYS OF THEIR APPLICATION - Google Patents

Description

The present application claims priority over US provisional application 61/506447, filed July 11, 2011, the entire contents of which are incorporated herein by reference.

Field of invention

The present invention relates to the field of vaccines, in particular norovirus vaccines. In addition, the present invention relates to methods for preparing vaccine compositions and methods for inducing and evaluating protective immune responses against norovirus in humans.

Background of the invention

Noroviruses are non-culturable human caliciviruses that have been found to be the single most important cause of epidemic outbreaks of non-bacterial gastroenteritis varieties (Glass et al., 2000; Hardy et al., 1999). The clinical significance of noroviruses was underestimated before sensitive molecular diagnostic assays were developed. Cloning the genome of the prototype genogroup I Norwalk virus (NV) and generating virus-like particles (VLPs) using a recombinant baculovirus expression system has led to the development of assays that detect widespread norovirus infection (Jiang et al. 1990; 1992).

Noroviruses are positive-polarity single-stranded RNA viruses that contain an unsegmented RNA genome. The viral genome encodes three open reading frames, of which the last two determine the production of a major capsid protein and a small structural protein, respectively (Glass et al. 2000). When the NV capsid protein and some other noroviruses are expressed at high levels in eukaryotic expression systems, it self-assembles into VLPs that structurally mimic native norovirus virions. When examined by transmission electron microscopy, VLPs are morphologically indistinguishable from infectious virions isolated from human fecal samples.

Immune responses to noroviruses are complex, and the correlates of protection are currently only being elucidated. Studies in human volunteers with native virus showed that mucosal immunological memory conferred short-term protection against infection and suggested that vaccine-mediated protection was possible (Lindesmith et al. 2003; Parrino et al. 1977; Wyatt et al, 1974).

Although norovirus cannot be cultivated in vitro, due to the availability of VLPs and their ability to be produced in large quantities, significant progress has been made in determining the antigenic and structural topography of the norovirus capsid. VLPs retain the original conformation of the viral capsid protein and lack infectious genetic material. Therefore, VLPs mimic the functional interactions of the virus with cellular receptors, thus eliciting an appropriate host immune response, but lack the ability to reproduce or cause infection. The Baylor College of Medicine collaborated with the NIH to study humoral, mucosal, and cellular immune responses to VLP NV in human volunteers in an investigator-sponsored phase I academic clinical trial. Orally administered VLPs have been shown to be safe and immunogenic in healthy adults (Ball et al. 1999; Tacket et al. 2003). But multiple doses with a relatively high amount of VLP were required to generate an immune response. In other centers, preclinical experiments in animal models have demonstrated an increase in immune responses to VLPs when administered intranasally with bacterial exotoxin adjuvants (Guerrero et al. 2001; Nicollier-Jamot et al. 2004; Periwal et al. 2003; Souza et al. ( 2007) Vaccine, Vol 25(50):8448-59). However, protective immunity against norovirus in humans remains unclear due to the fact that measures of protective immune response in humans are still not well defined (Herbst-Kralovetz et al. (2010) Expert Rev. Vaccines 9(3), 299-307).

Brief description of the present invention

The present invention is based in part on the discovery that a single dose of a norovirus vaccine induces a rapid, strong protective immune response against norovirus in humans when administered parenterally. Accordingly, the present invention provides a method for stimulating protective immunity against norovirus in a human, comprising parenterally administering to a human no more than a single dose of a vaccine composition, said composition comprising a genogroup I and/or genogroup II norovirus VLP, said composition inducing at least a three-fold increase in the titer of norovirus-specific serum antibodies compared to the titer in humans before the administration of the composition. In certain embodiments, the induction of an increase in the titer of norovirus-specific antibodies occurs within seven days after administration of a single dose of the composition. In some embodiments, the vaccine composition is administered to a human via an intravenous, subcutaneous, intradermal, or intramuscular route of administration. In one embodiment, the vaccine composition is administered to a human via the intramuscular route of administration.

Single dose vaccine compositions may contain genogroup I norovirus VLP, genogroup II norovirus VLP, or both, at a dose of from about 5 μg to about 150 μg. In embodiments in which single dose vaccine compositions contain both genogroup I and genogroup II norovirus VLPs, the dose of each genogroup VLP may be the same or

- 1 042694 various. In one embodiment, the composition contains no more than 50 µg of genogroup I norovirus VLP. In another embodiment, the composition contains no more than 25 µg of genogroup I norovirus VLP. In another embodiment, the composition contains no more than 150 µg of genogroup P norovirus VLP. implementation, the composition contains no more than 50 μg VLP of norovirus genogroup II. Norovirus VLPs can be monovalent VLPs or multivalent VLPs.

In some aspects of the present invention, the genogroup I norovirus VLPs in the vaccine compositions comprise a capsid protein derived from a genogroup I viral strain. In one embodiment, the genogroup I norovirus VLPs comprise a capsid protein from genogroup I norovirus 1. capsid protein from the Norwalk virus. In other aspects of the present invention, the genogroup II norovirus VLPs in the vaccine compositions comprise a capsid protein derived from a genogroup II viral strain. In some embodiments, the genogroup II norovirus VLPs comprise a capsid protein from genogroup II norovirus 4 genotype. In certain embodiments, the genogroup II norovirus VLPs are VLPs resulting from the expression of a genogroup II norovirus consensus sequence. In one specific embodiment, genogroup II norovirus VLPs comprise a capsid protein with the sequence of SEQ ID NO: 1.

In certain embodiments, the vaccine composition further comprises at least one adjuvant. The adjuvant is preferably not a bacterial exotoxin adjuvant. In one embodiment, the adjuvant is a toll-like receptor agonist such as monophosphoryl lipid A (MPL), flagellin, or CpG. In another embodiment, the adjuvant is aluminum hydroxide (eg, alum). In certain embodiments, the vaccine composition contains two adjuvants such as MPL and aluminum hydroxide. In some embodiments, the vaccine composition may further comprise a buffer such as L-histidine, imidazole, succinic acid, tris, and citric acid. The vaccine composition may be formulated as a dry powder or liquid. In one embodiment, the vaccine composition is formulated as a liquid (eg, an aqueous formulation).

Brief description of graphic materials

Fig. 1. Results of total Ig ELISA assays measuring pooled serum levels of IgG, IgA, and IgM in human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, 50, or 150 µg of each of the genogroup norovirus VLPs 1.1 and VLP of norovirus genogroup II.4. Geometric mean titer of antibodies to GI. 1 (A) and to GII.4 (B) is shown for each of the dosage levels on days 7 and 21 after the first immunization and on days 7 and 28 after the second immunization. Volunteers were immunized on days 0 and 28 of the study.

Fig. 2. Results of total Ig ELISA assays measuring pooled serum levels of IgG, IgA, and IgM in human volunteers immunized intramuscularly with placebo (saline) or with a vaccine formulation containing 5, 15, 50, or 150 µg of each of the genogroup norovirus VLPs 1.1 and VLP of norovirus genogroup II.4. The seroconversion factor for antibodies to GI.1 (A) and to GII.4 (B) 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. Volunteers were immunized on days 0 and 28 of the study.

Fig. 3. Results of total Ig ELISA assays measuring pooled serum levels of IgG, IgA, and IgM in human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, 50, or 150 μg of each of the genogroup norovirus VLPs 1.1 and VLP norovirus genogroup II.4. Percentages of serological response (i.e., a fourfold increase in antibody titer compared to pre-immunization titers) for antibodies to GI.1 (A) and to GII.4 (B) are 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. Volunteers were immunized on days 0 and 28 of the study.

Fig. 4. Results of ELISA assays measuring serum IgA in human volunteers immunized intramuscularly with a placebo (saline) or vaccine formulation containing 5, 15, or 50 µg of each of genogroup 1.1 norovirus VLP and genogroup II.4 norovirus VLP. Geometric mean titer of antibodies to GI. 1 (A) and to GII.4 (B) is shown for each of the dosage levels on days 7 and 21 after the first immunization and on days 7 and 28 after the second immunization. Volunteers were immunized on days 0 and 28 of the study.

Fig. 5. Results of ELISA assays measuring serum IgA in human volunteers immunized intramuscularly with a placebo (saline) or vaccine formulation containing 5, 15, or 50 μg each of genogroup 1.1 norovirus VLP and genogroup II.4 norovirus VLP. The seroconversion factor for antibodies to GI.1 (A) and to GII.4 (B) 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. Volunteers were immunized on days 0 and 28 of the study.

Fig. 6. Results of ELISA assays measuring serum IgA in human volunteers immunized intramuscularly with a placebo (saline) or vaccine formulation containing 5, 15, or 50 µg of each of genogroup 1.1 norovirus VLP and genogroup norovirus VLP

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II.4. Percentages of serologic response (i.e., a fourfold increase in antibody titer compared to pre-immunization titers) for anti-GI antibodies. 1 (A) and to GII.4 (B) are shown for each of the dosage levels on days 7 and 21 after the first immunization and on days 7 and 28 after the second immunization. Volunteers were immunized on days 0 and 28 of the study.

Fig. 7. Results of ELISA assays measuring serum IgG in human volunteers immunized intramuscularly with a placebo (saline) or vaccine formulation containing 5, 15, or 50 µg of each of genogroup 1.1 norovirus VLP and genogroup II.4 norovirus VLP. Geometric mean titer of antibodies to GI. 1 (A) and to GII.4 (B) is shown for each of the dosage levels on days 7 and 21 after the first immunization and on days 7 and 28 after the second immunization. Volunteers were immunized on days 0 and 28 of the study.

Fig. 8. Results of ELISA assays to measure serum IgG in human volunteers immunized intramuscularly with a placebo (saline) or vaccine formulation containing 5, 15, or 50 μg each of genogroup 1.1 norovirus VLP and genogroup II.4 norovirus VLP. The seroconversion factor for antibodies to GI.1 (A) and to GII.4 (B) 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. Volunteers were immunized on days 0 and 28 of the study.

Fig. 9. Results of ELISA assays to measure serum IgG in human volunteers immunized intramuscularly with a placebo (saline) or vaccine formulation containing 5, 15, or 50 µg of each of genogroup 1.1 norovirus VLP and genogroup II.4 norovirus VLP. Percentages of serologic response (i.e., a fourfold increase in antibody titer compared to pre-immunization titers) for anti-GI antibodies. 1 (A) and to GII.4 (B) are shown for each of the dosage levels on days 7 and 21 after the first immunization and on days 7 and 28 after the second immunization. Volunteers were immunized on days 0 and 28 of the study.

Fig. 10. Results of total Ig ELISA assays measuring pooled serum levels of IgG, IgA and IgM in human volunteers immunized with either a norovirus monovalent intranasal vaccine as described in El Kamary et al. (2010) J Infect Dis, Vol. 202(11): 1649-1658, (groups LV01-103), or a norovirus bivalent intramuscular vaccine as described in Example 1 (groups LV03-104), at the indicated time points. Human volunteers received either a placebo or two doses of the vaccine formulation either intramuscularly or intranasally. The bivalent intramuscular norovirus vaccine contained 5 μg each of genogroup 1.1 norovirus VLP and genogroup II.4 norovirus VLP. The monovalent intranasal vaccine contained 100 μg of norovirus genogroup 1.1. Volunteers receiving intranasal vaccine or placebo were challenged with live norovirus after the second immunization.

Fig. 11. FACS analysis of peripheral blood mononuclear cells obtained from human volunteers on day 0 before immunization with either a 5 μg dose of bivalent intramuscular norovirus vaccine (A) or placebo (B) and on day 7 after immunization. CD19+ PBMCs are mucosally targeted, as evidenced by the expression of the alpha4/beta7 homing receptor and the CCR10 chemokine receptor.

Detailed description of the invention

The present invention relates to methods for inducing protective immunity to norovirus infections in a subject. In particular, the present invention provides methods for inducing protective immunity against norovirus in a human by parenterally administering to a human no more than a single dose of a vaccine comprising a norovirus VLP and optionally at least one adjuvant, wherein the vaccine provides protection against or amelioration of at least one symptom of norovirus infections. The inventors have found that intramuscular administration of no more than a single dose of a vaccine composition containing norovirus VLP to humans induces rapid (i.e., within 7 days of immunization) serum seroconversion (i.e., at least a three-fold increase in antigen-specific titers). serum antibodies compared to pre-vaccination levels), indicating a protective immune response against norovirus infection and disease. Immune responses induced by this single dose vaccine composition achieve the same high level of antibody titers as observed in natural infection with live virus in human challenge studies. Notably, a booster dose of vaccine is not required because the immune response does not increase with an additional dose of vaccine.

The present invention provides a vaccine composition containing one or more norovirus antigens. Norovirus, norovirus (NOR), and grammatical equivalents are used herein to mean members of the genus Norovirus in the Caliciviridae family. In some embodiments, a norovirus may include a group of related non-enveloped positive polarity single-stranded RNA viruses that can be infectious to humans or non-human mammalian species. In some embodiments, norovirus can cause acute gastroenteritis in humans. Noroviruses may also be mentioned

- 3 042694 as small spherical structured viruses (SRSV) having a defined surface structure or irregular outline as seen by electron microscopy.

Noroviruses include at least five genogroups (GI, GII, GIII, GIV and GV). Noroviruses GI, GII, and GIV are infectious to humans, while noroviruses GIII mainly infect cattle. GV has recently been isolated from mice (Zheng et al. (2006) Virology, Vol 346: 312-323). GIII is typified by the Jena and Newbury strains, while GIV is typified by the Alphatron, Fort Lauderdale and Saint Cloud strains. Groups GI and GII can be further divided into genetic clusters or genotypes based on genetic classification (Ando et al. (2000) J. Infectious Diseases, Vol. 181(Supp2):S336-S348; Lindell et al. (2005) J. Clin Microbiol., Vol 43(3): 1086-1092). As used herein, the expression genetic clusters is used interchangeably with the expression genotypes. Genogroup I includes 8 GI clusters known to date (with the name of the prototype viral strain): GI.1 (Norwalk (NV-USA93)); GI.2 (Southhampton (SOV-GBR93)); GI.3 (Desert Shield (DSV-USA93)); GI.4 (Cruise Ship virus/Chiba (Chiba-JPN00)); GI.5 (318/Musgrove (Musgrov-GBR00)); GI.6 (Hesse (Hesse-DEU98)); GI.7 (WnchestGBR00) and GI.8 (Boxer-USA02). Genogroup II includes 19 GII clusters known to date (with the name of the prototype viral strain): GII.1 (Hawaii (Hawaii-USA94)); GII.2 (Snow Mountain/Melksham (Msham-GBR95)); GII.3 (Toronto (Toronto-CAN93)); GII.4 (Bristol/Lordsdale (BristolGBR93)); GII.5 (290/Hillingdon (Hilingd-GBR00)); GII.6 (269/Seacroft (Seacrof-GBR00)); GII.7 (273/Leeds (Leeds-GBR00)); GII.8 (539/Amsterdam (Amstdam-NLD99)); GII.9 (378 (VABeach-USA01)), GII.10 (ErfurtDEU01); GII.11 (SW9180JPN01); GII.12 (Wortley-GBR00); GII.13 (Faytvil-USA02); GII.14 (M7-USA03); GII.15 (J23-USA02); GII.16 (Tiffin-USA03); GII.17 (CSE1-USA03); GII.18 (QW101/2003/US) and GII.19 (QW170/2003/US).

Norovirus is also herein understood to mean recombinant norovirus virus-like particles (VLP rNOR). In some embodiments, recombinant expression of at least a norovirus capsid protein encoded by ORF2 in cells, for example from a baculovirus vector in Sf9 cells, can result in the capsid protein spontaneously self-assembling into VLPs. In some embodiments, recombinant expression of at least the norovirus proteins encoded by ORF1 and ORF2 in cells, for example, from a baculovirus vector in Sf9 cells, can lead to spontaneous self-assembly of the capsid protein into the VLP. VLPs are structurally similar to noroviruses, but they lack a viral RNA genome and are therefore not infectious. Accordingly, norovirus includes virions, which may be infectious or non-infectious particles, which include incomplete particles.

Non-limiting examples of noroviruses include genogroup 1 norovirus strain Hu/NoV/West Chester/200 I/USA, GenBank accession number AY502016; Chiba virus (CHV, GenBank AB042808); norovirus genogroup 2 strain Hu/NoV/Braddock Heights/1999/USA, GenBank accession number AY502015; norovirus genogroup 2 strain Hu/NoV/Fayette/1999/USA, GenBank accession number AY502014; norovirus genogroup 2 strain Hu/NoV/Fairfield/1999/USA, GenBank accession number AY502013; norovirus genogroup 2 strain Hu/NoV/Sandusky/1999/USA, GenBank accession number AY502012; norovirus genogroup 2 strain Hu/NoV/Canton/1999/USA, GenBank accession number AY502011; norovirus genogroup 2 strain Hu/NoV/Tiffin/1999/USA, GenBank accession number AY502010; norovirus genogroup 2 strain Hu/NoV/CS-E1/2002/USA, GenBank accession number AY50200; norovirus genogroup 1 strain Hu/NoV/Wisconsin/2001/USA, GenBank accession number AY502008; norovirus genogroup 1 strain Hu/NoV/CS-841/2001/USA, GenBank accession number AY502007; norovirus genogroup 2 strain Hu/NoV/Hiram/2000/USA, GenBank accession number AY502006; norovirus genogroup 2 strain Hu/NoV/Tontogany/1999/USA, GenBank accession number AY502005; Norwalk virus, complete genome, GenBank accession number NC.sub.--001959; norovirus Hu/GI/Otofuke/1979/JP, genomic RNA, complete genome, GenBank accession number AB187514; norovirus Hu/Hokkaido/133/2003/JP, GenBank accession number AB212306; norovirus Sydney 2212, GenBank accession number AY588132; Norwalk virus strain SN2000JA, GenBank accession number AB190457; Lordsdale virus, complete genome, GenBank accession number X86557; Norwalk-like virus, genomic RNA, Gifu'96, GenBank accession number AB045603; Norwalk virus strain Vietnam 026, complete genome, GenBank accession number AF504671; norovirus Hu/GII.4/2004/N/L, GenBank accession number AY883096; norovirus Hu/GII/Hokushin/03/JP, GenBank accession number AB195227; norovirus Hu/GII/Kamo/03/JP, GenBank accession number AB195228; norovirus Hu/GII/Sinsiro/97/JP, GenBank accession number AB195226; norovirus Hu/GII/Ina/02/JP, GenBank accession number AB195225; norovirus Hu/NLV/GII/Neustrelitz260/2000/DE, GenBank accession number AY772730; norovirus Hu/NLV/Dresden174/pUS-NorII/1997/GE, GenBank accession number AY741811; norovirus Hu/NLV/Oxford/B2S16/2002/UK, GenBank accession number AY587989; norovirus

Hu/NLV/Oxford/B4S7/2002/UK, number access V GenBank AY587987; norovirus Hu/NLV/Witney/B7S2/2003/UK, number access V GenBank AY588030; norovirus Hu/NLV/Banbury/B9S23/2003/UK, number access V GenBank AY588029; norovirus Hu/NLV/ChippingNorton/2003/UK, number access V GenBank AY588028; norovirus Hu/NLV/Didcot/B9S2/2003/UK, number access V GenBank AY588027; norovirus

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Hu/NLV/Oxford/B8S5/2002/UK, number access V GenBank AY588026; norovirus Hu/NLV/Oxford/B6S4/2003/UK, number access V GenBank AY588025; norovirus Hu/NLV/Oxford/B6S5/2003/UK, number access V GenBank AY588024; norovirus Hu/NLV/Oxford/B5S23/2003/UK, number access V GenBank AY588023; norovirus Hu/NLV/Oxford/B6S2/2003/UK, number access V GenBank AY588022; norovirus

Hu/NLV/Oxford/B6S6/2003/UK, GenBank accession number AY588021; Norwalk-like virus isolate Bo/Thirsk10/00/UK, GenBank accession number AY126468; Norwalk-like virus isolate Bo/Penrith55/00/UK, GenBank accession number AY126476; Norwalk-like virus isolate Bo/Aberystwyth24/00/UK, GenBank accession number AY126475; Norwalk-like virus isolate Bo/Dumfries/94/UK, GenBank accession number AY126474; norovirus NLV/IF2036/2003/Iraq, GenBank accession number AY675555; norovirus NLV/IF1998/2003/Iraq, GenBank accession number AY675554; norovirus NLV/BUDS/2002/USA, GenBank accession number AY660568; norovirus NLV/Paris Island/2003/USA, GenBank accession number AY652979; Snow Mountain virus, complete genome, GenBank accession number AY134748; Norwalk-like virus NLV/Fort Lauderdale/560/1998/US, GenBank accession number AF414426; Hu/Norovirus/hiroshima/1999/JP(9912-02F), GenBank accession number AB044366; Norwalk-like virus strain 11MSU-MW, GenBank accession number AY274820; Norwalk-like virus strain B-1SVD, GenBank accession number AY274819; norovirus genogroup 2 strain Hu/NoV/Farmington Hills/2002/USA, GenBank accession number AY502023; norovirus genogroup 2 strain Hu/NoV/CS-G4/2002/USA, GenBank accession number AY502022; norovirus genogroup 2 strain Hu/NoV/CS-G2/2002/USA, GenBank accession number AY502021; norovirus genogroup 2 strain Hu/NoV/CS-G1/2002/USA, GenBank accession number AY502020; norovirus genogroup 2 strain Hu/NoV/Anchorage/2002/USA, GenBank accession number AY502019; norovirus genogroup 2 strain Hu/NoV/CS-D1/2002/CAN, GenBank accession number AY502018; norovirus genogroup 2 strain Hu/NoV/Germanton/2002/USA, GenBank accession number AY502017; human calicivirus NLV/GII/Langen1061/2002/DE, complete genome, GenBank accession number AY485642; murine norovirus 1, polyprotein, GenBank accession number AY228235; Norwalk virus, GenBank accession number AB067536; human calicivirus NLV/Mex7076/1999, GenBank accession number AF542090; human calicivirus NLV/Oberhausen 455/01/DE, GenBank accession number AF539440; human calicivirus NLV/Herzberg 385/01/DE, GenBank accession number AF539439; human calicivirus NLV/Boxer/2001/US, GenBank accession number AF538679; Norwalk-like virus, genomic RNA, complete genome, GenBank accession number AB081723; Norwalk-like virus, genomic RNA, whole genome, isolate: Saitama U201, GenBank accession number AB039782; Norwalk-like virus, genomic RNA, whole genome, isolate: Saitama U18, GenBank accession number AB039781; Norwalk-like virus, genomic RNA, whole genome, isolate: Saitama U25, GenBank accession number AB039780; Norwalk virus strain: U25G, GenBank accession number AB067543; Norwalk virus strain: U201 GII, GenBank accession number AB067542; Norwalk-like viruses, strain 416/97003156/1996/LA, GenBank accession number AF080559; Norwalk-like viruses, strain 408/97003012/1996/FL, GenBank accession number AF080558; Norwalk-like virus NLV/Burwash Landing/331/1995/US, GenBank accession number AF414425; Norwalk-like virus NLV/Miami Beach/326/1995/US, GenBank accession number AF414424; Norwalk-like virus NLV/White River/290/1994/US, GenBank accession number AF414423; Norwalk-like virus NLV/New Orleans/306/1994/US, GenBank accession number AF414422; Norwalk-like virus NLV/Port Canaveral/301/1994/US, GenBank accession number AF414421; Norwalk-like virus NLV/Honolulu/314/1994/US, GenBank accession number AF414420; Norwalk-like virus

NLV/Richmond/283/1994/US, GenBank accession number AF414419; Norwalk-like virus

NLV/Westover/302/1994/US, GenBank accession number AF414418; Norwalk-like virus NLV/UK317/12700/1992/GB, GenBank accession number AF414417; Norwalk-like virus NLV/Miami/81/1986/US, GenBank accession number AF414416; strain Snow Mountain, GenBank accession number U70059; Desert Shield DSV395 virus, GenBank access number U04469; Norwalk virus, complete genome, GenBank accession number AF093797; Hawaii calicivirus, GenBank accession number U07611; Southampton virus, GenBank accession number L07418; Norwalk virus (SRSV-KY-89/89/J), GenBank accession number L23828; Norwalk virus (SRSV-SMA/76/US), GenBank accession number L23831; Camberwell virus, GenBank access number U46500; human calicivirus strain Melksham, GenBank accession number X81879; human calicivirus strain MX, GenBank accession number U22498; minireovirus TV24, GenBank access number U02030; and Norwalk-like virus NLV/Gwynedd/273/1994/US, GenBank accession number AF414409; the sequences of all of which (as entered on the filing date of this application) are incorporated herein by reference. Additional norovirus sequences are disclosed in the following patent publications: WO 2005/030806, WO 2000/79280, JP2002020399, US2003129588, US Pat. . For sequence comparisons and for a review of the genetic diversity and phylogenetic analysis of noroviruses, see also Green et al. (2000) J. Infect. Dis., Vol. 181(Suppl. 2):S322-330; Wang et al. (1994) J. Virol., Vol. 68:59825990; Chen et al. (2004) J. Virol., Vol. 78: 6469-6479; Chakravarty et al. (2005) J. Virol., Vol. 79:554-568;

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Hansman et al. (2006) J. Gen. Virol., Vol. 87:909-919; Bull et al. (2006) J. Clin. Micro., Vol. 44(2):327-333; Siebenga, et al. (2007) J. Virol., Vol. 81(18):9932-9941 and Fankhauser et al. (1998) J. Infect. Dis., Vol. 178:15711578. All nucleic acids and the corresponding amino acid sequences of each are incorporated by reference in their entirety. In some embodiments, a code may be used for identification purposes and is in the following order: host species from which the virus was isolated/abbreviated genus designation/abbreviated species designation/strain name/year of discovery/country of origin (Green et al., Human Caliciviruses , in Fields Virology Vol.1 841-874 (Knipe and Howley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)). Genotype II (GII.4) genotype 4 virus strains (eg, strains of Houston, Minerva (also known as Den Haag), and Laurens (also known as Yerseke)) are preferred in some embodiments. As new strains are identified and their genetic sequences become available, one skilled in the art will be able to apply VLPs using these current strains in the compositions and methods of the present invention using conventional techniques. Thus, the present invention contemplates VLPs derived from such strains as suitable antigens for use in the compositions and methods described herein.

Norovirus antigens can be in the form of peptides, proteins, or virus-like particles (VLPs). In a preferred embodiment, norovirus antigens include VLPs. As used herein, the expression virus-like particle(s) or VLP refers to a virus-like particle(s), fragment(s), complexes, or portion(s) thereof derived from the norovirus capsid protein coding sequence and comprising an antigenic characteristic(s). ), similar to those of norovirus infectious particles. Norovirus antigens may also be in the form of capsid monomers, capsid multimers, VLP protein or peptide fragments, or complexes or mixtures thereof. Norovirus antigenic proteins or peptides may also be in denatured form and may be prepared by methods known in the art.

The VLPs of the present invention can be formed from either a full length norovirus capsid protein, such as the VP1 and/or VP2 proteins, or certain derivatives of VP1 or VP2, by standard methods of 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 contains a truncated VP1 protein. In other embodiments, all VLPs contain truncated VP1 proteins. The truncation may be N- or C-terminal truncation. Truncated capsid proteins are suitable functional derivatives of the capsid protein. Functional derivatives of the capsid protein are capable of eliciting an immune response (if necessary, in the presence of a suitable adjuvant) in the same way that VLPs consisting of a full length capsid protein elicit an immune response.

VLPs may contain major VP1 proteins and/or minor VP2 proteins. In some embodiments, each VLP contains a VP1 and/or VP2 protein from a norovirus of only one genogroup, forming a monovalent VLP. As used herein, the term "monovalent" means that the antigenic proteins are derived from a norovirus of the same genogroup. For example, VLPs contain VP1 and/or VP2 from a strain of genogroup I virus (eg, VP1 and VP2 from Norwalk virus). Preferably the VLPs are predominantly composed of VP1 proteins. In one embodiment of the present invention, the antigen is a mixture of monovalent VLPs, where the composition comprises VLPs consisting of VP1 and VP2 from a norovirus of one genogroup mixed with VLPs consisting of VP1 and VP2 of a norovirus of another genogroup (e.g., Norwalk virus and Houston virus) obtained from several viral strains. By way of example only, the composition may comprise monovalent VLPs from one or more genogroup I norovirus strains together with monovalent VLPs from one or more genogroup II norovirus strains. Strains can be selected based on their predominance in the bloodstream at a given time. In certain embodiments, the norovirus VLP mixture comprises the GI.1 and GII.4 viral strains. More preferably, the norovirus VLP mixture contains a sequence of Norwalk strains and a consensus capsid protein sequence derived from genogroup II noroviruses. Capsid protein consensus sequences derived from circulating norovirus sequences and VLPs generated from such sequences are described in WO 2010/017542, which is incorporated herein by reference in its entirety. For example, in one embodiment, the consensus capsid protein sequence derived from genotype II (GII.4) genotype 4 viral strains contains the sequence SEQ ID NO: 1. Thus, in some embodiments, the vaccine composition contains a mixture of monovalent VLPs, VLPs contain a capsid protein from genogroup I norovirus (e.g., Norwalk), and other monovalent VLPs contain a consensus sequence capsid protein containing the sequence of SEQ ID NO: 1.

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MKMASSDANPSDGSTANLVPEVNNEVMALEPVVGAAIAAPV AGQQNVIDPWIRNNFVQAPGGEFTVSPRNAPGEILWSAPLGP DLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKIIFAAV PPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYH YNQSNDPTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPD FDFIFLVPPTVESRTKPFTVPILTVEEMTNSRFPIPLEKLFTGP SGAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHIAG TQEYTMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGVLTQ TTRGDGSTRGHKATVSTGSVHFTPKLGSVQFSTDTSNDFETG QNTKFTPVGVVQDGSTTHQNEPQQWVLPDYSGRDSHNVHL APAVAPTFPGEQLLFFRSTMPGCSGYPNMNLDCLLPQEWVQ HFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVA HTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRAL (SEQ ID NO: 1).

However, in an alternative embodiment of the present invention, the VLPs may be multivalent VLPs that contain, for example, VP1 and/or VP2 proteins from a norovirus of one genogroup mixed with VP1 and/or VP2 proteins from a norovirus of a second genogroup, where different VP1 and VP2 proteins are not VP1 and VP2 chimeric proteins, but linked together in a single capsid structure to form immunogenic VLPs. As used herein, the expression multivalent means that the antigenic proteins are derived from two or more genogroups or strains of norovirus. Multivalent VLPs may contain VLP antigens taken from two or more viral strains. By way of example only, the composition may comprise multivalent VLPs consisting of capsid monomers or multimers from one or more genogroup I norovirus strains (e.g., Norwalk virus) together with capsid monomers or multimers from one or more genogroup II norovirus strains (e.g., Houston virus) . Preferably, the multivalent VLPs contain capsid proteins from Norwalk and Houston strains of norovirus or other currently predominant circulating strains.

The combination of monovalent or multivalent VLPs in a composition will preferably not reduce the immunogenicity of each type of VLP. In particular, it is preferred that there is no interaction between the norovirus VLPs in the combination of the present invention, so that the combined VLP composition of the present invention is capable of stimulating immunity to infection by each norovirus genotype present in the vaccine. Accordingly, the immune response against a given VLP type in combination is at least 50% of the immune response against the same VLP type when measured alone, preferably 100% or substantially 100%. The immune response can be appropriately determined, for example, by means of humoral immune responses, as illustrated in the examples of this document.

As used herein, the term genogroup I norovirus VLPs refers to either monovalent or multivalent VLPs that contain a capsid protein derived from one or more strains of genogroup I norovirus. I (for example, the Norwalk virus). In other embodiments, the genogroup I norovirus VLPs comprise a consensus sequence capsid protein derived from different genogroup I strains. The genogroup I strains from which the consensus capsid protein sequence is derived may be in the same genotype or genetic cluster, or in different genotypes, or genetic clusters. Similarly, as used herein, the term genogroup II norovirus VLP refers to either monovalent or multivalent VLPs that contain a capsid protein derived from one or more strains of genogroup II norovirus. In some embodiments, the genogroup II norovirus VLPs comprise a full length capsid protein from a genogroup II norovirus (eg Laurens or Minerva virus). In other embodiments, the genogroup II norovirus VLPs comprise a consensus sequence capsid protein derived from various genogroup II strains. The strains of genogroup II from which the consensus sequence of the capsid protein is derived may belong to the same genotype or genetic cluster, or to different genotypes or genetic clusters. In one embodiment, the genogroup II norovirus VLPs comprise a consensus capsid protein sequence from genogroup II norovirus 4 genotype (GII.4). Thus, in some embodiments, genogroup II norovirus VLPs comprise

- 7 042694 capsid protein sequence SEQ ID NO: 1.

Multivalent VLPs can be generated by separate expression of individual capsid proteins followed by combination to produce VLPs. Alternatively, multiple capsid proteins can be expressed in the same cell from one or more DNA constructs. For example, host cells can be transformed or transfected with a variety of DNA constructs, with each vector encoding a different capsid protein. Alternatively, a single vector with multiple capsid protein genes driven by a common promoter or several separate promoters can be used. IRES elements can also be embedded in the vector if desired. With these expression strategies, co-expressed capsid proteins can be co-purified to further form VLPs, or they can spontaneously form multivalent VLPs that can then be purified.

A preferred method for making multivalent VLPs involves making capsid proteins or VLP derivatives such as VP1 proteins from noroviruses of different genotypes, mixing the proteins, and assembling the proteins to make multivalent VLPs. The VP1 proteins may be in the form of a crude extract, may be partially purified or purified prior to mixing. The assembled monovalent VLPs of different genogroups can be taken apart, mixed together and reassembled into multivalent VLPs. Preferably, the proteins or VLPs are at least partially purified prior to combination. Optionally, additional purification of multivalent VLPs can be carried out after assembly.

Suitably, the VLPs of the present invention are obtained by disassembling and reassembling VLPs to provide homogeneous and clean VLPs. In one embodiment, multivalent VLPs can be obtained by disassembling two or more VLPs, and then recombining the disassembled VLP components at any appropriate time prior to reassembly. This approach can be applied when VLPs are generated spontaneously from the expressed VP1 protein, which occurs, for example, in some yeast strains. In the case where expression of the VP1 protein does not spontaneously form a VLP, the production of VP1 proteins or capsomers can be combined prior to assembly into a VLP.

In the case where multivalent VLPs are used, the VLP components are preferably blended at the percentage they should be in the final blended VLPs. For example, a mixture of the same amount of partially purified VP1 protein from Norwalk and Houston viruses (or other strains of norovirus) provides multivalent VLPs with approximately equal amounts of each protein.

Compositions containing multivalent VLPs can be stabilized using solutions known in the art, such as those from WO 98/44944, WO 00/45841, incorporated herein by reference.

The compositions of the present invention may contain other proteins or protein fragments in addition to the norovirus VP1 and VP2 proteins or derivatives. Other proteins or peptides may also be administered with the composition of the present invention. Optionally, the composition may also be formulated or administered with antigens other than norovirus antigens. Accordingly, these antigens may confer protection against other diseases.

The VP1 protein or functional protein derivative is suitably capable of forming VLPs, and VLP formation can be assessed using standard techniques such as, for example, size exclusion chromatography, electron microscopy, and dynamic laser light scattering.

The antigen molecules of the present invention can be obtained by isolation and purification from the organisms in which they occur naturally, or they can be obtained using recombination techniques. Preferably norovirus VLP antigens are derived from insect cells such as Sf9 or H5 cells, although any suitable cell such as E. coli or yeast cells, e.g. S. cerevisiae, S. pombe, Pichia pastori or other expression systems based on Pichia, mammalian cell based expression systems such as CHO or HEK based expression systems. When obtained using a recombinant method or by synthesis, one or more insertions, deletions, inversions or substitutions of the amino acids that make up the peptide can be made. Each of the above antigens is preferably used in a substantially pure state.

Procedures for obtaining norovirus VLPs in insect cell culture have previously been disclosed in US Pat. No. 6,942,865, which is hereby incorporated by reference in its entirety. Briefly, cDNA is cloned from the 3' end of the genome containing the viral capsid gene (ORF2) and the small structural gene (ORF3). Recombinant baculoviruses carrying viral capsid genes are constructed from cloned cDNA. Norovirus VLPs are obtained in Sf9 or H5 insect cell cultures.

In some embodiments, the vaccine composition comprises one or more adjuvants in combination with a norovirus antigen. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and an immune response stimulator, such as proteins derived from Bordatella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are commercially available, such as incomplete Freund's adjuvant and complete adjuvant (Pifco Laboratories, Detroit, Michigan); adjuvant Merck Adjuvant 65 (Merck and Company, Inc., Rahway, New Jersey); aluminum salts such as aluminum hydroxide gel

- 8 042694 (alum) or aluminum phosphate; salts of calcium, iron or zinc; insoluble suspension of acylated tyrosine, acylated sugars; cationically or anionically derived polysaccharides; polyphosphazenes; biodegradable microspheres and Quil A.

Suitable adjuvants also include, without limitation, ton-like receptor (TLR) agonists, in particular toll-like type 4 receptor (TLR-4) agonists (e.g., monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogues ), aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligonucleotides, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, microparticles of copolymers (lactide and glycolide) (PLG), poloxamer particles, microparticles, liposomes, oil-in-water emulsions, MF59 and squalene. In some embodiments, the adjuvants are not exotoxins of bacterial origin. Preferred adjuvants include adjuvants that stimulate a Th1-type response such as 3DMPL or QS21.

Monophosphoryl lipid A (MPL), a non-toxic lipid A derivative derived from Salmonella, is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al. 2003). In preclinical studies in mice, intranasal administration of MPL has been shown to enhance secretory as well as systemic humoral responses (Baldridge et al. 2000; Yang et al. 2002). It has also been shown to be safe and effective as a vaccine adjuvant in over 120,000 clinical trials (Baldrick et al., 2002; Baldridge et al. 2004). MPL stimulates the induction of innate immunity via the TLR-4 receptor and thus is able to stimulate non-specific immune responses against a wide range of infectious pathogens, including gram-negative and gram-positive bacteria, viruses and parasites (Baldridge et al. 2004; Persing et al. 2002 ). The inclusion of MPL in vaccine formulations should ensure rapid induction of innate responses, stimulating the formation of non-specific immune responses, after the introduction of a viral challenge and, at the same time, enhance specific responses caused by the antigenic components of the vaccine.

In one embodiment, the present invention provides a composition containing monophosphoryl lipid A (MPL) or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) as an adaptive and innate immune stimulant. Chemically, 3D-MPL is a mixture of 3-de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred form of 3-de-O-acylated monophosphoryl lipid A is disclosed in EP 0689454 B1 (SmithKline Beecham Biologicals SA), which is incorporated herein by reference. In another embodiment, the present invention provides a composition comprising synthetic lipid A, lipid A mimetics or analogs, such as PET lipid A from BioMira, or synthetic derivatives designed to function as TLR-4 agonists.

In certain embodiments, the vaccine composition contains two adjuvants. The combination of adjuvants can be selected from the combinations described above. In one particular embodiment, the two adjuvants are MPL and aluminum hydroxide (eg, alum). In another specific embodiment, the two adjuvants are MPL and oil.

The term "effective amount of adjuvant" will be well understood by one skilled in the art and includes the amount of one or more adjuvants that is capable of stimulating an immune response to the administered antigen, i.e. an amount that enhances the immune response to the administered antigen composition as measured by IgA levels in nasal swabs, serum IgG or IgM levels, or B and T cell proliferation. Suitable effective increases in immunoglobulin levels include more than 5%, preferably more than 25% and in particular more than 50% compared to the same antigen composition without any adjuvant.

In one embodiment, the present invention provides a vaccine composition formulated for parenteral administration, wherein the composition comprises at least two types of norovirus VLPs in combination with aluminum hydroxide and a buffer. The buffer may be selected from the group consisting of L-histidine, imidazole, succinic acid, tris, citric acid, bis-tris, PIPES, MES, HEPES, glycinamide and tricine. In one embodiment, the buffer is L-histidine or imidazole. Preferably the buffer is present at a concentration of from about 15 mM to about 50 mM, more preferably from about 18 mM to about 40 mM, or most preferably from about 20 mM to about 25 mM. In some embodiments, the pH of the antigen or vaccine composition is from about 6.0 to about 7.0, or from about 6.2 to about 6.8, or about 6.5. The vaccine composition may be an aqueous formulation. In some embodiments, the vaccine composition is a lyophilized powder and is resuspended to form an aqueous formulation.

In certain embodiments, the vaccine composition further comprises at least one adjuvant in addition to two or more types of norovirus VLP, aluminum hydroxide, and a buffer. For example, the adjuvant may be a toll-like receptor agonist such as MPL, flagellin, CpG oligonucleotides, synthetic lipid A, or lipid A mimetics or analogs. In one specific embodiment, the adjuvant is MPL.

- 9 042694

The norovirus VLPs that are included in the vaccine compositions of the present invention may be any of the VLPs described herein. In one embodiment, each of the two types of norovirus VLPs contains a capsid protein of different genogroups (eg, genogroup I and genogroup II). For example, one type of norovirus VLPs contain a capsid protein derived from a genogroup I norovirus, and another type of norovirus VLPs contain a capsid protein derived from a genogroup II norovirus. In one embodiment, one type of norovirus VLPs contain a capsid protein from the Norwalk virus, and another type of norovirus VLPs contain a capsid protein with a consensus sequence derived from genotype 4 noroviruses of genogroup II (e.g., a capsid protein containing the sequence of SEQ ID NO: 1). The vaccine composition may contain from about 5 µg to about 200 µg of each type of norovirus VLP, more preferably from about 15 µg to about 50 µg of each type of norovirus VLP. In some embodiments, the dose of one type of norovirus VLP is different from the other type of norovirus VLP. For example, in certain embodiments, the vaccine composition contains from about 5 µg to about 15 µg of genogroup I VLP and from about 15 µg to about 50 µg of genogroup II VLP. In other embodiments, the vaccine composition contains from about 15 µg to about 50 µg of genogroup I VLP and from about 50 µg to about 150 µg of genogroup II VLP.

In some embodiments, the vaccine compositions further comprise a pharmaceutically acceptable salt, including, without limitation, sodium chloride, potassium chloride, sodium sulfate, ammonium sulfate, and sodium citrate. In one embodiment, the pharmaceutically acceptable salt is sodium chloride. The concentration of the pharmaceutically acceptable salt may be from about 10 mM to about 200 mM, with preferred concentrations ranging from about 100 mM to about 150 mM. Preferably, the vaccine compositions of the present invention contain less than 2 mM free phosphate. In some embodiments, the vaccine compositions contain less than 1 mM free phosphate. Vaccine compositions may also additionally contain other pharmaceutically acceptable excipients such as sugars (eg sucrose, trehalose, mannitol) and surfactants.

As discussed herein, the compositions of the present invention can be formulated for administration as vaccine formulations. As used herein, the expression vaccine refers to a formulation that contains norovirus VLPs or other norovirus antigens of the present invention as described above, which is in a form that can be administered to vertebrates, in particular humans, and which induces a protective immune response sufficient to induce immunity to prevent and/or alleviate the course of a norovirus infection or norovirus-induced disease and/or to reduce at least one symptom of a norovirus infection or disease.

As used herein, the term immune response refers to both a humoral immune response and a cell-mediated immune response. The humoral immune response includes stimulating the production of antibodies by B lymphocytes, which, for example, neutralize infectious agents, prevent the entry of infectious agents into cells, prevent the replication of said infectious agents, and/or protect host cells from infection and destruction. Cell-mediated immune response refers to an immune response mediated by T-lymphocytes and/or other cells such as macrophages against an infectious agent in vertebrates (e.g., humans) that prevents or alleviates infection or reduces at least one of its symptoms. . In particular, protective immunity or protective immune response refers to immunity or stimulation of the formation of an immune response against an infectious agent, manifested in vertebrates (for example, humans), which prevents or alleviates the infection or reduces at least one of its symptoms. In particular, the induction of a protective immune response upon administration of a vaccine is indicated by the elimination or reduction of one or more symptoms of acute gastroenteritis, or by a reduction in the duration or severity of such symptoms. Clinical symptoms of norovirus gastroenteritis include nausea, diarrhea, loose stools, vomiting, fever, and general malaise. A protective immune response that reduces or eliminates the symptoms of a disease will reduce or stop the spread of an epidemic of norovirus in a population. Vaccine preparation is generally described in Vaccine Design (The subunit and adjuvant approach (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). The compositions of the present invention can be formulated, for example, for delivery to one or more of the mucous membranes of the oral cavity, gastrointestinal tract and respiratory tract (eg, nasal cavity). The compositions of the present invention can be formulated, for example, for delivery by injection, such as parenteral injection (eg intravenous, subcutaneous, intradermal or intramuscular injection).

When the composition is intended to be delivered to the mucosa of the respiratory tract (e.g., nasal cavity), it is usually formulated as an aqueous solution for administration in the form of an aerosol or nasal drops, or alternatively, in the form of a dry powder, for example, for rapid accumulation

- 10 042694 in the nasal passage. Compositions for administration in the form of nasal drops may contain one or more adjuvants such as are commonly included in such compositions, for example, preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents, and the like. Viscosity modifiers can be microcrystalline cellulose, chitosan, starches, polysaccharides, and the like. Compositions for administration in the form of a dry powder may also contain one or more adjuvants such as are commonly included in such compositions, for example, mucoadhesive agents, excipients, and agents for imparting appropriate flow and volume characteristics to the powder. Fillers and powder flow and bulk agents may include mannitol, sucrose, trehalose, and xylitol.

When the composition is intended for parenteral injection, such as intravenous (i.v.), subcutaneous (s.c), intradermal or intramuscular (i.m.) injection, it is usually formulated in the form of a liquid suspension (i.e., an aqueous formulation) containing at least one norovirus VLP type; and optionally at least one adjuvant. In one embodiment, the adjuvant may be MPL. In another embodiment, a liquid vaccine formulated for parenteral administration may contain more than one adjuvant. In a preferred embodiment, the liquid vaccine formulated for parenteral administration (e.g., formulated for i.m., i.v., or s.c. administration) contains genogroup I and/or genogroup II norovirus VLPs with aluminum hydroxide (e.g., alum) and monophosphoryl lipid A (MPL) as adjuvants. In one embodiment, the parenteral liquid formulation comprises a norovirus genogroup antigen(s), such as one or more of the norovirus VLP types described herein, MPL, aluminum hydroxide, and a buffer. In another embodiment, the parenteral liquid formulation comprises the norovirus genogroup antigen(s), MPL, an oil, and a buffer. In certain embodiments, the buffer in parenteral vaccine formulations is L-histidine or imidazole. Parenteral administration of liquid vaccines can be done with a needle and syringe, as is well known in the art.

In certain embodiments, the vaccine composition of the present invention for stimulating a protective immune response against norovirus in humans comprises a genogroup I and/or genogroup II norovirus VLP at a dose of not more than 150 μg. For example, in some embodiments, the vaccine composition contains no more than 150 µg, no more than 100 µg, no more than 50 µg, no more than 25 µg, no more than 15 µg, or no more than 10 µg of genogroup I norovirus VLP. In other embodiments, the vaccine composition contains not more than 150 µg, not more than 100 µg, not more than 50 µg, not more than 25 µg, not more than 15 µg, or not more than 10 µg VLP of norovirus genogroup II. In certain embodiments, the vaccine composition contains no more than 150 μg of each of genogroup I and genogroup II norovirus VLPs. In such embodiments, the dosage of genogroup I norovirus VLP and genogroup II VLP may be the same or different. For example, in one embodiment, the vaccine composition may contain no more than 50 μg of genogroup I norovirus VLP and no more than 150 μg of genogroup II norovirus VLP. In another embodiment, the vaccine composition may contain no more than 25 μg of genogroup I norovirus VLP and no more than 50 μg of genogroup II norovirus VLP. In other embodiments, the vaccine composition may contain no more than 15 μg of genogroup I norovirus VLP and no more than 50 μg of genogroup II norovirus VLP. In still other embodiments, the vaccine composition may contain no more than 25 μg of genogroup I norovirus VLP and no more than 150 μg of genogroup II norovirus VLP.

Norovirus genogroup I and genogroup II VLPs can be obtained from any norovirus strain described herein. In one embodiment, the genogroup I norovirus VLPs are genogroup I genotype 1 (GI.1) VLPs (ie, contain the capsid protein from norovirus GI.1). In another embodiment, the genogroup I norovirus VLPs are Norwalk virus VLPs. In another embodiment, the genogroup II norovirus VLPs are genogroup II (GII.4) genotype 4 VLPs. In another embodiment, the genogroup II norovirus VLPs are VLPs resulting from the expression of a genogroup II norovirus consensus sequence. In a specific embodiment, genogroup II norovirus VLPs comprise a capsid protein with the sequence of SEQ ID NO: 1.

Vaccine compositions described earlier herein can be lyophilized and stored anhydrous until ready for use, at which time they are resuspended with a diluent. Alternatively, the various components of the composition may be stored separately in a kit (any or all of the components may be lyophilized). The components may be left in lyophilized form for dry formulations or may be resuspended for liquid formulations and either mixed prior to use or administered separately to the patient. In some embodiments, the vaccine compositions are stored in kits in the form of liquid formulations and may be accompanied by delivery devices such as syringes with needles. In other embodiments, liquid vaccine formulations may be stored in delivery devices in a kit. For example, the kit may contain pre-filled syringes, auto-injectors, or pen devices containing the liquid formulation of the vaccine composition described herein.

- 11 042694

Lyophilization of vaccines is well known in the art. Typically, the liquid antigen is lyophilized in the presence of agents to protect the antigen during the lyophilization process and to obtain a pellet with the desired powder characteristics. Sugars such as sucrose, mannitol, trehalose, or lactose (present at an initial concentration of 10-200 mg/ml) are commonly used to cryoprotect protein antigens and to produce a lyophilized pellet with the desired powder characteristics. Lyophilization of compositions theoretically results in a more stable composition.

The amount of antigen in each vaccine composition is chosen as the amount that induces a strong immune response without significant adverse side effects. This amount will vary depending on which specific antigen(s) are used, the route of administration and the adjuvants used. In general, the dose administered to a patient according to the present invention should be sufficient to elicit a protective immune response in the patient over time or to induce the production of antigen-specific antibodies. Thus, the composition is administered to a patient in an amount sufficient to stimulate the formation of an immune response to specific antigens and/or to prevent, alleviate, reduce or cure the symptoms and/or complications of a disease or infection and thereby reduce or stop the spread of the norovirus epidemic in the population. . An amount sufficient to accomplish the foregoing is defined as a therapeutically effective dose.

Vaccine compositions of the present invention may be administered via a mucosal or non-mucosal route. These routes of administration may include in vivo administration via parenteral injection (such as intravenous, subcutaneous, intradermal and intramuscular), or other conventional direct routes such as buccal/sublingual, rectal, oral, nasal, topical (such as transdermal and ophthalmic), vaginal, pulmonary, intra-arterial, intraperitoneal, intraocular or intranasal routes, or administration directly to a specific tissue. Other suitable routes of administration include transdermal, subdermal and suppository. In one embodiment, the vaccine is administered via a parenteral route such as intravenous, subcutaneous, intradermal, or intramuscular. In certain embodiments, the vaccine is administered via the intramuscular route of administration. Administration can be accomplished simply by direct administration with a needle, catheter, or similar device (eg, pre-filled syringes or auto-injectors) at the same time or at different times. Other parenteral formulations may be delivered subcutaneously or intradermally via microinjection or transdermal patch delivery methods.

The present invention provides methods for inducing protective immunity against norovirus in a subject, comprising parenterally administering to the subject no more than a single dose of a vaccine composition of the present invention, wherein said vaccine contains the genogroup I and/or genogroup II norovirus VLPs described herein and optionally at least one adjuvant. In such embodiments, the single-dose vaccine composition induces at least a three-fold increase in norovirus-specific serum antibody titer compared to the human titer prior to administration of the composition. In some embodiments, the single dose vaccine composition induces at least a six-fold increase in norovirus-specific serum antibody titer compared to the titer in a human prior to administration of the composition. In other embodiments, the single dose vaccine composition induces a norovirus-specific serum antibody titer comparable to the antibody titer induced by exposure to live norovirus in natural infection, i. more than a tenfold increase in the titer of norovirus-specific serum antibodies compared to the titer in humans before the administration of the composition. In certain embodiments, the single dose vaccine composition induces an increase in norovirus-specific serum antibody titer within seven days of administration of the composition. Preferably, the single dose vaccine composition is administered via the intravenous, subcutaneous or intramuscular routes of administration. In a particular embodiment, the single dose vaccine composition is administered intramuscularly to a human.

As described herein, in some embodiments, single dose vaccine compositions suitable for use in the method contain no more than 150 μg of each of genogroup I and/or genogroup II norovirus VLPs. For example, in some embodiments, the vaccine composition contains no more than 150 µg, no more than 100 µg, no more than 50 µg, no more than 25 µg, no more than 15 µg, or no more than 10 µg of genogroup I norovirus VLP. In other embodiments, the vaccine composition contains not more than 150 µg, not more than 100 µg, not more than 50 µg, not more than 25 µg, not more than 15 µg, or not more than 10 µg VLP of norovirus genogroup II. In certain embodiments, the vaccine composition contains no more than 50 μg of each of genogroup I and genogroup II norovirus VLPs. In embodiments in which the single dose vaccine composition comprises both genogroup I and genogroup II norovirus VLPs, the dose of genogroup I norovirus VLP and genogroup II VLP may be the same or different. For example, in one embodiment, the vaccine composition may contain no more than 50 μg of genogroup I norovirus VLP and no more than 150 μg of genogroup I norovirus VLP.

- 12 042694 group II. In another embodiment, the vaccine composition may contain no more than 25 μg of genogroup I norovirus VLP and no more than 50 μg of genogroup II norovirus VLP. In other embodiments, the vaccine composition may contain no more than 15 μg of genogroup I norovirus VLP and no more than 50 μg of genogroup II norovirus VLP. In still other embodiments, the vaccine composition may contain no more than 25 μg of genogroup I norovirus VLP and no more than 150 μg of genogroup II norovirus VLP.

In one embodiment of the method, the subject is a human and the vaccine provides protection against one or more symptoms of a norovirus infection. Although methods for inducing an immune response using norovirus antigens have been reported elsewhere (see U.S. Patent Application Publication No. US 2007/0207526), indicators of a protective immune response against norovirus in humans are still not well defined (Herbst-Kralovetz et al. (2010) Expert Rev. Vaccines 9(3), 299-307). Unlike some vaccines currently licensed in the US, where vaccine efficacy is correlated with serum antibodies, studies have shown that immune response markers, such as increased serum IgG titers to Norwalk virus, are not associated with protective immunity in humans (Johnson et al. al (1990) J. Infectious Diseases 161: 18-21). Moreover, another study investigating human response to Norwalk virus challenge showed that susceptibility to Norwalk infection is multifactorial and includes factors such as secretory status and mucosal immunological memory (Lindesmith et al. (2003) Nature Medicine 9:548-553).

Because norovirus cannot be cultured in vitro, virus neutralization assays are not currently available. A functional assay that serves as a replacement for the neutralization assay is the hemagglutination inhibition (HAI) assay (see Example 1). HAI measures the ability of norovirus vaccine-induced antibodies to inhibit the agglutination of antigen-coated erythrocytes by norovirus VLPs, since norovirus VLPs bind to erythrocyte antigens (eg, blood group antigens). This assay is also known as a carbohydrate blocking assay because it measures the functional ability of an antibody to block virus or VLP binding to blood group antigen carbohydrates on the surface of red blood cells. In this assay, a certain amount of norovirus VLP is mixed with a certain amount of red blood cells and serum from immunized subjects. If the serum sample contains functional antibodies, the antibodies will bind to the VLP and thus inhibit erythrocyte agglutination. As used herein, the term functional antibodies refers to antibodies capable of inhibiting the interaction between norovirus particles and erythrocyte antigens. In other words, the titer of functional antibodies is equal to the titer of antibodies that block blood group antigens (HBGA) or carbohydrates. Serum titer of norovirus-specific functional antibodies can be measured using the HAI assay described above. Serum titer of norovirus-specific functional antibodies can also be measured using an ELISA-based assay in which the carbohydrate Nantigen is bound to the wells of a microtiter plate and binding of the norovirus VLP to the H antigen is detected in the presence of serum (see example 1 and Reeck et al. (2010). ) J Infect Dis, Vol 202(8): 12121218). An increase in the level of norovirus-specific functional antibodies may serve as an indicator of a protective immune response. Thus, in one embodiment, the introduction of the vaccine stimulates the formation of protective immunity, which includes an increase in the serum titer of norovirus-specific functional antibodies compared with the serum titer in a person who did not receive the vaccine. The serum titer of norovirus-specific functional antibodies indicative of a protective immune response is preferably a geometric mean titer greater than 40, 50, 75, 100, 125, 150, 175, 200 as measured by HAI assay, or geometric mean titer blocking antibodies (BT) ₅₀ (50% inhibition of H antigen binding to norovirus VLP) is greater than 100, 150, 200, 250, 300, 350, 400, 450, or 500 as measured by the H antigen binding assay. In one embodiment, the norovirus-specific functional antibody serum titer is a geometric mean titer of greater than 40 as measured by the HAL assay In another embodiment, the norovirus-specific functional antibody serum titer is a geometric mean titer of greater than 100 as measured by HAL assay In another embodiment, the serum titer of norovirus-specific functional antibodies is a geometric mean _BT50 titer of greater than 100 as measured by an H-antigen binding assay. In yet another embodiment, the serum titer of norovirus-specific functional antibodies is a geometric mean BT ₅₀ titer of greater than 200 as measured by an H-antigen binding assay.

In a further aspect, protective immunity, including an IgA-mediated mucosal immune response and an IgG-mediated systemic immune response, is stimulated by administration of the vaccine by parenteral (preferably intramuscular) administration to a subject of no more than a single dose of an antigenic composition or a vaccine composition containing one or more how many types of norovirus antigens and optionally at least one effective adjuvant.

The inventors have found that parenteral administration of the norovirus vaccine compositions described herein induces a strong IgA-mediated response in addition to a strong IgG-mediated response. Typically, strong IgA-mediated responses are observed only when vaccines are administered via the mucosal route of administration.

In certain embodiments, the administration of the vaccine stimulates the formation of protective immunity, which includes an increase in the level of cells that secrete norovirus-specific IgA antibodies in the blood compared to the level in a person who did not receive the vaccine. In some embodiments, the administration of the vaccine stimulates the formation of protective immunity, which includes an increase in the level of cells that secrete norovirus-specific IgA antibodies in the blood compared to the level in a person before the vaccine is received. In one embodiment, cells secreting norovirus-specific IgA antibodies are CCR10+, CD19+, CD27+, CD62L+, and α4β7+. Antibody secreting cells with this marker profile are capable of homing to both peripheral lymphoid tissue, such as Peyer's patches in the intestine, and mucosal lymphoid tissue, such as the intestinal mucosa. In one embodiment, the number of CCR10+, CD19+, CD27+, CD62L+, and α4β7+ IgA class antibody-secreting cells is greater than about 500, about 700, about 1000, about 1500, or greater than about 2000 cells per 1x106 peripheral blood monocytes. In another embodiment, cells secreting norovirus-specific IgA class antibodies are CCR10+, CD19+, CD27+, CD62L- and α4β7+. Σ antibody-secreting cells with this marker profile are usually only homing to mucosal sites and may be indicative of a secondary B-cell response. In some embodiments in which the vaccine is administered intramuscularly, the number of CCR10+, CD19+, CD27+, CD62L-, and α4β7+ IgA class-secreting cells is greater than about 5000, about 6500, about 7000, about 10000, about 13000, about 15000 or more approximately 20,000 cells per 1x106 peripheral blood monocytes.

Similar results were obtained with vaccines against other viruses such as rotavirus. For rotavirus vaccine, there is controversy over whether serum antibodies are directly involved in protection or only reflect recent infection (Jiang, 2002; Franco, 2006). Determining such relationships with protection is particularly difficult for acute enteric infections such as rotavirus or norovirus because preclinical studies suggesting a protective effect may be multifactorial and take into account mucosal defenses (such as intestinal IgA mediated), production cytokines and cellular immunity. The difficulty of measuring such immune responses during clinical trials and the weak correlation with serum antibody measurements imply that the effectiveness of a vaccine against these types of viruses can only be detected by human challenge clinical experiments.

As indicated above, administration of the vaccine composition of the present invention prevents and/or reduces at least one symptom of a norovirus infection. The symptoms of norovirus infection are well known in the art and include nausea, vomiting, diarrhea, and stomach cramps. In addition, a patient with norovirus infection may experience low-grade fever, headache, chills, muscle pain, and weakness. The present invention also encompasses a method of inducing a protective immune response in a subject having a norovirus infection by administering to the subject a vaccine composition of the present invention in a manner that alleviates and/or reduces at least one symptom associated with a norovirus infection. Symptom reduction can be determined subjectively or objectively, for example, through the subject's self-assessment, clinician's assessment, or through appropriate analysis or measurement (for example, body temperature), including, for example, assessment of quality of life, slowing the progression of norovirus infection, or additional symptoms, reducing the severity of symptoms of norovirus infection, or appropriate tests (eg, antibody titer, antigen detection by RT-PCR, and/or B-cell or T-cell activation assay). An effective response can also be determined by direct measurement (eg, RT-PCR) of the viral load in stool samples, which reflects the amount of virus shed from the gut). Objective assessment includes assessment methods applied to both animals and humans.

The present invention also provides a method for stimulating the production of antibodies to one or more norovirus antigens, said method comprising administering the vaccine composition of the present invention described above to a subject. These antibodies can be isolated and purified using conventional methods from the prior art. Isolated antibodies specific for norovirus antigens can be used in the development of diagnostic immunoassays. These assays can be used to detect norovirus in clinical specimens and identify the specific virus causing the infection (for example, Norwalk, Houston, Snow Mountain, and

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etc.). Alternatively, isolated antibodies can be administered to subjects susceptible to norovirus infection to provide passive or short-term immunity.

The present invention will be further illustrated in more detail based on the specific embodiments described in the following examples. The examples are intended to serve purely illustrative purposes of the present invention and are not intended to limit its scope in any way.

Examples

Example 1 Dose Escalation Study to Investigate the Safety and Immunogenicity of Norovirus Bivalent Intramuscular Virus-Like Particle (VLP) Vaccine in Humans (Study LV03-104), Cohort A.

This example describes Cohort A of a randomized, multicentre, dose-escalation study investigating the safety and immunogenicity of four dosage levels of a VLP-based bivalent intramuscular (IM) norovirus vaccine with adjuvant monophosphoryl lipid A (MPL) and aluminum hydroxide (A10H) compared with placebo in adult subjects. Approximately 48 subjects aged 18 to 49 were included in the cohort. Subjects received two doses of vaccine or placebo via intramuscular (IM) injection using a 1.5-inch (38 mm) needle 28 days apart.

Norovirus bivalent VLP vaccine contained VLP 1 genotype genogroup I (GI.1) and genotype IV genogroup II (GII.4) as antigens and monophosphoryl lipid A (MPL) and aluminum hydroxide (AlOH) as adjuvants, sodium chloride (NaCl) and L-histidine (L-His) as a buffer (pH 6.3-6.7), ethanol and water for injection. The composition of the norovirus bivalent intramuscular vaccine based on VLP is set forth in Table 1. VLP GII.4 contained the sequence of the capsid protein SEQ ID NO: 1, obtained from three strains of GII.4.

Table 1

Final formulation of the finished dosage form for four compositions of norovirus bivalent IM vaccines based on VLP based on 0.5 ml

Compound VLP GI.1 (mcg) VLP GII.4 (µg) MPL (mcg) A1* (mg) NaCl (mg) L-His (mg) Ethanol (mg) Dosage 10 mcg 5 5 50 0.5 4.38 1.55 19.7 Dosage 30 mcg 15 15 50 0.5 4.38 1.55 19.7 Dosage 100 mcg 50 50 50 0.5 4.38 1.55 19.7 Dosage 300 mcg 150 150 50 0.5 4.38 1.55 19.7

* as aluminum hydroxide

The placebo was sterile normal saline injection (0.9% NaCl, no preservatives). Vaccine dose escalation was performed as follows: after appropriate screening for satisfactory health, cohort A subjects were sequentially assigned to each of four dose groups, ˜12 subjects each (dose groups A1, A2, A3, and A4). Dosage groups A1, A2, A3, and A4 represented bivalent antigen dosages of 5/5 µg, 15/15 µg, 50/50 µg, and 150/150 µg of norovirus GI.1 and GII.4 VLPs, respectively. Subjects in each dosage group were randomized 5:1 to receive vaccine or placebo. Subjects in the A1 dosage group received their respective randomized treatment (10 subjects received 5/5 μg of vaccine and 2 subjects received placebo). Subjects were observed for the purpose of assessing safety by reviewing symptoms recorded in the self-observation diary (days 0-7) and individual charts starting on visit day 7, 21, 28, 35, and 56. Safety data were reviewed by the chief safety monitor (CSM ). Once safety data from subjects in the A1 dosage group were available for review after 7 days post-dose 2 (study day 35) and deemed acceptable, subjects in the A2 dosage group were allowed to take their initial dose. The same rule was applied for dosing in subsequent dosage groups; that is, once safety data had been obtained from the dosage group for review after 7 days post dose 2 (study day 35), the next dosage group was allowed to take its initial dose.

At the end of cohort A enrollment, approximately 10 subjects in each dosage group received the vaccine (total of 40 vaccinated) and 2 subjects in each group received saline (total of approximately 8 recipients receiving saline control).

Subjects made daily entries in a self-monitoring diary of expected symptoms, including four local injection site reactions such as pain, tenderness, redness, and swelling, and 10 systemic manifestations or symptoms, including daily body temperature measured in the mouth. , headache, weakness, muscle pain, chills, joint pain, and gastrointestinal symptoms, namely nausea, vomiting, diarrhea, abdominal cramps/abdominal pain, day 0 to 7 after each dose of norovirus bivalent IM vaccine based on VLP or control . Redness and swelling at the injection site was measured and recorded daily for 7 days after each injection.

Individual cards were completed at each subsequent visit on days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 and during a subsequent phone call on day 265+14; Subjects were asked about past medical conditions, physician visits, any serious adverse events (SAEs), and the occurrence of any new significant medical conditions. Subjects were quantified with leukocyte count and platelet count, and assessed for serum urea, creatinine, glucose, AST, and ALT at screening and on days 21 and 35 (~7 days after each dose) to assess whether continued they meet the requirements, and to assess the safety, respectively.

Subjects had pre-vaccination blood samples taken on day 0 and on days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14 and 393+14 to measure serum antibodies (IgG, IgA and IgM individually and collectively) generated in response to a norovirus bivalent IM VLP-based vaccine by enzyme immunoassays (ELISA). Also measured the levels of antibodies that cause the activity of blocking carbohydrates in the blood serum and HAI in the blood serum. In Cohort A subjects, antibody-secreting cells (ASCs), homing markers, memory B cells, and cellular immune responses were analyzed.

The following methods were used to analyze blood samples collected from immunized or placebo treated individuals.

Measurements of Serum Antibody Levels by ELISA

Measurement of norovirus antibody levels by ELISA was performed in all subjects using purified recombinant norovirus VLPs (GI.1 and GII.4 separately) as target antigens in order to screen for code-assigned samples. Briefly, norovirus VLP in carbonate coating buffer pH 9.6 was used for coating on microtiter plates. Plates were washed, blocked, and incubated with 2-fold serial dilutions of test serum, followed by washing and incubation with enzyme-linked secondary antibody reagents specific for all human IgG, IgG1, IgG2, IgG3, IgG4, IgA, and IgM human. Appropriate substrate solutions were added, staining was developed, plates were read, and final titers of IgG, IgA, and IgM were determined against a control standard curve for each antibody class. Geometric mean titers (GMT), seroconversion factor (GMFR), and serological response rates were determined for each group. The serological response was characterized as a 4-fold increase in antibody titer compared to pre-immunization titers.

Activity of noroviruses to block carbohydrate blood group antigens (HBGA)

Blocking assays to determine the ability of serum antibodies to inhibit NV VLP binding to synthetic carbohydrate type 1 H antigens or type 3 H antigens were performed as previously described (Reeck et al. (2010) J Infect Dis, Vol. 202(8): 1212 -1218). Briefly, NV VLPs for blocking assays were incubated with an equal volume of serum and a two-fold serial dilution was made starting from an initial dilution of 1:25. Neutravidin-coated 96-well microtiter plates were washed and coated with 2.5 μg/mL of either synthetic type 1 H-antigen polyvalent-PAA-biotin complex or type 3 H-antigen polyvalent-PAAbiotin complex. Serum VLP solutions were added. The plates were washed and polyclonal rabbit serum specific for NV VLP was added, washed and then incubated with horseradish peroxidase-coupled goat anti-rabbit IgG. Staining was developed with tetramethylbenzidine peroxidase liquid substrate and stopped with 1M phosphoric acid. Optical density was measured at 450. Positive and negative controls were performed. Fifty percent blocking antibody titers (BT50) were determined and were characterized as the titers at which the OD reading (after removing control data) was 50% of the positive control. A value of 12.5 was assigned to samples with a BT50 less than 25. Geometric mean titers (GMT), seroconversion factor (GMFR), and serological response rates were determined for each group. The serological response was characterized as a 4-fold increase in antibody titer compared to pre-immunization titers. Blocking serum control samples were used as internal controls. An assay to confirm blocking specificity was performed using the same blocking assay protocol with the following features: after carbohydrate loading, sera were incubated directly on the plate without the first pre-incubation with VLP. After washing, the VLPs were incubated on the plate and detected in the same way as in the blocking assay.

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Norovirus Hemagglutination Inhibition (HAI) Assay Vaccine-induced antibodies were tested for their ability to inhibit hemagglutination of human erythrocyte O-type norovirus VLPs as described previously (E1 Kamary et al. (2010) J Infect Dis, Vol. 202(11): 1649 -58). HAI titers were calculated as the reciprocal of the highest dilution at which hemagglutination was inhibited to form a compact negative erythrocyte pattern and presented as GMT, GMFR and >4-fold increase.

Norovirus GI.1 and GII.4 VLPs were serially diluted separately and incubated with an equal volume of 0.5% human erythrocyte suspension in a 96-well V-bottom plate. The number of antigens, norovirus VLP corresponding to 4 HA units, was determined and confirmed by back titration. The test sera were heat inactivated at 56°C for 30 minutes and treated with a freshly prepared 25% kaolin suspension. In order to exclude serum inhibitors, the test samples were pre-adsorbed on erythrocytes. HAI analysis was performed as follows: Pretreated sera (two-fold dilution in PBS, pH 5.5) were added to 96-well V-bottom plates and incubated with equal volumes of antigen, norovirus GI.1 and GII.4 VLPs, respectively. containing 4 HA units. A 0.5% erythrocyte suspension was added to each well and the plates were incubated 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 (GMT), seroconversion factor (GMFR), and serological response rates were determined for each group. The serological response was characterized as a 4-fold increase in antibody titer compared to pre-immunization titers.

Antibody-secreting cell assays

PBMCs were isolated from approximately 60 ml of anticoagulant-suppressed blood on days 0, 7+3, 28+3, and 35+3 after administration of the VLP-based norovirus bivalent IM vaccine or placebo. Approximately 25 ml of blood was obtained for PBMC assays immediately after collection and 35 ml of blood for PBMC cryopreservation. ASC assays detect cells secreting anti-norovirus VLP antibodies (Tacket et al. (2000) J. Infect. Dis., Vol. 182:302-305; Tacket et al. (2003) Clin. Immunol., Vol. 108: 241-247; El Kamary et al (2010) J Infect Dis, Vol 202(11): 1649-58). Freshly collected PBMCs from a group of subjects were evaluated for ASC frequency and homing markers were determined. Frozen PBMCs from subjects in cohort A were evaluated for the incidence of ASC. Response rates and mean ASCs per 10 PBMCs are described at each time point for each group. A positive response was defined as the number of post-vaccination ASCs per 106 PBMCs that was at least 3 standard deviations (SD) greater than the pre-vaccination mean for all subjects (in logarithmic form), and the presence of at least 8 ASC spots, which corresponded to the mean. medium stimulated negative control wells (2 spots) plus 3 SD as determined in similar assays.

Measuring the number of virus-specific memory B cells specific for norovirus

Anticoagulant-suppressed blood samples were collected from Cohort A subjects only (approximately 25 mL on days 0, 28, 56, and 180) to measure memory B cell counts on days 0, 28, 56, and 180 post-vaccination using an antigen-preceded ELISpot assay. in vitro stimulation (Crotty et al. (2004) J. Immunol. Methods, Vol. 286:111-122.; Li et al. (2006) J. Immunol. Methods, Vol. 313:110-118). Peripheral blood mononuclear cells (5x106 cells/ml, 1 ml/well in 24-well plates) were incubated for 4 days with antigens, norovirus GI.1 and GII.4 VLPs, separately to allow clonal expansion of antigen- specific memory B cells and differentiation into antibody-secreting cells. Controls included cells incubated under the same conditions in the absence of antigen and/or cells incubated with unrelated antigen. Following stimulation, cells were washed, counted, and transferred to ELISpot plates coated with Norwalk VLPs. In order to determine the frequency of virus-specific memory B cells relative to total Ig-secreting B lymphocytes, expanded B cells were also added to anti-human IgG and anti-human IgA coated wells. Bound antibodies were detected with HRP-labeled anti-human IgG or anti-human IgA antibodies, followed by the application of the appropriate substrate. Conjugates to the IgA and IgG subclasses (IgA1, IgA2 and IgG1-4) are also used to determine antigen-specific responses mediated by antibodies of these subclasses, which may be associated with certain effector mechanisms and localization of immune priming. Spots were counted using an ELISpot reader. Proliferated cell populations for each subject were studied by flow cytometry to, among other things, confirm the memory B cell phenotype, ie. CD19+, CD27+, IgG+, IgM+, CD38+, IgD (Crotty et al. (2004) J. Immunol. Methods, Vol. 286:111-122.; Li et al. (2006) J. Immunol. Methods, Vol. 313 :110-118).

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Cellular immune responses

Anticoagulant-suppressed blood samples (approximately 25 ml on days 0, 28, 56, and 180) from cohort A subjects were collected as coded samples and PBMCs were isolated and frozen in liquid nitrogen for possible future evaluation of CMI antigen responses, VLP norovirus GI. 1 and GII.4. The assays that are performed include, among others, assays for PBMC proliferation and cytokine responses to antigens, norovirus GI.1 and GII.4 VLPs, by measuring levels of interferon (IFN)-y and interleukin (IL)-4 according to accepted techniques (Samandari et al (2000) J. Immunol., Vol 164:2221-2232 Tacket et al (2003) Clin Immunol., Vol 108:241-247). Also evaluate T-cell responses.

results

The safety assessment included local and systemic expected symptoms within 7 days and unexpected symptoms within 28 days after each dose. Monitored for serious adverse events for 12 months. Immunogenicity was assessed on the basis of serum obtained before and after each vaccination, for antibodies from ELISA for total Ig (IgG, IgA and IgM in total) and peripheral blood mononuclear cells (PBMC) against cells secreting antibodies of the IgG and IgA class (ASC) , via Elispot.

All four dosage groups were included in cohort A with safety data available after dose two for all four dosage groups (40 vaccinees in total). Among the 40 vaccinated, pain or tenderness was the most common local symptom reported after each dose, while swelling or redness was rare. No severe local symptoms were reported. Systemic symptoms, namely headache, myalgia or malaise, were reported after each dose in less than half of those vaccinated. None of the vaccinated had a fever. No related SAEs have been registered.

As shown in FIG. 1-3, in total Ig ELISA, strong anamnestic antibody-mediated responses (IgG, IgA, and IgM combined) were observed against both types of VLP antigens 7 days after the first dose at the lowest dose (5 μg VLP GI.1 + 5 μg VLP GII.4). The second dose did not enhance responses after dose one. Similar results were observed for responses mediated by antigen-specific serum IgG and serum IgA measured separately (FIGS. 4-9). Dose-dependent responses were observed for antibody-mediated responses to both antigens (FIGS. 1-9). However, the maximum response to VLP GI.1 appeared to be obtained with a lower dose than to obtain the maximum response to VLP GII.4 (15 μg compared to 50 μg). Remarkably, a single dose of intramuscularly administered bivalent norovirus vaccine induced an unexpectedly significantly higher titer of antigen-specific antibodies than that induced by two doses of intranasally administered VLP-based monovalent vaccines containing 20 times the VLP dose (Fig. 10; comparison of the 5 μg group LV03-104 with 100 µg group LV01-103). Moreover, a low dose (5 μg) of the bivalent norovirus IM vaccine produced the same norovirus-specific antibody titer as that induced in humans exposed to native norovirus (FIG. 10).

Strong IgG- and IgA-mediated Elispot responses were also observed 7 days after the first dose at the lowest dose (5 μg) for both types of VLP antigens (Table 2). Notably, antibody-secreting cell (ASC) mediated responses were biased towards IgA versus IgG, and ASC exhibited a mucosal homing receptor phenotype (alpha 4/beta 7) and a chemokine receptor (CCR10) phenotype as assessed by flow cytometry (Fig. 11; table 3). As shown in Table 3, more ASCs had mucosal homing markers (beta 7+, CD62L-) compared to dual mucosal/peripheral tissue homing markers (beta 7+, CD62L+). Table 4 shows the percentage of memory B cells per 106 peripheral blood monocytes responding to antigens, VLP. A greater percentage of antigen-specific memory B cells also express mucosal homing markers compared to dual mucosal/peripheral tissue or peripheral homing markers. Similar responses were also observed in recipients receiving doses of 15 μg and 50 μg (Tables 2-4).

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table 2

Day 7, characterization of the PBMC-mediated response. Approximate number of antibody-secreting cells (ASC)/million CD19+ cells

ASC/million CD19+ cells - Day 7 Vaccine response rate Vaccine-specific Norovirus-Specific Cells IgA GI.1 IgG GI.1 IgA GII.4 IgG GII.4 GI.1 specific GII.4 specific Percentage of the total population of circulating PBMCs Geometric mean A1 dose 5 mcg (n=5) 30947 13807 10947 3945 4.48% 1.49% 5.96% Standard deviation A1 dose 5 mcg 6674 9780 3651 2261 Geometric mean A2 dose 15 mcg (n=4) 25296 17004 7108 4336 4.23% 1.14% 5.37% Standard deviation A2 dose 15 mcg 10846 18770 6055 5697 Geometric mean AZ dose 50 mcg (n=4) 36158 20572 14103 2549 5.67% 1.67% 7.34% Standard deviation AZ dose 50 mcg 11470 418 7627 2230 Geometric mean A4 dose 150 mcg (n=4) 34183 9566 26213 11310 4.37% 3.75% 8.13% Standard deviation A4 dose 150 mcg 32938 4466 89769 15226 Placebo (n=2) 0 152 0 108 0.02% 0.01% 0.03%

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Table 3

ASC markers in vaccine and placebo recipients determined by flow cytometry - day 7

% of total CD19+ B cells that are CD27+ and CD38+ % of % of total CD27+, CD38+, CCR10+_ beta 7+, CD62L- % of % total CD27+, CD38+, CCR10+ beta 7+, CD62L+ Percentage of total ASCs CD27+, CD38+, CCR10+, beta 7+ CD62L(+) and (-) Total vaccine-specific per million cells* CD27+, CD38+, CCR10+, beta 7+ CD62L(+) and (' ) Percentage of vaccine-specific of total* circulating PBMCs with mucosal homing Geometric mean A1 dose 5 mcg (n=5) 25.10% 6.86% 1.06% 2.78% 1656 0.17% Standard deviation A1 dose 5 mcg 10.45 3.13 1.01 Geometric mean A2 dose 15 mcg (n=4) 12.99% 16.98% 2.43% 4.63% 1355 0.14% Standard deviation A2 15 mcg dose 9.13 1.56 0.23 Geometric mean AZ 50 mcg dose (n=4) 31.71% 26.43% 3.63% 12.01% 23915 2.39% Standard deviation AZ dose 50 mcg 6.32 1.82 1.38 Geometric mean A4 dose 150 mcg (n=4) 33.46% 30.06% 5.68% 15.74% 31350 3.14% Standard deviation A4 dose 150 mcg 9.86 2.97 1.70 Placebo (n=2) 1.26% 22.00% 0.87% 1.20% 5 0.001%

*in case most ASCs are norovirus-specific

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Table 4

Memory B cell responses in vaccine and placebo recipients - Day 7

% of total CD19+ B cells that are CD27+, CD38+, CD138+ % of % total CD27+, CD38+, CD138+, CCR10+ Beta 7+, CD62L- % of % total CD27+, CD38+, CD138+, CCR10+_B eta 7+, CD62L+ Percentage of total CD27+, CD38+, CD138+, CCR10+, Beta 7+ CD62L(+) and (-) memory cells Total vaccine-specific per million cells* CD27+, CD38+, CD138+, CCR10+, beta 7+_CD62L(+) and Percentage of vaccine-specific of total* circulating PBMCs with mucosal homing Geometric mean A1 dose 5 mcg But But But But But But Geometric mean A2 dose 15 mcg (n=4) 1.54% 11.58% 1.92% 0.21% 61 0.01% Standard deviation A2 dose 15 mcg 1.54 3.94 0.94 Geometric mean AZ dose 50 mcg (n=4) 3.31% 16.10% 4.60% 0.68% 1364 0.14% Standard deviation AZ dose 50 mcg 1.11 2.16 0.97 Geometric mean A4 150 mcg dose (n=4) 1.56% 16.90% 8.10% 0.39% 778 0.08% Standard deviation A4 dose 150 mcg 0.22 3.26 4.57 Placebo (n=1) 0.10% 12.50% 0.00% 0.01% 0 0%

*in case most ASCs are norovirus-specific

In the absence of a direct virus neutralization assay available due to the inability to culture norovirus in vitro, functional assays, which serve as a substitute for virus neutralization assays, have been performed to measure the amount of functional antibodies in the vaccinated.

The inhibition of GI.1 VLP binding to the H antigen mediated by vaccine-induced serum antibodies was measured using the carbohydrate H antigen blocking activity assay described above. Data are presented as seroconversion factor (GMFR) and serological response (4-fold increase) in Table. 5 and as geometric mean titer (GMT) in Table. 6. Surprisingly, after just one intramuscular injection of the vaccine formulation, significant carbohydrate blocking activity was observed for all dose groups; in fact, the administration of the second dose of the vaccine did not significantly increase the blocking activity compared to the levels after dose 1. The binding inhibition activity was maintained throughout the test period up to day 56 after dose 1.

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Table 5

Carbohydrate blocking activity (HBGA BT50). Seroconversion factor (GMFR) and serological response (4-fold increase) for GI norovirus antibodies. 1

Research Day 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) Vaccine based on VLP 5/5 mcg 9 26.6 (8.3, 85.1) 88.9 (51.8, 99.7) 9 25.1 (8.9, 70.3) 88.9 (51.8, 99.7) 9 19.7 (8.2, 47.1) 100.0 (66.4, 100.0) 9 20 (7.7, 51.7) 88.9 (51.8, 99.7) 9 16.6 (5.7, 48.1) 77.8 (40.0, 97.2) Vaccine based on VLP 15/15 mcg 8 33.2 (13.6, 80.8) 100.0 (63.1, 100.0) 8 25.5 (10.5, 61.8) 100.0 (63.1, 100.0) 8 18.5 (8.4, 40.6) 100.0 (63.1, 100.0) 7 22.2 (8.8, 56) 100.0 (59.0, 100.0) 7 8.4 (2.4, 29.6) 57.1 (18.4, 90.1) Vaccine based on VLP 50/50 mcg 1 0 38.6 (18.3, 81.6) 100.0 (69.2, 100.0) 1 0 27.9 (13.4, 58) 100.0 (69.2, 100.0) 1 0 20.9 (Yu, 43.5) 100.0 (69.2, 100.0) 1 0 19 (9.9, 36.4) 100.0 (69.2, 100.0) 9 10.2 (4.6, 22.8) 77.8 (40.0, 97.2) Vaccine based on VLP 150/150 mcg 7 30.6 (16.3, 57.6) 100.0 (59.0, 100.0) 8 19.4 (13.1, 28.5) 100.0 (63.1, 100.0) 8 16.3 (11.7, 22.6) 100.0 (63.1, 100.0) 8 18.8 (12.8, 27.5) 100.0 (63.1, 100.0) 8 23.8 (17, 33.3) 100.0 (63.1, 100.0) placebo 8 0.9 (0.8, 1) 0.0 (0.0, 36.9) 8 0.8 (0.7, 1.1) 0.0 (0.0, 36.9) 8 0.8 (0.6, 1.1) 0.0 (0.0, 36.9) 8 0.8 (0.7, 1.1) 0.0 (0.0, 36.9) 8 0.6 (0.3, 1.2) 0.0 (0.0, 36.9)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples were possibly mixed up; one of these data points was baseline data, which resulted in no fold data for the subject at any point in time.

Table 6

Carbohydrate blocking activity (HBGA BT50). Geometric mean titer (GMT) of anti-GI norovirus antibodies. 1

Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI)

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Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) Vaccine based on VLP 5/5 mcg 9 28.9 (12.7, 65.9) 9 768.5 (344.1, 1716) 9 723.7 (398.1, 1316) 9 568 (321.8, 1003) 9 577.1 (351.2, 948.3) 9 478.3 (293.3, 780.1) Vaccine based on VLP 15/15 mcg 8 24.9 (12.7, 48.7) 8 826.1 (524.9, 1300) 8 634.3 (285.9, 1407) 8 459.8 (225.3, 938.6) 7 610.3 (354.6, 1050) 7 230.9 (105.2, 506.7) Vaccine based on VLP 50/50 mcg 10 17.3 (9.9, 30.3) 10 669.2 (329.1, 1361) 10 483.7 (258.7, 904.2) 10 362.4 (192.9, 680.7) 10 328.9 (191.9, 563.8) 9 184 (97.2, 348.3) Vaccine based on VLP 150/150 mcg 8 15.5 (ND, 21.8) 7 435 (262.5, 720.8) 8 300.7 (173.9, 520) 8 252.7 (146.7, 435.2) 8 291.5 (171.5, 495.4) 8 369.7 (233.8, 584.6) placebo 8 29 (9.1, 92.8) 9 24.6 (9.8, 62.1) 9 22.5 (8.8, 57.3) 9 22.2 (8.9, 55.5) 8 24.6 (8.4, 72.6) 8 18.3 (10.1, 33.3)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples may have been mixed up.

Similarly, the carbohydrate blocking activity of anti-VLP GII.4 serum antibodies was measured. A significant response was observed in all dosing groups as measured by GMFR and serological response (Table 7) and GMT (Table 8). Similar to the antibody-mediated blocking of GI.1 binding described above, strong GII.4 VLP carbohydrate binding blocking activity was detected after just one dose, with the second dose not appearing to enhance the blocking activity.

Table 7

Carbohydrate blocking activity (HBGA BT50). Seroconversion factor (GMFR) and serological response (4-fold increase) for anti-norovirus antibodies GII.4

Research Day 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) processing group and N GMF R (95% CI) 4-fold increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMFR (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4-fold increase (95% CI) Vaccine based on VLP 5/5 mcg 9 5 (1.6, 16.1) 33.3 (7.5, 70.1) 9 5.9 (1.7, 20.3) 55.6 (21.2, 86.3) 9 4.7 (1.4, 15.7) 44.4 (13.7, 78.8) 9 4.7 (1.6, 13.8) 44.4 (13.7, 78.8) 9 5 (1.6, 15.9) 55.6 (21.2, 86.3) Vaccine based on VLP 15/15 mcg 8 11 (2.7, 45.3) 62.5 (24.5, 91.5) 8 9.2 (3, 27.9) 62.5 (24.5, 91.5) 8 7.4 (2.5, 21.8) 62.5 (24.5, 91.5) 7 7(2,1, 23) 57.1 (18.4, 90.1) 7 5.6 (2.3, 14) 57.1 (18.4, 90.1) Vaccine based on VLP 50/50 mcg 1 0 18.6 (4.9, 70.8) 70.0 (34.8, 93.3) 1 0 12.2 (3.8, 39.4) 70.0 (34.8, 93.3) 1 0 8.4 (2.9, 24.1) 70.0 (34.8, 93.3) 1 0 8.7 (2.9, 26.1) 70.0 (34.8, 93.3) 9 5.2 (2.2, 12) 66.7 (29.9, 92.5) Vaccine based on VLP 150/150 mcg 7 10.1 (2, 51.8) 57.1 (18.4, 90.1) 8 5.5 (1.9, 16.5) 50.0 (15.7, 84.3) 8 4.4 (1.6, 12.2) 50.0 (15.7, 84.3) 8 4.3 (1.7, 10.7) 37.5 (8.5, 75.5) 8 3.1 (1.3, 7.2) 25.0 (3.2, 65.1) placebo 8 1 (0.9, 1.1) 0.0 (0.0, 36.9) 8 1.1(1, 1.3) 0.0 (0.0, 36.9) 8 1.3 (0.9, 2D) 12.5 (0.3, 52.7) 8 1.8 (0.5, 6.7) 12.5 (0.3, 52.7) 8 2 (0.7, 6.1) 12.5 (0.3, 52.7)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples were possibly mixed up; one of these data points was base sample data, which resulted in no fold data for the subject at any point in time.

Table 8

Carbohydrate blocking activity (HBGA BT50). Geometric mean titer (GMT) of antibodies to noroviruses GII.4

Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) Vaccine based on VLP 5/5 mcg 9 40.3 (18.90) 9 202.1 (106.3, 384.3) 9 236.9 (133.4, 420.6) 9 189.7 (108.6, 331.3) 9 188.7 (R8.7, 300.1) 9 201.6 (R6.4, 349.5) Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) Vaccine based on VLP 15/15 mcg 8 23.7 (12.8, 43.8) 8 260.1 (95.1, 711.1) 8 218.1 (104.2, 456.3) 8 175.4 (82.7, 372) 7 182.3 (89.1, 372.8) 7 146.3 (92.4, 231.5) Vaccine based on VLP 50/50 mcg 10 28.4 (13.1, 61.5) 10 527.2 (271.1, 1025) 10 345.2 (195.5, 609.6) 10 238.3 (139.4, 407.3) 10 246.5 (138.7, 438.2) 9 160.2 (107.4, 238.8) Vaccine based on VLP 150/150 mcg 8 63 (24.8, 160.4) 7 721.8 (344.6, 1512) 8 347.7 (186.1, 649.5) 8 277 (145.6, 527) 8 267.9 (158.7, 452.2) 8 193.5 (121.6, 308.2) placebo 8 24.1 (12.9, 45) 9 22.8 (12.6, 41.6) 9 24.9 (12.7, 48.7) 9 29.1 (15.1, 56.1) 8 44 (12.6, 154.2) 8 48.2 (16.7, 139.5)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples may have been mixed up.

Hemagglutination inhibition (HAI) assays were also used to investigate the serum antibody response from vaccinated subjects against the target antigens, norovirus VLP. Similar to carbohydrate H antigen binding studies, just one dose of the VLP-based vaccine induced antibodies that inhibited hemagglutination in all dose groups, as measured by GMFR (Table 9), 4-fold magnification (Table 9), and GMT (Table 9). 10). Although the level of hemagglutination inhibition was maintained until the last day of the trial (28 days after dose 2, 56 days after dose 1), the second dose of the VLP-based vaccine did not appear to enhance the inhibition of hemagglutination mediated by vaccine-induced antibodies.

Table 9

Hemagglutination inhibition assay. Seroconversion factor (GMFR) and serological response (4-fold increase) for GI norovirus antibodies. 1

Research Day 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1)

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Processing group N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4x increase (95% CI) N GMF R (95% CI) 4-fold increase (95% CI) Vaccine based on VLP 5/5 mcg 9 5.4 (3, 9.8) 77.8 (40.0, 97.2) 9 7 (4.6, 10.7) 88.9 (51.8, 99.7) 9 6.1 (4.1, 9.3) 88.9 (51.8, 99.7) 9 6 (3.7, 9.5) 77.8 (40.0, 97.2) 9 6.3 (3.9, 10.3) 88.9 (51.8, 99.7) Vaccine based on VLP 15/15 mcg 8 8.9 (4.4, 18) 100.0 (63.1, 100.0) 8 9.5 (4, 22.5) 87.5 (47.3, 99.7) 8 7.1 (3.1, 16.1) 75.0 (34.9, 96.8) 7 8.5 (4D, 17.7) 85.7 (42.1, 99.6) 7 8.1 (4.4, 15.2) 100.0 (59.0, 100.0) Vaccine based on VLP 50/50 mcg 1 0 22.4 (I,6, 43) 100.0 (69.2, 100.0) 1 0 16.7 (9.3, 29.8) 100.0 (69.2, 100.0) 1 0 13.9 (8.1, 24) 100.0 (69.2, 100.0) 1 0 14.5 (9.3, 22.7) 100.0 (69.2, 100.0) 9 11.8 (6.3, 21.9) 100.0 (66.4, 100.0) Vaccine based on VLP 150/150 mcg 7 12.6 (5.7, 28) 85.7 (42.1, 99.6) 8 ID (6.4, 19.3) 100.0 (63.1, 100.0) 8 8.4 (5, 14) 100.0 (63.1, 100.0) 8 8.4 (5, 14) 100.0 (63.1, 100.0) 8 7.3 (4.5, H.9) 100.0 (63.1, 100.0) placebo 8 1 (0.8, 1.2) 0.0 (0.0, 36.9) 8 1 (0.9, 1.2) 0.0 (0.0, 36.9) 8 1 (0.9, 1D) 0.0 (0.0, 36.9) 8 0.9 (0.7, 1.1) 0.0 (0.0, 36.9) 8 1 (0.8, 1.2) 0.0 (0.0, 36.9)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples were possibly mixed up; one of these data points was baseline data, which resulted in no fold data for the subject at any point in time.

Table 10

Hemagglutination inhibition assay. Geometric mean titer (GMT) of anti-norovirus GI.1 antibodies

Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) Vaccine based on VLP 5/5 mcg 9 26.4 (14.1, 49.4) 9 143.5 (53.6, 383.8) 9 185.3 (88.6, 387.6) 9 162.1 (83.3, 315.6) 9 157 (80.3, 307.1) 9 167.4 (88.4, 316.8) Vaccine based on VLP 15/15 mcg 8 I,9 (6.1, 23.4) 8 105.3 (56.1, 197.6) 8 113.1 (42.5, 301.3) 8 84.2 (31.1, 227.6) 7 103.3 (43.3, 246.6) 7 99.2 (46.9, 209.7) Vaccine based on VLP 50/50 mcg 1 0 7.2 (5.3, 9.6) 1 0 160 (79.4, 322.6) 1 0 119.2 (60.4, 235.1) 1 0 99.7 (51.8, 191.8) 1 0 103.8 (58.4, 184.5) 9 81.1 (38.8, 169.5) Vaccine based on VLP 150/150 mcg 8 9.2 (7.5, 11.3) 7 114.1 (50.9, 255.7) 8 101.6 (62, 166.4) 8 77.2 (51.8, 115) 8 77.2 (51.8, 115) 8 67.3 (44.7, 101.3) placebo 8 16.8 (10.1, 28.1) 9 16.6(11.7, 23.7) 9 16.1 (10.7, 24.1) 9 16.6 (11.25) 8 15.4 (10, 23.7) 8 16.2 (10.8, 24.4)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples may have been mixed up.

Hemagglutination inhibition was also achieved when the target VLPs were assigned to the wrong virus. Serum antibodies induced by the vaccine inhibited hemagglutination of the VLP strain of the Houston virus, as measured by GMFR and serological reaction (Table 11) and GMT (Table 12). In this case, higher doses of the VLP-based vaccine provided stronger responses, as measured by 4-fold or GMT in particular. There was also a significant increase in GMFR and a 4-fold increase when the target VLPs were treated with the 2003 Cincinnati strain of the virus, measured only 7 days after dose 1 (Table 13).

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Table 11

Hemagglutination inhibition assay (Houston virus strain VLP). Seroconversion factor (GMFR) and serological response (4-fold increase) for anti-norovirus antibodies GII.4

Research Day 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMFR (95% CI) 4-fold increase (95% CI) N GMFR (95% CI) 4x increase (95% CI) N GMFR (95% CI) 4x increased shuttle (95% CI) N GMFR (95% CI) 4-fold lifted shuttle (95% CI) N GMFR (95% CI) 4x increase (95% CI) Vaccines based on VLP 5/5 mcg 9 1.2 (1, 1.5) 0.0 (0.0, 33.6) 9 1.3 (0.9, 1.7) 0.0 (0.0, 33.6) 9 1.3 (0.9, 1.7) 0.0 (0.0, 33.6) 9 1.3 (1, 1.7) 0.0 (0.0, 33.6) 9 1.3 (1, 1.6) 0.0 (0.0, 33.6) Vaccines based on VLP 15/15 mcg 8 1.7 (0.9, 3) 12.5 (0.3, 52.7) 8 1.6 (1.1, 2.4) 12.5 (0.3, 52.7) 8 1.5 (1.1, 2.2) 0.0 (0.0, 36.9) 7 1.5 (1, 2.2) 0.0 (0.0, 41.0) 7 1.6 (1.1, 2.4) 0.0 (0.0, 41.0) Vaccine based on VLP 50/50 mcg 1 0 2 (0.9, 4.3) 10.0 (0.3, 44.5) 1 0 1.6 (0.8, 3.1) 10.0 (0.3, 44.5) 1 0 1.7 (0.9, 3.1) 10.0 (0.3, 44.5) 1 0 1.5 (0.9, 2.4) 10.0 (0.3, 44.5) 9 1.3 (0.8, 2) 11.1 (0.3, 48.2) Vaccines based on VLP 150/150 mcg 7 4.3 (1.5, 12.4) 57.1 (18.4, 90.1) 8 2.6 (1.2, 5.8) 50.0 (15.7, 84.3) 8 1.9 (1.1, 3.3) 12.5 (0.3, 52.7) 8 1.9 (1.1, 3.5) 12.5 (0.3, 52.7) 8 1.7 (1.1, 2.6) 12.5 (0.3, 52.7) placeb o 8 1 (0.9, 1.1) 0.0 (0.0, 36.9) 8 1.1 (0.9, 1.3) 0.0 (0.0, 36.9) 8 1.1 (1, 1.2) 0.0 (0.0, 36.9) 8 1.2 (0.8, 1.9) 12.5 (0.3, 52.7) 8 1.1 (0.7, 1.8) 12.5 (0.3, 52.7)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples were possibly mixed up; one of these data points was baseline data, which resulted in no fold data for the subject at any point in time.

Table 12

Hemagglutination inhibition assay (Houston virus strain VLP). Geometric mean titer (GMT) of antibodies to noroviruses GII.4

Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI)

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Research Day Predose 1 7 days after dose 1 21 days after dose 1 28 days after dose 1 (before dose 2) 7 days after dose 2 (35 days after dose 1) 28 days after dose 2 (56 days after dose 1) Processing group N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) N GMT (95% CI) Vaccine based on VLP 5/5 mcg 9 143.5 (97.9, 210.3) 9 177.4 (122.7, 256.5) 9 180.8 (H4.4, 285.6) 9 180.8 (P4.4, 285.6) 9 189.1 (R9.2, 299.9) 9 180.8 (122.7, 266.3) Vaccine based on VLP 15/15 mcg 8 129.8 (86.4, 194.9) 8 218.3 (129.2, 368.8) 8 207.5 (134.8, 319.3) 8 200.2 (132.8, 301.7) 7 169.5 (R4.1, 252) 7 187.2 (P9.1, 294.2) Vaccine based on VLP 50/50 mcg 10 161.9 (R9.7, 219) 10 323.8 (156.5, 669.8) 10 252.5 (130.4, 489.2) 10 273.9 (150.9, 497.2) 10 242.5 (150.2, 391.5) 9 195.2 (125, 304.9) Vaccine based on VLP 150/150 mcg 8 210.6 (108.8, 407.5) 7 853.5 (297.4, 2449) 8 546.2 (237.6, 1255) 8 406.3 (201.5, 819.1) 8 406.3 (180.3, 915.4) 8 354.1 (184.6, 679.5) placebo 8 148.9 (73.8, 300.3) 9 150.1 (80D, 281.1) 9 157 (77.9, 316.5) 9 167.4 (91.4, 306.4) 8 183.6 (95, 354.5) 8 162.4 (88.5, 297.9)

Results based on data from all subjects receiving both doses of study product.

Excluded two data points from two subjects due to the fact that the samples may have been mixed up.

Table 13

Hemagglutination inhibition in the placebo group compared with the 50/50 µg VLP group

Hemagglutination inhibition assay (VLP of the 2003 Cincinnati virus strain). Seroconversion factor (GMFR) and geometric serological reaction (4-fold increase) for antibodies to noroviruses GII.4. Treatment group results, 7 days after dose 1 Processing group N GMFR (95% CI) 4-fold increase (95% CI) placebo 2 1.0 (0.6, 1.8) 0.0 (0.0, 84.2) Vaccine based on VLP 50/50 mcg 10 4.4(1.6, 11.9) 50.0(18.7.81.3)

The results of this study demonstrated that the VLP-based bivalent norovirus IM vaccine was generally well tolerated. Immunogenicity data suggest that a single dose of the vaccine may be sufficient to protect seropositive adults. The results of analyzes of carbohydrate blocking activity and hemagglutination inhibition provided additional evidence that a single dose of the vaccine induced strong anti-norovirus activity of serum antibodies. The strength and speed of the observed immune responses after a single parenteral dose in humans were significantly greater compared to the immune responses after multiple VLP-based nasal vaccines with higher VLP dosages previously reported (El Kamary et al. (2010) J Infect Dis, Vol 202(11): 16491658). These responses also outperformed those induced by orally administered norovirus VLPs (Tacket et al. (2003) Clin Immunol 108:241-247; Ball et al. (1999, Gastroenterology 117:40-48), as well as those induced by norovirus VLPs, produced by transgenic plants (Tacket et al. (2000) J Infect Dis 182:302-305) Specifically, this intramuscular vaccine formulation elicited anamnestic responses up to seven days post-immunization, and maximal serum antibody-mediated responses were observed after single dose, including a significant IgA-mediated response, and functional carbohydrate blocking activity, and hemagglutination inhibition activity.Thus, this bivalent norovirus vaccine induced a strong protective immune response in humans, which was superior to those induced by any currently available norovirus vaccine.

Example 2 Dose escalation. Safety and Immunogenicity Study of Virus-Like Particle (VLP) Intramuscular Norovirus Bivalent Vaccine in Humans (Study LV03-104).

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The following example represents the remaining planned portion of the clinical study described in Example 1, which conducted a randomized, multicentre, dose-escalation study in adults >18 years of age to study the safety and immunogenicity of four dosage levels of a norovirus bivalent intramuscular (IM) vaccine based on VLP. with adjuvant monophosphoryl lipid A (MPL) and aluminum hydroxide (AlOH) compared with placebo. Subjects received two doses of vaccine or placebo via intramuscular (IM) injection using a 1.5-inch (38 mm) needle 28 days apart. This example is intended to further illustrate the main ideas of the present invention.

Selection for Cohort A of the study is complete and has been described above in Example 1. Cohort B includes ~20 subjects aged 50-64 years. Cohort C includes ~30 subjects aged 65-85 years. A total of approximately 98 subjects were included in the study.

In cohort B, ~20 subjects aged 50-64 years were selected and randomized in a 1:1 ratio to receive either vaccine (N=10) or placebo (N=10). Once safety data have been received for review after 7 days post-dose 2 (study day 35) from Cohort B subjects, Cohort C subjects are eligible to receive their initial dose. In cohort C, ~30 subjects aged 65 to 85 years were selected and randomized in a 1:1:1 ratio to receive an MPL-AlOH adjuvanted vaccine (N=10), or an AlOH-adjuvanted vaccine alone, i.e. no MPL (N=10), or placebo (N=10). The concentrations of antigen, norovirus VLP, and AlOH in the two vaccine formulations evaluated are the same in cohort C; the difference is only in the presence or absence of MPL.

Norovirus bivalent VLP vaccine contains VLP 1 genotype genogroup I (GI.1) and genotype IV genogroup II (GII.4) as antigens and monophosphoryl lipid A (MPL) and aluminum hydroxide (AlOH) as adjuvants, sodium chloride (NaCl) and L-histidine (L-His) as a buffer (pH 6.3-6.7), ethanol and water for injection. VLP GII.4 contain the sequence of the capsid protein SEQ ID NO: 1, which was obtained from three strains of GII.4.

The single dose of vaccine selected for further evaluation in cohorts B and C is the lowest dose from cohort A that results in the strongest and most reproducible immune response, which is also generally well tolerated. Day 56 safety and immunogenicity data from cohort A subjects are reviewed by CSM/SMC, and the bivalent vaccine dosage is selected for evaluation in cohorts B and C.

Subjects record expected symptoms in a self-monitoring diary, including four local reactions at the injection site, such as pain, tenderness, redness, and swelling, and 10 systemic manifestations or symptoms, including daily body temperature measured in the mouth, headache, weakness , muscle pain, chills, joint pain and gastrointestinal symptoms, namely nausea, vomiting, diarrhea, abdominal cramps/abdominal pain, from day 0 to 7 after each dose of norovirus bivalent IM vaccine based on VLP or control. Redness and swelling at the injection site is measured and recorded daily for 7 days after each injection.

Individual cards are completed at each subsequent visit on days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14 and 393+14 and during the subsequent phone call on day 265+14; Subjects are asked about past medical conditions, physician visits, any serious adverse events (SAEs), and the occurrence of any new significant medical conditions. Subjects are quantified with leukocyte count and platelet count and assessed for serum urea, creatinine, glucose, AST, and ALT at screening and on days 21 and 35 (~7 days after each dose) to assess whether continued they meet the requirements, and to evaluate the safety accordingly.

Blood samples from subjects are collected prior to vaccination on day 0 and on days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14 and 393+14 to measure serum antibodies (IgG, IgA and IgM together and separately) generated in response to a norovirus bivalent IM vaccine based on VLP, by enzyme immunoassays (ELISA). Also measure the levels of antibodies that cause the activity of blocking carbohydrates in the blood serum and HAI in the blood serum.

The methods described above for cohort A are used to analyze blood samples collected from immunized individuals or individuals receiving placebo. The results of the study will be used in the development of a clinical protocol for the administration of the vaccine formulations of the present invention.

The present invention should not be limited in scope to the specific embodiments described, which are intended to be merely illustrative of certain aspects of the present invention, and functional equivalents of the methods and components are embraced by the scope of the present invention. Certainly, various modifications of the present invention in addition to those given 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 be covered by the scope of the appended claims.

All publications, patents, and patent applications referred to in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated herein by reference.

Citation or review of literary sources in this document should not be construed as an admission that they are prior art for the present invention.

Bibliography.

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8. Wyatt, RG, R Dolin, NR Blacklow, HL DuPont, RF Buscho, TS Thornhill, AZ Kapikian, and RM Chanock. Comparison of Three Agents of Acute Infectious Nonbacterial Gastroenteritis by Cross-challenge in Volunteers. J Infect Dis 1974; 129:709.

9. Ball, JM, DY Graham, AR Opekum, MA Gilger, RA Guerrero, and MK Estes. Recombinant Norwalk Virus-like Particles Given Orally to Volunteers: Phase I Study. Gastroenterology 1999; 117:40-48.

10. Tacket, CO, MB Sztein, GA Losonky, SS Wasserman, and MK Estes. Humoral, Mucosal, and Cellular Immune Responses to Oral Norwalk Virus-like Particles in Volunteers. Clin Immunol 2003; 108:241.

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Claims (19)

15. Kozlowski, PA, S Cu-Uvin, M. R. Neutra, and TP Flanigan. Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect Immun 1997; 65: 1387-1394. 16. Mestecky, J, SM Michalek, Z Moldoveanu, and MW Russell. Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans. Behring Inst Mitt 1997; 33-43. 17. Wu, HY, and MW Russell. Nasal lymphoid tissue, intranasal immunization, and compartmentalization of the common mucosal immune system. Immunol Res 1997; 16:187201. 18. Evans, JT, CW Cluff, D.A. Johnson, MJ Lacy, D.H. Persing, and JR Baldridge. Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi 529. Expert Rev Vaccines 2003; 2:219-229. 19. Baldridge, JR, Y Jorgensen, JR Ward, and JT Ulrich. Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration [In Process Citation], Vaccine 2000; 18:2416-2425. 20. Yang, QB, M Martin, SM Michalek, and J Katz. Mechanisms of monophosphoryl lipid A augmentation of host responses to recombinant HagB from Porphyromonas gingivalis. Infect Immun 2002; 70:3557-3565. 21. Baldrick, P, D Richardson, G Elliott, and A. W. Wheeler. Safety evaluation of monophosphoryl lipid A (MPL): an immunostimulatory adjuvant. Regul Toxicol Pharmacol 2002; 35:398-413. 22. Baldridge, JR, P McGowan, JT Evans, C Cluff, S Mossman, D Johnson, and D Persing. Taking a toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol Ther 2004; 4:1129-1138. 23. Persing, DH, RN Coler, MJ Lacy, DA Johnson, JR Baldridge, RM Hershberg, and SG reed. Taking toll: lipid A mimetics as adjuvants and immunomodulators. Trends Microbiol 2002; 10: S32-37. CLAIM 1. A composition for stimulating the formation of protective immunity against norovirus in a subject, wherein the composition comprises a virus-like particle (VLP) of genogroup I norovirus and a genogroup II norovirus VLP, wherein genogroup I and genogroup II norovirus VLPs are present in the composition in varying amounts, and wherein genogroup I norovirus VLPs is present in the composition in an amount of about 15-50 µg, and genogroup II norovirus VLP is present in the composition in an amount of about 50-150 µg. 2. The composition of claim 1 wherein the genogroup I norovirus VLP is present in the composition in an amount of approximately 15 µg. 3. The composition of claim 1, wherein the genogroup II norovirus VLP is present in the composition in an amount of approximately 50 μg. 4. The composition of claim 1, wherein the genogroup I norovirus VLP is present in the composition in an amount of about 15 μg and the genogroup II norovirus VLP is present in the composition in - 31 042694 in an amount of approximately 50 μg. 5. The composition of claim 1 wherein the genogroup I norovirus VLP is GI.1. 6. The composition of claim 1 wherein the genogroup II norovirus VLP is GII.4. 7. The composition of claim 1, wherein the composition further comprises an adjuvant. 8. The composition according to claim 7, wherein the adjuvant is not an endotoxin of bacterial origin. 9. The composition of claim 7 wherein the adjuvant is selected from the group consisting of monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, flagellin, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG -oligonucleotides, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, copolymer (lactide and glycolide) microparticles (PLG), poloxamer particles, microparticles, liposomes, oil-in-water emulsions, MF59, squalene, 3DMPL and QS21. 10. The composition of claim 1, wherein the composition further comprises aluminum hydroxide. 11. The composition of claim 1, wherein the composition comprises a buffer selected from the group consisting of buffers based on L-histidine, imidazole, succinic acid, tris, citric acid, bis-tris, PIPES, MES, HEPES, glycinamide and tricine. 12. The composition of claim 11, wherein the buffer is an L-histidine or imidazole buffer. 13. A method of stimulating the formation of protective immunity against norovirus in a subject, where the method includes administering to the subject a composition according to claim 1. 14. The method of claim 13, wherein the composition causes at least a three-fold increase in norovirus-specific serum antibody titer compared to the titer in the subject prior to administration of the composition. 15. The method of claim 13, wherein the composition elicits a serological response of at least 60% in the subject. 16. The method of claim 15 wherein the composition elicits a serological response of at least 65% in the subject. 17. The method of claim 15, wherein the serological response is an IgA or all Ig serological response. 18. The method of claim 13 wherein the composition is administered to the subject via a route selected from the group consisting of mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, and transdermal routes of administration. 19. The method of claim 18 wherein the composition is administered intramuscularly. -