Related U.S. Application Data 15 страница

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limited to, arsenic, lead, mercury, vinyl chloride, polychlorinated biphenyls, benzene, polycyclic aromatic hydrocarbons, cadmium, benzo(a)pyrene, benzo(b)fluoranthene, chloroform, DDT, P, P'-, aroclor 1254, aroclor 1260, dibenzo (a, h)anthracene, trichloroethylene, dieldrin, chromium hexavalent, and DDE, P, P'. Examples of such agents include those provided elsewhere herein.

In some embodiments, the В cell antigen is a self antigen. In other embodiments, the В cell antigen is an alloantigen, an allergen, a contact sensitizer, a degenerative disease antigen, a hapten, an infectious disease antigen, a cancer antigen, an atopic disease antigen, an autoimmune disease antigen, an addictive substance, a xenoantigen, or a metabolic disease enzyme or enzymatic product thereof. Examples of such antigens include those provided elsewhere herein.

As described above, the present invention provides vaccine nanocarriers comprising, for example, one or more immunomodulatory agents. In some embodiments, inventive nanocarriers comprising one or more immunomodulatory agents are used as vaccines. In some embodiments, antigen presentation to В cells can be optimized by presenting structurally intact immunomodulatory agents on the surface of nanocarriers. In some embodiments, structurally intact immunomodulatory agents are presented on the surface of vaccine nanocarriers at high copy number and/or density.

In some embodiments, an immunomodulatory agent may comprise isolated and/or recombinant proteins or peptides, inactivated organisms and viruses, dead organisms and virus, genetically altered organisms or viruses, and cell extracts. In some embodiments, an immunomodulatory agent may comprise nucleic acids, carbohydrates, lipids, and/or small molecules. In some embodiments, an immunomodulatory agent is one that elicits an immune response. In some embodiments, an immunomodulatory agent is an antigen. In some embodiments, an immunomodulatory agent is used for vaccines. Further description of immunomodulatory agents can be found in the section above entitled “B Cells. ”

As discussed above, a vaccine nanocarrier may comprise a single type of immunomodulatory agent that stimulates both В cells and T cells. In some embodiments, a vaccine nanocarrier comprises two types of immunomodulatory agents, wherein first type of immunomodulatory agent stimulates В cells, and the second type of immunomodulatory agent stimulates T cells. In some embodiments, a vaccine nanocarrier comprises greater than two types of immunomodulatory agents, wherein one or more types of immunomodulatory agents stimulate В cells, and one or more types of immunomodulatory agents stimulate T cells.

Targeting Moieties

As discussed above, inventive nanocarriers comprise one or more targeting moieties. For a discussion of general and specific properties of targeting moieties in accordance with the present invention, see the subheading entitled “Targeting Moieties” in the section above entitled “T Cells. ” In some embodiments, targeting moieties target particular cell types. In certain embodiments, a target is a В cell marker. In some embodiments, a В cell target is an antigen that is expressed in В cells but not in non-B cells. In some embodiments, a В cell target is an antigen that is more prevalent in В cells than in non-B cells.

In certain embodiments, a taiget is a SCS-Mph marker. In some embodiments, an SCS-Mph target is an antigen that is expressed in SCS-Mph but not in non-SCS-Mph. In some embodiments, an SCS-Mph target is an antigen that is more

prevalent in SCS-Mph than in non-SCS-Mph. Exemplary SCS-Mph markers are listed below in the section entitled “Subcapsular Sinus Macrophage Cells” and include those provided elsewhere herein.

In certain embodiments, a target is a FDC marker. In some embodiments, an FDC target is an antigen that is expressed in FDCs but not in non-FDCs. In some embodiments, an FDC target is an antigen that is more prevalent in FDCs than in non-FDCs. Exemplary FDC markers are listed below in the section entitled “Follicular Dendritic Cells” and include those provided elsewhere herein.

In some embodiments, a target is preferentially expressed in particular cell types. For example, expression of an SCS-Mph, FDC, and/or В cell target in SCS-Mph, FDCs, and/or В cells is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold overexpressed in SCS-Mph, FDCs, and/or В cells relative to a reference population. In some embodiments, a reference population may comprise non-SCS-Mph, FDCs, and/or В cells.

In some embodiments, expression of an SCS-Mph, FDC, and/or В cell target in activated SCS-Mph, FDCs, and/or В cells is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold overexpressed in activated SCS-Mph, FDCs, and/or В cells relative to a reference population. In some embodiments, a reference population may comprise non-activated SCS- Mph, FDCs, and/or В cells.

Subcapsular Sinus Macrophage Cells

The present invention encompasses the recognition that targeting of antigens to subcapsular sinus macrophages (SCS-Mph) is involved in efficient early presentation of lymph-borne pathogens, such as viruses, to follicular В cells (FIG. 2). As described in Example 1, following subcutaneous injection of vesicular stomatitis virus (VSV) or adenovirus (AdV) into the footpad of mice, viral particles were efficiently and selectively retained by CD169⁺SCS-Mph in the draining popliteal lymph nodes. VSV-specific В cell receptor (BCR) transgenic В cells in these lymph nodes were rapidly activated and generated extremely high antibody titers upon this viral challenge. Depletion of SCS-Mph by injection of liposomes laden with clodronate (which is toxic for Mph) abolished early В cell activation, indicating that SCS-Mph are essential for the presentation of lymph- borne particulate antigens to В cells.

В cells are more potently activated by polyvalent antigens that are presented to them on a fixed surface, rather than in solution. While not wishing to be bound by any one theory, the present invention suggests a reason why many enveloped viruses (such as VSV) elicit potent neutralizing antibody responses to their envelope glycoprotein: the antigenic protein is presented at a very high density on the surface of the viral particles, and the viral particles are presented to В cells in a relatively immotile manner, i. e., bound to the plasma membrane of SCS-Mph. The present invention encompasses the recognition that vaccine carriers that mimic viral particles by targeting SCS-Mph upon subcutaneous injection and presenting polyvalent conformationally intact antigens on their surface can stimulate a potent В cell response.

In some embodiments, SCS-Mph targeting is accomplished by moieties that bind CD169 (i. e., sialoadhesin), CDllb (i. e, CDllb/CD18, Mac-1, CR3 or < iM|! 2 integrin), and/or the mannose receptor (i. e, a multi-valent lectin),

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proteins which are all prominently expressed on SCS-Mph. Examples of such moieties include those provided elsewhere herein.

In some embodiments, SCS-Mph targeting can be accomplished by any targeting moiety that specifically binds to any entity (e. g., protein, lipid, carbohydrate, small molecule, etc. ) that is prominently expressed and/or present on macrophages (i. e., SCS-Mph markers). Exemplary SCS-Mph markers include, but are not limited to, CD4 (L3T4, W3/25, T4); CD9 (p24, DRAP-1, MRP-1); CDlla (LFA-la, aL Integrin chain); CD lib (aM Integrin chain, CR3, Mol, C3niR, Mac-1); CDllc (aX Integrin, pl50, 95, AXb2); CDwl2 (p90-120); CD13 (APN, gpl5O, EC3. 4. 11. 2); CD14 (LPS-R); CD15 (X-Hapten, Lewis, X, SSEA-1, 3-FAL); CD15s (Sialyl Lewis X); CD15u (3' sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA); CD16b (FcgRIIIb); CDwl7 (Lactosylceramide, LacCer); CD18 (Integrin 02, CDlla, b, c P-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein); CD29 (Platelet GPIIa, P-1 integrin, GP); CD31 (PECAM-1, Endocam); CD32 (FCyRII); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV, PASIV); CD37 (gp52-4O); CD38 (ADP-ribosyl cyclase, T10); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin, Leukosialin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49a (VLA-la, al Integrin); CD49b (VLA-2a, gpla, a2 Integrin); CD49c (VLA-3a, a3 Integrin); CD49e (VLA-5a, a5 Integrin); CD49f (VLA-6a, a6 Integrin, gplc); CD50 (ICAM-3); CD51 (Integrin a, VNR-a, Vitronectin-Ra); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58 (LFA-3); CD59 (lF5Ag, Hl9, Protectin, MACIF, MIRL, P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP Ma, P3 Integrin); CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP, MLA1, gp55, NGA, LAMP-3, ME491); CD64 (FcyRI); CD65 (Ceramide, VIM2); CD65s (Sialylated-CD65, VIM2); CD72 (Ly-19. 2, Ly-32. 2, Lyb-2); CD74 (Ii, invariant chain); CD75 (sialomasked Lactosamine); CD75S (a2, 6 sialylated Lac- tosamine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 (4F9, C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a (ILT5, LIR2, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc receptor, FcaR); CD91 (a2M-R, LRP); CDw92 (p70); CDw93 (GR11); CD95 (APO-1, FAS, TNFRSF6); CD97 (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, E2); CD99R (CD99 Mab restricted); CD100 (SEMA4D); CD101 (IGSF2, P126, V7); CD102 (ICAM-2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); CD112 (HveB, PRR2, PVRL2, Nectin2); CD114 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-frns, CSF-1R, M-CSFR); CD116 (GM-CSFRa); CDwll9 (IFNyR, IFNyRA); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFRp80); CD121b (Type 2 IL-1R); CD122 (IL2R0); CD123 (IL-3Ra); CD124 (IL-4Ra); CD127 (p90, IL-7R, IL-7Ra); CD128a (IL-8Ra, CXCR1, (Tentatively renamed as CD181)); CD128b (IL- 8Rb, CSCR2, (Tentatively renamed as CD182)); CD130 (gpl3O); CD131 (Common p subunit); CD132 (Common у chain, IL-2Ry); CDwl36 (MSP-R, RON, pl58-ron); CDwl37 (4-1BB, ILA); CD139; CD141 (Thrombomodulin, Fetomodulin); CD147 (Basigin, EMMPRIN, M6, 0X47); CD148 (HPTP-Т), p260, DEP-1); CD155 (PVR); CD156a (CD156, ADAM8, MS2); CD156b (TACE, ADAM17,

cSVP); CDwl56C (ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD165 (AD2, gp37); CD168 (RHAMM, IHABP, HMMR); CD169 (Sialoadhesin, Siglec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); CD172 (SIRP-la, MyD-1); CD172b (SIRPP); CD180 (RP105, Bgp95, Ly64); CD181 (CXCR1, (Formerly known as CD 128a)); CD 182 (CXCR2, (Formerly known as CD128b)); CD184 (CXCR4, NPY3R); CD191 (CCR1); CD192 (CCR2); CD195 (CCR5); CDwl97 (CCR7 (was CDwl97)); CDwl98 (CCR8); CD204 (MSR); CD205 (DEC-25); CD206 (MMR); CD207 (Langerin); CDw210 (CK); CD213a (CK); CDw217 (CK); CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD226 (DNAM-1, PTA1); CD230 (Prion Protein (PrP)); CD232 (VESP-R); CD244 (2B4, P38, NAIL); CD245 (p220/240); CD256 (APRIL, TALL2, TNF (ligand) superfamily, member 13); CD257 (BLYS, TALL1, TNF (ligand) superfamily, member 13b); CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNBF-R superfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member lOd); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD277 (BT3. 1, B7 family: Butyrophilin 3); CD280 (TEM22, ENDO180); CD281 (TLR1, TOLL-like receptor 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4, TOLL-like receptor 4); CD295 (LEPR); CD298 (ATP1B3, Na К ATPase, P3 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD300e (CMRF-35L1); CD302 (DCL1); CD305 (LAIR1); CD312 (EMR2); CD315 (CD9P1); CD317 (BST2); CD321 (JAMI); CD322 (JAM2); CDw328 (Siglec7); CDw329 (Si- glec9); CD68 (gp 110, Macrosialin); and/or mannose receptor; wherein the names listed in parentheses represent alternative names. Examples of such markers include those provided elsewhere herein.

DM-GRASP); CD227 (MUC1, PUM, PEM, EMA); CD253 (TRAIL, TNF (ligand) superfamily, member 10); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2, ICOSL); CD276 (B7H3); CD297 (ART4, ADP-ribosyltrans- ferase 4; and Dombrock blood group glycoprotein; wherein the names listed in parentheses represent alternative names. Examples of such markers include those provided elsewhere herein.

В Cell Targeting Moieties

In some embodiments, В cell targeting can be accomplished by moieties that bind the complement receptors, CR1 (i. e., CD35) or CR2 (i. e., CD21), proteins which are expressed on В cells as well as FDCs. In some embodiments, В cell targeting can be accomplished by В cell markers such as CD19, CD20, and/or CD22. In some embodiments, В cell targeting can be accomplished by В cell markers such as CD40, CD52, CD80, CXCR5, VLA-4, class II MHC, surface IgM or IgD, APRL, and/or BAFF-R. The present invention encompasses the recognition that simultaneous

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targeting of В cells by moieties specific for complement receptors or other APC-associated molecules boosts humoral responses.

In some embodiments, В cell targeting can be accomplished by any targeting moiety that specifically binds to any 5 entity (e. g., protein, lipid, carbohydrate, small molecule, etc. ) that is prominently expressed and/or present on В cells (i. e., В cell marker). Exemplary В cell markers include, but are not limited to, CDlc (M241, R7); CDld (R3); CD2 (E-rosette R, Til, LFA-2); CD5 (Tl, Tp67, Leu-1, Ly-1); 10 CD6 (T12); CD9 (p24, DRAP-1, MRP-1); CDlla (LFA-la, aL Integrin chain); CDllb (aM Integrin chain, CR3, Mol, C3niR, Mac-1); CDllc (aX Integrin, P150, 95, AXb2); CDwl7 (Lactosylceramide, LacCer); CD18 (Integrin |J2, CDlla, b, c P-subunit); CD19 (B4); CD20 (Bl, Bp35); 15 CD21 (CR2, EBV-R, C3dR); CD22 (BL-САМ, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2, Leu-20); CD24 (BBA-1, HSA); CD25 (Tac antigen, IL-2Ra, p55); CD26 (DPP IV ectoeneyme, ADA binding protein); CD27 (T14, S152); CD29 (Platelet GPIIa, 0-1 integrin, GP); CD31 20 (PECAM-1, Endocam); CD32 (FCyRII); CD35 (CR1, C3b/ C4b receptor); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39 (ATPdehydrogenase, NTPdehydroge- nase-1); CD40 (Bp50); CD44 (ECMRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; 25 CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, 0A3, Neurophilin); CD47R (MEM-133); CD48 (Blast- 1, Hulym3, BCM-1, OX-45); CD49b (VLA-2a, gpla, «2 Integrin); CD49c (VLA-3a, a3 Integrin); CD49d (VLA-4a, a4 Integrin); CD50 (ICAM-3); CD52 (CAMPATH-1, HES); 30 CD53 (OX-44); CD54 (ICAM-1); CD55 (DAF); CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD72 (Ly-19. 2, Ly-32. 2, Lyb-2); CD73 (Ecto-5'-nuciotidase); CD74 (Ii, invariant chain); CD75 (sialo-masked Lactosamine); CD75S (a2, 6 35 sialytated Lactosamine); CD77 (Pk antigen, BLA, СТН/ Gb3); CD79a (Iga, MB1); CD79b (IgP, B29); CD80; CD81 (TAPA-1); CD82 (4F9, C33, IA4, KAI1, R2); CD83 (HB15); CD84 (P75, GR6); CD85j (ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, TNFRSF6); CD98 40 (4F2, FRP-1, RL-388); CD99 (MIC2, E2); CD100 (SEMA4D); CD 102 (ICAM-2); CD 108 (SEMA7A, JMH blood group antigen); CDwll9 (IFNyR, IFNyRa); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFRp80); CD121b (Type 2 IL-1R); CD122 (IL2R0); CD124 (IL-4Ra); CD130 45 (gpl30); CD132 (Common у chain, IL-2Ry); CDwl37 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPRIN, M6, 0X47); CD150 (SLAM, IPO-3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD166 (ALCAM, KG-САМ, SC-1, BEN, DM-GRASP); CD167a (DDR1, trkE, cak); CD171 50 (L1CMA, NILE); CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD185 (CXCR5); CD192 (CCR2); CD196 (CCR6); CD197 (CCR7 (was CDwl97)); CDwl97 (CCR7, EBI1, BLR2); CD200 (OX2); CD205 (DEC-205); CDw210 (CK); CD213a (CK); 55 CDw217 (CK); CDw218a (IL18Ra); CDw218b (IL18RP) CD220 (Insulin R); CD221 (IGF1 R); CD222 (M6P-R, IGFII-R); CD224 (GGT); CD225 (Leul3); CD226 (DNAM- 1, PTA1); CD227 (MUC1, PUM, PEM, EMA); CD229 (Ly9); CD230 (Prion Protein (Prp)); CD232 (VESP-R); 60 CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL-R1, TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily, member 10d); CD265 65 (TRANCE-R, TNF-R superfamily, member 11a); CD267 (TACI, TNF-R superfamily, member 13B); CD268

(BAFFR, TNF-R superfamily, member 13C); CD269 (BCMA, TNF-R superfamily, member 16); CD275 (B7H2, ICOSL); CD277 (BT3. 1. B7 family: Butyrophilin 3); CD295 (LEPR); CD298 (ATP1B3 Na К ATPase ₽ 3 subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD305 (LAIR1); CD307 (IRTA2); CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321 (JAMI); CD322 (JAM2); CDw327 (Siglec6, CD33L); CD68 (gp 100, Macrosialin); CXCR5; VLA-4; class II MHC; surface IgM; surface IgD; APRL; and/or BAFF-R; wherein the names listed in parentheses represent alternative names. Examples of markers include those provided elsewhere herein.

In some embodiments, В cell targeting can be accomplished by any targeting moiety that specifically binds to any entity (e. g., protein, lipid, carbohydrate, small molecule, etc. ) that is prominently expressed and/or present on В cells upon activation (i. e., activated В cell marker). Exemplary activated В cell markers include, but are not limited to, CDla (R4, T6, HTA-1); CDlb (Rl); CD15s (Sialyl Lewis X); CD15u (3' sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1); CD69 (AIM, EA1, MLR3, gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80 (B7, B7-1, BB1); CD86 (B7-2/B70); CD97 (BL-KDD/F12); CD125 (IL-5Ra); CD126 (IL-6Ra); CD138 (Syndecan-1, Heparan sulfate proteoglycan); CD152 (CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4); CD253 (TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1); CD289 (TLR9, TOLL-like receptor 9); and CD312 (EMR2); wherein the names listed in parentheses represent alternative names. Examples of markers include those provided elsewhere herein.

Follicular Dendritic Cells

В cells that initially detect a previously unknown antigen typically express a В cell receptor (BCR, i. e., an antibody with a transmembrane domain) with suboptimal binding affinity for that antigen. However, В cells can increase by several orders of magnitude the affinity of the antibodies they make when they enter into a germinal center (GC) reaction. This event, which typically lasts several weeks, depends on FDC that accumulate, retain and present antigenic material to the activated В cells. В cells, while proliferating vigorously, repeatedly mutate the genomic sequences that encode the antigen binding site of their antibody and undergo class-switch recombination to form secreted high-affinity antibodies, mostly of the IgG isotype. GC reactions also stimulate the generation of long-lived memory В cells and plasma cells that maintain high protective antibody titers, often for many years. Vaccine carriers that target FDC upon subcutaneous injection and that are retained on the FDC surface for long periods of time are predicted to boost GC reactions in response to vaccination and improve the affinity and longevity of desired humoral immune responses.

In some embodiments, FDC targeting can be accomplished by moieties that bind the complement receptors, CR1 (i. e., CD35) or CR2 (i. e., CD21), proteins which are expressed on FDCs as well as В cells. Examples of moieties include those provided elsewhere herein.

Vaccine Nanocarriers Comprising Multiple Targeting Moieties

GC reactions and В cell survival not only require FDC, but also are dependent on help provided by activated CD4 T cells. Help is most efficiently provided when a CD4 T cell is first stimulated by a DC that presents a cognate peptide in MHC class II (pMHC) to achieve a follicular helper (T^) phenotype. The newly generated cell then migrates

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toward the В follicle and provides help to those В cells that present them with the same pMHC complex. For this, В cells first acquire antigenic material (e. g., virus or virus-like vaccine), internalize and process it (i. e., extract peptide that is loaded into MHC class II), and then present the pMHC to a cell.

Thus, the present invention encompasses the recognition that a vaccine that stimulates optimal humoral immunity can combine several features and components (FIG. 1): (a) antigenic material for CD4 T cells that is targeted to and presented by DCs; (b) high density surface antigens that can be presented in their native form by SCS-Mph to antigenspecific follicular В cells; (c) the capacity to be acquired and processed by follicular В cells for presentation to T^ cells (the present invention encompasses the recognition that В cells readily acquire and internalize particulate matter from SCS-Mph); (d) the ability to reach FDC and be retained on FDC in intact form and for long periods of time; and (e) adjuvant activity to render APC fully immunogenic and to avoid or overcome tolerance.

In some embodiments, a vaccine nanocarrier comprises at least one targeting moiety. In some embodiments, all of the targeting moieties of a vaccine nanocarrier are identical to one another. In some embodiments, a vaccine nanocarrier a number of different types of targeting moieties. In some embodiments, a vaccine nanocarrier comprises multiple individual targeting moieties, all of which are identical to one another. In some embodiments, a vaccine nanocarrier comprises exactly one type of targeting moiety. In some embodiments, a vaccine nanocarrier comprises exactly two distinct types of targeting moieties. In some embodiments, a vaccine nanocarrier comprises greater than two distinct types of targeting moieties.

In some embodiments, a vaccine nanocarrier comprises a single type of targeting moiety that directs delivery of the vaccine nanocarrier to a single cell type (e. g., delivery to SCS-Mph only). In some embodiments, a vaccine nanocarrier comprises a single type of targeting moiety that directs delivery of the vaccine nanocarrier to multiple cell types (e. g., delivery to both SCS-Mph and FDCs). In some embodiments, a vaccine nanocarrier comprises two types of targeting moieties, wherein the first type of targeting moiety directs delivery of the vaccine nanocarrier to one cell type, and the second type of targeting moiety directs delivery of the vaccine nanocarrier to a second cell type. In some embodiments, a vaccine nanocarrier comprises greater than two types of targeting moieties, wherein one or more types of targeting moieties direct delivery of the vaccine nanocarrier to one cell type, and one or more types of targeting moieties direct delivery of the vaccine nanocarrier to a second cell type. To give but one example, a vaccine nanocarrier may comprise two types of targeting moieties, wherein the first type of targeting moiety directs delivery of the vaccine nanocarrier to DCs, and the second type of targeting moiety directs delivery of the vaccine nanocarrier to SCS-Mph.

In some embodiments, a vaccine nanocarrier comprises at least one targeting moiety that is associated with the exterior surface of the vaccine nanocarrier. In some embodiments, the association is covalent. In some embodiments, the covalent association is mediated by one or more linkers. In some embodiments, the association is non-covalent. In some embodiments, the non-covalent association is mediated by charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding

interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof.

In some embodiments, a vaccine nanocarrier comprises a lipid membrane (e. g., lipid bilayer, lipid monolayer, etc. ), wherein at least one targeting moiety is associated with the lipid membrane. In some embodiments, at least one targeting moiety is embedded within the lipid membrane. In some embodiments, at least one targeting moiety is embedded within the lumen of a lipid bilayer. In some embodiments, at least one targeting moiety may be located at multiple locations of a vaccine nanocarrier. For example, a first targeting moiety may be embedded within a lipid membrane, and a second immunostimulatory agent may be associated with the exterior surface of a vaccine nanocarrier. To give another example, a first targeting moiety and a second targeting moiety may both be associated with the exterior surface of a vaccine nanocarrier.

Immunostimulatory Agents

As described above, in some embodiments, vaccine nanocarriers may transport one or more immuno stimulatory agents which can help stimulate immune responses. In some embodiments, a vaccine nanocarrier comprises a single type of immunostimulatory agent that stimulates both В cells and T cells. In some embodiments, a vaccine nanocarrier comprises two types of immuno stimulatory agents, wherein first type of immunostimulatory agent stimulates В cells, and the second type of immuno stimulatory agent stimulates T cells. In some embodiments, a vaccine nanocarrier comprises greater than two types of immunostimulatory agents, wherein one or more types of immuno stimulatory agents stimulate В cells, and one or more types of immunostimulatory agents stimulate T cells. See the section above for a more detailed description of immuno stimulatory agents that can be used in accordance with the present invention.

Assays for В Cell Activation

In some embodiments, various assays can be utilized in order to determine whether an immune response has been stimulated in a В cell or group of В cells (i. e., whether a В cell or group of В cells has become “activated”). In some embodiments, stimulation of an immune response in В cells can be determined by measuring antibody titers. In general, “antibody titer” refers to the ability of antibodies to bind and neutralize antigens at particular dilutions. For example, a high antibody titer refers to the ability of antibodies to bind and neutralize antigens even at high dilutions. In some embodiments, an immune response in В cells is said to be stimulated if antibody titers are measured to be positive at dilutions at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater, at least about 50-fold greater, at least about 100-fold greater, at least about 500- fold greater, at least about 1000 fold greater, or more than about 1000-fold greater than in non-immunized individuals or pre-immune serum.

In some embodiments, stimulation of an immune response in В cells can be determined by measuring antibody affinity. In particular, an immune response in В cells is said to be stimulated if an antibody has an equilibrium dissociation constant (K_r/) less than 10^-7 M, less than 10^-8M, less than 10^-9 M, less than 10^_1° M, less than 10^-11 M, less than 10^-12 M, or less.

In some embodiments, a T cell-dependent immune response in В cells is said to be stimulated if class-switch recombination has occurred. In particular, a switch from IgM to an IgG isotype or to IgA or to a mixture of these isotypes is indicative of a T cell dependent immune response in В cells.

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