Murine marginal zone B cells play a role in Vibrio cholerae LPS antibody responses.

The emergence of Vibrio cholerae (Vc) lipopolysaccharide (LPS) as a lead protective antigen for a cholera subunit vaccine has increased the interest in what type of B cell is best suited to generate anti-Vc LPS antibodies. A related question is what form of LPS is the most immunogenic. C57Bl/6 (B6) neonatal mice (10 days old) whose marginal zone (MZ) B cell compartment is still maturing and two lines of knockout mice that either lack the signaling mechanism required for the maturation of MZ B cells or that lack a receptor required for MZ B cell retention in the MZ were used to determine the role of MZ B cells in anti-Vc LPS antibody responses. Data support the conclusion that MZ B cells play a significant role in the anti-Vc LPS antibody response. Serum and vibriocidal antibody titers also depend on whether the Vc LPS is purified or bacterial cell-associated.

Acid-detoxified Inaba lipopolysaccharide (pmLPS) is a superior cholera conjugate vaccine immunogen than hydrazine-detoxified lipopolysaccharide and induces vibriocidal and protective antibodies.

Worldwide, in endemic areas of cholera, the group most burdened with cholera is children. This is especially vexing as young children (2-5 years of age) do not respond as well, or for as long as adults do, to the current killed oral cholera vaccines (OCV). Conjugate vaccines based on the hapten-carrier paradigm have been developed for several bacterial pathogens that cause widespread and severe diseases in young children. We and others have studied different formulations of Vibrio cholerae (Vc) O1 lipopolysaccharide (LPS, a T-independent antigen) conjugates. Detoxified LPS is a central component of a LPS-based conjugate vaccine. pmLPS, which is detoxified by acid treatment, is a superior immunogen compared with hydrazine-detoxified LPS (DetAcLPS) that has altered lipid A acyl chains. The other feature of pmLPS is the ability to link carrier proteins to a core region of sugar. pmLPS readily induced vibriocidal antibodies following one intraperitoneal dose in a MPL-type adjuvant One dose of the pmLPS conjugate was suggestive of being protective; a booster resulted in protective antibodies for infant mice challenged with virulent cholera.

A one dose experimental cholera vaccine.

The number of cholera vaccine doses required for immunity is a constraint during epidemic cholera. Protective immunity following one dose of multiple Vibrio cholerae (Vc) colonization factors (Inaba LPS El Tor, TcpA, TcpF, and CBP-A) has not been directly tested even though individual Vc colonization factors are the protective antigens. Inaba LPS consistently induced vibriocidal and protective antibodies at low doses. A LPS booster, regardless of dose, induced highly protective secondary sera. Vc protein immunogens emulsified in adjuvant were variably immunogenic. CBP-A was proficient at inducing high IgG serum titers compared with TcpA or TcpF. After one immunization, TcpA or TcpF antisera protected only when the toxin co-regulated pilus operon of the challenge Vc was induced by AKI culture conditions. CBP-A was not consistently able to induce protection independent of the challenge Vc culture conditions. These results reveal the need to understand how best to leverage the 'right' Vc immunogens to obtain durable immunity after one dose of a cholera subunit vaccine. The dominance of the protective anti-LPS antibody response over other Vc antigen antibody response needs to be controlled to find other protective antigens that can add to anti-LPS antibody-based immunity.

Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains.

Vibrio cholerae causes cholera, an enteric disease of humans that is a worldwide problem. The O1 serogroup of Vibrio cholerae contains two predominant serotypes (Inaba and Ogawa) of LPS, a proven protective antigen for humans and experimental animals. We generated B-cell hybridomas from mice immunized with either: (i) two doses of purified Inaba LPS; (ii) two doses of an Inaba hexasaccharide conjugate (terminal six perosamine bound to a protein carrier), (iii) four doses of purified Inaba LPS; or (iv) a low dose of purified Inaba LPS followed by a booster with the Inaba conjugate. We showed previously that the first and third immunization protocols induce vibriocidal antibodies, as does the fourth; the second protocol induces antibodies that bind Inaba and Ogawa LPS but are not vibriocidal. Anti-LPS mAbs derived from hybridomas resulting from each immunization protocol were characterized for binding to Inaba and Ogawa LPS, their vibriocidal or protective capacity, and the variable heavy chain family they expressed. LPS immunogens selected different LPS-specific B cells expressing six different Vh chain families. Protective and non-protective mAbs could express variable regions from the same family. One mAb was specific for Inaba LPS, the other mAbs were cross-reactive with both LPS serotypes. Sequence comparison suggests that the pairing of a specific light chain, somatic mutation, or the specific VDJ recombination can modulate the protective capacity of mAbs that express a common variable heavy chain family member.

Immunogenicity of synthetic saccharide fragments of Vibrio cholerae O1 (Ogawa and Inaba) bound to Exotoxin A.

Recombinant exotoxin A (rEPA) from Pseudomonas aeruginosa conjugated to Vibrio cholerae O1 serotype-specific polysaccharides (mono-, di- and hexasaccharide) were immunogenic in mice. Monosaccharide conjugates boosted the humoral responses to the hexasaccharide conjugates. Prior exposure to purified Ogawa lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to boost the vibriocidal response, but Inaba LPS did not prime for an enhanced vibriocidal response by a contra-serotype conjugate. Prior exposure to the carrier, and priming B cells with the LPS of either serotype, resulted in enhanced vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished response to the Inaba LPS. These studies demonstrate that the 'functional' B cell epitopes on the LPS differ from those of the neoglycoconjugates and that the order of immunization and the serotype of the boosting conjugate can influence the epitope specificity and function of the antisera.

Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide neoglycoconjugates.

Ogawa hexasaccharide neoglycoconjugates induce protective antibodies in mice. Similar Ogawa conjugates but with a longer linker that connects the carrier to shorter saccharides are immunogenic, but generally ineffective at inducing vibriocidal or protective antibodies. The efficacy of Ogawa hexasaccharide neoglycoconjugates of different linker lengths were tested. The majority of mice given immunizations separated by a 14-day gap did not produce vibriocidal or protective antibodies. Mice immunized 28 days apart with immunogens containing the shortest or medium length linker, but not the longest, produced vibriocidal and protective antibodies. A nonprotective, priming dose of purified Ogawa LPS followed 5 days later with a booster of the Ogawa neoglycoconjugates (di-, tetra-, or hexasaccharide) resulted in vibriocidal antibodies at day 10.

Effect of saccharide length on the immunogenicity of neoglycoconjugates from synthetic fragments of the O-SP of Vibrio cholerae O1, serotype Ogawa.

A synthetic hexasaccharide, identical to the terminal hexasaccharide of Ogawa LPS, coupled to bovine serum albumin induced protective antibodies in mice. To determine if there was a minimum saccharide length required for immunogenicity and efficacy, shorter (mono- to pentasaccharide) neoglycoconjugates (CHO-BSA) were tested in mice. The Ogawa CHO-BSA was inoculated at either a constant mass but differing moles, or equal moles but differing masses. Humoral responses were essentially the same when mice received 9 microg of the carbohydrate (0.007 mM with the pentasaccharide) in each of the neoglycoconjugates prepared from mono- through the pentasaccharide, or the same molar amount (0.007 mM), proportionally less by weight when going from the penta- to the monosaccharide. These data show that, within this dose range, the responses occurred virtually independently of the amount of immunogen. Humoral antibodies induced by these immunogens were generally not vibriocidal. Selected antisera induced by CHO-BSA immunogens were protective, but the ELISA titers of the sera were not predictive of the protective capacity. Purified, Ogawa LPS induced anti-Ogawa LPS IgM antibody titers similar to those induced by the Ogawa CHO-BSA conjugates. The anti-whole LPS sera were strongly vibriocidal, as were the previously reported sera induced by hexasaccharide conjugates. This suggests either that the shorter oligosaccharides lack a conformational epitope provided by the hexasaccharide or that the LPS has additional B cell epitopes or selects different B cells in the primary response.

A Vibrio cholerae classical TcpA amino acid sequence induces protective antibody that binds an area hypothesized to be important for toxin-coregulated pilus structure.

Vibrio cholerae is a gram-negative bacterium that has been associated with cholera pandemics since the early 1800s. Whole-cell, killed, and live-attenuated oral cholera vaccines are in use. We and others have focused on the development of a subunit cholera vaccine that features standardized epitopes from various V. cholerae macromolecules that are known to induce protective antibody responses. TcpA protein is assembled into toxin-coregulated pilus (TCP), a type IVb pilus required for V. cholerae colonization, and thus is a strong candidate for a cholera subunit vaccine. Polypeptides (24 to 26 amino acids) in TcpA that can induce protective antibody responses have been reported, but further characterization of their amino acid targets relative to tertiary or quaternary TCP structures has not been done. We report a refinement of the TcpA sequences that can induce protective antibody. One sequence, TcpA 15 (residues 170 to 183), induces antibodies that bind linear TcpA in a Western blot as well as weakly bind soluble TcpA in solution. These antibodies bind assembled pili at high density and provide 80 to 100% protection in the infant mouse protection assay. This is in sharp contrast to other anti-TcpA peptide sera (TcpA 11, TcpA 13, and TcpA 17) that bind very strongly in Western blot and solution assays yet do not provide protection or effectively bind TCP, as evidenced by immunoelectron microscopy. The sequences of TcpA 15 that induce protective antibody were localized on a model of assembled TCP. These sequences are centered on a site that is predicted to be important for TCP structure.

Synthetic fragments of Vibrio cholerae O1 Inaba O-specific polysaccharide bound to a protein carrier are immunogenic in mice but do not induce protective antibodies.

Development of Vibrio cholerae lipopolysaccharide (LPS) as a cholera vaccine immunogen is justified by the correlation of vibriocidal anti-LPS response with immunity. Two V. cholerae O1 LPS serotypes, Inaba and Ogawa, are associated with endemic and pandemic cholera. Both serotypes induce protective antibody following infection or vaccination. Structurally, the LPSs that define the serotypes are identical except for the terminal perosamine moiety, which has a methoxyl group at position 2 in Ogawa but a hydroxyl group in Inaba. The terminal sugar of the Ogawa LPS is a protective B-cell epitope. We chemically synthesized the terminal hexasaccharides of V. cholerae serotype Ogawa, which comprises in part the O-specific polysaccharide component of the native LPS, and coupled the oligosaccharide at different molar ratios to bovine serum albumin (BSA). Our initial studies with Ogawa immunogens showed that the conjugates induced protective antibody. We hypothesized that antibodies specific for the terminal sugar of Inaba LPS would also be protective. Neoglycoconjugates were prepared from synthetic Inaba oligosaccharides (disaccharide, tetrasaccharide, and hexasaccharide) and BSA at different levels of substitution. BALB/c mice responded to the Inaba carbohydrate (CHO)-BSA conjugates with levels of serum antibodies of comparable magnitude to those of mice immunized with Ogawa CHO-BSA conjugates, but the Inaba-specific antibodies (immunoglobulin M [IgM] and IgG1) were neither vibriocidal nor protective in the infant mouse cholera model. We hypothesize that the anti-Inaba antibodies induced by the Inaba CHO-BSA conjugates have enough affinity to be screened via enzyme-linked immunosorbent assay but not enough to be protective in vivo.

Distinctive maturation of in vitro versus in vivo anti-CD40 mAb-matured dendritic cells in mice.

Dendritic cells (DCs) can be matured by CD40 stimulation to upregulate their MHC class II/peptide complexes and costimulatory molecule surface expression to become adept at presenting antigen to and activating naive T lymphocytes. The use of anti-CD40 antibodies as adjuvants for DC-based therapy has been advanced. Little is known as to how DC biology in response to CD40 ligation differs between in vitro versus in vivo ligation. Therefore, the authors analyzed the expression kinetics of MHC class II (I-Ak)/HEL peptide "complex," total MHC class II, CD80, and CD86 on in vitro or in vivo CD40-stimulated DCs over a period of 5 days. MHC class II, "complex," and costimulatory molecule expression was elevated at 1 day in vitro and stayed high for the culture period, whereas in vivo expression of the cohort of molecules peaked earlier and then declined. When purified DCs were co-cultured in vitro with antigen-specific T cell hybridomas, the DCs had lower expression of total MHC class II and "complex," but did not reduce their CD80 and CD86 expression. The lower expression was dependent on cognate interaction as a non-antigen-specific T cell hybridoma was without effect. Blocking antigen-specific MHC class II/peptide-T cell receptor (TcR) complex interaction with antibody inhibited the reduction of MHC class II expression on CD40-stimulated DCs in vitro. Overall, their studies suggest distinct response of DCs to typical conditions that feature anti-CD40 monoclonal antibody (mAb)-activated DCs in vitro or in vivo.

IgA Fc receptor (FcalphaR) cross-linking recruits tyrosine kinases, phosphoinositide kinases and serine/threonine kinases to glycolipid rafts.

The human IgA Fc receptor (FcalphaR, CD89) triggers several important physiological functions, including phagocytosis, NADPH oxidase activation and antigen presentation. Efforts are underway to delineate FcalphaR signal-transduction pathways that control these functions. In a previous study, we demonstrated that cross-linking of FcalphaR increased its partitioning into membrane glycolipid rafts and was accompanied by gamma-chain-dependent recruitment and phosphorylation of the tyrosine kinases Lck/Yes-related novel protein tyrosine kinase (Lyn) and Bruton's tyrosine kinase (Btk). Here we have performed a more extensive characterization of signalling effectors recruited to rafts on FcalphaR cross-linking. We demonstrate that in addition to tyrosine kinases Lyn and Btk, FcalphaR cross-linking also recruits B-lymphocyte kinase (Blk) and spleen tyrosine kinase (Syk) to rafts. We show recruitment of phosphoinositide kinases, including 3-phosphoinositide 3-kinase and phospholipase Cgamma2, and serine/threonine kinases such as protein kinase C (PKC) alpha, PKCepsilon, and protein kinase B (PKB) alpha. This suggests that lipid rafts serve as sites for FcalphaR-triggered recruitment of multiple classes of signalling effectors. We further demonstrate that tyrosine kinases and PKCalpha have a sustained association with rafts, whereas phosphoinositide 3-kinase and its downstream effectors have a transient association with rafts. This is consistent with temporally regulated divergence of FcalphaR signalling pathways in rafts. Furthermore, we suggest the spatial separation of signalling effectors by transport of phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, PKBalpha and PKCepsilon to endocytic compartments containing internalized FcalphaR.

Induction of protective immunity by synthetic Vibrio cholerae hexasaccharide derived from V. cholerae O1 Ogawa lipopolysaccharide bound to a protein carrier.

Synthetic antigens that mimic the terminal hexasaccharide epitope of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, were conjugated to bovine serum albumin (BSA). Conjugates with carbohydrate-to-carrier molar ratios of 15.5:1, 9.2:1, and 4.6:1 were tested for immunogenicity and efficacy in mice. The role of preimmunity to BSA and the use of adjuvant in the generation of the serologic response to the O-specific polysaccharide and protection against virulent V. cholerae was examined. Preimmunity to BSA did not affect the anti-Ogawa titers but seemed to enhance the protective capacity of antiserum. All 3 conjugates were immunogenic, but adjuvant was effective at inducing higher and earlier antibody responses. In tertiary serum samples, a correlation was found between vibriocidal activity and protection. The protective capacity of antiserum was evident in serum from mice immunized with all conjugates, but it was highest in the groups that received the conjugate with the lowest level of substitution. Further studies are required to increase understanding of the reason for differential protection.