Coli Heat-labile Enterotoxin Research Articles

Immunity of two novel hepatitis C virus polyepitope vaccines

Hepatitis C virus (HCV) infection remains a serious public health burden around the world. So far there is no effective vaccine against this virus. Neutralizing antibody (NAb) responses to the epitopes within HCV E1 and E2 proteins are related to the resolution of hepatitis C infection. E. coli heat-labile enterotoxin B subunit (LTB) has been described as potent immunity adjuvants. In this study, we constructed recombinant pET vectors: pET-R9-Bp (B cell polyepitopes) expressing 7 epitopes from HCV E1 and E2 proteins including R9 (E2384-411aa)-Bp (E1313-327aa-E2396-424aa-E2436-447aa-E2523-540aa-E2610-627aa-E2631-648aa) and pET-LTB-R9-Bp expressing LTB adjuvant in combination with R9-Bp. Recombinant proteins R9-Bp and LTB-R9-Bp were expressed successfully in E. coli and purified by the Ni-NTA column. Both R9-Bp and LTB-R9-Bp in BALB/c mice induced robust humoral immune response in the context of intraperitoneal or intramuscular immunization but not oral immunization. Intraperitoneal administration of LTB-R9-Bp induced a higher antibody titer (peak titer: 1:341000) than that of R9-Bp (peak titer: 1:85000) after the second boost (P = 0.0036 or 0.0002). However, comparable antibody peak titers were elicited for both R9-Bp and LTB-R9-Bp in intramuscular immunization albeit with significant difference (P = 0.0032) a week after the second boost. In addition, both R9-Bp and LTB-R9-Bp induced the secretion of cytokines including IFN-γ and IL-4 at similar levels. anti-sera induced by both R9-Bp and LTB-R9-Bp recognized native HCV E1 and E2 proteins. Moreover, these HCV-specific antisera inhibited significantly the entry of HCV (P < 0.0001). Taken together, these findings showed that E. coli-based both R9-Bp and LTB-R9-Bp could become promising HCV vaccines.

Effects of therapeutic vaccination on the control of SIV in rhesus macaques with variable responsiveness to antiretroviral drugs.

A therapeutic vaccine that induces lasting control of HIV infection could eliminate the need for lifelong adherence to antiretroviral therapy. This study investigated a therapeutic DNA vaccine delivered with a single adjuvant or a novel combination of adjuvants to augment T cell immunity in the blood and gut-associated lymphoid tissue in SIV-infected rhesus macaques. Animals that received DNA vaccines expressing SIV proteins, combined with plasmids expressing adjuvants designed to increase peripheral and mucosal T cell responses, including the catalytic subunit of the E. coli heat-labile enterotoxin, IL-12, IL-33, retinaldehyde dehydrogenase 2, soluble PD-1 and soluble CD80, were compared to mock-vaccinated controls. Following treatment interruption, macaques exhibited variable levels of viral rebound, with four animals from the vaccinated groups and one animal from the control group controlling virus at median levels of 103 RNA copies/ml or lower (controllers) and nine animals, among all groups, exhibiting immediate viral rebound and median viral loads greater than 103 RNA copies/ml (non-controllers). Although there was no significant difference between the vaccinated and control groups in protection from viral rebound, the variable virological outcomes during treatment interruption enabled an examination of immune correlates of viral replication in controllers versus non-controllers regardless of vaccination status. Lower viral burden in controllers correlated with increased polyfunctional SIV-specific CD8+ T cells in mesenteric lymph nodes and blood prior to and during treatment interruption. Notably, higher frequencies of colonic CD4+ T cells and lower Th17/Treg ratios prior to infection in controllers correlated with improved responses to ART and control of viral rebound. These results indicate that mucosal immune responses, present prior to infection, can influence efficacy of antiretroviral therapy and the outcome of immunotherapeutic vaccination, suggesting that therapies capable of modulating host mucosal responses may be needed to achieve HIV cure.

An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity

ABSTRACTSoil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli. The binary toxin also shows strong nematicidal activity against three other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

Construction of an inactivated typhoid vaccine candidate expressing Escherichia coli heat-labile enterotoxin B subunit and evaluation of its immunogenicity in a murine model.

Purpose. Typhoid fever caused by Salmonella enterica serovar Typhi has contributed to the global public health burden, particularly in developing countries. In this study, an S. Typhi ghost was developed and its capacity as a vaccine candidate against typhoid fever was assessed.Methodology. An asd + plasmid pJHL187 harbouring a ghost cassette comprising the PhiX 174 Elysis gene tightly controlled under the convergent promotor system was transformed into an asd gene-deleted mutant S.Typhi strain (STG). The eltB gene encoding the E. coli heat-labile enterotoxin (LTB) protein was subcloned into a foreign antigen delivery cassette of pJHL187 to increase mucosal immunity.Results. The stringent repression and expression of the lethal E lysis gene in the system allowed stable production of the ghost strain and secretion of LTB, which was confirmed by immune blot analysis. The level of IgG and sIgA was significantly increased in the mice subcutaneously immunized with STG-LTB compared to the non-immunized mice (P<0.05). The CD3+CD4+ T cell subpopulation was augmented in the immunized group (P<0.05) and showed the increment of immunomodulatory cytokines IL-2, IL-6, IL-12, IL-17 and IFN-γ in in vitro restimulated splenocytes isolated from the inoculated mice. The serum bactericidal activity of antibodies generated in the rabbits injected with STG-LTB was proved by the elimination of approximately 87.5 % of wild-type S. Typhi in the presence of exogenous complement.Conclusion. The results demonstrated that the STG-LTB ghost effectively enhanced the immunological responses, meaning that STG-LTB is potentially available as a vaccine candidate against typhoid fever.

Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model.

Otitis media (OM) is a common pediatric disease, and nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic OM, recurrent OM, and OM associated with treatment failure. OM is also a polymicrobial disease, wherein an upper respiratory tract viral infection predisposes to ascension of NTHI from the nasopharynx, the site of colonization, to the normally sterile middle ear, resulting in disease. Using a clinically relevant viral-bacterial coinfection model of NTHI-induced OM, we performed transcutaneous immunization (TCI) via a band-aid delivery system to administer each of three promising NTHI vaccine candidates derived from bacterial adhesive proteins and biofilm mediators: recombinant soluble PilA (rsPilA), chimV4, and integration host factor. Each immunogen was admixed with the adjuvant LT(R192G/L211A), a double mutant of Escherichia coli heat-labile enterotoxin, and assessed for relative ability to prevent the onset of experimental OM. For each cohort, the presence of circulating immunogen-specific antibody-secreting cells and serum antibody was confirmed prior to intranasal NTHI challenge. After bacterial challenge, blinded video otoscopy and tympanometry revealed a significant reduction in the proportion of animals with signs of OM compared to levels in animals receiving adjuvant only, with an overall vaccine efficacy of 64 to 77%. These data are the first to demonstrate the efficacy afforded by TCI with a band-aid vaccine delivery system in a clinically relevant polymicrobial model of OM. The simplicity of TCI with a band-aid and the significant efficacy observed here hold great promise for reducing the global burden of OM in the pediatric population.

Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses

The Escherichia coli heat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant. Salmonella enterica serovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins. S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB in S. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with either S. Enteritidis-LTB ghosts or S. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n = 20 for each): group A, nonvaccinated control; group B, immunized with S. Enteritidis-LTB ghosts; and group C, immunized with S. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3(+) CD4(+) subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization with S. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization with S. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry.

Protection against Lethal Leptospirosis after Vaccination with LipL32 Coupled or Coadministered with the B Subunit of Escherichia coli Heat-Labile Enterotoxin

Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD(50)) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

6 Escherichia coli Heat-Labile Enterotoxin (LTS61K) Modulates Dendritic Cell Function and Attenuates Airway Inflammation in Mouse Model of Allergic Asthma

BackgroundEscherichia coli heat-labile enterotoxin (LT) with different mutant forms has been used as adjuvant for vaccines due to its ability to enhance immune response to specific antigen in vivo. We hypothesis that LTS61K or LTS61K mixed with dust mite allergen, Der p, (LTS61K/Der p) can modulate dendritic cells (DCs) s' functions thus alleviate allergen-induced airway inflammation.MethodsTwo protocols (ie, preventive and therapeutic protocol) were designed to evaluate the effects of LTS61K in Der p sensitized and challenged mouse model of asthma.ResultsBoth intranasal inoculations with LTS61K or LTS61K/Der p decreased allergen-induced airway inflammation and alleviated systemic TH2-type immune response. In addition, bronchoalveolar lavage (BAL) fluids and sera from LTS61K/Der p treated mice have higher concentrations of Der p-specific IgA than those of other groups. In the in vitro study, bone marrow-derived dendritic cells (BMDCs) and DC cell line, DC2.4 cells stimulated with LTS61K/Der p both secreted pro-inflammation cytokines IL-6 and TNF-a. In contrast, after LTS61K treatment, only BMDCs decreased production of IL-6 and TNF-a as well as decreased maturation. Furthermore, we found that pre-treatment BMDC with LTS61K inhibited Der p-induced NF-kB translocation which might explain the delayed maturation and decreased productions of IL-6 and TNF-a in LTS61K pre-treated BMDCs. Intratracheally adoptive transferred with LTS61K- or LTS61K/Der p-primed DC2.4 cells or BMDCS into Der p-sensitized mice decreased inflammatory cells infiltration and TH2-type chemokines in BAL fluids and alleviated airway inflammation.ConclusionsOur results show that LTS61K may influence DCs maturation and its cytokine production. On the other hands, LTS61K/Der p may induce more Der p-specific IgA production to decrease allergic TH2 cytokine responses and alleviate airway inflammation in murine model of asthma. These finding suggested that LTS61K may have clinical application as an immune-modulator effect on the diseases of allergy and asthma.

Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores

In an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model using Bacillus anthracis Ames strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-water emulsion, PA displayed on bacteriophage T4 by the intramuscular route, and PA mixed with Escherichia coli heat-labile enterotoxin administered by the needle-free transcutaneous route. Three of the vaccine formulations administered by the intramuscular or the transcutaneous route as a three-dose regimen induced 100% protection in the rabbit model. One of the formulations, liposomal PA, also induced significantly higher lethal toxin neutralizing antibodies than PA-Alhydrogel. Even 5 months after the second immunization of a two-dose regimen, rabbits vaccinated with liposomal PA were 100% protected from lethal challenge with Ames strain spores. In summary, the needle-free skin delivery and liposomal formulation that were found to be effective in two different animal model systems appear to be promising candidates for next-generation anthrax vaccine development.

Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.

Escherichia coliHeat-Labile Enterotoxin Promotes Protective Th17 Responses against Infection by Driving Innate IL-1 and IL-23 Production

Escherichia coli heat-labile enterotoxin (LT) is a powerful mucosal adjuvant; however, it is associated with toxic effects when delivered intranasally, and its mechanism of action is poorly understood. In this article, we demonstrate that LT acts as a highly effective adjuvant when administered parenterally, promoting Ag-specific IL-17, as well as IFN-γ, IL-4, and IL-10 production in response to coadministered Ags. We found that the adjuvant activity of LT was mediated in part by inducing dendritic cell (DC) activation; LT promoted CD80 and CD86 expression by DCs and enhanced IL-1α, IL-1β, and IL-23 production. An LT mutant, LTK63, that lacks enzyme activity was less effective than the wild-type toxin in promoting DC maturation and the development of Ag-specific Th17 cells. LT enhanced IL-23 and IL-1α production from DCs via activation of ERK MAPK and IL-1β secretion through activation of caspase-1 and the NLRP3 inflammasome. These cytokines played a major role in promoting Th17 responses by LT and LTK63. The induction of Th17 cells in vivo in response to LT and LTK63 as adjuvants was significantly reduced in IL-1RI-deficient mice. Finally, using a murine respiratory infection model, we demonstrated that LT can act as a highly effective adjuvant for a pertussis vaccine, promoting Ag-specific Th17 cells and protection against Bordetella pertussis challenge, which was significantly reduced in IL-17-defective mice. Our findings provide clear evidence that LT can promote protective immune responses in part through induction of innate IL-1 and, consequently, Th17 cells.

The Liposome PCR Assay Is More Sensitive than the Vibrio cholerae Enterotoxin and Escherichia coli Heat-Labile Enterotoxin Reversed Passive Latex Agglutination Test at Detecting Cholera Toxin in Feces and Water

Practical detection of cholera toxin (CT) by a liposome PCR (LPCR) immunoassay was compared to that of an established V. cholerae enterotoxin and Escherichia coli heat-labile enterotoxin reversed passive latex agglutination (VET-RPLA) assay. LPCR detected CT in the range of 10 pg/ml to 100 ng/ml in simulated feces and environmental water. Detection by VET-RPLA required at least 4 to 19 ng/ml CT.

Prophylactic Administration of Bacterially Derived Immunomodulators Improves the Outcome of Influenza Virus Infection in a Murine Model

Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection.

Cholera Toxin andEscherichia coliHeat-Labile Enterotoxin, but Not Their Nontoxic Counterparts, Improve the Antigen-Presenting Cell Function of Human B Lymphocytes

B lymphocytes play an important role in the immune response induced by mucosal adjuvants. In this study we investigated the in vitro antigen-presenting cell (APC) properties of human B cells upon treatment with cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) and nontoxic counterparts of these toxins, such as the B subunit of CT (CT-B) and the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Furthermore, forskolin (FSK), a direct activator of adenylate cyclase, and cyclic AMP (cAMP) analogues were used to investigate the role of the increase in intracellular cAMP caused by the A subunit of CT and LT. B lymphocytes were cultured with adjuvants and polyclonal stimuli necessary for activation of B cells in the absence of CD4 T cells. Data indicated that treatment with CT, LT, FSK, or cAMP analogues, but not treatment with CT-B or LTK63, upregulated surface activation markers on B cells, such as CD86 and HLA-DR, and induced inhibition of the proliferation of B cells at early time points, while it increased cell death in long-term cultures. Importantly, B cells treated with CT, LT, or FSK were able to induce pronounced proliferation of both CD4(+) and CD8(+) allogeneic T cells compared with untreated B cells and B cells treated with CT-B and LTK63. Finally, only treatment with toxins or FSK induced antigen-specific T-cell proliferation in Mycobacterium tuberculosis purified protein derivative or tetanus toxoid responder donors. Taken together, these results indicated that the in vitro effects of CT and LT on human B cells are mediated by cAMP.

Mechanisms of Immunization Against Cancer Using Chimeric Antigens

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

Protection against Shiga Toxin-ProducingEscherichia coliInfection by Transcutaneous Immunization with Shiga Toxin Subunit B

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.

Bile and unsaturated fatty acids inhibit the binding of cholera toxin and Escherichia coli heat-labile enterotoxin to GM1 receptor.

Cholera toxin (CT) is an archetypal bacterial toxin that binds with a high affinity to the receptor ganglioside GM1 on the intestinal epithelial surface and that causes the severe watery diarrhea characteristic of the disease cholera. Blockage of the interaction of CT with the GM1 receptor is an attractive approach for therapeutic intervention. We report here that crude bile prevents the interaction of CT with GM1 and reduces CT-mediated fluid accumulation in the rabbit intestine. The unsaturated fatty acids detected in crude bile, arachidonic, linoleic, and oleic acids, were found to be the most effective. Crude bile and the unsaturated fatty acids interacted with CT but not GM1 to prevent CT-GM1 binding. Neither crude bile nor the unsaturated fatty acids had any effect on the subunit structure of CT. The binding of CT to unsaturated fatty acids resulted in a shift of the apparent pI of CT from 6.8 to 8.2 and a marked decrease in intrinsic fluorescence. The Kd was calculated from fluorescence quenching assays. It was demonstrated by the rabbit ileal loop model that practically no fluid accumulated in the intestinal loops when CT was administered together with inhibitory concentrations of linoleic acid. The bile present in the intestine was sufficient to inhibit the activity of up to 300 ng CT. Bile and unsaturated fatty acids also inhibited the binding of Escherichia coli heat-labile enterotoxin (LT) to GM1, and no fluid accumulation was observed in rabbit ileal loops when LT was administered together with linoleic acid.

Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state.

The conserved membrane proximal external region (MPER) of the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 is the target of two broadly neutralizing antibodies, 2F5 and 4E10. However, no neutralizing antibodies have been elicited against immunogens bearing these epitopes. Given that structural and biochemical studies suggest that the lipid membrane of the virion is involved in their proper configuration, HIV-1 gp41 derivatives in a pre-fusion state were expressed on the surface of immature virus like particles (VLP) derived from Sf9 cells. Guinea pigs were immunized with three doses of VLPs or Sf9 cells presenting gp41 derivatives with or without E. coli heat-labile enterotoxin (LT) as an adjuvant. While immune sera contained high titer anti-VLP antibodies, the specific anti-gp41 antibody responses were low with no neutralizing antibodies detected. An explanation for this absence may be the low level of gp41 expression relative to the many other proteins derived from host cells which are incorporated onto the VLP surface. In addition, the anti-gp41 immune response was preferentially directed to the C-helical domain, away from the MPER. Future vaccine design needs to contend with the complexity of epitope display as well as immunodominance.

Short Amyloid-β (Aβ) Immunogens Reduce Cerebral Aβ Load and Learning Deficits in an Alzheimer's Disease Mouse Model in the Absence of an Aβ-Specific Cellular Immune Response

Amyloid-beta (Abeta) immunotherapy lowers cerebral Abeta and improves cognition in mouse models of Alzheimer's disease (AD). A clinical trial using active immunization with Abeta1-42 was suspended after approximately 6% of patients developed meningoencephalitis, possibly because of a T-cell reaction against Abeta. Nevertheless, beneficial effects were reported in antibody responders. Consequently, alternatives are required for a safer vaccine. The Abeta1-15 sequence contains the antibody epitope(s) but lacks the T-cell reactive sites of full-length Abeta1-42. Therefore, we tested four alternative peptide immunogens encompassing either a tandem repeat of two lysine-linked Abeta1-15 sequences (2xAbeta1-15) or the Abeta1-15 sequence synthesized to a cross-species active T1 T-helper-cell epitope (T1-Abeta1-15) and each with the addition of a three-amino-acid RGD (Arg-Gly-Asp) motif (R-2xAbeta1-15; T1-R-Abeta1-15). High anti-Abeta antibody titers were observed in wild-type mice after intranasal immunization with R-2xAbeta1-15 or 2xAbeta1-15 plus mutant Escherichia coli heat-labile enterotoxin LT(R192G) adjuvant. Moderate antibody levels were induced after immunization with T1-R-Abeta1-15 or T1-Abeta1-15 plus LT(R192G). Restimulation of splenocytes with the corresponding immunogens resulted in moderate proliferative responses, whereas proliferation was absent after restimulation with full-length Abeta or Abeta1-15. Immunization of human amyloid precursor protein, familial AD (hAPP(FAD)) mice with R-2xAbeta1-15 or 2xAbeta1-15 resulted in high anti-Abeta titers of noninflammatory T-helper 2 isotypes (IgG1 and IgG2b), a lack of splenocyte proliferation against full-length Abeta, significantly reduced Abeta plaque load, and lower cerebral Abeta levels. In addition, 2xAbeta1-15-immunized hAPP(FAD) animals showed improved acquisition of memory compared with vehicle controls in a reference-memory Morris water-maze behavior test that approximately correlated with anti-Abeta titers. Thus, our novel immunogens show promise for future AD vaccines.