Improving the antigenicity of sTn antigen by modification of its sialic acid residue for development of glycoconjugate cancer vaccines

Sialyl Tn (sTn) antigen is a sialylated disaccharide abundantly expressed by many tumors. To search for effective cancer immunotherapies based on sTn antigen, we designed and synthesized a series of unnatural N-acyl derivatives of sTn and studied their immunological properties. For this purpose, an efficient method was developed to synthesize the natural and unnatural forms of sTn antigen and their protein conjugates. The resultant glycoconjugates were used to immunize C57BL/6 mice, and the immune response was assessed by enzyme-linked immunosorbent assay (ELISA). Whereas the keyhole limpet hemocyanin (KLH) conjugate of sTn elicited low levels of IgM antibodies, the KLH conjugates of N-iso-butanoyl sTn and N-phenylacetyl sTn, especially the latter, induced high titers of antigen-specific IgG antibodies, showing a T-cell-dependent response that is critical for the antitumor activity. The results suggest that the modified forms of sTn, especially N-phenylacetyl sTn, have improved antigenicity and promising immunological properties for use as cancer vaccines.     

Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients

Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), do-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins.This work was supported by grants from the National Institutes of Health (CA33049, CA52491, CA08748) and the Chemotherapy Foundation     

A single-step method for the production of sugar hydrazides: intermediates for the chemoselective preparation of glycoconjugates

The ability to selectively conjugate carbohydrate molecules to a protein is a key step in the preparation of conjugate vaccines, while facile methods for linking carbohydrates to polymers or solid surfaces to produce diagnostic probes and functional microarrays are also sought. Here, we describe a simple, single-step method of producing glycosylhydrazides from unprotected sugars, which were then linked in a controlled manner to a desired carrier, through an appropriate linker. The method was chemoselective and did not require coupling reagents, and the native pyranose form of the reducing end residue was retained. Initially, mono- and disaccharide hydrazides were produced from the corresponding reducing sugars and linked to BSA through a bifunctional linker. Final exemplification of the procedure was demonstrated by the preparation of a LewisY tetrasaccharide protein conjugate, which was recognized by a LewisY monoclonal antibody indicating the preservation of the natural conformation of the tetrasaccharide in the final construct. It is envisaged that this method will have general applicability to a variety of functionally diverse reducing sugars and provide a route to highly defined glycoconjugates, without the need for elaborate synthetic strategies.     

Molecular modeling provides insights into the loading of sialic acid‐containing antigens onto CRM197: the role of chain flexibility in conjugation efficiency and glycoconjugate architecture

Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM₁₉₇. However, not only is there a surprisingly low limit for antigen chain attachment to CRM₁₉₇, but different serogroup saccharides have consistently different CRM₁₉₇ loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW?>?NmC?>?NmY) and the number of chains attached to CRM₁₉₇, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.

     

Production of antisera specific to aldosterone: effect of hapten density and of carrier proteins

In order to investigate the influence of hapten density and of carrier proteins on the immunological characteristics of antisera, 4 groups of rabbits were injected with different aldosterone-carboxymethoxime protein conjugates. Six animals immunized with an aldosterone rabbit serum albumin (RSA) conjugate carrying 15 steroid molecules (RSA-2 conjugate) showed markedly higher antibody titers than rabbits injected with a RSA conjugate carrying 8 aldosterone molecules (RSA-1 conjugate). Low antibody titers were found in 8 animals immunized with an aldosterone bovine gamma globulin (BGG) conjugate showing a molar incorporation of 15. In a group of rabbits which was first injected with the RSA-1 conjugate and re-immunized with the RSA-2 conjugate the magnitude of antibody production was not enhanced. No differences in antibody sensitivity or specificity were observed between the 4 groups. It was concluded from these experiments a) that the density of haptenic groups depending on the molar incorporation of haptens and on the molecular weight of the carrier protein had influenced the magnitude of antibody production, b) that hapten density or carrier proteins had no effect on antibody sensitivity or specificity, c) that the magnitude of antibody production cannot be altered by re-immunizing with a more potent antigen.     

不同蛋白载体的痢疾多糖结合疫苗小鼠免疫原性对比试验

试验中以小鼠为动物模型,对不同蛋白载体的痢疾多糖结合疫苗进行免疫效果观察。3种福氏2a痢疾结合疫苗和3种宋内氏痢疾结合疫苗分别皮下免疫NIH小鼠,同时设置O-SP(O-特异性多糖)对照组,免疫3针,在不同免疫针次间采血,用ELISA测定抗体滴度。单独使用福氏2aO-SP和宋内氏O-SP免疫后,小鼠血清中几乎没有抗LPS IgG抗体产生,而用结合疫苗免疫后,小鼠血清中产生了抗LPS IgG抗体,且第二次、第三次免疫后,小鼠血清中抗LPS IgG抗体水平有显著升高,表明结合疫苗具有加强免疫应答效应。三种不同的痢疾结合疫苗相比较,F2a-O-SP-rEPA结合疫苗较F2a-O-SP-TT结合疫苗和F2a-0-SP—DT结合疫苗的小鼠抗LPS IgG抗体水平高,S-O-SP-rEPA结合疫苗较S-O-SP-TT结合疫苗和S-O-SP—CRM9,结合疫苗的小鼠抗LPS IgG抗体水平高。以rEPA作为载体的痢疾结合疫苗比DT,TT作为载体的痢疾结合疫苗的免疫原性要强。     

Synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines

The peptides DRPVPY and MDWNMHAA, which were identified as mimics of the cell-surface polysaccharides of Streptococcus Group A and Shigella flexneri Y, respectively, were used in this study to develop experimental vaccines directed against these two bacteria. Both oligopeptides were synthesized employing the Fmoc solid-phase strategy and linked via the amino end to a bifunctional linker, diethylsquarate. These adducts were then conjugated to the two carrier proteins, bovine serum albumin (BSA) and tetanus toxoid (TT) to yield the peptide conjugate vaccines. The average level of incorporation of DRPVPY and MDWNMHAA on TT was 65% and 75%, respectively, whereas that of both peptide haptens on BSA was 100%. A polysaccharide conjugate against S. flexneri Y, which comprises about 10 tetrasaccharide repeating units, was also prepared based on reductive amination at the reducing end with 1,3-diaminopropane, followed by coupling of the aminated polysaccharide to diethylsquarate, and subsequent coupling of the adduct to TT. An average incorporation of 73% of polysaccharide haptens was achieved. The glycoconjugate and the oligopeptide conjugates were shown to bind effectively to the respective monoclonal antibodies directed against the cell-surface polysaccharides.     

New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development.

This review attempts to synthesize the new knowledge of pathogenesis of bacterial enteric infections and relate this information to vaccine development. Discussion focuses on human infections and to those in which significant strides have been made. As a general theme in the pathogenesis of bacterial enteric infections, pathogens can be characterized into 5 groups on the basis of their degree of ultimate invasiveness after ingestion by a susceptible hose: mucosal adherence and enterotoxin production; mucosal adherence and brush border dissolution -- enteropathogenic E. coli (EPEC) of "classical" serotypes; mucosal invasion and intraepithelial cell proliferation; mucosal translocation followed by bacterial proliferation in the lamina propria and mesenteric lymph nodes; and mucosal translocation followed by generalized infection. The review covers cholera (motility and chemotaxis, mucosal adhesion, flagellar sheath protein, hemagglutinins, outer membrane proteins, enterotoxin production, quality and duration of infection derived immunity, immune response in humans, LPS, flagellar sheath protein, cholera lectin, other cholera hemagglutinins, outer membrane protein, previous cholera vaccines, killer whole cell vaccines, toxoids, combination vaccines, attenuated versus cholerae vaccines): enterotoxigenic Escherichia coli (ETEC) (entertoxins, O:H serotypes and enterotoxin phenotypes, colonization factors, immune response in humans, vaccines against ETEC, and toxiods); EPEC (vaccines against EPEC); Shigella (smooth LPS O antigen, epithelial cell invasiveness, Shigella toxin, and Shigella vaccines); and typhoid fever (caccines against typhoid fever). The major attraction of a nonliving oral cholera vaccine is its safety. A review of available information leads to the conclusion that an oral vaccine consisting of a combination of antigens, intending to stimulate both antibacterial and antitoxic immunity, would be most likely to succeed. Current approaches to immunoprophylaxis of ETEC infection involve vaccines that stimulate antitoxic or antiadhesion immunity or both by means of killed antigens or attenuated strains. It is likely that the most effective vaccines will contain appropriate antigens intended to simultaneously stimulate both antibacterial and antitoxic immunity, thereby leading to a synergistic protective effect. Now that the speical enteroadhesive properties of EPEC have been characterized and shown to be associated with a plasmid, it should be possible to identify the phenotypic gene products responsible for this phenomenon. It is likely that fimbriae or outer membrane proteins will prove to be the organelle of adhesion. When such information becomes available, it should be possible to prepare oral vaccines consisting of the purified antigen. Efficacy has been shown for attenuated Shigella strains utilized as oral vaccines. The major thrust in the development of new immunization agensts against typhoid fever is to identify immunizing agents at least equal in efficacy to the parenteral acetone killed vaccine but which cause no adverse reactions.     

Synthesis and Evaluation of Protein Conjugates of GM3 Derivatives Carrying Modified Sialic Acids as Highly Immunogenic Cancer Vaccine Candidates

GM3, a sialylated trisaccharide antigen expressed by a number of tumors, is an attractive target in the design of therapeutic cancer vaccines. However, a serious problem associated with GM3 is that it is poorly immunogenic. To overcome this problem for the development of GM3-based cancer vaccines, four GM3 derivatives, including 5'-N-p-methylphenylacetyl, 5'-N-p-methoxyphenylacetyl, 5'-N-p-acetophenylacetyl and 5'-N-p-chlorophenylacetyl GM3, were synthesized and then coupled to a carrier protein, keyhole limpet haemocyanin (KLH). The resultant glycoconjugates were evaluated as vaccines in mouse and compared to the KLH conjugate of 5'-N-phenylacetyl GM3 (GM3NPhAc), a highly immunogenic GM3 derivative that was previously investigated as a vaccine candidate. All of the four new GM3 derivatives were proved to be more immunogenic than GM3NPhAc and elicit very strong T cell-dependent immune responses desirable for cancer immunotherapy. It was concluded that the new GM3 derivatives can form promising vaccine candidates that may be used to combine with cell glycoengineering for cancer immunotherapy.     

Haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (TLR2) and requires the presence of TLR2 for optimal immunogenicity

Conjugate vaccines consisting of the capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) covalently linked to carrier proteins, unlike pure PS, are immunogenic in infants and have significantly reduced Hib infections in the United States, but require multiple doses to induce protective anti-PS Ab titers. Hib-meningococcal outer membrane protein complex (OMPC) conjugate vaccine, however, elicits protective anti-PS Ab titers after one dose. We found that OMPC and Hib-OMPC engaged human Toll-like receptor 2 (TLR2) expressed in human embryonic kidney (HEK) cells, inducing IL-8 production, and engaged mouse TLR2 on bone marrow-derived dendritic cells, inducing TNF release. Hib conjugated to the carrier proteins CRM(197) and tetanus toxoid did not engage TLR2 on HEK or dendritic cells. Engagement of TLR2 by Hib-OMPC was MyD88 dependent, as Hib-OMPC-induced TNF production was ablated in MyD88 knockout (KO) mice. Hib-OMPC was significantly less immunogenic in TLR2 KO mice, inducing lower Hib PS IgG and IgM titers compared with those in wild-type mice. Splenocytes from OMPC-immunized TLR2 KO mice also produced significantly less IL-6 and TNF-alpha than those from wild-type mice. Hib-OMPC is unique among glycoconjugate vaccines by engaging TLR2, and the ability of Hib-OMPC to elicit protective levels of Abs after one dose may be related to TLR2-mediated induction and regulation of cytokines produced by T cells and macrophages in addition to the peptide/MHC II-dependent recruitment of T cell help commonly afforded by carrier proteins. TLR2 engagement by an adjuvant or carrier protein may be a useful strategy for augmentation of the anti-PS Ab response induced by glycoconjugate vaccines.     

Paracoccin, a GlcNAc-binding lectin from Paracoccidioides brasiliensis, binds to laminin and induces TNF-alpha production by macrophages

Paracoccidioides brasiliensis components interact with host cells and can influence the pathogenesis of paracoccidioidomycosis (PCM). Among the components released by P. brasiliensis, gp 43 and a heavily glycosylated antigen with MM>160 kDa are the most recognized by serum antibodies from patients with PCM. In order to isolate the high MM glycoconjugate, we carried out affinity chromatography of a crude exoantigen preparation on immobilized jacalin. The bound fraction (JBE, jacalin binding exoantigen) consisted of a major antigen of high MM and frequently of an additional 70-kDa minor protein. This protein, designated paracoccin, exhibited selective binding to immobilized GlcNAc, a property that was used for its purification. The structural data of paracoccin obtained by mass spectrometry of tryptic peptides did not match any known protein. Anti-paracoccin serum localized the lectin on the surface of P. brasiliensis yeasts, especially in the budding regions. Paracoccin was able to interact with laminin in a dose-dependent manner. This interaction was inhibited by GlcNAc, followed by D-glucose and D-mannose, but not by D-galactose, N-acetyl-galactosamine or L-fucose. Interestingly, paracoccin induced both resident and elicited mouse peritoneal cavity macrophages to release high and persistent levels of TNF-alpha in vitro, a fact that was associated with high nitric oxide production in elicited cells. Because binding to laminin can favor yeast adhesion and invasion of host tissues, and overproduction of NO has been associated with suppression of cell immunity, paracoccin is suggested to play an important role in PCM pathogenesis.     

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

The global spread of enteric disease, the increasingly limited options for antimicrobial treatment and the need for effective eradication programs have resulted in an increased demand for glycoconjugate enteric vaccines, made with carbohydrate-based membrane components of the pathogen, and their precise characterisation. A set of physico-chemical and immunological tests are employed for complete vaccine characterisation and to ensure their consistency, potency, safety and stability, following the relevant World Health Organization and Pharmacopoeia guidelines. Variable requirements for analytical methods are linked to conjugate structure, carrier protein nature and size and O-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide of Salmonella enterica subspecies enterica serovar Typhi capsular polysaccharide, by detergent precipitation, depolymerisation and HPAEC-PAD quantitation. Together with modern computational approaches, a more precise design of glycoconjugates is possible, allowing for improvements in solubility, structural conformation and stability, and immunogenicity of antigens, which may be applicable to a broad spectrum of vaccines. More validation experiments are required to establish the most effective and suitable methods for glycoconjugate analysis to bring uniformity to the existing protocols, although the need for product-specific approaches will apply, especially for the more complex vaccines. An overview of current and emerging analytical approaches for the characterisation of vaccines against Salmonella Typhi and Shigella species is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.     

Control of meningococcal meningitis with meningococcal vaccines.

The development of effective meinigococcal vaccines was based upon the finding that immunity to the meningococcus was directly correlated with serum bactericidal antibodies. Purified high molecular weight capsular polysaccharides of serogroups A and C meningococci stimulated the production of humoral antibodies which had group specific bactericidal activity. In controlled field trials in Army recruits, group C polysaccharide vaccines were highly effective in preventing group C disease. Following its use as a routine immunization in recruits in October 1971 group C meningococcal disease has been almost completely eliminated from Army training centers. Group A vaccine has been field tested in Egyptian school children with great success. Group B polysaccharide has failed to induce bactericidal antibodies in humans and, therefore, new research is underway to attempt to develop a cell wall protein antigen as a vaccine against group B disease.     

Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation

Novel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in order to confirm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was condensed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was confirmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS3 and SW1116 tumor cell lines, although the efficacy was different in individual cells. DU-257 was then conjugated through the linker to KM231 monoclonal antibody specifically reactive to GD3 antigen which was shown to be expressed on the surface of many malignant tumors such as SW1116. The conjugate retained its binding specificity for SW1116 cell with a similar activity with KM231. Furthermore, the conjugate showed significant growth inhibition on SW1116 cell at a concentration of 75 microg/mL while no effect on antigen negative cell, HeLaS3. These results suggest that the conjugate retained its anti-tumor effect only when it bound on and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLe(a) expression.     

Expanding polysaccharide–protein coupling of glycoconjugate vaccines

For the preparation of glycoconjugate vaccines, polysaccharide antigens can usually be chemically modified to generate reactive functional groups (e.g., the formation of aldehyde groups by periodate oxidation of adjacent diols) for covalent coupling with proteins. In a recent issue of JBC, Duke et al. showed that an alternative agent, galactose oxidase (GOase) isolated from the fungus Fusarium sp. can generate aldehyde groups in a unique chemoenzymatic approach to prepare a conjugate vaccine against Streptococcus pneumoniae. These findings introduce a new strategy for the design and development of glycoconjugate vaccines.     

病原细菌多糖疫苗和多糖结合疫苗研究进展

在许多病原细菌中,荚膜多糖和O抗原多糖能够刺激机体产生保护性抗体,因此利用病原细菌的多糖制成的疫苗能有效预防传染病,同时避免了病原细菌耐药性的出现。此类疫苗包括多糖疫苗和多糖结合疫苗。细菌体内糖基化的发现,使得利用生物法生产多糖结合疫苗成了多糖结合疫苗生产的热门方向。我们简要综述了多糖疫苗和多糖结合疫苗在研究和应用方面的主要进展。     

An asymmetric and slightly dimerized structure for the tetanus toxoid protein used in glycoconjugate vaccines

Tetanus toxoid protein has been characterized with regard oligomeric state and hydrodynamic (low-resolution) shape, important parameters with regard its use in glycoconjugate vaccines. From sedimentation velocity and sedimentation equilibrium analysis in the analytical ultracentrifuge tetanus toxoid protein is shown to be mostly monomeric in solution (～86%) with approximately 14% dimer. The relative proportions do not appear to change significantly with concentration, suggesting the two components are not in reversible equilibrium. Hydrodynamic solution conformation studies based on high precision viscometry, combined with sedimentation data show the protein to be slightly extended conformation in solution with an aspect ratio ～3. The asymmetric structure presents a greater surface area for conjugation with polysaccharide than a more globular structure, underpinning its popular choice as a conjugation protein for glycoconjugate vaccines.     

Difficulties in Establishing a Serological Correlate of Protection After Immunization with Haemophilus Influenzae Conjugate Vaccines

Abstract. in several studies the protective concentration of anti-Haemophilus infiuenzae type b (Hib) capsular polysaccharide (PS) antibodies has been concluded to be around 0·04 to 0·20 μg/ml. After the Finnish Hib polysaccharide vaccine trial it was estimated that 1 μg/ml has to be achieved to predict long term protection after vaccination. These estimates of protective anti-Hib PS antibody concentrations were based on the assumption that protection from invasive Hib disease is mediated by antibodies and the role of cell-mediated immunity is negligible. This assumption was justified since the Hib PS is a T cell-independent antigen. The matter becomes quite different when the character of the PS vaccine is altered by conjugating it to a protein carrier, so that it acquires the ability to stimulate T cells, and the immunological memory plays a role in the protection. The immunized infants are thought to be able to respond with a rapid and high antibody response after exposure to the organism. After immunization with conjugate vaccines, protection can be seen at a lower serum antibody concentration than after polysaccharide vaccine. In addition, higher avidity of anti-Hib PS antibodies is associated with the response to conjugate than PS vaccine, and there are differences between the conjugates. This might have an influence on the functional activity of the antibodies. Hib conjugate vaccines are also able to reduce the carriage rate of Hib. This should be kept in mind when estimating what is needed from protective immune response after immunization with Hib conjugate vaccines.     

Immune responses and protective efficacy of the gene vaccine expressing Ag85B and ESAT6 fusion protein from Mycobacterium tuberculosis

Genetic immunity is a new promising approach for the development of novel tuberculosis vaccines. In this study, it is shown that DNA vaccines expressing the fusion protein of antigen 85B (Ag85B) and early secreted antigenic target 6-kDa antigen (ESAT6) can induce high levels of specific IgG2a antibody subtype in the mice. With the prolongation of postimmunization time, the levels of IgG2a antibody decrease gradually. Although a high-level specific IgG2a antibody subtype is also elicited by classical BCG, the ratio of antibody subtypes IgG2a to IgG1 changes 4 weeks after immunization, and IgG1 is gradually shifted to the main antibody subtype. DNA vaccines also elicit cellular immunity as shown by specific spleen lymphocytes proliferation to Ag85B or ESAT6 protein and the production of high levels of IFN-gamma and IL-2, which is similar to that elicited by BCG. Vaccination of mice with DNA vaccines expressing the fusion protein Ag85B-ESAT6 results in a significant level of protection against the subsequent high-dose challenge with virulent Mycobacterium tuberculosis (MTB) H37Rv. Dramatic reduction in the number of MTB colony-forming units in the spleens and lungs is observed. Pathological examination showed that recombinant plasmid and BCG groups have only minor damage and organizational structures that are kept relatively complete, while in the control group, spleens and lungs are damaged seriously. Therefore, although the reducing degree of mycobacterial loads in the organ of mice immunized with recombinant plasmid is not more than that of BCG, through the analysis of pathological changes, we may conclude that the protective effect provided by DNA vaccine expressing the Ag85B-ESAT6 fusion protein is equivalent to that afforded by the classical BCG.     

Hyodeoxycholate-6-O-glucuronide cannot be quantitated with 3 alpha-hydroxysteroid dehydrogenase

The reaction of the 6-hydroxylated bile acid, hyodeoxycholic acid, and its 6-O-glucuronide conjugate with 3 alpha-hydroxysteroid dehydrogenase was examined. A standard end-point assay and determination of the initial rates of reaction showed only minimal activity of the enzyme toward hyodeoxycholate-6-glucuronide in spite of the presence of a free 3 alpha-hydroxyl group. It was established that 6-hydroxylation itself did not significantly affect the enzyme reaction. It is concluded that the 6-glucuronide either blocks or hinders enzyme access to the 3-hydroxyl group.