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At least 883 records · Page 49Linked to original sources
PubMed2000

One-step single-chain Fv recombinant antibody-based purification of gp96 for vaccine development.

Heat shock proteins such as gp96 (grp94) isolated from tumor or infected cells are able to induce specific cytotoxic T-cell responses and protective immunity. To facilitate rapid and efficient isolation, we generated gp96-specific recombinant single-chain Fv (scFv) antibodies from a semisynthetic phage display library. When immobilized on Sepharose beads, these antibodies allow a high-yield, one-step purification of native gp96 molecules from both mouse and human tumor cell lysates. gp96 molecules eluted from these affinity columns under mild conditions are still capable of generating antigen-specific CTL responses in mice. Thus, scFv-purified gp96 is still associated with peptides; however, in contrast to conventionally purified gp96, scFv-isolated gp96 is free of contaminating material such as mitogenic concanavalin A and proteolytic cathepsins. With the help of these high-yield antibody columns, it is now possible to rapidly isolate immunogenic gp96-peptide complexes from small amounts of tumor material to a purity that allows their use in cancer immunotherapy protocols.

Amino Acid Sequence↗
PubMedDate not supplied

Leprosy immunopathogenesis and vaccine development.

The truly effective immunity against intracellular parasites, including mycobacteria, is mediated by monocyte/macrophages, and in the immunologically responding (resistant) host these phagocytes need minimal antigenic stimulus, specific or non-specific, to become activated and be microbicidal. T-cell mediated delayed hypersensitivity (DTH) causes tissue damage and destruction, which is particularly unwelcome in leprosy because of its nerve-damaging potential. Gamma interferon (INF-gamma), the terminal lymphokine of a DTH response, promotes mycobacterial survival and growth. There are T-cells (TH1 subtypes) that produce DH response either independent of, or, only partly dependent on INF-gamma; this type of DH peaking at 24 hours appears similar to the Jones-Mote type rather than to the tuberculin type of DTH peaking at 48-72 hours and is devoid of the necrotic component of tuberculin type of DTH. M. leprae antigens normally elicit this Jones-Mote type of DH. Suppressor T-cells are associated with a protective immune response, while helper T-cells mediating DTH are harmful. In view of this immunobiology, it would appear that pathogenic mycobacteria that generate a tuberculin type DTH response should not be used as immunogens in leprosy.

Bacterial Vaccines↗
PubMed1999

Secretory immunoglobulin A: from mucosal protection to vaccine development.

Immune responses taking place in mucosal tissues are typified by secretory immunoglobulin A (S-IgA) molecules, which are assembled from proteins expressed in two cell lineages. The heavy and light chains as well as the J chain are produced in plasma cells, whereas the secretory component (SC) is associated to the immunoglobulin complex during transcytosis across the epithelial layer. S-IgA antibodies represent the predominant immunoglobulin class in external secretions, and the best defined entity providing specific immune protection for mucosal surfaces by blocking attachment of bacteria and viruses. S-IgA constitutes greater than 80% of all antibodies produced in mucosa-associated lymphoid tissues in humans. The existence of a common mucosal immune system permits immunization on one mucosal surface to induce secretion of antigen-specific S-IgA at distant sites. In addition, S-IgA antibodies not only function in external secretions, but also exert their antimicrobial properties within the epithelial cell during transport across the epithelium. Passive mucosal delivery of monoclonal IgA molecules neutralizes pathogens responsible for gastrointestinal and respiratory infections. Mucosal and systemic immunity can be achieved by orally administered recombinant S-IgA molecules carrying a protective bacterial epitope within the SC polypeptide primary sequence.

Animals↗
PubMed2005

Immunologic uniqueness of the genital tract: challenge for vaccine development.

Although the genital tract is considered to be a component of the mucosal immune system, it displays several distinct features not shared by other typical mucosal tissues and external secretions. Both male and female genital tract tissues lack inductive mucosal sites analogous to intestinal Peyer's patches. Consequently, local humoral and cellular immune responses stimulated by infections [with e.g. Neisseria gonorrhoeae, Chlamydia trachomatis, papilloma virus, and human immunodeficiency virus (HIV-1)] are weak or absent, and repeated local intravaginal immunizations result in minimal humoral responses. In contrast to typical external secretions such as intestinal fluid that contain secretory immunoglobulin A (S-IgA) as the dominant isotype, semen and cervico-vaginal fluid contain more IgG than IgA. Furthermore, irrespective of the route of infection, humoral immune responses to HIV-1 are dominated by specific IgG and low or absent IgA antibodies in all external secretions. Because a significant proportion of IgG in genital tract secretions is derived from the circulation, systemic immunization may provide protective IgG antibody-mediated immunity in the genital tract. Furthermore, combined systemic and mucosal (oral, rectal, and especially intranasal) immunization may induce protective humoral responses in both the systemic and mucosal compartments of the immune system.

Antibody Formation↗
PubMed1993

Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development.

The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).

AIDS Vaccines↗
PubMed2006

Seroepidemiology of Klebsiella pneumoniae in an Australian Tertiary Hospital and its implications for vaccine development.

The aim of this study was to determine the diversity of Klebsiella pneumoniae capsular serotypes in an Australian setting. Consecutive (n = 293) nonrepetitive isolates of K. pneumoniae from a large teaching hospital laboratory were analyzed. The majority of isolates were from urinary specimens (60.8%); the next most common source was sputum (14.3%), followed by blood (14%). Serotyping revealed a wide range of capsule types. K54 (17.1%), K28 (4.1%), and K17 (3.1%) were the most common, and K54 isolates displayed a high degree of clonality, suggesting a common, nosocomial source. In vitro, one K54 isolate was more adherent to urinary catheters and HEp-2 cells than four other tested isolates; it was slightly more resistant to chlorhexidine but was more susceptible to drying than heavily encapsulated strains. This is the first seroprevalence survey of K. pneumoniae to be performed on Australian isolates, and the high level of diversity of serotypes suggests that capsule-based immunoprophylaxis might not be useful for Australia. In addition there are significant differences in the predominance of specific serotypes compared to the results of surveys performed overseas, which has important implications for capsule-based immunoprophylaxis aimed at a global market.

Antibodies, Bacterial↗
PubMed2003

Shigellosis: innate mechanisms of inflammatory destruction of the intestinal epithelium, adaptive immune response, and vaccine development.

Acute infectious colitis remains a major pediatric issue of worldwide impact because it still represents a significant public health burden among the larger group of diarrheal diseases with the highest mortality rate. It is also a relevant model of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. Among cases of acute colitis of infectious origin, shigellosis is certainly the one that has benefited the most from a significant research effort. Shigella, the causative agent, is a Gram-negative bacterium that has the capacity to invade, disrupt, and cause inflammatory destruction of the intestinal epithelial barrier. The molecular and cellular bases of this invasive phenotype essentially encompass crossing of the epithelial lining, apoptotic killing of macrophages, entry into epithelial cells, and escape into the cytoplasm, followed by cell-to-cell spread. Intracellular colonization is likely to protect the micro-organisms from killing by humoral and cellular effectors of the innate immune response. Concurrently, the capacity of Shigella to reprogram invaded epithelial cells to produce proinflammatory mediators plays a major role in the strong inflammatory profile of the disease. This profile is likely to impact on the nature and quality of the adaptive response, which is dominated by humoral protection at the mucosal level.

Adaptation, Physiological↗
PubMed2000

Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development.

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules. In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.

Animals↗
PubMed2000

Capsular polysaccharide typing of domestic mastitis-causing Staphylococcus aureus strains and its potential exploration of bovine mastitis vaccine development. I. Capsular polysaccharide typing, isolation and purification of the strains.

One hundred seven isolates of Staphylococcus aureus from bovine mastitis were investigated for colony morphology in serum-soft agar (SSA), autoagglutination in salt, and capsular serotype. Capsular polysaccharide (CP) was purified and quantified from the extracts of clinical isolates. Overall, 89 isolates (83.2%) were diffuse in the SSA, without any difference in the proportion of diffuse colony between type 5 and type 8 strains. Some strains exhibited compact colonies in the SSA and expressed CP as determined by an enzyme-linked immunosorbent assay, indicating that compact morphology does not exclude encapsulation. The majority of the strains (11/12) showed autoagglutination in the salt aggregation test. The serotype 336 accounted for 46.7% of the isolates followed by serotype 5 (12.1%) and serotype 8 (12.1%). Particularly, twenty-six (24.3%) isolates reacted with two serotypes; 7 for type 8/336 and 19 for type 5/336. Five isolates (4.7%) were nontypeable with monoclonal antibodies specific for CP serotype 5, 8, or 336. The CP concentration in culture supernatants varied with the serotypes, and the total amount of CP produced by cells grown in a liquid medium was much less than that produced by cells grown on a solid medium. The Western blotting indicated that the CP bands of S. aureus serotype 5 and 8 were ranged in the molecular mass of 58-84 kilodalton (kDa), with additional bands in the region of approximately >/= 48 or </= 84 kDa.

Agglutination Tests↗
PubMed1997

Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3-like superfamily: implications for vaccine development.

Recent data demonstrate that HLA class I alleles can be grouped into superfamilies based on similarities of their peptide-binding motifs. In this study, we have tested the immunogenicity and antigenicity of peptides capable of degenerate binding to multiple HLA class I molecules of the A3-like superfamily. The assay systems utilized included both primary in vitro cultures of lymphocytes from healthy donors, as well as in vitro restimulation of lymphocytes from HIV-infected individuals. Several of the peptides capable of binding more than one HLA A3-like class I molecule were also found to be immunogenic in the context of this same group of A3-like molecules (degenerate CTL recognition). Furthermore, some of the CTL lines thus generated demonstrated promiscuous recognition of the cognate epitope in the context of MHC molecules from more than one member of the superfamily. The fine Ag specificity of this phenomenon was further analyzed using two promiscuous CTL clones derived from A3 and A11 individuals, respectively, and specific for an epitope in the HIV-1 reverse transcriptase. By the use of single-amino acid-substitution analogues, it was demonstrated that the fine specificity of the TCR is largely maintained between MHC-matched and MHC-mismatched presentation of peptide within the A3-like superfamily. These results indicate that the similar peptide-binding specificities among different members of the A3-like superfamily can be reflected in a remarkable similarity in the peptide-MHC complex structures engaged by the TCR and responsible for T cell activation.

AIDS Vaccines↗
PubMed1998

Stepwise iodination. A general procedure for detoxification of proteins suitable for vaccine development and antiserum production.

By gradual incorporation of stable iodine into toxins and whole venoms it is possible to abolish completely the lesion and lethal properties of the native components. Allergen extracts can be turned anallergic. Physiological proteins with strong biological activity can also be rendered innocuous. Tyrosine and histidine are the reactive groups that incorporate the hapten. Within the same batch of protein, there is a defined ratio hapten/protein to achieve the desired modified properties of the derivative. The iodinating solutions are easily prepared, can be accurately standardized and have unlimited shelf lives. The derivatives are obtained in a short time. The cost of the entire procedure is very low. The method was applied to tityustoxin and whole venom of the scorpion Tityus serrulatus; crotoxin and whole venom of Crotalus durissus terrificus; to five bothropic venoms; to allergenic extracts of Schistosoma mansoni; to cholera and tetanus toxin; and to insulin, kallikrein and tonin. The derivatives obtained were stable, did not show any reversion to toxicity, generated antibodies against the native antigens and gave active protection when injected into animals. No local or systemic side effects were observed, even after prolonged use. The injections were also apparently painless. By extensive haptenization self proteins can be rendered non-self, able to generate antibodies against both the derivative and the native unmodified protein, and iodination was very convenient for this purpose. A new schedule for immunization, only feasible with completely toxoided venoms is presented. It is based on a clonal expansion induced by a small dose, followed by an exponential saturation dose of the same toxoid. The attainment of higher levels of antibodies against the native antigen, in the generated sera is unmatched by other procedures.

Animals↗
PubMed2000

T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: characterization of the fine antigenic specificity of the clones and implications for vaccine development.

Excreted-secreted Ags (ESA) of Toxoplasma gondii (Tg) play an important role in the stimulation of the host immune system in both acute and chronic infections. To identify the parasite Ag(s) involved in the maintenance of T cell-mediated long term immunity, 40 ESA-specific T cell clones were derived from three chronically infected healthy subjects. All the clones were CD4+ and recognized both ESA and live tachyzoites in a HLA-DR-restricted manner. Conversely, CD4+ tachyzoite-specific T cell clones from the same subjects proliferated in response to ESA, pointing to shared immunodominant Ags between ESA and Tg tachyzoites. By T cell blot analysis using SDS-PAGE-fractionated parasite extracts, the following patterns of reactivity were detected. Of 25 clones, 6 recognized Tg fractions in the 24- to 28-kDa range and proliferated to purified GRA2, 5 reacted with Tg fractions in the 30- to 33-kDa range; and 4 of them proved to be specific for rSAg1. Although surface Ag (SAg1) is not a member of ESA, small amounts of this protein were present in ESA preparation by Western blot. Of 25 clones, 8 responded to Tg fractions in the 50- to 60-kDa range but not to the 55-kDa recombinant rhoptries-2 parasite Ag, and 6 did not react with any Tg fraction but proliferated in response to either ESA or total parasite extracts. In conclusion, CD4+ T cells specific for either ESA (GRA2) or SAg1 may be involved in the maintenance of long term immunity to Tg in healthy chronically infected individuals.

Animals↗
PubMed2005

High epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenicity: a novel strategy for modern vaccine development and a preliminary investigation about B cell discrimination of monomeric proteins.

Although early studies have shown a close correlation between epitope density and epitope-specific humoral immune responses, few attempts have been made to quantitatively compare the antigenic and immunogenic differences between protein molecules bearing low or high degrees of epitope density, nor have studies quantitatively investigated the mechanism of B cell discrimination of monomeric antigens. In this study, we prepared glutathione S-transferase (GST) fusion proteins bearing various copies of the M2e epitope from the influenza virus M2 protein [GST-(M2e)8, GST-(M2e)4 and GST-(M2e)1], which were used to detect and compare the real-time kinetic binding with M2e-specific mAb by surface plasma resonance. Our data show clearly that fusion proteins bearing higher M2e epitope density resulted in higher average avidity for M2e-specific mAb. Furthermore, it was observed that fusion proteins bearing high M2e epitope density could induce polyclonal antibodies (pAb) with enhanced an average affinity constant (KA) for M2e epitope peptide compared to fusion proteins bearing low epitope density. The average KA of pAb induced by GST-(M2e)8 (3.08 x 10(8) M(-1) or 9.96 x 10(8) M(-1)) was up to two orders of magnitude greater than the average KA of pAb induced by GST-(M2e)1 (2.00 x 10(6) M(-1) or 3.43 x 10(6) M(-1)). Thus, the data presented here demonstrate that high epitope density in a single protein molecule significantly enhances antigenicity and immunogenicity. These findings enrich our knowledge of how epitope density might relate to the recognition, activation and antibody production processes of epitope-specific immature B cells.

Animals↗
PubMed1996

Immunity and vaccine development in Pasteurella multocida infections.

The role of LPS in immunity was studied using monoclonal antibodies (MAbs) and active immunisation experiments. A panel of six MAbs produced against Pasteurella multocida serotype B:2 reacted with the LPS of serotypes B:2 and B:5, but not with other serotypes. The MAbs could opsonise P. multocida for phagocytosis by mouse macrophages, but were not bactericidal in the presence of complement. They conferred only partial passive protection in mice. Similar results showing only partial protection were obtained when purified LPS was used to actively immunise mice prior to challenge, suggesting that LPS plays a partial role in immunity to infection. The aroA gene from P. multocida serotypes A:1 and A:3 was cloned and inactivated by insertion of a kanamycin resistance gene. The mutated gene was re-introduced onto the chromosome by allelic exchange. The resultant aroA mutants were highly attenuated in a mouse model system, with a 6-log decrease in ID50. Virulence could be restored by complementation with a functional aroA gene. Mice immunised with two doses of the live mutants were protected against lethal challenge with the homologous parental strain, but not against the heterologous strain. P. multocida A:1 and A:3 expressed unique proteins when grown in iron-restricted medium. Moreover, the outer membrane (OM) fractions of these cells contained novel proteins of 75 kDa, 85 kDa and 94 kDa molecular mass. Mice were immunised with OM fractions prepared from serotype A:3 grown in iron-restricted (OM Fe-) or iron-replete (OM Fe+) media. When low challenge doses were used, both immunogens protected mice against serotype A:3, but only the OM Fe- fraction protected mice against heterologous challenge with serotype A:1. When higher challenge doses were used, only partial protection was observed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1997

Expression and immune response to foreign epitopes in bacteria. Perspectives for live vaccine development.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different regions of a carrier protein. By using as carriers, two bacterial envelope proteins, the LamB and MalE proteins of E. coli K12, we were able to express the same epitope in different sites of the two proteins and in different compartments of the bacteria. This allowed us to analyze the influence of the localization in E. coli cells of a foreign B-cell epitope on the induction of specific antibody responses, and the role of the molecular environment on the immunological properties of foreign B- or T-cell epitopes, using either purified hybrid proteins or live recombinant bacteria. Several LamB and MalE hybrid proteins were expressed in the aroA attenuated strain of S. typhimurium, SL3261. Immunizations of mice with live recombinant bacteria by the intravenous route showed that it was possible to induce humoral responses against inserted foreign sequences. In order to improve the in vivo stability of the plasmids carrying the different contructions, and to increase the amounts of recombinant LamB and MalE hybrid proteins expressed in vivo, the LamB and malE genes were placed under the control of the anaerobically inducible pnirBpromoter control. The genetic factors susceptible of influencing the immune response to recombinant Salmonella in mice were also studied.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Fusogenic potential of prokaryotic membrane lipids. Implication in vaccine development.

Development of protective immunity against many pathogens, particularly viruses, requires fine orchestration of both humoral- and cell mediated-immunity. The immunization of animals with soluble antigens usually leads to the induction of humoral immune responses. In contrast, the activation of a cell-mediated immune response against exogenous antigens has always been a challenge, requiring special strategies to expose them to the proteasome, a multifunctional protease complex in the cytosol of the target cells. The degradation of the protein by the cytosolic proteolytic system forms a cardinal step for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we report that a potent primary CTL response against a soluble protein, ovalbumin, can be induced in mice by encapsulating it in the liposomes comprised of Escherichia coli membrane lipids. These lipids were shown to induce strong membrane-membrane fusion as evident from resonance energy transfer and content mixing assays. Furthermore, the fusion of these liposomes with living cells (J774 A1) was demonstrated to result in effective transfer of a fluorescent lipid probe to the plasma membrane of the cells. Moreover, ricin A, a protein synthesis inhibitor that does not cross plasma membrane, was demonstrated to gain access to the cytosol when it was encapsulated in these liposomes. Finally, the liposomes were demonstrated to behave like efficient vehicles for the in vivo delivery of the antigens to the target cells resulting in the elicitation of antigen reactive CD8+ T cell responses.

Animals↗
PubMed2002

Analysis of a human sperm CD52 glycoform in primates: identification of an animal model for immunocontraceptive vaccine development.

Sperm agglutination antigen-1 (SAGA-1) is a human male reproductive tract glycoform of CD52. Unique modification of CD52 N-linked oligosaccharide chains in the epididymis and vas deferens results in the appearance of a carbohydrate epitope that is localized over the entire surface of human spermatozoa. SAGA-1 was characterized by the sperm-inhibitory murine monoclonal antibody (mAb) S19, and it is the target antigen of a human mAb (H6-3C4) associated with antibody-mediated infertility. Collectively, sperm surface localization, antibody inhibition of sperm function, and potential reproductive-tissue specificity identify SAGA-1 as an attractive candidate contraceptive immunogen. To establish an animal model for the study of SAGA-1 in immunologic infertility and immunocontraceptive development, we investigated the appearance of the S19 carbohydrate epitope in nonhuman primates. The S19 mAb demonstrated little to no immunoreactivity by Western blot analysis with protein extracts of spermatozoa from the baboon, marmoset, bonnet, cynomolgus, and pigtailed macaques. Immunohistochemical analysis identified CD52 in the bonnet monkey epididymis; however, the N-linked carbohydrate moiety recognized by the S19 mAb, and unique to SAGA-1, was absent. In contrast, the S19 carbohydrate epitope was identified in chimpanzee sperm extracts by Western blot analysis and in chimpanzee epididymal tissue sections by immunohistochemical analysis, indicating that it is conserved in this close relative of the human. Chimpanzee testis, seminal vesicle, and prostate do not express the S19 epitope. Although anti-CD52 immunoreactivity was identified in the spleen, the carbohydrate moiety recognized by the S19 mAb was absent, corroborating data in the human that demonstrated tissue-specific glycosylation of sperm CD52. Immunofluorescent analysis indicated that the chimpanzee homologue of sperm CD52 was present over the entire spermatozoon. In addition, the S19 mAb agglutinated chimpanzee spermatozoa in a manner similar to the effect observed on human spermatozoa. These data indicate that the distinctive carbohydrate moiety of human sperm CD52 is present in the chimpanzee, and they identify the chimpanzee as the most appropriate primate model to study the potential of this unique CD52 glycoform as a contraceptive immunogen.

Animals↗