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PubMed2002

Increased immunogenicity of HIV envelope subunit complexed with alpha2-macroglobulin when combined with monophosphoryl lipid A and GM-CSF.

Critical to the success of HIV-1 subunit vaccines is the development of strategies to augment vaccine immunogenicity. Successful adjuvants must not only improve immunogenicity above current adjuvant levels, but must also decrease the dose of immunogen required for optimal immunogenicity. We have evaluated activated alpha2-macroglobulin (alpha2M*) and a squalene-based stable emulsion containing monophosphoryl lipid A (MPL-SE) with granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvants to enhance the immunogencity of candidate HIV immunogens. Balb/c mice were subcutaneously immunized on days 0, 14 and 28 with 100-0.1 microg of HIV-1 envelope gp120 C4-V3 immunogens from either HIV IIIB (C4-V3(IIIB)) or SHIV 89.6P (C4-V3(89.6P)). Immunogens were tested covalently coupled to alpha2M*, formulated with MPL-SE/GM-CSF, or as a combination of both. Using CFA/IFA, only 50 and 100 microg, but not lower doses of C4-V3(IIIB) peptides, induced antibody responses. In contrast, peak antibody responses were detected in mice immunized with 10 microg of C4-V3 peptide coupled to alpha2M* (alpha2M*-peptide). Similar to CFA/IFA, MPL-SE/GM-CSF induced optimal antibody responses at 50 and 100 microg of C4-V3 immunogen. However, the combination of MPL-SE/GM-CSF with alpha2M*-C4-V3 peptide decreased the dose of C4-V3 required for optimal response to 5 microg for C4-V3(IIIB), and to 0.1 microg for C4-V3(89.6P). Taken together, HIV envelope gp120 C4-V3 peptides covalently complexed with alpha2M* and formulated with MPL-SE/GM-CSF resulted in a subunit HIV immunogen capable of inducing anti-HIV envelope antibody responses at doses up to 100-fold less than those needed with CFA/IFA or MPL-SE/GM-CSF alone.

AIDS Vaccines↗

PubMed2005

Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K(D) approximately 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni(2+)-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

Animals↗

PubMed2003

Tumour-induced polarization of tumour vaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies.

Previously we have shown that vaccination with the poorly immunogenic B16BL6-D5 melanoma (D5) elicits a dominant type 2 (T2) cytokine response that fails to protect the host from a subsequent tumour challenge. Here we investigated whether the inherent immunogenicity of a tumour can be correlated with its ability to bias the anti-tumour cytokine response towards either a type 1 (T1) or a T2 profile. The immune response to six tumours of different inherent immunogenicity was assayed. By isolating l-selectinlow T cells from tumour vaccine draining lymph nodes (TVDLN), it was possible to detect tumour-specific cytokine responses from both immunogenic, poorly immunogenic and non-immunogenic tumours. Immunogenic tumours (MCA-304, MCA-309, MPR-4) induced a predominant tumour-specific T1 cytokine response. In contrast, weakly (MCA-310, MPR-3) and poorly/non-immunogenic tumours (MPR-5, D5) sensitized T cells with a predominant tumour-specific T2 cytokine response. A significant correlation (P < 0.025) between immunogenicity and the ratio of tumour-specific interferon-gamma : interleukin-4 (IL-4) secretion by TVDLN T cells was identified. We then documented that non-therapeutic T cells primed by the poorly immunogenic D5, recognized "tumour-rejection" antigens and that reprogramming their cytokine response, by in vitro culture with IL-12 and anti-IL-4, to a T1 profile uncovered therapeutic efficacy. In contrast, TVDLN T cells primed by a therapeutic vaccine lose therapeutic efficacy when cultured with IL-4. These results provide insights into the development of a protective anti-tumour immune response and strengthen the hypothesis that a T1 cytokine response is critical for T-cell-mediated tumour regression.

Animals↗

PubMed2002

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology.

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Amino Acid Sequence↗

PubMed1969

The immunogenicity of dinitrophenyl amino acids.

Numerous dinitrophenyl amino acid preparations injected intradermally induced contact hypersensitivity to dinitrochlorobenzene, delayed type skin reactions to DNP-amino acids, and anti-DNP antibodies in guinea pigs. Some DNP-amino adds induced precipitating anti-DNP antibodies in rabbits as well. Some of the DNP-ammo acids studied were regularly immunogenic, possible immunogenic impurities having been excluded by extensive purification procedures. Others were either constantly nonimmunogenic or irregularly immunogenic, e.g., their immunogenicity varying from one preparation lot to another. By means of extensive chemical analyses and the establishment of dose-response curves, we were able to demonstrate in most cases that the immunogenicity was not due to contamination with unreacted dinitrofluorobenzene or other DNP derivatives, to photodecomposition or other degradation products, or to DNP-protein contaminants. Nevertheless, the irregular immunogenicity of several DNP-amino acid preparations can only be explained by a highly immunogenic impurity (or impurities) which we were unable to detect analytically. The regular immunogenicity of some other DNP-amino acids (e.g. di-DNP-L-histidine) appears to be based on a "transconjugation" phenomenon, the DNP group being able to split off from its amino acid carrier and to conjugate secondarily with proteins in vivo and in vitro. Accordingly, the interpretation of some recent data concerning the immunogenicity of low molecular weight hapten-amino acids may have to be reevaluated.

Alkanes↗

PubMed2005

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins.

AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design. METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA. RESULTS: The results in mice showed that the immu-nogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing. CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic dom-ains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

Animals↗

PubMed2003

An overview of scientific and regulatory issues for the immunogenicity of biological products.

Immunogenicity of biological products can occur pre-clinically and clinically when products elicit immune responses in animals or humans receiving the products. This is a concern for manufacturers, regulatory agencies and clinicians as immune responses can result in effects on product effectiveness and safety. The clinical sequelae of immunogenicity range from no effects to serious, life-threatening syndromes. However, although many biological products are immunogenic to some extent, it is quite rare that immunogenicity leads to serious adverse events. Whilst there are methods to detect immunogenicity, they currently rely on detecting the humoral rather than the cellular response of the immune system. The design and validation of assays such as immuno-assays and bio-assays are critical for a meaningful assessment of immunogenicity. There are a growing number of computational and laboratory-based methods for the prediction of immunogenicity, as well as methods to reduce potential immunogenicity and these may lead to less immunogenic biological products in future.

Animals↗

PubMed1987

In situ augmentation of class I major histocompatibility antigen expression on immunogenic variants of a spontaneous murine mammary carcinoma.

The relationship between expression of cell surface glycoproteins encoded by the major histocompatibility complex (MHC) and immunogenicity of a recently obtained spontaneous murine mammary adenocarcinoma (designated CBA.SP1) was examined. Immunogenic and nonimmunogenic variant clones were isolated from a subclone of the parent tumor after treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the DNA hypomethylating agent and "gene activator," 5-aza-2'-deoxycytidine (5-aza-dCyd). All clones from the untreated tumor population were tumorigenic in normal syngeneic recipients. In contrast, immunogenic variant clones, isolated at high frequencies after drug treatment [ranging from 5% (5-aza-dCyd treated) to greater than 90% (MNNG treated)], were rejected in normal syngeneic mice but grew progressively in T-cell deficient nude mice. Consistent with our previous report (J. Natl. Cancer Inst., 75: 291, 1985), all 5-aza-dCyd induced immunogenic clones expressed elevated levels of class I (particularly Dk) MHC antigens. However some (three out of nine) nonimmunogenic clones also showed enhanced class I MHC expression, implying that not all high MHC expressors were immunogenic. In contrast to 5-aza-dCyd induced variants, only 50% of MNNG induced immunogenic variants showed elevated levels of Dk or Dk and Kk antigens in vitro. Strong augmentation of class I MHC antigens in situ was observed on all immunogenic, but not nonimmunogenic, clones following transplant into syngeneic mice; no increase in MHC expression on variants during progressive growth in athymic nude mice occurred. Although no class II (Ak or Ek) antigens were detected on the parent line or any of the immunogenic variants, a strong infiltration of host I-A bearing cells occurred during immune rejection of SP1 variants. These results are consistent with the hypothesis that induction of class I MHC antigen expression on certain low MHC expressing tumors, although not the sole requirement for immunogenicity, can facilitate immune rejection of the SP1 tumor and, conversely, that the reduced level of MHC observed in certain clinical cancers may significantly affect the immunological aspects of the tumor-host relationship.

Adenocarcinoma↗

PubMedDate not supplied

The immunogenic properties of human melanomas and melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last years significant progress has been achieved in the identification of melanoma-associated antigens (MAA) recognized by cytotoxic T lymphocytes (CTL). These antigens belong to three main groups: tumor-associated testis-specific antigens (MAGE, BAGE, GAGE and PRAME), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2) and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). For the identification of these antigens, CTL cultures from mainly only 4 different melanoma patients have been used. These patients developed a strong anti-melanoma response resulting in long-lasting disease-free periods, pointing to the importance of the identification of highly immunogenic melanomas. In each of these patients, the immune response was observed against a unique set of 4 to 6 individual antigenic epitopes, on one hand suggesting the low immunogenicity of the individual antigens, and on the other pointing to the importance of the identification of additional highly immunogenic melanomas for the discovery of new MAA. The analysis of the available data on the immunogenic and protective properties of individual MAA confirms their low immunogenicity. In our study, we focused on the identification of especially highly immunogenic melanomas among a panel of 40 newly established melanoma cell lines. So far, only two such melanoma cell lines, FM3 and FM57 have been identified in this panel. The immunogenic properties of uncloned FM3 cells and several FM3 clones have been further investigated. It was found that the immunogenic properties of melanoma cells are mainly determined by the expression of progression-associated antigens as well as by ecto-ATPase, a molecule which is able to modulate cell adhesion. Cloning the cultures of PBL, stimulated with uncloned FM3 or with the highly immunogenic FM3 clone, FM3.29, has permitted us to identify the immune response against eight different MAA, five of these probably representing not previously described antigens. (Tab. 2, Fig. 2, Ref. 68.)

Antigens, Neoplasm↗

PubMed2005

Immunogenicity and safety of a combination pneumococcal-meningococcal vaccine in infants: a randomized controlled trial.

CONTEXT: The success of conjugate vaccines in decreasing invasive disease due to Streptococcus pneumoniae and group C Neisseria meningitidis has placed pressure on crowded infant immunization schedules, making development of combination vaccines a priority. OBJECTIVE: To determine the safety and immunogenicity of a combination 9-valent pneumococcal-group C meningococcal conjugate candidate vaccine (Pnc9-MenC) administered as part of the routine UK infant immunization schedule at ages 2, 3, and 4 months. DESIGN, SETTING, AND PARTICIPANTS: Phase 2 randomized controlled trial conducted from August 2000 to January 2002 and enrolling 240 healthy infants aged 7 to 11 weeks from 2 UK centers, with home follow-up visits at ages 2, 3, 4, and 5 months. INTERVENTION: Pnc9-MenC (n = 120) or monovalent group C meningococcal conjugate vaccine (MenC) (n = 120) administered in addition to routine immunizations (diphtheria and tetanus toxoids and whole-cell pertussis [DTwP], Haemophilus influenzae type b [Hib] polyribosylribitol phosphate-tetanus toxoid protein conjugate, oral polio vaccine). MAIN OUTCOME MEASURES: Group C meningococcal immunogenicity measured by serum bactericidal titer (SBT) 1 month following the third dose; rates of postimmunization reactions. RESULTS: MenC component immunogenicity was reduced in the Pnc9-MenC vs the MenC group (geometric mean SBT, 179 [95% confidence interval {CI}, 133-243] vs 808 [95% CI, 630-1037], respectively; P<.001). The proportion with group C meningococcal SBT greater than 1:8 was lower in the Pnc9-MenC vs the MenC group (95% vs 100%, P = .05). The geometric mean concentration of antibodies to concomitantly administered Hib vaccine was reduced in the Pnc9-MenC vs the MenC group (2.11 [95% CI, 1.57-2.84] microg/mL vs 3.36 [95% CI, 2.57-4.39] microg/mL; P = .02), as were antibodies against diphtheria (0.74 [95% CI, 0.63-0.87] microg/mL vs 1.47 [95% CI, 1.28-1.69] microg/mL; P<.001). Pnc9-MenC was immunogenic for each of 9 contained pneumococcal serotypes, with responses greater than 0.35 microg/mL observed in more than 88% of infants. Increased irritability and decreased activity were observed after the third dose in the Pnc9-MenC group. CONCLUSIONS: Pnc9-MenC combination vaccine administered to infants at ages 2, 3, and 4 months demonstrated reduced group C meningococcal immunogenicity compared with MenC vaccine. The immunogenicity of concomitantly administered Hib and DTwP vaccines was also diminished. The Pnc9-MenC vaccine was safe and immunogenic for all contained pneumococcal serotypes. The reduced MenC immunogenicity may limit the development of the Pnc9-MenC vaccine.

Antibodies, Bacterial↗

PubMed1989

Lack of correlation between DNA-methylating activity and appearance of the immunogenic phenotype in clones of a murine lymphoma treated with mutagens.

The relationship between induction of novel immunogenicity by xenogenizing chemicals and DNA-methylating activity in murine tumors was investigated at the clonal level in L1210Ha cells treated with 5-azacytidine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 1-(p-chlorophenyl)-3,3-dimethyltriazene (DM-Cl). Cells were exposed to the drugs in vitro, cloned by limiting dilution, and assayed for transplantation immunogenicity and 5-methylcytosine content. The results showed that 0% (0/29, 5-azacytidine), 6.8% (2/29, MNNG) and 87.5% (28/32, DM-Cl) of the resulting clones were highly immunogenic, as judged by their tumorigenicity in intact compared to immunodepressed hosts. Frequency distribution analysis of the 5-methylcytosine content of drug-treated and parental clones showed that the methylation pattern was not significantly modified by tumor exposure to either 5-azacytidine or MNNG, and the two immunogenic clones induced by MNNG had methylcytosine levels very close to the 50th percentile value. In contrast, the extent of DNA methylation was increased in the cells treated with DM-Cl, but no obvious association was found between methylation status and immunogenicity of the drug-treated clones. In four 5-azacytidine-treated clones that displayed little or no immunogenicity, additional rounds of drug exposure led to progressive DNA demethylation, but failed, as a rule, to enhance tumor cell immunogenicity. Taken together, the present data indicate that, at least for the examined tumor, immunogenic variants are generated by mutagen treatment at high (MNNG) or very high (DM-Cl) frequencies under conditions in which hypomethylation-induced antigen amplification is unlikely.

5-Methylcytosine↗

PubMed1999

[Incidence of immunogenic hyperthyroidism after radioiodine therapy of focal thyroid gland autonomy. Results of a multicenter study].

BACKGROUND: There are case reports in the literature that patients occasionally develop immunogenic hyperthyroidism 2 to 14 months following iodine-131-therapy of focal, non-immunogenic, autonomous thyroid nodules with a prevalence between 0.05 and 2.5%. Purpose of this multicenter evaluation was to assess the appearance of this phenomenon in a larger patient population. PATIENTS AND METHODS: So far 2867 patients out of 4 university hospitals are included in our study focusing on the appearance of pathologically elevated levels of thyrotropin-receptor-antibodies (TRAb) combined with hyperthyroidism following iodine-therapy. Records of the patients were screened for pre- and post-therapeutic biochemical tests, scintigraphic uptake patterns and ultrasound findings of the thyroid. RESULTS: Nineteen of 2867 patients with pretherapeutically scintigraphic "hot nodules" developed recurrent hyperthyroidism suggestive for immunogenic genesis 2 to 12 months following iodine-131-therapy (elevated TRAb-levels, homogeneous uptake in Tc-99m-pertechnetate scans). Pretherapeutically, 9 of these patients presented with a strictly focal scintigraphic uptake-pattern, 10 cases with a mixed disseminated-focal pattern. Because of missing pretherapeutic TRAb-tests in 8/9 patients presenting with a strictly focal scintigraphic uptake pattern, postradiogenic immunogenic hyperthyroidism could be reliably assessed in 1 case only. CONCLUSION: One could speculate that iodine-131-therapy may stimulate immunogenic mechanisms finally leading to immunogenic hyperthyroidism. Posttherapeutically observed hyperthyroidism following iodine-treatment might be based on an exacerbation of a preexisting--clinically not relevant/detectable--immunothyropathia. Also pretherapeutic TRAb-negative immunogenic hyperthyroidism could not be definitely excluded. Our multicenter data collected in a large patient population show similar results to the case reports of immunogenic hyperthyroidism following iodine-131-treatment in smaller populations. Therfore, the occurrence of this phenomenon plays a minor role regarding to its prevalence. Therapeutical consequences in treatment of functional thyroid autonomy are not recommended.

Adult↗

PubMed1995

Biodegradable delivery system for a birth control vaccine: immunogenicity studies in rats and monkeys.

PURPOSE: The purpose of this study was to develop a single administration delivery system for a model birth control vaccine, in order to reduce the need for multiple injections and enhance immunogenicity. METHODS: The immunogen-loaded microspheres were prepared by solvent evaporation method and characterized for loading levels, size distribution and in vitro release kinetics. The microspheres were immunized intramuscularly in wistar rats and bonnet monkeys, and the antibody response was compared to that obtained with the same total dose of the immunogen on alum given at a monthly interval. RESULTS: Results indicated that a single injection of the immunogen entrapped in the microspheres generated a response comparable to that obtained by the same immunogen on alum injected at a monthly interval. The antibodies generated by the microspheres in the monkeys also had a good bioneutralization capacity indicating immunogen integrity during the microencapsulation process. CONCLUSIONS: Biodegradable microspheres served as an effective delivery system for a model immunogen used in this study to reduce the need for frequent immunizations and enhance immunogenicity.

Animals↗

PubMed1998

Targeted in vivo delivery of therapeutic gene into experimental squamous cell carcinomas using anti-epidermal growth factor receptor antibody: immunogene approach.

The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.

Animals↗

PubMed1971

Failure of synthetic polynucleotides to affect the immunogenicity of mycobacterial ribonucleic Acid and ribosomal protein preparations.

The synthetic polyribonucleotides adenylic acid (poly A), uridylic acid (poly U), cytidylic acid (poly C), and inosinic acid (poly I), whether single- or double-stranded (poly A:U, poly I:C), cannot replace mycobacterial ribonucleic acid (RNA) in the production of a high immune response in CF-1 mice against tuberculous disease. These conclusions are based on the results of several types of experiments. (i) Poly A and poly U, used either singly or in combination, did not increase the immunogenicity of mycobacterial RNA preparations whether emulsified in Freund's incomplete adjuvant (FIA) or not emulsified. (ii) Mycobacterial ribosomal protein, extracted with 2-chloroethanol, was not immunogenic; the addition of poly A:U to the protein did not produce an immune response and FIA did not affect these results. (iii) The RNA left after the protein was extracted was partially immunogenic when emulsified in FIA even though it was partially degraded. (iv) Mycobacterial RNA prepared with ethyl alcohol and partially degraded with ribonuclease had a significantly lower immunogenic activity, and the original higher immune response was not restored by the addition of poly A:U. (v) Mycobacterial RNA totally degraded by weak alkali was not immunogenic, the original immunogenic activity was not restored by the addition of poly A:U or poly I:C, and FIA again did not influence the results. These findings suggest that (i) protein, polypeptides, or other antigenic fragments, if present, are not the specific immunogens; and (ii) mycobacterial RNA is responsible for the high immunogenic activity of mycobacterial ribosomal and RNA preparations. In addition, since the double-stranded forms of these synthetic polynucleotides markedly potentiate the formation of circulating antibodies, these results also reemphasize the lack of correlation between conventional antibody formation and immunity against tuberculosis.

Journal Article↗

PubMed1972

Purification of immunogenically active ribonucleic acid preparations of Salmonella typhimurium: molecular-sieve and anion-exchange chromatography.

Immunogenic Salmonella typhimurium ribonucleic acid (RNA) preparations, prepared by differential centrifugation, phenol extraction at 65 C, and ethanol precipitation from 0.5% sodium dodecyl sulfate solution, maintained their immunogenicity through lyophilization. As measured by survival, differential pathogen counts 5 days postchallenge, or clearance of the infecting organism from the tissues, immunization with 50 mug (dry weight) of the lyophilized preparation proved as effective as immunization with 0.1 LD(50) of attenuated S. typhimurium cells. Chromatography of the immunogenic fraction through Biogel P-6 (exclusion limit > 4,600) or through Biogel P-300 (exclusion limit > 300,000) resulted in only one immunogenically active protein of the eluate found in the void volume of the columns. Diethylaminoethyl (DEAE) cellulose anion-exchange chromatography of the RNA preparations showed that the immunogenic activity was eluted from the column at 0.8 to 1.0 m NaCl in a linear 0.1 to 2.0 m NaCl gradient. Nonimmunogenic, protein-containing minor peaks were eluted at 0.1 to 0.5 m NaCl. Serial fractionation of the crude RNA preparations over Biogel P-6 to DEAE cellulose to Biogel P-300 molecular-sieve or anion-exchange columns did not alter the immunogenicity of the RNA preparation. Incorporation of the column fractions into Freund's incomplete adjuvant did not increase their relative effectiveness in eliciting anti-salmonella resistance. Chemical analysis of the immunogenic preparations indicated that they were lacking in detectable protein, lipid, and deoxyribonucleic acid. These results suggest that the immunogenic moiety of the crude nucleic acid fraction is either RNA or an as yet undefined polysaccharide of greater than 300,000 molecular weight.

Acrylamides↗

PubMed1964

FURTHER STUDIES ON A LABILE IMMUNOGENIC PARTICULATE SUBSTANCE ISOLATED FROM MYCOBACTERIUM TUBERCULOSIS.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Further studies on a labile immunogenic particulate substance isolated from Mycobacterium tuberculosis. J. Bacteriol. 87:278-285. 1964.-A particulate fraction which was highly immunogenic for mice was collected by ultracentrifugation from mycobacteria disrupted in 0.25 m sucrose buffer. The active immunogenic material was present in the gelatinous pellet obtained after centrifugation at 40,000 rev/min (144,000 x g) for 3 hr. This active material could be prepared free from whole cells and cell walls. This was done either by several centrifugations at lower speeds, or by filtering the supernatant fluid from the 10,000 rev/min centrifugation through a Millipore filter (porosity 0.5 mu). No microorganisms were found on slides or in cultures made from these filtrates. The immunogenic moiety in the particulate fraction was found to be very labile. Temperatures higher than 0 to 4 C inactivated the immunogenic activity. There was an irreversible linear decrease in activity as the temperature increased. If fractions were frozen or lyophilized, the activity remained as high as the original material for 4 weeks, and then rapidly decreased. The immunogenic material also was very sensitive to the hydrogen-ion concentration; the optimal activity was found at pH 6.8 to 7.0. The activity decreased rapidly at more acid or alkaline pH values. Also, particulate fraction prepared in sucrose buffer at pH 7.3 and 7.6 was much less active than that prepared in sucrose buffer at pH 7.0. Immunogenic activity was decreased if the particulate fraction was dialyzed overnight against 0.01 m phosphate buffer or distilled water at 4 C. The detergent sodium lauryl sulfate inactivated immunogenic activity. Moreover, the use of a Waring Blendor to blend the ruptured cell mass before centrifugation decreased the activity. Finally, a markedly lower activity resulted if both the 20,000 and 40,000 rev/min centrifugations were done the day after the rupture of the cells. Some refinements in technique which are used now in the preparation of the particulate fraction are detailed.

Animals↗

PubMed1964

EFFECT OF MITOCHONDRIAL STABILIZERS ON THE IMMUNOGENICITY OF THE PARTICULATE FRACTION ISOLATED FROM MYCOBACTERIUM TUBERCULOSIS.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of mitochondrial stabilizers on the immunogenicity of the particulate fraction isolated from Mycobacterium tuberculosis. J. Bacteriol. 87:1346-1354. 1964.-A number of substances which have been used to stabilize mammalian mitochondrial preparations were tested to determine whether they would similarly affect the immunogenicity of a particulate fraction prepared from ruptured viable attenuated mycobacterial cells. The use of 0.44 m sucrose and the presence of 3 x 10(-2)m MgCl(2) during the preparatory processes markedly increased the immunogenicity of the particulate fraction. The increase was so great that immunogenic preparations were then consistently obtained which, in adequate dosage, were more immunogenic in CF-1 male mice than were viable attenuated mycobacterial cells. On the other hand, adenosine triphosphate (ATP), citrate, and polyvinylpyrrolidone when present during the preparatory processes reduced the immunogenicity. The addition of MgCl(2), ethylene-diaminetetraacetate, or ATP to the particulate fraction after it had been prepared did not increase its immunogenicity. When the particles were prepared in the 0.44 m sucrose buffer alone, incorporated in Freund's adjuvant, and injected intraperitoneally, immunogenicity was increased. However, this increase was not significantly greater than that obtained when the particles were prepared in the sucrose buffer containing MgCl(2). The immune state engendered in mice by the intraperitoneal injection of the particulate fraction persisted for at least 12 weeks.

Adenosine Triphosphate↗