Neonatal administration of idiotype or antiidiotype primes for protection against Escherichia coli K13 infection in mice.

Antibodies directed against the capsular polysaccharides (Ps) of encapsulated pathogenic bacteria can protect the host against infection with such organisms. The immune response to Ps, however, does not develop until relatively late in ontogeny. We have, therefore, studied alternative ways to stimulate anti-Ps antibody responses in neonates, namely priming with idiotype (Id) and anti-Id. We believe that these studies provide the first demonstration of the use of an anti-Id antibody to prime for protection against a bacterial infection and the first demonstration of the ability of a monoclonal anti-Id to prime for protection against any microbial infection. We have used a monoclonal IgM Id, anti-K13 capsular antibody, and a monoclonal IgG1 anti-Id in studies of the effects of administration of anti-Id or Id within 24 h after birth on the ability of mice to respond to subsequent immunization and challenge with live bacteria. These studies show that neonatal administration of 1 micrograms of Id or 50 ng of anti-Id lead to significantly enhanced protection in mice immunized at 4 wk of age and challenged at 5 wk with an intraperitoneal injection of 20-30 LD50 of E. coli 06:K13:H1, as compared with unprimed or antigen (Ps)-primed controls. Mice primed at birth, immunized at 12 wk of age, a time when they can respond fully to Ps itself, and challenged 1 wk later, were still significantly protected by anti-Id priming but no longer showed the effects of Id. We conclude that administration of protective Id early in life may serve a dual function in providing immediate passive protection as well as priming for protective antibodies upon subsequent antigen exposure.

VH gene family expression in mice with the xid defect.

Preferential use of particular VH gene families in the response to specific antigens has been demonstrated in several systems. The lack of responses to certain types of antigens, therefore, could be the result of deletion of or failure to express some VH genes. Because CBA/N mice, which carry the X-linked immunodeficiency (xid) gene defect, have been shown to be unresponsive to thymus-independent polysaccharide antigens, it was of interest to examine if this unresponsiveness could be accounted for by abnormal expression of particular VH gene families. Using in situ hybridization on B cell colonies, we determined the expression of nine VH gene families in CBA/CaHN females (genotypically normal), CBA/N males (xid) and females (xid), and (CBA/N x CBA/CaHN)F1 males (xid) and females (phenotypically normal). Our results indicate that VH gene family expression, including the S107 family, in CBA/N males and F1 males, is similar to that of CBA/CaHN and F1 females with predominant expression of J558, the largest gene family, in all individuals. Interestingly, CBA/N female mice, which carry two defective X chromosomes, as a group expressed significantly reduced levels of the J558 gene family, and as individuals showed variation in which family was predominantly expressed. We conclude that the unresponsiveness of mice with the xid defect to polysaccharide antigens can not attributed to a failure to express the nine VH gene families that we examined. Our findings do not support previous studies (Primi, D., and P.-A. Cazenave 1986. J. Exp. Med. 165:357), which found an absence of expression of the S107 family in xid mice.

T cell regulation of IgG subclass antibody production in response to T-independent antigens.

The effect of T lymphocytes on the IgM, IgG3, IgG1, IgG2b, and IgG2a responses of B lymphocytes to the type-2 T-independent antigens, trinitrophenylated (TNP)-Ficoll, and TNP-Levan, was investigated. T cell-bearing nu/+ mice were found to produce substantially higher IgG2 serum anti-TNP antibody than their athymic counterparts, and nu/nu and nu/+ IgG2a titers exhibiting more disparity than nu/nu and nu/+ IgG2b titers. The Igm, IgG3, and IgG1 anti-TNP levels in nu/nu and nu/+ mice were indistinguishable. By cell transfer experiments, it was determined that this variance in nude and heterozygote IgG2 responses could not be explained by B cell differences between the two strains or by suppressive effects on IgG2 production within nu/nu mice. Rather, the difference was shown to be the result of the absence of T cells at the time B cells were responding to antigen. In the absence of T cells, the strength of the nu/nu anti-TNP antibody response was found to be in the following order: IgM > IgG3 > IgG1 > IgG2b > IgG2a, a heirarchy identical with the recently proposed heavy chain gene order. The possibilities that T cells influence IgG2 production via their specific recognition of IgG2-bearing B cells or via signals to increase heavy chain switching of responding B cell clones are discussed.

Anti-immunoglobulin antibodies. I. Expression of cross-reactive idiotypes and Ir gene control of the response to IgG2a of the b allotype.

The anti-allotype antibody response to the b allotypic form of IgG2a is regulated by major histocompatibility complex (MHC)-encoded immune response (Ir) genes. Mice of d, b, p, q, r, and s haplotypes make a strong anti-allotype response on immunization with the CBPC101 myeloma protein (IgG2ab), whereas mice of the k, m, a, a1, u, and z haplotypes made no, or a very poor, response. All responder strains produce anti-IgG2ab antibodies which share common idiotypes (Id) without relation to the allelic forms of the Ig heavy-chain-constant region genes that the responding mice possess. Isoelectric focusing analysis of the anti-allotype antibodies produced in various strains of mice showed that they are of limited heterogeneity and quite similar from strain to strain. Five out of six hybridoma products with specificity for CBPC101 allotype expressed cross-reactive idiotypes (IdX). Two of hybridoma products expressing IdX identify CH3-domain determinants, and one has been assigned a CH2-domain specificity.

Regulation of the anti-inulin antibody response by a nonallotype-linked gene.

The antibody response to the inulin [(In), beta-(2 leads to 1) fructosan] determinant of bacterial levan [(BL), a beta-(2 leads to 6) polyfructosan that contains beta-(2 leads to 1) branch points] requires the presence of the a haplotype of the Igh gene complex. BALB/c (Igh a) mice immunized with BL produce IgG anti-In antibodies of a single spectrotype by isoelectric focusing analysis. C57BL/6 mice, which possess the b haplotype of the Igh gene complex and which fail to produce anti-In antibodies, nevertheless possess a gene, spectrotype regulation gene 1 (Sr-1), that regulates the isoelectric focusing (IEF) pattern of anti-In antibodies in mice of the a haplotype. Thus, the IEF patterns of anti-In antibodies of (BALB/c x C57BL/6)F1 mice and of B.C8 mice (C57BL/Ka . Igh-Ca) are considerably more complex than those of BALB/c. Backcross analysis indicates that Sr-1 is not linked to the Igh complex, the major histocompatibility complex, or to the genes that code for coat color. Studies of the heterogeneity of anti-In antibodies in recombinant inbred lines and their progeny from matings to BALB/c and C.B20 (BALB/c . Igh-Cb) suggest the existence of other regulatory genes.

Idiotype-antiidiotype regulation. V. The requirement for immunization with antigen or monoclonal antiidiotypic antibodies for the activation of beta 2 leads to 6 and beta 2 leads to 1 polyfructosan-reactive clones in BALB/c mice treated at birth with minute amounts of anti-A48 idiotype antibodies.

The anti-beta 2 leads to 6 fructosan antibodies sharing the idiotypes (Id) of ABPC48 (A48) monoclonal protein represent a silent fraction of the anti-beta 2 leads to 6 fructosan repertoire, since these antibodies cannot be detected during a conventional immune response elicited by bacterial levan (BL). However, the administration at birth of minute amounts of anti-A48 Id antibodies causes a long-lasting activation of A48 Id+-bearing clones. This activation is related to direct interaction of anti-A48 Id antibodies with precursors bearing the A48 Id+ immunoglobulin receptor, since an A48 Id+ response can be transferred with highly purified B cells in lethally irradiated mice. The maturation of these precursors into A48 Id+ anti-beta 2 leads to 6 fructosan antibody-secreting cells requires challenge by the antigen. Isoelectric focusing (IEF) data showed that in 1-mo-old mice an UPC10 (U10)-like spectrotype was observed, whereas in 3-mo-old mice, a new spectrotype binding BL rather than inulin (In) was identified. This spectrotype was observed only in CXBJ mice, the single strain in which an A48 Id+ response was observed. The antigenic challenge can be replaced by a monoclonal anti-A48 Id antibody (i.e., 17-38). Interestingly, in 1-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of A48 Id- anti-beta 2 leads to 6 fructosan-reactive clones with BALB/c type IEF spectrotypes, whereas in 3-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of W3082 IdX+ anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan-reactive clones. In these animals, inhibition of A48 Id+ anti-beta 2 leads to 6 fructosan clones was observed. This antibody probably represents a homobody carrying the internal image of the antigen, which through its paratope suppresses the A48 Id+ response and through its Id activates an A48 Id- anti-beta 2 leads to 6 fructosan response in 1-mo-old mice and in 3-mo-old mice leads to an anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan response dominated by the W3082 IdX.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune response to a thymus-dependent form of B512 dextran requires the presence of Lyb-5+ lymphocytes.

Studies of the ontogeny of the immune response to B512 dextran (Dex) show that antibody responses equal to those of adult mice are not attained until 12 wk of age. We have examined the anti-Dex response after immunization with a thymus-dependent antigen isomaltohexaosyl-keyhole limpet hemocyanin (IM6-KLH) and have shown that the development of the cross-reacting anti-Dex response parallels the development of Lyb-5+ B cells. Adult levels of anti-Dex antibody after immunization with IM6-KLH are achieved in mice between 3 and 12 wk of age, a time when Lyb-5+ cells have reached adult levels. Neonatal mice, immunized at 1 d or 1 wk after birth, failed to produce a significant amount of anti-Dex antibodies, although they did produce IM6-specific antibodies after immunization with IM6-KLH. Data, which support the conclusion from these experiments that Lyb-5+ cells are required for an anti-polysaccharide response even when the immunizing antigen is thymus-dependent, include the failure of IM6-KLH to stimulate a normal anti-Dex response in mice with the xid defect and the direct demonstration in normal adult mice that elimination of Lyb-5+ cells from spleens of mice primed with IM6-KLH abolishes the ability of these cells to transfer an anti-Dex response. The data imply that the expressed B cell repertoire in adult animals is skewed such that the vast majority of B cells capable of responding to polysaccharide determinants are in the Lyb-5+ subset.

Augmentation of in vitro humoral immune responses in the mouse by an antibody to IgD.

Heterologous anti-delta-chain antibodies have an adjuvant effect on specific in vivo humoral immune responses to simultaneously, or subsequently, injected antigens in the rat and rhesus monkey. We have used a hybridoma-secreted antibody that binds murine delta-chain of the allotype (4.22aM delta a) to study this phenomenon in the mouse and to investigate the mechanism of this effect. Injection of 4.22aM delta a into BALB/c mice removes almost all surface IgD (sIgD) from splenic B lymphocites. sIgD does not reappear until the serum level of 4.22aM delta a decreased 5-7 d after injection. 4.22aM delta a fails to induce detectable proliferation or to raise total serum Ig levels substantially above control values. However, 4.22aM dalta a injected 24 h before antigen elicits an approximately twofold enhancement of serum IgM and a 3- to 10-fold enhancement of serum IgG anti-trintriphenyl (TNP) antibodies in response to immunization with optimal doses of TNP-Ficoll or TNP-sheep red blood cells (TNP-SRBC). 4.22aM delta a injected 1 wk before or 3 d after TNP-SRBC, however, has no effect on IgG anti-TNP levels. The adjuvant effect of anti-delta-chain antibody was markedly decreased when suboptimal antigen doses were used. Furthermore, even in the case of TNP-Ficoll, a relatively T-independent antigen, the ability of 4.22aM dalta a to enhance the anti-TNP antibody response was T cell dependent. Our data suggest that the binding of anti-delta-chain antibody to cell sIgD may partially activate B lymphocytes and make them more capable of differentiating into antibody-secreting cells when stimulated by antigen-specific T cell help.

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

The regulation of biologic products derived from bioengineered plants.

Recently, there has been a large increase in the number and types of biological products--from therapeutic antibodies to vaccines for the prevention of infectious diseases--that are produced in bioengineered plant systems. We anticipate that this technology will be used increasingly on a commercial scale for the manufacture of human and animal products. These production systems have the capacity to produce very large quantities of products at lower costs and with reduced risks compared with mammalian systems.

Immunoglobulin isotype switching in xid mice.

Mice with the x-linked immunodeficiency mutation (xid) are unresponsive to polysaccharide antigens, lack a subset of B cells, and have low serum IgM (2-20% of normal) and IgG3 (3% of normal). Because of the disproportionate reduction of IgG3, the ability of B cells from xid mice to switch to gamma 3 was examined. Switching was indirectly measured by comparing IgG3 production and C gamma 3 mRNA steady state levels of purified B cells activated to switch to IgG3 by LPS in bulk culture. Direct measurement of switching was achieved by enumerating on a percentage basis switched cells in a filter disk culture assay and by FACS analysis. In both bulk culture and the filter disk assay, switching to gamma 3 was equivalent between xid and non-xid B cells.

Overcoming obstacles to monoclonal antibody product development and approval.

Using current monoclonal antibody technology one can now produce a humanized antibody to virtually any target antigen that can be identified. Consequently, one would expect there to be more approved monoclonal antibody products. Inadequate product development at both the preclinical and clinical stages has contributed to the overall lack of success. This article discusses some of the obstacles to successful product development and offers suggestions to overcoming them. The key to monoclonal antibody development, as with other biological products, is understanding the properties of the product itself, to have some proof of concept before embarking on clinical studies, and to adequately design and power the pivotal trial.

A simple method for coating native polysaccharides onto nitrocellulose.

A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disks. These were analyzed by blotting with size-matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS.

A quick method for the preparation of stable DNP red cell conjugates.

Another procedure for preparing dinitrophenyl (DNP) sheep erythrocytes (SRBC) using the DNP-alanylglycylglycyl hapten has been developed. These DNP-SRBC also are stable for as long as 1 month, but the conjugation procedure is much simpler to carry out than the previously reported method.

An avidin-biotin based ELISA for quantitation of antibody to bacterial polysaccharides.

A solid phase immunoassay utilizing avidin-biotin binding has been developed for measuring anticapsular polysaccharide antibodies. Capsular polysaccharides of Escherichia coli K1, Haemophilus influenzae type b, Staphylococcus aureus types 5 and 8, and levan from Aerobacter levanicum have been biotinylated through -OH or COOH groups with retention of antigenicity. Polysaccharides were immobilized on avidin-coated microtiter wells for use in an enzyme-linked immunosorbent assay (ELISA) to detect antibody. Two preparations of biotinylated S. aureus type 8 polysaccharide were equivalent as antigens in ELISA. Specificity was demonstrated by absorption of antisera, by competitive inhibition with purified antigens, and by reaction with specific monoclonal or myeloma antibodies. Reproducibility of the assay for H. influenzae type b and S. aureus type 8 antibody was demonstrated by replicate titrations of high and low level antisera.

Comparison of RIA and IRMA methods for measurement of carcinoembryonic antigen (CEA).

We present data comparing the Roche CEA radioimmunoassay (RIA) utilizing ultrafiltration and the Abbott CEA immunoradiometric assay (IRMA) methods for standard curve sensitivity, analytical reproducibility, recovery (pre and post extraction), dilution linearity, and patient correlation. The Roche intra- and inter-assay precision figures for this assay were 8% and 15%, respectively, for a CEA concentration of 4.0 ng/mL. The Abbott assay gave comparable precision values of 9% and 16%, respectively, for a CEA concentration of 2.0 ng/mL. Recovery for plasma spiked with CEA stock preparations was commercial-source-dependent when assayed by the Roche assay, but independent when measured by the Abbott assay. The Roche assay did not recover both preparations quantitatively, while the Abbott assay did. Plasma dilution studies over a wide range of CEA concentrations gave linear results for both assays provided the Roche assay utilizes the extraction method with dialyzate dilution for CEA values greater than 20 ng/mL. A difference (60%) was observed in linear regression slopes of the measured values between the Roche "indirect" and "direct" assays. Exposure of CEA to perchloric acid seems to play a critical role in recovery of CEA material when assayed by the Roche method. A large number of the patient samples assayed gave widely discrepant results when comparing the RIA to the IRMA methods. The potential significance of these discrepancies is discussed.

Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Immunogenicity: concepts/issues/concerns.

A human protein made in cell culture via recombinant DNA technology, theoretically, should not be immunogenic in humans. In practice, however, fully human proteins, partially human proteins and modified human proteins have all been shown to be immunogenic in some patients under some circumstances. The variables that contribute to the immunogenicity of therapeutic proteins and the measurement of antibodies to these proteins are discussed.