Regeneration of natural antibody repertoire after massive ablation of lymphoid system: robust selection mechanisms preserve antigen binding specificities

Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5(+) B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.     

The human repertoire of antibody specificities against Thomsen-Friedenreich and Tn-carcinoma-associated antigens as defined by human monoclonal antibodies

Summary Human monoclonal antibodies specific for tumour-associated Thomsen-Friedenreich (TF) [Gal(1–3)GalNAc()-O-] and Tn [GalNAc()-O-] glycoproteins were prepared using peripheral blood lymphocytes from healthy blood donors. The B lymphocytes were either directly transformed with Epstein-Barr virus (EBV) or transformed after an in vitro stimulation period with synthetic glycoproteins. The EBV-transformed lymphocytes were subsequently fused with a mouse-human heteromyeloma to secure antibody production and stability. IgM antibodies exhibiting different patterns of specificity for synthetic TF and Tn antigens were obtained, including antibodies specific for the and forms of different Gal(1–3)GalNAc-O- and GalNAc-O- conjugates and antibodies agglutinating neuraminidase-treated erythrocytes. Several of the human monoclonal antibodies showed an increased binding to cultured carcinoma cells as compared to melanoma cells. This straightforward approach for the production of human monoclonal antibodies demonstrates the possibility of investigating the reactivity pattern of tumour-binding antibodies from peripheral blood lymphocytes. The binding patterns of these monoclonal antibodies show that healthy donors carry different fine specificities against synthetic TF/Tn antigens and that these antibodies react with different tumour cells.     

Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen.

Carcinomas of the gastrointestinal tract are not curable by standard therapies. Thus, new therapeutic approaches for this disease are needed. This study proposes the use of anti-Id mAb as Ag substitutes to induce anti-tumor immunity in gastrointestinal cancer patients. Recently, we have generated and characterized one monoclonal anti-Id antibody, designated 3H1 (Ab2), which mimics biologically and antigenically a distinct and specific epitope of the 180,000 m.w. carcinoembryonic antigen (CEA) primarily expressed in high density by human pancreatic and colonic tumor cells. This epitope is unique to CEA and not present on other CEA-related lower m.w. members of the Ag family also found on normal tissues. The antigenic determinant as defined by the mAb 8019 (Ab1) against which the Ab2, 3H1 was raised, is absent on normal adult tissues by immunoperoxidase staining and haematopoietic cells including granulocytes by flow cytometry analysis. Anti-Id (Ab2) 3H1 induced CEA-specific antibodies in mice and rabbits. The immune sera from both mice and rabbits competed with Ab1 for binding to the colon carcinoma cell line LS174T and inhibited the binding of radioiodinated Ab1 to Ab2. This indicates that anti-anti-Id (Ab3) in mice and rabbits share idiotopes with Ab1 (8019). Furthermore, monoclonal Ab3 that bind to CEA have been generated from mice immunized with 3H1. The Ab3 (both polyclonal as well as monoclonal) immunoprecipitated the same 180,000 m.w. CEA as Ab1 (8019) by Western blotting analysis and showed almost identical immuno-staining patterns as Ab1 on colonic adenocarcinoma tissue sections from several patients. Collectively these data suggest that Ab2 3H1 could potentially be used clinically as a network Ag for immunotherapy of patients with CEA positive tumors.     

The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level.

The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Regulation fo immunoglobulin variable region gene assembly: development of the primary antibody repertoire

The immune system can generate an almost infinite number of different antibody specificities, the sum of which is the antibody repertoire. This article considers aspects of the mechanism and control of immunoglobulin variable (V) region gene assembly with a focus on how these factors may affect generation of the antibody repertoire in normal and disease states. New model systems to study the mechanism and control of V gene assembly are described, in particular the introduction of V gene recombination substrates into Abelson murine leukemia virus-transformed pre-B cells. Finally, a model is presented which suggests that control of V gene assembly is mediated by regulating accessibility of substrate gene segments to recombinational machinery.     

Efficient construction of a highly useful phage-displayed human antibody repertoire

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.     

The challenges of modelling antibody repertoire dynamics in HIV infection

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selection and compare this to the observed temporal patterns of antibody evolution in HIV infection. We describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.     

Doxorubicin entrapped within liposome-associated antigens results in a selective inhibition of the antibody response to the linked antigen

The generation of an immune response can dramatically alter the circulation lifetime of a targeted liposome, particularly when the response is generated against the surface-coupled ligand. Following repeated administrations, rapid elimination of the carrier system is observed, thereby limiting potential applications for targeted liposomes in a therapeutic setting. In this study, we have investigated whether the encapsulation of a toxic drug within the carrier could prevent an immune response against a surface-bound protein. Liposome clearance and humoral immune response were monitored throughout multiple administrations of liposomes containing doxorubicin with surface-conjugated ovalbumin. The results show that low doses of encapsulated doxorubicin can prevent humoral immunity against repeated administration of liposomes conjugated with ovalbumin. The immunosuppressive effect was specific for the ovalbumin coupled to the liposome surface. This selective suppression of immunity against a surface conjugated protein could prove advantageous for safe repeated administration of protein containing liposomal systems.     

Stability engineering of the human antibody repertoire

Human monoclonal antibodies often display limited thermodynamic and colloidal stabilities. This behavior hinders their production, and places limitations on the development of novel formulation conditions and therapeutic applications. Antibodies are highly diverse molecules, with much of the sequence variation observed within variable domain families and, in particular, their complementarity determining regions. This has complicated the development of comprehensive strategies for the stability engineering of the human antibody repertoire. Here we provide an overview of the field, and discuss recent advances in the development of robust and aggregation resistant antibody therapeutics.     

The C-terminal antibody binding domain of Candida albicans mp58 represents a protective epitope during candidiasis

The 58-kDa surface mannoprotein of Candida albicans (mp58) elicits strong antibody responses during infection. Epitope mapping with sera from patients with candidiasis and control individuals indicated the presence of multiple IgG-reactive continuous epitopes on the protein, expanding both the amino- and carboxy-terminal domains and several internal regions. These immunoreactive regions were similar to the ones previously identified using sera from immunized animals. Two of the epitopic regions (including the C-terminal domain) showed increased reactivity with antibodies present in sera from patients with candidiasis as compared to control individuals. Patients who survived the infection displayed increased antibody reactivity towards the C-terminal epitope as compared to those succumbing to candidiasis. A monoclonal antibody directed towards this epitopic region conferred protection in serum therapy experiments in a murine model of hematogenously disseminated candidiasis. Together, these observations indicate the carboxy-terminal antibody binding domain of C. albicans mp58 represents a protective epitope during candidiasis.     

The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies

The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nm range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.     

Analysis of the antibody repertoire of lymphoma patients

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.     

The T cell repertoire to a multideterminant antigen. Clonal heterogeneity of the T cell response, variation between syngeneic individuals, and in vitro selection of T cell specificities.

The specificity of lysozyme-induced T cell proliferative responses by individual B10.A mice was compared by using a panel of three peptides. A surprising degree of variation in the focus of the responses was observed among individual animals, both in the newly isolated lymph node cell populations and in long term T cell lines. The responses to each determinant after hen egg-white lysozyme immunization were not equal and in examining the mice as a group some determinants tended to be dominant. However, despite each animal favoring a restricted number of determinants, the responding T cell populations were still highly heterogeneous. The data suggest that many determinants are involved in the response to the whole Ag. The role of one or more dominant determinants can be overestimated because the degree of heterogeneity in long term T cell lines appears to be less than in freshly obtained lymph node cells, indicating that a process of in vitro selection occurs. We observed that the T cells responsive to one peptide, 46-61, appeared to have a selective advantage in vitro culture.     

The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.