CH1 Exon Research Articles

A novel SARS-CoV-2 Beta RBD DNA vaccine directly targeted to antigen-presenting cells induces strong humoral and T cell responses

Throughout the COVID-19 pandemic, several variants of concern (VoC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have evolved, affecting the efficacy of the approved COVID-19 vaccines. To address the need for vaccines that induce strong and persistent cross-reactive neutralizing antibodies and T cell responses, we developed a prophylactic SARS-CoV-2 vaccine candidate based on our easily and rapidly adaptable plasmid DNA vaccine platform. The vaccine candidate, referred to here as VB2129, encodes a protein homodimer consisting of the receptor binding domain (RBD) from lineage B.1.351 (Beta) of SARS-CoV-2, a VoC with a severe immune profile, linked to a targeting unit (human LD78β/CCL3L1) that binds chemokine receptors on antigen-presenting cells (APCs) and a dimerization unit (derived from the hinge and CH3 exons of human IgG3). Immunogenicity studies in mice demonstrated that the APC-targeted vaccine induced strong antibody responses to both homologous Beta RBD and heterologous RBDs derived from Wuhan, Alpha, Gamma, Delta, and Omicron BA.1 variants, as well as cross-neutralizing antibodies against these VoC. Overall, preclinical data justify the exploration of VB2129 as a potential booster vaccine that induces broader antibody- and T cell-based protection against current and future SARS-CoV-2 VoC.

Immunoglobulin T genes in Actinopterygii

In teleost fishes, there are three immunoglobulin isotypes named immunoglobulin M (IgM), D (IgD), and T (IgT). IgT was the last to be described in teleost fishes, and it is specific to them. From recent fish genomes, we identified and studied the immunoglobulin heavy chain genes in Actinopterygii. For this analysis, a custom bioinformatics and machine learning pipeline, we call CHfinder, was developed that identifies the exons coding for the CH domains of fish immunoglobulins. Some IgT in teleost and holostean fish found from this systematic study have not been previously described. Phylogenetic analysis of the deduced amino acid sequences of the IgT CH1 exons reveals they are similar to the CH1 of IgM. This analysis also shows that the other three domains (CH2, CH3, and CH4) were not the result of recent IgM duplication processes in Actinopterygii, demonstrating that it is an immunoglobulin of earlier origin. The bioinformatics program, CHfinder, is publicly available at https://github.com/compimmuno/CHfinder.

Genetic removal of the CH1 exon leads to the production of hypofunctional heavy chain-only IgG2a in rats

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

Allelic polymorphism of Immunoglobulin heavy chain genes in the Antarctic teleost Trematomus bernacchii

IgM represents the main immunoglobulin isotype shared by all teleost fish. However, Antarctic fish IgM possess a peculiar hinge region, which connects the CH2 and CH3 domains, not seen in any other teleost species. In the present study allelic polymorphism of IgM gene of the Antarctic teleost Trematomus benacchii was investigated. By nucleotide sequencing the entire Immunoglobulin heavy chain constant region from ten T. bernacchii individuals, 47 positions were found to be polymorphic. The largest number of polymorphic positions, accounting for 51% of the total, was found to fall within the hinge region. This region not only displayed extensive nucleotide variation, but also length diversity; in fact several sequences were one amino acid shorter as resulting from the usage of a different splice acceptor site of the CH3 exon, as demonstrated by genomic DNA analysis. The Ka/Ks ratios of the polymorphic positions showed typical values higher than 1, indicative of positive selection acting to polymorphic codons to favor amino acid replacements and maintain allelic variants.

Diverse splicing pathways of the membrane IgHM pre-mRNA in a Chondrostean, the Siberian sturgeon

Teleosts and tetrapods have evolved different splice patterns to generate their membrane-bound IgM. In the tetrapod lineage, the first transmembrane exon is spliced to an internal cryptic site located close to the end of the fourth constant exon. Because teleosts lack this site they use the regular 3′-splice site of the CH3 exon instead. We characterized the μm splicing patterns in a Chondrostean, the Siberian sturgeon. We observed a surprising diversity of splice patterns, the TM1 exon being spliced to a cryptic site at the end of CH4, to a cryptic site in CH3 or to the 3′-end of CH1. These different pathways lead to mIGHM transcripts encoding four, two or one complete C-domain(s), respectively. The short variant CH1-TM1 was found only in VH2 positive transcripts, while the two other variants were observed for IgHM transcripts expressing all VH families. These results shed light on the evolution of IgM splicing pathways.

Structural Evidence for a New IgG1 Antibody Sequence Allele of Cattle

Analysis of the heavy-chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV-1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1(a) (clone 2) and IgG1(b) (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216-230. The Thr224 and Thr226 of IgG1(a) were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1(b) were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three-dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro-Ala-Ser-Ser indicated a potential structure-enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O-linked glycosylation sites and increasing the susceptibility to degradation by protease-secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1(d). Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1(c) that is distinct in both sequence and structure from the known sequence variants.

Selective IgG2 deficiency due to a point mutation causing abnormal splicing of the C 2 gene

The mechanism underlying selective IgG subclass deficiency is largely unknown in humans. We have previously reported the acquisition of a complete IgG2 deficiency in a leukemia patient after bone marrow transplantation. Southern blot analysis showed a deletion including the Cgamma2 and Cgamma4 genes on one chromosome in the donor, suggesting that the remaining Cgamma2 gene allele was silent. In the patient and his two IgG2 deficient brothers, the silent Cgamma2 gene showed both germ-line transcription and switch recombination and no structural defects were found in the intronic promoter or the switch region of the gene. However, an A-->G transition in the fourth nucleotide in the 5' portion of intron 1 was identified. Transfection of artificial constructs into the human B cell lines demonstrated that this A-->G transition inactivated the normal splice site, and instead, a cryptic splice site in the CH1 exon was used in RNA post-transcriptional processing, leading to a 16 bp deletion of the gamma2 CH1 exon. This aberrantly spliced RNA that is mostly derived from germ-line transcription in vivo was also detected in both homozygous and heterozygous individuals carrying this mutation. These findings suggest a novel genetic mechanism as the cause of IgG subclass deficiency in selected patients.

Anti-idiotypic DNA vaccines for lymphoma immunotherapy require the presence of both variable region genes for tumor protection.

Vaccination with immunogenic formulations of lymphoma-derived immunoglobulin can elicit strong anti-idiotypic immune responses which have proved effective in murine B cell tumor challenge experiments and suggested possible benefits in recent human clinical trials. Naked plasmid DNA vaccines encoding the Id determinants as scFv fragments provide the most promising alternative to protein immunization. With this approach the addition of an immunogenic domain linked to the scFv has proved essential for the induction of a protective immune response. In this study we have produced a scFv gene construct linked to the CH3 exon of the human IgG1 constant region and tested its efficacy in inducing protective immunity against the mouse BCL1 lymphoma. We have also generated a second construct in which the BCL1 VL gene was deleted to investigate whether the VH region domain contains sufficient antigenic determinants for a protective immune response. Both constructs induced anti-idiotypic antibodies that specifically reacted with the BCL1 IgM protein in ELISA and with BCL1 tumor cells in flow cytometry assays. Protection against tumor challenge was fully achieved with the complete scFv construct whereas immunization with the construct lacking the VL gene resulted in only a slight prolongation of the survival. We therefore conclude that a plasmid DNA vaccine containing the VH and VL genes of the lymphoma Ig linked to the human IgG1 CH3 exon is highly effective in inducing a protective immune response in the BCL1 model. We also demonstrated that VH gene immunization can induce strong anti-idiotypic antibody responses.

Construction and Characterization of a Chimeric Fusion Protein Consisting of an Anti-idiotype Antibody Mimicking a Breast Cancer-Associated Antigen and the Cytokine GM-CSF

Anti-idiotype antibody, 11D10 mimics biologically and antigenically a distinct and specific epitope of the high molecular weight human milk fat globule (HMFG), a cancer-associated antigen present in over 90% of breast tumor samples. To augment the immunogenicity of 11D10 without the aid of a carrier protein or adjuvant, we made a chimeric 11D10-GM-CSF fusion protein for use as a vaccine. An expression plasmid for 11D10 was made by ligation of the DNA sequences of the 11D10 light-chain variable region upstream of the human kappa constant region. The heavy-chain plasmid carrying GM-CSF was made by ligation of the heavy-chain variable region sequences upstream of the human gamma1 constant region CH1 fused to the DNA fragment encoding the mature GM-CSF peptide 3' to the CH3 exon. NS1 plasmacytoma cells were transfected with the light and heavy-chain vectors by electroporation. Fusion protein secreted in the culture medium was purified and was characterized by gel electrophoresis as well as by determination of the biological activity of the fused GM-CSF. In nonreducing SDS-polyacrylamide gels, a single band approximately 200 Kd reacted with anti-human kappa, anti-human lambda1 and anti-GM-CSF antibodies. In reducing polyacrylamide gels, a approximately 74 kd protein reacted with anti-human lambda1 and anti-GM-CSF antibodies. The fusion protein induced proliferation of GM-CSF dependent NFS-60 cells. These results suggest that the protein is a chimeric anti-idiotype antibody consisting of 11D10 variable domains, human kappa and lambda1 constant domains and that the GM-CSF moiety fused to the constant region lambda1 is biologically active.

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes

Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

Comparison of llama V H sequences from conventional and heavy chain antibodies

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the V HH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama V HH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the V L. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H 2 antibodies. The repertoire of the llama V HHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human V Hs. The variability plot of the amino acids in the V HH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama V HHs of the homodimeric heavy-chain H 2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H 2L 2 immunoglobulins.

Genetic polymorphism of the IGHG3 gene in cattle

We cloned and analyzed the gene (IGHG3) encoding cattle IgG3 using a spleen cDNA library and a genomic phage clone. IGHG3 differs most remarkably from IGHG1 and IGHG2 in the structure of its hinge. The IGHG3 hinge is encoded by two exons, one of which encodes a 22 amino acid 3' extension of the CH1 exon. The IGHG3 gene is highly homologous to the C-gamma pseudogene previously reported. Seventy-one DNA samples from different cattle were tested by pulsed-field gel electrophoresis and Southern blot to detect genetic polymorphisms. This analysis revealed two different alleles which we designated IGHG3(a) and IGHG3(b). The two putative alleles differ from each other by a 6 amino acid substitution in the coding region and an 84 base pair insertion in the intron between the CH2 and CH3 exons. Our data also indicate that the cattle heavy chain locus spans approximately 400 kilobases from IGHM to IGHA and encodes only three IgG subclasses, all of which are transcriptionally active.

Deletion of the 3' splice site of the leader-variable region intron of immunoglobulin heavy chain genes induces a direct splicing of leader to constant region, resulting in the production of truncated mu-chains.

An Abelson virus-transformed immature B cell line, AT8-1-12-5-2, produced truncated mu-chains. Sequencing analysis of the VHDJH complex on the expressed H-chain allele revealed the deletion of 75 nucleotides that involved leader-variable region intron and the 5' end of the variable region, which resulted in the loss of the 3' splice site of leader-variable region intron. Sequence studies of a leader- and CH1-containing cDNA clone showed that leader region was directly spliced to the CH1 exon, resulting in the production of the truncated mu-chains without variable portion. Our results demonstrated for the first time that the only loss of the 3' splice site of leader-variable region intron could induce an aberrant splicing between leader and constant region.

Structure of Abnormal Heavy Chains in Human Heavy-chain-deposition Disease

The sequences of two immunoglobulin gamma 1 heavy chains involved in the formation of non-amyloid tissue deposits were determined in two patients (RIC and THR) affected with plasma cell monoclonal proliferation and heavy-chain-deposition disease. The proliferating plasma cells of patients RIC and THR synthesized truncated gamma 1 chains of 45 kDa and 24 kDa, respectively, carrying internal deletions of the first constant (CH1) domain (RIC) or of the CH1, hinge and CH2 domains(THR). The shortened gamma chains were associated with lambda light chains in the monoclonal IgG component present in the serum from both patients but not in tissue deposits which lacked any detectable light chains. Bone marrow plasma cells from RIC contained short gamma 1 heavy-chain transcripts in which a VDJ exon related to the VH2 subgroup was directly joined to the hinge exon; plasma cells from THR contained short gamma 1 transcripts with a VDJ exon related to the VH3 subgroup joined to the CH3 exon. In both cases, the truncated transcripts carried precise deletions of complete exons and used regular splice sites at the variable/constant junction, consistent with the hypothesis that they originated from abnormal splicing of primary transcripts from the productively rearranged heavy-chain genes. Striking abnormalities of the variable regions were found, especially in framework regions, including replacement of an invariant tryptophan residue in protein from THR, hydrophobic residues most likely exposed to the solvent and inversion of charged amino acids probably exposed on the surface of the molecule.

Structure of Abnormal Heavy Chains in Human Heavy-chain-deposition Disease

The sequences of two immunoglobulin gamma 1 heavy chains involved in the formation of non-amyloid tissue deposits were determined in two patients (RIC and THR) affected with plasma cell monoclonal proliferation and heavy-chain-deposition disease. The proliferating plasma cells of patients RIC and THR synthesized truncated gamma 1 chains of 45 kDa and 24 kDa, respectively, carrying internal deletions of the first constant (CH1) domain (RIC) or of the CH1, hinge and CH2 domains(THR). The shortened gamma chains were associated with lambda light chains in the monoclonal IgG component present in the serum from both patients but not in tissue deposits which lacked any detectable light chains. Bone marrow plasma cells from RIC contained short gamma 1 heavy-chain transcripts in which a VDJ exon related to the VH2 subgroup was directly joined to the hinge exon; plasma cells from THR contained short gamma 1 transcripts with a VDJ exon related to the VH3 subgroup joined to the CH3 exon. In both cases, the truncated transcripts carried precise deletions of complete exons and used regular splice sites at the variable/constant junction, consistent with the hypothesis that they originated from abnormal splicing of primary transcripts from the productively rearranged heavy-chain genes. Striking abnormalities of the variable regions were found, especially in framework regions, including replacement of an invariant tryptophan residue in protein from THR, hydrophobic residues most likely exposed to the solvent and inversion of charged amino acids probably exposed on the surface of the molecule.

Complete variable region deletion in Aα heavy chain disease protein (roul). Correlation with light chain secretion

In a patient affected with chronic lymphocytic leukemia with lymphocyte surface μ and κ determinants and vacuolated bone marrow plasma cells, the serum contained polymers of a truncated μ chain and normal-sized κ chains. These light chains were present as monomers and covalent dimers in studies performed under dissociating conditions, but they were linked by non-covalent bridges to a portion of the serum short μ chains. The patient's urine contained a κ type Bence-Jones protein. Study of a messenger RNA and complementary DNA from blood cells showed the abnormal μ chain to lack the entire variable region, likely due to a direct splicing of the leader peptide exon onto the CH1 exon. The production of light chains, a rare event in heavy chain diseases, appears to correlate with the occurrence of a heavy chain deletion restricted to the variable domain, likely because the non-covalently linked light chains allow these unusual heavy chains to be secreted.

Exon skipping without splice site mutation accounting for abnormal immunoglobulin chains in nonsecretory human myeloma

The proliferating plasma cells of patient COM with nonsecretory myeloma synthesized truncated 42 kDa gamma 1 chains made of a complete constant region but devoid of variable domain. In the absence of light chain expression, the shortened gamma chains were retained intracellularly and were subsequently degraded within 12 h. COM neoplastic plasma cells contained short gamma 1 heavy chain transcripts in which the leader peptide exon was directly joined to the CH1 exon using the regular splice sites. However, study of the productive gamma gene showed that the skipped variable exon was bounded by normal splicing signals and that the adjacent intron organization was not altered. Since this unusual splicing pattern was maintained when COM gamma gene was transfected in murine plasmocytoma cells, exon skipping possibly relates to the modified structure of COM variable region. The latter showed a 2-base pair deletion introducing a translation frameshift in the VH region and a DNA insertion at the VH-DJH junction consisting in a perfect duplication of the first 54 nucleotides of the recombined DJH segment. The lack of light chain production by COM cells was explained by alterations of the variable region of the rearranged kappa gene leading to abnormally spliced transcripts.

Characterisation of rainbow trout cdnas encoding a secreted and membrane-bound Ig heavy chain and the genomic intron upstream of the first constant exon

Two different rainbow trout cDNA sequences encoding a heavy chain secreted Ig (Hs) and a part of a membrane-bound heavy chain Ig (Hm) are reported. The sequences were most similar to those encoding the Ig heavy chains (IgH) of other teleost fish. As in the Hm of the other teleost fish the rainbow trout Hm results from the splicing of the 3' end of the third constant exon (CH3) to the sequence encoding the membrane-bound domain. Analysis of a rainbow trout IgH genomic clone revealed that a joining heavy chain (JH) segment, different to the one observed in the cDNA, is located 825 bp 5' of the CH1 exon. The sequence also contains possible enhancer-like and octamer-like motifs.

Sequence of the CH1 and hinge-CH2 exons of the human immunoglobulin IGHA2 A2m(2) allele: comparison with the nonallelic and allelic IGHA genes

Sequence of the CH1 and hinge-CH2 exons of the human immunoglobulin IGHA2 A2m(2) allele: comparison with the nonallelic and allelic IGHA genes