Myfanwy B. Spellerberg

DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma

Vaccination with idiotypic protein protects against B-cell lymphoma, mainly through anti-idiotypic antibody. For use in patients, DNA vaccines containing single-chain Fv derived from tumor provide a convenient alternative vaccine delivery system. However, single-chain Fv sequence alone induces low anti-idiotypic response and poor protection against lymphoma. Fusion of the gene encoding fragment C of tetanus toxin to single-chain Fv substantially promotes the anti-idiotypic response and induces strong protection against B-cell lymphoma. The same fusion design also induces protective immunity against a surface Ig-negative myeloma. These findings indicate that fusion to a pathogen sequence allows a tumor antigen to engage diverse immune mechanisms that suppress growth. This fusion design has the added advantage of overcoming potential tolerance to tumor that may exist in patients.

Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv–FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.

Immunogenetic analysis of the immune response to pneumococcal polysaccharide

Pneumococcal serotype‐specific anti‐capsular polysaccharide antibodies protect against invasive pneumococcal disease. Within an individual the diversity of these antibodies is limited. To evaluate the repertoire of antibodies to pneumococcus and determine whether oligoclonality is seen both between serotypes and between individuals, we sampled the B cell repertoire induced by polysaccharide and conjugate vaccine in adult volunteers. Fifteen hybridomas secreting pneumococcus‐specific monoclonal antibodies were generated from five volunteers. Ten were isotype switched, six were IgG2 and four were IgA. These included two isotype switch variants of the same clone. VH3 and Vκ2 were used by 10 / 15 and 7 / 13 of the sequenced clones, respectively, with identical genes, VH3 – 48 and Vκ2‐A17 used by a number of volunteers to a variety of serotypes. VDJ junctional characteristics and complementarity‐determining region (CDR) 3 length were variable. High levels of somatic mutation in CDR1 and 2, inconsistent with a primary response, were found in 10 / 11 of the isotype‐switched antibodies, including those induced by plain polysaccharide antigens. These data suggest that wild‐type infection or nasopharyngeal carriage of Streptococcus pneumoniae in adults may induce memory and the response to subsequent immunization with plain polysaccharide or conjugate pneumococcal vaccines may have the characteristics of a secondary response.

Manipulation of pathogen-derived genes to influence antigen presentation via DNA vaccines

To gain insight into the routes of presentation of pathogen sequences via DNA vaccines, we have compared the abilities of sequences encoding fragment C of tetanus toxin (FrC) and influenza A virus nucleoprotein (NP) to induce antibody or cytotoxic T-cell (CTL) responses in vivo. Strong antibody and CTL responses were induced against FrC targeted to the endoplasmic reticulum (ER) and both were reduced by removal of the leader sequence. In contrast, targeting of NP to the ER generated only a modest antibody response, likely due to misfolding in this site. Removal of the leader sequence led to anti-NP antibodies via cross-priming. For NP, induction of CTLs was not influenced by the leader sequence. Exogenous FrC or NP delivered as proteins were unable to induce CTLs. Routes to induction of optimal immune responses via DNA evidently differ according to the nature of the encoded pathogen sequence. Understanding processing pathways for pathogen sequences should assist rational design of DNA vaccines.

Correlation of 9G4 idiotope with disease activity in patients with systemic lupus erythematosus

OBJECTIVE To compare the levels of the 9G4 idiotope (9G4 Id) in systemic lupus erythematosus (SLE) patients with a detailed disease activity index, the British Isles Lupus Assessment Group (BILAG) index, and serological parameters of disease activity by ds DNA antibody levels and serum C3 concentrations. METHODS In a cross sectional analysis serum samples from 190 patients with SLE were studied and a further 55 serial bleeds from 14 patients. An enzyme linked immunosorbent assay was used to measure the 9G4 Id, and anti dsDNA and anti-myeloperoxidase (MPO) antibodies. The C3 levels were measured by laser nephelometer. RESULTS Seventy six of 190 (40%) of the patients tested had raised 9G4 Id levels. In the cross sectional study 9G4 Id levels were found to correlate with disease activity in the BILAG cardiovascular/respiratory renal, and haematological systems and with global BILAG score (p<0.01). In the serial bleeds 9G4 Id levels correlated with anti-dsDNA antibody and C3 levels, but not with anti-MPO antibodies. No correlations were found with treatment. In six cases the 9G4 Id levels correlated well with global BILAG scores and dsDNA antibody levels. In four cases the BILAG global and 9G4 Id levels alone correlated well. CONCLUSIONS Raised levels of the 9G4 Id are present in a substantial proportion of serum samples from patients with lupus, correlate with various aspects of disease activity in SLE. The Id is detectable on anti-dsDNA antibodies, though it must also be present on other immunoglobulins whose specificities remain unknown.

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: a possible link between infection and lupus.

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.

The immunoglobulin VH gene, VH4-21, specifically encodes autoanti-red cell antibodies against the I or i antigens.

Most autoanti‐red cell antibodies found in patients with cold agglutinin disease are specific for the I or i carbohydrate antigenic determinants. However, antibodies specific for other antigens such as Pr or Sa can also be found, and these are identified by their pattern of reactivity with enzyme‐treated red cells. Recently, it has been shown that the vast majority of anti‐Ii antibodies react with a monoclonal anti‐idiotypic antibody (9G4); this reactivity arises from restriction of the immunoglobulin heavy chains used to encode the antibodies to a single VH4–21 gene, VH4–21. The 9G4 antibody appears specific for this gene product, and we have used it to analyse VH4–21 gene involvement in encoding a spectrum of red cell antibodies of various specificities. The results support the strong association between usage of this gene and anti‐Ii specificity and indicate that it is not generally used by other specificities. In particular, it is striking that the unsubstituted type 2 oligosaccharide antigens (I and i) induce a highly restricted autoantibody response very different from that induced by the sialylated type 2 antigens (Sia‐b,‐1 and lb). The 9G4 antibody therefore provides a simple tool for discrimination between these autoanti‐red cell antibodies, which should be of use in red cell serology.

Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment

BACKGROUND: The V4‐34 gene segment is commonly used by human monoclonal IgM alloantibodies against blood group antigens and by cold‐reactive red cell autoantibodies with anti‐I or anti‐i specificity. This study was conducted to determine whether cold agglutinin activity is found among the V4‐34‐encoded alloantibodies. STUDY DESIGN AND METHODS: Fifty‐ four human IgM monoclonal antibodies (MoAbs) against Rh system antigens were tested for cold agglutinin activity against red cells lacking the relevant Rh system antigen and for reactivity with tissue I and/or i antigens using immunohistochemistry. The findings were correlated with the utilization of the V4‐34 segment as determined in an enzyme‐linked immunosorbent assay with an antibody (9G4) that is specific for this gene product and were also correlated with other serologic properties. RESULTS: Of the MoAbs, 59 percent were 9G4‐positive. Of the 9G4‐ positive subset, 16 and 44 percent agglutinated native adult (express I) and cord (express i) cells, respectively, at 4 degrees C; these levels rose to 84 and 94 percent, respectively, with the use of papain‐ treated cells. The red cell antigens recognized at 4 degrees C were cleaved by endo‐beta‐galactosidase, which is consistent with their being I and i. Of the 9G4‐positive subset, 53 percent bound to tissue i antigen. These reactivities were not found among 9G4‐negative MoAbs. Endo‐beta‐galactosidase treatment of red cells enhanced Rh system antibody agglutination by 9G4‐negative MoAbs. CONCLUSION: Anti‐I/i reactivity is common among IgM Rh system MoAbs and is shown only by the V4‐34‐encoded subset. This finding has implications for the use of MoAbs for Rh system typing of blood.

Differential Usage of an Autoantibody-associated VH Gene, VH4-21, by Human B-cell Tumors

Selection of immunoglobulin variable region genes for recombination in B cells takes place from among those VH and VL gene segments available in the unrearranged germ line repertoire. In the case of neoplastic B cells, there is apparent deviation in the use of V-genes from that expected on a random basis, both for VH and for VL. Also, the preferred V-genes, and their patterns of mutation, differ among the various categories of B-cell tumor possibly reflecting the distinct origins and clonal histories on the individual tumor cells. This review focuses on a single VH gene, VH4-21, which is a member of the VH4 family, and which appears selectively to encode immunoglobulins with autoantibody activity, particularly anti-red cell antibodies. The pattern of usage of this VH gene by B-cell tumors demonstrates clear asymmetry among different tumor types. Also, the mutations detected in this relatively non-polymorphic gene indicate that antigen, possibly autoantigen, may influence the behavior of the tumor cell.

Pattern of usage of the VH4-21 gene by B lymphocytes in a patient with EBV infection indicates ongoing mutation and class switching.

Following infection with EBV, patients have selectively raised serum levels of immunoglobulins encoded by the VH4-21 gene. In order to follow the detailed pattern of usage of the VH4-21 gene by blood B lymphocytes of a typical patient during infection, EBV lines were established, and transformed B cells were hybridized and cloned. In addition, to widen the genetic analysis, cDNA preparations from the EBV transformants using the gene were also analysed by polymerase chain reaction, cloning and sequencing. The majority (12/15) of the clonally distinct sequences derived from IgM utilized the VH4-21 gene in germ line configuration; however, 3/15 showed replacement mutations. For one of these, a heterogeneous pattern of mutation within the clone indicated ongoing mutation, and one sequence contained a stop codon. Three distinct clones which had rearranged to C gamma were obtained, and all were extensively mutated, with some evidence for a role for antigen in selection. Following resolution of the infection, no VH4-21-encoded products were detectable by this approach. It appears therefore that infection with EBV leads to selective activation, mutation and class switching of the VH4-21 gene, with the unusual feature that B cells harbouring deleterious mutations in the functional gene are able to survive in the circulation.