Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens.

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA. The antibody titers in animals injected with Vaxfectin-pDNA remained higher than in the naked pDNA controls for at least 9 months. The enhancement in antibody titers was dependent on the Vaxfectin dose and was accomplished without diminishing the strong anti-NP cytolytic T cell response typical of pDNA-based vaccines. In rabbits, complexing pDNA with Vaxfectin enhanced antibody titers up to 50-fold with needle and syringe injections and also augmented humoral responses when combined with a needle-free injection device. Vaxfectin did not facilitate transfection and/or increase synthesis of beta-galactosidase reporter protein in muscle tissue. ELISPOT assays performed on bone marrow cells from vaccinated mice showed that Vaxfectin produced a three- to five-fold increase in the number of NP-specific plasma cells. Thus, Vaxfectin should be a useful adjuvant for enhancing pDNA-based vaccinations.

Sodium phosphate enhances plasmid DNA expression in vivo.

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182.

Effect of a genetic deficiency of terminal deoxynucleotidyl transferase on autoantibody production by C57BL6 Fas(lpr) mice.

Terminal deoxynucleotidyl transferase (TdT) adds nontemplate coded nucleotides (N additions) between the recombining ends of immunoglobulin and T cell receptor genes. These nucleotides add significant diversity to the Ig and TCR repertoires. Amino acids coded for by these nucleotides play a key role in the binding of self antigens by autoantibodies and autoreactive T cells. To determine the effect of a lack of N additions on autoantibody production, we bred the TdT knockout genotype onto the autoimmune C57BL/6-Fas(lpr) background. TdT-deficient mice had significantly lower sera anti-DNA and rheumatoid factor activity than their TdT-producing littermates. C57BL/6-Fas(lpr) TdT-deficient mice had shorter VH CDR3 regions and fewer VH CDR3 arginines [0.6% versus 4. 7%] than their TdT-producing littermates. These data indicate that the absence of TdT limited the production of anti-DNA antibodies and rheumatoid factors in C57BL/6-Fas(lpr) mice, likely due to constraints on Ig diversity secondary to the lack of TdT-derived N additions.

The influence of DNA sequence on the immunostimulatory properties of plasmid DNA vectors.

To determine the influence of DNA sequence on immunostimulatory properties of vaccine vectors, we tested the induction of in vitro and in vivo immune responses by plasmids modified to contain extended runs of dG sequences. Studies with oligonucleotides indicate that dG sequences can directly stimulate B cells as well as enhance the activity of immunostimulatory CpG motifs because of interaction with the macrophage scavenger receptor (MSR); this receptor can bind a variety of polyanions including dG sequences. To modify vectors, we introduced stretches of 20-60 dG residues into the pCMV-beta and pSG5rab.gp vectors and measured the ability of these plasmids to induce IL-12 and IFN-gamma production by murine splenocytes. The induction of in vivo antibody responses to rabies glycoprotein was also assessed with the pSG5rab.gp vectors. In in vitro cultures, cytokine production induced by plasmids with and without dG sequences was similar. Furthermore, the addition of dG sequences to pSG5rab.gp vectors failed to enhance the anti-rabies glycoprotein response to immunization. To assess further mechanisms by which plasmids stimulate macrophages, we measured the effects of MSR ligands on in vitro cytokine induction. In in vitro cultures, poly(G), dG30, and fucoidan inhibited IL-12 induction by plasmids. IL-12 induction was also inhibited by mammalian DNA but was unaffected by polyanions that are not MSR ligands. Together, these results suggest that the addition of 20 to 60-base dG sequences to plasmids does not significantly affect their properties as immunostimulators or vaccines. Furthermore, these results suggest that MSR ligands can block cytokine induction by plasmid DNA whether or not the plasmid contains extended runs of dG.

Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA.

To elucidate the mechanism of Ag drive in the anti-DNA response, the Ab response to bacterial DNA has been analyzed in normal and autoimmune mice. Preautoimmune NZB/W mice immunized with Escherichia coli dsDNA produce Abs that resemble spontaneous autoantibodies and bind mammalian dsDNA. In contrast, normal mice, when immunized similarly, produce Abs that bind only bacterial dsDNA. To characterize further the responsiveness of NZB/W mice to bacterial DNA, we determined the molecular properties of mAbs from preautoimmune NZB/W mice immunized with E. coli DNA. Of nine Abs studied, all were IgM and all bound mammalian ssDNA, while four had appreciable reactivity with mammalian dsDNA. The induced anti-dsDNA resembled spontaneous anti-DNA from autoimmune mice in V gene utilization and V(H) CDR3 arginine content. These Abs lacked evidence of somatic mutation, however, indicating that affinity maturation via somatic mutation is not essential for dsDNA reactivity. The findings suggest that preautoimmune NZB/W mice have immunoregulatory defects that allow activation of mammalian dsDNA reactive B cells by bacterial DNA.

Influence of VH CDR3 arginine and light chain pairing on DNA reactivity of a bacterial DNA-induced anti-DNA antibody from a BALB/c mouse.

Anti-DNA induced in BALB/c mice by immunization with bacterial (Escherichia coli) DNA resemble spontaneous anti-DNA from lupus mice in V gene use and cross-reactivity with other nuclear Ags, but lack the high V(H) CDR3 arginine content seen in anti-DNA from lupus mice. Moreover, the induced anti-DNA bind bacterial and mammalian single-stranded (ss) DNA and bacterial double-stranded (ds) DNA, but do not bind mammalian dsDNA. This reactivity profile is in contrast to that of the spontaneously arising anti-DNA of lupus mice, among which mammalian dsDNA reactive Abs are prominent. In this study we demonstrate that the addition of arginine to V(H) CDR3 of an induced anti-DNA confers the mammalian dsDNA binding characteristic of anti-DNA from lupus mice. The ability to confer mammalian dsDNA binding is dependent on both the position of the arginine in V(H) CDR3 and the light chain with which the heavy chain is paired, suggesting the light chain plays a more substantial role in DNA binding by this Ab than has previously been reported for other anti-DNA. Our data support the argument that V(H) CDR3 arginines tend to confer antimammalian dsDNA reactivity, leading to censure of B cells expressing these Abs and provides an explanation for the absence of arginine-rich V(H) CDR3 in the bacterial DNA-induced response.

Differences in V kappa gene utilization and VH CDR3 sequence among anti-DNA from C3H-lpr mice and lupus mice with nephritis.

To investigate the molecular properties of anti-DNA from lpr mice that express high levels of anti-DNA without immune-mediated nephritis, the sequences of VH and V kappa genes encoding 11 monoclonal anti-DNA antibodies derived from C3H-lpr/lpr (C3H-lpr) mice were studied. All of the C3H-lpr monoclonal anti-DNA bound single-stranded DNA while five also bound double-stranded DNA. Two of the hybridomas were clonally related as determined by Southern analysis and sequencing. Sequence analysis of C3H-lpr anti-DNA revealed the use of VH genes that encode anti-DNA from the MRL-lpr/lpr and (NZB X NZW) F1 mouse models of lupus, although differences occurred in the VH CDR3 amino acid content. In contrast, the V kappa genes from C3H-lpr mice lacked significant identity with previously reported V kappa genes for anti-DNA from lupus models. These results indicate that anti-DNA from C3H-lpr mice differ from anti-DNA from lupus mice with nephritis in patterns of V gene expression and suggest a molecular basis for the lack of pathogenicity of anti-DNA in these mice.

Binding analysis of antibodies produced by precursor and branchpoint intermediates of an anti-influenza hemagglutinin B cell clone. Parallel replacement mutations do not confer increased avidity for hemagglutinin.

We have previously described a set of secondary (2 degrees) hybridomas that are specific for the Sb site of influenza hemagglutinin (HA(Sb)) and share the H37-68 ld (68ld). Cells producing 68ld Abs dominated the 2 degree response of one BALB/c mouse, but were virtually absent from the primary (1 degree) and 2 degree responses of other BALB/c mice. To understand the basis for the idiosyncratic nature of this response, we have assessed the functional importance of a conserved DJ junctional sequence. We find that substitutions of conserved residues within this sequence drastically reduced HA(Sb) binding, providing an explanation for the absence of this ld from 1 degree responses. We have also examined intraclonal affinity maturation by generating Abs inferred to have been produced by the unmutated precursors and branchpoint intermediates of the largest 68ld clone. Interestingly, unmutated Abs of this clone bound HA(Sb) well, comparable to that of some 2 degree 68ld hybridoma Abs. Abs produced by branchpoint intermediates of this clone have a lower affinity for HA(Sb) than their unmutated precursor, despite containing parallel amino acid replacement mutations. Thus, expansion of this clone did not follow the paradigm of stepwise increases in affinity. Together, these data underscore the interconnective nature of precursor frequency and somatic mutation in the formation of the 2 degree response to a given Ag.

The BALB/c secondary response to the Sb site of influenza virus hemagglutinin. Nonrandom silent mutation and unequal numbers of VH and Vk mutations.

We have determined the nucleotide sequences of the expressed VH and Vk genes from 13 secondary (2 degrees) hemagglutinin (HA) (Sb) specific hybridomas derived from a single mouse. These antibodies share an Id, H37-68 (68Id) that dominates the 2 degrees HA(Sb) response in this mouse, but is rare or absent from 2 degrees responses of other mice. We find that these antibodies derive from five clones. The H chains of these antibodies are encoded by a single VH gene joined to a variety of DH and JH genes. The length of complementarity-determining region (CDR) 3 and sequence of the D-J junction are restricted, suggesting selection on CDR3 of the H chain. The L chains are more diverse. In the presented examples, they are encoded by the Vk21C and Vk21E genes and a Vk9 gene, and are joined to Jk1, 2, or 4. Each antibody is extensively mutated. The nature and distribution of the mutations suggests that 68Id-producing cells have been selected by Ag, although there are differences regarding the domain (VH, Vk, or both) in which mutations were selected. The implications of these findings on the idiosyncratic nature of the 68Id antibody response to HA(Sb) are discussed. There are two unusual characteristics regarding somatic mutation in these hybridomas. Whereas the expressed VH and Vk21 genes appear to have accumulated mutations at a high rate (1 to 1.5 x 10(-3)/base pairs/division, the expressed Vk9 genes appear to have accumulated mutations at a 5 to 15-fold lower rate than the expressed VH genes in the same cells. There is also a surprisingly high number of parallel silent somatic mutations in the VH genes, of which all but one are clustered to a 28-bp region in framework region 2 and CDR 2-encoding segments. The probability that this could have occurred by a random mutational process is essentially zero.

The octamer motif is a B-lymphocyte-specific regulatory element of the HLA-DR alpha gene promoter.

The human class II gene, HLA-DR alpha, contains an octanucleotide sequence ATTTGCAT located approximately 40 base pairs upstream of the transcription initiation site. We have investigated the transcriptional function of the DR alpha octamer in human B-lymphoblastoid cells and non-B cells. Deletion and substitution mutagenesis of the octamer sequence greatly reduced the activity of the DR alpha promoter in both in vivo and in vitro cell-free transcription systems of B-cell origin. Conversely, these mutations did not affect promoter activity in several non-B-cell lines that express the DR alpha gene. Removal of octamer-binding proteins by in vivo titration with an octamer-containing competitor plasmid reduced DR alpha promoter activity in B-lymphoblastoid cells. These results suggest that a protein-octamer interaction, most likely involving the B-cell-specific octamer binding protein (OTF-2), is required for DR alpha promoter function in B-lymphoblastoid cells but not in non-B cells.