Combined Skin and Muscle DNA Priming Provides Enhanced Humoral Responses to a Human Immunodeficency Virus Type 1 Clade C Envelope Vaccine.

Intradermal (i.d.) and intramuscular (i.m.) injections when administered with or without electroporation (EP) have the potential to tailor the immune response to DNA vaccination. This Phase I randomized controlled clinical trial in human immunodeficiency virus type 1-negative volunteers investigated whether the site and mode of DNA vaccination influences the quality of induced cellular and humoral immune responses following the DNA priming phase and subsequent protein boost with recombinant clade C CN54 gp140. A strategy of concurrent i.d. and i.m. DNA immunizations administered with or without EP was adopted. Subtle differences were observed in the shaping of vaccine-induced virus-specific CD4+ and CD8+ T cell-mediated immune responses between groups receiving: i.d.EP + i.m., i.d. + i.m.EP, and i.d.EP + i.m.EP regimens. The DNA priming phase induced 100% seroconversion in all of the groups. A single, non-adjuvanted protein boost induced a rapid and profound increase in binding antibodies in all groups, with a trend for higher responses in i.d.EP + i.m.EP. The magnitude of antigen-specific binding immunoglobulin G correlated with neutralization of closely matched clade C 93MW965 virus and Fc-dimer receptor binding (FcγRIIa and FcγRIIIa). These results offer new perspectives on the use of combined skin and muscle DNA immunization in priming humoral and cellular responses to recombinant protein.

Identification of RNA cargoes by antibody-positioned RNA amplification.

The interactions between various RNA-binding proteins (RBPs) and the RNA transcripts they bind strongly influence posttranscriptional control of gene expression in vertebrates. The hundreds of vertebrate RBPs that have been identified within the genome, often with multiple RNA recognition motifs, are capable of recognizing specific target RNA sequences mediating the maturation, movement, and translational state of their RNA cargoes. To identify the cargoes associated with a specific RBP, we have developed a technique called antibody-positioned RNA amplification (APRA), which positions an oligonucleotide with a degenerate priming sequence in proximity to the RNAs sequestered by a specific RBP. The conjugation of the priming oligonucleotide to the antibody by itself does not interfere with the antibody's intrinsic affinity for the target RBP epitope, thus enabling RNA targets to be reverse-transcribed and amplified via a T7 bacteriophage RNA polymerase promoter sequence located upstream of the degenerate priming sequence in the oligonucleotide. By identifying the mRNA transcripts associated with the RBP in situ, we may be able to ascertain the significance of their temporal expression and physiological activities within the vast transcriptional networks regulating functional responses to stimuli.

Vaccination-induced changes in human B-cell repertoire and pneumococcal IgM and IgA antibody at different ages.

It is well known that older people are more susceptible to morbidity and mortality from infectious diseases, particularly from pulmonary diseases such as pneumococcal pneumonia where vaccines do not provide efficient protection as in younger populations. We have previously shown that the B-cell repertoire in the old is reduced and hypothesise that this may contribute to the impaired humoral responses of the elderly. Here, we investigated the repertoire and antibody responses to winter vaccination in two age groups, aged 18-49 and 65-89. We found that the serum IgM and IgA pneumococcal responses were significantly impaired in the older group, with no difference in IgG levels. IGHM spectratype analysis seems to be the most promising in terms of its predictive ability for vaccine responses. Spectratypes showed a clear change in the repertoire at day 7 after vaccination, with a return to the baseline levels at day 28. The changes at day 7 reflected expansion of IGH sequences that have smaller, more hydrophilic, CDR3 regions, and these changes were attenuated in the older group. The older group was more likely to have spectratypes indicative of a reduced diversity at day 0 and day 28. On average, the baseline repertoire in the older group was comprised of larger CDR3 regions than in the younger group. In conclusion, IgA and IgM responses are significantly impaired in the elderly pneumococcal response and are likely key mediators of protection. Hydrophilicity and/or small size of the IGH CDR3 appear to be important in these responses.

Improved genotyping of the human minor histocompatibility antigen HB-1 by polymerase chain reaction with sequence-specific primers using a complementary oligonucleotide.

Single nucleotide polymorphisms of minor histocompatibility antigens (mHags) have been genotyped by allele-specific polymerase chain reaction with sequence-specific primers (PCR-SSP). Because discriminating the genotype of HB-1 Y by PCR-SSP under various PCR conditions was difficult, we optimized the use of oligonucleotides complementary to the allele-specific forward primer to improve the specificity of the HB-1 Y PCR-SSP. Specific allele discrimination was possible with an annealing temperature between 61°C and 63°C and in the presence of a threefold excess of a 15-bp complementary oligonucleotide. In conclusion, the inclusion of a complementary oligonucleotide in the PCR-SSP assay may improve its specificity and selectivity for genotyping several mHags for which optimizing PCR conditions have been difficult.

Safety evaluation of a canine hepatitis DNA vaccine.

DNA vaccines are a novel disease prevention methodology, however their safety has not been well described. We evaluated the safety and efficacy of the DNA vaccine pVAX1-CpG-Loop against infectious canine hepatitis. We demonstrated that pVAX1-CpG-Loop could not be recovered from tissues of vaccinated mice nor from F1 progeny and following vaccination there were no apparent changes in hematologic markers compared to unvaccinated controls. Most important, vaccinated mice were protected from viral challenge. The only detectable effects of the vaccination were elevated AST levels 4 weeks post vaccination and liver lymphocyte infiltration and hydropic degeneration which normalized 6 months later.

Primer sets for cloning the human repertoire of T cell Receptor Variable regions.

BACKGROUND: Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. RESULTS: Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. CONCLUSION: This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

Enhanced Th2 immunity after DNA prime-protein boost immunization with amyloid beta (1-42) plus CpG oligodeoxynucleotides in aged rats.

Generation and accumulation of fibrillar amyloid beta (Abeta) is widely considered as the pathogenic basis of neurodegeneration in Alzheimer's disease (AD). Both active immunization with fibrillar Abeta and passive immunization with anti-Abeta antibodies in transgenic mouse models of AD result in prevention/dissociation of Abeta plaque formation and restoration of cognitive functions. However, similar immunization studies in humans had to be halted because 6% of the AD patients developed acute meningoencephalitis, likely due to anti-Abeta specific autoimmune Th1 cells. Hence, making Abeta immunotherapy successful requires production of strong antibody responses without Th1-type immunity. In an attempt to develop safer vaccines, we examined the influence of oligodeoxynucleotides as adjuvant on the Th1 and Th2 immune response to Abeta in aged rats. We further investigated whether a DNA prime-protein boost strategy could elicit a more robust Th2 response. The results of the present study showed that all the animals injected with either Abeta peptide alone or Abeta encoding plasmid alone or plasmid DNA prime followed by peptide boost have elicited specific anti-Abeta antibodies. When co-administered, synthetic oligodeoxynucleotides (ODN) further enhanced the anti-Abeta titres. More importantly, the IgG subclasses of the antibodies generated by DNA prime-peptide boost regimen with ODN as adjuvant were primarily of IgG2b and IgG1 isotypes, suggesting that heterologous immunization strategy along with ODN would be advantageous in eliciting more beneficial Th2-type humoral immune response.

Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats.

Rats are widely used as a model in the study of many important diseases, yet their utility in research is limited by the lack of robust technology for the production of rat recombinant antibodies. Here we have identified and compiled putative rat immunoglobulin sequences by bioinformatic analysis of recently available genomic and EST databases, and used this information to design PCR primers to amplify rat V(H) and V(L) domain coding DNA representing the expressed repertoire of the animal. These primer sets were successfully tested and used to produce a scFv phage display library from rats that had been infected by the blood fluke, Schistosoma mansoni. The library was selected for scFvs that bind to extracellular loop epitopes on either of two known immunogenic S. mansoni membrane proteins, Sm23 and SmTsp2, both of the tetraspanin protein family. Two unique scFvs were identified that bind to each tetraspanin and shown to have excellent specificity for the target, including recognition of the native tetraspanins produced by the fluke. The primers and methods developed for rat scFvs should be of use to others seeking to characterize the antibody repertoire and/or prepare recombinant antibodies from this model.

Immunization with plasmid DNA encoding a truncated, secreted form of the bovine viral diarrhea virus E2 protein elicits strong humoral and cellular immune responses.

The major protective antigen of bovine viral diarrhea virus (BVDV), the E2 protein, is cell-associated and not expressed on the cell surface. In this study we evaluated a DNA vaccine encoding various secreted versions of E2. In vitro analysis demonstrated that deletion of the transmembrane anchor and addition of the signal sequence of bovine herpesvirus-1 (BHV-1) (gDsDeltaE2) resulted in efficient secretion of E2 into the culture medium. In contrast, full-length E2, either without or with gDs (gDsE2), as well as truncated E2 without gDs (DeltaE2), remained entirely cell-associated. Mice immunized with plasmid encoding gDsDeltaE2 developed significantly higher IgG and virus neutralizing antibody titres compared to animals vaccinated with plasmid encoding E2, DeltaE2 or gDsE2. To optimize secretion of E2, the efficiency of gDs was compared with that of the tissue plasminogen activator signal (tPAs) sequence. In addition, the effect of the plasmid backbone was assessed by comparing two vectors. Four plasmids, pMASIA-gDsDeltaE2, pMASIA-tPAsDeltaE2, pSLKIA-gDsDeltaE2 and pSLKIA-tPAsDeltaE2, were constructed and administered intradermally to mice. The mice immunized with pMASIA-tPAsDeltaE2 developed the strongest and most balanced immune responses. Vaccination of cattle confirmed that pMASIA-tPAsDeltaE2 elicited both strong humoral and cellular immune responses and thus could be a candidate DNA vaccine against BVDV.

Differential prevention of experimental autoimmune encephalomyelitis with antigen-specific DNA vaccination.

We compared the potential therapeutic effect of vaccination with DNA constructs encoding two encephalitogenic proteins, PLP and MOG, on the outcome of subsequent sensitization of EAE induced in SJL/J and C57/B6 mice. Early sensitization for EAE (4 weeks after DNA vaccination) caused recipient animals to develop enhanced disease with DNA-encoding PLP but not with DNA-encoding MOG. Late sensitization (more than 10 weeks) resulted in an amelioration of EAE in animals vaccinated with both PLP and MOG DNA constructs. These results, confirming the DNA-mediated ameliorating effect on EAE, also indicate significant differences in the kinetics of development of EAE tolerance in response to vaccination with different DNA-encoding myelin antigens. Since PLP and MOG require different MHC presentation and induce different EAE models, the results point to potential differences in immune system requirements for efficient DNA-induced amelioration of the autoimmune response.

Induction of an antitumor immunological response by an intratumoral injection of dendritic cells pulsed with genetically engineered Semliki Forest virus to produce interleukin-18 combined with the systemic administration of interleukin-12.

OBJECT: The aim of this study was to investigate further immunogene treatment of malignant brain tumor to improve its therapeutic efficacy. METHODS: Intratumoral dendritic cells pulsed with Semliki Forest virus (SFV)-interleukin-18 (IL-18) and/or systemic IL-12 were injected into mice bearing the B16 brain tumor. To study the immune mechanisms involved in tumor regression, we monitored the growth of implanted B16 brain tumor cells in T cell-depleted mice and IFNgamma-neutralized mice. To analyze the protective immunity created by tumor inoculation, B16 cells were injected into the left thighs of mice that had received an inoculation, and tumor growth was monitored. The local delivery of dendritic cells pulsed with IL-18 bound by SFV combined with the systemic administration of IL-12 enhanced the induction of the T helper type 1 response from tumor-specific CD4+ and CD8+ T cells and natural killer cells as well as antitumor immunity. Interferon-gamma is partly responsible for this IL-18-mediated antitumor immunity. Furthermore, the protective immunity is mediated mainly by CD8+ T cells. CONCLUSIONS: Immunogene therapy that combines the local administration of dendritic cells pulsed with IL-18 bound by SFV and the systemic administration of IL-12 may be an excellent candidate for the development of a new treatment protocol. A self-replicating SFV system may therefore open a novel approach for the treatment of malignant brain tumor.

Osteoclast stimulating and differentiating factors in human cholesteatoma.

OBJECTIVES: To investigate the expression of osteoclast-activating and differentiating factors and to study the occurrence of osteoclast precursor cells and osteoclasts in acquired human cholesteatoma tissue. METHODS: We examined 21 cholesteatoma samples versus 18 normal auditory canal skin specimens for the expression of osteoprotegerin ligand (OPGL), osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Immunohistochemistry and computer-assisted microscopy using markers CD4, CD11a, CD11b, CD14, CD51, CD68, and TRAP obtained the detection of osteoclast cell lineage. RESULTS: An increased expression of the investigated cytokines M-CSF, OPG, and OPGL was demonstrated by immunohistochemistry and RT-PCR in cholesteatoma tissue compared with normal external meatal skin. Several CD4-positive cells exhibited a co-expression for OPGL within the perimatrix of cholesteatoma. The presence of osteoclast precursor cells was confirmed in all samples of cholesteatoma tissue. CONCLUSIONS: This study reveals that the number of osteoclast precursor cells is markedly increased in the perimatrix of cholesteatoma tissue. Our results support a concept described for inflammatory arthritis: the inflammation related to cholesteatoma induces bone resorption by release of OPGL from activated T-cells and triggers osteoclastogenesis. This could be a major target for drugs to inhibit osteoclast formation and bone resorption and may be an adjunct in cholesteatoma management.

The morphine-binding site on human activated T-cells is not related to the mu opioid receptor.

Mitogen activation of human T-lymphocytes induces a morphine-binding site. Morphine binding is displaceable by beta-endorphin (1--31) and (--)-naloxone but not DAMGO. This site is not stereoselective for (--)-morphine. T-lymphocytes, expressing this binding site, were assayed by reverse-transcription polymerase chain reaction (RT-PCR) for expression of hMOR-1 mRNA. Several primer sets were used and each assay compared with cells known to express human or mouse MOR-1 mRNA. Neither hMOR-1 nor any homologous receptor was detected in human T-lymphocytes. Therefore, the morphine-binding site on mitogen-activated T-lymphocytes is unlikely to be closely related to hMOR-1.

The role of fibroblasts from oropharyngeal mucosa in producing proinflammatory and mitogenic cytokines without prior stimulation.

Cytokine production by fibroblasts is not only important for immunological and inflammatory reactions in the epidermis and mucosa, but also for growth and differentiation of epithelial cells. To characterize the role of fibroblasts in the oropharyngeal mucosa, the expression of a panel of cytokines and cytokine receptors by fibroblasts isolated from normal human oropharyngeal mucosa was investigated by enzyme-linked immunosorbent assay (ELISA), reverse transcribed polymerase chain reaction (RT-PCR) and flow cytometry (FACS). Oropharyngeal fibroblasts produced the proinflammatory cytokines interleukin 1 alpha (IL-1 alpha), IL-6 and IL-8 without addition of phorbol-12-myristate-13-acetate (PMA) or biological response modifiers, suggesting an active involvement of these cells in host defence mechanisms. Keratinocyte growth factor (KGF), a growth factor for epithelial cells, and the angiogenetic fibroblast growth factors acidic and basic FGF (aFGF, bFGF) were also synthesized. Expression of receptors for IL-1, IL-4, IL-6, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) was found. These results indicate that oral fibroblasts are capable of producing a number of cytokines without the need for additional stimuli and emphasize their active regulatory role in the maintenance of the oral mucosa.

In vitro induction of the expression of multiple IgA isotype genes in rabbit B cells by TGF-beta and IL-2.

The rabbit genome has 13 different Calpha genes that are expressed at different levels in mucosal tissues. To analyze the factors involved in the differential expression of these Calpha genes, we cloned and sequenced the promoters of the Ialpha regions that control the expression of sterile mRNA. We found that all Calpha genes, including Calpha3 and Calpha8, which are not expressed, and Calpha4, which is expressed at high levels, have similar nucleotide sequences in the Ialpha region, and all contain the recognition elements for TGF-beta in the promoter. B lymphocytes from popliteal lymph nodes or Peyer's patch activated in vitro could be induced by TGF-beta to express sterile IgA transcripts of all IgA isotypes, except Calpha2, Calpha3, and Calpha8. Many single B lymphocytes transcribed sterile mRNA of more than one IgA isotype, which demonstrates that transcription of sterile mRNA alone does not regulate the IgA isotype switch. The addition of IL-2 led to the expression of transcripts of mature IgA of all isotypes, except Calpha2, Calpha3, and Calpha8. The predominantly expressed isotype in these experiments was Calpha4. With the use of an IgA4-specific mAb we found that IgA4+ plasma cells are unevenly distributed throughout the small intestine such that many of the IgA+ plasma cells in the duodenum-jejunum produced IgA4, whereas in the lower part of the ileum IgA4-producing cells were almost absent. Because the microbial flora varies throughout the intestine, we suggest that the microbial flora creates different local environments and thus affects either isotype switching or homing of IgA-expressing cells.

The PCR-SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR-SSP primers and primer mixes.

An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class II means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class II sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class II typing set. The SSP Manager is available on request from the authors.

RHD/CE typing by polymerase chain reaction using sequence-specific primers.

BACKGROUND: Current DNA-based Rh system typing strategies may detect the two RH genes and their prevalent alleles, but they are known to fail sometimes, when rare RH alleles (e.g., D category phenotypes) are encountered. It is almost impossible to find a single DNA-based method that can accommodate the great heterogeneity within the human Rh system. STUDY DESIGN AND METHODS: An easy-to-perform DNA-based method for the detection of the two RH genes and their alleles, including variant RHD alleles, was developed. By the use of one RHD/C-, seven RHD-, and four RHCE-specific polymerase chain reactions, all triggered to work at identical thermocycling conditions, the DNA of 77 blood donors carrying weak D and that of 200 random donors with common D phenotype was investigated. In addition, 77 selected samples of ccDee and rare Rh system phenotypes were examined. RESULTS: Among 77 samples of weak D, one Rh33 and six DVI categories were detected, one of which showed new RHD-specific nucleotide patterns. In DFR and CCee samples, novel variant RHD alleles were found. RHD DNA types of 200 random donors were found to be concordant with their D phenotype. For RHE and RHe genotyping, a full correlation with serologic phenotypes was found. Our method for genotyping RHC and RHc failed in some cases, because of an already published RHc allelic variation, which we have called RHc(cyt48). An estimate of the frequency of this RHc(cyt48) allele in a white population was made. CONCLUSION: The presented exon-scanning RHD/CE polymerase chain reaction using sequence-specific primers complements current DNA-based Rh system typing strategies and is superior in the detection of variant RHD alleles.

Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA.

A monoclonal antibody against DNA established from a mouse strain that spontaneously develops systemic lupus erythematosus was characterized by migration shift immuno-capillary electrophoresis. The minimal size for DNA binding antibody was > 16 bases and the interaction with a double-stranded 32-mer oligonucleotide was almost one order of magnitude stronger than the interaction with a single-stranded oligonucleotide. The binding was highly dependent on the ionic strength conditions with an increase in binding with a decrease in ionic strength. The estimate of the dissociation constant for the antibody binding of a single stranded 32-mer oligonucleotide was 0.62 microM at pH 7.90. This value was in good agreement with the value of 0.44 microM measured by an independent method using biosensor (surface plasmon resonance) technology.

Sequence-specific priming and exonuclease-released fluorescence detection of HLA-DQB1 alleles.

Molecular typing of HLA DQB1 alleles, employing sequence-specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence-based oligonucleotide probe assay to assign the most common DQB1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB1 locus using SSOP and conventional SSP-based approaches. We used a panel of 14 DQB1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single-base allelic differences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB1 *0401/0402 and DQB1 *0302/ 0303). Sequence-specific priming and exonuclease-released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large numbers of samples, for which high throughput is critical and for which gel-based approaches are difficult to perform. This technique may also be useful for small-scale class I and class II molecular typing in clinically oriented laboratories.

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an essential component for the normal processive DNA synthesis by DNA polymerase delta and is also required for DNA excision repair. Human PCNA autoantisera has been shown to inhibit the function of PCNA in vitro, in contrast to induced antibodies. A monoclonal IgG2 PCNA antibody, 74B1, that effectively inhibited DNA replication in vitro was identified. The inhibitory effect was dose dependent and using synthetic overlapping peptides of PCNA the 74B1 epitope was mapped to aa 121-135, a region of the PCNA protein containing the interdomain connector implicated in intermolecular interactions. Interestingly, a neighboring and partly overlapping peptide, aa 111-125, contains an immunodominant region recognized by a number of monoclonal PCNA antibodies with no inhibitory effect on DNA synthesis. The 74B1 antibody is a potentially useful antibody in future studies of PCNA and its interaction in complexes associated with cell cycle progress, DNA replication, and DNA repair.