Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt­TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

Increased cytotoxic capacity of tumor antigen specific human T cells after in vitro stimulation with IL21 producing dendritic cells.

Monocyte derived dendritic cells (moDC) electroporated with tumor associated antigen derived mRNA can elicit specific T cells against tumor cells in vivo. IL21 has been shown to enhance activation and cytotoxicity in CD8+ T cells. We therefore investigated in vitro effects on human CD8+ T-cells after stimulation with IL21 mRNA electroporated moDC. Codon modification of the IL21 gene significantly enhanced IL21 production upon electroporation of moDC. Tumor associated antigen specific CTL induction efficiency was significantly enhanced when codon modified IL21 mRNA was co-electroporated with tumor associated antigen mRNA. Tumor associated antigen specific T cells induced by codon modified IL21-DC demonstrated increased cytotoxic capacity and killing compared to control cultures. In conclusion, ectopic expression of codon modified IL21 by moDC enhances the priming efficiency of the DC as well as the cytotoxic potential of the induced CTL.

A polymorphism in the coding region of Il12b promotes IL-12p70 and IL-23 heterodimer formation.

IL-12 and IL-23 are heterodimeric cytokines involved in the induction of Th1 and Th17 immune responses. Previous work indicated that a region on chromosome 11 encoding the IL-12p40 subunit regulates strain differences in susceptibility to murine trinitrobenzene sulfonic acid-induced colitis. In addition, this region determines strain differences in LPS-induced IL-12 responses. In this study, we investigated how polymorphisms in the coding region of murine Il12b influence IL-12 and IL-23 heterodimer formation. Transfection studies using constructs containing IL-12p35 linked to IL-12p40 from the colitis-resistant C57BL/6 strain or to the polymorphic p40 variant from the colitis-susceptible SJL/J strain demonstrated that SJL/J-derived p40 constructs synthesized significantly more IL-12p70 than did constructs harboring the C57BL/6-p40 variant. This could not be attributed to differences in synthesis rate or secretion, implicating a greater affinity of SJL/J-derived IL-12p40 for its IL-12p35 subunit. This greater affinity is also associated with increased IL-23 synthesis. In addition, C57BL/6 mice transgenic for the SJL/J 40 variant synthesized significantly more IL-12p70 upon LPS challenge and were more prone to develop colonic inflammation than did C57BL/6 mice transgenic for the C57BL/6-p40 variant. The more efficient binding of the polymorphic Il12b variant to p35 and p19 is most likely due to conformational changes following differential glycosylation as a consequence of the polymorphism. The high synthesis rate of the mature cytokines resulting from this efficient binding can lead to rapid proinflammatory skewing of immune responses and distortion of the homeostatic balance underlying the greater susceptibility for colitis.

Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system.

Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR(+) T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19(+) artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4(+) and CD8(+) T cells expressing CAR to redirect specificity for CD19(+) tumor cells.

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

Metabolism of recombination coding ends in scid cells.

V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3' overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.

Gamma-irradiation directly affects the formation of coding joints in SCID cell lines.

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.

Targeting and subsequent selection of somatic hypermutations in the human V kappa repertoire.

The number and distribution of nucleotide substitutions in human VkappaJkappa genes were examined using a PCR technique that analyzed nonproductive and productive rearrangements amplified from genomic DNA of individual B cells. The results indicate that the mutational mechanism introduces replacement (R) mutations comparably throughout the length of the VkappaJkappa rearrangement, but tends to target specific triplets. Moreover, hotspots of mutational activity were identified in complementarity determining regions (CDR). A marked increase in the frequency of R mutations in CDR was noted when productive were compared to nonproductive rearrangements, indicating that these were selected into the expressed repertoire. Of note, amino acids encoded by codons adjacent to hotspots of mutation were also positively selected implying that similar regions were targeted for hypermutation and subsequent selection. In contrast to the distribution of CDR mutations, R mutations in the framework (FR) regions tended to be eliminated from productive VkappaJkappa rearrangements, implying that the somatic hypermutational machinery frequently introduced amino acid changes that were deleterious to the structural integrity of the kappa chain protein. The difference in the ratio of R to silent mutations in CDR and FR in the expressed repertoire, therefore, reflects the summation of positive selection of R mutations in the CDR and the elimination of R mutations in the FR. The data indicate that the balance between targeted mutation of VkappaJkappa rearrangements and subsequent selection and elimination governs the pattern of mutations manifest within the expressed kappa repertoire.

Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation.

Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-C mu, VHDJH-C delta, VHDJH-C gamma, VHDJH-C alpha, VHDJH-C epsilon, and V lambda J lambda-C lambda transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80: CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the lambda-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and C lambda. These mutations were more abundant in secondary VHDJH-C gamma than primary VHDJH-C mu transcripts and in V(D)J-C than V lambda J lambda-C lambda transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 x 10(-4) base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities.

Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium.

In an attempt to study codon usage effects of DNA vaccines on the induction of MHC class I-restricted T cell responses against an intracellular bacterium, Listeria monocytogenes, we designed two plasmid DNA vaccines encoding an H-2Kd-restricted epitope of listeriolysin O (LLO) of L. monocytogenes, LLO 91-99. One DNA vaccine, p91wt, carries the wild-type DNA sequence encoding LLO 91-99, and the other one, p91mam, possesses the altered DNA sequence in which the codon usage was optimized for murine system. Our read-through analyses with LLO 91-99/luciferase fusion genes confirmed that the optimized 91mam DNA sequence showed extremely higher translation efficiency than the wild-type sequence in murine cells. Consistent with this, i.m. injections of p91mam, but not of p91wt, into BALB/c mice were capable of inducing specific CTL- and IFN-gamma-producing CD8+ T cells able to confer partial protection against listerial challenge. Taken together, these observations suggest that optimization of codon should be taken into consideration in the construction of DNA vaccines against nonviral pathogens.

Analysis of BR96 binding sites for antigen and anti-idiotype by codon-based scanning mutagenesis.

We performed a scanning mutagenesis study of heavy chain complementarity-determining region (CDR) residues to identify how mutations affected binding of the anti-carcinoma mAb BR96 to Ag, Lewis Y, and to an anti-Id Ab (anti-Id). By ELISA, we demonstrated that the anti-Id bound close to the Ag binding site of BR96, but the anti-Id and Ag sites were not identical. Immunoblot analysis and screening of light and heavy chain CDR libraries with multiple mutations in each CDR suggested that the heavy chain had greater involvement in anti-Id binding. We then analyzed contributions of individual residues in the heavy chain CDRs to binding of Ag and anti-Id. In a filamentous phage vector containing BR96 V region sequences, mutations were introduced by codon-based mutagenesis at single positions within the three heavy chain CDRs. The resulting libraries of Fab fragments had all amino acids represented at a CDR position. We evaluated the expressed Fabs for binding to Ag and anti-Id by plaque lift assay. We identified the positions with mutations that had the greatest negative effect on binding to the anti-Id and to Ag and analyzed them on the basis of the BR96 x-ray structure. The residues most important for binding to the anti-Id were located in heavy chain CDR1 and CDR2 and were peripheral to the residues within the Lewis Y binding pocket.

Identification of L2 open reading frame gene products of bovine papillomavirus type 1 using monoclonal antibodies.

Four hybridoma cell lines producing monoclonal antibodies (MAbs) to bovine papillomavirus type 1 (BPV-1) L2 open reading frame (ORF) gene products have been established from mice immunized with a BPV-1 L2-beta-galactosidase fusion protein. Hybridomas were selected and cloned (from over 700 hybridomas) on the basis of specific reactivity of supernatant fluids with BPV-1 L2 epitopes on disrupted BPV-1 particles and L2-beta-galactosidase fusion proteins by ELISA and Western blotting, and with acetone-fixed frozen sections of BPV-1-induced fibropapillomas by immunofluorescence. These MAbs were not reactive with intact BPV-1 particles or BPV-1 L1-beta-galactosidase fusion proteins by ELISA or with beta-galactosidase by ELISA and Western blotting. The four MAbs detected viral structural proteins of Mr 76K, 68K and possibly 55K in purified BPV-1 preparations by Western blotting. Two of the four MAbs were cross-reactive with BPV-2-induced fibropapillomas. These findings suggest that (i) the BPV-1 L2 ORF encodes the minor capsid protein(s), (ii) the gene products of the BPV-1 L2 ORF have Mr values of 76K, 68K and possibly 55K, (iii) minor capsid epitopes are internal to the BPV-1 particle, and (iv) MAbs reactive with genetically engineered truncated BPV-1 L2 ORF gene products can distinguish between BPV-1 and BPV-2 productive infections.

The type-specific epitopes of the Epstein-Barr virus nuclear antigen 2 are near the carboxy terminus of the protein.

The Epstein-Barr virus nuclear antigen 2 (EBNA 2) shows serotype variation and two serologically distinct groups of viruses have been identified. These correspond to the two hybridization groups of viruses (A and B) that are distinguished by a highly substituted nucleic acid sequence in the middle of the open reading frame of the EBNA 2 gene. An epitope survey of the EBNA 2-coding region was carried out using a new prokaryotic expression vector tailored to express DNA fragments from the M13 sequencing libraries of the B95-8 (type A) and Jijoye (type B) prototype virus strains. Short overlapping stretches of EBNA 2 sequence were expressed as fusion proteins and used in Western blotting with human sera that contained serotype-specific antibodies. The type A-specific epitope was located between residues 378 and 435 of the B95-8 EBNA 2 polypeptide and the type B-specific epitope mapped between residues 390 and 454, at the carboxy terminus of the Jijoye polypeptide chain. All of the type-specific anti-EBNA 2 sera tested reacted with fusion proteins containing one or other of these epitopes. Despite the direct correlation between the hybridization and serological phenotypes, the type-specific epitopes appear to lie in the relatively conserved carboxy-terminal region of EBNA 2. There was no indication that the residues of the non-homologous region contributed to the formation of antibody-combining sites.