Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals.

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates.

γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue.

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples.

Reduced frequencies of polyfunctional CMV-specific T cell responses in infants with congenital CMV infection.

Purpose: CMV infection remains a priority for vaccine development. Vaccination of infants could modify congenital infection and provide lifetime immunity. Properties of CMV-specific T cells associated with control of viral replication in early life have not been fully defined.

Ex vivo detection of CD8 T cells specific for H-Y minor histocompatibility antigens in allogeneic hematopoietic stem cell transplant recipients.

Immunologic disparities between minor histocompatibility antigen (mHAg) genes on Y (H-Y) and X (H-X) chromosomes contribute to graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects observed in male recipients of a female donor (FtoM) hematopoietic stem cell transplant (HCT). Using in silico prediction tools, a panel of HLA-A0201 restricted H-Y peptides was synthesized. Expression of CD137 was monitored on CD8(+) T cells after brief stimulation with the H-Y peptides in FtoM HLA-A0201 HCT recipients (N=29), and control groups (HLA-A0201 MtoM [N=18], non-HLA-A0201 FtoM [N=14], and HLA-A0201 female volunteers [N=25]).

Detection and preliminary characterization of CD8+T lymphocytes specific for Wilms' tumor antigen in patients with non-Hodgkin lymphoma.

Wilms' tumor antigen (WT1) is overexpressed in many different solid tumors and hematologic malignancies. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). In a cross-sectional survey study, we investigated the immune recognition of WT1 by patients with NHL.

Recombinant modified vaccinia virus ankara (MVA) expressing wild-type human p53 induces specific antitumor CTL expansion.

The p53 gene product is an attractive target for tumor immunotherapy. The present study aims to understand the potential of MVAp53 vaccine to induce expansion of p53-specific cytotoxic T lymphocyte ex vivo in cancer patients. The result indicated that 14 of 23 cancer patients demonstrated p53-specific IFN-γ production, degranulation, cell proliferation, and lysis of p53 overexpressed human tumor cell lines.

Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice.

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags.

Modified vaccinia Ankara expressing survivin combined with gemcitabine generates specific antitumor effects in a murine pancreatic carcinoma model.

Survivin is overexpressed by 70-80% of pancreatic cancers, and is associated with resistance to chemotherapy and a poor prognosis. Gemcitabine has been a standard treatment for patients with advanced pancreatic cancer for a decade. Recent reports have demonstrated that gemcitabine treatment attenuates the tumor-suppressive environment by eliminating CD11b(+)/Gr-1(+) myeloid-derived suppressor cells (MDSCs).

Heterologous prime/boost immunization with p53-based vaccines combined with toll-like receptor stimulation enhances tumor regression.

The p53 gene product is overexpressed in approximately 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated that a modified vaccinia Ankara (MVA) vaccine expressing human p53 (MVA-p53) was moderately active when given as a homologous prime/boost in a human p53 knock in (Hupki) mouse model. We needed to improve upon the inefficient homologous boosting approach, because development of neutralizing immunity to the vaccine viral vector backbone suppresses its immunogenicity.

CD154 expression is associated with neutralizing antibody titer levels postinfluenza vaccination in stem cell transplant patients and healthy adults.

We undertook a prospective longitudinal study to examine humoral and cellular immune responses to influenza vaccination in hematopoietic cell transplant (HCT) patients and healthy adults. Healthy volunteers and HCT patients had blood samples taken prior to influenza vaccination and 30, 90, and 180 days postvaccination. Serum from pre- and postvaccination time points were tested for influenza A IgG and IgM by ELISA as well as tested for neutralizing antibody (NAb) titers via hemagglutination inhibition assay.

Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines.

We have engineered recombinant (r) Modified Vaccinia Ankara (MVA) to express multiple antigens under the control of either of two related vaccinia synthetic promoters (pSyn) with early and late transcriptional activity or the modified H5 (mH5) promoter which has predominant early activity. We sequentially passaged these constructs and analyzed their genetic stability by qPCR, and concluded that rMVA expressing multiple antigens using the mH5 promoter exhibit remarkable genetic stability and maintain potent immunogenicity after serial passage. In contrast, rMVA expressing antigens using engineered vaccinia synthetic E/L (pSyn I or II) promoters are genetically unstable.

Mamu-A01/K(b) transgenic and MHC Class I knockout mice as a tool for HIV vaccine development.

We have developed a murine model expressing the rhesus macaque (RM) Mamu-A01 MHC allele to characterize immune responses and vaccines based on antigens of importance to human disease processes. Towards that goal, transgenic (Tg) mice expressing chimeric RM (alpha1 and alpha2 Mamu-A01 domains) and murine (alpha3, transmembrane, and cytoplasmic H-2K(b) domains) MHC Class I molecules were derived by transgenesis of the H-2K(b)D(b) double MHC Class I knockout strain. After immunization of Mamu-A01/K(b) Tg mice with rVV-SIVGag-Pol, the mice generated CD8(+) T-cell IFN-gamma responses to several known Mamu-A01 restricted epitopes from the SIV Gag and Pol antigen sequence.

A novel approach to evaluate the immunogenicity of viral antigens of clinical importance in HLA transgenic murine models.

Transgenic (Tg) mice expressing HLA class I alleles and lacking murine MHC class I represent a useful model for the pre-clinical evaluation of human vaccines, which focus on induction of CD8(+) T-cell responses. We have developed a platform to be used in Tg mice for exploring the immunogenicity of T-cell targets, whose immunologic epitopes have yet to be defined. To test the attributes of the evaluation system in the context of an important human pathogen, we have explored multiple antigens from cytomegalovirus (CMV).

Functional characterization of BK virus-specific CD4+ T cells with cytotoxic potential in seropositive adults.

BK polyomavirus (BKV) reactivation is associated with a failure of T cell immunity in kidney transplant patients, and may lead to BKV-associated nephropathy (BKVN) and loss of the allograft. BKV reactivation in hematopoietic stem cell transplant recipients is associated with hemorrhagic cystitis. We have investigated T cell responses to overlapping peptide mixtures corresponding to the whole BKV major T antigen (TAg) and major capsid protein (VP1) in peripheral blood mononuclear cell samples from a cohort of healthy BKV-seropositive subjects.

Cross-reactive CTL recognizing two HLA-A*02-restricted epitopes within the BK virus and JC virus VP1 polypeptides are frequent in immunocompetent individuals.

Two HLA-A*02-restricted epitopes have been identified within the VP1 polypeptide of a human polyomavirus, BK virus, which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Immunization of transgenic mice with recombinant modified vaccinia Ankara expressing BKV VP1 (rMVA-BKV VP1) elicited functional CTL populations recognizing the sequences LLMWEAVTV (amino acids residues 108-116, BKV VP1p108) and AITEVECFL (residues 44-52, BKV VP1p44) and cross-reactive to the previously described JC virus VP1 homologs. Flow-based analyses of PBMC from a panel of thirty healthy HLA-A*02 human volunteers indicated that the majority of these subjects harbored functional CTL populations recognizing the BKV epitopes and cross-reactive with the JCV homologs.