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. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

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. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.

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. METHODS: CMV-specific CD4 and CD8 T cell responses were investigated in infants with congenital CMV infection and compared to adults with primary or chronic infection. PBMC were stimulated with UL83 (pp65) or UL122 (IE-2) peptide pools then stained with antibodies to markers of T cell subset (CD4 or CD8), phenotype (CD45RA, CCR7), or function (MIP1ß, CD107, IFNγ, IL2) for flow cytometry analysis. RESULTS: Detection of CMV pp65-specific CD4 T cells was less common in infants than adults. Responder cells were primarily effector memory (EM, CD45RA-CCR7-) in adults, but mixed memory subsets in infants. Detection of CMV pp65-specific CD8 T cells did not differ between the groups, but infants had lower frequencies of total responding cells and of MIP1ß- or CD107-expressing cells. Responder cells were EM or effector memory RA (CD45RA + CCR7-) in all groups. Polyfunctional T cells were less commonly detected in infants than adults. Responses to IE-2 were detected in adults but not infants. All infants had detectable circulating CMV DNA at initial study (versus 60 % of adults with primary infection) despite longer duration of CMV infection. CONCLUSIONS: Reduced frequencies and altered functional profile of CMV-specific CD4 and CD8 T cell responses were detected in infants compared to adults, and were associated with persistent CMV DNA in peripheral blood.

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]). Specific H-Y responses were significantly greater in HLA-A0201 FtoM than controls. CD8(+) T-cell responses to novel H-Y epitopes were shared among multiple patients, showing marked CD45RA(+)CD27 cytolytic effector profiles. These data represent a proof of concept for our in silico/ex vivo CD8(+) T-cell based approach of prediction and validation of H-Y mHAgs in HCT recipients, which may facilitate prospective studies to identify targets/biomarkers of GVHD/GVL.

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. Utilizing a WT1 overlapping peptide library, we discovered that a large percentage of patients with NHL of all grades maintain WT1-specific T cells. Ex vivo frequencies of these T cells measured from unfractionated samples by the CD137 activation marker assay were high in many patients (some > 1% CD8+). Using standard in vitro techniques we discovered that they were cytotoxic to WT1 peptide library-loaded T2 cells and WT1 antigen-primed autologous Epstein-Barr virus-transformed B cell lines (EBV-LCLs) and expressed interferon gamma (IFN-γ). In addition, we detected WT1 mRNA transcripts in diseased lymph node tissues of patients with NHL utilizing real-time quantitative polymerase chain reaction (RT-qPCR) technology. These results are the first example of strong T cell reactivity against WT1 in patients with NHL which also demonstrate strong cytotoxicity against peptide-loaded tumor cells. The potential for developing WT1 as a target for immunotherapy in NHL deserves further exploration.

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. These experiments show that MVAp53 stimulation has the potential to induce the expansion of p53-specific cytotoxic T lymphocyte from the memory T cell repertoire. The data suggest that MVAp53 vaccine is an ideal candidate for cancer immunotherapy.

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. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

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). We hypothesize that a cancer vaccine targeting survivin can achieve enhanced efficacy when combined with gemcitabine. In this study, we tested this hypothesis using modified vaccinia Ankara (MVA) expressing full-length murine survivin. The poorly immunogenic mouse pancreas adenocarcinoma cell line, Pan02, which expresses murine survivin and is syngeneic to C57BL/6, was used for this study. Immunization with MVA-survivin resulted in a modest therapeutic antitumor effect on established Pan02 tumors. When administered with gemcitabine, MVA-survivin immunization resulted in significant tumor regression and prolonged survival. The enhanced vaccine efficacy was associated with decreased CD11b(+)/Gr-1(+) MDSCs. To analyze the survivin-specific immune response to MVA-survivin immunization, we utilized a peptide library of 15mers with 11 residues overlapping from full-length murine survivin. Splenocytes from mice immunized with MVA-survivin produced intracellular γ-interferon in response to in vitro stimulation with the overlapping peptide library. Increased survivin-specific CD8(+) T cells that specifically recognized the Pan02 tumor line were seen in mice treated with MVA-survivin and gemcitabine. These data suggest that vaccination with MVA-survivin in combination with gemcitabine represents an attractive strategy to overcome tumor-induced peripheral immune tolerance, and this effect has potential for clinical benefit in pancreatic cancer.

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. Polychromatic flow cytometry was used to examine CD4(+) T cells for levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and CD154 (CD40 ligand) expression after stimulation with inactivated flu virus. In healthy subjects, we found a significant increase in Influenza A IgG and IgM levels as well as an increase in NAb titers pre- and post-influenza vaccination. Notably, NAb titers of most HCT patients did not rise to a protective level postvaccination. CD4(+) T cell expression of CD154 and cytokine responses were significantly reduced in HCT recipients compared to healthy adults. A lack of B cell reconstitution and dysfunctional CD4 T cell costimulation (as marked by low CD154 expression) is associated with low NAb levels postvaccination in HCT patients.

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. To enhance specificity, we examined the combination of 2 different vaccine vectors provided in sequence as a heterologous prime/boost. Hupki mice were evaluated as a human p53 tolerant model to explore the capacity of heterologous p53 immunization to reject human p53-expressing tumors. We employed attenuated recombinant Listeria monocytogenes expressing human p53 (LmddA-LLO-p53) in addition to MVA-p53. Heterologous p53 immunization resulted in a significant increase in p53-specific CD8 and CD4 T cells compared with homologous single vector p53 immunization. Heterologous p53 immunization induced protection against tumor growth but had only a modest effect on established tumors. To enhance the immune response we used synthetic double-strand RNA (polyinsosinic:polycytidylic acid) and unmethylated CpG-containing oligodeoxynucleotide to activate the innate immune system via Toll-like receptors. Treatment of established tumor-bearing Hupki mice with polyinsosinic:polycytidylic acid and CpG-oligodeoxynucleotide in combination with heterologous p53 immunization resulted in enhanced tumor rejection relative to treatment with either agent alone. These results suggest that heterologous prime/boost immunization and Toll-like receptor stimulation increases the efficacy of a cancer vaccine, targeting a tolerized tumor antigen.

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. Progressive accumulation of antigen loss variants resulted in a viral preparation with lower immunogenicity after serial passage. Metabolic labeling, followed by cold chase revealed little difference in stability of proteins expressed from mH5 or pSyn promoter constructs. We conclude that maintenance of genetic stability which is achieved using mH5, though not with pSyn promoters, is linked to timing, not the magnitude of expression levels of foreign antigen, which is more closely associated with immunogenicity of the vaccine.

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. Fusion peptides of highly recognized CTL epitopes from SIV Pol and Gag and a strong T-help epitope were shown to be immunogenic and capable of limiting an rVV-SIVGag-Pol challenge. Mamu-A01/K(b) Tg mice provide a model system to study the Mamu-A01 restricted T-cell response for various infectious diseases which are applicable to a study in RM.

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). A panel of recombinant modified vaccinia Ankara (MVA) vectors, expressing various CMV proteins (CMV-MVA) was used to immunize HLA-A*0201, B*0702 and A*1101 Tg mice. Immune splenocytes were in vitro stimulated (IVS) either using syngeneic lipo-polysaccharide activated lymphoblasts or Tg HLA-I matched human EBV-transformed B-lymphoblastoid cells (LCL), both loaded with peptide libraries, encompassing the CMV protein under investigation. IVS performed with peptide library loaded lymphoblasts failed to provide a reliable stimulation. In contrast, the usage of LCL as antigen presenting cells (APC) of CMV peptide libraries resulted in a consistent and specific amplification of the Tg T-cell response in animals immunized with CMV-MVAs. The LCL IVS method reliably allowed defining the immunogenicity and immunodominant CD8(+) T-cell regions of uncharacterized CMV antigens. The combination of CMV-MVA vectors, unbiased pools of CMV-specific peptide libraries presented by Tg HLA-I matched LCL constitutes a valid tool for the pre-clinical evaluation of model candidate vaccines. This convenient method could find application to investigate the immunogenicity profile of cancer antigens or proteins from infectious human pathogens.

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. The majority of these individuals possessed populations of both CD8(+) and CD4(+) T cells specific for these BKV antigens. After expansion in culture, the majority of the BKV-specific CD4(+) T cells, in addition to expressing CD40L (CD154), secreted both interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, contained both granzyme A and granzyme B, and degranulated/mobilized CD107 in response to antigen-specific stimulation. These T cells thus represent potentially functional BKV-specific cytotoxic CD4(+) T lymphocytes. Secretion of both TNF-alpha and IFN-gamma by CD154(+)CD4(+) T cells on BKV-specific stimulation was associated with higher levels of granzyme B and a higher proportion of degranulating cells compared with CD154(+)CD4(+) T cells producing only IFN-gamma or neither cytokine. These healthy subjects also harbored populations of functional CD8(+) T cells specific for one or more of three newly defined HLA-A 02-restricted cytotoxic T lymphocyte epitopes within the BKV TAg as well as two HLA-A 02-restricted epitopes within the BKV VP1 we have previously described. The BKV-specific CD4(+) T cells characterized in this study may play a part in maintaining persistent memory T cell responses to the virus and thus contribute to the immune control of BKV in healthy individuals.

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. CTL recognizing the JCV VP1p100 and JCV VP1p36 epitopes have previously been associated with prolonged survival in progressive multifocal leukoencephalopathy patients. These findings suggest that infection with BKV or JCV could potentially induce cross-protective T-cell immunity against diseases associated with these viruses.