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Purpose: To evaluate CD4+ helper functions and antitumor effect of promiscuous universal cancer peptides (UCP) derived from telomerase reverse transcriptase (TERT).Experimental Design: To evaluate the widespread immunogenicity of UCPs in humans, spontaneous T-cell responses against UCPs were measured in various types of cancers using T-cell proliferation and ELISPOT assays. The humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice were used to study the CD4+ helper effects of UCPs on antitumor CTL responses. UCP-based antitumor therapeutic vaccine was evaluated using HLA-A*0201–positive B16 melanoma that express TERT.Results: The presence of a high number of UCP-specific CD4+ T cells was found in the blood of patients with various types of cancer. These UCP-specific T cells mainly produce IFN-γ and TNF-α. In HLA transgenic mice, UCP vaccinations induced high avidity CD4+ TH1 cells and activated dendritic cells that produced interleukin-12. UCP-based vaccination breaks self-tolerance against TERT and enhances primary and memory CTL responses. Furthermore, the use of UCP strongly improves the efficacy of therapeutic vaccination against established B16-HLA-A*0201 melanoma and promotes tumor infiltration by TERT-specific CD8+ T cells.Conclusions: Our results showed that UCP-based vaccinations strongly stimulate antitumor immune responses and could be used to design efficient immunotherapies in multiple types of cancers. Clin Cancer Res; 18(22); 6284–95. ©2012 AACR.

Purpose:Human telomerase reverse transcriptase (hTERT) is highly expressed in >85% of human tumors and is thus considered as a good tumor-associated antigen candidate for vaccine development. We conducted a phase I study to investigate the safety, tolerability, clinical response, and immunogenicity of INVAC-1, a DNA plasmid encoding a modified hTERT protein in patients with relapsed or refractory solid tumors.Patients and Methods:INVAC-1 was either administered by intradermal route followed by electroporation or by Tropis, a needle-free injection system. Safety and tolerability were monitored by clinical and laboratory assessments. Progression-free survival and overall survival were reported using Kaplan–Meier survival analysis. Immunogenicity was studied by ELISpot, Luminex, and Flow Cytometry.Results:Twenty-six patients were treated with INVAC-1 administered at three dose levels (100, 400, and 800 μg). Vaccination was well tolerated and no dose-limiting toxicity was reported. One treatment-related grade 3 SAE was reported. Fifty-eight percent of patients experienced disease stabilization. PFS was 2.7 months, median OS was 15 months, and 1-year survival was reached for 65% of patients. INVAC-1 vaccination stimulated specific anti-hTERT CD4 T-cell response as well as cytotoxic CD8 T-cell response. No evidence of peripheral vaccine-induced immunosuppression was observed.Conclusions:INVAC-1 vaccination was safe, well tolerated, and immunogenic when administered intradermally at the three tested doses in patients with relapsed or refractory cancers. Disease stabilization was observed for the majority of patients (58%) during the treatment period and beyond.See related commentary by Slingluff Jr, p. 529

Histological diagnosis, age, number of cycles received and Overall Survival

Immunophenotyping of circulating CD4 and CD8 T cells

Number and percentage of patients reporting studytreatment-related AEs per cycle

PDF file - 57K, UCPs mediated helper effect on CTLs in other models of vaccinations

PDF file - 65K, UCP2 and UCP4 peptides increase self/TERT pY572 specific CTL responses

PDF file - 26K, UCP2 specific CD4 T cells recognize the mTERT-derived counterpart peptide p568

Clinical features of patients with OS 1 year

Flow Chart

PDF file - 51K, Isolation of UCP4-specific CD4 Th1 cell clone from a cancer patient

Kaplan-Meier representation of Overall Survival and Progression Free Survival

This file compiles all the results identified as "data not shown" in the manuscript

PDF file - 38K, Comparison of T-cell help mediated by UCP2 and a HTLV-1 Tax-derived peptide on self/TERT pY988 specific CTL response

Flow Cytometry antibody references

Best Overall Response

Purpose: Human telomerase reverse transcriptase (hTERT) is highly expressed in >85% of human tumors and is thus considered as a good tumor-associated antigen candidate for vaccine development. We conducted a phase I study to investigate the safety, tolerability, clinical response, and immunogenicity of INVAC-1, a DNA plasmid encoding a modified hTERT protein in patients with relapsed or refractory solid tumors. Patients and Methods: INVAC-1 was either administered by intradermal route followed by electroporation or by Tropis, a needle-free injection system. Safety and tolerability were monitored by clinical and laboratory assessments. Progression-free survival and overall survival were reported using Kaplan–Meier survival analysis. Immunogenicity was studied by ELISpot, Luminex, and Flow Cytometry. Results: Twenty-six patients were treated with INVAC-1 administered at three dose levels (100, 400, and 800 μg). Vaccination was well tolerated and no dose-limiting toxicity was reported. One treatment-related grade 3 SAE was reported. Fifty-eight percent of patients experienced disease stabilization. PFS was 2.7 months, median OS was 15 months, and 1-year survival was reached for 65% of patients. INVAC-1 vaccination stimulated specific anti-hTERT CD4 T-cell response as well as cytotoxic CD8 T-cell response. No evidence of peripheral vaccine-induced immunosuppression was observed. Conclusions: INVAC-1 vaccination was safe, well tolerated, and immunogenic when administered intradermally at the three tested doses in patients with relapsed or refractory cancers. Disease stabilization was observed for the majority of patients (58%) during the treatment period and beyond. See related commentary by Slingluff Jr, p. 529

Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 μg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.

International audience; Human cells are stressed by numerous mechanisms that can lead to leakage of mitochondrial DNA (mtDNA) to the cytoplasm and ultimately apoptosis. this agonist DNA constitutes a danger to the cell and is counteracted by cytoplasmic DNases and APOBEC3 cytidine deamination of DNA. To investigate APOBEC3 editing of leaked mtDNA to the cytoplasm, we performed a PCR analysis of APOBEC3 edited cytoplasmic mtDNA (cymtDNA) at the single cell level for primary CD4 + T cells and the established P2 EBV blast cell line. Up to 17% of primary CD4 + T cells showed signs of APOBEC3 edited cymtDNA with ~50% of all mtDNA sequences showing signs of APOBEC3 editing-between 1500-5000 molecules. Although the P2 cell line showed a much lower frequency of stressed cells, the number of edited mtDNA molecules in such cells was of the same order. Addition of the genotoxic molecules, etoposide or actinomycin D increased the number of cells showing APOBEC3 edited cymtDNA to around 40%. These findings reveal a very dynamic image of the mitochondrial network, which changes considerably under stress. APOBEC3 deaminases are involved in the catabolism of mitochondrial DNA to circumvent chronic immune stimulation triggered by released mitochondrial DNA from damaged cells. The APOBEC3 (A3) locus encodes a series of seven genes encoding six functional endogenous cytidine deam-inases with substrate specificity for single stranded DNA (ssDNA) 1. They leave DNA peppered with uracil residues. This process is referred to as genetic editing as it occurs post replication. A3 enzymes leave a telltale editing signature in DNA: most A3 enzymes preferentially edit a cytidine residue in the context of 5′TpC with the exception of A3G, which prefers 5′CpC dinucleotides 2-6. The antiviral role of these A3 enzymes was initially highlighted by their impact on HIV and HBV replication 6-9 and the fact that several A3 genes can be up-regulated by interferon α 10-12. A3 can restrict the transposition of SINE and LINE retroelements 13,14 and mitochondrial DNA (mtDNA) in cell lines and tissues, all of which show the A3 editing signature-5′TpC and 5′CpC are preferentially deam-inated 15. For the latter, the target was cytoplasmic mtDNA (cymtDNA) in keeping with the observation that all A3 enzymes are unable to access the mitochondrial network 15. In addition, A3A and to a lesser extent A3B, were shown to target chromosomal DNA non-specifically, leading to hypermutated DNA with up to 70% of cyti-dine residues deaminated 15,16. A3A and A3B-induced editing occur predominantly on the lagging strand during DNA replication 17,18. Interestingly, A3A and A3B can edit 5-methylcytidine (5MeC) residues in ssDNA which makes sense given that 5MeC is found exclusively in the nucleus 16,19-21. Uracil bases in DNA are excised by uracil N-glycosidase (UNG) that initiates DNA damage responses, which can result in either DNA repair or catabolism.

APOBEC3 (A3) enzymes are best known for their role as antiviral restriction factors and as mutagens in cancer. Although four of them, A3A, A3B, A3F and A3G, are induced by type-1-interferon (IFN-I), their role in inflammatory conditions is unknown. We thus investigated the expression of A3, and particularly A3A and A3B because of their ability to edit cellular DNA, in Systemic Lupus Erythematosus (SLE), a chronic inflammatory disease characterized by high IFN-α serum levels. In a cohort of 57 SLE patients, A3A and A3B, but also A3C and A3G, were upregulated ~ 10 to 15-fold (> 1000-fold for A3B) compared to healthy controls, particularly in patients with flares and elevated serum IFN-α levels. Hydroxychloroquine, corticosteroids and immunosuppressive treatment did not reverse A3 levels. The A3AΔ3B polymorphism, which potentiates A3A, was detected in 14.9% of patients and in 10% of controls, and was associated with higher A3A mRNA expression. A3A and A3B mRNA levels, but not A3C or A3G, were correlated positively with dsDNA breaks and negatively with lymphopenia. Exposure of SLE PBMCs to IFN-α in culture induced massive and sustained A3A levels by 4 h and led to massive cell death. Furthermore, the rs2853669 A > G polymorphism in the telomerase reverse transcriptase (TERT) promoter, which disrupts an Ets-TCF-binding site and influences certain cancers, was highly prevalent in SLE patients, possibly contributing to lymphopenia. Taken together, these findings suggest that high baseline A3A and A3B levels may contribute to cell frailty, lymphopenia and to the generation of neoantigens in SLE patients. Targeting A3 expression could be a strategy to reverse cell death and the generation of neoantigens.

Discoverer of the human immunodeficiency virus

International audience; The incidence of developing cancer should increase with the body mass, yet is not the case, a conundrum referred to as Peto's paradox. Elephants have a lower incidence of cancer suggesting that these animals have probably evolved different ways to protect themselves against the disease. The paradox is worth revisiting with the realization that most mammals encode an endogenous APOBEC3 cytidine deaminase capable of mutating single stranded DNA. Indeed, the mutagenic activity of some APOBEC3 enzymes has been shown to introduce somatic mutations into genomic DNA. These enzymes are now recognized as causal agent responsible for the accumulation of CG-> TA transitions and DNA breaks leading to chromosomal rearrangements in human cancer genomes. Here, we identified an elephant A3Z1 gene, related to human APOBEC3A and showed that it could efficiently deaminate cytidine, 5-methylcytidine and produce DNA breaks leading to massive apoptosis, similar to other mammalian APOBEC3A enzymes where body mass varies by up to four orders of magnitude. Consequently, it could be considered that eAZ1 might contribute to cancer in elephants in a manner similar to their proposed role in humans. If so, eAZ1 might be particularly well regulated to counter Peto's paradox. The APOBEC3 (A3) locus is bounded by two conserved genes, chromobox 6 and 7 (CBX6 and CBX7) in most pla-cental mammals and encodes a family of cytidine deaminases capable of converting cytidine residues to uridine in single strand DNA (ssDNA). The mutagenic activity of these enzymes is involved with the restriction of retro-viruses and DNA viruses, as well as endogenous retroelements and retrotransposons through hypermutation of viral DNA in a process called editing 1. The A3 repertoire is extremely variable among mammals, the locus being shaped through extensive gene duplications and functionalization in the context of a virus-host arms race. A3 enzymes are made up of three related, but distinct zinc-finger domains referred to as Z1, Z2 and Z3 2-4 presumably already present in the genome of the ancestor of placental mammals 5. The last few years has seen the identification of two human endogenous A3 cytidine deaminases, APOBEC3A (A3A) and APOBEC3B (A3B) capable of introducing multiple mutations in chromosomal DNA 6-9. These findings are grounded by the analysis of many cancer genomes, revealing far more mutations and rearrangements than hitherto imagined, where CG-> TA transitions appears to be the dominant mutations 10-13. Human A3A is composed of a single Z1 domain, while A3B is composed of a double Z2Z1 domain, although only the C terminal Z1 domain being catalytically functional 6. A3A and A3B enzymes are both localized in the nucleus and can edit cytidine residues to uridine in ssDNA during transcription and replication, following DNA repair, and leave TpC to TpT signature mutations that show up in cancer genomes 6,8,9. Both enzymes can mutate 5-methylcytidine (5MeC) to thymidine leaving another distinct signature in cancer genomes 6,14-16. Although A3A and A3B are accepted as intrinsic mutators of cellular chromosomal DNA, analyzed in several cancer types 8,11,17 , debate still persists regarding the contribution of each enzyme in the accumulation of mutations paving the way for oncogenesis. While, it has been described that A3A and A3B could be enzymat-ically active in different cancers 18 , A3A is the more active of the two enzymes and as a consequence, only A3A can produce double stranded breaks (DSBs), at least in an experimental setting 6,7,19. Editing frequencies of >0.5 can be found which is why the phenomenon is referred to as hyperediting or hypermutation 9. Accumulation of substitutions localized in the A3B C-terminal domain attenuated the activity of the enzyme compared to A3A 6 .

BackgroundAPOBEC1 (A1) enzymes are cytidine deaminases involved in RNA editing. In addition to this activity, a few A1 enzymes have been shown to be active on single stranded DNA. As two human ssDNA cytidine deaminases APOBEC3A (A3A), APOBEC3B (A3B) and related enzymes across the spectrum of placental mammals have been shown to introduce somatic mutations into nuclear DNA of cancer genomes, we explored the mutagenic threat of A1 cytidine deaminases to chromosomal DNA.ResultsMolecular cloning and expression of various A1 enzymes reveal that the cow, pig, dog, rabbit and mouse A1 have an intracellular ssDNA substrate specificity. However, among all the enzymes studied, mouse A1 appears to be singular, being able to introduce somatic mutations into nuclear DNA with a clear 5’TpC editing context, and to deaminate 5-methylcytidine substituted DNA which are characteristic features of the cancer related mammalian A3A and A3B enzymes. However, mouse A1 activity fails to elicit formation of double stranded DNA breaks, suggesting that mouse A1 possess an attenuated nuclear DNA mutator phenotype reminiscent of human A3B.ConclusionsAt an experimental level mouse APOBEC1 is remarkable among 12 mammalian A1 enzymes in that it represents a source of somatic mutations in mouse genome, potentially fueling oncogenesis. While the orderRodentiais bereft of A3A and A3B like enzymes it seems that APOBEC1 may well substitute for it, albeit remaining much less active. This modifies the paradigm that APOBEC3 and AID enzymes are the sole endogenous mutator enzymes giving rise to off-target editing of mammalian genomes.

International audience; Numerous human APOBEC3 cytidine deaminases have proven to be, inter alia, host cell restriction factors for retroviruses and hepadnaviruses. Although they can bind to genomic RNA and become encapsidated, they are only catalytically active on single-stranded DNA. As there are many cellular deoxyribonucleases (DNases), we hypothesized that a parallel could be struck between APOBEC3 and DNases. For human hepatitis B virus (HBV), we show that DNase I can considerably reduce the virion genome copy number from a variety of transfected or infected cells. DNASE1 is overexpressed and encapsidated in HBV particles in vitro in hypoxic environments and in vivo in cirrhotic patient livers as well as in the serum of infected patients. The use of CoCl2 and dimethyloxalylglycine, mimetic agents used to induce hypoxia by inhibiting prolyl hydroxylase enzymes that stabilize hypoxia-inducible factor (HIF)-1α, showed that the formation of HIF-1α/HIF-1β heterodimers results in the induction of DNASE1. Indeed, transfection with HIF-1α and HIF-1β expression constructs upregulated DNASE1. These findings suggest that human DNase I can impact HBV replication through the catabolism of the DNA genome within the capsid. The activity of DNases in general may explain in part the high frequency of empty or 'light' hepatitis B virions observed in vivo.

International audience; Cancer immunotherapy is seeing an increasing focus on vaccination with tumor-associated antigens (TAAs). Human telomerase (hTERT) is a TAA expressed by most tumors to overcome telomere shortening. Tolerance to hTERT can be easily broken both naturally and experimentally and hTERT DNA vaccine candidates have been introduced in clinical trials. DNA prime/boost strategies have been widely developed to immunize efficiently against infectious diseases. We explored the use of a recombinant measles virus (MV) hTERT vector to boost DNA priming as recombinant live attenuated measles virus has an impressive safety and efficacy record. Here, we show that a MV-TERT vector can rapidly and strongly boost DNA hTERT priming in MV susceptible IFNAR/CD46 mouse models. The cellular immune responses were Th1 polarized. No humoral responses were elicited. The 4 kb hTERT transgene did not impact MV replication or induction of cell-mediated responses. These findings validate the MV-TERT vector to boost cell-mediated responses following DNA priming in humans.

// Helene C. Laude 1 , Vincent Caval 1 , Mohamed S. Bouzidi 1 , Xiongxiong Li 1, 2 , Florence Jamet 1 , Michel Henry 1 , Rodolphe Suspene 1 , Simon Wain-Hobson 1 and Jean-Pierre Vartanian 1 1 Molecular Retrovirology Unit, Institut Pasteur, CNRS UMR 3569, France 2 Lanzhou Institute of Biological Products Co., Ltd (LIBP), subsidiary company of China National Biotec Group Company Limited (CNBG), Lanzhou 730046, China Correspondence to: Jean-Pierre Vartanian, email: jean-pierre.vartanian@pasteur.fr Keywords: rabbit; animal model; APOBEC3A; cytidine deaminase; cancer Received: March 15, 2018 Accepted: May 24, 2018 Published: June 12, 2018 ABSTRACT APOBEC3 are cytidine deaminases that convert cytidine to uridine residues. APOBEC3A and APOBEC3B enzymes able to target genomic DNA are involved in oncogenesis of a sizeable proportion of human cancers. While the APOBEC3 locus is conserved in mammals, it encodes from 1–7 genes. APOBEC3A is conserved in most mammals, although absent in pigs, cats and throughout Rodentia whereas APOBEC3B is restricted to the Primate order. Here we show that the rabbit APOBEC3 locus encodes two genes of which APOBEC3A enzyme is strictly orthologous to human APOBEC3A. The rabbit enzyme is expressed in the nucleus and the cytoplasm, it can deaminate cytidine, 5-methcytidine residues, nuclear DNA and induce double-strand DNA breaks. The rabbit APOBEC3A enzyme is negatively regulated by the rabbit TRIB3 pseudokinase protein which is guardian of genome integrity, just like its human counterpart. This indicates that the APOBEC3A/TRIB3 pair is conserved over approximately 100 million years. The rabbit APOBEC3A gene is widely expressed in rabbit tissues, unlike human APOBEC3A . These data demonstrate that rabbit could be used as a small animal model for studying APOBEC3 driven oncogenesis.

International audience; Human APOBEC3A (A3A) cytidine deaminase shows pro-apoptotic properties resulting from hypermutation of genomic DNA, induction of double-stranded DNA breaks (DSBs) and G1 cell cycle arrest. Given this, we evaluated the antitumor efficacy of A3A by intratumoral electroporation of an A3A expression plasmid. DNA was repeatedly electroporated into B16OVA, B16Luc tumors of C57BL/6J mice as well as the aggressive fibrosarcoma Sarc2 tumor of HLA-A*0201/DRB1*0101 transgenic mice using noninvasive plate electrodes. Intratumoral electroporation of A3A plasmid DNA resulted in regression of ~50% of small B16OVA melanoma tumors that did not rebound in the following 2 months without treatment. Larger or more aggressive tumors escaped regression when so treated. As APOBEC3A was much less efficient in provoking hypermutation and DSBs in B16OVA cells compared with human or quail cells, it is likely that APOBEC3A would be more efficient in a human setting than in a mouse model.

The mouse is widely used as a model for DNA therapy and vaccination even though the efficiency of DNA delivery in higher mammals and humans is much less. The human APOBEC3 (A3) enzymes impact viral genomes by cytidine deamination, which introduces multiple uridine residues into single-stranded DNA, a process known as genetic editing. This initiates rapid DNA catabolism via a uracil DNA glycosylase dependent pathway. In tissue culture, A3A, A3C and A3B can hyperedit transfected plasmid DNA. We explored plasmid catabolism in vivo initiated by A3A, the most efficient of the human enzymes and one that is functionally conserved across most mammals. As rodents do not encode an A3A enzyme, it was possible to explore DNA degradation in the mouse model. Human A3A genetically edits co-electroporated luciferase plasmid DNA in mouse skeletal muscle that initiates DNA degradation resulting in approximately fourfold decrease in bioluminescence. Part of the degradation occurs in the nucleus as indicated by complex hyperedited DNA molecules. As human A3A is strongly upregulated by interferon α and DNA sensing pathways, it is a strong candidate enzyme for restricting plasmid DNA in higher mammals.

International audience; Background: The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5'TpC context. These can be copied as G->A transitions in the 5'GpA context.Methods: DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA.Results: Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5'GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR.Conclusions: Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼10(4)-10(5) base substitutions per cancer genome, or 0.003-0.03 kb(-1). By contrast, the 3DPCR error rate is of the order of 4-20 kb(-1) owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5'GpC represent artefacts.

2642 Background: INVAC-1 is an optimized DNA plasmid encoding an inactive form of human Telomerase Reverse Transcriptase (hTERT), a universal tumor antigen expressed in most of human tumors with little or no expression in somatic cells. We report here the final results of a First-In-Human Phase I study evaluating INVAC-1 as a single agent in patients (pts) with advanced solid tumors, ended in June 2018. Methods: A two center Phase I trial evaluated INVAC-1 given monthly for a minimum of 3 cycles and up to 9 cycles by intradermal injection followed by electroporation (n = 20) or using a needle-free injection system (n = 6). Primary objectives included safety, tolerability and dose limiting toxicities to identify the maximum tolerated dose and recommended phase 2 dose. Secondary objectives included immune response (assessed by IFN-γ Elispot) and anti-tumor activity. Immuno-monitoring included detection of autoantibodies, lymphocyte phenotyping and inflammatory cytokine levels in blood. Anti-tumor activity was evaluated through RECIST 1.1 adapted to immune response, and plasma circulating tumor DNA (ctDNA). Results: 26 pts with refractory/progressive tumors were enrolled and treated with 3 escalating doses of 100, 400 and 800 µg. 15 pts experienced stable disease according to RECIST. For 11 of them, the treatment was extended, up to 9 months. INVAC-1 was well tolerated with no dose-limiting toxicities. No significant biological signs of autoimmunity were observed. No significant modification in inflammatory plasma cytokines levels was observed after INVAC-1 administration. INVAC-1 triggered de novo or enhanced pre-existing CD4/CD8 specific anti-hTERT response in 63% of pts. This specific anti-hTERT immune response was enhanced ex vivo by adding the immune checkpoint inhibitor nivolumab. ctDNA was evaluated in 17 pts. We observed a ctDNA decrease in 6 cases, a stable level in 5 cases and an increase in 6 cases. Conclusions: Results indicate that INVAC-1 was well tolerated and immunogenic at the doses and schedule tested. Disease stabilization was obtained for the majority of pts (58%) according to RECIST criteria or ctDNA levels. Clinical trial information: NCT02301754.

International audience; The human APOBEC3 gene cluster locus encodes polynucleotide cytidine deaminases. Although many act as viral restriction factors through mutation of single-stranded DNA, recent reports have shown that human APOBEC3A was capable of efficiently hypermutating nuclear DNA and inducing DNA breaks in genomic DNA. In addition, the enzyme was unique in efficiently deaminating 5-methylcytidine in single-stranded DNA. To appreciate the evolutionary relevance of these activities, we analyzed A3A-related enzymes from the rhesus and tamarin monkey, horse, sheep, dog, and panda. All proved to be orthologous to the human enzyme in all these activities revealing strong conservation more than 148 My. Hence, their singular role in DNA catabolism is a well-established mechanism probably outweighing any deleterious or pathological roles such as genomic instability and cancer formation.

International audience; Background: The replication of HBV involves the production of covalently closed circular DNA (cccDNA) from the HBV genome through the repair of virion relaxed circular DNA (rcDNA) in the virion. As cccDNA is the transcription template for HBV genomes, it needs to be eliminated from hepatocytes if the eradication of chronic HBV infection is to be achieved. PCR quantitation of cccDNA copy number is the technique of choice for evaluating the efficiency of treatment regimens. The PCR target commonly used to identify cccDNA spans the gapped region of rcDNA and is considered to accurately distinguish between cccDNA and rcDNA. There is however, a potentially confounding issue in that PCR can generate larger targets from collections of small DNA fragments, a phenomenon known as PCR recombination.Results: The impact of PCR recombination towards the amplification of this cccDNA specific target was explored by mixing three marked, yet overlapping HBV DNA fragments. Thirteen of sixteen possible recombinants were identified by sequencing with frequencies ranging from 0.6 to 23%. To confirm this finding in vivo, HBV positive sera were treated with DNase I and submitted to quantitative real-time PCR. Under these conditions, it was possible to amplify the cccDNA specific segment without difficulty. As the virion contains uniquely rcDNA, amplification of the cccDNA target resulted from PCR recombination.Conclusions: PCR quantitation of cccDNA may be more difficult than hitherto thought. Current detection protocols need to be investigated so as to help in the management of chronic HBV infection.

The APOBEC3 locus consists of seven genes (A3A-A3C, A3DE, A3F-A3H) that encode DNA cytidine deaminases. These enzymes deaminate single-stranded DNA, the result being DNA peppered with CG →TA mutations preferentially in the context of 5'TpC with the exception of APOBEC3G (A3G), which prefers 5'CpC dinucleotides. Hepatitis B virus (HBV) DNA is vulnerable to genetic editing by APOBEC3 cytidine deaminases, A3G being a major restriction factor. APOBEC3DE (A3DE) stands out in that it is catalytically inactive due to a fixed Tyr320Cys substitution in the C-terminal domain. As A3DE is closely related to A3F and A3G, which can form homo- and heterodimers and multimers, the impact of A3DE on HBV replication via modulation of other APOBEC3 restriction factors was investigated. A3DE binds to itself, A3F, and A3G and antagonizes A3F and, to a lesser extent, A3G restriction of HBV replication. A3DE suppresses A3F and A3G from HBV particles, leading to enhanced HBV replication. Ironically, while being part of a cluster of innate restriction factors, the A3DE phenotype is proviral. As the gorilla genome encodes the same Tyr320Cys substitution, this proviral phenotype seems to have been selected for.

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation. In vivo , for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the one in vitro was observed, along with a considerable proportion of noncanonical assortment.

Human T-cell leukemia virus type 1 (HTLV-1) induces cell proliferation after infection, leading to efficient transmission by cell-to-cell contact. After a long latent period, a fraction of carriers develop adult T-cell leukemia (ATL). Genetic changes in the tax gene in ATL cells were reported in about 10% of ATL cases. To determine genetic changes that may occur throughout the provirus, we determined the entire sequence of the HTLV-1 provirus in 60 ATL cases. Abortive genetic changes, including deletions, insertions, and nonsense mutations, were frequent in all viral genes except the HBZ gene, which is transcribed from the minus strand of the virus. G-to-A base substitutions were the most frequent mutations in ATL cells. The sequence context of G-to-A mutations was in accordance with the preferred target sequence of human APOBEC3G (hA3G). The target sequences of hA3G were less frequent in the plus strand of the HBZ coding region than in other coding regions of the HTLV-1 provirus. Nonsense mutations in viral genes including tax were also observed in proviruses from asymptomatic carriers, indicating that these mutations were generated during reverse transcription and prior to oncogenesis. The fact that hA3G targets the minus strand during reverse transcription explains why the HBZ gene is not susceptible to such nonsense mutations. HTLV-1-infected cells likely take advantage of hA3G to escape from the host immune system by losing expression of viral proteins.