THE POSSIBLE ROLE OF ENDOGENOUS RETROVIRUSES IN TUMOUR DEVELOPMENT AND INNATE SIGNALLING

Endogenous retroviruses (ERVs) are fossils of ancient retroviral infection in the germline. In primates they represent around 5% of the genome sequence. During time spent in the genome, being transmitted in a Mendelian fashion, copies of ERVs have accumulated mutations, which rendered them inactive. However, some of them (the most recently integrated ones) are still able to transcribe and produce viral proteins, although few are capable of re-infection. In the past often considered as unharmful ‘junk DNA’, recent evidence link ERVs with cancer and several inflammatory diseases. For example, a few reports demonstrate that ERVs are involved in tumour development using shRNA knock-down and over-expression systems, and their overexpression tends to correlate with inflammation status, generating the hypothesis that they can act as pathogen-associated molecular patterns (PAMPs) and bind to innate sensors. Focusing on the Human (Homo sapiens) and the rhesus macaque (Macaca mulatta), the main aims of this thesis are to look for further evidence linking ERVs to tumour development, with possible implications for therapies, and test the hypothesis that ERVs are PAMPs by seeing if individuals with higher levels of ERV expression exhibit a higher innate immune response. The work on ERVs in cancer involved the human ERV type-K HML2 lineage (HERV-K (HML2)), an ERV lineage found in humans, in Merlin-deficient tumours. These are schwannomas that arise from Schwann cells and for which effective drug therapy is urgently needed. The work on ERVs in inflammation involved the Papio cynocephalus ERV (PcEV), in rhesus macaques infected with simian immunodeficiency virus (SIV) infection. The main outcomes are as follows: regarding HERV-K (HML2) in human schwannomas, (i) HERV-K (HML2) proteins are overexpressed in schwannoma compared to Schwann cells; (ii) these proteins are released from the tumour; (iii) regulation of HERV-K (HML2) expression in the tumour appears to involve the transcription factor TEAD; (iv) schwannomas are potentially treatable using anti-HERV-K (HML2) monoclonal antibodies and antiretroviral drugs since both decreased proliferation in vitro. Regarding PcEV in SIV-infected macaques: (i) PcEV is transcriptionally active; (ii) PcEV can be retrieved at low levels in the blood of some macaque animals; (iii) the levels of PcEV in cells correlates strongly with the strength of the innate response as measured by cellular levels of STAT1 transcripts – an interferon-stimulated gene (ISG). Other recent research has shown that human ERV lineages, namely HERV-W and HERV-H, have been co-opted and are involved in placentation and pluripotency during development, respectively. The present work suggests that ERVs are involved in a wide range of biological process and supports the need for further research into the biological significance of ERVs for their hosts

Variable Baseline Papio cynocephalus Endogenous Retrovirus (PcEV) Expression Is Upregulated in Acutely SIV-Infected Macaques and Correlated to STAT1 Expression in the Spleen

Retroviral replication leaves a DNA copy in the host cell chromosome, which over millions of years of infection of germline cells has led to 5% of the human genome sequence being comprised of endogenous retroviruses (ERVs), distributed throughout an estimated 100,000 loci. Over time these loci have accrued mutations such as premature stop codons that prevent continued replication. However, many loci remain both transcriptionally and translationally active and ERVs have been implicated in interacting with the host immune system. Using archived plasma and tissue samples from past macaque studies, experimentally infected with simian immunodeficiency virus (SIV), the expression of one macaque ERV in response to acute viral infection was explored together with a measure of the innate immune response. Specifically, RNA levels were determined for (a) Papio cynocephalus Endogenous Retrovirus (PcEV), an ERV (b) STAT1, a key gene in the interferon signaling pathway, and (c) SIV, an exogenous pathogen. Bioinformatic analysis of DNA sequences of the PcEV loci within the macaque reference genome revealed the presence of open reading frames (ORFs) consistent with potential protein expression but not ERV replication. Quantitative RT-PCR analysis of DNase-treated RNA extracts from plasma derived from acute SIV-infection detected PcEV RNA at low levels in 7 of 22 macaques. PcEV RNA levels were significantly elevated in PBMC and spleen samples recovered during acute SIV infection, but not in the thymus and lymph nodes. A strong positive correlation was identified between PcEV and STAT1 RNA levels in spleen samples recovered from SIV-positive macaques. One possibility is that SIV infection induces PcEV expression in infected lymphoid tissue that contributes to induction of an antiviral response

Identification and Phenotype of MAIT Cells in Cattle and Their Response to Bacterial Infections

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections

Simultaneous aerosol and intramuscular immunization with influenza vaccine induces powerful protective local T cell and systemic antibody immune responses in pigs

A vaccine providing both powerful Ab and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. In this study, we evaluated i.m., aerosol (Aer), and simultaneous systemic and respiratory immunization (SIM) by both routes in Babraham pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization, pigs were challenged with H1N1pdm09 virus. i.m.-immunized pigs generated a high titer of neutralizing Abs but poor T cell responses, whereas Aer induced powerful respiratory tract T cell responses but a low titer of Abs. SIM pigs combined high Ab titers and strong local T cell responses. SIM showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study

The HIV-1 Antisense Protein (ASP) induces CD8 T cell responses during chronic infection

International audienceBackground : CD8+ T cells recognize HIV-1 epitopes translated from a gene’s primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3’ end of HIV-1’s proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection.Results : A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A*02 HIV-infected patient.Conclusion : Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines

Identification and Phenotype of MAIT Cells in Cattle and Their Response to Bacterial Infections

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections