Prevalence of Pre-Existing Neutralizing Antibodies Against Adeno-Associated Virus Serotypes 1, 2, 5, 6, 8, and 9 in Sera of Different Pig Strains.

Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5-6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes. These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were <3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.

Intrastriatal Administration of AAV5-miHTT in Non-Human Primates and Rats Is Well Tolerated and Results in miHTT Transgene Expression in Key Areas of Huntington Disease Pathology.

Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies.

Magnitude and Functional Profile of the Human CD4+ T Cell Response throughout Primary Immunization with Tick-Borne Encephalitis Virus Vaccine.

Tick-borne encephalitis (TBE) is a viral infection of the CNS caused by TBE virus. With no specific treatment available, the only protection is a formalin-inactivated whole virus vaccine. Primary immunization with European TBE vaccines, as recommended by the manufacturers, consists of three vaccine doses administered within a 1-y period. Protection from vaccination is believed to be mediated by Abs, yet T cells may also have a protective role. We set out to characterize the human CD4+ T cell response throughout primary TBE immunization. The responses were evaluated before vaccination and 1 mo after each vaccine dose. A heterogeneous magnitude of CD4+ T cell-mediated memory responses was observed in regard to lymphoblast expansion and cytokine production (IFN-γ, IL-2, and TNF), with the highest median magnitude detected after the second dose of vaccine. Stimulation with an overlapping peptide library based on structural TBE virus proteins E and C revealed that CD4+ T cells concomitantly producing IL-2 and TNF dominated the responses from vaccinees after each vaccine dose, whereas a control cohort of TBE patients responded mainly with all three cytokines. CD107a expression was not upregulated upon peptide stimulation in the vaccinees. However, CD154 (CD40L) expression on cytokine-positive memory CD4+ T cells significantly increased after the second vaccine dose. Taken together, TBE vaccination induced CD4+ T cell responses dominated by IL-2 and TNF production together with CD154 upregulation and a lower IFN-γ response compared with TBE patients. This response pattern was consistent after all three doses of TBE vaccine.

Therapeutic hFIX Activity Achieved after Single AAV5-hFIX Treatment in Hemophilia B Patients and NHPs with Pre-existing Anti-AAV5 NABs.

Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.

Breadth and Dynamics of HLA-A2- and HLA-B7-Restricted CD8+ T Cell Responses against Nonstructural Viral Proteins in Acute Human Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV) is a leading cause of viral meningoencephalitis in many parts of Europe and eastwards in Asia, with high morbidity and often long-term neurologic sequelae. With no treatment available, studies of the immune response to TBEV are essential for the understanding of the immunopathogenesis of tick-borne encephalitis and for the development of therapeutics. We have previously demonstrated that CD8+ T cell responses in peripheral blood in patients with acute TBEV peak at around 7 d after hospitalization in the neuroinvasive phase of the disease. In this study, we identified six novel TBEV HLA-A2- and HLA-B7-restricted epitopes, all derived from the nonstructural proteins of TBEV. This identification allowed for a comprehensive phenotypic and temporal analysis of the HLA-A2- and HLA-B7-restricted Ag-specific CD8+ T cell response during the acute stages of human TBEV infection. HLA-A2- and HLA-B7-restricted TBEV epitope-specific effector cells predominantly displayed a CD45RA-CCR7-CD27+CD57- phenotype at day 7, which transitioned into separate distinct phenotypes for HLA-A2- and HLA-B7-restricted TBEV-specific CD8+ T cells, respectively. At day 21, the most prevalent phenotype in the HLA-A2-restricted CD8+ T cell populations was CD45RA-CCR7-CD27+CD57+, whereas the HLA-B7-restricted CD8+ T cell population was predominantly CD45RA+CCR7-CD27+CD57+ Almost all TBEV epitope-specific CD8+ T cells expressed α4 and ß1 integrins at days 7 and 21, whereas the bulk CD8+ T cells expressed lower integrin levels. Taken together, human TBEV infection elicits broad responses to multiple epitopes, predominantly derived from the nonstructural part of the virus, establishing distinct maturation patterns for HLA-A2- and HLA-B7-restricted TBEV epitope-specific CD8+ T cells.

Specificity and dynamics of effector and memory CD8 T cell responses in human tick-borne encephalitis virus infection.

Tick-borne encephalitis virus (TBEV) is transferred to humans by ticks. The virus causes tick-borne encephalitis (TBE) with symptoms such as meningitis and meningoencephalitis. About one third of the patients suffer from long-lasting sequelae after clearance of the infection. Studies of the immune response during TBEV-infection are essential to the understanding of host responses to TBEV-infection and for the development of therapeutics. Here, we studied in detail the primary CD8 T cell response to TBEV in patients with acute TBE. Peripheral blood CD8 T cells mounted a considerable response to TBEV-infection as assessed by Ki67 and CD38 co-expression. These activated cells showed a CD45RA-CCR7-CD127- phenotype at day 7 after hospitalization, phenotypically defining them as effector cells. An immunodominant HLA-A2-restricted TBEV epitope was identified and utilized to study the characteristics and temporal dynamics of the antigen-specific response. The functional profile of TBEV-specific CD8 T cells was dominated by variants of mono-functional cells as the effector response matured. Antigen-specific CD8 T cells predominantly displayed a distinct Eomes+Ki67+T-bet+ effector phenotype at the peak of the response, which transitioned to an Eomes-Ki67-T-bet+ phenotype as the infection resolved and memory was established. These transcription factors thus characterize and discriminate stages of the antigen-specific T cell response during acute TBEV-infection. Altogether, CD8 T cells responded strongly to acute TBEV infection and passed through an effector phase, prior to gradual differentiation into memory cells with distinct transcription factor expression-patterns throughout the different phases.

Alternative peptide repertoire of HLA-E reveals a binding motif that is strikingly similar to HLA-A2.

The non-classical HLA-E is a conserved class I molecule that mainly presents monomorphic leader peptides derived from other HLA class I molecules. These leader peptides comprise an optimized sequence for tight and deep binding into the HLA-E groove. In a TAP-deficient environment, as it can be generated during viral infection or in tumor tissue, loading of the classical leader peptide sequences is hampered leading to an alternative HLA-E peptide repertoire. In this study, we characterized this alternative peptide repertoire using cells in which TAP activity is inhibited. We identified more than 500 unique peptide sequences carried by HLA-E and found that their binding motif is different from the dominant leader peptides. Hydrophobic amino acids were only found at positions 2 and 9, in close resemblance to the peptide binding motif of HLA-A*0201. HLA-E-eluted peptides were indeed able to bind this classical HLA class I molecule. Our findings suggest that the dominant leader peptides uniquely conform to HLA-E, but that in their absence a peptide pool is presented like that of HLA-A*0201.

Promiscuous binding of invariant chain-derived CLIP peptide to distinct HLA-I molecules revealed in leukemic cells.

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.

A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens.

Deficiencies in MHC class I antigen presentation are a common feature of tumors and allows escape from cytotoxic T lymphocyte (CTL)-mediated killing. It is crucial to take this capacity of tumors into account for the development of T-cell-based immunotherapy, as it may strongly impair their effectiveness. A variety of escape mechanisms has been described thus far, but progress in counteracting them is poor. Here we review a novel strategy to target malignancies with defects in the antigenic processing machinery (APM). The concept is based on a unique category of CD8+ T-cell epitopes that is associated with impaired peptide processing, which we named TEIPP. We characterized this alternative peptide repertoire emerging in MHC-I on tumors lacking classical antigen processing due to defects in the peptide transporter TAP (transporter associated with peptide processing). These TEIPPs exemplify interesting parallels with the folktale figure Cinderella: they are oppressed and neglected by a stepmother (like functional TAP prevents TEIPP presentation), until the suppression is released and Cinderella/TEIPP achieves unexpected recognition. TEIPP-specific CTLs and their cognate peptide-epitopes provide a new strategy to counteract immune evasion by APM defects and bear potential to targeting escape variants observed in a wide range of cancers.

Strategies to counteract MHC-I defects in tumors.

Defects in MHC-I antigen presentation represent a common feature of cancer and allow evasion from T cell recognition. Recent findings from immunotherapy in melanoma suggested that irreversible MHC-I defects enable escape from immune pressure. Although loss of antigen presentation is known for many years, strategies to counteract these defects are scarce and largely unexamined. Now that the first forms of T-cell-based immunotherapy show clinical efficacy and reach FDA approval, this issue deserves urgent awareness. Here we describe possible roads leading to corrections of MHC-I defects in tumors and describe a salvage pathway for CTL by targeting novel tumor antigens that we recently uncovered.

CD8+ T cell responses against TAP-inhibited cells are readily detected in the human population.

Target cell recognition by CTLs depends on the presentation of peptides by HLA class I molecules. Tumors and herpes viruses have adopted strategies to greatly hamper this peptide presentation at the important bottleneck, the peptide transporter TAP. Previously, we described the existence of a CD8(+) CTL subpopulation that selectively recognizes such TAP-deficient cells in mouse models. In this study, we show that the human counterpart of this CTL subset is readily detectable in healthy subjects. Autologous PBMC cultures were initiated with dendritic cells rendered TAP-impaired by gene transfer of the viral evasion molecule UL49.5. Strikingly, specific reactivity to B-LCLs expressing one of the other viral TAP-inhibitors (US6, ICP47, or BNLF2a) was already observed after three rounds of stimulation. These short-term T cell cultures and isolated CD8(+) CTL clones derived thereof did not recognize the normal B-LCL, indicating that the cognate peptide-epitopes emerge at the cell surface upon an inhibition in the MHC class I processing pathway. A diverse set of TCRs was used by the clones, and the cellular reactivity was TCR-dependent and HLA class I-restricted, implying the involvement of a broad antigenic peptide repertoire. Our data indicate that the human CD8(+) T cell pool comprises a diverse reactivity to target cells with impairments in the intracellular processing pathway, and these might be exploited for cancers that are associated with such defects and for infections with immune-evading herpes viruses.

Active DNA photolyase encoded by a baculovirus from the insect Chrysodeixis chalcites.

The genome of Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) contains two open reading frames, Cc-phr1 and Cc-phr2, which encode putative class II CPD-DNA photolyases. CPD-photolyases repair UV-induced pyrimidine cyclobutane dimers using visible light as an energy source. Expression of Cc-phr2 provided photolyase deficient Escherichia coli cells with photoreactivating activity indicating that Cc-phr2 encodes an active photolyase. In contrast, Cc-phr1 did not rescue the photolyase deficiency. Cc-phr2 was overexpressed in E. coli and the resulting photolyase was purified till apparent homogeneity. Spectral measurements indicated the presence of FAD, but a second chromophore appeared to be absent. Recombinant Cc-phr2 photolyase was found to bind specifically F0 (8-hydroxy-7,8-didemethyl-5-deazariboflavin), which is an antenna chromophore present in various photolyases.. After reconstitution, FAD and F0 were present in approximately equimolar amounts. In reconstituted photolyase the F0 chromophore is functionally active as judged from the increase in the in vitro repair activity. This study demonstrates for the first time that a functional photolyase is encoded by an insect virus, which may have implications for the design of a new generation of baculoviruses with improved performance in insect pest control.