Advances in mRNA Vaccines for Infectious Diseases

During the last two decades, there has been broad interest in RNA-based technologies for the development of prophylactic and therapeutic vaccines. Preclinical and clinical trials have shown that mRNA vaccines provide a safe and long-lasting immune response in animal models and humans. In this review, we summarize current research progress on mRNA vaccines, which have the potential to be quick-manufactured and to become powerful tools against infectious disease and we highlight the bright future of their design and applications

811. Correction of Laminin-5 β3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Mutations in any of the genes encoding the laminin 5 heterotrimer (|[alpha]|3, |[beta]|3 and |[gamma]|2) cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. We and others have shown that expression of a retrovirally transferred |[beta]|3-chain cDNA in keratinocytes from affected patients reconstitutes normal synthesis, assembly and secretion of laminin 5, and corrects the adhesion defect in vitro and in vivo. We have recently started a phase-I clinical trial of gene therapy of JEB based on transplantation of cultured skin derived from autologous epidermal stem cells transduced with a MLV-derived retroviral vector. Since gamma- retroviral vectors have raised safety concerns for the genotoxic risk associated with the insertion of LTR elements into the human genome, we developed an alternative gene transfer strategy based on LTR- modified, HIV-derived lentiviral vectors. Two self-inactivating (SIN) lentiviral vectors were built, in which expression of either GFP or a LAMB3 cDNA is under the control of either a constitutive promoter (PGK) or the keratinocyte-specific, 2.2-kb promoter-enhancer of keratin 14 (K14). In a third construct, expression of the transgene is under the control of the viral LTR, modified by replacing the U3 region with two K14 enhancer elements. Analysis in human keratinocyte cultures and in full-thickness human skin equivalents reconstituted onto immunodeficient mice showed that GFP expression directed by the K14 elements is tissue-specific and restricted to the basal layer of the epidermis. Expression of laminin5 from the three alternative vectors was evaluated in keratinocyte cultures derived from skin biopsies of JEB patients. Biochemical and cell kinetics assays demonstrated transduction of epidermal clonogenic stem/progenitor cells and full phenotypic correction of JEB keratinocytes with all vectors. Southern blot analysis of individual cell clones showed that LTR-modified lentiviral vectors are genetically stable and integrate in multiple copies in the human genome. This study shows that the use of lentiviral vectors transcriptionally targeted to the basal keratinocytes by the insertion of restricted enhancer elements is an effective, and potentially safer, alternative for gene therapy of JEB

NadA3 structures reveal undecad coiled coils and LOX1 binding regions competed by meningococcus B vaccine-elicited human antibodies

Neisseria meningitidis serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine.IMPORTANCE The bacterial microbe Neisseria meningitidis serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA

725. Correction of Laminin-5-Deficient Junctional Epidermolysis Bullosa by Transplantation of Genetically Modified Epidermal Stem Cells. A Phase-I Clinical Trial

Mutations in genes encoding the laminin-5 heterotrimer, a key component of the epidermal-dermal junction, cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Epidermal stem cells isolated from patients affected by |[beta]|3 chain-deficient JEB were transduced with a retroviral vector expressing a |[beta]|3 cDNA, and used to generate uniformly transduced cultured skin implants. The transgene was steadily expressed for >160 cell doublings in culture, leading to restoration of normal laminin 5 levels, assembly of functional hemidesmosomes, and full phenotypic correction. Cloning and sequencing of vector integrations showed that <20 stem cells are responsible for long-term maintenance of a transplantable skin culture. A phase-I clinical trial started in October 2005, aimed at proving the safety of the transduction/transplantation procedure, and analyzing persistence of transgene expression and long-term survival of transduced stem cells. The first patient was a 30-yr-old male affected by non-lethal JEB, carrying a null mutation in one LAMB3 allele and a point mutation (E212K) in the other one. The mutation affects the assembly of the laminin-5 heterotrimer, present at residual levels (<5%) in vitro and in vivo. Six genetically modified, cultured epidermal sheets of 100 sq cm were transplanted on both legs after removal of the outer skin layer using a minimally invasive technique. The procedure was well tolerated, and the patient discharged after five days. Engraftment was completed after 10 days, and transplanted skin remained stable on both legs in the absence of blistering or inflammation for the duration of the follow-up (4 months at the time of writing). 3-mm punch biopsies were taken 1 and 3 months after transplantation, and analyzed for vector presence by quantitative PCR and for protein expression by immunohistochemistry. A vector signal compatible with full transduction of the transplanted epidermis was observed at both time points. Synthesis and assembly of normal levels of heterotrimeric laminin-5 and |[alpha]|6|[beta]|4 integrin was observed at the level of the basal lamina in all biopsies, together with the development of a firmly adherent, fully differentiated epidermis. Epidermal stem cells (p64+) were detected in the basal layer of the transplanted skin in normal numbers. These data show that gene therapy of JEB by transplantation of genetically corrected stem cells is feasible, and leads to full phenotypic correction of the adhesion defect in vivo. Safety studies are under way, which include detection or humoral or cytotoxic immune responses against laminin-5, and ex vivo cloning and sequencing of the integrated proviruses

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs

High-Definition Mapping of Retroviral Integration Sites Defines the Fate of Allogeneic T Cells After Donor Lymphocyte Infusion

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction

Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients

Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human beta-globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34(+) cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for a future clinical translation

Structures of NHBA elucidate a broadly conserved epitope identified by a vaccine induced antibody

Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel β-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal β-hairpin fragment and the β-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the β-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects

Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted lentiviral vectors.

International audienceDeficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204