Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo ß7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Genetic immunization: bacteria as DNA vaccine delivery vehicles.

The so-called DNA vaccination represents one of the most notable tools under development in the field of vaccinology. The concept of administering the gene coding for any given protective antigen and make responsible vaccinee's own cells to produce the protein appeals as too simple to be true. Indeed, the implementation of this approach for mass vaccination should overcome several bottlenecks, such as need of high dosages and poor immunogenicity. In this context, the use of live attenuated bacteria as delivery system for plasmid DNA has emerged as a promising alternative to overcome many of those pitfalls. In addition, this approach is not only amenable for mucosal administration, but allows to specifically target professional antigen presenting cells. This results in their transfection, as well as in their activation and maturation, due to their built-in adjuvant properties resulting from the stimulation of pattern recognition receptors. This chapter discusses the specific features that should be taken into consideration when designing a plasmid vector, current candidate bacterial carriers for DNA delivery and main safety issues.

Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

The generation of tissue resident memory (TRM) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8+ T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8+ T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8+ T cell expression of CXCR3+, CD103+, CD49a+, CD69+, CD127+ homing, retention and survival markers. Furthermore, memory CD8+ T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8+ T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces.

Dynamic changes in viral population structure and compartmentalization during chronic hepatitis C virus infection in children.

Classic phylogenetic and modern population-based clustering methods were used to analyze hepatitis C virus (HCV) evolution in plasma and to assess viral compartmentalization within peripheral blood mononuclear cells (PBMCs) in 6 children during 3.2-9.6yr of follow-up. Population structure analysis of cloned amplicons encompassing hypervariable region 1 led to the distinction of two evolutionary patterns, one highly divergent and another one genetically homogeneous. Viral adaptability was reflected by co-evolution of viral communities switching rapidly from one to another in the context of divergence and stability associated with highly homogeneous communities which were replaced by new ones after long periods. Additionally, viral compartmentalization of HCV in PBMCs was statistically demonstrated, suggesting their role as a pool of genetic variability. Our results support the idea of a community-based structure of HCV viral populations during chronic infection and highlight a role of the PBMC compartment in the persistence of such structure.

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit.

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

Phylogenetic analysis of previously nontypeable hepatitis C virus isolates from Argentina.

Phylogenetic analysis of hepatitis C virus isolates from Argentina that were previously nontypeable by restriction fragment length polymorphism (RFLP) analysis revealed that they belong to genotype 1a. A substitution at position 107 (G-->A), which is the landmark of these strains, was shown to be distributed among isolates worldwide. The RFLP patterns obtained for these isolates should be added to the ones reported for genotype 1 isolates.

Efficient systemic and mucosal responses against the HIV-1 Tat protein by prime/boost vaccination using the lipopeptide MALP-2 as adjuvant.

A major goal of HIV-1 vaccine development is the induction of mucosal immune responses able to stop or reduce viral infection directly at the portal of entry. We established a heterologous prime/boost vaccination protocol based on intradermal priming with the HIV-1 Tat protein and intranasal boosting with the Tat protein co-administered with the mucosal adjuvant MALP-2. Strong Tat-specific humoral responses were elicited in vaccinated mice at both systemic and mucosal levels. The cellular responses were characterized by a Th1 dominant helper pattern. The heterologous prime/boost regimen was also able to induce Tat-specific CTL, which were absent in animals receiving the homologous prime boost scheme. Thus, the heterologous prime/boost protocol was the only regimen able to evoke both CTL and sIgA responses. This suggests that a similar approach can be exploited to develop multi-component vaccines against HIV-1 infections able to induce both systemic and mucosal immune responses.