Genetic Diversity of the Hemagglutinin Genes of Influenza a Virus in Asian Swine Populations.

Swine influenza (SI) is a major respiratory disease of swine; SI is due to the influenza A virus of swine (IAV-S), a highly contagious virus with zoonotic potential. The intensity of IAV-S surveillance varies among countries because it is not a reportable disease and causes limited mortality in swine. Although Asia accounts for half of all pig production worldwide, SI is not well managed in those countries. Rigorously managing SI on pig farms could markedly reduce the economic losses, the likelihood of novel reassortants among IAV-S, and the zoonotic IAV-S infections in humans. Vaccination of pigs is a key control measure for SI, but its efficacy relies on the optimal antigenic matching of vaccine strains with the viral strains circulating in the field. Here, we phylogenetically reviewed the genetic diversity of the hemagglutinin gene among IAVs-S that have circulated in Asia during the last decade. This analysis revealed the existence of country-specific clades in both the H1 and H3 subtypes and cross-border transmission of IAVs-S. Our findings underscore the importance of choosing vaccine antigens for each geographic region according to both genetic and antigenic analyses of the circulating IAV-S to effectively manage SI in Asia.

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis.

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

Successful Transfer of Umbilical Cord Blood CD34+ Hematopoietic Stem and Progenitor-derived NK Cells in Older Acute Myeloid Leukemia Patients.

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units.Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting.Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 104 T cells/kg and <3 × 105 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months.Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential "off-the-shelf" translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107-18. ©2017 AACR.

Ex vivo generation of interstitial and Langerhans cell-like dendritic cell subset-based vaccines for hematological malignancies.

Autologous, patient-specific, monocyte-derived dendritic cell (MoDC) vaccines have been successfully applied in the clinical studies so far. However, the routine application of this strategy has been hampered by the difficulties in generating sufficient numbers of DC and the poor DC vaccine quality because of pathology or prior treatment received by the patients. The immunotherapeutic potential of other subsets of DC has not been thoroughly investigated because of their rarity in tissues and difficulties associated with their ex vivo generation. The high expansion and differentiation potential of CD34 hematopoietic progenitor cells (HPC), isolated from umbilical cord blood (UCB), into different DC subsets make them an attractive alternative DC source for cancer immunotherapy. Therefore, the aim of this study was to generate a large number of different DC subsets from CD34 HPC and evaluate their functionality in comparison with MoDC. Our culture protocol generated a clinically relevant number of mature CD1a myeloid DC and CD207 Langerhans cells (LC)-like DC subsets from CD34 HPC with >95% purity. Both DC subsets exhibited a cytokine profile that favors cytotoxic T-cell responses. Furthermore, UCB-DC and UCB-LC demonstrated superior induction of proliferation of both allogeneic as well as viral antigen-specific CD8 T cells, both in vitro and in vivo. Additional studies revealed that UCC-DC and UCB-LC can efficiently expand minor histocompatibility antigen (MiHA) HA-1-specific cytotoxic T cells in the peripheral blood of leukemia patients and prime MiHA HA-1-specific and HA-2-specific cytotoxic T cells in vitro. These preclinical findings support the pharmaceutical development of the described culture protocol for clinical evaluation.

The aryl hydrocarbon receptor antagonist StemRegenin 1 promotes human plasmacytoid and myeloid dendritic cell development from CD34+ hematopoietic progenitor cells.

The superiority of dendritic cells (DCs) as antigen-presenting cells has been exploited in numerous clinical trials, where generally monocyte-derived DCs (Mo-DCs) are injected to induce immunity in patients with cancer or infectious diseases. Despite promising expansion of antigen-specific T cells, the clinical responses following vaccination have been limited, indicating that further improvements of DC vaccine potency are necessary. Pre-clinical studies suggest that vaccination with combination of primary DC subsets, such as myeloid and plasmacytoid blood DCs (mDCs and pDCs, respectively), may result in stronger clinical responses. However, it is a challenge to obtain high enough numbers of primary DCs for immunotherapy, since their frequency in blood is very low. We therefore explored the possibility to generate them from hematopoietic progenitor cells (HPCs). Here, we show that by inhibiting the aryl hydrocarbon receptor with its antagonist StemRegenin 1 (SR1), clinical-scale numbers of functional BDCA2(+)BDCA4(+) pDCs, BDCA1(+) mDCs, and BDCA3(+)DNGR1(+) mDCs can be efficiently generated from human CD34(+) HPCs. The ex vivo-generated DCs were phenotypically and functionally comparable to peripheral blood DCs. They secreted high levels of pro-inflammatory cytokines such as interferon (IFN)-α, interleukin (IL)-12, and tumor necrosis factor (TNF)-α and upregulated co-stimulatory molecules and maturation markers following stimulation with Toll-like receptor (TLR) ligands. Further, they induced potent allogeneic T-cell responses and activated antigen-experienced T cells. These findings demonstrate that SR1 can be exploited to generate high numbers of functional pDCs and mDCs from CD34(+) HPCs, providing an alternative option to Mo-DCs for immunotherapy of patients with cancer or infections.

Natural killer cells generated from cord blood hematopoietic progenitor cells efficiently target bone marrow-residing human leukemia cells in NOD/SCID/IL2Rg(null) mice.

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34⁺ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rg(null) mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.

CD40-targeted adenoviral cancer vaccines: the long and winding road to the clinic.

The ability of dendritic cells (DCs) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DCs for clinical administration, their loading with tumor associated antigens (TAAs) and their activation, is laborious and expensive, and, as a result of inter-individual variability in the personalized vaccines, remains poorly standardized. An attractive alternative approach is to load resident DCs in vivo by targeted delivery of TAAs, using viral vectors and activating them simultaneously. To this end, we have constructed genetically-modified adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAAs to the CD40 receptor on DCs. Pre-clinical human and murine studies conducted so far have clearly demonstrated the suitability of a 'two-component' (i.e. Ad and adaptor molecule) configuration for targeted modification of DCs in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in the clinical translation of this approach.

Potent antitumor immunity generated by a CD40-targeted adenoviral vaccine.

In situ delivery of tumor-associated antigen (TAA) genes into dendritic cells (DC) has great potential as a generally applicable tumor vaccination approach. Although adenoviruses (Ad) are an attractive vaccine vehicle in this regard, Ad-mediated transduction of DCs is hampered by the lack of expression of the Ad receptor CAR on the DC surface. DC activation also requires interaction of CD40 with its ligand CD40L to generate protective T-cell-mediated tumor immunity. Therefore, to create a strategy to target Ads to DCs in vivo, we constructed a bispecific adaptor molecule with the CAR ectodomain linked to the CD40L extracellular domain via a trimerization motif (CFm40L). By targeting Ad to CD40 with the use of CFm40L, we enhanced both transduction and maturation of cultured bone marrow-derived DCs. Moreover, we improved transduction efficiency of DCs in lymph node and splenic cell suspensions in vitro and in skin and vaccination site-draining lymph nodes in vivo. Furthermore, CD40 targeting improved the induction of specific CD8(+) T cells along with therapeutic efficacy in a mouse model of melanoma. Taken together, our findings support the use of CD40-targeted Ad vectors encoding full-length TAA for in vivo targeting of DCs and high-efficacy induction of antitumor immunity.

Delivery route, MyD88 signaling and cross-priming events determine the anti-tumor efficacy of an adenovirus based melanoma vaccine.

Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intranasal or intraperitoneal delivery in the B16F10 melanoma model. The intradermal route induced superior systemic anti-melanoma immunity which was MyD88 signaling-dependent. Predominant transduction of non-professional antigen-presenting cells at the dermal vaccination sites and draining lymph nodes, suggested a role for cross-presentation, which was confirmed in vitro. We conclude that the dermis provides an optimal route of entry for Ad-based vaccines for high-efficacy systemic anti-tumor immunization and that this immunization likely involves cross-priming events in the draining lymph nodes.

Selective transduction of dendritic cells in human lymph nodes and superior induction of high-avidity melanoma-reactive cytotoxic T cells by a CD40-targeted adenovirus.

Targeted delivery of tumor antigen genes to dendritic cells (DCs) using adenoviral (Ad) vectors holds great potential for cancer immunotherapy. We previously showed that CD40 targeting of Ad vectors enhanced specific transduction of DC in human skin, while simultaneously ensuring their stable maturation and superior allogeneic T-cell stimulatory capacity. In this study, we evaluated whether CD40-targeted Ad encoding the full-length melanoma antigen recognized by T cells-1 (CD40-Ad-MART-1) could be used to efficiently and selectively transduce conventional and plasmacytoid DC to prime melanoma-specific CD8(+) T-effector cells in human melanoma-draining sentinel lymph nodes (SLNs). CD40 targeting of Ad was achieved using a bispecific fusion protein, binding and neutralizing the Ad fiber knob through soluble coxsackie and adenovirus receptor while retargeting the virus to hCD40 through the tumor necrosis factor-like domain of mCD40L. Selective transduction of conventional and plasmacytoid DC subsets by CD40-Ad was observed in suspensions of human melanoma-draining SLN. Moreover, CD40-Ad-MART-1 enhanced the expansion of functional MART-1-specific CD8(+) T cells from SLN with concomitant decreases in CD4:CD8 T-cell ratios and CD4(+)CD25(hi)FoxP3(+) regulatory T-cell rates. Additional studies revealed that transduction and activation of monocyte-derived DCs with CD40-Ad-MART-1 significantly enhanced their priming efficiency of functional CD8(+) effector T cells with high avidity. These findings provide preclinical evidence of possible efficacy of this approach for cancer immunotherapy.

Pathogenesis of Herpesvirus anguillae (HVA) in juvenile European eel Anguilla anguilla after infection by bath immersion.

A clinical infection in post-larval (glass) European eels Anguilla anguilla was successfully induced after artificial bath immersion with Herpesvirus anguillae (HVA), isolated from diseased European eel. HVA caused a clinical infection after 7 d post-inoculation (pi); virus was detected by polymerase chain reaction (PCR) from Day 1 pi; virus isolation was positive from Day 7 pi, and HVA antigen was detected by immunohistochemistry in gills and stomach from Day 4 pi. Tissue changes were found by histological examination in gills and skin from Day 4 pi. In general, there was good correlation in the timing of the clinical signs, PCR, virus isolation, immunohistochemistry and histopathology results, although PCR, histopathology and immunohistochemistry were the first positive tests. HVA was first detected in skin and stomach, followed by gills, and later heart and intestine, whereas HVA was detected persistently in gills and skin. Koch's postulates were fulfilled. For diagnosis of HVA infections, clinical pathology combined with virus isolation and/or PCR are recommended.