Human induced pluripotent stem cell-derived macrophages ameliorate liver fibrosis.

With an increasing number of patients with degenerative hepatic diseases, such as liver fibrosis, and a limited supply of donor organs, there is an unmet need for therapies that can repair or regenerate damaged liver tissue. Treatment with macrophages that are capable of phagocytosis and anti-inflammatory activities such as secretion of matrix metalloproteinases (MMPs) provide an attractive cellular therapy approach. Human induced pluripotent stem cells (iPSCs) are capable of efficiently generating a large-scale, homogenous population of human macrophages using fully defined feeder- and serum-free differentiation protocol. Human iPSC-macrophages exhibit classical surface cell markers and phagocytic activity similar to peripheral blood-derived macrophages. Moreover, gene and cytokine expression analysis reveal that these macrophages can be efficiently polarized to pro-inflammatory M1 or anti-inflammatory M2 phenotypes in presence of LPS + IFN-γ and IL-4 + IL-13, respectively. M1 macrophages express high level of CD80, TNF-α, and IL-6 while M2 macrophages show elevated expression of CD206, CCL17, and CCL22. Here, we demonstrate that treatment of liver fibrosis with both human iPSC-derived macrophage populations and especially M2 subtype significantly reduces fibrogenic gene expression and disease associated histological markers including Sirius Red, αSMA and desmin in immunodeficient Rag2-/- γc-/- mice model, making this approach a promising cell-based avenue to ameliorate fibrosis.

Development of innate immune cells from human pluripotent stem cells.

Mouse and human pluripotent stem cells have been widely used to study the development of the hematopoietic and immune systems. Although not all cells can be derived with the same efficiency, immune cells such as natural killer (NK) cells and macrophages can be easily produced from PSCs to enable development of new cell-based therapies. NK cells and macrophages are part of the innate immune system, the first line of defense against malignancies and infectious disease. Human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived NK cells can be produced at a clinical scale suitable for translation into clinical trials. Additionally, PSCs can be genetically modified to produce hESC/iPSC-derived human NK cells with enhanced antitumor activity. These engineered NK cells can express a stabilized version of the high-affinity Fc receptor CD16, chimeric antigen receptors, or other strategies to enable more potent and targeted cellular immunotherapies. Moreover, macrophages can also be routinely and efficiently produced from hESCs and iPSCs as a tool to expand our knowledge of the basic biology of these cells. hESC- and iPSC-derived macrophages can also be employed as a novel approach for cancer immunotherapy, as well as a strategy to repair or regenerate diseased and damaged tissues and organs.

Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses.

The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs.IMPORTANCE Infections by a large number of human papillomaviruses lead to malignant and nonmalignant disease. Current commercial vaccines based on virus-like particles (VLPs) effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so-called neutralization epitopes within its N terminus. We designed polytopes comprising the L2 epitope amino acids 20 to 38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile archaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising a heptamerized L2 8-mer thioredoxin single-peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.

Broadly neutralizing antiviral responses induced by a single-molecule HPV vaccine based on thermostable thioredoxin-L2 multiepitope nanoparticles.

Vaccines targeting the human papillomavirus (HPV) minor capsid protein L2 are emerging as chemico-physically robust and broadly protective alternatives to the current HPV (L1-VLP) vaccines. We have previously developed a trivalent L2 vaccine prototype exploiting Pyrococcus furiosus thioredoxin (PfTrx) as a thermostable scaffold for the separate presentation of three distinct HPV L2(20-38) epitopes. With the aim of achieving a highly immunogenic, yet simpler and more GMP-production affordable formulation, we report here on a novel thermostable nanoparticle vaccine relying on genetic fusion of PfTrx-L2 with the heptamerizing coiled-coil polypeptide OVX313. A prototype HPV16 monoepitope version of this nanoparticle vaccine (PfTrx-L2-OVX313; median radius: 8.6 ± 1.0 nm) proved to be approximately 10-fold more immunogenic and with a strikingly enhanced cross-neutralization capacity compared to its monomeric counterpart. Vaccine-induced (cross-)neutralizing responses were further potentiated in a multiepitope derivative displaying eight different L2(20-38) epitopes, which elicited neutralizing antibodies against 10 different HPVs including three viral types not represented in the vaccine. Considering the prospective safety of the PfTrx scaffold and of the OVX313 heptamerization module, PfTrx-OVX313 nanoparticles lend themselves as robust L2-based immunogens with a high translational potential as a 3rd generation HPV vaccine, but also as a novel and extremely versatile peptide-antigen presentation platform.

Human papillomavirus first and second generation vaccines-current status and future directions.

It has been more than 10 years that the first prophylactic papillomavirus vaccine became available, although distribution has been mainly limited to the more affluent countries. The first two vaccines have been a great success, hundreds of millions of women and a much smaller number of men have been vaccinated ever since. In a few countries with high vaccination coverage, in particular Australia but also parts of Great Britain and others, clinical impact of vaccination programs is already visible and there are indications for herd immunity as well. Vaccine efficacy is higher than originally estimated and the vaccines have an excellent safety profile. Gardasil9 is a second generation HPV virus-like particle vaccine that was licensed in 2015 and there are more to come in the near future. Currently, burning questions in respect to HPV vaccination are the duration of protection - especially in regard to cross-protection - reduction of the three-dose regimen and its impact on cross-protection; and duration of response, as well as protection against oropharyngeal HPV infections. Furthermore, researchers are seeking to overcome limitations of the VLP vaccines, namely low thermal stability, cost, invasive administration, limited coverage of non-vaccine HPV types, and lack of therapeutic efficacy. In this review we summarize the current status of licensed VLP vaccines and address questions related to second and third generation HPV vaccines.

Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris.

Internal grafting of designed peptides to scaffold proteins is a valuable strategy for a variety of applications including recombinant peptide antigen construction. A peptide epitope from human papillomavirus (HPV) minor capsid protein L2 displayed on thioredoxin (Trx) has been validated preclinically as a broadly protective and low-cost alternative HPV vaccine. Focusing on thioredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus (PfTrx) as a scaffold, we have constructed a modified Pichia pastoris expression vector and used a PfTrx fusion derivative containing three tandemly repeated copies of a 19 amino acids peptide epitope from HPV-L2 for expression optimization and biochemical-immunological characterization of the Pichia-produced PfTrx-L2 antigen. We show that PfTrx-L2 is produced at high levels (up to 100 mg from a 100 ml starting culture using a multi-cycle induction protocol) and secreted into the culture medium as a highly enriched (>70% pure), non-glycosylated polypeptide that can be purified to homogeneity in a single step. Oxidation and aggregation state, thermal stability and immunogenicity of the endotoxin-free PfTrx-L2 antigen produced in P. pastoris were tested and found to be identical to those of the same antigen produced in Escherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastoris represents a cost- and time-effective alternative to E. coli production. Specifically designed for peptide antigens, the PfTrx-expression vector and conditions described herein are easily transferable to a variety of applications centred on the use of structurally constrained bioactive peptides as immune as well as target-specific binder reagents.

Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.

Immunization with M2e-displaying T7 bacteriophage nanoparticles protects against influenza A virus challenge.

Considering the emergence of highly pathogenic influenza viruses and threat of worldwide pandemics, there is an urgent need to develop broadly-protective influenza vaccines. In this study, we demonstrate the potential of T7 bacteriophage-based nanoparticles with genetically fused ectodomain of influenza A virus M2 protein (T7-M2e) as a candidate universal flu vaccine. Immunization of mice with non-adjuvanted T7-M2e elicited M2e-specific serum antibody responses that were similar in magnitude to those elicited by M2e peptide administered in Freund's adjuvant. Comparable IgG responses directed against T7 phage capsomers were induced following vaccination with wild type T7 or T7-M2e. T7-M2e immunization induced balanced amounts of IgG(1) and IgG(2a) antibodies and these antibodies specifically recognized native M2 on the surface of influenza A virus-infected mammalian cells. The frequency of IFN-γ-secreting T cells induced by T7-M2e nanoparticles was comparable to those elicited by M2e peptide emulsified in Freund's adjuvant. Emulsification of T7-M2e nanoparticles in Freund's adjuvant, however, induced a significantly stronger T cell response. Furthermore, T7-M2e-immunized mice were protected against lethal challenge with an H1N1 or an H3N2 virus, implying the induction of hetero-subtypic immunity in our mouse model. T7-M2e-immunized mice displayed considerable weight loss and had significantly reduced viral load in their lungs compared to controls. We conclude that display of M2e on the surface of T7 phage nanoparticles offers an efficient and economical opportunity to induce cross-protective M2e-based immunity against influenza A.

Effect of LIGHT adjuvant on kinetics of T-cell responses induced by HSV-1 DNA immunization.

BACKGROUND: Studies on efficacy of various vaccines that prevent or reduce the primary and recurrent HSV-1 infection have demonstrated the importance of cellular immunity for protection against the infection. We previously used DNA vaccination to induce cellular immunity against HSV-1 infection in mice. OBJECTIVE: The aim of our study was to evaluate the effect of LIGHT; a member of TNF super family, on the kinetic of CTL response induced by HSV-1 glycoprotein B based DNA vaccine. METHODS: Using a granzyme B ELISA for detection and analysis of CD8+ T cells, CTL activity was determined in the spleen of BALB/c mice at various time points after primary and booster dose of vaccination. The kinetics of CTL response to primary and secondary HSV-1 infection and DNA vaccination were compared to those induced by DNA vaccination in combination with LIGHT adjuvant in the present study. RESULTS: In primary and secondary immunization, the CTL activity in the HSV injected group peaked 7 days and 12 hours post immunization, respectively. After 5 days, LIGHT could neither accelerate the CTL response compared to DNA vaccination alone nor could enhance the CTL activity in the primary and the first peak of memory response, the amount of granzyme B induced by the LIGHT containing vaccine was significantly higher than that induced by the vaccine without the adjuvant. CONCLUSION: Although LIGHT enhances the cellular response in the booster dose of vaccination, it does not accelerate the CTL response.

Impact of timing strategy of LIGHT, a new TNF superfamily on immune platform induced by HSV-1 gB DNA vaccine.

Although the role of various cytokines on stimulating the immune responses is characterized well, the importance of LIGHT, a member of TNF superfamily, is less clear. In the current study, we administrated LIGHT expression plasmid as an adjuvant to HSV-1 gB DNA vaccine. HSV-1 gB DNA can elicit vigorous humoral and cell mediated immunity in BALB/c mice. LIGHT could potentiate the proliferation of T lymphocytes and induction of T CD8(+) cells performing by measuring Granzyme B, a specific marker of CMI immunity and virus neutralization antibody titer. In this study, timing effect of cytokine administration on the resultant immune pattern was evaluated in three different timing groups. The group received LIGHT 3 days before DNA vaccine, demonstrated significant increase in cell mediated immunity. So, utilization of an adjuvant to DNA vaccine can significantly influences the induced immune response consequently and this phenomenon could be important to obtain the optimal response in DNA vaccine strategy. Given the growing use of plasmid-based immune adjuvants to improve the immunogenicity and efficacy of DNA vaccines, these findings support the need for further detailed study of this class of agent.

Evaluation of humoral and cellular immune responses against HSV-1 using genetic immunization by filamentous phage particles: a comparative approach to conventional DNA vaccine.

Phage display is based on expressing peptides as a fusion to one of the phage coat proteins. To date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. In recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as DNA delivery vehicles to mammalian cells. In this study, recombinant filamentous phage whole particles were used for vaccination of mice. BALB/c mice were inoculated with filamentous phage particles containing expression cassette of Herpes simplex virus 1 (HSV-1) glycoprotein D that has essential roles in the virus attachment and entry. Both humoral and cellular immune responses were measured in the immunized mice and compared to conventional DNA vaccination. A dose-response relationship was observed in both arms of immune responses induced by recombinant filamentous phage inoculation. The results were similar to those from DNA vaccination. Filamentous phages can be considered as suitable alternative candidate vaccines because of easier and more cost-effective production and purification over plasmid DNA or bacteriophage lambda particles.