Live Vaccinia Virus-Coated Microneedle Array Patches for Smallpox Vaccination and Stockpiling

Although smallpox has been eradicated globally, the potential use of the smallpox virus in bioterrorism indicates the importance of stockpiling smallpox vaccines. Considering the advantages of microneedle-based vaccination over conventional needle injections, in this study, we examined the feasibility of microneedle-based smallpox vaccination as an alternative approach for stockpiling smallpox vaccines. We prepared polylactic acid (PLA) microneedle array patches by micromolding and loaded a second-generation smallpox vaccine on the microneedle tips via dip coating. We evaluated the effect of excipients and drying conditions on vaccine stability in vitro and examined immune responses in female BALB/c mice by measuring neutralizing antibodies and interferon (IFN)-γ-secreting cells. Approximately 40% of the virus titer was reduced during the vaccine-coating process, with or without excipients. At −20 °C, the smallpox vaccine coated on the microneedles was stable up to 6 months. Compared to natural evaporation, vacuum drying was more efficient in improving the smallpox vaccine stability. Microneedle-based vaccination of the mice elicited neutralizing antibodies beginning 3 weeks after immunization; the levels were maintained for 12 weeks. It significantly increased IFN-γ-secreting cells 12 weeks after priming, indicating the induction of cellular immune responses. The smallpox-vaccine-coated microneedles could serve as an alternative delivery system for vaccination and stockpiling.

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Oxidative Stress Compromises Lymphocyte Function in Neonatal Dairy Calves

Antioxidants ◽

10.3390/antiox10020255 ◽

2021 ◽

Vol 10 (2) ◽

pp. 255

Author(s):

Wilmer Cuervo ◽

Lorraine M. Sordillo ◽

Angel Abuelo

Keyword(s):

Oxidative Stress ◽

Immune Responses ◽

Immune Cell ◽

Lymphocyte Activation ◽

Dairy Calves ◽

Lymphocyte Function ◽

Newborn Calves ◽

Ifn Γ ◽

The Impact

Dairy calves are unable to mount an effective immune response during their first weeks of life, which contributes to increased disease susceptibility during this period. Oxidative stress (OS) diminishes the immune cell capabilities of humans and adult cows, and dairy calves also experience OS during their first month of life. However, the impact that OS may have on neonatal calf immunity remains unexplored. Thus, we aimed to evaluate the impact of OS on newborn calf lymphocyte functions. For this, we conducted two experiments. First, we assessed the association of OS status throughout the first month of age and the circulating concentrations of the cytokines interferon-gamma (IFN-γ) and interleukin (IL) 4, as well as the expression of cytokine-encoding genes IFNG, IL2, IL4, and IL10 in peripheral mononuclear blood cells (PBMCs) of 12 calves. Subsequently, we isolated PBMCs from another 6 neonatal calves to investigate in vitro the effect of OS on immune responses in terms of activation of lymphocytes, cytokine expression, and antibody production following stimulation with phorbol 12-myristate 13-acetate or bovine herpesvirus-1. The results were compared statistically through mixed models. Calves exposed to high OS status in their first month of age showed higher concentrations of IL-4 and expression of IL4 and IL10 and lower concentrations of IFN-γ and expression of IFNG and IL2 than calves exposed to lower OS. In vitro, OS reduced lymphocyte activation, production of antibodies, and protein and gene expression of key cytokines. Collectively, our results demonstrate that OS can compromise some immune responses of newborn calves. Hence, further studies are needed to explore the mechanisms of how OS affects the different lymphocyte subsets and the potential of ameliorating OS in newborn calves as a strategy to augment the functional capacity of calf immune cells, as well as enhance calves’ resistance to infections.

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Optimized Adenovirus-Antibody Complexes Stimulate Strong Cellular and Humoral Immune Responses against an Encoded Antigen in Naïve Mice and Those with Preexisting Immunity

Clinical and Vaccine Immunology ◽

10.1128/cvi.05319-11 ◽

2011 ◽

Vol 19 (1) ◽

pp. 84-95 ◽

Cited By ~ 6

Author(s):

Jin Huk Choi ◽

Joe Dekker ◽

Stephen C. Schafer ◽

Jobby John ◽

Craig E. Whitfill ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

Immune Responses ◽

Neutralizing Antibodies ◽

T Cell Responses ◽

Transduction Efficiency ◽

Virus Antibody ◽

Cell Responses

ABSTRACTThe immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity.In vitroandin vivoassays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 × 1011adenovirus particles mixed with different concentrations of neutralizing antibodies. Complexes formed at concentrations of 500 to 0.05 times the 50% neutralizing dose (ND50) elicited strong virus- and transgene-specific T cell responses. The 0.05-ND50formulation elicited measurable anti-transgene antibodies that were similar to those of virus alone (P= 0.07). This preparation also elicited very strong transgene-specific memory T cell responses (28.6 ± 5.2% proliferation versus 7.7 ± 1.4% for virus alone). Preexisting immunity significantly reduced all responses elicited by these formulations. Although lower concentrations (0.005 and 0.0005 ND50) of antibody did not improve cellular and humoral responses in naïve animals, they did promote strong cellular (0.005 ND50) and humoral (0.0005 ND50) responses in mice with preexisting immunity. Some virus-antibody complexes may improve the potency of adenovirus-based vaccines in naïve individuals, while others can sway the immune response in those with preexisting immunity. Additional studies with these and other virus-antibody ratios may be useful to predict and model the type of immune responses generated against a transgene in those with different levels of exposure to adenovirus.

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The effect of Kefir on The Immune Response of Healthy Volunteers In Vitro

Journal of Agromedicine and Medical Sciences ◽

10.19184/ams.v3i2.5067 ◽

2017 ◽

Vol 3 (2) ◽

pp. 28

Author(s):

Desie Dwi Wisudanti

Keyword(s):

Immune Response ◽

T Cells ◽

Immune Responses ◽

Healthy Volunteers ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Fermented Milk ◽

Bioactive Components ◽

Ifn Γ

Kefir is a functional foodstuff of probiotics, made from fermented milk with kefir grains containing various types of beneficial bacteria and yeast. There have been many studies on the effects of oral kefir on the immune system, but few studies have shown the effect of bioactive components from kefir (peptides and exopolysaccharides/ kefiran), on immune responses. The purpose of this study was to prove the effect of kefir supernatant from milk goat on healthy immune volunteer response in vitro. The study was conducted on 15 healthy volunteers, then isolated PBMC from whole blood, then divided into 5 groups (K-, P1, P2, P3 and P4) before culture was done for 4 days. The harvested cells from culture were examined for the percentage of CD4+ T cells, CD8+ T cells, IFN-γ, IL-4 using flowsitometry and IL-2 levels, IL-10 using the ELISA method. The results obtained that kefir do not affect the percentage of CD4+ T cells and CD8+ T cells. The higher the concentration of kefir given, the higher levels of secreted IFN- γ and IL-4, but a decrease in IL-2 levels. Significant enhancement occurred at levels of IL-10 culture PBMC given kefir with various concentrations (p <0.01), especially at concentrations of 1%. These results also show the important effects of kefir bioactive components on immune responses. The conclusion of this study is that kefir can improve the immune response, through stimulation of IL-10 secretion in vitro.

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Immunostimulatory Effects of Recombinant Erysipelothrix rhusiopathiae Expressing Porcine Interleukin-18 in Mice and Pigs

Clinical and Vaccine Immunology ◽

10.1128/cvi.00342-12 ◽

2012 ◽

Vol 19 (9) ◽

pp. 1393-1398 ◽

Cited By ~ 2

Author(s):

Yohsuke Ogawa ◽

Yu Minagawa ◽

Fang Shi ◽

Masahiro Eguchi ◽

Yoshihiro Muneta ◽

...

Keyword(s):

Immune Responses ◽

Peritoneal Macrophages ◽

Erysipelothrix Rhusiopathiae ◽

Interleukin 18 ◽

Murine Splenocytes ◽

Content Type ◽

Immunostimulatory Effect ◽

Ifn Γ ◽

Acquired Immune Responses

ABSTRACTInterleukin-18 (IL-18), which was originally called gamma interferon (IFN-γ)-inducing factor, has been shown to play an important role in innate and acquired immune responses. In this study, attenuatedErysipelothrix rhusiopathiaestrains were engineered to produce porcine IL-18 (poIL-18) and evaluated for their potential immunostimulatory effect in animals. Recombinant poIL-18 was successfully expressed in the recombinantE. rhusiopathiaestrains YS-1/IL-18 and KO/IL-18. The culture supernatant of YS-1/IL-18 was confirmed to induce IFN-γ production in murine splenocytesin vitro, and this production was inhibited by incubation with anti-poIL-18 monoclonal antibodies. Furthermore, more IFN-γ production was induced upon stimulation of splenocytes with concanavalin A for splenocytes from mice that were intraperitoneally inoculated with YS-1/IL-18 than for splenocytes from control mice inoculated with the parent strain YS-1. Peritoneal macrophages from mice preinoculated with YS-1/IL-18 exhibited enhanced phagocytosis ofSalmonella entericasubsp.entericaserovar Typhimurium compared with peritoneal macrophages from control mice preinoculated with YS-1. We also confirmed the immunostimulatory effect on humoral immune responses against antigens ofE. rhusiopathiaeandMycoplasma hyopneumoniaein gnotobiotic pigs that were orally preinoculated with KO/IL-18. Thus, these results provide evidence thatE. rhusiopathiaeis a promising vector for the expression of host cytokines and suggest the potential utility ofE. rhusiopathiaevector-encoded cytokines in the activation of host innate and acquired immune responses.

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Simultaneous Administration of Recombinant Measles Viruses Expressing Respiratory Syncytial Virus Fusion (F) and Nucleo (N) Proteins Induced Humoral and Cellular Immune Responses in Cotton Rats

Vaccines ◽

10.3390/vaccines7010027 ◽

2019 ◽

Vol 7 (1) ◽

pp. 27 ◽

Cited By ~ 2

Author(s):

Yoshiaki Yamaji ◽

Akihito Sawada ◽

Yosuke Yasui ◽

Takashi Ito ◽

Tetsuo Nakayama

Keyword(s):

Respiratory Syncytial Virus ◽

Immune Responses ◽

Neutralizing Antibodies ◽

Simultaneous Administration ◽

Cellular Immune Responses ◽

Immunization Group ◽

Cotton Rats ◽

Ifn Γ ◽

Syncytial Virus ◽

Cellular Immune

We previously reported that recombinant measles virus expressing the respiratory syncytial virus (RSV) fusion protein (F), MVAIK/RSV/F, induced neutralizing antibodies against RSV, and those expressing RSV-NP (MVAIK/RSV/NP) and M2-1 (MVAIK/RSV/M2-1) induced RSV-specific CD8+/IFN-γ+ cells, but not neutralizing antibodies. In the present study, MVAIK/RSV/F and MVAIK/RSV/NP were simultaneously administered to cotton rats and immune responses and protective effects were compared with MVAIK/RSV/F alone. Sufficient neutralizing antibodies against RSV and RSV-specific CD8+/IFN-γ+ cells were observed after re-immunization with simultaneous administration. After the RSV challenge, CD8+/IFN-γ+ increased in spleen cells obtained from the simultaneous immunization group in response to F and NP peptides. Higher numbers of CD8+/IFN-γ+ and CD4+/IFN-γ+ cells were detected in lung tissues from the simultaneous immunization group after the RSV challenge. No detectable RSV was recovered from lung homogenates in the immunized groups. Mild inflammatory reactions with the thickening of broncho-epithelial cells and the infiltration of inflammatory cells were observed in lung tissues obtained from cotton rats immunized with MVAIK/RSV/F alone after the RSV challenge. No inflammatory responses were observed after the RSV challenge in the simultaneous immunization groups. The present results indicate that combined administration with MVAIK/RSV/F and MVAIK/RSV/NP induces humoral and cellular immune responses and shows effective protection against RSV, suggesting the importance of cellular immunity.

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MVA Vectored Vaccines Encoding Rift Valley Fever Virus Glycoproteins Protect Mice against Lethal Challenge in the Absence of Neutralizing Antibody Responses

Vaccines ◽

10.3390/vaccines8010082 ◽

2020 ◽

Vol 8 (1) ◽

pp. 82 ◽

Cited By ~ 2

Author(s):

Elena López-Gil ◽

Sandra Moreno ◽

Javier Ortego ◽

Belén Borrego ◽

Gema Lorenzo ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Cellular Immunity ◽

Rift Valley ◽

Neutralizing Antibodies ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Fever Virus ◽

Valley Fever

In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be achieved by the activation of cellular responses mainly directed against Gc epitopes. The involvement of cellular immunity was stressed by the fact that protection of mice was strain dependent. Furthermore, our data suggest that the rMVA based single dose vaccination elicits suboptimal humoral immune responses against Gn antigen since disease in mice was exacerbated upon virus challenge in the presence of rMVAGnGc or rMVAGn immune serum. Thus, Gc-specific cellular immunity could be an important component in the protection after the challenge observed in BALB/c mice, contributing to the elimination of infected cells reducing morbidity and mortality and counteracting the deleterious effect of a subneutralizing antibody immune response.

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Inflammation restraining effects of prostaglandin E2 on natural killer–dendritic cell (NK-DC) interaction are imprinted during DC maturation

Blood ◽

10.1182/blood-2010-09-307835 ◽

2011 ◽

Vol 118 (9) ◽

pp. 2473-2482 ◽

Cited By ~ 36

Author(s):

Catharina H. M. J. Van Elssen ◽

Joris Vanderlocht ◽

Tammy Oth ◽

Birgit L. M. G. Senden-Gijsbers ◽

Wilfred T. V. Germeraad ◽

...

Keyword(s):

Dendritic Cell ◽

Immune Responses ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

T Helper 1 ◽

Ifn Γ ◽

Dc Maturation

Abstract Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer–dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood–derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44+ NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.

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dSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.

Blood ◽

10.1182/blood.v108.11.1727.1727 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1727-1727

Author(s):

Manuel Schmidt ◽

Javier de Cristobal ◽

Astrid Sander ◽

Bernadette Brzezicha ◽

Sven A. König Merediz ◽

...

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Neutralizing Antibodies ◽

De Novo ◽

Tumor Cell Line ◽

Control Group ◽

Γδt Cells ◽

Ifn Γ

Abstract Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis of the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.

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Phagocytosis May Be a Regulatory Mechanism for Myeloid Dendritic Cells to Terminate Type 1 CD8+ T Cells.

Blood ◽

10.1182/blood.v114.22.1654.1654 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1654-1654

Author(s):

Young-June Kim ◽

Hal E. Broxmeyer

Keyword(s):

T Cells ◽

Immune Responses ◽

Cd8 T Cells ◽

Phagocytic Activity ◽

Regulatory Mechanism ◽

Gm Csf ◽

T Cell Immune Responses ◽

Ifn Γ

Abstract Abstract 1654 Poster Board I-680 CD8+ cytotoxic T cells are often ‘exhausted’ by programmed death-1 (PD-1) signaling, and subsequently the functions of these cells are terminated especially in a tumor environment or upon chronic HIV or HCV infection. Subsets of myeloid cells referred to as myeloid derived suppressor cells (MDSC) or regulatory dendritic cells (DCs) have been implicated in inducing exhaustion or termination of effector CD8+ T cells. To this end, we developed various myeloid-derived dendritic cell (DC) types in vitro from human CD14+ monocytes using M-CSF or GM-CSF in the presence of IL-4 with/without other cytokines, and characterized these DCs with respect to their capacity to induce PD-1 expression on and exhaustion of CD8+ T cells. We then assessed their impact on longevity of CD8+ T cells following coculture. Myeloid DCs developed in vitro with M-CSF and IL-4 for 5 days (referred to as M-DC) did not express ligand for PD-1 (PD-L1) nor did they induce PD-1 on CD8+ T cells. Thus, using M-DCs as starting cells, we sought determinant factors that could modulate M-DCs to express PD-L1 and thereby induce exhaustion of CD8+ T cells. In order to better monitor exhaustion processes, we incubated human peripheral CD8+ T cells for 5 days in the presence of IL-15, an important cytokine for maintaining viability, before coculture. M-DCs showed little impact on exhaustion or longevity of the CD8+ T cells. IL-10 converted M-DC into a distinct myeloid DC subset (referred to as M-DC/IL-10) with an ability to express PD-L1 as well as to induce PD-1 on cocultured CD8+ T cells. M-DC/IL-10 cells markedly suppressed proliferation of cocultured CD8+ T cells. M-DC/IL-10 cells were morphologically unique with many granules and filamentous structures around the cell periphery. These IL-10 effects on M-DC were completely abrogated in the presence of TNF-á. M-DC/IL-10 cells could be further differentiated into another myeloid DC subset in the presence of IFN-γ (referred to as M-DC/IL-10/IFN-γ) with an ability to express even higher levels of PD-L1 compared to M-DC/IL-10 cells. The most remarkable effect of M-DC/IL-10/IFN-γ cells on cocultured CD8+ T cells was a dramatic loss of CD8+ T cells. Light and confocal microscopic observations indicated that loss of CD8+ T cells was due to phagocytosis by M-DC/IL-10/IFN-γ cells. As IFN-γ, a type 1 cytokine which is induced in CD8+ T cells by IL-12 is essential for phagocytosis, we tested whether IL-12 treatment of CD8+ T cells could further enhance phagocytosis induced by M-DC/IL-10/IFN-γ cells. Indeed, IL-12 treatment greatly increased numbers of phagocytosed CD8+ T cells. In contrast, IL-4 treated CD8+ T cells became resistant to phagocytosis, suggesting IFN-γ producing (type1) CD8+ T cells may be primary target cells for the M-DC/IL-10 cells mediated phagocytosis. CD4+ T cells were not as susceptible as CD8+ T cells to phagocytosis. We failed to detect such phagocytic activity induced by prototype DCs generated with GM-CSF and IL-4. Phagocytic activity was not inhibited by various arginase-1 inhibitors suggesting that nitric oxide signaling may not mediate phagocytic activity. Neutralizing antibody to PD-L1 slightly but significantly lowered phagocytic activity suggesting that PD-L1/PD-1 interaction may be partially involved in this process. Myeloid DCs are thought to be immunogenic, actively inducing T cell immune responses. Our results demonstrate that myeloid DCs may play suppressive roles as well through induction of phagocytic activity, especially against IFN-γ producing CD8+ T cells. This may serve as a regulatory mechanism for type 1 CD8+ T cell immune responses in an IL-10 enriched microenvironment. Disclosures No relevant conflicts of interest to declare.

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Nanoparticle Encapsulated L-Asparaginase

Blood ◽

10.1182/blood.v122.21.2669.2669 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2669-2669

Author(s):

Inanc Ortac ◽

Laura Ruff ◽

Yasan Yeh ◽

Sadik Esener ◽

Bradley T Messmer

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Repeated Administration ◽

Therapeutic Modality ◽

Silica Layer ◽

Amino Acid Depletion ◽

Extended Circulation

Abstract Amino acid depletion with catabolizing enzymes is a potential therapeutic modality for many cancer types. L-asparaginase has been used to treat Acute Lymphoblastic Leukemia (ALL) for many decades and is part of treatment regimen that has a roughly 80% cure rate. The current frontline formulation is L-asparaginase from E.coli that has been conjugated to polyethylene glycol (PEG) for extended circulation half life and protection from immune responses. However, neutralizing antibodies still occur in a subset of patients, necessitating a switch to a different L-asparaginase, such as the less active enzyme from Erwinia chrysanthemi. This complicates therapy, risks severe allergic reactions, and if antibodies develop to the second enzyme the options become limited. The threat of immune reactions, as well as other side effects, has largely restricted L-asparaginase therapy to ALL, despite in vitro evidence that many other cancers might be sensitive to asparagine depletion. Our group has developed a novel nanoparticle enzyme encapsulation technology that overcomes the limitation of immune responses to enzymes or PEG. These particles, termed Synthetic Hollow Enzyme Loaded Porous nanoShells (SHELS), are produced in a two-step process where the first creates silica shells with large pores through which the enzyme can be loaded, and a second sealing step that encapsulates the mesoporous shell and the loaded enzyme cargo with a nanoporous silica layer. The work below was done with 200nm particles that have a total shell wall thickness of approximately 10nm. SHELS were loaded with L-asparaginase (Elspar). No loss of activity was seen when enzyme activity was measured in vitro. Groups of mice were injected IM with 5 IU of either free or SHELS encapsulated L-asparaginase. In each case, half of the mice were passively immunized with rabbit polyclonal anti-asparaginase antibodies to a blood concentration of ∼0.5 mg/ml. Serum asparaginase levels pre and post injection were measured by HPLC at 2, 5 and 8 days. Both free and encapsulated enzyme produced complete depletion of asparagine at day 2, but by day 5 asparagine levels had returned to baseline in the animals given free enzyme, whereas those given encapsulated enzyme maintained depletion at day 5 with restoration to baseline at day 8. There was no asparagine depletion in immunized animals with the free L-asparaginase, but SHELS encapsulated asparaginase produced an identical depletion both in the presence or absence of the neutralizing antibodies, indicating that the SHELS effectively shield the enzyme from the antibodies. Experiments in tumor models are ongoing as well as dose response and repeated administration studies. Disclosures: No relevant conflicts of interest to declare.

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