Army Liposome Formulation (ALF) family of vaccine adjuvants.

Introduction: From its earliest days, the US. military has embraced the use of vaccines to fight infectious diseases. The Army Liposome Formulation (ALF) has been a pivotal innovation as a vaccine adjuvant that provides excellent safety and potency and could lead to dual-use military and civilian benefits. For protection of personnel against difficult disease threats found in many areas of the world, Army vaccine scientists have created novel liposome-based vaccine adjuvants.Areas covered: ALF consists of liposomes containing saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA) as an immunostimulant. ALF exhibited safety and strong potency in many vaccine clinical trials. Improvements based on ALF include: ALF adsorbed to aluminum hydroxide (ALFA); ALF containing QS21 saponin (ALFQ); and ALFQ adsorbed to aluminum hydroxide (ALFQA). Preclinical safety and efficacy studies with ALF, LFA, ALFQ, and ALFQA are discussed in preparation for upcoming vaccine trials targeting malaria, HIV-1, bacterial diarrhea, and opioid addiction.Expert opinion: The introduction of ALF in the 1980s stimulated commercial interest in vaccines to infectious diseases, and therapeutic vaccines to cancer, and Alzheimer's disease. It is likely that ALF, ALFA, and ALFQ, will provide momentum for new types of modern vaccines with improved efficacy and safety.

Isolation of human lymphocytes with high yield and viability from the gastrointestinal and female reproductive tract of a humanized DRAG mouse.

The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets. Therefore, human-immune-system humanized mice are surrogate models to investigate the human mucosal immune cell responses during the course of the disease. The current protocols used to harvest the immune cells from the mucosal tissues, however, result in low recovery of cells with poor viability. We have established a novel protocol, which results in a high yield of human lymphocytes with high viability to overcome this issue. The immune cells obtained from a single DRAG mouse by our protocol were sufficient for conducting functional assays and for flow cytometry analyses including phenotypic, exhaustion, and functional panels.

Tracking Human Immunodeficiency Virus-1 Infection in the Humanized DRAG Mouse Model.

Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP) models for studying human immunodeficiency virus (HIV)-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several different types of humanized mice have been developed with varying levels of reconstitution of human CD45+ cells. In this study, we utilized humanized Rag1KO.IL2RγcKO.NOD mice expressing HLA class II (DR4) molecule (DRAG mice) infused with HLA-matched hematopoietic stem cells from umbilical cord blood to study early events after HIV-1 infection, since the mucosal tissues of these mice are highly enriched for human lymphocytes and express the receptors and coreceptors needed for HIV-1 entry. We examined the various tissues on days 4, 7, 14, and 21 after an intravaginal administration of a single dose of purified primary HIV-1. Plasma HIV-1 RNA was detected as early as day 7, with 100% of the animals becoming plasma RNA positive by day 21 post-infection. Single cells were isolated from lymph nodes, bone marrow, spleen, gut, female reproductive tissue, and brain and analyzed for gag RNA and strong stop DNA by quantitative (RT)-PCR. Our data demonstrated the presence of HIV-1 viral RNA and DNA in all of the tissues examined and that the virus was replication competent and spread rapidly. Bone marrow, gut, and lymph nodes were viral RNA positive by day 4 post-infection, while other tissues and plasma became positive typically between 7 and 14 days post-infection. Interestingly, the brain was the last tissue to become HIV-1 viral RNA and DNA positive by day 21 post-infection. These data support the notion that humanized DRAG mice could serve as an excellent model for studying the trafficking of HIV-1 to the various tissues, identification of cells harboring the virus, and thus could serve as a model system for HIV-1 pathogenesis and reservoir studies.

Effect of cytokines on Siglec-1 and HIV-1 entry in monocyte-derived macrophages: the importance of HIV-1 envelope V1V2 region.

Monocytes and monocyte-derived macrophages express relatively low levels of CD4. Despite this, macrophages can be effectively infected with human immunodeficiency virus type 1. Macrophages have a critical role in human immunodeficiency virus type 1 transmission; however, the mechanism or mechanisms of virus infection are poorly understood. We report that growth factors, such as granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor affect the phenotypic profile and permissiveness of macrophages to human immunodeficiency virus type 1. Human immunodeficiency virus type 1 infection of monocyte-derived macrophages derived from granulocyte macrophage and macrophage colony-stimulating factors was predominantly facilitated by the sialic acid-binding immunoglobulin-like lectin-1. The number of sialic acid-binding immunoglobulin-like lectin receptors on macrophage colony-stimulating factor-derived monocyte-derived macrophages was significantly greater than on granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages, and correspondingly, human immunodeficiency virus type 1 infection was greater in the macrophage colony-stimulating factor-derived monocyte-derived macrophages. Single-genome analysis and quantitative reverse transcriptase-polymerase chain reaction revealed that the differences in infectivity was not due to differences in viral fitness or in viral variants with differential infectivity but was due to reduced viral entry into the granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages. Anti-sialic acid-binding immunoglobulin-like lectin, trimeric glycoprotein 145, and scaffolded V1V2 proteins were bound to sialic acid-binding immunoglobulin-like lectin and significantly reduced human immunodeficiency virus type 1 entry and infection. Furthermore, sialic acid residues present in the V1V2 region of the envelope protein mediated human immunodeficiency virus type 1 interaction with sialic acid-binding immunoglobulin-like lectin and entry into macrophage colony-stimulating factor-derived monocyte-derived macrophages. Removal of sialic acid residues or glycans from scaffolded V1V2 protein decreased human immunodeficiency virus type 1 infectivity. These results highlight the importance of sialic acids on the V1V2 region in binding to sialic acid-binding immunoglobulin-like lectin and suggest that the unusually long surface-exposed sialic acid-binding immunoglobulin-like lectin might aid in the capture and entry of human immunodeficiency virus type 1 into monocyte-derived macrophages.

Vaccine induction of antibodies against a structurally heterogeneous site of immune pressure within HIV-1 envelope protein variable regions 1 and 2.

The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, which correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1-V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field-isolate HIV-1-infected CD4(+) T cells. Crystal structures of two of the V2 antibodies demonstrated that residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the ß strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.

Designing a soluble near full-length HIV-1 gp41 trimer.

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.

An anti-phosphoinositide-specific monoclonal antibody that neutralizes HIV-1 infection of human monocyte-derived macrophages.

HIV-1 entry into cells requires the interaction of both HIV-1 envelope proteins and membrane lipids. We investigated the mechanism of neutralization of HIV-1 infection of primary monocyte-derived macrophages (MDM) by a murine monoclonal antibody (mAb) WR321. WR321 specifically binds phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. These phosphoinositides are present not only on the inner surface of the plasma membranes of cells but also on the surface of virions. HIV-1 acquires these lipids during the budding process. Pre-incubation of WR321 with the virus but not with MDM neutralized HIV-1 infection of MDM. Our results demonstrate that WR321 was internalized only when it was bound to HIV-1. WR321 did not prevent the entry of HIV-1 into MDM. However, once WR321 was internalized along with HIV-1 the mAb acted intracellulary to prevent the release of virions from MDM and also triggered the release of ß-chemokines.

Liposome-encapsulated HIV-1 Gag p24 containing lipid A induces effector CD4+ T-cells, memory CD8+ T-cells, and pro-inflammatory cytokines.

Liposomal lipid A is an effective adjuvant for the delivery of antigens and for the induction of both cellular and humoral immunity. In this study, we demonstrate that following the third immunization with HIV-1 Gag p24 encapsulated in liposomes containing lipid A [L(p24+LA)], central memory CD8+ T-cells were localized in the spleen and lymph nodes of mice while effector memory CD8+ T-cells and effector CD4+ T-cells were found in the PBMC. Effector CD4+ T-cells were also detected in the spleen and lymph nodes. The predominant cytokine secreted from splenic lymphocytes and lymph nodes was IFN-gamma. In contrast, IL-6 and IL-10 were the major cytokines produced from PBMC. The peptide stimulation indicated that the cytokine responses observed were T-cell specific. The results demonstrate the importance of the adjuvant liposomal lipid A for the induction of HIV-1 Gag p24 -specific CD8+ T-cells, effector CD4+ T-cells, and cytokines with a Th-1 type profile after immunization with L(p24+LA).

Human immunodeficiency virus type 1 Gag p24 alters the composition of immunoproteasomes and affects antigen presentation.

Proteasomes are the major source of proteases responsible for the generation of peptides bound to major histocompatibility complex class I molecules. Antigens, adjuvants, and cytokines can modulate the composition and enzymatic activity of proteasomes and thus alter the epitopes generated. In the present study, we examined the effect of human immunodeficiency virus type 1 (HIV-1) p24 on proteasomes from a dendritic cell line (JAWS II), from a macrophage cell line (C2.3), and from murine primary bone marrow-derived macrophages and dendritic cells. HIV-1 p24 downregulated PA28beta and the beta2i subunit of the immunoproteasome complex in JAWS II cells but did not decrease the immunoproteasome subunits in macrophages, whereas in primary dendritic cells, PA28alpha, beta2i, and beta5i were downregulated. Exposure of JAWS II cells and primary dendritic cells to HIV-1 p24 for 90 min significantly decreased the presentation of ovalbumin to a SIINFEKL-specific CD8(+) T-cell hybridoma. The decrease in antigen presentation and the downmodulation of the immunoproteasome subunits in JAWS II cells and primary dendritic cells could be overcome by pretreating the cells with gamma interferon for 6 h or by exposing the cells to HIV-1 p24 encapsulated in liposomes containing lipid A. These results suggest that early antigen processing kinetics could influence the immunogenicity of CD8(+) T-cell epitopes generated.

Dengue virus infection promotes translocation of high mobility group box 1 protein from the nucleus to the cytosol in dendritic cells, upregulates cytokine production and modulates virus replication.

High mobility group box 1 (HMGB1) protein functions in regulation of transcription, cellular activation and pro-inflammatory responses. However, the potential role of HMGB1 during viral infection has not been investigated. This study attempted to elucidate whether the HMGB1-mediated inflammatory response contributes to the pathogenesis of dengue virus (DENV) infection. Our data showed that HMGB1 was released at low DENV infection levels (m.o.i. of 1) under non-necrotic conditions by human dendritic cells (DCs). When DENV-infected DCs were co-cultured with autologous T cells, there was increased production of HMGB1 by both cell types. HMGB1 regulated tumour necrosis factor alpha, interleukin (IL)-6, IL-8 and alpha interferon secretion in DENV-infected DCs. Additionally, increased HMGB1 production was associated with reduced DENV replication titres in DCs. These results suggest that HMGB1 production influences DENV infection in susceptible hosts.

Regulation of arachidonate remodeling enzymes impacts eosinophil survival during allergic asthma.

Although the role of arachidonic acid (AA) metabolism to eicosanoids has been well established in allergy and asthma, recent studies in neoplastic cells have revealed that AA remodeling through phospholipids impacts cell survival. This study tests the hypothesis that regulation of AA/phospholipid-remodeling enzymes, cytosolic phospholipase A(2) alpha(cPLA(2)-alpha, gIValphaPLA(2)) and CoA-independent transacylase (CoA-IT), provides a mechanism for altered eosinophil survival during allergic asthma. In vitro incubation of human eosinophils (from donors without asthma) with IL-5 markedly increased cell survival, induced gIValphaPLA(2) phosphorylation, and increased both gIValphaPLA(2) and CoA-IT activity. Furthermore, treatment of eosinophils with nonselective (ET18-O-CH(3)) and selective (SK&F 98625) inhibitors of CoA-IT triggered apoptosis, measured by changes in morphology, membrane phosphatidylserine exposure, and caspase activation, completely reversing IL-5-induced eosinophil survival. To determine if similar activation occurs in vivo, human blood eosinophils were isolated from either normal individuals at baseline or from subjects with mild asthma, at both baseline and 24 hours after inhaled allergen challenge. Allergen challenge of subjects with allergic asthma induced a marked increase in cPLA(2) phosphorylation, augmented gIValphaPLA(2) activity, and increased CoA-IT activity. These findings indicate that both in vitro and in vivo challenge of eosinophils activated gIValphaPLA(2) and CoA-IT, which may play a key role in enhanced eosinophil survival.

The differential effect of dexamethasone on granulocyte apoptosis involves stabilization of Mcl-1L in neutrophils but not in eosinophils.

In the absence of activation signals, circulating human neutrophils and eosinophils undergo spontaneous apoptosis. The glucocorticoid dexamethasone (Dex) accelerates apoptosis in inflammatory cells such as eosinophils, but uniquely delays neutrophil apoptosis. Corresponding to the opposite effects of Dex on granulocyte apoptosis, we demonstrate that in neutrophils and eosinophils Dex oppositely affects expression of the anti-apoptotic Bcl-2 family protein Mcl-1L. Mcl-1L expression declines over time in vitro; however, Dex maintains Mcl-1L expression in neutrophils. In contrast, Dex accelerates Mcl-1L protein loss in eosinophils. Neither Mcl-1S, a pro-apoptotic splice variant, nor Bax were affected. Dex treatment in the presence of a translation inhibitor stabilized existing Mcl-1L protein in neutrophils, while Mcl-1L stability in eosinophils was unaffected. Accordingly, delay of neutrophil apoptosis by Dex was prevented by antisense Mcl-1L siRNA. Our findings suggest that regulation of Mcl-1L degradation plays an important role in the opposite effects of Dex on granulocyte apoptosis.

Human dendritic cells and macrophages exhibit different intracellular processing pathways for soluble and liposome-encapsulated antigens.

The intracellular fates of soluble and liposomal antigens in human macrophages and dendritic cells are not well defined. Previous studies using murine macrophages have demonstrated that liposomal antigens can enter the MHC class I pathway. The Golgi complex is a major organelle in this pathway. Phagocytosis of the antigens is followed by translocation of antigen-derived peptides to the trans-Golgi where they can complex with MHC class I molecules. In contrast, soluble antigens are normally processed through the MHC class II pathway. Therefore, in the present study, ovalbumin and a synthetic Ebola peptide were used either in a soluble form or encapsulated in liposomes to investigate the intracellular trafficking and localization of these antigens to the Golgi complex in human macrophages and dendritic cells. While liposome-encapsulated antigens were transported to the trans-Golgi region in 59-78% of macrophages, soluble antigens remained diffuse throughout the cytoplasm with only 3-11% of the macrophages exhibiting trans-Golgi localization. The majority of dendritic cells localized both soluble (Ebola, 75%; ovalbumin, 84%) and liposomal antigens (58% and 65%), and irradiated Ebola virus to the trans-Golgi. These studies demonstrate that the intracellular fate of soluble and liposomal antigens can differ depending upon the antigen-presenting cell.

Functional microtubules are required for antigen processing by macrophages and dendritic cells.

Antigen-presenting cells readily phagocytose antigens and channel them through various membrane-bound organelles within the cell. In previous studies, we demonstrated that macrophages concentrated and localized particulate antigens to the trans-Golgi prior to displaying the MHC-class I-antigenic peptides on the cell surface. In this study, we evaluated the importance of cytoskeletal elements in the intracellular trafficking of soluble and liposome-encapsulated ovalbumin in murine bone marrow-derived macrophages and human dendritic cells. F-actin, as identified by staining with fluorescein phalloidin, was observed at the point of contact between soluble or liposomal antigen and the cell membrane, suggesting that a rearrangement of the cytoskeleton occurs to facilitate the uptake of the antigens. Cells were incubated with colchicine, a microtubule depolymerizing agent, or paclitaxel, a microtubule polymerizing agent, before the addition of Texas Red-labeled ovalbumin or liposome-encapsulated Texas Red-labeled ovalbumin. Colchicine disrupted the trans-Golgi, whereas the trans-Golgi complexes were intact in paclitaxel treated cells. In either paclitaxel or colchicine-treated macrophages, internalized liposomal ovalbumin was not concentrated in the area of the trans-Golgi as determined by staining with fluorescent ceramide. In contrast, soluble ovalbumin was concentrated in the region of the trans-Golgi in 15% of the dendritic cells treated with paclitaxel, whereas 6% of the dendritic cells were able to concentrate liposomal antigen. In colchicine-treated dendritic cells, both soluble and liposomal antigens were internalized but did not localize to the area of the trans-Golgi. These data suggest that trafficking of soluble and liposome-encapsulated ovalbumin requires a functional microtubule-dependent translocation system.

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.