Design of lipid nanocapsule delivery vehicles for multivalent display of recombinant Env trimers in HIV vaccination.

Immunization strategies that elicit antibodies capable of neutralizing diverse virus strains will likely be an important part of a successful vaccine against HIV. However, strategies to promote robust humoral responses against the native intact HIV envelope trimer structure are lacking. We recently developed chemically cross-linked lipid nanocapsules as carriers of molecular adjuvants and encapsulated or surface-displayed antigens, which promoted follicular helper T-cell responses and elicited high-avidity, durable antibody responses to a candidate malaria antigen. To apply this system to the delivery of HIV antigens, Env gp140 trimers with terminal his-tags (gp140T-his) were anchored to the surface of lipid nanocapsules via Ni-NTA-functionalized lipids. Initial experiments revealed that the large (409 kDa), heavily glycosylated trimers were capable of extracting fluid phase lipids from the membranes of nanocapsules. Thus, liquid-ordered and/or gel-phase lipid compositions were required to stably anchor trimers to the particle membranes. Trimer-loaded nanocapsules combined with the clinically relevant adjuvant monophosphoryl lipid A primed high-titer antibody responses in mice at antigen doses ranging from 5 µg to as low as 100 ng, whereas titers dropped more than 50-fold over the same dose range when soluble trimer was mixed with a strong oil-in-water adjuvant comparator. Nanocapsule immunization also broadened the number of distinct epitopes on the HIV trimer recognized by the antibody response. These results suggest that nanocapsules displaying HIV trimers in an oriented, multivalent presentation can promote key aspects of the humoral response against Env immunogens.

Importance of MUC1 and spontaneous mouse tumor models for understanding the immunobiology of human adenocarcinomas.

Many important aspects of cancer biology, such as cancer initiation, progression, and metastasis, have been studied in animal models, mostly mice. As long as cancer was considered primarily a genetic disease, the study of transplantable mouse tumors, or even human tumor xenografts in immunocompromised mice, appeared to suffice. Many important genetic events that lead to transformation and in vivo tumor growth were elucidated. However, many even more important factors that determine whether or not the genetic potential of a tumor cell will be realized, such as the host response to the tumor and the tumor microenvironment that influences this response over a long period of time of tumor development, remained untested and unappreciated. This is slowly changing with the advent of molecular techniques that have spurred efforts to engineer better mouse models of human tumors. In this review, we show results of our efforts to combine a genetic mouse model of spontaneous human adenocarcinomas based on a Kras mutation, with an important human molecule MUC1 that is abnormally expressed on human adenocarcinomas, promoting oncogenesis, proinflammatory tumor microenvironment, and immunosurveillance.

In vivo expression of HGF/NK1 and GLP-1 From dsAAV vectors enhances pancreatic ß-cell proliferation and improves pathology in the db/db mouse model of diabetes.

OBJECTIVE: The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of ß-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in ß-cells, improves pathology in the db/db mouse model of type 2 diabetes. RESEARCH DESIGN AND METHODS; The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 ß-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous ß-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice. RESULTS: Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant ß-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance ß-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice. CONCLUSIONS: A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of ß-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles.

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.

Regulation of maturation and activating potential in CD8+ versus CD8- dendritic cells following in vivo infection with vaccinia virus.

DC maturation is known to be a necessary step in the generation of an effective immune response. We have used vaccinia virus (VACV) as a model to investigate the regulation of DC subsets in vivo following infection. While a number of in vitro studies have shown that DC infected with VACV fail to undergo maturation, the effect of VACV infection on the maturation of and cytokine production by DC subsets in vivo remains less defined. We have found that following systemic infection with vaccinia virus, both CD8+ and CD8- dendritic cells are infected. The number of infected DC peaked at 6 h and was highly decreased by 24 h post-infection. In both subsets, there was evidence of generalized upregulation of costimulatory molecules. Surprisingly, this included vaccinia infected DC, suggesting the regulation of DC maturation in vivo is much more complex and likely influenced by DC extrinsic signals. However, while we observed generalized upregulation of costimulatory molecules, IL-12 production was restricted to a subset of non-infected cells in both the CD8+ and CD8- DC populations. Importantly, the control of IL-12 production was differentially dependent on MyD88 signaling. IL-12 production was ablated in the absence of MyD88 in CD8- DC, while it was unchanged in CD8+ DC. These findings provide new insights into the control of DC maturation in vivo and demonstrate that the regulation of maturation in vivo following virus infection can be differentially controlled in distinct types of DC.

Cancer vaccines: accomplishments and challenges.

Advancements in knowledge in diverse fields of science, including genetics, cell biology, molecular biology and biochemistry, have shed light on the origins of cancer and cell intrinsic properties that allow it to grow, invade and metastasize. Many therapies currently in use or under development are based on this knowledge. Advances in immunology, on the other hand, have shed light on how the host responds to these malignant properties of cancer. Based on that knowledge, immunotherapy, in particular vaccines directed at improving the host response against cancer, is being developed as an alternative therapeutic approach. In this review, we address main issues that have driven development of cancer vaccines and the challenges that have been met and/or are anticipated.

Distinct pathways for signaling maturation in macrophages and dendritic cells after infection with paramyxovirus simian virus 5.

Professional antigen-presenting cells are critical components of both the innate and adaptive immune responses. Although dendritic cells (DCs) are generally thought to be the primary activators of naive T cells, macrophages have also been shown to fulfill this role. As with DCs, the capacity to induce optimal activation of T cells requires that macrophages undergo a process that results in the increased expression of costimulatory molecules, such as CD40, CD80, and CD86, and the production of cytokines. In this study we analyzed the effect of infection of macrophages generated from BALB/c mice with the paramyxovirus simian virus 5 (SV5). Here we have shown that bone marrow-derived macrophages (BMMs) are not productively infected at any multiplicity of infection tested. Analysis of activation markers revealed that SV5-infected BMMs robustly upregulated CD40 and modestly upregulated CD86, but did not upregulate the expression of CD80. Further, SV5-infected BMMs secreted low levels of interferon-beta and interleukin (IL)-12p40, but high levels of tumor necrosis factor-alpha and IL-6. Intriguingly, upregulation of these molecules on BMMs, unlike our previous results using bone marrow-derived dendritic cells, was not dependent on live virus. These findings provide evidence that different professional antigen-presenting cells can detect and respond to virus via distinct mechanisms.

Ligation of CD80 is critical for high-level CD25 expression on CD8+ T lymphocytes.

CD80 and CD86 have been shown to play a critical role in the optimal activation of T cells. Although these two molecules bind the same ligand, CD28, the question of whether CD80 and CD86 provide unique signals or serve redundant roles remains controversial. Previous studies have suggested that CD80 binding to CD28 may be superior to CD86 for the activation of naive CD8+ T cells. This study provides a potential mechanism to explain these observations. Our study demonstrates a previously unappreciated role for CD80, its superiority over CD86 in promoting CD25 expression, increasing both the number of cells that express CD25 and the level expressed on a per cell basis. These findings provide new insights into the role of CD80 vs CD86 and have important implications for the design of vaccines and immunotherapeutics aimed at the generation of a robust CD8+ T cell response in vivo.