Guy T. Layton

Matrix Metalloproteinases 9 and 2 Are Necessary for the Migration of Langerhans Cells and Dermal Dendritic Cells from Human and Murine Skin

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.

Protection from Plasmodium berghei infection by priming and boosting T cells to a single class I-restricted epitope with recombinant carriers suitable for human use

The desirability of inducing cytotoxic T cell responses to defined epitopes in humans has led to the development of a variety of recombinant delivery systems. Recombinant protein particles derived from a yeast retrotransposon (Ty) and the modified Ankara vaccinia (MVA) virus can deliver large epitope strings or even whole proteins. Both have previously been administered safely in humans. Immunization with recombinant Ty and MVA containing a single Plasmodium berghei class I‐binding epitope provided 95 % sterile protection against malaria in mice. The sequence of immunization, Ty followed by MVA, was critical to elicit high levels of IFN‐γ‐producing cells and protection. The reciprocal sequence (MVA/TY) or homologous boosting was not protective. Both constructs (Ty and MVA) contain the H‐2Kd‐restricted pb9 CTL epitope from the circumsporozoite protein of P. berghei among a string of 8 – 15 human P. falciparum‐derived CTL epitopes restricted through 7 common HLA alleles as well as widely recognized CD4 T cell epitopes. Thus, the novel recombinant Ty/MVA prime/boost combination with these constructs provides a safe alternative for evaluation for human vaccination against P. falciparum malaria.

Yeast Retrotransposon Particles as Antigen Delivery Systems

The development of technologies to produce recombinant proteins for use in the pharmaceutical industry has made substantial advances, in particular in the area of generating antigens containing multiple copies of important immunological regions. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles. Ty-fusion proteins retain this ability to form particles, and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to induce potent immune responses. In particular, hybrid VLPs carrying the core protein p24 of HIV (p24-VLPs) have been shown to induce antibody and T-cell proliferative responses in both experimental animals and human volunteers, and immunization of rabbits with VLPs carrying the principal neutralizing determinant of HIV (V3-VLPs) resulted in the induction of neutralizing antibody responses and T-cell proliferation. Further studies with V3-VLPs have shown that this particulate antigen stimulates enhanced V3-specific lymphoproliferative responses as compared to whole recombinant gp120 or to V3 peptide conjugated to albumin. The V3-VLPs also induce potent CTL responses following immunization of mice in the absence of adjuvant. These responses are MHC class I restricted and are mediated by CD8-positive cells. These observations therefore demonstrate that hybrid Ty-VLPs induce both humoral and cellular immune responses against HIV and suggest that these immunogens may be important in combatting AIDS and other infections.

Hybrid Ty Virus-Like Particles

Vaccines need to activate antigen presenting cells, overcome genetic restriction in T-cell responses and elicit both T and B memory cells. In order to produce recombinant vaccines which can do this, considerable effort has been put into developing particulate antigen presentation systems to generate polyvalent, high molecular weight antigens which should maximally stimulate the immune system. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles (VLPs). Ty-fusion proteins retain this ability to form particles and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to elicit potent immune responses. Hybrid VLPs carrying human immunodeficiency virus (HIV) antigens stimulate the three main components of the immune system, namely antibody synthesis, T-cell proliferative responses and cytotoxic T-lymphocyte (CTL) responses.

Induction of neutralizing antibody and T-cell responses to Varicella-zoster virus (VZV) using Ty-virus-like particles carrying fragments of glycoprotein E (gE)

During infection with Varicella-zoster virus (VZV), the envelope proteins are highly immunogenic and glycoprotein E (gE) is one of the most abundant and antigenic. We have previously identified the immunodominant regions of gE and mapped the B-cell epitopes. In this study, we have evaluated the immunogenicity of recombinant hybrid Ty-virus-like particles (VLPs) carrying amino acids (1-134) or (101-161) of gE which contain the immunodominant sequences. VZV-specific antibodies were detected by ELISA in sera from mice and guinea pigs immunized with either gE(1-134)-VLPs or gE (101-161)-VLPs. The dominant B-cell epitopes, mapped by pepscan analysis of the sera, were found in peptides spanning amino acids 41-60, 56-75, 101-120, 116-135, 131-150 and 141-161. These sera also showed neutralizing activity against VZV in vitro. Epitopes recognized by neutralizing MAbs were mapped to both gE sequences (3B3 MAb recognizing amino acids 141-161 and IFB9 MAb recognizing amino acids 71-90). Lymphocyte proliferative responses to VZV were detected in four different mouse strains immunized with either gE(1-134)-VLPs or gE(101-134)-VLPs in alum. All mouse strains immunized with gE(1-134)-VLPs recognized epitopes in amino acids 11-30 and 71-90 and all those immunized with gE(101-161)-VLPs recognized epitopes in amino acids 91-110 and 106-125. These results indicate that VLPs carrying these gE sequences can prime potent humoral and cellular anti-VZV responses in small animals and warrant further investigation as potential vaccine candidates against varicella-zoster infections.

Inhibition of TCR‐mediated shedding of L‐selectin (CD62L) on human and mouse CD4+ T cells by metalloproteinase inhibition: analysis of the regulation of Th1/Th2 function

The generation of a productive primary immune response is dependent on the ability of naïve T lymphocytes to recirculate through peripheral lymph organs to encounter specific antigen. The process of naïve CD4+ T cell entry into lymph nodes correlates with cell surface expression of L‐selectin (CD62L), which mediates early tethering and rolling events to endothelium prior to entry. Here, we demonstrate that surface expression of CD62L enhances CD4+ T cell activation in vitro. The synthetic hydroxamate metalloproteinase inhibitor (BB‐3103), specifically inhibits activation‐induced shedding of CD62L from CD4+ T cells by TCR cross‐linking and lowers proliferation in part by reducing rapid tyrosine phosphorylation of zeta‐associated protein 70 kDa (ZAP‐70) and by increasing cytosolic free Ca2+ concentration mobilization. BB‐3103 also inhibited the proliferative response of both murine CD4+ Th1 and Th2 subsets in vitro but the inhibitory effects were sustained only in Th2‐type cells. Similarly, BB‐3103 mediated prolonged inhibition of allergen‐dependent peripheral T cell proliferation in atopic dermatitis patientsbut not in healthy controls. Analysis of CD62L expression on murine CD4+ T cell subsets revealed that surface expression was maintained on Th1 cells but not Th2 cells. The differential effects of BB‐3103 on primed effector CD4+ T cells may provide new insights into generating therapeutic agents capable of redressing the Th2/Th1 imbalance in allergic diseases.

The effects of adjuvants on CTL induction by V3:Ty-virus-like particles (V3-VLPs) in mice

We have previously described the generation of HIV-1 V3-specific cytotoxic T-lymphocytes (CTL) responses in BALB/c (H-2d) mice following immunization with Ty-virus-like particles carrying the V3 loop of gp120 (V3-VLPs) without adjuvant. In this study the effects of various adjuvants on CTL induction by V3-VLPs was examined. Mice immunized with V3-VLPs formulated in aqueous-based adjuvants, Detox, gamma-inulin, galactosaminylmuramyl dipeptide and Chemivax generated V3-specific CTL responses, although at reduced levels when compared to the no adjuvant group. V3-VLPs prepared in Alhydrogel, algamulin or as an oil emulsion in SAF-MF failed to generate V3-specific CTL responses. The mechanism whereby alum prevented the induction of a CTL response was investigated further. Immunization with V3-VLPs prepared in non-saturating doses of alum or alum plus EDTA primed for strong CTL responses, indicating that free VLPs do, but alum-bound VLPs do not enter the MHC class I processing pathway of antigen-presenting cells (APCs). Furthermore, V3-VLPs with very low doses of alum led to an enhancement of the CTL response. The formulation of hybrid Ty-VLPs in oil based or precipitating adjuvants, therefore, inhibits access to the MHC class I processing pathway of APCs. The intact particulate structure of hybrid VLPs is therefore strictly necessary for CTL induction.

Generation of diversity in the hierarchy of T-cell epitope responses following different routes of immunization with simian immunodeficiency virus protein.

ObjectivesTo examine whether the route of immunization determines the hierarchy of T-cell epitope proliferative responses in macaques. DesignMacaques were immunized with a recombinant simian immunodeficiency virus (SIV) p27 core protein by the intramuscular, male and female genital or rectal route, each of which was augmented by oral immunization, and by the novel targeted lymph-node immunization route. Overlapping peptides were used to identify the proliferative T-cell epitopes and to determine their hierarchy in the circulation, spleen and lymph nodes. MethodsT-cell epitope mapping of the proliferative responses was studied in short-term cell lines. Dendritic cells and macrophages were enriched by metrizamide gradient and adherence to plastic, respectively. ResultsIntramuscular immunization elicited in the circulating T cells a hierarchy of T-cell epitopes within four peptides in the following descending order of frequency: peptides 121–140 (57.9%), 41–60 (28.9%), 61–80 (18.9%) and 101–120 (5.4%). The hierarchy of these four T-cell epitope responses differed significantly with each of the five routes of immunization, when circulating (P<0.001), splenic (P<0.02-<0.001) or iliac lymph-node cells (P<0.001) were analysed. The effect of antigen-presenting cells was then investigated and enriched dendritic cells were more effective than macrophages in processing and presenting the p27 antigen and the immunodominant (121–140) and 61–80 T-cell epitopes. ConclusionsThe route of immunization may determine the hierarchy of T-cell epitopes in the lymph nodes draining the mucosa in the circulating and splenic lymphocytes. The diversity of T-cell epitopes may affect the control of HIV at different anatomical sites, the administration route of the vaccine, and selection of polypeptides or recombinant antigens for immunization.

Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients

Yeast Ty virus‐like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI). The antigenicity of hybrid VLPs containing fragments of the varicella‐zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV‐VLPs together with control antigens. PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV‐VLPs. As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples. The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty‐VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination. J. Med. Virol. 59:78–83, 1999.

Cloning, expression, and immunogenicity of the assembly protein of varicella-zoster virus and detection of the products of open reading frame 33

Herpesviruses produce assembly proteins (AP) that act as scaffolding proteins for the assembly of the viral capsids. The products of the assemblin gene, which encodes both maturational protease and AP, have been established for herpes simplex virus type 1 (HSV‐1) and human cytomegalovirus (CMV). We cloned an inframe ORF (encoding amino acids 304–605), found within the ORF 33 assemblin gene of VZV, into a yeast expression vector. The 34‐kDa AP was expressed as a fusion protein with the particle‐forming Ty p1 protein, resulting in high‐level production of hybrid AP‐virus‐like particles (AP‐VLPs). When AP‐VLPs were injected into mice and rabbits, antibodies were produced that reacted with, but that did not neutralise, native VZV. Three of four inbred strains of mice immunised with AP‐VLPs produced a VZV‐specific T‐cell response. The mouse and rabbit sera reacted with six bands on native VZV by Western blot analysis. The dominant bands were found at 34 and 38 kDa. Bands were also seen at 66, 63, 41, and 31 kDa. The 38‐kDa protein may represent the mature AP derived from the 41‐kDa precursor AP, itself the release product from the full‐length 66‐kDa assemblin. The 34‐kDa protein probably represents the product of the inframe co‐translational gene within ORF 33 encoding amino acids 304–605. The genetic organisation and proteolytic maturation of VZV assemblin are, therefore, analogous to those of other herpesviruses.J. Med. Virol. 53:332–339, 1997.