Systemic gene transfer of binding immunoglobulin protein (BiP) prevents disease progression in murine collagen-induced arthritis.

Summary Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production.

Recent developments in mucosal delivery of pDNA vaccines.

The development of DNA vaccination to mucosal surfaces has continued apace over the last 2 years, with the investigation of several novel delivery vehicles. There have been advances in the understanding of the basic immunological mechanisms behind the induction of immune responses by plasmid DNA. The mechanistic insights are paving the way for the design of a second generation of mucosally delivered DNA vaccines. This article reviews the recent progress in the field of microparticle, cationic lipid and bacterial delivery systems. All these mechanisms afford some protection from environmental degradation and facilitate DNA uptake. These methods have been compared with respect to transfection efficiency, ability to elicit a full range of immune responses and their relative safety for in vivo applications.

The cellular basis of immune induction at mucosal surfaces by DNA vaccination.

The nasal mucosa provides a simple, non-invasive route to deliver DNA encoding the gene of interest to stimulate mucosal and systemic immune responses. However, unlike the intradermal or intramuscular routes for plasmid DNA (pDNA) delivery, immune responsiveness to antigen exposure at the respiratory mucosa is tightly regulated, consistent with the balance between active immunity and non-responsiveness to pathogenic or inert environmental antigens. We have characterised the antigen presenting cell types, their distribution and activation status following nasal vaccination with pDNA-cytofectin complexes encoding model antigens. We demonstrate that nasal immunisation is associated with expression of the encoded protein in a small population of dendritic cells and macrophages at the site of pDNA delivery, in the draining lymph nodes (LN) and in the spleen. Antigen expression by nasal dendritic cells was associated with up-regulation of surface MHC class II and CD86 expression and functional activation of T-lymphocytes. The results highlight the potential of intranasal vaccination with pDNA, provided the activation / costimulatory requirements for an active immune response are achieved.

Intranasal immunization with plasmid DNA-lipid complexes elicits mucosal immunity in the female genital and rectal tracts.

The development of vaccines against pathogens transmitted across the genito-rectal mucosa that effectively stimulate both secretory IgA Abs and cytotoxic T lymphocytes in the genital tract and CTL in the draining lymph nodes (LN) has proven a major challenge. Here we report a novel, noninvasive approach of genetic vaccination via the intranasal route. Such vaccination elicits immune responses in the genital and rectal mucosa, draining LNs, and central lymphoid system. Intranasal immunization with plasmid DNA-lipid complexes encoding the model Ag firefly luciferase resulted in dissemination of the DNA and the encoded transcript throughout the respiratory and gastrointestinal tracts, draining LNs, and spleen. Complexing the plasmid DNA with the lipid DMRIE/DOPE enhanced expression of the encoded protein in the respiratory tract, increased specific secretory IgA Ab in the vaginal and rectal tracts, and increased the circulating levels of specific IgA and IgG. In addition, intranasal DNA immunization resulted in generation of Ag-specific CTL that were localized in the genital and cervical LNs and spleen. These results suggest that intranasal immunization with plasmid DNA-lipid complexes may represent a generic immunization strategy against pathogens transmitted across the genito-rectal and other mucosal surfaces.

Mucosal immunization with DNA-liposome complexes.

The mucosal surfaces represent the primary site for transmission of several viruses including HIV. To prevent mucosal transmission and dissemination to the regional lymph nodes, an effective HIV vaccine may need to stimulate immune responses at the genital and rectal mucosa. Optimal induction of mucosal immunity in general requires targeting antigens to the specialized antigen presenting cells of mucosal associated lymphoid tissues. The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding the introduced gene to stimulate mucosal immunity. As a first step to evaluate the feasibility of this approach, we have investigated as a model system, systemic and mucosal immune responses elicited to firefly luciferase generated by DNA immunization. Incorporating DNA into liposomes with cationic lipids enhanced luciferase expression in nasal tissue, and was associated with induction of a humoral response in serum and vaginal fluids and also a proliferative and cytotoxic T lymphocyte response in the spleen and iliac lymph nodes draining the genital and rectal mucosa.

Mucosal or targeted lymph node immunization of macaques with a particulate SIVp27 protein elicits virus-specific CTL in the genito-rectal mucosa and draining lymph nodes.

The major routes of HIV transmission are through the rectal and cervico-vaginal mucosa. To prevent dissemination of HIV to the regional lymph nodes (LNs), an effective vaccine may need to stimulate CTL in the rectal or genital tract and the draining LNs. We report that mucosal immunization by the recto-oral and vagino-oral route or s.c. immunization targeting the iliac LNs with a particulate SIVp27:Ty-VLP vaccine elicits SIVgag-specific CTL in the regional LNs as well as in the spleen and PBMC. Targeted LN immunization with this vaccine elicited MHC class I-restricted CD8+ CTL responses, and the highest frequency of CTL was found in the iliac LNs. Moreover, SIVgag-specific CTL activity was detected in short term T cell lines established in mononuclear cells eluted from the rectal and cervico-vaginal mucosa. The relative frequency of CTL in short term cell lines prepared from the rectal mucosa (21/113 or 18.6%) was similar to that obtained from the cervico-vaginal mucosa (16/79 or 20.3%). Examination of the relative frequency of CTL to the T cell epitopes residing within SIVp27 showed a higher frequency in iliac LN cells to peptide aa 41-70 than in that to peptide aa 121-150, and this was significant after both recto-oral (chi-squared 6.500, p < 0.02) and vagino-oral (chi-squared = 10.391, p < 0.01) immunization. In contrast, the relative frequency of CTL in PBMC to peptide aa 41-70 (15.5%) was comparable to that elicited by peptide aa 121-150 (17.6%). This study provides novel evidence that mucosal or targeted LN immunization can generate anti-SIV CTL in the rectal and genital mucosa, in the draining LNs, and in the central lymphoid system.

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp 120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load ( > 90%) or transient viremia, indicating that all seven TILN-immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of IgA antibody-secreting cells to p27 (P < 0.02), CD8-suppressor factor (P < 0.01), and the chemokines RANTES and MIP-1 beta (P < 0.01).

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

OBJECTIVES: To examine whether the route of immunization determines the hierarchy of T-cell epitope proliferative responses in macaques. DESIGN: Macaques 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. METHODS: T-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. RESULTS: Intramuscular 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. CONCLUSIONS: The 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.

Targeted lymph node immunization with simian immunodeficiency virus p27 antigen to elicit genital, rectal, and urinary immune responses in nonhuman primates.

A s.c. route of immunization was developed in non-human primates, which targets the genitourinary-rectal associated lymphoid tissue. A vaccine consisting of rSIV gag p27, expressed as hybrid Ty virus-like particles (p27: Ty-VLP) was administered in the proximity of the internal iliac lymph nodes. Secretory IgA and IgG Abs to the p27 Ag were elicited in the vaginal, male urethral, rectal and seminal fluids, urine and serum. Two or more immunodominant B cell epitopes were identified within peptides 51-90 and 121-170 of the sequence of p27, using serum or biliary IgA and IgG Abs. CD4+ T cell proliferative responses to p27 were elicited predominantly in the targeted internal iliac, as well as the inferior mesenteric lymph nodes and the spleen, but not in the unrelated lymph nodes. These cells were then studied for helper function in p27 specific B cell Ab synthesis. Specific IgA and IgG Abs were detected in the same lymphoid tissues as those that displayed proliferative responses. However, cross-over reconstitution experiments between splenic and iliac lymph node B and CD4+ T cells suggest that the iliac B cells are essential for specific IgA Ab synthesis, whereas splenic B cells preferentially synthesize IgG Ab. The targeted lymph node (TLN) route of immunization gave comparable B cell, proliferative T cell, and Th cell responses to the vaginal, male genitourinary, and rectal mucosal routes, which were augmented by oral immunization. However, the TLN route induced urinary and seminal fluid sIgA and IgG Abs in addition to genital and rectal Abs. Generating secretory IgA and IgG Abs at the mucosal surfaces, and T and B cell immunity in the regional draining lymph nodes, spleen and circulation by TLN immunization may prevent transmission of virus through the mucosa, dissemination of the virus, and the formation of a latent reservoir of infection.

Mucosal model of genital immunization in male rhesus macaques with a recombinant simian immunodeficiency virus p27 antigen.

Human immunodeficiency virus (HIV) can be transmitted through infected seminal fluid or vaginal or rectal secretions during heterosexual or homosexual intercourse. To prevent mucosal transmission and spread to the regional lymph nodes, an effective vaccine may need to stimulate immune responses at the genitourinary mucosa. In this study, we have developed a mucosal model of genital immunization in male rhesus macaques, by topical urethral immunization with recombinant simian immunodeficiency virus p27gag, expressed as a hybrid Ty virus-like particle (Ty-VLP) and covalently linked to cholera toxin B subunit. This treatment was augmented by oral immunization with the same vaccine but with added killed cholera vibrios. Polymeric secretory immunoglobulin A (sIgA) and IgG antibodies to p27 were induced in urethral secretions, urine, and seminal fluid. This raises the possibility that the antibodies may function as a primary mucosal defense barrier against SIV (HIV) infection. The regional lymph nodes which constitute the genital-associated lymphoid tissue contained p27-specific CD4+ proliferative and helper T cells for antibody synthesis by B cells, which may function as a secondary immune barrier to infection. Blood and splenic lymphocytes also showed p27-sensitized CD4+ T cells and B cells in addition to serum IgG and IgA p27-specific antibodies; this constitutes a third level of immunity against dissemination of the virus. A comparison of genito-oral with recto-oral and intramuscular routes of immunization suggests that only genito-oral immunization elicits specific sIgA and IgG antibodies in the urine, urethra, and seminal fluid. Both genito-oral and recto-oral immunizations induced T-cell and B-cell immune responses in regional lymph nodes, with preferential IgA antibody synthesis. The mucosal route of immunization may prevent not only virus transmission through the genital mucosa but also dissemination and latency of the virus in the draining lymph nodes.

T- and B-cell functions and epitope expression in nonhuman primates immunized with simian immunodeficiency virus antigen by the rectal route.

Transmission of human immunodeficiency virus (HIV) in North America and Europe occurs most commonly through the rectal mucosa during homosexual intercourse. The simian immunodeficiency virus (SIV) macaque model has been used to investigate rectal immunization. The vaccine used was a recombinant SIV gag p27 expressed as hybrid Ty virus-like particles (Ty-VLP). Sequential ororectal (OR) mucosal immunization was compared with i.m. immunization. Whereas both routes of immunization induced serum IgA and IgG p27 antibodies, only OR immunization induced rectal secretory IgA antibodies. Specific CD4+ T-cell proliferative responses to stimulation with p27 were found after i.m. immunization only in the blood and spleen, but after OR immunization they were found in the internal iliac and inferior mesenteric lymph nodes in addition to the blood and spleen. T-cell epitope mapping of the proliferative responses of short-term cell lines (STCLs) grown from peripheral blood or lymphoid cells revealed a major epitope within the polypeptide 121-150 after either route of immunization. Two minor T-cell epitopes were found within peptide 41-80 in STCLs from splenic and circulating cells. B-cell epitope mapping of serum or biliary IgA and IgG antibodies revealed two overlapping or adjacent immunodominant epitopes to the T-cell epitopes within the polypeptides 121-170 and 51-90. The results suggest that rectal augmented by oral immunization with a recombinant particulate antigen in nonhuman primates elicits secretory IgA and to a lesser extent IgG responses in the draining lymph nodes and the rectal mucosa, whereas systemic immunization targets predominantly splenic and circulating T- and B-cell responses. These findings may have important implications in the strategy of designing vaccines in prevention of homosexual transmission of HIV infection.

Induction of mucosal and systemic immunity to a recombinant simian immunodeficiency viral protein.

Heterosexual transmission through the cervico-vaginal mucosa is the principal route of human immunodeficiency virus (HIV) infection in Africa and is increasing in the United States and Europe. Vaginal immunization with simian immunodeficiency virus (SIV) had not yet been studied in nonhuman primates. Immune responses in macaques were investigated by stimulation of the genital and gut-associated lymphoid tissue with a recombinant, particulate SIV antigen. Vaginal, followed by oral, administration of the vaccine elicited three types of immunity: (i) gag protein p27-specific, secretory immunoglobulin A (IgA) and immunoglobulin G (IgG) in the vaginal fluid, (ii) specific CD4+ T cell proliferation and helper function in B cell p27-specific IgA synthesis in the genital lymph nodes, and (iii) specific serum IgA and IgG, with CD4+ T cell proliferative and helper functions in the circulating blood.

Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion.

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.

Vaccination and protection from a lethal viral infection: identification, incorporation, and use of a cytotoxic T lymphocyte glycoprotein epitope.

The outcome of infection by lymphocytic choriomeningitis virus (LCMV) is determined largely by the cytotoxic T lymphocyte (CTL) response of the host. In H-2b mice, the anti-glycoprotein (GP) response is directed to at least two epitopes, one located at GP aa 272-286 and a second in GP-1. Here we show that the second epitope can be minimally identified by amino acid residues GP 34-40 (AVYNFAT). The epitope is restricted by the Db class I glycoprotein. Characterization of these CTL epitopes allowed us to address the role(s) played by each epitope when expressed singly in the control of a lethal challenge with LCMV. Here we show that a single immunization with a recombinant vaccinia virus (VV) vaccine expressing LCMV GP aa 1-59 confers protection to H-2b mice from lethal LCMV infection. In contrast, a VV expressing LCMV GP aa 272-293, although recognized by CTL, does not protect. We show that the success or failure of protective immunization is determined by the ability of the immunizing sequences to prime for CTL in vivo. Although the GP 278-286 epitope when contained as a "minigene" fails to induce CTL, when incorporated in the normal GP "backbone" it successfully elicits CTL. These observations suggest that the "minimal" recognition sequence alone may not be sufficient to induce a protective CTL response in vivo. Thus a single CTL epitope can protect against a lethal virus infection, but to achieve an effective vaccine, the immunizing sequences must be carefully selected.

Cytotoxic T lymphocyte control of acute lymphocytic choriomeningitis virus infection: interferon gamma, but not tumour necrosis factor alpha, displays antiviral activity in vivo.

Virus-specific cytotoxic T lymphocytes (CTL) mediate their antiviral activity either by direct lysis of infected cells, or by the release of soluble lymphokines, or by a combination of the two. We have examined the role played by interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF alpha) in virus clearance. In vitro the amount of IFN-gamma synthesized by some lymphocytic choriomeningitis virus-specific H-2-restricted CTL clones was quantitatively too small to correlate with a direct antiviral activity in vivo. However, treatment of mice with a neutralizing monoclonal antibody to IFN-gamma significantly inhibited the clearance of virus from the spleens of acutely infected mice given adoptive transfers of immune spleen cells. Additionally, mice treated with exogenous recombinant murine IFN-gamma 24 h before or at the same time as virus inoculation showed reduced virus titres in their spleens. Hence, IFN-gamma displayed a direct antiviral effect in vivo. In contrast, treatment of mice with recombinant TNF alpha had no effect on virus clearance and thus TNF alpha is unlikely to play a significant role in this acute viral infection.