Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8+ CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5–12 log10) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8+ cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1+ humans.

Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice

IL-2 and -15 belong to the four α-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with 105 plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-γ as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4(+) T cell responses to human cytonnegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4(+) CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HICMV isolates revealed that the gB and gH epitope sequences recognized by human CD4(+) T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4(+) CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse pepticle-sensitized Patr-DR7(+) cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class 11 lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Recombinant influenza viruses as a vaccine vector for HIV

 Live recombinant influenza viruses were successfully used as HIV vaccine vectors in a mouse model. Following intranasal prime-boost vaccination, HIV-specific CD8+ T cell responses were detected in the spleen, broncho-alveolar lavage, mediastinal and inguinal lymph nodes. HIV+α4β7+ CD8+ T cells contributed to protection in pseudo-challenge experiments using recombinant vaccinia virus expressing HIV antigens. This research highlights the importance of mucosal CD8+ T cells in viral immunity and emphasizes the need for additional studies to provide key insights to underpin future vaccine development.

Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Vaccinia locomotion in host cells: Evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 μM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages.

We investigated the relationship between the fusion selectivity of the envelope glycoprotein (env) and the tropism of different human immunodeficiency virus type 1 (HIV-1) isolates for CD4+ human T-cell lines vs. primary macrophages. Recombinant vaccinia viruses were prepared encoding the envs from several well-characterized HIV-1 isolates with distinct cytotropisms. Cells expressing the recombinant envs were mixed with various CD4+ partner cell types; cell fusion was monitored by a quantitative reporter gene assay and by syncytia formation. With CD4+ continuous cell lines as partners (T-cell lines, HeLa cells expressing recombinant CD4), efficient fusion occurred with the envs from T-cell line-tropic isolates (IIIB, LAV, SF2, and RF) but not with the envs from macrophage-tropic isolates (JR-FL, SF162, ADA, and Ba-L). The opposite selectivity pattern was observed with primary macrophages as cell partners; stronger fusion occurred with the envs from the macrophage-tropic than from the T-cell line-tropic isolates. All the envs showed fusion activity with peripheral blood mononuclear cells as partners, consistent with the ability of this cell population to support replication of all the corresponding HIV-1 isolates. These fusion selectivities were maintained irrespective of the cell type used to express env, thereby excluding a role for differential host cell modification. We conclude that the intrinsic fusion selectivity of env plays a major role in the tropism of a HIV-1 isolate for infection of CD4+ T-cell lines vs. primary macrophages, presumably by determining the selectivity of virus entry and cell fusion.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

Similar T-cell immune responses induced by group M consensus env immunogens with wild-type or minimum consensus variable regions.

Consensus HIV-1 genes can decrease the genetic distances between candidate immunogens and field virus strains. To ensure the functionality and optimal presentation of immunologic epitopes, we generated two group-M consensus env genes that contain variable regions either from a wild-type B/C recombinant virus isolate (CON6) or minimal consensus elements (CON-S) in the V1, V2, V4, and V5 regions. C57BL/6 and BALB/c mice were primed twice with CON6, CON-S, and subtype control (92UG37_A and HXB2/Bal_B) DNA and boosted with recombinant vaccinia virus (rVV). Mean antibody titers against 92UG37_A, 89.6_B, 96ZM651_C, CON6, and CON-S Env protein were determined. Both CON6 and CON-S induced higher mean antibody titers against several of the proteins, as compared with the subtype controls. However, no significant differences were found in mean antibody titers in animals immunized with CON6 or CON-S. Cellular immune responses were measured by using five complete Env overlapping peptide sets: subtype A (92UG37_A), subtype B (MN_B, 89.6_B and SF162_B), and subtype C (Chn19_C). The intensity of the induced cellular responses was measured by using pooled Env peptides; T-cell epitopes were identified by using matrix peptide pools and individual peptides. No significant differences in T-cell immune-response intensities were noted between CON6 and CON-S immunized BALB/c and C57BL/6 mice. In BALB/c mice, 10 and eight nonoverlapping T-cell epitopes were identified in CON6 and CON-S, whereas eight epitopes were identified in 92UG37_A and HXB2/BAL_B. In C57BL/6 mice, nine and six nonoverlapping T-cell epitopes were identified after immunization with CON6 and CON-S, respectively, whereas only four and three were identified in 92UG37_A and HXB2/BAL_B, respectively. When combined together from both mouse strains, 18 epitopes were identified. The group M artificial consensus env genes, CON6 and CON-S, were equally immunogenic in breadth and intensity for inducing humoral and cellular immune responses.

Multi-envelope HIV-1 vaccine devoid of SIV components controls disease in macaques challenged with heterologous pathogenic SHIV

A central obstacle to the design of a global HIV-1 vaccine is virus diversity. Pathogen diversity is not unique to HIV-1, and has been successfully conquered in other fields by the creation of vaccine cocktails. Here we describe the testing of an HIV-1 envelope cocktail vaccine. Six macaques received the vaccine, delivered by successive immunizations with recombinant DNA, recombinant vaccinia virus and recombinant envelope proteins. Following vaccination, animals developed a diversity of anti-envelope antibody binding and neutralizing activities toward proteins and viruses that were not represented by sequence in the vaccine. T-cells were also elicited, as measured by gamma-interferon production assays with envelope-derived peptide pools. Vaccinated and control animals were then challenged with the heterologous pathogenic SHIV, 89.6P. Vaccinated monkeys experienced significantly lower virus titers and better maintenance of CD4+ T-cells than unvaccinated controls. The B- and T-cell immune responses were far superior post-challenge in the vaccinated group. Four of six vaccinated animals and only one of six control animals survived a 44-week observation period post-challenge. The present report is the first to describe pathogenic SHIV disease control mediated by a heterologous HIV-1 vaccine, devoid of 89.6 or SIV derivatives.

The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5′ extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans ≈25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.