Scott Thomson

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one‐step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with ‘null’ mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR‐related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement (‘null’) and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild‐type CFTR activity. The long‐term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype‐phenotype correlations.

Expression of CTLA-4 by human monocytes

Cytotoxic T lymphocyte‐associated molecule‐4 (CTLA‐4) is a receptor present on T cells that plays a critical role in the downregulation of antigen‐activated immune responses. CTLA‐4 interacts with the ligands CD80 and CD86 on antigen‐presenting cells (APC), and also directs the assembly of inhibitory signalling complexes that lead to quiescence or anergy. In this study, we show that human monocytes constitutively express CTLA‐4. About 3% of monocytes expressed CTLA‐4 on the cell surface, whereas the intracellular expression was higher and present in about 20% of the monocytes. The sequences of the cDNAs from human monocytes were identical to the sequences of CTLA‐4 from T cells. Expression of CTLA‐4 was also confirmed in the activated myelomonocytic cell lines U937 and THP‐1. Monocytes, but not T cells, activated by interferon (IFN)‐γ also secreted soluble CTLA‐4 in vitro. The CTLA‐4 expression was upregulated upon treatment with phorbol 12‐myristate 13‐acetate (PMA) and IFN‐γ. This increased expression could be partially abolished by staurosporine, an inhibitor of protein kinase C (PKC). Ligation of CTLA‐4 in the monocyte‐like cell‐line U937 with antibodies against CTLA‐4 partially inhibited the proliferation of cells and the upregulation of cell‐surface markers CD86, CD54, HLA‐DR and HLA‐DQ induced by IFN‐γ and Staphylococcus aureus, Cowan I strain (SAC). Ligation of CTLA‐4 suppressed the PMA‐stimulated activation of transcription activator protein 1 (AP‐1) and nuclear factor (NF)‐κB in the U937 cell line, indicating the involvement of an inhibitory signal transduction. These data provide the first evidence that CTLA‐4 is constitutively expressed by monocytes and thus might be important for the regulation of immune mechanisms associated with monocytes.

Genetic vaccination strategies for enhanced cellular, humoral and mucosal immunity

Summary: In this article, we describe several novel genetic vaccination strategies designed to facilitate the development of different types of immune responses. These include: the consecutive use of DNA and fowlpoxvirus vectors in “prime‐boost” strategies which induce greatly enhanced and sustained levels of both cell‐mediated immunity and humoral immunity, including mucosal responses; ii) the co‐expression of genes encoding cytokines and cell‐surface receptors, and the use of immunogenic carrier molecules, for immune modulation and/or Improved targeting of vector‐expressed vaccine antigens; acid iii) the expression of minimal immunogenic arnino acid sequences, particularly cytotoxic CD8+ T‐cell determinants, in “polytope” vector vaccines. The capacity to modulate and enhance specific immune responses by the use of approaches such as these may underpin the development of vaccines against diseases for which no effective strategies are currently available.

Induction of Therapeutic T-Cell Responses to Subdominant Tumor-associated Viral Oncogene after Immunization with Replication-incompetent Polyepitope Adenovirus Vaccine

The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed in various EBV-associated malignancies have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and potential threat from their oncogenic potential. Here we have developed a replication- incompetent adenoviral vaccine that encodes multiple HLA class I-restricted CTL epitopes from LMP1 and LMP2 as a polyepitope. Immunization with this polyepitope vaccine consistently generated strong LMP-specific CTL responses in HLA A2/Kb mice, which can be readily detected by both ex vivo and in vivo T-cell assays. Furthermore, a human CTL response to LMP antigens can be rapidly expanded after stimulation with this recombinant polyepitope vector. These expanded T cells displayed strong lysis of autologous target cells sensitized with LMP1 and/or LMP2 CTL epitopes. More importantly, this adenoviral vaccine was also successfully used to reverse the outgrowth of LMP1-expressing tumors in HLA A2/Kb mice. These studies demonstrate that a replication-incompetent adenovirus polyepitope vaccine is an excellent tool for the induction of a protective CTL response directed toward multiple LMP CTL epitopes restricted through common HLA class I alleles prevalent in different ethnic groups where EBV-associated malignancies are endemic.

Efficacy of DNA and fowlpox virus priming/boosting vaccines for simian/human immunodeficiency virus

ABSTRACT Further advances are required in understanding protection from AIDS by T-cell immunity. We analyzed a set of multigenic simian/human immunodeficiency virus (SHIV) DNA and fowlpox virus priming and boosting vaccines for immunogenicity and protective efficacy in outbred pigtail macaques. The number of vaccinations required, the effect of DNA vaccination alone, and the effect of cytokine (gamma interferon) coexpression by the fowlpox virus boost was also studied. A coordinated induction of high levels of broadly reactive CD4 and CD8 T-cell immune responses was induced by sequential DNA and fowlpox virus vaccination. The immunogenicity of regimens utilizing fowlpox virus coexpressing gamma interferon, a single DNA priming vaccination, or DNA vaccines alone was inferior. Significant control of a virulent SHIV challenge was observed despite a loss of SHIV-specific proliferating T cells. The outcome of challenge with virulent SHIVmn229 correlated with vaccine immunogenicity except that DNA vaccination alone primed for protection almost as effectively as the DNA/fowlpox virus regimen despite negligible immunogenicity by standard assays. These studies suggest that priming of immunity with DNA and fowlpox virus vaccines could delay AIDS in humans.

A randomized, placebo-controlled phase I trial of DNA prime, recombinant fowlpox virus boost prophylactic vaccine for HIV-1.

An HIV-vaccine consisting of a DNA prime, recombinant fowlpox virus (rFPV) boost was evaluated in a double-blind placebo controlled trial. One milligram of pHIS–HIV-B expressing mutated gag, pol, env, vpu, tat and rev was administered at weeks 0 and 4 boosted by 5 × 107 pfu rFPV–HIV-B expressing gag/pol at week 8. The vaccine regimen was safe, but there was no difference between vaccine (n = 18) and placebo recipients (n = 6) for Gag or Pol-specific T-cell immune responses at week 9.

Comparative Efficacy of Subtype AE Simian-Human Immunodeficiency Virus Priming and Boosting Vaccines in Pigtail Macaques

ABSTRACT Vaccination against AIDS is hampered by great diversity between human immunodeficiency virus (HIV) strains. Heterologous B-subtype-based simian-human immunodeficiency virus (SHIV) DNA prime and poxvirus boost vaccine regimens can induce partial, T-cell-mediated, protective immunity in macaques. We analyzed a set of DNA, recombinant fowlpox viruses (FPV), and vaccinia viruses (VV) expressing subtype AE HIV type 1 (HIV-1) Tat, Rev, and Env proteins and SIV Gag/Pol in 30 pigtail macaques. SIV Gag-specific CD4 and CD8 T-cell responses were induced by sequential DNA/FPV vaccination, although lower FPV doses, VV/FPV vaccination, and DNA vaccines alone were not as consistently immunogenic. The SHIV AE DNA prime, FPV boost regimens were significantly less immunogenic than comparable B-subtype SHIV vaccination. Peak viral load was modestly (0.4 log10 copies/ml) lower among the AE subtype SHIV-immunized animals compared to controls following the virulent B subtype SHIV challenge. Protection from persistent high levels of viremia and CD4 T-cell depletion was less in AE subtype compared to B subtype SHIV-vaccinated macaques. Gag was highly immunodominant over the other AE subtype SHIV vaccine proteins after vaccination, and this immunodominance was exacerbated after challenge. Interestingly, the lower level of priming of immune responses did not blunt postchallenge Gag-specific recall responses, despite more modest protection. These studies suggest priming of T-cell immunity to prevent AIDS in humans is possible, but differences in the immunogenicity of various subtype vaccines and broad cross-subtype protection are substantial hurdles.

Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in mammalian cells

1 To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full‐length cDNA encoding wild‐type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells. 2 Like human CFTR, murine CFTR formed Cl− channels that were regulated by cAMP‐dependent phosphorylation and intracellular ATP. However, murine CFTR Cl− channels had a reduced single‐channel conductance and decreased open probability (Po) compared with those of human CFTR. 3 Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant. 4 For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long‐lived closed state. 5 5′‐Adenylylimidodiphosphate (AMP‐PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl− channels open, although AMP‐PNP increased the Po of murine CFTR. 6 The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.

Constitutive transduction of peptide transporter and HLA genes restores antigen processing function and cytotoxic T cell-mediated immune recognition of human melanoma cells

Potentiation of immunogenicity of malignant cells by gene transduction provides a unique opportunity for immune targeting of human cancers in vivo. This approach is undoubtedly influenced by the ability of the malignant cells to process and present endogenously target epitopes on their cell surface for immune recognition by cytotoxic T lymphocytes (CTLs). In the present study, we have investigated potential immune‐resistance pathways in human malignant melanoma by analyzing the major histocompatibility complex (MHC) gene expression and function in a panel of tumour cell lines. Our analysis showed that a large proportion of these cell lines consistently display a functional defect in the endogenous processing of CTL epitopes and are recognised poorly by specific T cells in spite of high levels of target antigen expression in the tumour cells. Molecular characterisation of this defect revealed that tumour cells under‐expressed peptide transporters and surface‐assembled MHC class I molecules, which constitute essential components of the class I processing pathway. Induction of peptide transporter and surface class I following treatment of these tumour cells with interferon gamma (IFN‐γ) suggested a transcriptional defect in the expression of antigen‐processing genes. Endogenous processing function in these tumour cells was restored completely following simultaneous transduction of cells with peptide transporter and HLA class I genes. Our findings provide a rationale for focussing on strategies designed to improve antigen‐processing function in tumour cells and, thus, may strongly influence future strategies for melanoma‐specific immunotherapy. Int. J. Cancer 75:590–595, 1998.

Novel Approach to the Formulation of an Epstein-Barr Virus Antigen-Based Nasopharyngeal Carcinoma Vaccine

ABSTRACT Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.