Ian A. Ramshaw

Expression of Mouse Interleukin-4 by a Recombinant Ectromelia Virus Suppresses Cytolytic Lymphocyte Responses and Overcomes Genetic Resistance to Mousepox

ABSTRACT Genetic resistance to clinical mousepox (ectromelia virus) varies among inbred laboratory mice and is characterized by an effective natural killer (NK) response and the early onset of a strong CD8+ cytotoxic T-lymphocyte (CTL) response in resistant mice. We have investigated the influence of virus-expressed mouse interleukin-4 (IL-4) on the cell-mediated response during infection. It was observed that expression of IL-4 by a thymidine kinase-positive ectromelia virus suppressed cytolytic responses of NK and CTL and the expression of gamma interferon by the latter. Genetically resistant mice infected with the IL-4-expressing virus developed symptoms of acute mousepox accompanied by high mortality, similar to the disease seen when genetically sensitive mice are infected with the virulent Moscow strain. Strikingly, infection of recently immunized genetically resistant mice with the virus expressing IL-4 also resulted in significant mortality due to fulminant mousepox. These data therefore suggest that virus-encoded IL-4 not only suppresses primary antiviral cell-mediated immune responses but also can inhibit the expression of immune memory responses.

The prime-boost strategy: exciting prospects for improved vaccination

Abstract Unprecedented levels of cell-mediated immunity can be stimulated by the consecutive use of DNA vaccines and attenuated poxvirus vectors encoding similar heterologous antigens. This may offer a means of preventive vaccination against diseases for which no effective treatment is available.

Cytokines and immunity to viral infections

In this review, we discuss two broad approaches we have taken to study the role of cytokines and chemokines in antiviral immunity. Firstly, recombinant vaccinia viruses were engineered to exit encoding cytokines and chemokines of interest. Potent antiviral activity was mediated by many of these encoded factors, including IL‐2, IL12. IFN‐γ. TNF‐α. CD40L. Mig and Crg‐2, In some cases, liosi defense mechanisms were induced (IL‐2, IL‐t2. Mig and Crg‐2), whilst for others, a direct antiviral effect was demonstrated (IFN‐γ. TNF‐α and CD40L), In sharp contrast, vector‐directed expression of IL 4, a type 2 factor, greatly increased virus virulence, due 10 a downregulation of host type 1 immune responses. Our second experimental approach involved the use of strains of mice deficient for the production of particular cytokines or their receptors, often in combination with our engineered viruses. Mice deficient in either IFN‐γ, IFN‐γR, IFN‐α/βR, TNFFRs, CD40 or IL‐6 were, in general, highly susceptible to poxvirus infection. Surprisingly, not only the TNFR1, but also the TNFR2, was able to mediate the antiviral effects of TNF‐α in viv, whilst the antiviral activity observed following CD40‐CD40L interaction is a newly defined function which may involve apoptosis of infected cells. Through the use of perforin‐deficient mice, we were able to demonstrate a requirement for this molecule in the clearance of some viruses. such as ectromelia virus, whilst for others, such as vaccinia virus, perforin was less important but IFN‐γ was essential.

A case for cytokines as effector molecules in the resolution of virus infection.

Some cytokines are known to have potent antiviral activity in vitro, and recent work shows that severely immunodeficient mice, which lack conventional effector T cells, can still recover from virus infection provided these factors are present at sites of virus replication. Here Alistair Ramsay, Janet Ruby and Ian Ramshaw discuss these findings and raise fundamental questions concerning the physiological role of cytotoxic T cells in the resolution of virus infection.

Cytokines and murine autoimmune encephalomyelitis: Inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system

To examine the complex role of cytokines in the pathogenesis of actively induced murine EAE we measured the levels of a number of cytokines (IL‐6, IFN7 and TNF) in the spinal cord and CSF of mice with active experimental autoimmune encephalomyelitis (EAE) and found them all to be elevated. We next treated mice with antibodies to these three cytokines, which were over expressed in the CNS, to determine if they would alter disease and found the following: anti‐IL‐6 had no significant effect on disease, anti‐IFNγ exacerbated disease, and anti‐TNF either enhanced, had no effect or inhibited EAE depending on the antibody used. We then treated mice with exogenous cytokines, delivered using a recombinant vaccinia virus system, and found that the IL‐6 and TNF virus constructs inhibited EAE whereas the IFN1β construct had no effect on disease. Other cytokine recombinant viruses were also tested and it was found that the IL‐1β, IL‐2 and IL‐10 viruses inhibited EAE while an IL‐4 virus either had no effect or enhanced disease. We do not know the mechanism of action of the various cytokines in this system, but irrespective of the mechanism(s), this work clearly demonstrates that delivery of select cytokines using recombinant virus‐cytokine constructs can provide a powerful means of downregulating experimental organ‐specific autoimmune disease.

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.

DNA vaccination against virus infection and enhancement of antiviral immunity following consecutive immunization with DNA and viral vectors.

Recent demonstrations of the immunogenicity of antigens encoded in DNA plasmids following delivery by various routes have heralded a new era in vaccine development. In this article, we review progress in DNA‐based antiviral immunoprophylaxis. Preclinical studies have already established the immunogenicity of DNA plasmids encoding protective antigens from a wide variety of viral pathogens and work published in recent months has raised real prospects of broadly protective DNA vaccination against infections with influenza virus and HIV. We also describe a consecutive immunization protocol consisting of a priming dose of vaccine antigen encoded in DNA plasmids followed by a booster with the same antigen encoded in recombinant fowlpox virus vectors. We have used this strategy to generate protective antiviral cell‐mediated immunity and sustained, high‐level antibody responses both systemically and at mucosae. and to elucidate immunological mechanisms underlying the development of immunity to antigens delivered in DNA vectors.

Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine

ABSTRACT Despite eradication attempts, measles remains a global health concern. Here we report results that demonstrate that a single-dose DNA immunization followed by multiple boosters, delivered orally as a plant-derived vaccine, can induce significantly greater quantities of measles virus-neutralizing antibodies than immunization with either DNA or plant-derived vaccines alone. This represents the first demonstration of an enhanced immune response to a prime-boost vaccination strategy combining a DNA vaccine with edible plant technology.

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.