Geoffrey L. Smith

Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-γ-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.

Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species soecificity

Vaccinia virus (VV) and other orthopoxviruses express a soluble type I interferon (IFN) receptor that for VV strain Western Reserve is encoded by gene B18R. The 60-65 kDa glycoprotein is related to the interleukin-1 receptors and is a member of the immunoglobulin superfamily, unlike other type I IFN receptors, which belong to the class II cytokine receptor family. The receptor has high affinity (KD, 174 pM) for human IFN alpha and, unlike other type I IFN receptors, has broad species specificity, binding to human, rabbit, bovine, rat, and mouse type I IFNs. This may have aided VV replication in multiple host species during evolution. A VV B18R deletion mutant is attenuated in a murine intranasal model. This type I IFN receptor represents the fourth VV protein that interferes with IFN and the fourth soluble cytokine receptor expressed by poxviruses.

A soluble receptor for interleukin-1β encoded by vaccinia virus: A novel mechanism of virus modulation of the host response to infection

Vaccinia virus gene B15R is shown to encode an abundant, secretory glycoprotein that functions as a soluble interleukin-1 (IL-1) receptor. This IL-1 receptor has novel specificity since, in contrast with cellular counterparts, it binds only IL-1 beta and not IL-1 alpha or the natural competitor IL-1 receptor antagonist. The vaccinia IL-1 beta receptor is secreted when expressed in a baculovirus system and competitively inhibited binding of IL-1 beta to the natural receptor on T cells. Deletion of B15R from vaccinia virus accelerated the appearance of symptoms of illness and mortality in intranasally infected mice, suggesting that the blockade of IL-1 beta by vaccinia virus can diminish the systemic acute phase response to infection and modulate the severity of the disease. The IL-1 beta binding activity is present in other orthopoxviruses.

The formation and function of extracellular enveloped vaccinia virus.

Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.

Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

The Poxvirus Protein A52R Targets Toll-like Receptor Signaling Complexes to Suppress Host Defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor κB (NF-κB) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor–associated kinase 2 (IRAK2) and tumor necrosis factor receptor–associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen.

Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like–interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain–containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain–containing adaptor inducing IFN-β (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor κB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-β by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.

Enhancement of MHC class I-restricted peptide-specific T cell induction by a DNA prime/MVA boost vaccination regime

Human immunodeficiency virus (HIV) vaccine candidates were previously constructed as a string of cytotoxic T lymphocyte (CTL) epitopes delivered and expressed using DNA and modified virus Ankara (MVA; an attenuated vaccinia virus) vectors. These vaccines were shown to induce interferon (IFN)-gamma-producing and cytolytic CD8+ T cells after a single vaccine administration. In the course of this work, immunization protocols were sought which would improve the levels of induced HIV-specific T cells. It was found that previous immunological exposure to MVA reduced the efficiency of subsequent priming and boosting using the same vaccine vehicle. However, a combined regime whereby the animals were first primed with the DNA vaccine and then boosted with MVA was the most potent protocol for the induction of both interferon-gamma-producing and cytolytic T cells against two CTL epitopes simultaneously. The general applicability of this novel vaccination method for induction of major histocompatibility complex class I-restricted T cells is discussed.

Vaccinia virus immune evasion

Vaccinia virus and other poxviruses express a wide variety of proteins which are nonessential for virus replication in culture but help the virus to evade the host response to infection. Examples include proteins which oppose apoptosis. Synthesise steroids, capture chemokines, counteract complement, interfere with interferon and intercept interleukins. This review provides an overview of such proteins, with an emphasis on work from our laboratory, and illustrates how the study of these proteins can increase our understanding of virus pathogenesis, the function of the immune system and how to make safer and more immunogenic poxvirus‐based vaccines.