Geoffrey Lilley Smith

Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine

Modified virus Ankara (MVA) is a vaccinia virus (VV) strain that was attenuated by serial passage through chick embryo fibroblasts (CEFs) and contains six large genomic deletions compared with parental virus. MVA replicates well in CEFs, but poorly in most mammalian cells. Recombinant MVA is a promising human vaccine candidate due to its restricted host range, immunogenicity and avirulence in animal models, and excellent safety record as a smallpox vaccine. Here we present a further characterization of MVA and demonstrate that: (i) MVA can replicate, albeit poorly, in transformed human cell lines, but not in primary human fibroblasts although there is limited cell-to-cell spread; (ii) MVA is a potent inducer of type I interferon (IFN) from primary human cells, which may restrict virus spread in vivo; and (iii) unlike other VV strains, MVA does not express soluble receptors for IFN-gamma, IFN-alpha/beta, tumour necrosis factor and CC chemokines, but does express a soluble interleukin-1beta receptor. This provides a plausible and testable explanation for the good immunogenicity of MVA despite its poor replication in mammals. The implications of these findings for the use of MVA as a safe and immunogenic human vaccine candidate are discussed.

Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA.

To test the capacity of poxviruses for added foreign DNA, a recombinant was constructed that contains 24 700 bp of bacteriophage lambda DNA inserted within the vaccinia virus thymidine kinase (TK) gene. The recombinant is stable, infectious and replicates in tissue culture at the same rate and to the same titer as standard vaccinia virus. This size flexibility of the poxvirus genome and the lack of stringent packaging requirements are useful features for an infectious eukaryotic cloning vector.

Vaccinia Virus Expression Vectors

Introduction. Recombinant DNA technology has made possible the expression of heterologous genes in a variety of animal viruses (for review, see Rigby, 1983). The availability of a wide variety of animal virus vectors enables a foreign gene to be expressed in different cell types, at different levels and with different consequences for the host cell. Factors influencing the choice of virus vector include the types of cells or animals to be infected, whether infection leading to cell transformation or lysis is required, the number of genes to be expressed, and whether virus infectivity is to be retained. Small DNA viruses (e.g. papovaviruses) may have restricted host range and limited capacity for foreign DNA due to severe packaging constraints imposed by the icosahedral virus capsid. Consequently these virus vectors are mostly replication-defective, requiring helper virus or special cell lines for their replication. In contrast, larger viruses (poxviruses and herpesviruses) have a greater capacity for foreign DNA without destruction of infectivity and can have a wide host range.

Recognition of influenza A virus nucleoprotein by human cytotoxic T lymphocytes

A recombinant vaccinia virus (NP-VAC) containing cDNA corresponding to segment 5, the nucleoprotein (NP) gene of influenza A/PR/8/34 virus was used to examine the specificity of human influenza virus immune cytotoxic T lymphocytes (CTL). Effector cell preparations from two donors recognized autologous lymphocytes that had been infected with NP-VAC. Lysis was specific because cells infected with vaccinia virus were not killed and recognition was HLA-restricted. In one donor, the influenza virus-specific CTL response changed with time so that his effector cells no longer recognized autologous lymphocytes infected with NP-VAC. However, a component that was NP-specific remained because these CTL lysed the more sensitive autologous B lymphoblastoid cells that had been infected with NP-VAC. In four other donors, no NP-specific CTL response could be detected using autologous lymphocyte targets. Thus NP, an internal virus protein, is one antigen that is recognized by human influenza A virus-specific CTL, but it is likely that other individual virus components contribute to the total CTL response.

Immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus Ankara-based multi-CTL epitope vaccine for human immunodeficiency virus type 1 in mice

A vaccine against human immunodeficiency virus (HIV) is still awaited. Although the correlates of protection remain elusive, it is likely that CD8+ T cells play an important role in the control of this infection. To firmly establish the importance of these cells in protective immunity, a means of efficient elicitation of CD8+ T cell responses in the absence of antibody is needed and, when available, might represent a crucial step towards a protective vaccine. Here, a novel vaccine candidate was constructed as a multi-cytotoxic T lymphocyte (CTL) epitope gene delivered and expressed using modified vaccinia virus Ankara (MVA). The immunogen consists of 20 human, one murine and three rhesus macaque epitopes. The non-human epitopes were included so that the vaccine can be tested for immunogenicity and optimal vaccination doses, routes and regimes in experimental animals. Mice were immunized intravenously (i.v.) or intramuscularly (i.m.) using a single dose of 10(6) p.f.u. of the recombinant MVA and the induction of CTL was assessed. It was demonstrated that both administration routes induced specific CTL responses and that the i.v. route was moderately more immunogenic than the i.m. route. The frequencies of ex vivo splenocytes producing interferon-y upon MHC class I-restricted peptide stimulation were determined using an ELISPOT assay. Also, the correct processing and presentation of some HLA-restricted epitopes in human cells was confirmed.