Safety and immunogenicity of the malaria candidate vaccines FP9 CS and MVA CS in adult Gambian men.

We assessed the safety and immunogenicity of prime-boost vectors encoding the Plasmodium falciparum circumsporozoite (CS) protein expressed either in the attenuated fowl-pox virus (FP9) or modified vaccinia virus Ankara (MVA). Thirty-two adult Gambians in groups of four to eight received one, two or three doses of FP9 CS and/or MVA CS. No serious adverse event was observed following vaccination. The most immunogenic regimen was two doses of FP9 followed by a single dose of MVA 4 weeks later (an average of 1000 IFN-gamma spot forming units/million PBMCs). This level of effector T-cell responses appears higher than that seen in previously reported studies of CS-based candidate malaria vaccines.

Safety of recombinant fowlpox strain FP9 and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers.

The ability to generate potent antigen-specific T cell responses by vaccination has been a major hurdle in vaccinology. Vaccinia virus and avipox viruses have been shown to be capable of expressing antigens in mammalian cells and can induce a protective immune response against several mammalian pathogens. We report on two such vaccine constructs, modified vaccinia virus Ankara and FP9 (an attenuated fowlpox virus) both expressing the pre-erythrocytic malaria antigen thrombospondin-related adhesion protein and a string of CD8+ epitopes (ME-TRAP). In prime-boost combinations in a mouse model MVA and FP9 are highly immunogenic and induce substantial protective efficacy. A series of human clinical trials using the recombinant MVA and FP9 malaria vaccines encoding ME-TRAP, both independently and in prime-boost combinations with or without the DNA vaccine DNA ME-TRAP, has shown them to be both immunogenic for CD8+ T cells and capable of inducing protective efficacy. We report here a detailed analysis of the safety profiles of these viral vectors and show that anti-vector antibody responses induced by the vectors are generally low to moderate. We conclude that these vectors are safe and show acceptable side effect profiles for prophylactic vaccination.

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121.

The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture.

Fowlpox virus encodes nonessential homologs of cellular alpha-SNAP, PC-1, and an orphan human homolog of a secreted nematode protein.

The genome of fowlpox virus (FWPV), type species of the Avipoxviridae, is considerably rearranged compared with that of vaccinia virus (the prototypic poxvirus and type species of the Orthopoxviridae) and is 30% larger. It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probably involved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five open reading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been found in poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2 encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in the exocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2. ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematode Caenorhabditis elegans and to a 43-kDa antigen from the parasitic nematode Trichinella spiralis. ORF-5 encodes a homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesterase activity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and are conserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virus replication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late in infection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.

Glutamate-induced apoptosis results in a loss of striatal neurons in the parkinsonian rat.

The motor symptoms of Parkinson's disease are caused by an increase in activity of striatal neurons which project to the globus pallidus. The discharge activity of these striatal cells is normally regulated by a balance between an inhibitory nigral dopamine input and an excitatory cortical glutamate input. The loss of nigrostriatal dopamine in Parkinson's disease allows the cortical glutamatergic input to dominate (see Fig. 1). Pharmacological or surgical manipulations which redress this imbalance in activity in the striatum, or prevent its propagation throughout the basal ganglia, alleviate the motor symptoms of Parkinsonism. We present evidence to suggest the existence of an endogenous mechanism which compensates for the striatal imbalance during the early stages of Parkinsonism. In the rat rendered parkinsonian by systemic administration of reserpine, selective deletion of striatal neurons was observed. The dying striatal neurons exhibited all of the morphological and biochemical hallmarks of apoptosis. This apoptotic cell death was blocked by either administration of glutamate antagonists or decortication. Our data demonstrate that unchecked endogenous glutamate can induce apoptosis of striatal projection neurons in vivo. This observation may have relevance to the neurophysiological mechanisms which maintain the balance of neural activity within the CNS and to the pathology of neurological diseases.