Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8(+) T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8(+) T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of greater than or equal to1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8(+) T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8(+) T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8(+) T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro. (C) 2004 Elsevier Inc. All rights reserved.

Presence of a novel small RNA-containing virus in a laboratory culture of the endoparasitic wasp Venturia canescens (Hymenoptera : Ichneumonidae)

Parasitoid wasps use a variety of mechanisms to alter their host's physiology to the benefit of the developing endoparasite inside the host larva. Association of certain wasps with viruses and virus-like particles (VLPs) that contribute to their success in parasitism is one of the fascinating evolutionary adaptations conferring active or passive protection for the endoparasite from the host immune system. Venturia canescens has been shown to produce VLPs that provide protection for the developing parasitoid egg inside the host, Ephestia kuehniella. Here, we report on the presence of a novel small RNA-containing virus from V. canescens, designated as VcSRV, occurring in the ovaries of the wasp. The virus particles are found together with VcVLPs in the lumen of the calyx region of the ovaries and are injected together with the egg and VcVLPs into E kuehniella larvae where they enter hemocytes. Alignment of the RNA-dependent RNA polymerase gene of VcSRV indicates that the virus most likely belongs to the recently described genus Iflavirus. (c) 2004 Elsevier Ltd. All rights reserved.

Identification of functional sequences in the pregenomic RNA promoter of the Banana streak virus Cavendish strain (BSV-Cav)

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and regain-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-l-like element, the region around-141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease. (C) 2004 Elsevier B.V. All rights reserved.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and anti-viral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Sequence variation in the Newcastle disease virus genome

Full-length genome sequences of five virulent and five avirulent strains of Newcastle disease virus isolated between 1998 and 2002 in Victoria and New South Wales, Australia were determined. Comparisons between these strains revealed that coding sequence variability in the haemagglutinin-neuraminidase (HN), matrix (M) and phosphoprotein (P) gene sequences appeared to be more variable than in the fusion (F), nucleocapsid (N) and RNA dependent-RNA replicase (L) genes. Sequence analysis of a number of other isolates made during the recent virulent NDV outbreaks, also identified the presence of a number of variants with altered F gene cleavage sites, which resulted in altered biological properties of those viruses. Quasispecies analysis of a number of field isolates indicated the presence of virulent virus in one particular isolate. Gene sequence analysis of the progenitor virus isolated in 1998 showed very little sequence variation when compared to that of a progenitor-like virus isolated in 2001 demonstrating that in the field. viral genome sequence variation appears to be biologically restricted to that of a consensus sequence. (c) 2005 Elsevier B.V. All rights reserved.

Evidence of a spotted fever-like rickettsia and a potential new vector from northeastern Australia

A spotted fever-like rickettsia was identified in a Hemaphysalis tick by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA, ompA, and ompB genes. A comparison of these nucleotide sequences with those of other spotted fever group (SFG) rickettsiae revealed that the Hemaphysalis tick rickettsia was distinct from other previously reported strains. Phylogenetic analysis based on both ompA and ompB also indicates that the strain’s closest relatives are the agents of Thai tick typhus (Rickettsia honei strain TT-118) and Flinders Island spotted fever (R. honei). This study represents the first report of an R. honei-like agent from a Hemaphysalis tick in Australia and of a spotted fever group rickettsia from Cape York Peninsula, Queensland.

Francisella-like endosymbionts of ticks

Ticks affect human and animal health both directly by their blood feeding and indirectly by transmission of many disease-causing bacteria, such as Rickettsia, Ehrlichia, Borrelia, Coxiella, Cowdria, Anaplasma, Aegyptionella, and Tularemia, as well as many viruses (Piesman and Gage, 1996). In addition to these infectious agents, ticks harbor bacterial endosymbionts, such as Wolbachia persica, which was first isolated from the soft tick now classified as Argus arboreus (Suitor and Weiss, 1961).

Semliki Forest virus as an expression vector in insect cell lines

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins. (C) 1997 Elsevier Science B.V.

Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies

Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coil with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens. (C) 1998 Academic Press.